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Bio X Cell anti cd40
Interfering with systemic immune responses prevents effective immunotherapy (A) MMTV-PyMT tumor-bearing mice treated with alloIgG, <t>anti-CD40,</t> IFNγ and daily FTY720 or ethanol control starting one day before therapy. (B) Mice with orthotopic 4T1 tumors treated as in A. (C) Immunofluorescence of 4T1 tumors 14 days after therapy. (D–E) 4T1 lung metastases 20 days after therapy. (F) T cells from spleen and lymph nodes of mice with 4T1 tumors, treated with alloIgG, anti-CD40, IFNγ and FTY720 were transferred with IL-2 into naïve Balb/c mice. Controls only received IL-2. Recipients were challenged s.c. with 4T1 cells the next day. (G) MC38 tumor-bearing mice untreated or treated with anti-PD-1 and ethanol control or FTY720. All p-values reflect two-tailed, heteroskedastic t-tests in R. Error bars represent S.D.
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1) Product Images from "Systemic Immunity is Required for Effective Cancer Immunotherapy"

Article Title: Systemic Immunity is Required for Effective Cancer Immunotherapy

Journal: Cell

doi: 10.1016/j.cell.2016.12.022

Interfering with systemic immune responses prevents effective immunotherapy (A) MMTV-PyMT tumor-bearing mice treated with alloIgG, anti-CD40, IFNγ and daily FTY720 or ethanol control starting one day before therapy. (B) Mice with orthotopic 4T1 tumors treated as in A. (C) Immunofluorescence of 4T1 tumors 14 days after therapy. (D–E) 4T1 lung metastases 20 days after therapy. (F) T cells from spleen and lymph nodes of mice with 4T1 tumors, treated with alloIgG, anti-CD40, IFNγ and FTY720 were transferred with IL-2 into naïve Balb/c mice. Controls only received IL-2. Recipients were challenged s.c. with 4T1 cells the next day. (G) MC38 tumor-bearing mice untreated or treated with anti-PD-1 and ethanol control or FTY720. All p-values reflect two-tailed, heteroskedastic t-tests in R. Error bars represent S.D.
Figure Legend Snippet: Interfering with systemic immune responses prevents effective immunotherapy (A) MMTV-PyMT tumor-bearing mice treated with alloIgG, anti-CD40, IFNγ and daily FTY720 or ethanol control starting one day before therapy. (B) Mice with orthotopic 4T1 tumors treated as in A. (C) Immunofluorescence of 4T1 tumors 14 days after therapy. (D–E) 4T1 lung metastases 20 days after therapy. (F) T cells from spleen and lymph nodes of mice with 4T1 tumors, treated with alloIgG, anti-CD40, IFNγ and FTY720 were transferred with IL-2 into naïve Balb/c mice. Controls only received IL-2. Recipients were challenged s.c. with 4T1 cells the next day. (G) MC38 tumor-bearing mice untreated or treated with anti-PD-1 and ethanol control or FTY720. All p-values reflect two-tailed, heteroskedastic t-tests in R. Error bars represent S.D.

Techniques Used: Mouse Assay, Immunofluorescence, Two Tailed Test

2) Product Images from "Protection from tumor recurrence following adoptive immunotherapy varies with host conditioning regimen despite initial regression of autochthonous murine brain tumors"

Article Title: Protection from tumor recurrence following adoptive immunotherapy varies with host conditioning regimen despite initial regression of autochthonous murine brain tumors

Journal: Cancer immunology, immunotherapy : CII

doi: 10.1007/s00262-014-1635-7

Host conditioning with anti-CD40 induces high-level T cell accumulation in the lymphoid organs and brains of SV11 mice at early time points. Groups of mice received either anti-CD40, WBI, or no conditioning regimen with TCR-IV T-cell ACT. Representative plots show MHC tetramer staining (mean±SEM) of TCR-IV T cells on days +4 and +5 in a spleen and c brain. Quantification of TCR-IV T-cell accumulation (mean±SEM) in b spleen and d brain of mice that received ACT with the indicated treatments. n=3 mice/group (except n=2 for day +4 control group). Data shown are from one experiment and representative of two independent experiments. Asterisks above connecting lines indicate significant differences between time points. Asterisks next to vertical brackets indicate significant differences between treatment groups. * p
Figure Legend Snippet: Host conditioning with anti-CD40 induces high-level T cell accumulation in the lymphoid organs and brains of SV11 mice at early time points. Groups of mice received either anti-CD40, WBI, or no conditioning regimen with TCR-IV T-cell ACT. Representative plots show MHC tetramer staining (mean±SEM) of TCR-IV T cells on days +4 and +5 in a spleen and c brain. Quantification of TCR-IV T-cell accumulation (mean±SEM) in b spleen and d brain of mice that received ACT with the indicated treatments. n=3 mice/group (except n=2 for day +4 control group). Data shown are from one experiment and representative of two independent experiments. Asterisks above connecting lines indicate significant differences between time points. Asterisks next to vertical brackets indicate significant differences between treatment groups. * p

Techniques Used: Mouse Assay, Activated Clotting Time Assay, Staining

Anti-CD40-enhanced ACT promotes initial regression of established tumors. a H E brain sections on day +10 post-ACT following conditioning with anti-CD40 (left), control IgG (middle), or WBI (right). Representative low-power images (top row, scale bar = 1mm) and high-power images (bottom row, scale bar = 50μm) that show established tumor refractory to therapy (middle column) or tumor stromal condensation indicative of tumor regression (left and right columns). b For each mouse, the largest cross-sectional tumor area (mm 2 ) observed in H E sections was plotted. Data is pooled from multiple experiments with a total of 6–10 mice/group. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparison test. ** p
Figure Legend Snippet: Anti-CD40-enhanced ACT promotes initial regression of established tumors. a H E brain sections on day +10 post-ACT following conditioning with anti-CD40 (left), control IgG (middle), or WBI (right). Representative low-power images (top row, scale bar = 1mm) and high-power images (bottom row, scale bar = 50μm) that show established tumor refractory to therapy (middle column) or tumor stromal condensation indicative of tumor regression (left and right columns). b For each mouse, the largest cross-sectional tumor area (mm 2 ) observed in H E sections was plotted. Data is pooled from multiple experiments with a total of 6–10 mice/group. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparison test. ** p

Techniques Used: Activated Clotting Time Assay, Mouse Assay

Anti-CD40-enhanced ACT promotes increased survival but short-term surveillance against tumor recurrence. a Groups of mice received the indicated conditioning with or without ACT and were monitored for tumor recurrence. The percentage of surviving mice versus age is plotted. b Statistical differences in survival were calculated using the log-rank test. Data are pooled from multiple experiments with 5–8 mice/group.
Figure Legend Snippet: Anti-CD40-enhanced ACT promotes increased survival but short-term surveillance against tumor recurrence. a Groups of mice received the indicated conditioning with or without ACT and were monitored for tumor recurrence. The percentage of surviving mice versus age is plotted. b Statistical differences in survival were calculated using the log-rank test. Data are pooled from multiple experiments with 5–8 mice/group.

Techniques Used: Activated Clotting Time Assay, Mouse Assay

Donor T cells fail to persist in anti-CD40-conditioned SV11 mice following acute tumor regression. Groups of SV11 mice received either anti-CD40, control IgG, or WBI conditioning prior to ACT with CD90.1 + TCR-IV T cells. On day +30, cells from a spleens, cLN (not shown), and b brains were stained for CD90.1 and CD8. Values on dot plots indicate percent CD90.1 + of total CD8 + cells (mean±SEM). c Total CD90.1 + cells in spleens, cLN, and brains on day +30 are plotted. Representative histograms of CD44, CD62L, and KLRG1 expression gated on live CD45.2 + CD8 + CD90.1 + TCR-IV T cells (open histogram) or CD45.2 + CD8 + CD90.1 − T cells (filled histogram, spleen only) are shown on day +30 in d spleen and e brain. Values indicate the percent of TCR-IV T cells within the indicated gate (mean±SEM). Samples with
Figure Legend Snippet: Donor T cells fail to persist in anti-CD40-conditioned SV11 mice following acute tumor regression. Groups of SV11 mice received either anti-CD40, control IgG, or WBI conditioning prior to ACT with CD90.1 + TCR-IV T cells. On day +30, cells from a spleens, cLN (not shown), and b brains were stained for CD90.1 and CD8. Values on dot plots indicate percent CD90.1 + of total CD8 + cells (mean±SEM). c Total CD90.1 + cells in spleens, cLN, and brains on day +30 are plotted. Representative histograms of CD44, CD62L, and KLRG1 expression gated on live CD45.2 + CD8 + CD90.1 + TCR-IV T cells (open histogram) or CD45.2 + CD8 + CD90.1 − T cells (filled histogram, spleen only) are shown on day +30 in d spleen and e brain. Values indicate the percent of TCR-IV T cells within the indicated gate (mean±SEM). Samples with

Techniques Used: Mouse Assay, Activated Clotting Time Assay, Staining, Expressing

3) Product Images from "Systemic Immunity is Required for Effective Cancer Immunotherapy"

Article Title: Systemic Immunity is Required for Effective Cancer Immunotherapy

Journal: Cell

doi: 10.1016/j.cell.2016.12.022

Interfering with systemic immune responses prevents effective immunotherapy (A) MMTV-PyMT tumor-bearing mice treated with alloIgG, anti-CD40, IFNγ and daily FTY720 or ethanol control starting one day before therapy. (B) Mice with orthotopic 4T1 tumors treated as in A. (C) Immunofluorescence of 4T1 tumors 14 days after therapy. (D–E) 4T1 lung metastases 20 days after therapy. (F) T cells from spleen and lymph nodes of mice with 4T1 tumors, treated with alloIgG, anti-CD40, IFNγ and FTY720 were transferred with IL-2 into naïve Balb/c mice. Controls only received IL-2. Recipients were challenged s.c. with 4T1 cells the next day. (G) MC38 tumor-bearing mice untreated or treated with anti-PD-1 and ethanol control or FTY720. All p-values reflect two-tailed, heteroskedastic t-tests in R. Error bars represent S.D.
Figure Legend Snippet: Interfering with systemic immune responses prevents effective immunotherapy (A) MMTV-PyMT tumor-bearing mice treated with alloIgG, anti-CD40, IFNγ and daily FTY720 or ethanol control starting one day before therapy. (B) Mice with orthotopic 4T1 tumors treated as in A. (C) Immunofluorescence of 4T1 tumors 14 days after therapy. (D–E) 4T1 lung metastases 20 days after therapy. (F) T cells from spleen and lymph nodes of mice with 4T1 tumors, treated with alloIgG, anti-CD40, IFNγ and FTY720 were transferred with IL-2 into naïve Balb/c mice. Controls only received IL-2. Recipients were challenged s.c. with 4T1 cells the next day. (G) MC38 tumor-bearing mice untreated or treated with anti-PD-1 and ethanol control or FTY720. All p-values reflect two-tailed, heteroskedastic t-tests in R. Error bars represent S.D.

Techniques Used: Mouse Assay, Immunofluorescence, Two Tailed Test

4) Product Images from "Concomitant or delayed anti-TNF differentially impact on immune-related adverse events and antitumor efficacy after anti-CD40 therapy"

Article Title: Concomitant or delayed anti-TNF differentially impact on immune-related adverse events and antitumor efficacy after anti-CD40 therapy

Journal: Journal for Immunotherapy of Cancer

doi: 10.1136/jitc-2020-001687

Delayed anti-TNF reduces irAEs while maintaining antitumor efficacy in DT+ anti-CD40 treated MC38 tumor-bearing mice. (A) Schematic representation of the treatment protocol. C57BL/6 FoxP3 DTR mice were injected s.c. with 1×10 6 MC38 tumor cells. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with PBS or DT and 3 days later treated i.p. with cIg, anti-CD40 or anti-CD40+anti TNF (concomitant), for three doses given 3 days apart. In one group, anti-TNF treatment (delayed) was given at the time of the second dose of anti-CD40 and then given for one more dose. (B) Mean tumor size represented as mean±SEM (n=8/group). (C) Individual tumor growth curves of the indicated groups. Data representative of two experiments. (D) Corresponding survival curves. Statistical comparisons between tumor sizes and survival curves were performed with Mann-Whitney analysis at the final measurement of the PBS+ CIg-treated group or log-rank analysis, respectively, *p
Figure Legend Snippet: Delayed anti-TNF reduces irAEs while maintaining antitumor efficacy in DT+ anti-CD40 treated MC38 tumor-bearing mice. (A) Schematic representation of the treatment protocol. C57BL/6 FoxP3 DTR mice were injected s.c. with 1×10 6 MC38 tumor cells. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with PBS or DT and 3 days later treated i.p. with cIg, anti-CD40 or anti-CD40+anti TNF (concomitant), for three doses given 3 days apart. In one group, anti-TNF treatment (delayed) was given at the time of the second dose of anti-CD40 and then given for one more dose. (B) Mean tumor size represented as mean±SEM (n=8/group). (C) Individual tumor growth curves of the indicated groups. Data representative of two experiments. (D) Corresponding survival curves. Statistical comparisons between tumor sizes and survival curves were performed with Mann-Whitney analysis at the final measurement of the PBS+ CIg-treated group or log-rank analysis, respectively, *p

Techniques Used: Mouse Assay, Injection, MANN-WHITNEY

Anti-CD40 induces liver damage and release of inflammatory cytokines within 24 hours of administration. From the same experiments as described in figure 1 , sera from (A) 4T1.2 and (B) MC38 tumor-bearing mice were collected 24 hours after the start of anti-CD40 treatment. ALT activity, TNF, IL-6 and IFN-γ levels were measured. Data pooled from two independent experiments (n=4–7/group/experiment; mean±SEM) with significant differences determined by Kruskal-Wallis test with Dunn’s post hoc analysis, *p
Figure Legend Snippet: Anti-CD40 induces liver damage and release of inflammatory cytokines within 24 hours of administration. From the same experiments as described in figure 1 , sera from (A) 4T1.2 and (B) MC38 tumor-bearing mice were collected 24 hours after the start of anti-CD40 treatment. ALT activity, TNF, IL-6 and IFN-γ levels were measured. Data pooled from two independent experiments (n=4–7/group/experiment; mean±SEM) with significant differences determined by Kruskal-Wallis test with Dunn’s post hoc analysis, *p

Techniques Used: Mouse Assay, Activity Assay

Anti-TNF but not anti-IL-6R reduces physical and biochemical irAEs induced by anti-CD40. (A) Schematic representation of the treatment protocol. (B–D, H) BALB/c or (C–E, I) C57BL/6 FoxP3 DTR mice were injected with 1×10 5 4T1.2 or 1×10 6 MC38 tumor cells, respectively. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with DT and 3 days later treated i.p. with cIg or anti-CD40 and continued for two more doses given 3 days apart. In, some groups, mice were additionally treated with anti-TNF or anti-IL-6R at the same time as anti-CD40. (B, E) Clinical score and (C, F) weight change are shown. (D, G) 24 hours after the start of anti-CD40 treatment, sera were collected and ALT activity was measured. (H–I) From the same sera samples, IL-6, TNF and IFN-γ levels were measured. Data pooled from two experiments for BALB/c and performed once for C57BL/6 (n=5–7/group/experiment; mean±SEM). Kruskal-Wallis test with Dunn’s post hoc analysis was used to compare the indicated groups (clinical score and weight was compared at day 5), *p
Figure Legend Snippet: Anti-TNF but not anti-IL-6R reduces physical and biochemical irAEs induced by anti-CD40. (A) Schematic representation of the treatment protocol. (B–D, H) BALB/c or (C–E, I) C57BL/6 FoxP3 DTR mice were injected with 1×10 5 4T1.2 or 1×10 6 MC38 tumor cells, respectively. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with DT and 3 days later treated i.p. with cIg or anti-CD40 and continued for two more doses given 3 days apart. In, some groups, mice were additionally treated with anti-TNF or anti-IL-6R at the same time as anti-CD40. (B, E) Clinical score and (C, F) weight change are shown. (D, G) 24 hours after the start of anti-CD40 treatment, sera were collected and ALT activity was measured. (H–I) From the same sera samples, IL-6, TNF and IFN-γ levels were measured. Data pooled from two experiments for BALB/c and performed once for C57BL/6 (n=5–7/group/experiment; mean±SEM). Kruskal-Wallis test with Dunn’s post hoc analysis was used to compare the indicated groups (clinical score and weight was compared at day 5), *p

Techniques Used: Mouse Assay, Injection, Activity Assay

Concomitant anti-TNF negatively impacts antitumor efficacy of DT+ anti-CD40 treated MC38 tumor-bearing mice. (A, B) From the same experiments as described in figure 3 (A) 4T1.2 or (B–E) MC38 tumor growth curves. (A) Mean tumor size represented as mean±SEM (n=6/group). Data representative of two experiments. (B) Mean tumor size represented as mean±SEM (n=5–7/group). Experiment performed once. (C, D) In a similar treatment protocol as figure 4B, individual tumor growth curves of MC38 tumor-bearing mice treated with (C) DT+ anti-CD40 or (D) DT+ anti-CD40+ anti-TNF are shown (n=20/group). (E) Corresponding survival of pooled experiments from B to D. Statistical comparisons between the indicated groups were performed with a log-rank analysis, respectively, *p
Figure Legend Snippet: Concomitant anti-TNF negatively impacts antitumor efficacy of DT+ anti-CD40 treated MC38 tumor-bearing mice. (A, B) From the same experiments as described in figure 3 (A) 4T1.2 or (B–E) MC38 tumor growth curves. (A) Mean tumor size represented as mean±SEM (n=6/group). Data representative of two experiments. (B) Mean tumor size represented as mean±SEM (n=5–7/group). Experiment performed once. (C, D) In a similar treatment protocol as figure 4B, individual tumor growth curves of MC38 tumor-bearing mice treated with (C) DT+ anti-CD40 or (D) DT+ anti-CD40+ anti-TNF are shown (n=20/group). (E) Corresponding survival of pooled experiments from B to D. Statistical comparisons between the indicated groups were performed with a log-rank analysis, respectively, *p

Techniques Used: Mouse Assay

Concomitant compared with delayed anti-TNF is more effective at reducing both physical and biochemical irAEs in DT+anti-CD40 treated 4T1.2 tumor-bearing mice. (A) Schematic representation of the treatment protocol. BALB/c FoxP3 DTR mice were injected with 1×10 5 4T1.2 tumor cells. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with PBS or DT and 3 days later treated i.p. with cIg, anti-CD40 or anti-CD40+ anti-TNF (concomitant). One group was treated with anti-TNF 24 hours later (delayed). (B) Weight change and (C) clinical score are shown. (D–F) Indicated organs were taken 96 hours after the start of anti-CD40 treatment. The organs were fixed in paraformaldehyde for 24 hours (colon) or 72 hours (liver), embedded, H E stained and sliced. (D) Representative images from H E-stained sections of colon (scale bar 100 µm), liver (scale bar 300 µm) and necropsy pictures of the liver. Histological (E) colon and (F) liver scores. Data representative of two experiments (n=6–8/group). Kruskal-Wallis test with Dunn’s post hoc analysis (clinical score and weight was compared at day 4), colon and liver scores of indicated groups were compared with the corresponding DT+ cIg treated group, *p
Figure Legend Snippet: Concomitant compared with delayed anti-TNF is more effective at reducing both physical and biochemical irAEs in DT+anti-CD40 treated 4T1.2 tumor-bearing mice. (A) Schematic representation of the treatment protocol. BALB/c FoxP3 DTR mice were injected with 1×10 5 4T1.2 tumor cells. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with PBS or DT and 3 days later treated i.p. with cIg, anti-CD40 or anti-CD40+ anti-TNF (concomitant). One group was treated with anti-TNF 24 hours later (delayed). (B) Weight change and (C) clinical score are shown. (D–F) Indicated organs were taken 96 hours after the start of anti-CD40 treatment. The organs were fixed in paraformaldehyde for 24 hours (colon) or 72 hours (liver), embedded, H E stained and sliced. (D) Representative images from H E-stained sections of colon (scale bar 100 µm), liver (scale bar 300 µm) and necropsy pictures of the liver. Histological (E) colon and (F) liver scores. Data representative of two experiments (n=6–8/group). Kruskal-Wallis test with Dunn’s post hoc analysis (clinical score and weight was compared at day 4), colon and liver scores of indicated groups were compared with the corresponding DT+ cIg treated group, *p

Techniques Used: Mouse Assay, Injection, Staining

Transient Treg depletion and anti-CD40 treatment display different therapeutic index against established MC38 and 4T1.2 tumors. (A) Schematic representation of the treatment protocol. (B) BALB/c or (C) C57BL/6 FoxP3 DTR mice were injected with 1×10 5 4T1.2 or 1×10 6 MC38 tumor cells, respectively. When tumors reached a mean size of 40 mm 2 , mice were treated intraperitoneally (i.p.) with PBS or DT and 3 days later treated i.p. with control IgG (cIg) or anti-CD40. Antibody treatment continued for two more doses given 3 days apart. Mean tumor size (mm 2 ) with their corresponding weight change and clinical score are shown. Data representative of two experiments (n=5–7/group; mean±SEM). Mice were monitored for tumor growth and development of irAEs and euthanized when tumor size or clinical signs of illness reached cumulative ethical limits. Statistical comparisons between tumor sizes were performed with Mann-Whitney U test analysis at the final measurement of the PBS+ CIg-treated group, while a Kruskal-Wallis test with Dunn’s post hoc analysis was used to compare weight change and clinical score between the indicated groups (at day 2), *p
Figure Legend Snippet: Transient Treg depletion and anti-CD40 treatment display different therapeutic index against established MC38 and 4T1.2 tumors. (A) Schematic representation of the treatment protocol. (B) BALB/c or (C) C57BL/6 FoxP3 DTR mice were injected with 1×10 5 4T1.2 or 1×10 6 MC38 tumor cells, respectively. When tumors reached a mean size of 40 mm 2 , mice were treated intraperitoneally (i.p.) with PBS or DT and 3 days later treated i.p. with control IgG (cIg) or anti-CD40. Antibody treatment continued for two more doses given 3 days apart. Mean tumor size (mm 2 ) with their corresponding weight change and clinical score are shown. Data representative of two experiments (n=5–7/group; mean±SEM). Mice were monitored for tumor growth and development of irAEs and euthanized when tumor size or clinical signs of illness reached cumulative ethical limits. Statistical comparisons between tumor sizes were performed with Mann-Whitney U test analysis at the final measurement of the PBS+ CIg-treated group, while a Kruskal-Wallis test with Dunn’s post hoc analysis was used to compare weight change and clinical score between the indicated groups (at day 2), *p

Techniques Used: Mouse Assay, Injection, MANN-WHITNEY

5) Product Images from "TFH-derived dopamine accelerates productive synapses in germinal centres"

Article Title: TFH-derived dopamine accelerates productive synapses in germinal centres

Journal: Nature

doi: 10.1038/nature23013

Effect of ICOSL on CD40L presentation and reception in SLB model for T FH cell- GC B cell interaction. a , Activated human T cells that express ICOS and CD40L were incubated with SLB containing ICAM-1 and UCHT1 (anti-CD3) as a basal condition with a ring of ICAM-1 surrounding a central cluster enriched in T cell receptor enriched extracellular vesicles by 15 minutes 26 . This condition resulted in low presentation of CD40L in punctate structures detected by anti-CD40L mAb that accumulated in the same central synapse with the TCR enriched extracellular vesicles. Addition of ICOSL the SLB resulted in strong central accumulation of fluorescent ICOSL with the TCR enriched extracellular vesicles, but no increase in CD40L presentation. Addition of CD40 the SLB resulted in a significant increase in CD40L accumulation, which we refer to as reception because its receptor dependent. When ICOSL and CD40 were added the reception of CD40L was further significantly enhanced over the level observed with CD40 alone. Thus, ICOSL ligation in the centre of the immunological synapse increases CD40L reception. All levels are shown in gray scale except CD40L panels, for which the pseudocolor scale is indicated. Scale bar 5 µm. b , Human T FH cells were incubated with SLB containing ICAM-1 and UCHT1 (anti-CD3). Addition of ICOSL resulted in increased accumulation of CgB at the synapse centre. Addition of CD40 did not further increased CgB accumulation.
Figure Legend Snippet: Effect of ICOSL on CD40L presentation and reception in SLB model for T FH cell- GC B cell interaction. a , Activated human T cells that express ICOS and CD40L were incubated with SLB containing ICAM-1 and UCHT1 (anti-CD3) as a basal condition with a ring of ICAM-1 surrounding a central cluster enriched in T cell receptor enriched extracellular vesicles by 15 minutes 26 . This condition resulted in low presentation of CD40L in punctate structures detected by anti-CD40L mAb that accumulated in the same central synapse with the TCR enriched extracellular vesicles. Addition of ICOSL the SLB resulted in strong central accumulation of fluorescent ICOSL with the TCR enriched extracellular vesicles, but no increase in CD40L presentation. Addition of CD40 the SLB resulted in a significant increase in CD40L accumulation, which we refer to as reception because its receptor dependent. When ICOSL and CD40 were added the reception of CD40L was further significantly enhanced over the level observed with CD40 alone. Thus, ICOSL ligation in the centre of the immunological synapse increases CD40L reception. All levels are shown in gray scale except CD40L panels, for which the pseudocolor scale is indicated. Scale bar 5 µm. b , Human T FH cells were incubated with SLB containing ICAM-1 and UCHT1 (anti-CD3). Addition of ICOSL resulted in increased accumulation of CgB at the synapse centre. Addition of CD40 did not further increased CgB accumulation.

Techniques Used: Incubation, Ligation

Dopamine is released from T FH cells upon cognate interactions. a-c Flow cytometric quantification of dopamine content in FSK-stimulated T FH cells after 30 min incubation with anti-CD3/CD28 beads (1:1) or autologous or allogeneic GC B cells (1:2) (n=3) ( b ) also showing changes in DA content in GC B cells (autologous or allogeneic) cultured separately (“nil”), or together with T FH cells (n=5); and with or without ICAM-1 (5 μg/ml) and LFA-1 (10 μg/ml) block ( c ) (n=3); Mann-Whitney test. d , Flow cytometric plots showing plasma cells (PCs), identified as CD27 hi CD38 hi , induced in cultures of GC B cells stimulated for five days with anti-CD40 (2 μg/ml), IL-21 (20 ng/ml) and different concentrations of freshly-prepared DA (n=5). e , Fold changes in PC differentiation from GC B cells stimulated for 2h with or without DA (5μM) and Haloperidol (Haldol, 50nM), and cultured in the presence of anti-CD40 (2 μg/ml) and IL-21 (20 ng/ml) for 5d; two tailed student t-test. a-c, e: Bars represent median values and each dot represents a single experiment conducted in triplicates (n=5). Two tailed student t-test; ns, not significant, *p ≤ 0.05, ***p ≤ 0.001.
Figure Legend Snippet: Dopamine is released from T FH cells upon cognate interactions. a-c Flow cytometric quantification of dopamine content in FSK-stimulated T FH cells after 30 min incubation with anti-CD3/CD28 beads (1:1) or autologous or allogeneic GC B cells (1:2) (n=3) ( b ) also showing changes in DA content in GC B cells (autologous or allogeneic) cultured separately (“nil”), or together with T FH cells (n=5); and with or without ICAM-1 (5 μg/ml) and LFA-1 (10 μg/ml) block ( c ) (n=3); Mann-Whitney test. d , Flow cytometric plots showing plasma cells (PCs), identified as CD27 hi CD38 hi , induced in cultures of GC B cells stimulated for five days with anti-CD40 (2 μg/ml), IL-21 (20 ng/ml) and different concentrations of freshly-prepared DA (n=5). e , Fold changes in PC differentiation from GC B cells stimulated for 2h with or without DA (5μM) and Haloperidol (Haldol, 50nM), and cultured in the presence of anti-CD40 (2 μg/ml) and IL-21 (20 ng/ml) for 5d; two tailed student t-test. a-c, e: Bars represent median values and each dot represents a single experiment conducted in triplicates (n=5). Two tailed student t-test; ns, not significant, *p ≤ 0.05, ***p ≤ 0.001.

Techniques Used: Flow Cytometry, Incubation, Cell Culture, Blocking Assay, MANN-WHITNEY, Two Tailed Test

Dopamine induces ICOSL upregulation on human GC B cells. a , Gating of GC B cells and fluorescence intensity of specified proteins 30 minutes after stimulation with DA (10μM) (n=3). b , Fold changes of surface ICOSL expression with medium control set as unit 1 (n=8). c , Survival of GC B cells after DA stimulation (n=8). d , Fold changes of surface ICOSL expression on GC B cells stimulated with DA (10μM), DA agonist SKF38393 (10nM), Haloperidol (50nM) and DA antagonist SKF83566 (10nM) for 30 min, with medium control set as unit 1 (n=5). e , f , Representative histograms ( e ) and quantification ( f ) of surface and intracellular ICOSL on naïve, memory and GC B cells (n=4); Mann-Whitney test. g , RNA counts per million (CPM) of indicated transcripts in human GC B cells stimulated with or without DA (5μM) for 2h (n=3). h , Fold changes of surface ICOSL expression on human GC B cells treated with cycloheximide (CHX, 10 μg/ml) and stimulated with DA (10μM) for 30 min. i, j, Fold changes of surface ICOSL expression on human GC B cells stimulated with DA (10μM), anti-CD40 (1 μg/ml) or recombinant CD40L (10 μg/ml) ( i ), IL-21 (10, 50 or 100 ng/ml) or IL-4 (10 νγ/ml) ( j ) for 30 min (n=5). b, d, h-j , Bars represent medians and each dot represents a single experiment conducted in triplicate (n=10); two tailed student t-test. ns, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001.
Figure Legend Snippet: Dopamine induces ICOSL upregulation on human GC B cells. a , Gating of GC B cells and fluorescence intensity of specified proteins 30 minutes after stimulation with DA (10μM) (n=3). b , Fold changes of surface ICOSL expression with medium control set as unit 1 (n=8). c , Survival of GC B cells after DA stimulation (n=8). d , Fold changes of surface ICOSL expression on GC B cells stimulated with DA (10μM), DA agonist SKF38393 (10nM), Haloperidol (50nM) and DA antagonist SKF83566 (10nM) for 30 min, with medium control set as unit 1 (n=5). e , f , Representative histograms ( e ) and quantification ( f ) of surface and intracellular ICOSL on naïve, memory and GC B cells (n=4); Mann-Whitney test. g , RNA counts per million (CPM) of indicated transcripts in human GC B cells stimulated with or without DA (5μM) for 2h (n=3). h , Fold changes of surface ICOSL expression on human GC B cells treated with cycloheximide (CHX, 10 μg/ml) and stimulated with DA (10μM) for 30 min. i, j, Fold changes of surface ICOSL expression on human GC B cells stimulated with DA (10μM), anti-CD40 (1 μg/ml) or recombinant CD40L (10 μg/ml) ( i ), IL-21 (10, 50 or 100 ng/ml) or IL-4 (10 νγ/ml) ( j ) for 30 min (n=5). b, d, h-j , Bars represent medians and each dot represents a single experiment conducted in triplicate (n=10); two tailed student t-test. ns, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001.

Techniques Used: Fluorescence, Expressing, MANN-WHITNEY, Recombinant, Two Tailed Test

Regulation of ICOSL upregulation in mouse and human B cells a, Fold changes of surface ICOSL expression on mouse GC B cells that were treated with anti-CD40 (10 μg/ml) and DA (0.5, 1, 5, 10μM) for 30 minutes, with medium control set as unit 1 (n=5). b, Representative histogram and quantification of surface and intracellular ICOSL on GC and non-GC B cells (n=5). **p ≤ 0.01; nonparametric Mann-Whitney test (U test). c, RNA counts per million of ICOSL, CD40, BCL6, IL21R, CD86, BAFFR and FAS mRNA in human memory B cells stimulated with or without DA (5μM) for 2h (n=3). d , Fold changes of surface ICOSL expression on mouse GC B cells that were treated with cycloheximide (CHX, 10 μg/ml) for 4h, with medium control set as unit 1. Bars represent median values and each dot represents a single mouse. e, Fold changes of surface ICOSL expression on mouse GC B cells that were stimulated with BAFF (100ng/ml), LPS (1 or 10 μg/ml), anti-CD40 (10 μg/ml) and anti-IgM (1 or 10 μg/ml) for 30 min and 4h. Unit 1 set on medium control. f, Fold changes of surface ICOSL expression on mouse GC B cells that were treated with actinomycin D (ActD, 5 μg/ml), anti-CD40 (10 μg/ml) for 4h, with medium control set as unit 1. Bars represent median and each dot represent a single mouse (n=5). d-f , ns, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001; two tailed student t-test. g , Representative histogram of surface ICOSL expression on human GC B cells that were stimulated with DA (10μM) or anti-CD40 (1 μg/ml) for 30 min. h , Fold changes of surface ICOSL expression on human GC B cells stimulated with several concentrations of anti-CD40 for 4 and 8 hours, with medium control set as unit 1 (n=3). i , Bar plot showing survival of GC B in the presence of anti-CD40 (1 μg/ml) after 4 or 8 hours of stimulation (n=4). *p ≤ 0.05 and ***p ≤ 0.001; nonparametric Mann-Whitney test (U test).
Figure Legend Snippet: Regulation of ICOSL upregulation in mouse and human B cells a, Fold changes of surface ICOSL expression on mouse GC B cells that were treated with anti-CD40 (10 μg/ml) and DA (0.5, 1, 5, 10μM) for 30 minutes, with medium control set as unit 1 (n=5). b, Representative histogram and quantification of surface and intracellular ICOSL on GC and non-GC B cells (n=5). **p ≤ 0.01; nonparametric Mann-Whitney test (U test). c, RNA counts per million of ICOSL, CD40, BCL6, IL21R, CD86, BAFFR and FAS mRNA in human memory B cells stimulated with or without DA (5μM) for 2h (n=3). d , Fold changes of surface ICOSL expression on mouse GC B cells that were treated with cycloheximide (CHX, 10 μg/ml) for 4h, with medium control set as unit 1. Bars represent median values and each dot represents a single mouse. e, Fold changes of surface ICOSL expression on mouse GC B cells that were stimulated with BAFF (100ng/ml), LPS (1 or 10 μg/ml), anti-CD40 (10 μg/ml) and anti-IgM (1 or 10 μg/ml) for 30 min and 4h. Unit 1 set on medium control. f, Fold changes of surface ICOSL expression on mouse GC B cells that were treated with actinomycin D (ActD, 5 μg/ml), anti-CD40 (10 μg/ml) for 4h, with medium control set as unit 1. Bars represent median and each dot represent a single mouse (n=5). d-f , ns, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001; two tailed student t-test. g , Representative histogram of surface ICOSL expression on human GC B cells that were stimulated with DA (10μM) or anti-CD40 (1 μg/ml) for 30 min. h , Fold changes of surface ICOSL expression on human GC B cells stimulated with several concentrations of anti-CD40 for 4 and 8 hours, with medium control set as unit 1 (n=3). i , Bar plot showing survival of GC B in the presence of anti-CD40 (1 μg/ml) after 4 or 8 hours of stimulation (n=4). *p ≤ 0.05 and ***p ≤ 0.001; nonparametric Mann-Whitney test (U test).

Techniques Used: Expressing, MANN-WHITNEY, Mann-Whitney U-Test, Two Tailed Test

Effects of ICOS ligation at the immunological synapse. a , Representative images of ICAM-1 ring (white) around CD40L (pseudocolor scale) in the presence or absence of CD40 and ICOSL at physiological densities on the supported lipid bilayer (SLB) containing ICAM-1 and UCHT1. Scale bar 5 µm. b , c , Plots represent CD40L MFI of individual activated human T ( b ) or T FH ( c ) cells forming synapses (n=3). d , Representative images of chromogranin B stain in the presence or absence of ICOSL at the immunological synapse. e , Plots represent CgB fluorescent intensity of individual activated T FH and non-T FH cells forming synapses (n=3). b,c,e , ns, not significant, (***p ≤ 0.001) and (****p ≤ 0.0001) nonparametric Mann-Whitney test (U test). f , Representative images of CgB + T FH cells (red) forming synapses with allogeneic B cells (green).
Figure Legend Snippet: Effects of ICOS ligation at the immunological synapse. a , Representative images of ICAM-1 ring (white) around CD40L (pseudocolor scale) in the presence or absence of CD40 and ICOSL at physiological densities on the supported lipid bilayer (SLB) containing ICAM-1 and UCHT1. Scale bar 5 µm. b , c , Plots represent CD40L MFI of individual activated human T ( b ) or T FH ( c ) cells forming synapses (n=3). d , Representative images of chromogranin B stain in the presence or absence of ICOSL at the immunological synapse. e , Plots represent CgB fluorescent intensity of individual activated T FH and non-T FH cells forming synapses (n=3). b,c,e , ns, not significant, (***p ≤ 0.001) and (****p ≤ 0.0001) nonparametric Mann-Whitney test (U test). f , Representative images of CgB + T FH cells (red) forming synapses with allogeneic B cells (green).

Techniques Used: Ligation, Staining, MANN-WHITNEY, Mann-Whitney U-Test

6) Product Images from "ISCOMATRIX vaccines mediate CD8+ T-cell cross-priming by a MyD88-dependent signaling pathway"

Article Title: ISCOMATRIX vaccines mediate CD8+ T-cell cross-priming by a MyD88-dependent signaling pathway

Journal: Immunology and Cell Biology

doi: 10.1038/icb.2011.71

In vivo DC activation and cytokine responses to ISCOMATRIX adjuvant administration. ( a ) Flt3L-derived DCs were cultured overnight in the presence of IMX (5 μg ml −1 ), CpG (1 μ M ) or LPS (1 μg ml −1 ). CD40, CD69, CD80 and CD86 upregulation (black lines) was monitored on conventional (CD11c + CD45RA − ) DCs by flow cytometry, as compared with an isotype control antibody (dashed lines). ( b ) CD8 and double-negative (DN) lymphoid DCs were distinguished from ‘tissue-derived' MigDCs based on the expression of CD8 and CD205 (left dot plot). 53 Right histogram; expression of CD40, CD80, CD86 and MHC class II expression (black lines) on DCs following a single subcutaneous dose of ISCOMATRIX adjuvant, as compared with DCs isolated from untreated DLN (gray lines). Isotype control antibody staining is shown as dashed lines. Data are representative of 3–5 experiments. ( c ) Activation marker expression by CD8 DCs isolated from the DLN of untreated mice, or 24 h after a subcutaneous dose of IMX (5 μg) or LPS (3 μg). The mean linear fluorescence (MLF) is shown on the y-axis, with isotype controls MLF values subtracted for each sample. ( d ) Cytokine levels in the serum collected 6 or 24 h after subcutaneous IMX or LPS administration: shown are the levels of IL—1β,IL-5,IL-6, IL-10, IL-12/23(p40), granulocyte-CSF, keratinocyte chemoattractant (KC or CXCL1), monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1α (MIP-1α or CCL3) and β (MIP-1β or CCL4), Rantes (or C) and IFN-γ. Error bars show the s.e.m. ( n =6 mice per treatment group). Student's t -test was used to calculate statistical significance ( * P
Figure Legend Snippet: In vivo DC activation and cytokine responses to ISCOMATRIX adjuvant administration. ( a ) Flt3L-derived DCs were cultured overnight in the presence of IMX (5 μg ml −1 ), CpG (1 μ M ) or LPS (1 μg ml −1 ). CD40, CD69, CD80 and CD86 upregulation (black lines) was monitored on conventional (CD11c + CD45RA − ) DCs by flow cytometry, as compared with an isotype control antibody (dashed lines). ( b ) CD8 and double-negative (DN) lymphoid DCs were distinguished from ‘tissue-derived' MigDCs based on the expression of CD8 and CD205 (left dot plot). 53 Right histogram; expression of CD40, CD80, CD86 and MHC class II expression (black lines) on DCs following a single subcutaneous dose of ISCOMATRIX adjuvant, as compared with DCs isolated from untreated DLN (gray lines). Isotype control antibody staining is shown as dashed lines. Data are representative of 3–5 experiments. ( c ) Activation marker expression by CD8 DCs isolated from the DLN of untreated mice, or 24 h after a subcutaneous dose of IMX (5 μg) or LPS (3 μg). The mean linear fluorescence (MLF) is shown on the y-axis, with isotype controls MLF values subtracted for each sample. ( d ) Cytokine levels in the serum collected 6 or 24 h after subcutaneous IMX or LPS administration: shown are the levels of IL—1β,IL-5,IL-6, IL-10, IL-12/23(p40), granulocyte-CSF, keratinocyte chemoattractant (KC or CXCL1), monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1α (MIP-1α or CCL3) and β (MIP-1β or CCL4), Rantes (or C) and IFN-γ. Error bars show the s.e.m. ( n =6 mice per treatment group). Student's t -test was used to calculate statistical significance ( * P

Techniques Used: In Vivo, Activation Assay, Derivative Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing, Isolation, Staining, Marker, Mouse Assay, Fluorescence

ISCOMATRIX vaccines are dependent on a MyD88-signaling axis in vivo ( a ) CD8 + T-cell responses were compared in wild type (WT) and MyD88-deficient mice (MyD88 KO) vaccinated with an ISCOMATRIX vaccine on day −7, 0, with the magnitude of the CD8 + T-cell response shown relative to WT mice. ( b , c ) Same as in ( a ) except the CD8 + T-cell response was evaluated in TRIF or TLR4-deficient mice. ( d ) Purified CD8 and MigDCs from wild type or MyD88-deficient (KO) mice were isolated from the DLN 24 h after vaccine administration. MHC class I cross-presentation was assessed by co-culturing each population with 5 × 10 4 CFSE-labeled OT-1 cells and quantifying proliferation 60 h later, as described. ( e ) CD40, CD80 and CD86 expression (black lines) was assessed for CD8 DCs isolated from the DLN of WT or MyD88 KO mice dosed with ISCOMARTIX adjuvant, compared with CD8 DCs from untreated WT mice (gray lines). Dashed lines illustrate the median fluorescence for each marker. ( f ) Schematic illustrating the interaction between DCs, T cells and NK cells in the DLN following ISCOMATRIX vaccine delivery. ISCOMATRIX vaccines initiate a localized inflammatory response at the subcutaneous injection site, and efficient DC activation and MHC class I cross-presentation in the DLN (MyD88-independent). Although the precise DC activation signal(s) is currently unknown, a distinct pro-inflammatory milieu was detected locally and systemically following ISCOMATIRX adjuvant administration. In the DLN, NK cell activation and CD8 + T-cell cross-priming was dependent on DCs, as well as an intact MyD88 signaling network. Cross-primed CD8 + T cells exhibit potent antitumor activity in prophylactic tumor challenge models. However, in the case of pre-established tumor burden, the effectiveness of the vaccine is likely to be blunted by immune suppressive networks, such as myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg) and tumor-derived factors that prevent complete tumor eradication.
Figure Legend Snippet: ISCOMATRIX vaccines are dependent on a MyD88-signaling axis in vivo ( a ) CD8 + T-cell responses were compared in wild type (WT) and MyD88-deficient mice (MyD88 KO) vaccinated with an ISCOMATRIX vaccine on day −7, 0, with the magnitude of the CD8 + T-cell response shown relative to WT mice. ( b , c ) Same as in ( a ) except the CD8 + T-cell response was evaluated in TRIF or TLR4-deficient mice. ( d ) Purified CD8 and MigDCs from wild type or MyD88-deficient (KO) mice were isolated from the DLN 24 h after vaccine administration. MHC class I cross-presentation was assessed by co-culturing each population with 5 × 10 4 CFSE-labeled OT-1 cells and quantifying proliferation 60 h later, as described. ( e ) CD40, CD80 and CD86 expression (black lines) was assessed for CD8 DCs isolated from the DLN of WT or MyD88 KO mice dosed with ISCOMARTIX adjuvant, compared with CD8 DCs from untreated WT mice (gray lines). Dashed lines illustrate the median fluorescence for each marker. ( f ) Schematic illustrating the interaction between DCs, T cells and NK cells in the DLN following ISCOMATRIX vaccine delivery. ISCOMATRIX vaccines initiate a localized inflammatory response at the subcutaneous injection site, and efficient DC activation and MHC class I cross-presentation in the DLN (MyD88-independent). Although the precise DC activation signal(s) is currently unknown, a distinct pro-inflammatory milieu was detected locally and systemically following ISCOMATIRX adjuvant administration. In the DLN, NK cell activation and CD8 + T-cell cross-priming was dependent on DCs, as well as an intact MyD88 signaling network. Cross-primed CD8 + T cells exhibit potent antitumor activity in prophylactic tumor challenge models. However, in the case of pre-established tumor burden, the effectiveness of the vaccine is likely to be blunted by immune suppressive networks, such as myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg) and tumor-derived factors that prevent complete tumor eradication.

Techniques Used: In Vivo, Mouse Assay, Purification, Isolation, Labeling, Expressing, Fluorescence, Marker, Injection, Activation Assay, Activity Assay, Derivative Assay

7) Product Images from "CD70 encoded by modified vaccinia virus Ankara enhances CD8 T‐cell‐dependent protective immunity in MHC class II‐deficient mice"

Article Title: CD70 encoded by modified vaccinia virus Ankara enhances CD8 T‐cell‐dependent protective immunity in MHC class II‐deficient mice

Journal: Immunology

doi: 10.1111/imm.12884

Up‐regulation of CD 86 and CD 70 on splenic antigen‐presenting cells ( APC s) after modified vaccinia virus Ankara ( MVA ) immunization. C57 BL /6 mice were immunized intravenously (i.v.) with 5 × 10 7 TCID 50 modified vaccinia virus Ankara–Bavarian Nordic ® ( MVA ‐ BN ® ) or pIC + anti‐ CD 40. Expression of CD 86 and CD 70 on splenic APC subsets was analysed after 41 hr by 11‐colour flow cytometry. Cells were gated as shown in the exemplary dot plots (a) and were defined as follows: plasmacytoid dendritic cells (live CD 90.2 − CD 335 − CD 45R + CD 317 + ), B cells (live CD 90.2 − CD 335 − CD 45R + CD 317 − ), conventional dendritic cells (live CD 90.2 − CD 335 − CD 45R − CD 317 − CD 11c + ), CD 8 + conventional dendritic cells (live CD 90.2 − CD 335 − CD 45R − CD 317 − CD 11c + CD 172a − CD 8α + ), CD 8 − conventional dendritic cells (live CD 90.2 − CD 335 − CD 45R − CD 317 − CD 11c + CD 172a + CD 8α − ), CD 11c − CD 11b + ((live CD 90.2 − CD 335 − CD 45R − CD 317 − CD 11c − CD 11b + ) and CD 11c − CD 11b − (live CD 90.2 − CD 335 − CD 45R − CD 317 − CD 11c − CD 11b − ). (b) Bar graphs show the mean fluorescence intensity ( MFI ) ± SEM of CD 86 and the relative (r) MFI of CD 70 of three mice per group. The rMFI was calculated by subtracting the MFI of the fluorescence‐minus‐one control from the MFI of stained samples. Data are representative of three independent experiments for MVA ‐ BN ® and one for pIC + anti‐CD40.
Figure Legend Snippet: Up‐regulation of CD 86 and CD 70 on splenic antigen‐presenting cells ( APC s) after modified vaccinia virus Ankara ( MVA ) immunization. C57 BL /6 mice were immunized intravenously (i.v.) with 5 × 10 7 TCID 50 modified vaccinia virus Ankara–Bavarian Nordic ® ( MVA ‐ BN ® ) or pIC + anti‐ CD 40. Expression of CD 86 and CD 70 on splenic APC subsets was analysed after 41 hr by 11‐colour flow cytometry. Cells were gated as shown in the exemplary dot plots (a) and were defined as follows: plasmacytoid dendritic cells (live CD 90.2 − CD 335 − CD 45R + CD 317 + ), B cells (live CD 90.2 − CD 335 − CD 45R + CD 317 − ), conventional dendritic cells (live CD 90.2 − CD 335 − CD 45R − CD 317 − CD 11c + ), CD 8 + conventional dendritic cells (live CD 90.2 − CD 335 − CD 45R − CD 317 − CD 11c + CD 172a − CD 8α + ), CD 8 − conventional dendritic cells (live CD 90.2 − CD 335 − CD 45R − CD 317 − CD 11c + CD 172a + CD 8α − ), CD 11c − CD 11b + ((live CD 90.2 − CD 335 − CD 45R − CD 317 − CD 11c − CD 11b + ) and CD 11c − CD 11b − (live CD 90.2 − CD 335 − CD 45R − CD 317 − CD 11c − CD 11b − ). (b) Bar graphs show the mean fluorescence intensity ( MFI ) ± SEM of CD 86 and the relative (r) MFI of CD 70 of three mice per group. The rMFI was calculated by subtracting the MFI of the fluorescence‐minus‐one control from the MFI of stained samples. Data are representative of three independent experiments for MVA ‐ BN ® and one for pIC + anti‐CD40.

Techniques Used: Modification, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Fluorescence, Staining

8) Product Images from "Concomitant or delayed anti-TNF differentially impact on immune-related adverse events and antitumor efficacy after anti-CD40 therapy"

Article Title: Concomitant or delayed anti-TNF differentially impact on immune-related adverse events and antitumor efficacy after anti-CD40 therapy

Journal: Journal for Immunotherapy of Cancer

doi: 10.1136/jitc-2020-001687

Delayed anti-TNF reduces irAEs while maintaining antitumor efficacy in DT+ anti-CD40 treated MC38 tumor-bearing mice. (A) Schematic representation of the treatment protocol. C57BL/6 FoxP3 DTR mice were injected s.c. with 1×10 6 MC38 tumor cells. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with PBS or DT and 3 days later treated i.p. with cIg, anti-CD40 or anti-CD40+anti TNF (concomitant), for three doses given 3 days apart. In one group, anti-TNF treatment (delayed) was given at the time of the second dose of anti-CD40 and then given for one more dose. (B) Mean tumor size represented as mean±SEM (n=8/group). (C) Individual tumor growth curves of the indicated groups. Data representative of two experiments. (D) Corresponding survival curves. Statistical comparisons between tumor sizes and survival curves were performed with Mann-Whitney analysis at the final measurement of the PBS+ CIg-treated group or log-rank analysis, respectively, *p
Figure Legend Snippet: Delayed anti-TNF reduces irAEs while maintaining antitumor efficacy in DT+ anti-CD40 treated MC38 tumor-bearing mice. (A) Schematic representation of the treatment protocol. C57BL/6 FoxP3 DTR mice were injected s.c. with 1×10 6 MC38 tumor cells. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with PBS or DT and 3 days later treated i.p. with cIg, anti-CD40 or anti-CD40+anti TNF (concomitant), for three doses given 3 days apart. In one group, anti-TNF treatment (delayed) was given at the time of the second dose of anti-CD40 and then given for one more dose. (B) Mean tumor size represented as mean±SEM (n=8/group). (C) Individual tumor growth curves of the indicated groups. Data representative of two experiments. (D) Corresponding survival curves. Statistical comparisons between tumor sizes and survival curves were performed with Mann-Whitney analysis at the final measurement of the PBS+ CIg-treated group or log-rank analysis, respectively, *p

Techniques Used: Mouse Assay, Injection, MANN-WHITNEY

Anti-CD40 induces liver damage and release of inflammatory cytokines within 24 hours of administration. From the same experiments as described in figure 1 , sera from (A) 4T1.2 and (B) MC38 tumor-bearing mice were collected 24 hours after the start of anti-CD40 treatment. ALT activity, TNF, IL-6 and IFN-γ levels were measured. Data pooled from two independent experiments (n=4–7/group/experiment; mean±SEM) with significant differences determined by Kruskal-Wallis test with Dunn’s post hoc analysis, *p
Figure Legend Snippet: Anti-CD40 induces liver damage and release of inflammatory cytokines within 24 hours of administration. From the same experiments as described in figure 1 , sera from (A) 4T1.2 and (B) MC38 tumor-bearing mice were collected 24 hours after the start of anti-CD40 treatment. ALT activity, TNF, IL-6 and IFN-γ levels were measured. Data pooled from two independent experiments (n=4–7/group/experiment; mean±SEM) with significant differences determined by Kruskal-Wallis test with Dunn’s post hoc analysis, *p

Techniques Used: Mouse Assay, Activity Assay

Anti-TNF but not anti-IL-6R reduces physical and biochemical irAEs induced by anti-CD40. (A) Schematic representation of the treatment protocol. (B–D, H) BALB/c or (C–E, I) C57BL/6 FoxP3 DTR mice were injected with 1×10 5 4T1.2 or 1×10 6 MC38 tumor cells, respectively. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with DT and 3 days later treated i.p. with cIg or anti-CD40 and continued for two more doses given 3 days apart. In, some groups, mice were additionally treated with anti-TNF or anti-IL-6R at the same time as anti-CD40. (B, E) Clinical score and (C, F) weight change are shown. (D, G) 24 hours after the start of anti-CD40 treatment, sera were collected and ALT activity was measured. (H–I) From the same sera samples, IL-6, TNF and IFN-γ levels were measured. Data pooled from two experiments for BALB/c and performed once for C57BL/6 (n=5–7/group/experiment; mean±SEM). Kruskal-Wallis test with Dunn’s post hoc analysis was used to compare the indicated groups (clinical score and weight was compared at day 5), *p
Figure Legend Snippet: Anti-TNF but not anti-IL-6R reduces physical and biochemical irAEs induced by anti-CD40. (A) Schematic representation of the treatment protocol. (B–D, H) BALB/c or (C–E, I) C57BL/6 FoxP3 DTR mice were injected with 1×10 5 4T1.2 or 1×10 6 MC38 tumor cells, respectively. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with DT and 3 days later treated i.p. with cIg or anti-CD40 and continued for two more doses given 3 days apart. In, some groups, mice were additionally treated with anti-TNF or anti-IL-6R at the same time as anti-CD40. (B, E) Clinical score and (C, F) weight change are shown. (D, G) 24 hours after the start of anti-CD40 treatment, sera were collected and ALT activity was measured. (H–I) From the same sera samples, IL-6, TNF and IFN-γ levels were measured. Data pooled from two experiments for BALB/c and performed once for C57BL/6 (n=5–7/group/experiment; mean±SEM). Kruskal-Wallis test with Dunn’s post hoc analysis was used to compare the indicated groups (clinical score and weight was compared at day 5), *p

Techniques Used: Mouse Assay, Injection, Activity Assay

Concomitant anti-TNF negatively impacts antitumor efficacy of DT+ anti-CD40 treated MC38 tumor-bearing mice. (A, B) From the same experiments as described in figure 3 (A) 4T1.2 or (B–E) MC38 tumor growth curves. (A) Mean tumor size represented as mean±SEM (n=6/group). Data representative of two experiments. (B) Mean tumor size represented as mean±SEM (n=5–7/group). Experiment performed once. (C, D) In a similar treatment protocol as figure 4B, individual tumor growth curves of MC38 tumor-bearing mice treated with (C) DT+ anti-CD40 or (D) DT+ anti-CD40+ anti-TNF are shown (n=20/group). (E) Corresponding survival of pooled experiments from B to D. Statistical comparisons between the indicated groups were performed with a log-rank analysis, respectively, *p
Figure Legend Snippet: Concomitant anti-TNF negatively impacts antitumor efficacy of DT+ anti-CD40 treated MC38 tumor-bearing mice. (A, B) From the same experiments as described in figure 3 (A) 4T1.2 or (B–E) MC38 tumor growth curves. (A) Mean tumor size represented as mean±SEM (n=6/group). Data representative of two experiments. (B) Mean tumor size represented as mean±SEM (n=5–7/group). Experiment performed once. (C, D) In a similar treatment protocol as figure 4B, individual tumor growth curves of MC38 tumor-bearing mice treated with (C) DT+ anti-CD40 or (D) DT+ anti-CD40+ anti-TNF are shown (n=20/group). (E) Corresponding survival of pooled experiments from B to D. Statistical comparisons between the indicated groups were performed with a log-rank analysis, respectively, *p

Techniques Used: Mouse Assay

Concomitant compared with delayed anti-TNF is more effective at reducing both physical and biochemical irAEs in DT+anti-CD40 treated 4T1.2 tumor-bearing mice. (A) Schematic representation of the treatment protocol. BALB/c FoxP3 DTR mice were injected with 1×10 5 4T1.2 tumor cells. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with PBS or DT and 3 days later treated i.p. with cIg, anti-CD40 or anti-CD40+ anti-TNF (concomitant). One group was treated with anti-TNF 24 hours later (delayed). (B) Weight change and (C) clinical score are shown. (D–F) Indicated organs were taken 96 hours after the start of anti-CD40 treatment. The organs were fixed in paraformaldehyde for 24 hours (colon) or 72 hours (liver), embedded, H E stained and sliced. (D) Representative images from H E-stained sections of colon (scale bar 100 µm), liver (scale bar 300 µm) and necropsy pictures of the liver. Histological (E) colon and (F) liver scores. Data representative of two experiments (n=6–8/group). Kruskal-Wallis test with Dunn’s post hoc analysis (clinical score and weight was compared at day 4), colon and liver scores of indicated groups were compared with the corresponding DT+ cIg treated group, *p
Figure Legend Snippet: Concomitant compared with delayed anti-TNF is more effective at reducing both physical and biochemical irAEs in DT+anti-CD40 treated 4T1.2 tumor-bearing mice. (A) Schematic representation of the treatment protocol. BALB/c FoxP3 DTR mice were injected with 1×10 5 4T1.2 tumor cells. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with PBS or DT and 3 days later treated i.p. with cIg, anti-CD40 or anti-CD40+ anti-TNF (concomitant). One group was treated with anti-TNF 24 hours later (delayed). (B) Weight change and (C) clinical score are shown. (D–F) Indicated organs were taken 96 hours after the start of anti-CD40 treatment. The organs were fixed in paraformaldehyde for 24 hours (colon) or 72 hours (liver), embedded, H E stained and sliced. (D) Representative images from H E-stained sections of colon (scale bar 100 µm), liver (scale bar 300 µm) and necropsy pictures of the liver. Histological (E) colon and (F) liver scores. Data representative of two experiments (n=6–8/group). Kruskal-Wallis test with Dunn’s post hoc analysis (clinical score and weight was compared at day 4), colon and liver scores of indicated groups were compared with the corresponding DT+ cIg treated group, *p

Techniques Used: Mouse Assay, Injection, Staining

Transient Treg depletion and anti-CD40 treatment display different therapeutic index against established MC38 and 4T1.2 tumors. (A) Schematic representation of the treatment protocol. (B) BALB/c or (C) C57BL/6 FoxP3 DTR mice were injected with 1×10 5 4T1.2 or 1×10 6 MC38 tumor cells, respectively. When tumors reached a mean size of 40 mm 2 , mice were treated intraperitoneally (i.p.) with PBS or DT and 3 days later treated i.p. with control IgG (cIg) or anti-CD40. Antibody treatment continued for two more doses given 3 days apart. Mean tumor size (mm 2 ) with their corresponding weight change and clinical score are shown. Data representative of two experiments (n=5–7/group; mean±SEM). Mice were monitored for tumor growth and development of irAEs and euthanized when tumor size or clinical signs of illness reached cumulative ethical limits. Statistical comparisons between tumor sizes were performed with Mann-Whitney U test analysis at the final measurement of the PBS+ CIg-treated group, while a Kruskal-Wallis test with Dunn’s post hoc analysis was used to compare weight change and clinical score between the indicated groups (at day 2), *p
Figure Legend Snippet: Transient Treg depletion and anti-CD40 treatment display different therapeutic index against established MC38 and 4T1.2 tumors. (A) Schematic representation of the treatment protocol. (B) BALB/c or (C) C57BL/6 FoxP3 DTR mice were injected with 1×10 5 4T1.2 or 1×10 6 MC38 tumor cells, respectively. When tumors reached a mean size of 40 mm 2 , mice were treated intraperitoneally (i.p.) with PBS or DT and 3 days later treated i.p. with control IgG (cIg) or anti-CD40. Antibody treatment continued for two more doses given 3 days apart. Mean tumor size (mm 2 ) with their corresponding weight change and clinical score are shown. Data representative of two experiments (n=5–7/group; mean±SEM). Mice were monitored for tumor growth and development of irAEs and euthanized when tumor size or clinical signs of illness reached cumulative ethical limits. Statistical comparisons between tumor sizes were performed with Mann-Whitney U test analysis at the final measurement of the PBS+ CIg-treated group, while a Kruskal-Wallis test with Dunn’s post hoc analysis was used to compare weight change and clinical score between the indicated groups (at day 2), *p

Techniques Used: Mouse Assay, Injection, MANN-WHITNEY

9) Product Images from "Protection from tumor recurrence following adoptive immunotherapy varies with host conditioning regimen despite initial regression of autochthonous murine brain tumors"

Article Title: Protection from tumor recurrence following adoptive immunotherapy varies with host conditioning regimen despite initial regression of autochthonous murine brain tumors

Journal: Cancer immunology, immunotherapy : CII

doi: 10.1007/s00262-014-1635-7

Host conditioning with anti-CD40 induces high-level T cell accumulation in the lymphoid organs and brains of SV11 mice at early time points. Groups of mice received either anti-CD40, WBI, or no conditioning regimen with TCR-IV T-cell ACT. Representative plots show MHC tetramer staining (mean±SEM) of TCR-IV T cells on days +4 and +5 in a spleen and c brain. Quantification of TCR-IV T-cell accumulation (mean±SEM) in b spleen and d brain of mice that received ACT with the indicated treatments. n=3 mice/group (except n=2 for day +4 control group). Data shown are from one experiment and representative of two independent experiments. Asterisks above connecting lines indicate significant differences between time points. Asterisks next to vertical brackets indicate significant differences between treatment groups. * p
Figure Legend Snippet: Host conditioning with anti-CD40 induces high-level T cell accumulation in the lymphoid organs and brains of SV11 mice at early time points. Groups of mice received either anti-CD40, WBI, or no conditioning regimen with TCR-IV T-cell ACT. Representative plots show MHC tetramer staining (mean±SEM) of TCR-IV T cells on days +4 and +5 in a spleen and c brain. Quantification of TCR-IV T-cell accumulation (mean±SEM) in b spleen and d brain of mice that received ACT with the indicated treatments. n=3 mice/group (except n=2 for day +4 control group). Data shown are from one experiment and representative of two independent experiments. Asterisks above connecting lines indicate significant differences between time points. Asterisks next to vertical brackets indicate significant differences between treatment groups. * p

Techniques Used: Mouse Assay, Activated Clotting Time Assay, Staining

Anti-CD40-enhanced ACT promotes initial regression of established tumors. a H E brain sections on day +10 post-ACT following conditioning with anti-CD40 (left), control IgG (middle), or WBI (right). Representative low-power images (top row, scale bar = 1mm) and high-power images (bottom row, scale bar = 50μm) that show established tumor refractory to therapy (middle column) or tumor stromal condensation indicative of tumor regression (left and right columns). b For each mouse, the largest cross-sectional tumor area (mm 2 ) observed in H E sections was plotted. Data is pooled from multiple experiments with a total of 6–10 mice/group. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparison test. ** p
Figure Legend Snippet: Anti-CD40-enhanced ACT promotes initial regression of established tumors. a H E brain sections on day +10 post-ACT following conditioning with anti-CD40 (left), control IgG (middle), or WBI (right). Representative low-power images (top row, scale bar = 1mm) and high-power images (bottom row, scale bar = 50μm) that show established tumor refractory to therapy (middle column) or tumor stromal condensation indicative of tumor regression (left and right columns). b For each mouse, the largest cross-sectional tumor area (mm 2 ) observed in H E sections was plotted. Data is pooled from multiple experiments with a total of 6–10 mice/group. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparison test. ** p

Techniques Used: Activated Clotting Time Assay, Mouse Assay

Anti-CD40-enhanced ACT promotes increased survival but short-term surveillance against tumor recurrence. a Groups of mice received the indicated conditioning with or without ACT and were monitored for tumor recurrence. The percentage of surviving mice versus age is plotted. b Statistical differences in survival were calculated using the log-rank test. Data are pooled from multiple experiments with 5–8 mice/group.
Figure Legend Snippet: Anti-CD40-enhanced ACT promotes increased survival but short-term surveillance against tumor recurrence. a Groups of mice received the indicated conditioning with or without ACT and were monitored for tumor recurrence. The percentage of surviving mice versus age is plotted. b Statistical differences in survival were calculated using the log-rank test. Data are pooled from multiple experiments with 5–8 mice/group.

Techniques Used: Activated Clotting Time Assay, Mouse Assay

Donor T cells fail to persist in anti-CD40-conditioned SV11 mice following acute tumor regression. Groups of SV11 mice received either anti-CD40, control IgG, or WBI conditioning prior to ACT with CD90.1 + TCR-IV T cells. On day +30, cells from a spleens, cLN (not shown), and b brains were stained for CD90.1 and CD8. Values on dot plots indicate percent CD90.1 + of total CD8 + cells (mean±SEM). c Total CD90.1 + cells in spleens, cLN, and brains on day +30 are plotted. Representative histograms of CD44, CD62L, and KLRG1 expression gated on live CD45.2 + CD8 + CD90.1 + TCR-IV T cells (open histogram) or CD45.2 + CD8 + CD90.1 − T cells (filled histogram, spleen only) are shown on day +30 in d spleen and e brain. Values indicate the percent of TCR-IV T cells within the indicated gate (mean±SEM). Samples with
Figure Legend Snippet: Donor T cells fail to persist in anti-CD40-conditioned SV11 mice following acute tumor regression. Groups of SV11 mice received either anti-CD40, control IgG, or WBI conditioning prior to ACT with CD90.1 + TCR-IV T cells. On day +30, cells from a spleens, cLN (not shown), and b brains were stained for CD90.1 and CD8. Values on dot plots indicate percent CD90.1 + of total CD8 + cells (mean±SEM). c Total CD90.1 + cells in spleens, cLN, and brains on day +30 are plotted. Representative histograms of CD44, CD62L, and KLRG1 expression gated on live CD45.2 + CD8 + CD90.1 + TCR-IV T cells (open histogram) or CD45.2 + CD8 + CD90.1 − T cells (filled histogram, spleen only) are shown on day +30 in d spleen and e brain. Values indicate the percent of TCR-IV T cells within the indicated gate (mean±SEM). Samples with

Techniques Used: Mouse Assay, Activated Clotting Time Assay, Staining, Expressing

10) Product Images from "Concomitant or delayed anti-TNF differentially impact on immune-related adverse events and antitumor efficacy after anti-CD40 therapy"

Article Title: Concomitant or delayed anti-TNF differentially impact on immune-related adverse events and antitumor efficacy after anti-CD40 therapy

Journal: Journal for Immunotherapy of Cancer

doi: 10.1136/jitc-2020-001687

Delayed anti-TNF reduces irAEs while maintaining antitumor efficacy in DT+ anti-CD40 treated MC38 tumor-bearing mice. (A) Schematic representation of the treatment protocol. C57BL/6 FoxP3 DTR mice were injected s.c. with 1×10 6 MC38 tumor cells. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with PBS or DT and 3 days later treated i.p. with cIg, anti-CD40 or anti-CD40+anti TNF (concomitant), for three doses given 3 days apart. In one group, anti-TNF treatment (delayed) was given at the time of the second dose of anti-CD40 and then given for one more dose. (B) Mean tumor size represented as mean±SEM (n=8/group). (C) Individual tumor growth curves of the indicated groups. Data representative of two experiments. (D) Corresponding survival curves. Statistical comparisons between tumor sizes and survival curves were performed with Mann-Whitney analysis at the final measurement of the PBS+ CIg-treated group or log-rank analysis, respectively, *p
Figure Legend Snippet: Delayed anti-TNF reduces irAEs while maintaining antitumor efficacy in DT+ anti-CD40 treated MC38 tumor-bearing mice. (A) Schematic representation of the treatment protocol. C57BL/6 FoxP3 DTR mice were injected s.c. with 1×10 6 MC38 tumor cells. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with PBS or DT and 3 days later treated i.p. with cIg, anti-CD40 or anti-CD40+anti TNF (concomitant), for three doses given 3 days apart. In one group, anti-TNF treatment (delayed) was given at the time of the second dose of anti-CD40 and then given for one more dose. (B) Mean tumor size represented as mean±SEM (n=8/group). (C) Individual tumor growth curves of the indicated groups. Data representative of two experiments. (D) Corresponding survival curves. Statistical comparisons between tumor sizes and survival curves were performed with Mann-Whitney analysis at the final measurement of the PBS+ CIg-treated group or log-rank analysis, respectively, *p

Techniques Used: Mouse Assay, Injection, MANN-WHITNEY

Anti-CD40 induces liver damage and release of inflammatory cytokines within 24 hours of administration. From the same experiments as described in figure 1 , sera from (A) 4T1.2 and (B) MC38 tumor-bearing mice were collected 24 hours after the start of anti-CD40 treatment. ALT activity, TNF, IL-6 and IFN-γ levels were measured. Data pooled from two independent experiments (n=4–7/group/experiment; mean±SEM) with significant differences determined by Kruskal-Wallis test with Dunn’s post hoc analysis, *p
Figure Legend Snippet: Anti-CD40 induces liver damage and release of inflammatory cytokines within 24 hours of administration. From the same experiments as described in figure 1 , sera from (A) 4T1.2 and (B) MC38 tumor-bearing mice were collected 24 hours after the start of anti-CD40 treatment. ALT activity, TNF, IL-6 and IFN-γ levels were measured. Data pooled from two independent experiments (n=4–7/group/experiment; mean±SEM) with significant differences determined by Kruskal-Wallis test with Dunn’s post hoc analysis, *p

Techniques Used: Mouse Assay, Activity Assay

Anti-TNF but not anti-IL-6R reduces physical and biochemical irAEs induced by anti-CD40. (A) Schematic representation of the treatment protocol. (B–D, H) BALB/c or (C–E, I) C57BL/6 FoxP3 DTR mice were injected with 1×10 5 4T1.2 or 1×10 6 MC38 tumor cells, respectively. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with DT and 3 days later treated i.p. with cIg or anti-CD40 and continued for two more doses given 3 days apart. In, some groups, mice were additionally treated with anti-TNF or anti-IL-6R at the same time as anti-CD40. (B, E) Clinical score and (C, F) weight change are shown. (D, G) 24 hours after the start of anti-CD40 treatment, sera were collected and ALT activity was measured. (H–I) From the same sera samples, IL-6, TNF and IFN-γ levels were measured. Data pooled from two experiments for BALB/c and performed once for C57BL/6 (n=5–7/group/experiment; mean±SEM). Kruskal-Wallis test with Dunn’s post hoc analysis was used to compare the indicated groups (clinical score and weight was compared at day 5), *p
Figure Legend Snippet: Anti-TNF but not anti-IL-6R reduces physical and biochemical irAEs induced by anti-CD40. (A) Schematic representation of the treatment protocol. (B–D, H) BALB/c or (C–E, I) C57BL/6 FoxP3 DTR mice were injected with 1×10 5 4T1.2 or 1×10 6 MC38 tumor cells, respectively. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with DT and 3 days later treated i.p. with cIg or anti-CD40 and continued for two more doses given 3 days apart. In, some groups, mice were additionally treated with anti-TNF or anti-IL-6R at the same time as anti-CD40. (B, E) Clinical score and (C, F) weight change are shown. (D, G) 24 hours after the start of anti-CD40 treatment, sera were collected and ALT activity was measured. (H–I) From the same sera samples, IL-6, TNF and IFN-γ levels were measured. Data pooled from two experiments for BALB/c and performed once for C57BL/6 (n=5–7/group/experiment; mean±SEM). Kruskal-Wallis test with Dunn’s post hoc analysis was used to compare the indicated groups (clinical score and weight was compared at day 5), *p

Techniques Used: Mouse Assay, Injection, Activity Assay

Concomitant anti-TNF negatively impacts antitumor efficacy of DT+ anti-CD40 treated MC38 tumor-bearing mice. (A, B) From the same experiments as described in figure 3 (A) 4T1.2 or (B–E) MC38 tumor growth curves. (A) Mean tumor size represented as mean±SEM (n=6/group). Data representative of two experiments. (B) Mean tumor size represented as mean±SEM (n=5–7/group). Experiment performed once. (C, D) In a similar treatment protocol as figure 4B, individual tumor growth curves of MC38 tumor-bearing mice treated with (C) DT+ anti-CD40 or (D) DT+ anti-CD40+ anti-TNF are shown (n=20/group). (E) Corresponding survival of pooled experiments from B to D. Statistical comparisons between the indicated groups were performed with a log-rank analysis, respectively, *p
Figure Legend Snippet: Concomitant anti-TNF negatively impacts antitumor efficacy of DT+ anti-CD40 treated MC38 tumor-bearing mice. (A, B) From the same experiments as described in figure 3 (A) 4T1.2 or (B–E) MC38 tumor growth curves. (A) Mean tumor size represented as mean±SEM (n=6/group). Data representative of two experiments. (B) Mean tumor size represented as mean±SEM (n=5–7/group). Experiment performed once. (C, D) In a similar treatment protocol as figure 4B, individual tumor growth curves of MC38 tumor-bearing mice treated with (C) DT+ anti-CD40 or (D) DT+ anti-CD40+ anti-TNF are shown (n=20/group). (E) Corresponding survival of pooled experiments from B to D. Statistical comparisons between the indicated groups were performed with a log-rank analysis, respectively, *p

Techniques Used: Mouse Assay

Concomitant compared with delayed anti-TNF is more effective at reducing both physical and biochemical irAEs in DT+anti-CD40 treated 4T1.2 tumor-bearing mice. (A) Schematic representation of the treatment protocol. BALB/c FoxP3 DTR mice were injected with 1×10 5 4T1.2 tumor cells. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with PBS or DT and 3 days later treated i.p. with cIg, anti-CD40 or anti-CD40+ anti-TNF (concomitant). One group was treated with anti-TNF 24 hours later (delayed). (B) Weight change and (C) clinical score are shown. (D–F) Indicated organs were taken 96 hours after the start of anti-CD40 treatment. The organs were fixed in paraformaldehyde for 24 hours (colon) or 72 hours (liver), embedded, H E stained and sliced. (D) Representative images from H E-stained sections of colon (scale bar 100 µm), liver (scale bar 300 µm) and necropsy pictures of the liver. Histological (E) colon and (F) liver scores. Data representative of two experiments (n=6–8/group). Kruskal-Wallis test with Dunn’s post hoc analysis (clinical score and weight was compared at day 4), colon and liver scores of indicated groups were compared with the corresponding DT+ cIg treated group, *p
Figure Legend Snippet: Concomitant compared with delayed anti-TNF is more effective at reducing both physical and biochemical irAEs in DT+anti-CD40 treated 4T1.2 tumor-bearing mice. (A) Schematic representation of the treatment protocol. BALB/c FoxP3 DTR mice were injected with 1×10 5 4T1.2 tumor cells. When tumors reached a mean size of 40 mm 2 , mice were treated i.p. with PBS or DT and 3 days later treated i.p. with cIg, anti-CD40 or anti-CD40+ anti-TNF (concomitant). One group was treated with anti-TNF 24 hours later (delayed). (B) Weight change and (C) clinical score are shown. (D–F) Indicated organs were taken 96 hours after the start of anti-CD40 treatment. The organs were fixed in paraformaldehyde for 24 hours (colon) or 72 hours (liver), embedded, H E stained and sliced. (D) Representative images from H E-stained sections of colon (scale bar 100 µm), liver (scale bar 300 µm) and necropsy pictures of the liver. Histological (E) colon and (F) liver scores. Data representative of two experiments (n=6–8/group). Kruskal-Wallis test with Dunn’s post hoc analysis (clinical score and weight was compared at day 4), colon and liver scores of indicated groups were compared with the corresponding DT+ cIg treated group, *p

Techniques Used: Mouse Assay, Injection, Staining

Transient Treg depletion and anti-CD40 treatment display different therapeutic index against established MC38 and 4T1.2 tumors. (A) Schematic representation of the treatment protocol. (B) BALB/c or (C) C57BL/6 FoxP3 DTR mice were injected with 1×10 5 4T1.2 or 1×10 6 MC38 tumor cells, respectively. When tumors reached a mean size of 40 mm 2 , mice were treated intraperitoneally (i.p.) with PBS or DT and 3 days later treated i.p. with control IgG (cIg) or anti-CD40. Antibody treatment continued for two more doses given 3 days apart. Mean tumor size (mm 2 ) with their corresponding weight change and clinical score are shown. Data representative of two experiments (n=5–7/group; mean±SEM). Mice were monitored for tumor growth and development of irAEs and euthanized when tumor size or clinical signs of illness reached cumulative ethical limits. Statistical comparisons between tumor sizes were performed with Mann-Whitney U test analysis at the final measurement of the PBS+ CIg-treated group, while a Kruskal-Wallis test with Dunn’s post hoc analysis was used to compare weight change and clinical score between the indicated groups (at day 2), *p
Figure Legend Snippet: Transient Treg depletion and anti-CD40 treatment display different therapeutic index against established MC38 and 4T1.2 tumors. (A) Schematic representation of the treatment protocol. (B) BALB/c or (C) C57BL/6 FoxP3 DTR mice were injected with 1×10 5 4T1.2 or 1×10 6 MC38 tumor cells, respectively. When tumors reached a mean size of 40 mm 2 , mice were treated intraperitoneally (i.p.) with PBS or DT and 3 days later treated i.p. with control IgG (cIg) or anti-CD40. Antibody treatment continued for two more doses given 3 days apart. Mean tumor size (mm 2 ) with their corresponding weight change and clinical score are shown. Data representative of two experiments (n=5–7/group; mean±SEM). Mice were monitored for tumor growth and development of irAEs and euthanized when tumor size or clinical signs of illness reached cumulative ethical limits. Statistical comparisons between tumor sizes were performed with Mann-Whitney U test analysis at the final measurement of the PBS+ CIg-treated group, while a Kruskal-Wallis test with Dunn’s post hoc analysis was used to compare weight change and clinical score between the indicated groups (at day 2), *p

Techniques Used: Mouse Assay, Injection, MANN-WHITNEY

11) Product Images from "CD4+ T Cells in the Blood of MS Patients Respond to Predicted Epitopes From B cell Receptors Found in Spinal Fluid"

Article Title: CD4+ T Cells in the Blood of MS Patients Respond to Predicted Epitopes From B cell Receptors Found in Spinal Fluid

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.00598

CD4 + T cell responses against idiotope peptides. A total of 500,000 PBMC were left unstimulated, or stimulated with EBNA-1 peptide mix, insulin peptide mix, anti CD3/CD28 beads (not shown), or one of 22 idiotope peptides for 12 h in presence of anti-CD40 antibodies and analyzed by flow cytometry. We gated on CD3 + CD4 + CD8 − T cells and assayed for the activation marker CD154 among all CD4 + cells, CD45RO + memory-, or CD45RO − naive cells. Activated cells are presented as proportions of all CD4 + cells (upper three panels) or proportion of memory cells (lower panel). Responses were deemed positive (arrows) if the proportion of CD154 + cells were 3x higher than in unstimulated (negative) wells.
Figure Legend Snippet: CD4 + T cell responses against idiotope peptides. A total of 500,000 PBMC were left unstimulated, or stimulated with EBNA-1 peptide mix, insulin peptide mix, anti CD3/CD28 beads (not shown), or one of 22 idiotope peptides for 12 h in presence of anti-CD40 antibodies and analyzed by flow cytometry. We gated on CD3 + CD4 + CD8 − T cells and assayed for the activation marker CD154 among all CD4 + cells, CD45RO + memory-, or CD45RO − naive cells. Activated cells are presented as proportions of all CD4 + cells (upper three panels) or proportion of memory cells (lower panel). Responses were deemed positive (arrows) if the proportion of CD154 + cells were 3x higher than in unstimulated (negative) wells.

Techniques Used: Flow Cytometry, Activation Assay, Marker

Flow cytometry based idiotope-specific T cell activation assay. (A) IGHV amino acid sequences [mean 1,079 ( SD = 1,213) per patient] from nine MS patients were run through predictive models to identify likely antigenic idiotopes based on HLA class II affinity, cathepsin cleavage and frequency classification (FC) of T cell exposed motifs (TCEM). (B) 500,000 PBMC were stimulated with synthetic idiotope peptides predicted to be stimulatory, tolerogenic, or inert as well as positive and negative controls for 12 h in presence of anti-CD40 antibodies. B cells or other professional APCs with idiotope peptides bound to their HLA class II receptor may activate cognate CD4 + T cells. (C) CD4 + CD45RO + memory T cells specifically activated by idiotope peptides were detected by surface expression of CD154, upregulated upon TCR stimulation. The example shows a detected memory T cell response to idiotope peptide 12 in patient MS-11.
Figure Legend Snippet: Flow cytometry based idiotope-specific T cell activation assay. (A) IGHV amino acid sequences [mean 1,079 ( SD = 1,213) per patient] from nine MS patients were run through predictive models to identify likely antigenic idiotopes based on HLA class II affinity, cathepsin cleavage and frequency classification (FC) of T cell exposed motifs (TCEM). (B) 500,000 PBMC were stimulated with synthetic idiotope peptides predicted to be stimulatory, tolerogenic, or inert as well as positive and negative controls for 12 h in presence of anti-CD40 antibodies. B cells or other professional APCs with idiotope peptides bound to their HLA class II receptor may activate cognate CD4 + T cells. (C) CD4 + CD45RO + memory T cells specifically activated by idiotope peptides were detected by surface expression of CD154, upregulated upon TCR stimulation. The example shows a detected memory T cell response to idiotope peptide 12 in patient MS-11.

Techniques Used: Flow Cytometry, Activation Assay, Expressing

12) Product Images from "Systemic Immunity is Required for Effective Cancer Immunotherapy"

Article Title: Systemic Immunity is Required for Effective Cancer Immunotherapy

Journal: Cell

doi: 10.1016/j.cell.2016.12.022

Interfering with systemic immune responses prevents effective immunotherapy (A) MMTV-PyMT tumor-bearing mice treated with alloIgG, anti-CD40, IFNγ and daily FTY720 or ethanol control starting one day before therapy. (B) Mice with orthotopic 4T1 tumors treated as in A. (C) Immunofluorescence of 4T1 tumors 14 days after therapy. (D–E) 4T1 lung metastases 20 days after therapy. (F) T cells from spleen and lymph nodes of mice with 4T1 tumors, treated with alloIgG, anti-CD40, IFNγ and FTY720 were transferred with IL-2 into naïve Balb/c mice. Controls only received IL-2. Recipients were challenged s.c. with 4T1 cells the next day. (G) MC38 tumor-bearing mice untreated or treated with anti-PD-1 and ethanol control or FTY720. All p-values reflect two-tailed, heteroskedastic t-tests in R. Error bars represent S.D.
Figure Legend Snippet: Interfering with systemic immune responses prevents effective immunotherapy (A) MMTV-PyMT tumor-bearing mice treated with alloIgG, anti-CD40, IFNγ and daily FTY720 or ethanol control starting one day before therapy. (B) Mice with orthotopic 4T1 tumors treated as in A. (C) Immunofluorescence of 4T1 tumors 14 days after therapy. (D–E) 4T1 lung metastases 20 days after therapy. (F) T cells from spleen and lymph nodes of mice with 4T1 tumors, treated with alloIgG, anti-CD40, IFNγ and FTY720 were transferred with IL-2 into naïve Balb/c mice. Controls only received IL-2. Recipients were challenged s.c. with 4T1 cells the next day. (G) MC38 tumor-bearing mice untreated or treated with anti-PD-1 and ethanol control or FTY720. All p-values reflect two-tailed, heteroskedastic t-tests in R. Error bars represent S.D.

Techniques Used: Mouse Assay, Immunofluorescence, Two Tailed Test

13) Product Images from "IL-23R+ innate lymphoid cells induce colitis via interleukin-22-dependent mechanism"

Article Title: IL-23R+ innate lymphoid cells induce colitis via interleukin-22-dependent mechanism

Journal: Mucosal immunology

doi: 10.1038/mi.2013.33

Interleukin (IL)-22 requires innate cells to drive pathogenesis but not p40. ( a ) Rag −/− IL2Rγc −/− mice were hydrodynamically injected with either IL-22 or mock-expressing plasmid 2 days before anti-CD40 injection.
Figure Legend Snippet: Interleukin (IL)-22 requires innate cells to drive pathogenesis but not p40. ( a ) Rag −/− IL2Rγc −/− mice were hydrodynamically injected with either IL-22 or mock-expressing plasmid 2 days before anti-CD40 injection.

Techniques Used: Mouse Assay, Injection, Expressing, Plasmid Preparation

Interleukin (IL)-22 upregulates neutrophil-recruiting chemokines, downregulates matrix metalloproteinase (MMP)-2 and MMP-9. Relative mRNA expression of indicated genes in the colon lamina propria at the end of day 7 of anti-CD40-induced colitis. CXCL,
Figure Legend Snippet: Interleukin (IL)-22 upregulates neutrophil-recruiting chemokines, downregulates matrix metalloproteinase (MMP)-2 and MMP-9. Relative mRNA expression of indicated genes in the colon lamina propria at the end of day 7 of anti-CD40-induced colitis. CXCL,

Techniques Used: Expressing

Interleukin (IL)-22 is pathogenic during α-CD40-induced colitis. ( a ) Anti-CD40-injected Rag −/− mice were injected with either IL-17RA blocking or isotype immunoglobulin G (IgG) daily until day 5 of colitis. Weight loss was measured
Figure Legend Snippet: Interleukin (IL)-22 is pathogenic during α-CD40-induced colitis. ( a ) Anti-CD40-injected Rag −/− mice were injected with either IL-17RA blocking or isotype immunoglobulin G (IgG) daily until day 5 of colitis. Weight loss was measured

Techniques Used: Injection, Mouse Assay, Blocking Assay

Neutrophil recruitment is interleukin (IL)-22 dependent. ( a ) IL-23Rag −/− and IL23R −/+ Rag −/− mice colon lamina propria (LP) lymphocytes were stained with Gr-1 and CD11b antibodies following the day of anti-CD40
Figure Legend Snippet: Neutrophil recruitment is interleukin (IL)-22 dependent. ( a ) IL-23Rag −/− and IL23R −/+ Rag −/− mice colon lamina propria (LP) lymphocytes were stained with Gr-1 and CD11b antibodies following the day of anti-CD40

Techniques Used: Mouse Assay, Staining

14) Product Images from "In situ vaccination with defined factors overcomes T cell exhaustion in distant tumors"

Article Title: In situ vaccination with defined factors overcomes T cell exhaustion in distant tumors

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI128562

CMP is active across tumor models but inactive in unmatched distant tumors. ( A ) Average growth curves of animals bearing bilateral MC38, MB49, and Hep-55.1c tumors ( n = 10/group). ( B ) Kaplan-Meier curves show survival rates of C57BL/6 animals bearing established orthotopic KPC pancreatic tumors treated intravenously with anti-CD40 and MPL ( n = 10/group). ( C ) Bilateral tumor model with distant B16F10 tumors and either MC38, Hep55.1c, or B16F10 (positive control) as the treated tumor ( n = 10/group). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001, by unpaired, 2-tailed Student’s t test.
Figure Legend Snippet: CMP is active across tumor models but inactive in unmatched distant tumors. ( A ) Average growth curves of animals bearing bilateral MC38, MB49, and Hep-55.1c tumors ( n = 10/group). ( B ) Kaplan-Meier curves show survival rates of C57BL/6 animals bearing established orthotopic KPC pancreatic tumors treated intravenously with anti-CD40 and MPL ( n = 10/group). ( C ) Bilateral tumor model with distant B16F10 tumors and either MC38, Hep55.1c, or B16F10 (positive control) as the treated tumor ( n = 10/group). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001, by unpaired, 2-tailed Student’s t test.

Techniques Used: Positive Control

Agents that promote phagocytosis and APC activation, but not direct tumor cell lysis, control local and distant tumors. ( A ) Bone marrow–derived myeloid cells were treated with MPL or vehicle for 16 hours and incubated with FITC-labeled latex beads. Flow cytometry was performed to determine the fraction of CD11c hi cells that phagocytosed FITC-labeled beads and the median fluorescence intensity (MFI) of CD86 expression ( n = 3 – 5/group). ( B ) C57BL/6 mice were implanted intradermally with 5 × 10 5 B16F10 cells. On day 8, FITC-labeled latex beads were coinjected intratumorally with vehicle, MPL, or anti-CD40. Twenty-four hours later, the CD11c hi cell population was analyzed in tumors (left) for phagocytosis ( n = 5/group) and in DLNs (right) for CD86 expression ( n = 4/group). ( C ) Treatment schedule: intratumoral biweekly treatments, with or without intraperitoneal anti–PD-1, were started once bilateral tumors were established; treatment was continued for 4 weeks. ( D ) Individual growth curves of treated and distant tumors in animals treated with MPL and anti-CD40 ( n = 10/group). ( E ) Average tumor growth curves comparing MPL and anti-CD40 with constituent monotherapies ( n = 10/group). ( F ) Viability of B16F10 cells treated in vitro with MPL, anti-CD40, or gemcitabine for 72 hours. ( G ) Growth of treated and distant tumors upon addition of anti–PD-1 ( n = 10/group). * P ≤ 0.0, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001, by unpaired, 2-tailed Student’s t test.
Figure Legend Snippet: Agents that promote phagocytosis and APC activation, but not direct tumor cell lysis, control local and distant tumors. ( A ) Bone marrow–derived myeloid cells were treated with MPL or vehicle for 16 hours and incubated with FITC-labeled latex beads. Flow cytometry was performed to determine the fraction of CD11c hi cells that phagocytosed FITC-labeled beads and the median fluorescence intensity (MFI) of CD86 expression ( n = 3 – 5/group). ( B ) C57BL/6 mice were implanted intradermally with 5 × 10 5 B16F10 cells. On day 8, FITC-labeled latex beads were coinjected intratumorally with vehicle, MPL, or anti-CD40. Twenty-four hours later, the CD11c hi cell population was analyzed in tumors (left) for phagocytosis ( n = 5/group) and in DLNs (right) for CD86 expression ( n = 4/group). ( C ) Treatment schedule: intratumoral biweekly treatments, with or without intraperitoneal anti–PD-1, were started once bilateral tumors were established; treatment was continued for 4 weeks. ( D ) Individual growth curves of treated and distant tumors in animals treated with MPL and anti-CD40 ( n = 10/group). ( E ) Average tumor growth curves comparing MPL and anti-CD40 with constituent monotherapies ( n = 10/group). ( F ) Viability of B16F10 cells treated in vitro with MPL, anti-CD40, or gemcitabine for 72 hours. ( G ) Growth of treated and distant tumors upon addition of anti–PD-1 ( n = 10/group). * P ≤ 0.0, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001, by unpaired, 2-tailed Student’s t test.

Techniques Used: Activation Assay, Lysis, Derivative Assay, Incubation, Labeling, Flow Cytometry, Cytometry, Fluorescence, Expressing, Mouse Assay, In Vitro

15) Product Images from "Dendritic cell paucity leads to dysfunctional immune surveillance in pancreatic cancer"

Article Title: Dendritic cell paucity leads to dysfunctional immune surveillance in pancreatic cancer

Journal: Cancer cell

doi: 10.1016/j.ccell.2020.02.008

Enhancing cDC infiltration and activation in established PDAC leads to disease stabilization (A) Schematic of Flt3L administration in ultrasound-diagnosed KPPC-OG mice. n=5–8 mice/group. (B) Density of (LEFT) CD103 + cDC1s, CD11b + cDC2s in tumors, and (RIGHT) migratory cDC1, cDC2 populations in respective dLNs of KPPC-OG mice treated with Flt3L. n=5–8 mice/group. (C) Density of CD8 + T cells, CD4 + T H cells and frequency of CD4 + T REG in tumors of KPPC-OG mice treated with Flt3L. n=5–8 mice/group. (D) Density of cDC1s and cDC2s in tumors of KPPC-OG mice treated as described. n=7–8 mice/group. (E) Density of CD8 + T cells and CD4 + T H cells and frequency of CD4 + T REGS in tumors of KPPC-OG mice treated as described. n=7–8 mice/group. (F) Density of OVA-specific CD8 + T cells in tumors of treated KPPC-OG mice. n=5–8 mice/group. (G) Representative immunohistochemistry of CD8 + T cells (brown) and CK19 + tumor lesions (pink) in KPPC-OG mice treated as indicated. RIGHT: cumulative CD8 + T cell density within 50 μm of CK19 + lesions. n=5–8 mice/group. (H) Density of tumor-infiltrating NK cells, NKT cells and γδ-T cells in treated KPPC-OG mice. n=5–8 mice/group. (I) Representative flow histogram indicating ZsGreen in migratory cDC1s from draining nodes of KPPC-Z treated as indicated. RIGHT: absolute number of migratory cDC subsets that have ZsGreen. n=3–4 mice/group. (J) Density of OVA-specific CD8 + T cell in draining lymph nodes of treated KPPC-OG mice. n=5–8 mice/group. (K) Tumor growth quantified by ultrasound measurements over 2 weeks of treatment. RIGHT: Individual traces of untreated and anti-CD40 plus Flt3L combination cohorts. n=5–8 mice/group. (L) Representative Masson’s trichrome staining with quantification in KPPC-OG mice treated as denoted. n=5–8 mice/group. (M) Representative α-SMA staining with quantification in KPPC-OG mice treated as denoted. n=5–8 mice/group. .
Figure Legend Snippet: Enhancing cDC infiltration and activation in established PDAC leads to disease stabilization (A) Schematic of Flt3L administration in ultrasound-diagnosed KPPC-OG mice. n=5–8 mice/group. (B) Density of (LEFT) CD103 + cDC1s, CD11b + cDC2s in tumors, and (RIGHT) migratory cDC1, cDC2 populations in respective dLNs of KPPC-OG mice treated with Flt3L. n=5–8 mice/group. (C) Density of CD8 + T cells, CD4 + T H cells and frequency of CD4 + T REG in tumors of KPPC-OG mice treated with Flt3L. n=5–8 mice/group. (D) Density of cDC1s and cDC2s in tumors of KPPC-OG mice treated as described. n=7–8 mice/group. (E) Density of CD8 + T cells and CD4 + T H cells and frequency of CD4 + T REGS in tumors of KPPC-OG mice treated as described. n=7–8 mice/group. (F) Density of OVA-specific CD8 + T cells in tumors of treated KPPC-OG mice. n=5–8 mice/group. (G) Representative immunohistochemistry of CD8 + T cells (brown) and CK19 + tumor lesions (pink) in KPPC-OG mice treated as indicated. RIGHT: cumulative CD8 + T cell density within 50 μm of CK19 + lesions. n=5–8 mice/group. (H) Density of tumor-infiltrating NK cells, NKT cells and γδ-T cells in treated KPPC-OG mice. n=5–8 mice/group. (I) Representative flow histogram indicating ZsGreen in migratory cDC1s from draining nodes of KPPC-Z treated as indicated. RIGHT: absolute number of migratory cDC subsets that have ZsGreen. n=3–4 mice/group. (J) Density of OVA-specific CD8 + T cell in draining lymph nodes of treated KPPC-OG mice. n=5–8 mice/group. (K) Tumor growth quantified by ultrasound measurements over 2 weeks of treatment. RIGHT: Individual traces of untreated and anti-CD40 plus Flt3L combination cohorts. n=5–8 mice/group. (L) Representative Masson’s trichrome staining with quantification in KPPC-OG mice treated as denoted. n=5–8 mice/group. (M) Representative α-SMA staining with quantification in KPPC-OG mice treated as denoted. n=5–8 mice/group. .

Techniques Used: Activation Assay, Mouse Assay, Immunohistochemistry, Staining

16) Product Images from "Enhancing Antitumor Immune Responses by Optimized Combinations of Cell-penetrating Peptide-based Vaccines and Adjuvants"

Article Title: Enhancing Antitumor Immune Responses by Optimized Combinations of Cell-penetrating Peptide-based Vaccines and Adjuvants

Journal: Molecular Therapy

doi: 10.1038/mt.2016.134

Identification of the most potent CPP truncations in vitro (proliferation assay) and in vivo (CD8 T cell immune response after vaccination). ( a ) Processing of each ZEBRA CPP truncation (conjugated to OVACD8 epitope) by BMDCs and antigen presentation to OVA 257–264 specific, MHC class I restricted (H-2K b ) OT-I T cells in vitro . CFSE-labeled splenocytes from OT-I mice were cocultured for 4 days alone, or with mature BMDCs that had been loaded with each ZEBRA CPP truncation (Z13– Z20). Negative controls include OT1 incubated with non-loaded BMDCs or BMDCs loaded with irrelevant peptide (gp100 peptide). A positive control was performed by stimulating OT1 cells with BMDCs loaded with OVACD8 peptide. Except when indicated, proliferation was assessed by CFSE dilution analyzed by flow cytometry, gating on live CD8 T cells. Data from one experiment representative of two independent experiments. ( b ) C57BL/6 mice were vaccinated by s.c. injection at wk0, and wk2 with 10 nmoles of each ZEBRA CPP truncation (Z12–Z20) conjugated to OVACD8 epitope and mixed with 100 μg of anti-CD40. Mice were also injected with 50 μg of Hiltonol i.m. (same side as the s.c. injection). Adjuvant control mice received anti-CD40 and Hiltonol. Mice were bled 1 week after the second vaccination for assessing OVA-specific CD8 T cells. Data from two independent experiments are shown. One representative dot-plot of each group is shown. * P
Figure Legend Snippet: Identification of the most potent CPP truncations in vitro (proliferation assay) and in vivo (CD8 T cell immune response after vaccination). ( a ) Processing of each ZEBRA CPP truncation (conjugated to OVACD8 epitope) by BMDCs and antigen presentation to OVA 257–264 specific, MHC class I restricted (H-2K b ) OT-I T cells in vitro . CFSE-labeled splenocytes from OT-I mice were cocultured for 4 days alone, or with mature BMDCs that had been loaded with each ZEBRA CPP truncation (Z13– Z20). Negative controls include OT1 incubated with non-loaded BMDCs or BMDCs loaded with irrelevant peptide (gp100 peptide). A positive control was performed by stimulating OT1 cells with BMDCs loaded with OVACD8 peptide. Except when indicated, proliferation was assessed by CFSE dilution analyzed by flow cytometry, gating on live CD8 T cells. Data from one experiment representative of two independent experiments. ( b ) C57BL/6 mice were vaccinated by s.c. injection at wk0, and wk2 with 10 nmoles of each ZEBRA CPP truncation (Z12–Z20) conjugated to OVACD8 epitope and mixed with 100 μg of anti-CD40. Mice were also injected with 50 μg of Hiltonol i.m. (same side as the s.c. injection). Adjuvant control mice received anti-CD40 and Hiltonol. Mice were bled 1 week after the second vaccination for assessing OVA-specific CD8 T cells. Data from two independent experiments are shown. One representative dot-plot of each group is shown. * P

Techniques Used: Conditioned Place Preference, In Vitro, Proliferation Assay, In Vivo, Labeling, Mouse Assay, Incubation, Positive Control, Flow Cytometry, Cytometry, Injection

TAA-specific CD8 T cell immune responses elicited by vaccination with CPP truncations and effect of CPP truncations on control of melanoma lung metastasis growth. ( a , b ) Mice were vaccinated three times (wk0, wk2, and wk5) by s.c. injection (right flank) of 10 nmoles of Z13OVACD4gp100CD8, Z14OVACD4gp100CD8, Z18OVACD4gp100CD8 and i.m. injection of 50 μg of Hiltonol (right hind leg) ( a ) or four times (wk0, wk2, wk4, and wk7) by s.c. injection (right flank) of 10 nmoles of same constructs mixed with 20 μg of MPLA ( b ). After 1 week from the last vaccination, spleen cells were restimulated in vitro for 7 days with gp100 25-33 peptide and multimer staining was performed. ( c ) Mice were vaccinated twice (d-21 and d-7) by s.c. injection of 2 nmoles of Z13OVACD4gp100, Z14OVACD4gp100 or Z18OVACD4gp100 mixed with 100 μg of anti-CD40 and by i.m. injection of 50 μg of Hiltonol (right hind leg) and implanted i.v. with 1 × 10 5 B16-OVA melanoma tumor cells at day 0. Mice were euthanized at day 17 and lung recovered. Control mice group was injected with anti-CD40 and Hiltonol only. Number of metastasis foci was counted for each lung (5–7 mice/group). Two representative lung pictures of each group are shown. * P
Figure Legend Snippet: TAA-specific CD8 T cell immune responses elicited by vaccination with CPP truncations and effect of CPP truncations on control of melanoma lung metastasis growth. ( a , b ) Mice were vaccinated three times (wk0, wk2, and wk5) by s.c. injection (right flank) of 10 nmoles of Z13OVACD4gp100CD8, Z14OVACD4gp100CD8, Z18OVACD4gp100CD8 and i.m. injection of 50 μg of Hiltonol (right hind leg) ( a ) or four times (wk0, wk2, wk4, and wk7) by s.c. injection (right flank) of 10 nmoles of same constructs mixed with 20 μg of MPLA ( b ). After 1 week from the last vaccination, spleen cells were restimulated in vitro for 7 days with gp100 25-33 peptide and multimer staining was performed. ( c ) Mice were vaccinated twice (d-21 and d-7) by s.c. injection of 2 nmoles of Z13OVACD4gp100, Z14OVACD4gp100 or Z18OVACD4gp100 mixed with 100 μg of anti-CD40 and by i.m. injection of 50 μg of Hiltonol (right hind leg) and implanted i.v. with 1 × 10 5 B16-OVA melanoma tumor cells at day 0. Mice were euthanized at day 17 and lung recovered. Control mice group was injected with anti-CD40 and Hiltonol only. Number of metastasis foci was counted for each lung (5–7 mice/group). Two representative lung pictures of each group are shown. * P

Techniques Used: Conditioned Place Preference, Mouse Assay, Injection, Construct, In Vitro, Staining

17) Product Images from "Agonistic Antibody to CD40 Boosts the Antitumor Activity of Adoptively Transferred T Cells In Vivo"

Article Title: Agonistic Antibody to CD40 Boosts the Antitumor Activity of Adoptively Transferred T Cells In Vivo

Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

doi: 10.1097/CJI.0b013e31824e7f43

Anti-CD40 monoclonal antibody (mAb) induces expansion of adoptively transferred T cells independent of tumor antigen cross-presentation. B6 mice (5 mice per group) were subcutaneously inoculated with B16 or MC38 tumor cells on day -7 and treated by intravenous injection of pmel-1 T cells on day 0. Recombinant human interleukin-2 (RhIL-2) was administered intraperitoneally for 3 days after T cell transfer. Anti-CD40 mAb was injected intraperitoneally on day 0. Peripheral blood was subjected to flow cytometric analysis for percentage of pmel-1 cells on day 4. Results shown are representative of two independent experiments with similar results.
Figure Legend Snippet: Anti-CD40 monoclonal antibody (mAb) induces expansion of adoptively transferred T cells independent of tumor antigen cross-presentation. B6 mice (5 mice per group) were subcutaneously inoculated with B16 or MC38 tumor cells on day -7 and treated by intravenous injection of pmel-1 T cells on day 0. Recombinant human interleukin-2 (RhIL-2) was administered intraperitoneally for 3 days after T cell transfer. Anti-CD40 mAb was injected intraperitoneally on day 0. Peripheral blood was subjected to flow cytometric analysis for percentage of pmel-1 cells on day 4. Results shown are representative of two independent experiments with similar results.

Techniques Used: Mouse Assay, Injection, Recombinant, Flow Cytometry

Anti-CD40 monoclonal antibody (mAb) induces expansion of adoptively transferred T cells and enhances antitumor activity. B6 mice (5–10 mice per group) were subcutaneously inoculated with B16 tumor cells on day -7 and treated by intravenous injection of pmel-1 T cells on day 0. Recombinant human IL-2 (rhIL-2) was administered intraperitoneally on days 0–3. Anti-CD40 mAb was injected intraperitoneally on day 0. A, Peripheral blood was subjected to flow cytometric analysis for percentage of pmel-1 cells at the indicated time points. B, Tumor growth is shown as the area calculated by multiplying the perpendicular diameters of the tumors. Results shown are representative of three independent experiments with similar results. ** P
Figure Legend Snippet: Anti-CD40 monoclonal antibody (mAb) induces expansion of adoptively transferred T cells and enhances antitumor activity. B6 mice (5–10 mice per group) were subcutaneously inoculated with B16 tumor cells on day -7 and treated by intravenous injection of pmel-1 T cells on day 0. Recombinant human IL-2 (rhIL-2) was administered intraperitoneally on days 0–3. Anti-CD40 mAb was injected intraperitoneally on day 0. A, Peripheral blood was subjected to flow cytometric analysis for percentage of pmel-1 cells at the indicated time points. B, Tumor growth is shown as the area calculated by multiplying the perpendicular diameters of the tumors. Results shown are representative of three independent experiments with similar results. ** P

Techniques Used: Activity Assay, Mouse Assay, Injection, Recombinant, Flow Cytometry

18) Product Images from "Systemic Immunity is Required for Effective Cancer Immunotherapy"

Article Title: Systemic Immunity is Required for Effective Cancer Immunotherapy

Journal: Cell

doi: 10.1016/j.cell.2016.12.022

Interfering with systemic immune responses prevents effective immunotherapy (A) MMTV-PyMT tumor-bearing mice treated with alloIgG, anti-CD40, IFNγ and daily FTY720 or ethanol control starting one day before therapy. (B) Mice with orthotopic 4T1 tumors treated as in A. (C) Immunofluorescence of 4T1 tumors 14 days after therapy. (D–E) 4T1 lung metastases 20 days after therapy. (F) T cells from spleen and lymph nodes of mice with 4T1 tumors, treated with alloIgG, anti-CD40, IFNγ and FTY720 were transferred with IL-2 into naïve Balb/c mice. Controls only received IL-2. Recipients were challenged s.c. with 4T1 cells the next day. (G) MC38 tumor-bearing mice untreated or treated with anti-PD-1 and ethanol control or FTY720. All p-values reflect two-tailed, heteroskedastic t-tests in R. Error bars represent S.D.
Figure Legend Snippet: Interfering with systemic immune responses prevents effective immunotherapy (A) MMTV-PyMT tumor-bearing mice treated with alloIgG, anti-CD40, IFNγ and daily FTY720 or ethanol control starting one day before therapy. (B) Mice with orthotopic 4T1 tumors treated as in A. (C) Immunofluorescence of 4T1 tumors 14 days after therapy. (D–E) 4T1 lung metastases 20 days after therapy. (F) T cells from spleen and lymph nodes of mice with 4T1 tumors, treated with alloIgG, anti-CD40, IFNγ and FTY720 were transferred with IL-2 into naïve Balb/c mice. Controls only received IL-2. Recipients were challenged s.c. with 4T1 cells the next day. (G) MC38 tumor-bearing mice untreated or treated with anti-PD-1 and ethanol control or FTY720. All p-values reflect two-tailed, heteroskedastic t-tests in R. Error bars represent S.D.

Techniques Used: Mouse Assay, Immunofluorescence, Two Tailed Test

19) Product Images from "Tumor cure by radiation therapy and checkpoint inhibitors depends on pre-existing immunity"

Article Title: Tumor cure by radiation therapy and checkpoint inhibitors depends on pre-existing immunity

Journal: Scientific Reports

doi: 10.1038/s41598-018-25482-w

FTY-720 immunosuppression at tumor implantation eliminates tumor control by subsequent radiation therapy and checkpoint inhibition. ( a ) BALB/c mice were randomized to receive CD8 depleting antibodies 2 days prior to implantation with CT26 tumors. Graphs show quantitative analysis of i) CD8 + , ii) CD4 + and iii) CD11b + cells over time following CD8 depletion. ( b ) i) BALB/c mice were randomized to receive FTY-720 1day prior to implantation with CT26 tumors. Graphs show quantitative analysis of ii) CD8 + , iii) CD4 + and iv) CD11b + cells over time following FTY-720 administration. ( c ) i) C57BL/6 mice were implanted with Panc02-SIY tumors and analyzed for SIY-specific T cells in the spleen by ICS for IFNγ, or in the blood using an SIY-pentamer. ii) Time course of the SIY-specific response in the peripheral blood of mice implanted with Panc02-SIY through tumor growth. Mice were randomized to treatment with FTY-720 or left untreated, then vaccinated 1day later with iii) subcutaneous injection of Panc02-SIY, iv) subcutaneous injection of Dec205-Ova + anti-CD40, or v) intravenous administration of Lm-SIY. Graphs show antigen-specific cells in blood, LN or spleen, respectively. d) i) Mice were implanted with CT26 and treated with anti-CTLA4 on d7 and radiation on day 14. Groups were randomized to treatment with i-ii) FTY-720 1day prior to tumor implantation or iii-iv) 1day prior to radiation. ( e ) Mice were implanted with CT26 and i) left untreated; ii) treated with anti-PD1 on d7, 14 and 21; iii) treated with radiation on day 14; iv) treated with the combination; iv) treated with FTY720 1day prior to implantation and radiation on d14. v) treated with FTY720 1day prior to implantation, anti-PD1 on d7, 14 and 21 and radiation on d14. Key: *p
Figure Legend Snippet: FTY-720 immunosuppression at tumor implantation eliminates tumor control by subsequent radiation therapy and checkpoint inhibition. ( a ) BALB/c mice were randomized to receive CD8 depleting antibodies 2 days prior to implantation with CT26 tumors. Graphs show quantitative analysis of i) CD8 + , ii) CD4 + and iii) CD11b + cells over time following CD8 depletion. ( b ) i) BALB/c mice were randomized to receive FTY-720 1day prior to implantation with CT26 tumors. Graphs show quantitative analysis of ii) CD8 + , iii) CD4 + and iv) CD11b + cells over time following FTY-720 administration. ( c ) i) C57BL/6 mice were implanted with Panc02-SIY tumors and analyzed for SIY-specific T cells in the spleen by ICS for IFNγ, or in the blood using an SIY-pentamer. ii) Time course of the SIY-specific response in the peripheral blood of mice implanted with Panc02-SIY through tumor growth. Mice were randomized to treatment with FTY-720 or left untreated, then vaccinated 1day later with iii) subcutaneous injection of Panc02-SIY, iv) subcutaneous injection of Dec205-Ova + anti-CD40, or v) intravenous administration of Lm-SIY. Graphs show antigen-specific cells in blood, LN or spleen, respectively. d) i) Mice were implanted with CT26 and treated with anti-CTLA4 on d7 and radiation on day 14. Groups were randomized to treatment with i-ii) FTY-720 1day prior to tumor implantation or iii-iv) 1day prior to radiation. ( e ) Mice were implanted with CT26 and i) left untreated; ii) treated with anti-PD1 on d7, 14 and 21; iii) treated with radiation on day 14; iv) treated with the combination; iv) treated with FTY720 1day prior to implantation and radiation on d14. v) treated with FTY720 1day prior to implantation, anti-PD1 on d7, 14 and 21 and radiation on d14. Key: *p

Techniques Used: Tumor Implantation, Inhibition, Mouse Assay, Injection

20) Product Images from "Migratory dendritic cells acquire and present lymphatic endothelial cell-archived antigens during lymph node contraction"

Article Title: Migratory dendritic cells acquire and present lymphatic endothelial cell-archived antigens during lymph node contraction

Journal: Nature Communications

doi: 10.1038/s41467-017-02247-z

Archived antigen acquisition and DC antigen presentation. a Representative image of raw data from wild-type mice challenged with antigen (10 μg/site) alone, antigen with poly(I:C) (2 μg/site), and anti-CD40 (2 μg/site) or antigen (10 μg/site) with vaccinia virus (1 × 10 4 CFU). One week after challenge, mice were harvested and flow cytometry was performed to visualize antigen acquisition. Shown are cells gated as in Supplementary Fig. 1B (CD45 − , PDPN + , CD31 + ). b Number of antigen-positive lymphatic endothelial cells per lymph node was calculated from each challenge in a . c Mice were immunized weekly as indicated with antigen and poly(I:C)/aCD40 (as above). LECs were gated as CD45 − , CD31 + , PDPN + , blood endothelial cells (BECs) were gated as CD45 − , CD31 + , PDPN − , FRC/marginal reticular cells (MRC)/FDCs were gated as CD45 − , CD31 − , PDPN + , macrophages (Mac) were gated as CD11c − , B220 − , CD11b hi , F4/80 + , DCs were gated as CD11c hi , B220 − , F4/80 − , B cells were gated as B220 + , and T cells were gated as CD3 + cells (Supplementary Fig. 1 ). The percent of antigen-positive of each cell type was calculated as a percent of the total cell type. Error bars shown in figure are mean ± standard deviation from two independent experiments with three mice per group per time point. d Schematic of the experimental design. e Wild-type mice were irradiated and K bm8 (cannot effectively present the dominant ovalbumin peptide) bone marrow was used to reconstitute the hematopoietic compartment. OT1 T cells were transferred into these mice 2 weeks after immunization with ovalbumin (10 μg/site), poly(I:C)/anti-CD40 (2 μg/site), and 1 day later WT BMDCs were transferred subcutaneously (1 × 10 6 per site). OT1 division of unimmunized mice that received BMDCs is shown in black, mice immunized, but did not receive BMDCs is shown in light gray, and mice immunized and received BMDCs is shown in dark gray. f Quantification of e . Shown in e is a representative experiment and in f quantification is shown from two independent experiments with three chimeric mice per group, where error bars are mean ± standard error of the mean. Statistical analysis was done using an unpaired t -test
Figure Legend Snippet: Archived antigen acquisition and DC antigen presentation. a Representative image of raw data from wild-type mice challenged with antigen (10 μg/site) alone, antigen with poly(I:C) (2 μg/site), and anti-CD40 (2 μg/site) or antigen (10 μg/site) with vaccinia virus (1 × 10 4 CFU). One week after challenge, mice were harvested and flow cytometry was performed to visualize antigen acquisition. Shown are cells gated as in Supplementary Fig. 1B (CD45 − , PDPN + , CD31 + ). b Number of antigen-positive lymphatic endothelial cells per lymph node was calculated from each challenge in a . c Mice were immunized weekly as indicated with antigen and poly(I:C)/aCD40 (as above). LECs were gated as CD45 − , CD31 + , PDPN + , blood endothelial cells (BECs) were gated as CD45 − , CD31 + , PDPN − , FRC/marginal reticular cells (MRC)/FDCs were gated as CD45 − , CD31 − , PDPN + , macrophages (Mac) were gated as CD11c − , B220 − , CD11b hi , F4/80 + , DCs were gated as CD11c hi , B220 − , F4/80 − , B cells were gated as B220 + , and T cells were gated as CD3 + cells (Supplementary Fig. 1 ). The percent of antigen-positive of each cell type was calculated as a percent of the total cell type. Error bars shown in figure are mean ± standard deviation from two independent experiments with three mice per group per time point. d Schematic of the experimental design. e Wild-type mice were irradiated and K bm8 (cannot effectively present the dominant ovalbumin peptide) bone marrow was used to reconstitute the hematopoietic compartment. OT1 T cells were transferred into these mice 2 weeks after immunization with ovalbumin (10 μg/site), poly(I:C)/anti-CD40 (2 μg/site), and 1 day later WT BMDCs were transferred subcutaneously (1 × 10 6 per site). OT1 division of unimmunized mice that received BMDCs is shown in black, mice immunized, but did not receive BMDCs is shown in light gray, and mice immunized and received BMDCs is shown in dark gray. f Quantification of e . Shown in e is a representative experiment and in f quantification is shown from two independent experiments with three chimeric mice per group, where error bars are mean ± standard error of the mean. Statistical analysis was done using an unpaired t -test

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Standard Deviation, Irradiation

21) Product Images from "The Zinc Finger Protein Zbtb18 Represses Expression of Class I Phosphatidylinositol 3-Kinase Subunits and Inhibits Plasma Cell Differentiation"

Article Title: The Zinc Finger Protein Zbtb18 Represses Expression of Class I Phosphatidylinositol 3-Kinase Subunits and Inhibits Plasma Cell Differentiation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.2000367

Zbtb18 overexpression negatively regulates PI3K signaling in B cells. ( A and B ) Immunoblot analysis of p-Akt, total Akt, and GAPDH in Ctrl/RFP– or Zbtb18/GFP–transduced activated B cells. The schematic of the experiment is shown in (A). Left, Quantification of p-Akt normalized against total Akt and GAPDH. Figure shows data from four independent experiments performed. Each circle represents data from one mouse transduced and stimulated independently. Right, A representative immunoblots from the above. ( C ) Intracellular stain of p-AKT in Ctrl/RFP– or Zbtb18/GFP–transduced B cells before (day 0) or 3 d post coincubation in the presence IL-4 and anti-CD40. Histograms are pregated on B220 + CD138 − cells. Cells were preincubated with 40LB prior to stimulation and mixed, as in (A). Left, A representative flow cytometry plot. Right, Average MFI of p-AKT from technical replicates ( n = 3). The figure shows the result from one independent experiment out of four performed. ( D ) WEHI cells were stably transduced with control/RFP and Zbtb18/GFP and cocultured for 2 d. Cells were stimulated with anti-IgM and anti-CD40 for the indicated time points, fixed, and stained for intracellular p-Akt. Left, A representative histogram pregated on RFP + (control)- or GFP + (Zbtb18)-transduced cells. Right, quantification of average p-Akt MFI in technical replicates ( n = 4) from one representative experiment out of three performed. ( E and F ) WEHI cells stably transduced with control/RFP and Zbtb18/GFP were cocultured for 2 d. RFP + and GFP + cells were sorted and subjected to immunoblot analysis to detect levels of p-Akt, total Akt, and GAPDH. A representative immunoblots is shown in (E). In (F), quantification of p-Akt normalized against total Akt and GAPDH. (F) shows pooled data from three independent experiments (color coded). One circle represents data from one biological replicate. ( G ) Control/GFP– or Zbtb18/GFP–transduced splenocytes were cotransduced with control-shRNA/RFP or PTEN-shRNA/RFP, as indicated. Cells were stimulated for 3 d posttransduction with anti-CD40 and IL-4. Frequencies of PCs in GFP + RFP + cotransduced B cells were determined by flow cytometry. The figure shows one out of three independent experiments performed. Each circle represents one technical replicate. ( H ) B cells transduced with control/RFP or Zbtb18/GFP were sorted, cocultured, and incubated with LPS with or witho ut increasing concentrations of FOXO1 inhibitor and analyzed by flow cytometry 3 d later. The frequencies of GFP + and RFP + cells in the non-PCs (B220 high CD138 − ) and PCs (B220 low CD138 high ) populations are shown. Figure shows one out of three independent experiments performed ( n = 3, technical replicates). * p
Figure Legend Snippet: Zbtb18 overexpression negatively regulates PI3K signaling in B cells. ( A and B ) Immunoblot analysis of p-Akt, total Akt, and GAPDH in Ctrl/RFP– or Zbtb18/GFP–transduced activated B cells. The schematic of the experiment is shown in (A). Left, Quantification of p-Akt normalized against total Akt and GAPDH. Figure shows data from four independent experiments performed. Each circle represents data from one mouse transduced and stimulated independently. Right, A representative immunoblots from the above. ( C ) Intracellular stain of p-AKT in Ctrl/RFP– or Zbtb18/GFP–transduced B cells before (day 0) or 3 d post coincubation in the presence IL-4 and anti-CD40. Histograms are pregated on B220 + CD138 − cells. Cells were preincubated with 40LB prior to stimulation and mixed, as in (A). Left, A representative flow cytometry plot. Right, Average MFI of p-AKT from technical replicates ( n = 3). The figure shows the result from one independent experiment out of four performed. ( D ) WEHI cells were stably transduced with control/RFP and Zbtb18/GFP and cocultured for 2 d. Cells were stimulated with anti-IgM and anti-CD40 for the indicated time points, fixed, and stained for intracellular p-Akt. Left, A representative histogram pregated on RFP + (control)- or GFP + (Zbtb18)-transduced cells. Right, quantification of average p-Akt MFI in technical replicates ( n = 4) from one representative experiment out of three performed. ( E and F ) WEHI cells stably transduced with control/RFP and Zbtb18/GFP were cocultured for 2 d. RFP + and GFP + cells were sorted and subjected to immunoblot analysis to detect levels of p-Akt, total Akt, and GAPDH. A representative immunoblots is shown in (E). In (F), quantification of p-Akt normalized against total Akt and GAPDH. (F) shows pooled data from three independent experiments (color coded). One circle represents data from one biological replicate. ( G ) Control/GFP– or Zbtb18/GFP–transduced splenocytes were cotransduced with control-shRNA/RFP or PTEN-shRNA/RFP, as indicated. Cells were stimulated for 3 d posttransduction with anti-CD40 and IL-4. Frequencies of PCs in GFP + RFP + cotransduced B cells were determined by flow cytometry. The figure shows one out of three independent experiments performed. Each circle represents one technical replicate. ( H ) B cells transduced with control/RFP or Zbtb18/GFP were sorted, cocultured, and incubated with LPS with or witho ut increasing concentrations of FOXO1 inhibitor and analyzed by flow cytometry 3 d later. The frequencies of GFP + and RFP + cells in the non-PCs (B220 high CD138 − ) and PCs (B220 low CD138 high ) populations are shown. Figure shows one out of three independent experiments performed ( n = 3, technical replicates). * p

Techniques Used: Over Expression, Western Blot, Staining, Flow Cytometry, Stable Transfection, Transduction, shRNA, Incubation

Zbtb18 regulates differentiation, but not survival of PCs. ( A – C ) Splenocytes derived from WT mice (upper panel) or Bcl2-Tg mice (lower panel) were transduced with control/RFP or Zbtb18/GFP retroviral vectors, sorted, and incubated with anti-CD40 and IL-4, as described in Fig. 1A . Cocultured cells were analyzed by flow cytometry before (A) and 3 d (B) poststimulation. The relative frequencies of GFP + and RFP + cells in the non-PC and PCs compartments are shown (C). (A)–(C) show the results of one representative experiment out of three independent experiments performed. Each circle represents a technical replicate. ( D and E ) Control/GFP– or Zbtb18/GFP–transduced splenocytes were incubated with anti-CD40 and IL-4. After 3 d, cells were sorted (GFP + B220 + live cells) and subjected to qPCR analysis of antiapoptotic genes (D). Expression of mRNA is presented relative to the abundance of GAPDH ( n = 3). Live count (trypan blue) was performed to determine viability (E). Data in (D) and (E) show the results from one representative experiment out of three independent experiments performed. Each circle represents a technical replicate. ( F and G ) Cells derived from Blimp-1–Venus–transgenic mice were labeled with the cell tracer dye CTV and stimulated with IL-4 and anti-CD40 for 3 d. Cells were sorted based on CTV dilutions (divisions [Div] 0, 2, and 5) and Blimp-1 expression and each fraction was analyzed by qPCR for expression of the indicated genes. Left, Flow cytometry showing sorting strategy to purify dividing cells. Right, Expression of mRNA is presented relative to the abundance of GAPDH and HPRT. Data shown in (F) and (G) are from one representative experiment out of three independent experiments performed. Each circle represents one technical replicate. ( H ) Cell division and PC differentiation in CTV-labeled splenic B cells from BM chimeras transduced with control/GFP or Zbtb18/GFP retroviral vector followed by 3 d incubation with anti-CD40 and IL-4. Left, Representative flow cytometry plots showing cell division. Cells were pregated on GFP + B220 + cells. Right, Frequencies of cells in each cell division of total B cells. Data shown in (H) are from one representative experiment out of three independent experiments performed ( n = 3, technical replicates). * p
Figure Legend Snippet: Zbtb18 regulates differentiation, but not survival of PCs. ( A – C ) Splenocytes derived from WT mice (upper panel) or Bcl2-Tg mice (lower panel) were transduced with control/RFP or Zbtb18/GFP retroviral vectors, sorted, and incubated with anti-CD40 and IL-4, as described in Fig. 1A . Cocultured cells were analyzed by flow cytometry before (A) and 3 d (B) poststimulation. The relative frequencies of GFP + and RFP + cells in the non-PC and PCs compartments are shown (C). (A)–(C) show the results of one representative experiment out of three independent experiments performed. Each circle represents a technical replicate. ( D and E ) Control/GFP– or Zbtb18/GFP–transduced splenocytes were incubated with anti-CD40 and IL-4. After 3 d, cells were sorted (GFP + B220 + live cells) and subjected to qPCR analysis of antiapoptotic genes (D). Expression of mRNA is presented relative to the abundance of GAPDH ( n = 3). Live count (trypan blue) was performed to determine viability (E). Data in (D) and (E) show the results from one representative experiment out of three independent experiments performed. Each circle represents a technical replicate. ( F and G ) Cells derived from Blimp-1–Venus–transgenic mice were labeled with the cell tracer dye CTV and stimulated with IL-4 and anti-CD40 for 3 d. Cells were sorted based on CTV dilutions (divisions [Div] 0, 2, and 5) and Blimp-1 expression and each fraction was analyzed by qPCR for expression of the indicated genes. Left, Flow cytometry showing sorting strategy to purify dividing cells. Right, Expression of mRNA is presented relative to the abundance of GAPDH and HPRT. Data shown in (F) and (G) are from one representative experiment out of three independent experiments performed. Each circle represents one technical replicate. ( H ) Cell division and PC differentiation in CTV-labeled splenic B cells from BM chimeras transduced with control/GFP or Zbtb18/GFP retroviral vector followed by 3 d incubation with anti-CD40 and IL-4. Left, Representative flow cytometry plots showing cell division. Cells were pregated on GFP + B220 + cells. Right, Frequencies of cells in each cell division of total B cells. Data shown in (H) are from one representative experiment out of three independent experiments performed ( n = 3, technical replicates). * p

Techniques Used: Derivative Assay, Mouse Assay, Transduction, Incubation, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing, Transgenic Assay, Labeling, Plasmid Preparation

Zbtb18 suppresses differentiation of human PCs. ( A ) qPCR analysis of the transcript abundance of Zbtb18 in human naive cell (CD19 + IgD + CD27 − ), memory cell (CD19 + CD27 + CD38 − ), and PC (CD19 + IgD − CD27 + CD38 + ) sorted from human peripheral blood samples. Expression of mRNA is presented relative to the abundance of RPL13A. Data are from two biological replicates analyzed with two technical replicates each. The experiment is one out of two independent experiments performed. ( B ) Human B cells were purified from healthy donors, stimulated, and electroporated with control (Ctrl) or Zbtb18 RNA. Left, Representative example of flow cytometry analysis of cells 5 d poststimulation pregated on CD19 + cells. Right, Frequencies of PCs (CD38 + CD27 + ). Data are from pooled samples from four independent experiments performed. Each experiment is color coded. ( C ) Left, Schematic illustration of point mutations in Zbtb18 identified in cancer patients. Red lines highlight mutations that target residues within the zinc finger motif or that lead to loss of the entire domain. Right, B cells from mouse spleen were transduced with control/RFP or Zbtb18-Myc–Tagged/GFP WT or mutants, sorted, coincubated, and stimulated with IL-4 and anti-CD40 for 3 d (as in Fig. 1A ). Shown are the frequencies of non-PC and PC populations in GFP- and RFP-expressing cells. Data show one representative experiment out of three independent experiments performed. Each circle represents a technical replicate. ( D ) Anti-Myc intracellular stain splenocytes transduced with Zbtb18-Myc–tagged WT and mutants. Ctrl B cells transduced with empty (GFP-only) vector. Histograms are pregated on untransduced (GFP − , black) and transduced (GFP + , green) fractions. Left, Representative histograms. Right, MFIs of Myc. Figure shows data from one representative experiment out of three independent experiments performed. Each circle represents a technical replicate. ( E ) Chip qPCR analysis for binding of Zbtb18-Myc (WT) or the indicated mutated forms to class I PI3K genes. β-actin was used as a nonspecific binding gene. Ctrl B cells transduced with empty (GFP) vector. Analysis was performed on sorted cells (live B220 + GFP + ) 2 d after anti-CD40 and IL-4 stimulation. Figure shows one representative experiment out of three independent experiments performed ( n = 4, technical replicates). * p
Figure Legend Snippet: Zbtb18 suppresses differentiation of human PCs. ( A ) qPCR analysis of the transcript abundance of Zbtb18 in human naive cell (CD19 + IgD + CD27 − ), memory cell (CD19 + CD27 + CD38 − ), and PC (CD19 + IgD − CD27 + CD38 + ) sorted from human peripheral blood samples. Expression of mRNA is presented relative to the abundance of RPL13A. Data are from two biological replicates analyzed with two technical replicates each. The experiment is one out of two independent experiments performed. ( B ) Human B cells were purified from healthy donors, stimulated, and electroporated with control (Ctrl) or Zbtb18 RNA. Left, Representative example of flow cytometry analysis of cells 5 d poststimulation pregated on CD19 + cells. Right, Frequencies of PCs (CD38 + CD27 + ). Data are from pooled samples from four independent experiments performed. Each experiment is color coded. ( C ) Left, Schematic illustration of point mutations in Zbtb18 identified in cancer patients. Red lines highlight mutations that target residues within the zinc finger motif or that lead to loss of the entire domain. Right, B cells from mouse spleen were transduced with control/RFP or Zbtb18-Myc–Tagged/GFP WT or mutants, sorted, coincubated, and stimulated with IL-4 and anti-CD40 for 3 d (as in Fig. 1A ). Shown are the frequencies of non-PC and PC populations in GFP- and RFP-expressing cells. Data show one representative experiment out of three independent experiments performed. Each circle represents a technical replicate. ( D ) Anti-Myc intracellular stain splenocytes transduced with Zbtb18-Myc–tagged WT and mutants. Ctrl B cells transduced with empty (GFP-only) vector. Histograms are pregated on untransduced (GFP − , black) and transduced (GFP + , green) fractions. Left, Representative histograms. Right, MFIs of Myc. Figure shows data from one representative experiment out of three independent experiments performed. Each circle represents a technical replicate. ( E ) Chip qPCR analysis for binding of Zbtb18-Myc (WT) or the indicated mutated forms to class I PI3K genes. β-actin was used as a nonspecific binding gene. Ctrl B cells transduced with empty (GFP) vector. Analysis was performed on sorted cells (live B220 + GFP + ) 2 d after anti-CD40 and IL-4 stimulation. Figure shows one representative experiment out of three independent experiments performed ( n = 4, technical replicates). * p

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Purification, Flow Cytometry, Transduction, Staining, Plasmid Preparation, Chromatin Immunoprecipitation, Binding Assay

Zbtb18 directly binds and suppresses expression of PI3K class I genes. ( A ) Splenocytes were transduced with control (RFP + ) or Zbtb18 (GFP + ) vectors. Cells were analyzed by flow cytometry and mixed to achieve a 1:1 ratio of B220 + -GFP + – and RFP + -expressing cells, followed by stimulation with anti-CD40 and IL-4. Two days after stimulation, RFP + or GFP + B220 + CD138 – live cells were sorted by flow cytometry and subjected to Chip qPCR analysis of the ATAC-seq differential peaks of Pik3r1 , Pik3r5 , and Pik3r6 genes. Data are from pooled samples from three independent experiments performed (total n = 3). ( B ) De novo motif–enrichment analysis in repressed ATAC-seq peaks of Zbtb18 versus control (Ctrl) cells. Motif density distribution relative to the peak summit for Zbtb18, Pu.1 and Oct2 motifs in the repressed ATAC-seq peaks (bottom). ( C ) Chip qPCR analysis of interactions between Myc-tagged Zbtb18 (WT), or ΔZF with the indicated genes. Ctrl B cells transduced with empty GFP-expressing vector. Conserved Zbtb18-binding motif identified in the ATAC-seq peaks of the indicated genes are shown in red. Analysis was performed on transduced sorted cells (live B220 + GFP + cells) 2 d after anti-CD40 and IL-4 stimulation. Data show pooled samples collected in three independent experiments performed (total n = 3, biological replicates). ( D ) Immunoblot analysis of p85a, p101, and GAPDH in Ctrl/RFP– or Zbtb18/GFP–transduced B cells 2 d after being cocultured with or without anti-CD40 and IL4 stimulation (left) [as in (A) above]. Cells were sorted prior to analysis on day 2 (gated on live B220 + CD138 − GFP + or RFP + cells). Quantification of p85a or p101 expression normalized with GAPDH (right). Figure shows combined data collected in two independent experiments performed. Each circle represents data from one experiment. ( E ) qPCR analysis of the indicated genes in control- and Zbtb18-transduced B cells, sorted (as GFP + B220 + CD138 − live cells) 2 d poststimulation with anti-CD40 and IL-4. Expression of mRNA is presented relative to the abundance of GAPDH. Data in (E) show the results from one representative experiment out of three independent experiments performed. Each circle represents a technical replicate. * p
Figure Legend Snippet: Zbtb18 directly binds and suppresses expression of PI3K class I genes. ( A ) Splenocytes were transduced with control (RFP + ) or Zbtb18 (GFP + ) vectors. Cells were analyzed by flow cytometry and mixed to achieve a 1:1 ratio of B220 + -GFP + – and RFP + -expressing cells, followed by stimulation with anti-CD40 and IL-4. Two days after stimulation, RFP + or GFP + B220 + CD138 – live cells were sorted by flow cytometry and subjected to Chip qPCR analysis of the ATAC-seq differential peaks of Pik3r1 , Pik3r5 , and Pik3r6 genes. Data are from pooled samples from three independent experiments performed (total n = 3). ( B ) De novo motif–enrichment analysis in repressed ATAC-seq peaks of Zbtb18 versus control (Ctrl) cells. Motif density distribution relative to the peak summit for Zbtb18, Pu.1 and Oct2 motifs in the repressed ATAC-seq peaks (bottom). ( C ) Chip qPCR analysis of interactions between Myc-tagged Zbtb18 (WT), or ΔZF with the indicated genes. Ctrl B cells transduced with empty GFP-expressing vector. Conserved Zbtb18-binding motif identified in the ATAC-seq peaks of the indicated genes are shown in red. Analysis was performed on transduced sorted cells (live B220 + GFP + cells) 2 d after anti-CD40 and IL-4 stimulation. Data show pooled samples collected in three independent experiments performed (total n = 3, biological replicates). ( D ) Immunoblot analysis of p85a, p101, and GAPDH in Ctrl/RFP– or Zbtb18/GFP–transduced B cells 2 d after being cocultured with or without anti-CD40 and IL4 stimulation (left) [as in (A) above]. Cells were sorted prior to analysis on day 2 (gated on live B220 + CD138 − GFP + or RFP + cells). Quantification of p85a or p101 expression normalized with GAPDH (right). Figure shows combined data collected in two independent experiments performed. Each circle represents data from one experiment. ( E ) qPCR analysis of the indicated genes in control- and Zbtb18-transduced B cells, sorted (as GFP + B220 + CD138 − live cells) 2 d poststimulation with anti-CD40 and IL-4. Expression of mRNA is presented relative to the abundance of GAPDH. Data in (E) show the results from one representative experiment out of three independent experiments performed. Each circle represents a technical replicate. * p

Techniques Used: Expressing, Transduction, Flow Cytometry, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Plasmid Preparation, Binding Assay

Zbtb18 suppresses PCs responses. ( A – C ) Splenocytes were transduced with Zbtb18/GFP or control/RFP retroviral vectors. B220 + RFP + and GFP + live cells were sorted by flow cytometry, mixed in a 1:1 ratio (day 0), and incubated with anti-CD40 and IL-4 for 3 d. A schematic illustration of the experiment is shown in (A). An example for flow cytometry gating scheme at day 0 (B) and day 3 (C) are shown. Red arrows show gating strategy. The ratio between Zbtb18/GFP + and Ctrl/RFP + cells in the non-PCs (B220 high CD138 − ) and PCs (B220 low CD138 high ) compartments is shown (C, right). (A)–(C) show the result of one representative experiment out of at least three independent experiments performed. Each circle represents one technical replicate. ( D – F ) CD45.2 + splenocytes were transduced with control/Thy1.1 or with Zbtb18/GFP and then cultured in the presence of 40LB stroma cells for 2 d. Transduced cells were analyzed by flow cytometry and cotransferred in a 1:1 ration into CD45.1 + MD4 mice. The next day, mice were immunized with SRBCs, and 12 d later, their spleens were analyzed by flow cytometry. A schematic illustration of the experiment is shown in (D). The gating scheme is shown (plots are pregated on live CD45.1 neg cells) (E). The ratios between CD45.2 + Zbtb18/GFP– and CD45.2 + control/Thy1.1–expressing cells were determined in the indicated populations (F). (D)–(F) show pooled data from two independent experiments performed. Each circle represents data from one mouse. ** p
Figure Legend Snippet: Zbtb18 suppresses PCs responses. ( A – C ) Splenocytes were transduced with Zbtb18/GFP or control/RFP retroviral vectors. B220 + RFP + and GFP + live cells were sorted by flow cytometry, mixed in a 1:1 ratio (day 0), and incubated with anti-CD40 and IL-4 for 3 d. A schematic illustration of the experiment is shown in (A). An example for flow cytometry gating scheme at day 0 (B) and day 3 (C) are shown. Red arrows show gating strategy. The ratio between Zbtb18/GFP + and Ctrl/RFP + cells in the non-PCs (B220 high CD138 − ) and PCs (B220 low CD138 high ) compartments is shown (C, right). (A)–(C) show the result of one representative experiment out of at least three independent experiments performed. Each circle represents one technical replicate. ( D – F ) CD45.2 + splenocytes were transduced with control/Thy1.1 or with Zbtb18/GFP and then cultured in the presence of 40LB stroma cells for 2 d. Transduced cells were analyzed by flow cytometry and cotransferred in a 1:1 ration into CD45.1 + MD4 mice. The next day, mice were immunized with SRBCs, and 12 d later, their spleens were analyzed by flow cytometry. A schematic illustration of the experiment is shown in (D). The gating scheme is shown (plots are pregated on live CD45.1 neg cells) (E). The ratios between CD45.2 + Zbtb18/GFP– and CD45.2 + control/Thy1.1–expressing cells were determined in the indicated populations (F). (D)–(F) show pooled data from two independent experiments performed. Each circle represents data from one mouse. ** p

Techniques Used: Transduction, Flow Cytometry, Incubation, Cell Culture, Mouse Assay, Expressing

22) Product Images from "Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice"

Article Title: Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1510518113

Combination therapy with vaccination reverses CD8 T-cell anergy and restores T-cell function in a spontaneous adenocarcinoma model. ( A ) Naive Pmel TCR transgenic CD8 T cells were purified by negative selection and adoptively transferred on day 6 into B16F10 tumor-bearing wild-type C57BL/6 mice. Mice were treated with IgG or aOX40 on days 7 and 11; with aCTLA-4 on days 7, 9, and 11; or with monotherapy or combination therapy and vaccine (gp100/anti-CD40) on day 7. Blood was analyzed by flow cytometry on day 14. ( B – D ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET-1 mice on day 0. Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28 along with mono- or combination therapy (50 µg aOX40 or control rat IgG on days 28 and 29 and 200 µg aCTLA-4 on days 28, 30, and 32). Splenocytes were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict ( B ) the total number of OT-I + cells; the total number of Ki-67 + , granzyme B + (GrzB), KLRG-1 + , CD62L + , and CD25 + OT-I; ( C and D ) and the percentage ( C ) and total number ( D ) of IFNγ-, TNFα-, and IL-2--producing OT-I cells. Data are shown for one representative mouse ( C ) or for all mice in each cohort for donor OT-I cells ( B and D ). Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group). * P
Figure Legend Snippet: Combination therapy with vaccination reverses CD8 T-cell anergy and restores T-cell function in a spontaneous adenocarcinoma model. ( A ) Naive Pmel TCR transgenic CD8 T cells were purified by negative selection and adoptively transferred on day 6 into B16F10 tumor-bearing wild-type C57BL/6 mice. Mice were treated with IgG or aOX40 on days 7 and 11; with aCTLA-4 on days 7, 9, and 11; or with monotherapy or combination therapy and vaccine (gp100/anti-CD40) on day 7. Blood was analyzed by flow cytometry on day 14. ( B – D ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET-1 mice on day 0. Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28 along with mono- or combination therapy (50 µg aOX40 or control rat IgG on days 28 and 29 and 200 µg aCTLA-4 on days 28, 30, and 32). Splenocytes were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict ( B ) the total number of OT-I + cells; the total number of Ki-67 + , granzyme B + (GrzB), KLRG-1 + , CD62L + , and CD25 + OT-I; ( C and D ) and the percentage ( C ) and total number ( D ) of IFNγ-, TNFα-, and IL-2--producing OT-I cells. Data are shown for one representative mouse ( C ) or for all mice in each cohort for donor OT-I cells ( B and D ). Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group). * P

Techniques Used: Cell Function Assay, Transgenic Assay, Purification, Selection, Mouse Assay, Flow Cytometry, Cytometry

( A and C ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET mice (day 0). Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28, along with mono- or combination therapy: 50 µg aOX40 or control rat IgG on days 28 and 29, and 200 µg aCTLA-4 on days 28, 30, and 3. ( A ) Model of treatment. ( B ) Naive OT-I CD8 T cells (2.5 × 10 6 ) were purified by negative selection and adoptively transferred into TRAMP/POET or wild-type C57BL/6 mice. Three days later, lymph nodes were processed, and OX40 and CTLA-4 expression was analyzed by flow cytometry on donor OT-1 cells. For CTLA-4 expression on donor OT-1 cells, the unstained control is shown in gray; the wild-type control is shown as a solid black line; TRAMP/POET mice are shown as a purple line. For OX40 expression ( Middle and Right ), images represent OT-I + (Thy1.1 + ) CD8 T cells versus OX40 expression; the numbers shown are the percentage of OX40 + OT-I cells. ( C ) Splenocytes and prostate-draining lymph nodes (pooled for each group) were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group).
Figure Legend Snippet: ( A and C ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET mice (day 0). Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28, along with mono- or combination therapy: 50 µg aOX40 or control rat IgG on days 28 and 29, and 200 µg aCTLA-4 on days 28, 30, and 3. ( A ) Model of treatment. ( B ) Naive OT-I CD8 T cells (2.5 × 10 6 ) were purified by negative selection and adoptively transferred into TRAMP/POET or wild-type C57BL/6 mice. Three days later, lymph nodes were processed, and OX40 and CTLA-4 expression was analyzed by flow cytometry on donor OT-1 cells. For CTLA-4 expression on donor OT-1 cells, the unstained control is shown in gray; the wild-type control is shown as a solid black line; TRAMP/POET mice are shown as a purple line. For OX40 expression ( Middle and Right ), images represent OT-I + (Thy1.1 + ) CD8 T cells versus OX40 expression; the numbers shown are the percentage of OX40 + OT-I cells. ( C ) Splenocytes and prostate-draining lymph nodes (pooled for each group) were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group).

Techniques Used: Purification, Selection, Mouse Assay, Expressing, Flow Cytometry, Cytometry

23) Product Images from "Amplifying IFNγ signaling in DC by CD11c-specific loss of SOCS1 increases innate immunity to infection while decreasing adaptive immunity"

Article Title: Amplifying IFNγ signaling in DC by CD11c-specific loss of SOCS1 increases innate immunity to infection while decreasing adaptive immunity

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1700909

Diminished CD8 T cell responses to infection in mice lacking SOCS1 in DC a) Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl littermates were primed with 1×10 5 cfu ΔactA QV, 5μg anti-DEC205-OVA with 25 μg anti-CD40 or 1×10 6 pfu VV-OVA. Spleens were harvested 7 days post priming and IFNγ + SIINFEKL-specific CD8 T cells determined by using ICS. i) Representative flow-cytometry plots of IFNγ expression in response to SIINFEKL peptide. Quantitation of ii) the percent and iii) absolute numbers of IFNγ + SIINFEKL-specific CD8 T cells per spleen. b) Quantitation of the percent of IFNγ + i) B8R 20–27 -specific or ii) A42R 88–96 -specific CD8 T cells in spleens from mice immunized with ΔactA QV. c) Percent IFNγ + LLO 190-201 –specific CD4 T cells 7 days post immunization with ΔactA QV. d) OVA-specific CD90.1 + OT1 CD8 T cells were isolated from OT1 transgenic mice and 10,000 cells per mouse adoptively transferred to Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice. The following day mice were immunized with 1×10 5 cfu ΔactA QV as above, spleens harvested 7 days later, cells stained and CD90.1 + (OT1) CD8 T cells analyzed. i) Representative flow-cytometry plot of CD90.1 + CD8 T cell expansion in Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice. ii) The percent and iii) total number of OT1 cells per mouse. e) CD8 T cells from Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice were purified and transferred to Rag1 -/- mice. 24hr later mice were immunized with 1×10 5 cfu ΔactA QV. Spleens were harvested 7 post infection and splenocytes stimulated in vitro with the indicated peptide. i) Percent and ii) absolute numbers of antigen-specific CD8 T cells as well as iii) percent of LLO 190-201 CD4 T cell responses are shown. Each symbol represents one mouse, n=3-5 mice per group. Data represents the mean ± SEM of each group. The displayed experiments are representative of 3 to 5 independent repeats. Statistics calculated by Student's t test; ns = no significant differences observed between the groups analyzed; ** = p
Figure Legend Snippet: Diminished CD8 T cell responses to infection in mice lacking SOCS1 in DC a) Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl littermates were primed with 1×10 5 cfu ΔactA QV, 5μg anti-DEC205-OVA with 25 μg anti-CD40 or 1×10 6 pfu VV-OVA. Spleens were harvested 7 days post priming and IFNγ + SIINFEKL-specific CD8 T cells determined by using ICS. i) Representative flow-cytometry plots of IFNγ expression in response to SIINFEKL peptide. Quantitation of ii) the percent and iii) absolute numbers of IFNγ + SIINFEKL-specific CD8 T cells per spleen. b) Quantitation of the percent of IFNγ + i) B8R 20–27 -specific or ii) A42R 88–96 -specific CD8 T cells in spleens from mice immunized with ΔactA QV. c) Percent IFNγ + LLO 190-201 –specific CD4 T cells 7 days post immunization with ΔactA QV. d) OVA-specific CD90.1 + OT1 CD8 T cells were isolated from OT1 transgenic mice and 10,000 cells per mouse adoptively transferred to Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice. The following day mice were immunized with 1×10 5 cfu ΔactA QV as above, spleens harvested 7 days later, cells stained and CD90.1 + (OT1) CD8 T cells analyzed. i) Representative flow-cytometry plot of CD90.1 + CD8 T cell expansion in Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice. ii) The percent and iii) total number of OT1 cells per mouse. e) CD8 T cells from Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice were purified and transferred to Rag1 -/- mice. 24hr later mice were immunized with 1×10 5 cfu ΔactA QV. Spleens were harvested 7 post infection and splenocytes stimulated in vitro with the indicated peptide. i) Percent and ii) absolute numbers of antigen-specific CD8 T cells as well as iii) percent of LLO 190-201 CD4 T cell responses are shown. Each symbol represents one mouse, n=3-5 mice per group. Data represents the mean ± SEM of each group. The displayed experiments are representative of 3 to 5 independent repeats. Statistics calculated by Student's t test; ns = no significant differences observed between the groups analyzed; ** = p

Techniques Used: Infection, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Quantitation Assay, Isolation, Transgenic Assay, Staining, Purification, In Vitro

Increased innate response and TipDC activity in mice lacking SOCS1 in DC a) i) Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice were immunized with anti-DEC205-OVA and anti-CD40 to generate an equivalent priming event then boosted 21 days later with 1×10 5 cfu ΔactA QV. ii) Spleens were harvested 5 days post-boost and IFNγ + SIINFEKL-specific CD8 T cells determined by using ICS. b) Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice were left untreated or primed with 1×10 5 cfu ΔactA QV and challenged 21 days later with 1×10 5 cfu wt L. monocytogenes . Three days later i) spleens and ii) livers were harvested, homogenized, plated on BHI plates and cfu numbers determined after overnight incubation at 37°C. c) Spleens from naïve mice were harvested, stained and analyzed by flow-cytometry. i) Summary of the absolute number of CD11b + Ly6C high cells and ii) the absolute number of Ly6G + Ly6C int cells gated in the population. d) Mice were left untreated (0hr) or immunized with 1×10 4 cfu wt, spleens removed at 24hr and 48hr post-infection, dissociated, stained and iNOS synthesis determined by ICS. i) Representative expression of iNOS and MHCII in CD3 - CD19 - CD11b + Ly6C high monocytes over time, and ii) summary of the number of iNOS + MHCII + Tip-DC/spleen. Each symbol represents one mouse. Data represents the mean ± SEM of each group. Results shown are representative of three independent experiments. Statistics calculated by Student's t test; ns = no significant differences observed between the groups analyzed; * = p
Figure Legend Snippet: Increased innate response and TipDC activity in mice lacking SOCS1 in DC a) i) Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice were immunized with anti-DEC205-OVA and anti-CD40 to generate an equivalent priming event then boosted 21 days later with 1×10 5 cfu ΔactA QV. ii) Spleens were harvested 5 days post-boost and IFNγ + SIINFEKL-specific CD8 T cells determined by using ICS. b) Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice were left untreated or primed with 1×10 5 cfu ΔactA QV and challenged 21 days later with 1×10 5 cfu wt L. monocytogenes . Three days later i) spleens and ii) livers were harvested, homogenized, plated on BHI plates and cfu numbers determined after overnight incubation at 37°C. c) Spleens from naïve mice were harvested, stained and analyzed by flow-cytometry. i) Summary of the absolute number of CD11b + Ly6C high cells and ii) the absolute number of Ly6G + Ly6C int cells gated in the population. d) Mice were left untreated (0hr) or immunized with 1×10 4 cfu wt, spleens removed at 24hr and 48hr post-infection, dissociated, stained and iNOS synthesis determined by ICS. i) Representative expression of iNOS and MHCII in CD3 - CD19 - CD11b + Ly6C high monocytes over time, and ii) summary of the number of iNOS + MHCII + Tip-DC/spleen. Each symbol represents one mouse. Data represents the mean ± SEM of each group. Results shown are representative of three independent experiments. Statistics calculated by Student's t test; ns = no significant differences observed between the groups analyzed; * = p

Techniques Used: Activity Assay, Mouse Assay, Incubation, Staining, Flow Cytometry, Cytometry, Infection, Expressing

24) Product Images from "Protection from tumor recurrence following adoptive immunotherapy varies with host conditioning regimen despite initial regression of autochthonous murine brain tumors"

Article Title: Protection from tumor recurrence following adoptive immunotherapy varies with host conditioning regimen despite initial regression of autochthonous murine brain tumors

Journal: Cancer immunology, immunotherapy : CII

doi: 10.1007/s00262-014-1635-7

Host conditioning with anti-CD40 induces high-level T cell accumulation in the lymphoid organs and brains of SV11 mice at early time points. Groups of mice received either anti-CD40, WBI, or no conditioning regimen with TCR-IV T-cell ACT. Representative plots show MHC tetramer staining (mean±SEM) of TCR-IV T cells on days +4 and +5 in a spleen and c brain. Quantification of TCR-IV T-cell accumulation (mean±SEM) in b spleen and d brain of mice that received ACT with the indicated treatments. n=3 mice/group (except n=2 for day +4 control group). Data shown are from one experiment and representative of two independent experiments. Asterisks above connecting lines indicate significant differences between time points. Asterisks next to vertical brackets indicate significant differences between treatment groups. * p
Figure Legend Snippet: Host conditioning with anti-CD40 induces high-level T cell accumulation in the lymphoid organs and brains of SV11 mice at early time points. Groups of mice received either anti-CD40, WBI, or no conditioning regimen with TCR-IV T-cell ACT. Representative plots show MHC tetramer staining (mean±SEM) of TCR-IV T cells on days +4 and +5 in a spleen and c brain. Quantification of TCR-IV T-cell accumulation (mean±SEM) in b spleen and d brain of mice that received ACT with the indicated treatments. n=3 mice/group (except n=2 for day +4 control group). Data shown are from one experiment and representative of two independent experiments. Asterisks above connecting lines indicate significant differences between time points. Asterisks next to vertical brackets indicate significant differences between treatment groups. * p

Techniques Used: Mouse Assay, Activated Clotting Time Assay, Staining

Anti-CD40-enhanced ACT promotes initial regression of established tumors. a H E brain sections on day +10 post-ACT following conditioning with anti-CD40 (left), control IgG (middle), or WBI (right). Representative low-power images (top row, scale bar = 1mm) and high-power images (bottom row, scale bar = 50μm) that show established tumor refractory to therapy (middle column) or tumor stromal condensation indicative of tumor regression (left and right columns). b For each mouse, the largest cross-sectional tumor area (mm 2 ) observed in H E sections was plotted. Data is pooled from multiple experiments with a total of 6–10 mice/group. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparison test. ** p
Figure Legend Snippet: Anti-CD40-enhanced ACT promotes initial regression of established tumors. a H E brain sections on day +10 post-ACT following conditioning with anti-CD40 (left), control IgG (middle), or WBI (right). Representative low-power images (top row, scale bar = 1mm) and high-power images (bottom row, scale bar = 50μm) that show established tumor refractory to therapy (middle column) or tumor stromal condensation indicative of tumor regression (left and right columns). b For each mouse, the largest cross-sectional tumor area (mm 2 ) observed in H E sections was plotted. Data is pooled from multiple experiments with a total of 6–10 mice/group. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparison test. ** p

Techniques Used: Activated Clotting Time Assay, Mouse Assay

Anti-CD40-enhanced ACT promotes increased survival but short-term surveillance against tumor recurrence. a Groups of mice received the indicated conditioning with or without ACT and were monitored for tumor recurrence. The percentage of surviving mice versus age is plotted. b Statistical differences in survival were calculated using the log-rank test. Data are pooled from multiple experiments with 5–8 mice/group.
Figure Legend Snippet: Anti-CD40-enhanced ACT promotes increased survival but short-term surveillance against tumor recurrence. a Groups of mice received the indicated conditioning with or without ACT and were monitored for tumor recurrence. The percentage of surviving mice versus age is plotted. b Statistical differences in survival were calculated using the log-rank test. Data are pooled from multiple experiments with 5–8 mice/group.

Techniques Used: Activated Clotting Time Assay, Mouse Assay

Donor T cells fail to persist in anti-CD40-conditioned SV11 mice following acute tumor regression. Groups of SV11 mice received either anti-CD40, control IgG, or WBI conditioning prior to ACT with CD90.1 + TCR-IV T cells. On day +30, cells from a spleens, cLN (not shown), and b brains were stained for CD90.1 and CD8. Values on dot plots indicate percent CD90.1 + of total CD8 + cells (mean±SEM). c Total CD90.1 + cells in spleens, cLN, and brains on day +30 are plotted. Representative histograms of CD44, CD62L, and KLRG1 expression gated on live CD45.2 + CD8 + CD90.1 + TCR-IV T cells (open histogram) or CD45.2 + CD8 + CD90.1 − T cells (filled histogram, spleen only) are shown on day +30 in d spleen and e brain. Values indicate the percent of TCR-IV T cells within the indicated gate (mean±SEM). Samples with
Figure Legend Snippet: Donor T cells fail to persist in anti-CD40-conditioned SV11 mice following acute tumor regression. Groups of SV11 mice received either anti-CD40, control IgG, or WBI conditioning prior to ACT with CD90.1 + TCR-IV T cells. On day +30, cells from a spleens, cLN (not shown), and b brains were stained for CD90.1 and CD8. Values on dot plots indicate percent CD90.1 + of total CD8 + cells (mean±SEM). c Total CD90.1 + cells in spleens, cLN, and brains on day +30 are plotted. Representative histograms of CD44, CD62L, and KLRG1 expression gated on live CD45.2 + CD8 + CD90.1 + TCR-IV T cells (open histogram) or CD45.2 + CD8 + CD90.1 − T cells (filled histogram, spleen only) are shown on day +30 in d spleen and e brain. Values indicate the percent of TCR-IV T cells within the indicated gate (mean±SEM). Samples with

Techniques Used: Mouse Assay, Activated Clotting Time Assay, Staining, Expressing

25) Product Images from "Migratory dendritic cells acquire and present lymphatic endothelial cell-archived antigens during lymph node contraction"

Article Title: Migratory dendritic cells acquire and present lymphatic endothelial cell-archived antigens during lymph node contraction

Journal: Nature Communications

doi: 10.1038/s41467-017-02247-z

Archived antigen acquisition and DC antigen presentation. a Representative image of raw data from wild-type mice challenged with antigen (10 μg/site) alone, antigen with poly(I:C) (2 μg/site), and anti-CD40 (2 μg/site) or antigen (10 μg/site) with vaccinia virus (1 × 10 4 CFU). One week after challenge, mice were harvested and flow cytometry was performed to visualize antigen acquisition. Shown are cells gated as in Supplementary Fig. 1B (CD45 − , PDPN + , CD31 + ). b Number of antigen-positive lymphatic endothelial cells per lymph node was calculated from each challenge in a . c Mice were immunized weekly as indicated with antigen and poly(I:C)/aCD40 (as above). LECs were gated as CD45 − , CD31 + , PDPN + , blood endothelial cells (BECs) were gated as CD45 − , CD31 + , PDPN − , FRC/marginal reticular cells (MRC)/FDCs were gated as CD45 − , CD31 − , PDPN + , macrophages (Mac) were gated as CD11c − , B220 − , CD11b hi , F4/80 + , DCs were gated as CD11c hi , B220 − , F4/80 − , B cells were gated as B220 + , and T cells were gated as CD3 + cells (Supplementary Fig. 1 ). The percent of antigen-positive of each cell type was calculated as a percent of the total cell type. Error bars shown in figure are mean ± standard deviation from two independent experiments with three mice per group per time point. d Schematic of the experimental design. e Wild-type mice were irradiated and K bm8 (cannot effectively present the dominant ovalbumin peptide) bone marrow was used to reconstitute the hematopoietic compartment. OT1 T cells were transferred into these mice 2 weeks after immunization with ovalbumin (10 μg/site), poly(I:C)/anti-CD40 (2 μg/site), and 1 day later WT BMDCs were transferred subcutaneously (1 × 10 6 per site). OT1 division of unimmunized mice that received BMDCs is shown in black, mice immunized, but did not receive BMDCs is shown in light gray, and mice immunized and received BMDCs is shown in dark gray. f Quantification of e . Shown in e is a representative experiment and in f quantification is shown from two independent experiments with three chimeric mice per group, where error bars are mean ± standard error of the mean. Statistical analysis was done using an unpaired t -test
Figure Legend Snippet: Archived antigen acquisition and DC antigen presentation. a Representative image of raw data from wild-type mice challenged with antigen (10 μg/site) alone, antigen with poly(I:C) (2 μg/site), and anti-CD40 (2 μg/site) or antigen (10 μg/site) with vaccinia virus (1 × 10 4 CFU). One week after challenge, mice were harvested and flow cytometry was performed to visualize antigen acquisition. Shown are cells gated as in Supplementary Fig. 1B (CD45 − , PDPN + , CD31 + ). b Number of antigen-positive lymphatic endothelial cells per lymph node was calculated from each challenge in a . c Mice were immunized weekly as indicated with antigen and poly(I:C)/aCD40 (as above). LECs were gated as CD45 − , CD31 + , PDPN + , blood endothelial cells (BECs) were gated as CD45 − , CD31 + , PDPN − , FRC/marginal reticular cells (MRC)/FDCs were gated as CD45 − , CD31 − , PDPN + , macrophages (Mac) were gated as CD11c − , B220 − , CD11b hi , F4/80 + , DCs were gated as CD11c hi , B220 − , F4/80 − , B cells were gated as B220 + , and T cells were gated as CD3 + cells (Supplementary Fig. 1 ). The percent of antigen-positive of each cell type was calculated as a percent of the total cell type. Error bars shown in figure are mean ± standard deviation from two independent experiments with three mice per group per time point. d Schematic of the experimental design. e Wild-type mice were irradiated and K bm8 (cannot effectively present the dominant ovalbumin peptide) bone marrow was used to reconstitute the hematopoietic compartment. OT1 T cells were transferred into these mice 2 weeks after immunization with ovalbumin (10 μg/site), poly(I:C)/anti-CD40 (2 μg/site), and 1 day later WT BMDCs were transferred subcutaneously (1 × 10 6 per site). OT1 division of unimmunized mice that received BMDCs is shown in black, mice immunized, but did not receive BMDCs is shown in light gray, and mice immunized and received BMDCs is shown in dark gray. f Quantification of e . Shown in e is a representative experiment and in f quantification is shown from two independent experiments with three chimeric mice per group, where error bars are mean ± standard error of the mean. Statistical analysis was done using an unpaired t -test

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Standard Deviation, Irradiation

26) Product Images from "Cis-Acting Pathways Selectively Enforce the Non-Immunogenicity of Shed Placental Antigen for Maternal CD8 T Cells"

Article Title: Cis-Acting Pathways Selectively Enforce the Non-Immunogenicity of Shed Placental Antigen for Maternal CD8 T Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084064

Pregnancy does not induce DC activation in the spleen, nor does it inhibit adjuvant-induced DC activation. (A) Induction of CD86 expression by splenic DC subsets in virgin and pregnant mice by anti-CD40 Abs or poly(I:C). Data show n = 4–12 mice per group (mean±SD), from at least 2 independent experiments per group. *, P
Figure Legend Snippet: Pregnancy does not induce DC activation in the spleen, nor does it inhibit adjuvant-induced DC activation. (A) Induction of CD86 expression by splenic DC subsets in virgin and pregnant mice by anti-CD40 Abs or poly(I:C). Data show n = 4–12 mice per group (mean±SD), from at least 2 independent experiments per group. *, P

Techniques Used: Activation Assay, Expressing, Mouse Assay

Non-immunogenic responses of maternal CD8 T cells to shed placental mOVA. (A) Effects of mating, pregnancy, adjuvant (anti-CD40 Abs +poly(I:C)), sOVA, and mOVA on the expansion of OVA-specific CD8 T cells. Adjuvant±sOVA was injected 6 days prior to sacrifice. Representative dot plots and mean±SEM of the percentage of CD44 hi APC-conjugated K b /OVAp-tetramer + cells of total splenic CD8 T cells. Pregnant mice were killed on E17.5 to 1 day after delivery. Mated mice that failed to become pregnant were killed on what would have been E17.5-21.5. Aside from group 3 (one experiment), data are from at least 4 independent experiments per group. In addition (*), n = 7 group 2 and n = 4 group 3 mice showed no difference compared to n = 9 virgin, untreated (group 1) mice when analyzed with PE-conjugated K b /OVAp-tetramers (2-8 experiments). a, P
Figure Legend Snippet: Non-immunogenic responses of maternal CD8 T cells to shed placental mOVA. (A) Effects of mating, pregnancy, adjuvant (anti-CD40 Abs +poly(I:C)), sOVA, and mOVA on the expansion of OVA-specific CD8 T cells. Adjuvant±sOVA was injected 6 days prior to sacrifice. Representative dot plots and mean±SEM of the percentage of CD44 hi APC-conjugated K b /OVAp-tetramer + cells of total splenic CD8 T cells. Pregnant mice were killed on E17.5 to 1 day after delivery. Mated mice that failed to become pregnant were killed on what would have been E17.5-21.5. Aside from group 3 (one experiment), data are from at least 4 independent experiments per group. In addition (*), n = 7 group 2 and n = 4 group 3 mice showed no difference compared to n = 9 virgin, untreated (group 1) mice when analyzed with PE-conjugated K b /OVAp-tetramers (2-8 experiments). a, P

Techniques Used: Injection, Mouse Assay

27) Product Images from "Protection from tumor recurrence following adoptive immunotherapy varies with host conditioning regimen despite initial regression of autochthonous murine brain tumors"

Article Title: Protection from tumor recurrence following adoptive immunotherapy varies with host conditioning regimen despite initial regression of autochthonous murine brain tumors

Journal: Cancer immunology, immunotherapy : CII

doi: 10.1007/s00262-014-1635-7

Host conditioning with anti-CD40 induces high-level T cell accumulation in the lymphoid organs and brains of SV11 mice at early time points. Groups of mice received either anti-CD40, WBI, or no conditioning regimen with TCR-IV T-cell ACT. Representative plots show MHC tetramer staining (mean±SEM) of TCR-IV T cells on days +4 and +5 in a spleen and c brain. Quantification of TCR-IV T-cell accumulation (mean±SEM) in b spleen and d brain of mice that received ACT with the indicated treatments. n=3 mice/group (except n=2 for day +4 control group). Data shown are from one experiment and representative of two independent experiments. Asterisks above connecting lines indicate significant differences between time points. Asterisks next to vertical brackets indicate significant differences between treatment groups. * p
Figure Legend Snippet: Host conditioning with anti-CD40 induces high-level T cell accumulation in the lymphoid organs and brains of SV11 mice at early time points. Groups of mice received either anti-CD40, WBI, or no conditioning regimen with TCR-IV T-cell ACT. Representative plots show MHC tetramer staining (mean±SEM) of TCR-IV T cells on days +4 and +5 in a spleen and c brain. Quantification of TCR-IV T-cell accumulation (mean±SEM) in b spleen and d brain of mice that received ACT with the indicated treatments. n=3 mice/group (except n=2 for day +4 control group). Data shown are from one experiment and representative of two independent experiments. Asterisks above connecting lines indicate significant differences between time points. Asterisks next to vertical brackets indicate significant differences between treatment groups. * p

Techniques Used: Mouse Assay, Activated Clotting Time Assay, Staining

Anti-CD40-enhanced ACT promotes initial regression of established tumors. a H E brain sections on day +10 post-ACT following conditioning with anti-CD40 (left), control IgG (middle), or WBI (right). Representative low-power images (top row, scale bar = 1mm) and high-power images (bottom row, scale bar = 50μm) that show established tumor refractory to therapy (middle column) or tumor stromal condensation indicative of tumor regression (left and right columns). b For each mouse, the largest cross-sectional tumor area (mm 2 ) observed in H E sections was plotted. Data is pooled from multiple experiments with a total of 6–10 mice/group. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparison test. ** p
Figure Legend Snippet: Anti-CD40-enhanced ACT promotes initial regression of established tumors. a H E brain sections on day +10 post-ACT following conditioning with anti-CD40 (left), control IgG (middle), or WBI (right). Representative low-power images (top row, scale bar = 1mm) and high-power images (bottom row, scale bar = 50μm) that show established tumor refractory to therapy (middle column) or tumor stromal condensation indicative of tumor regression (left and right columns). b For each mouse, the largest cross-sectional tumor area (mm 2 ) observed in H E sections was plotted. Data is pooled from multiple experiments with a total of 6–10 mice/group. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparison test. ** p

Techniques Used: Activated Clotting Time Assay, Mouse Assay

Anti-CD40-enhanced ACT promotes increased survival but short-term surveillance against tumor recurrence. a Groups of mice received the indicated conditioning with or without ACT and were monitored for tumor recurrence. The percentage of surviving mice versus age is plotted. b Statistical differences in survival were calculated using the log-rank test. Data are pooled from multiple experiments with 5–8 mice/group.
Figure Legend Snippet: Anti-CD40-enhanced ACT promotes increased survival but short-term surveillance against tumor recurrence. a Groups of mice received the indicated conditioning with or without ACT and were monitored for tumor recurrence. The percentage of surviving mice versus age is plotted. b Statistical differences in survival were calculated using the log-rank test. Data are pooled from multiple experiments with 5–8 mice/group.

Techniques Used: Activated Clotting Time Assay, Mouse Assay

Donor T cells fail to persist in anti-CD40-conditioned SV11 mice following acute tumor regression. Groups of SV11 mice received either anti-CD40, control IgG, or WBI conditioning prior to ACT with CD90.1 + TCR-IV T cells. On day +30, cells from a spleens, cLN (not shown), and b brains were stained for CD90.1 and CD8. Values on dot plots indicate percent CD90.1 + of total CD8 + cells (mean±SEM). c Total CD90.1 + cells in spleens, cLN, and brains on day +30 are plotted. Representative histograms of CD44, CD62L, and KLRG1 expression gated on live CD45.2 + CD8 + CD90.1 + TCR-IV T cells (open histogram) or CD45.2 + CD8 + CD90.1 − T cells (filled histogram, spleen only) are shown on day +30 in d spleen and e brain. Values indicate the percent of TCR-IV T cells within the indicated gate (mean±SEM). Samples with
Figure Legend Snippet: Donor T cells fail to persist in anti-CD40-conditioned SV11 mice following acute tumor regression. Groups of SV11 mice received either anti-CD40, control IgG, or WBI conditioning prior to ACT with CD90.1 + TCR-IV T cells. On day +30, cells from a spleens, cLN (not shown), and b brains were stained for CD90.1 and CD8. Values on dot plots indicate percent CD90.1 + of total CD8 + cells (mean±SEM). c Total CD90.1 + cells in spleens, cLN, and brains on day +30 are plotted. Representative histograms of CD44, CD62L, and KLRG1 expression gated on live CD45.2 + CD8 + CD90.1 + TCR-IV T cells (open histogram) or CD45.2 + CD8 + CD90.1 − T cells (filled histogram, spleen only) are shown on day +30 in d spleen and e brain. Values indicate the percent of TCR-IV T cells within the indicated gate (mean±SEM). Samples with

Techniques Used: Mouse Assay, Activated Clotting Time Assay, Staining, Expressing

28) Product Images from "A multikinase and DNA-PK inhibitor combination immunomodulates melanomas, suppresses tumor progression, and enhances immunotherapies"

Article Title: A multikinase and DNA-PK inhibitor combination immunomodulates melanomas, suppresses tumor progression, and enhances immunotherapies

Journal: Cancer immunology research

doi: 10.1158/2326-6066.CIR-17-0009

Reg cooperated with anti-CD40 and STING agonist immunotherapies ( A–B ) and c-di-GMP (STING agonist, S) and tumor volume ( A ) and survival ( B ) were monitored ( n = 5–6/group). Differences for tumor volumes were determined on days 16–28 by one-way ANOVA with Tukey post-test comparing vehicle to all groups, and comparing 40/S or Reg to 40/S+Reg. All survival comparisons except Reg vs. 40/S were significant according to log-rank tests. On day 21, intratumoral CD45 + leukocytes ( C ), CD4 + T cells ( D–E ) and CD8 + T cells ( F–G ) were measured. Intratumoral CD4 + ( H ) and CD8 + ( I ) T cells were stimulated with PMA and ionomycin and evaluated for cytokine production. Differences in C–I were assessed by one-way ANOVA. ( J–K ) B16-F1 tumor-bearing mice were treated with anti-CD8 to deplete CD8 + T cells, Reg (5 mg/kg), anti-CD40, and c-di-GMP and tumor volume ( J ) and survival ( K ) were monitored ( n = 5–7/group). Reg and 40/S+Reg groups were significantly different compared to associated anti-CD8-treated mice on days 19–29 by one-way ANOVA with Tukey post-test. Log-rank tests were used to evaluate survival comparisons. A–G are representative of two independent experiments; H–K were performed once. All results show mean ± SEM. *, P
Figure Legend Snippet: Reg cooperated with anti-CD40 and STING agonist immunotherapies ( A–B ) and c-di-GMP (STING agonist, S) and tumor volume ( A ) and survival ( B ) were monitored ( n = 5–6/group). Differences for tumor volumes were determined on days 16–28 by one-way ANOVA with Tukey post-test comparing vehicle to all groups, and comparing 40/S or Reg to 40/S+Reg. All survival comparisons except Reg vs. 40/S were significant according to log-rank tests. On day 21, intratumoral CD45 + leukocytes ( C ), CD4 + T cells ( D–E ) and CD8 + T cells ( F–G ) were measured. Intratumoral CD4 + ( H ) and CD8 + ( I ) T cells were stimulated with PMA and ionomycin and evaluated for cytokine production. Differences in C–I were assessed by one-way ANOVA. ( J–K ) B16-F1 tumor-bearing mice were treated with anti-CD8 to deplete CD8 + T cells, Reg (5 mg/kg), anti-CD40, and c-di-GMP and tumor volume ( J ) and survival ( K ) were monitored ( n = 5–7/group). Reg and 40/S+Reg groups were significantly different compared to associated anti-CD8-treated mice on days 19–29 by one-way ANOVA with Tukey post-test. Log-rank tests were used to evaluate survival comparisons. A–G are representative of two independent experiments; H–K were performed once. All results show mean ± SEM. *, P

Techniques Used: Mouse Assay

29) Product Images from "Transient cell-in-cell formation underlies tumor resistance to immunotherapy"

Article Title: Transient cell-in-cell formation underlies tumor resistance to immunotherapy

Journal: bioRxiv

doi: 10.1101/2020.09.10.287441

Tumor cells remaining after T-cell killing organize in a cell-in-cell formation a. Representative images of B16F10 tumor cells sorted from tumors relapsed after immunotherapy. b. Transmitting electron microscopy images of relapsed B16F10 tumor cells. c. Histological sections of B16F10 tumor untreated, and five days following immunotherapy. d. Mean percentage of cell-in-cell and single cells in untreated B16F10 tumor-bearing mice, treated with anti-CD40, TNFα, and anti-TRP1 (Ab-IT), or treated with gp100-reactive (gp100 ACT) (n=4). e. Representative images of B16F10 labeled with H2B-tdTomato and H2B-GFP freshly isolated from tumor-bearing mice treated anti-CD40, TNFα, and anti-TRP1. f. Mean percentage of cell-in-cell formation following incubation of B16F10 cells with immune cells (n=3). g. Representative 3D projection and horizontal sections (Z-stack) of B16F10 cells incubated with gp100-reactive CD8 + T cells. h. Mean percentage of cell-in-cell and single cells in tumor-bearing NSG -/- mice treated with anti-CD40, TNFα, and anti-TRP1 antibodies (n=3). i. Representative staining of gp100 expression in B16F10 tumor cells following incubation with gp100-reactive CD8 + T cells. j. Representative images of MHC-I expression in B16F10 tumor cells following incubation with gp100-reactive CD8 + T cells. k. SEM analysis of B16F10 incubated with gp100-reactive CD8 + T cells. l. Percentages of B16F10confluence tumor cells following incubation with gp100- and Trp2-reactive T cells (n=3). m. Mean percentage of cellin-cell in B16F10 culture following one (1 st ) or two (2 nd ) cycles of incubation with gp100-reactive CD8 + T cells (n=3). n. Mean percentage of confluence change over time of B16F10 control (WT) or cell-in-cell tumors (n=3). Experiments were repeated independently at least three times. Statistical significance was calculated using ANOVA with Tukey’s correction for multiple comparisons (*** denotes p
Figure Legend Snippet: Tumor cells remaining after T-cell killing organize in a cell-in-cell formation a. Representative images of B16F10 tumor cells sorted from tumors relapsed after immunotherapy. b. Transmitting electron microscopy images of relapsed B16F10 tumor cells. c. Histological sections of B16F10 tumor untreated, and five days following immunotherapy. d. Mean percentage of cell-in-cell and single cells in untreated B16F10 tumor-bearing mice, treated with anti-CD40, TNFα, and anti-TRP1 (Ab-IT), or treated with gp100-reactive (gp100 ACT) (n=4). e. Representative images of B16F10 labeled with H2B-tdTomato and H2B-GFP freshly isolated from tumor-bearing mice treated anti-CD40, TNFα, and anti-TRP1. f. Mean percentage of cell-in-cell formation following incubation of B16F10 cells with immune cells (n=3). g. Representative 3D projection and horizontal sections (Z-stack) of B16F10 cells incubated with gp100-reactive CD8 + T cells. h. Mean percentage of cell-in-cell and single cells in tumor-bearing NSG -/- mice treated with anti-CD40, TNFα, and anti-TRP1 antibodies (n=3). i. Representative staining of gp100 expression in B16F10 tumor cells following incubation with gp100-reactive CD8 + T cells. j. Representative images of MHC-I expression in B16F10 tumor cells following incubation with gp100-reactive CD8 + T cells. k. SEM analysis of B16F10 incubated with gp100-reactive CD8 + T cells. l. Percentages of B16F10confluence tumor cells following incubation with gp100- and Trp2-reactive T cells (n=3). m. Mean percentage of cellin-cell in B16F10 culture following one (1 st ) or two (2 nd ) cycles of incubation with gp100-reactive CD8 + T cells (n=3). n. Mean percentage of confluence change over time of B16F10 control (WT) or cell-in-cell tumors (n=3). Experiments were repeated independently at least three times. Statistical significance was calculated using ANOVA with Tukey’s correction for multiple comparisons (*** denotes p

Techniques Used: Electron Microscopy, Mouse Assay, Labeling, Isolation, Incubation, Staining, Expressing

Relapsed tumor cells share neoantigens with primary tumors and are equally susceptible to killing by T cells. a. Tumor size following treatments with anti-CD40, TNFα, and tumor-binding antibodies (n=4). b. Mean percentages of T cells out of CD45 + cells in relapsed tumors (n=4). c. Mean percentage of TRP2- and gp100-reactive T-cell clones in relapsed tumors (n=4). d. Representative Flow cytometry of MHC-I and MHC-II expression in relapsed B16F10 cells. e. Representative staining of TRP2 and gp100 in relapsed B16F10 cells. f. Illustration of neoantigen discovery pipeline. g. Neoantigen burden in B16F10 cells isolated from untreated tumors, or following immunotherapy treated tumors (IT). h. Allele frequency comparison of 25 neoantigens with the highest MHC-I affinity. i. RNAseq expression level of B16F10-known neoantigens. j. Tumor growth following treatments with anti-CD40, TNFα, and anti-TRP1 tumor-binding antibodies (n=4). k. Tumor size following adoptive transfer of gp100-reactive CD8 + or TRP1-reactive CD4 + T cell (n=4). l. Mean percentages of apoptotic tumor cells after incubation overnight with gp100-reactive CD8 + T cells (n=5). m. Tumor size following adoptive transfer of gp100-reactive T cell (n=4). n. Principal Component Analysis (PCA) of B16F10 tumor cell lines freshly isolated from treated mice. Experiments were repeated independently at least three times. Statistical significance was calculated using ANOVA with Tukey’s correction for multiple comparisons (*** denotes p
Figure Legend Snippet: Relapsed tumor cells share neoantigens with primary tumors and are equally susceptible to killing by T cells. a. Tumor size following treatments with anti-CD40, TNFα, and tumor-binding antibodies (n=4). b. Mean percentages of T cells out of CD45 + cells in relapsed tumors (n=4). c. Mean percentage of TRP2- and gp100-reactive T-cell clones in relapsed tumors (n=4). d. Representative Flow cytometry of MHC-I and MHC-II expression in relapsed B16F10 cells. e. Representative staining of TRP2 and gp100 in relapsed B16F10 cells. f. Illustration of neoantigen discovery pipeline. g. Neoantigen burden in B16F10 cells isolated from untreated tumors, or following immunotherapy treated tumors (IT). h. Allele frequency comparison of 25 neoantigens with the highest MHC-I affinity. i. RNAseq expression level of B16F10-known neoantigens. j. Tumor growth following treatments with anti-CD40, TNFα, and anti-TRP1 tumor-binding antibodies (n=4). k. Tumor size following adoptive transfer of gp100-reactive CD8 + or TRP1-reactive CD4 + T cell (n=4). l. Mean percentages of apoptotic tumor cells after incubation overnight with gp100-reactive CD8 + T cells (n=5). m. Tumor size following adoptive transfer of gp100-reactive T cell (n=4). n. Principal Component Analysis (PCA) of B16F10 tumor cell lines freshly isolated from treated mice. Experiments were repeated independently at least three times. Statistical significance was calculated using ANOVA with Tukey’s correction for multiple comparisons (*** denotes p

Techniques Used: Binding Assay, Clone Assay, Flow Cytometry, Expressing, Staining, Isolation, Adoptive Transfer Assay, Incubation, Mouse Assay

a. Gating strategy on immune and tumor cells following a second treatment with tumor-binding antibodies, TNFα, and anti-CD40 agonist. b . immunohistochemistry of total single cells obtained from tumors treated twice with tumorbinding antibodies, TNFα, and anti-CD40 agonist. c. and d. Volcano plots comparing gene expression in B16F10 tumor cells sorted from relapsed tumors in vivo and B16F10 cell line established from relapsed tumors and cultured in vitro . Scale bar = 20 μm.
Figure Legend Snippet: a. Gating strategy on immune and tumor cells following a second treatment with tumor-binding antibodies, TNFα, and anti-CD40 agonist. b . immunohistochemistry of total single cells obtained from tumors treated twice with tumorbinding antibodies, TNFα, and anti-CD40 agonist. c. and d. Volcano plots comparing gene expression in B16F10 tumor cells sorted from relapsed tumors in vivo and B16F10 cell line established from relapsed tumors and cultured in vitro . Scale bar = 20 μm.

Techniques Used: Binding Assay, Immunohistochemistry, Expressing, In Vivo, Cell Culture, In Vitro

30) Product Images from "Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice"

Article Title: Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1510518113

Combination therapy with vaccination reverses CD8 T-cell anergy and restores T-cell function in a spontaneous adenocarcinoma model. ( A ) Naive Pmel TCR transgenic CD8 T cells were purified by negative selection and adoptively transferred on day 6 into B16F10 tumor-bearing wild-type C57BL/6 mice. Mice were treated with IgG or aOX40 on days 7 and 11; with aCTLA-4 on days 7, 9, and 11; or with monotherapy or combination therapy and vaccine (gp100/anti-CD40) on day 7. Blood was analyzed by flow cytometry on day 14. ( B – D ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET-1 mice on day 0. Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28 along with mono- or combination therapy (50 µg aOX40 or control rat IgG on days 28 and 29 and 200 µg aCTLA-4 on days 28, 30, and 32). Splenocytes were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict ( B ) the total number of OT-I + cells; the total number of Ki-67 + , granzyme B + (GrzB), KLRG-1 + , CD62L + , and CD25 + OT-I; ( C and D ) and the percentage ( C ) and total number ( D ) of IFNγ-, TNFα-, and IL-2--producing OT-I cells. Data are shown for one representative mouse ( C ) or for all mice in each cohort for donor OT-I cells ( B and D ). Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group). * P
Figure Legend Snippet: Combination therapy with vaccination reverses CD8 T-cell anergy and restores T-cell function in a spontaneous adenocarcinoma model. ( A ) Naive Pmel TCR transgenic CD8 T cells were purified by negative selection and adoptively transferred on day 6 into B16F10 tumor-bearing wild-type C57BL/6 mice. Mice were treated with IgG or aOX40 on days 7 and 11; with aCTLA-4 on days 7, 9, and 11; or with monotherapy or combination therapy and vaccine (gp100/anti-CD40) on day 7. Blood was analyzed by flow cytometry on day 14. ( B – D ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET-1 mice on day 0. Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28 along with mono- or combination therapy (50 µg aOX40 or control rat IgG on days 28 and 29 and 200 µg aCTLA-4 on days 28, 30, and 32). Splenocytes were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict ( B ) the total number of OT-I + cells; the total number of Ki-67 + , granzyme B + (GrzB), KLRG-1 + , CD62L + , and CD25 + OT-I; ( C and D ) and the percentage ( C ) and total number ( D ) of IFNγ-, TNFα-, and IL-2--producing OT-I cells. Data are shown for one representative mouse ( C ) or for all mice in each cohort for donor OT-I cells ( B and D ). Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group). * P

Techniques Used: Cell Function Assay, Transgenic Assay, Purification, Selection, Mouse Assay, Flow Cytometry, Cytometry

( A and C ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET mice (day 0). Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28, along with mono- or combination therapy: 50 µg aOX40 or control rat IgG on days 28 and 29, and 200 µg aCTLA-4 on days 28, 30, and 3. ( A ) Model of treatment. ( B ) Naive OT-I CD8 T cells (2.5 × 10 6 ) were purified by negative selection and adoptively transferred into TRAMP/POET or wild-type C57BL/6 mice. Three days later, lymph nodes were processed, and OX40 and CTLA-4 expression was analyzed by flow cytometry on donor OT-1 cells. For CTLA-4 expression on donor OT-1 cells, the unstained control is shown in gray; the wild-type control is shown as a solid black line; TRAMP/POET mice are shown as a purple line. For OX40 expression ( Middle and Right ), images represent OT-I + (Thy1.1 + ) CD8 T cells versus OX40 expression; the numbers shown are the percentage of OX40 + OT-I cells. ( C ) Splenocytes and prostate-draining lymph nodes (pooled for each group) were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group).
Figure Legend Snippet: ( A and C ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET mice (day 0). Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28, along with mono- or combination therapy: 50 µg aOX40 or control rat IgG on days 28 and 29, and 200 µg aCTLA-4 on days 28, 30, and 3. ( A ) Model of treatment. ( B ) Naive OT-I CD8 T cells (2.5 × 10 6 ) were purified by negative selection and adoptively transferred into TRAMP/POET or wild-type C57BL/6 mice. Three days later, lymph nodes were processed, and OX40 and CTLA-4 expression was analyzed by flow cytometry on donor OT-1 cells. For CTLA-4 expression on donor OT-1 cells, the unstained control is shown in gray; the wild-type control is shown as a solid black line; TRAMP/POET mice are shown as a purple line. For OX40 expression ( Middle and Right ), images represent OT-I + (Thy1.1 + ) CD8 T cells versus OX40 expression; the numbers shown are the percentage of OX40 + OT-I cells. ( C ) Splenocytes and prostate-draining lymph nodes (pooled for each group) were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group).

Techniques Used: Purification, Selection, Mouse Assay, Expressing, Flow Cytometry, Cytometry

31) Product Images from "B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model"

Article Title: B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model

Journal: Diabetologia

doi: 10.1007/s00125-018-4597-z

Fewer repopulated B cells express co-stimulatory molecules. Splenic B cells, from mice aged 6–8 ( b , c ) or 12–15 weeks ( d , e ) at the time of depletion, were analysed for co-stimulatory molecules 8 and 12 weeks post treatment with control IgG (black circles) or 2H7 anti-CD20 depleting antibody (white squares). The B cells were either unstimulated or were stimulated with 5 μg/ml LPS or anti-CD40 for 24 h. ( a ) Representative flow plots (12 weeks after depletion) of CD80 and CD86 expression on unstimulated or stimulated B cells. ( b – e ) Frequency of cells expressing CD80 ( b , d ) and CD86 ( c , e ) at 8 and 12 weeks after depletion. Horizontal lines represent the medians. Each time point includes a minimum of seven mice from at least two independent experiments. * p
Figure Legend Snippet: Fewer repopulated B cells express co-stimulatory molecules. Splenic B cells, from mice aged 6–8 ( b , c ) or 12–15 weeks ( d , e ) at the time of depletion, were analysed for co-stimulatory molecules 8 and 12 weeks post treatment with control IgG (black circles) or 2H7 anti-CD20 depleting antibody (white squares). The B cells were either unstimulated or were stimulated with 5 μg/ml LPS or anti-CD40 for 24 h. ( a ) Representative flow plots (12 weeks after depletion) of CD80 and CD86 expression on unstimulated or stimulated B cells. ( b – e ) Frequency of cells expressing CD80 ( b , d ) and CD86 ( c , e ) at 8 and 12 weeks after depletion. Horizontal lines represent the medians. Each time point includes a minimum of seven mice from at least two independent experiments. * p

Techniques Used: Mouse Assay, Flow Cytometry, Expressing

Repopulated B cells are not enriched for IL-10. B cells from the spleen were analysed for intracytoplasmic cytokines 8 and 12 weeks post treatment with control IgG (black circles) or 2H7 anti-CD20 depleting antibody (white squares) in mice aged 6–8 ( b , c ) or 12–15 weeks ( d , e ). B cells were either unstimulated or stimulated with 5 μg/ml LPS or anti-CD40. ( a ) Representative flow plots from 12 weeks post depletion. ( b – e ) Frequency of IL-10-producing B cells ( b , d ) and TGF-β-producing B cells ( c , e ) 8 and 12 weeks post depletion. Horizontal lines represent the medians. Each time point includes a minimum of seven mice, from at least two independent experiments. * p
Figure Legend Snippet: Repopulated B cells are not enriched for IL-10. B cells from the spleen were analysed for intracytoplasmic cytokines 8 and 12 weeks post treatment with control IgG (black circles) or 2H7 anti-CD20 depleting antibody (white squares) in mice aged 6–8 ( b , c ) or 12–15 weeks ( d , e ). B cells were either unstimulated or stimulated with 5 μg/ml LPS or anti-CD40. ( a ) Representative flow plots from 12 weeks post depletion. ( b – e ) Frequency of IL-10-producing B cells ( b , d ) and TGF-β-producing B cells ( c , e ) 8 and 12 weeks post depletion. Horizontal lines represent the medians. Each time point includes a minimum of seven mice, from at least two independent experiments. * p

Techniques Used: Mouse Assay, Flow Cytometry

Related Articles

Flow Cytometry:

Article Title: Activation-induced cytidine deaminase deficiency accelerates autoimmune diabetes in NOD mice
Article Snippet: Immune cells isolated from spleens, PLNs, and thymi were stained with fluorochrome-labeled anti-BrdU and analyzed by flow cytometry. .. Flow cytometrically sorted splenic B cells (stained for B220+ CD19+ ) from 8-week-old female AID–/– NOD and AID+/+ NOD mice were seeded at 5 × 104 cells/well and cultured with polyclonal goat anti-mouse IgM (3 μg/ml, MilliporeSigma) and different concentrations of monoclonal rat anti-mouse CD40 (FGK4.5, BioXcell). ..

Staining:

Article Title: Activation-induced cytidine deaminase deficiency accelerates autoimmune diabetes in NOD mice
Article Snippet: Immune cells isolated from spleens, PLNs, and thymi were stained with fluorochrome-labeled anti-BrdU and analyzed by flow cytometry. .. Flow cytometrically sorted splenic B cells (stained for B220+ CD19+ ) from 8-week-old female AID–/– NOD and AID+/+ NOD mice were seeded at 5 × 104 cells/well and cultured with polyclonal goat anti-mouse IgM (3 μg/ml, MilliporeSigma) and different concentrations of monoclonal rat anti-mouse CD40 (FGK4.5, BioXcell). ..

Mouse Assay:

Article Title: Activation-induced cytidine deaminase deficiency accelerates autoimmune diabetes in NOD mice
Article Snippet: Immune cells isolated from spleens, PLNs, and thymi were stained with fluorochrome-labeled anti-BrdU and analyzed by flow cytometry. .. Flow cytometrically sorted splenic B cells (stained for B220+ CD19+ ) from 8-week-old female AID–/– NOD and AID+/+ NOD mice were seeded at 5 × 104 cells/well and cultured with polyclonal goat anti-mouse IgM (3 μg/ml, MilliporeSigma) and different concentrations of monoclonal rat anti-mouse CD40 (FGK4.5, BioXcell). ..

Article Title: Divergent Roles of Interferon-γ and Innate Lymphoid Cells in Innate and Adaptive Immune Cell-Mediated Intestinal Inflammation
Article Snippet: .. Anti-CD40-Mediated Colitis Mice were injected i.p. with the anti-CD40 monoclonal antibody (clone FGK45, BioXcell; 150μg/mouse). .. After antibody injection, mice were observed daily and sacrificed after 5 days for subsequent immunological and histopathological analyses.

Article Title: Amplifying IFNγ signaling in DC by CD11c-specific loss of SOCS1 increases innate immunity to infection while decreasing adaptive immunity
Article Snippet: Cre- SOCS1fl/fl and Cre+ SOCS1fl/fl littermates made equivalent responses to vaccination with anti-DEC205-OVA and anti-CD40 , suggesting that DC function is intact when antigen is delivered via this route. .. Furthermore, the success of this vaccination strategy was not due to the activity of anti-CD40, since delivery of anti-CD40 to mice vaccinated with ΔactA QV L. monocytogenes was not able to restore T cell responses in Cre+ SOCS1fl/fl mice ( ). .. Interestingly, the CD4+ T cell response to Listeria -associated antigen was not diminished in Cre+ SOCS1fl/fl mice , suggesting that the failure in response was associated with intracellular antigens from the infectious agents that are cross-presented by DC to CD8+ T cells.

Article Title: Systemic Immunity is Required for Effective Cancer Immunotherapy
Article Snippet: Balb/c mice were injected orthotopically into the fourth mammary fat pad with 105 4T1 tumor cells. .. On days 12 and 14, mice were injected intratumorally with 100μg anti-CD40 (clone FGK4.5; BioXCell) and 1μg IFNγ (Biolegend) and 400μg allo-IgG along with daily i.p. injections of FTY720 (3mg/kg). .. On day seven, mice were euthanized and the draining lymph nodes and spleens were removed and mechanically dissociated to obtain single cell suspension.

Article Title: Divergent Roles of Interferon-γ and Innate Lymphoid Cells in Innate and Adaptive Immune Cell-Mediated Intestinal Inflammation
Article Snippet: Cells were acquired on a LSRII SORP (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA). .. Mice were injected i.p. with the anti-CD40 monoclonal antibody (clone FGK45, BioXcell; 150μg/mouse). .. After antibody injection, mice were observed daily and sacrificed after 5 days for subsequent immunological and histopathological analyses.

Cell Culture:

Article Title: Activation-induced cytidine deaminase deficiency accelerates autoimmune diabetes in NOD mice
Article Snippet: Immune cells isolated from spleens, PLNs, and thymi were stained with fluorochrome-labeled anti-BrdU and analyzed by flow cytometry. .. Flow cytometrically sorted splenic B cells (stained for B220+ CD19+ ) from 8-week-old female AID–/– NOD and AID+/+ NOD mice were seeded at 5 × 104 cells/well and cultured with polyclonal goat anti-mouse IgM (3 μg/ml, MilliporeSigma) and different concentrations of monoclonal rat anti-mouse CD40 (FGK4.5, BioXcell). ..

Injection:

Article Title: Divergent Roles of Interferon-γ and Innate Lymphoid Cells in Innate and Adaptive Immune Cell-Mediated Intestinal Inflammation
Article Snippet: .. Anti-CD40-Mediated Colitis Mice were injected i.p. with the anti-CD40 monoclonal antibody (clone FGK45, BioXcell; 150μg/mouse). .. After antibody injection, mice were observed daily and sacrificed after 5 days for subsequent immunological and histopathological analyses.

Article Title: Systemic Immunity is Required for Effective Cancer Immunotherapy
Article Snippet: Balb/c mice were injected orthotopically into the fourth mammary fat pad with 105 4T1 tumor cells. .. On days 12 and 14, mice were injected intratumorally with 100μg anti-CD40 (clone FGK4.5; BioXCell) and 1μg IFNγ (Biolegend) and 400μg allo-IgG along with daily i.p. injections of FTY720 (3mg/kg). .. On day seven, mice were euthanized and the draining lymph nodes and spleens were removed and mechanically dissociated to obtain single cell suspension.

Article Title: Divergent Roles of Interferon-γ and Innate Lymphoid Cells in Innate and Adaptive Immune Cell-Mediated Intestinal Inflammation
Article Snippet: Cells were acquired on a LSRII SORP (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA). .. Mice were injected i.p. with the anti-CD40 monoclonal antibody (clone FGK45, BioXcell; 150μg/mouse). .. After antibody injection, mice were observed daily and sacrificed after 5 days for subsequent immunological and histopathological analyses.

Activity Assay:

Article Title: Amplifying IFNγ signaling in DC by CD11c-specific loss of SOCS1 increases innate immunity to infection while decreasing adaptive immunity
Article Snippet: Cre- SOCS1fl/fl and Cre+ SOCS1fl/fl littermates made equivalent responses to vaccination with anti-DEC205-OVA and anti-CD40 , suggesting that DC function is intact when antigen is delivered via this route. .. Furthermore, the success of this vaccination strategy was not due to the activity of anti-CD40, since delivery of anti-CD40 to mice vaccinated with ΔactA QV L. monocytogenes was not able to restore T cell responses in Cre+ SOCS1fl/fl mice ( ). .. Interestingly, the CD4+ T cell response to Listeria -associated antigen was not diminished in Cre+ SOCS1fl/fl mice , suggesting that the failure in response was associated with intracellular antigens from the infectious agents that are cross-presented by DC to CD8+ T cells.

Purification:

Article Title: B cell receptor and CD40 Signaling are Rewired for Synergistic induction of the c-Myc transcription factor in Germinal Center B cells
Article Snippet: For GC B cell purification from B1-8i mice, biotin conjugated IgD and CD38 antibodies were added to B cell purification cocktail; for GC B cell purification from MEG mice, biotin-CD38 antibody was added to the B cell purification cocktail (GC B cell purity ≥ 90%, example of purity examination by flow cytometry is shown in ). .. Purified B cells and GC B cells were warmed to 37 °C with 5% CO2 in B cell medium (RPMI-1640 medium supplemented with 10% FBS, penicillin/streptomycin, glutamine and 50 μM β-mercaptoethanol) for 30min and stimulated with 200ng/ml NP-Ficoll (LGC Biosearch Technologies), 20 μg/ml goat anti-mouse IgM (μ chain specific, Jackson ImmunoResearch) or 20 μg/ml anti-CD40 antibody (FGK45, Bio X Cell or prepared in our lab) as indicated in figure legends. ..

Isolation:

Article Title: MEK inhibition suppresses B regulatory cells and augments anti-tumor immunity
Article Snippet: Isolated B cells were counted and resuspended at 1*106 cells/ml in complete media, and 500 ul (5*105 cells) were plated in wells of a 48-well flat bottom plate. .. The isolated B cells were stimulated with goat anti-mouse IgM (1 mg/ml, catalog 1021–01, SouthernBiotech) and anti-CD40 (8 mg/ml, clone FGK4.5/ FGK45, Bio X Cell) or isotype control. ..

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    Bio X Cell anti cd40 mab
    341G2 h2 Mediates Super-agonistic Activity In Vivo (A) OTI cells (1 × 10 5 ) were adoptively transferred into hCD40Tg mice 1 day before treatment with 30 μg <t>anti-CD40</t> mAbs as indicated. Mice were bled on day 5 and SIINFEKL + cells were expressed as a percentage of total CD8 + T cells. Means ± SEM, n = 5, data representative of three experiments. Each dot represents one mouse. Two-tailed, non-paired Student’s t test, ∗ p
    Anti Cd40 Mab, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio X Cell anti cd40
    Combination therapy with vaccination reverses CD8 T-cell anergy and restores T-cell function in a spontaneous adenocarcinoma model. ( A ) Naive Pmel TCR transgenic CD8 T cells were purified by negative selection and adoptively transferred on day 6 into B16F10 tumor-bearing wild-type C57BL/6 mice. Mice were treated with IgG or aOX40 on days 7 and 11; with aCTLA-4 on days 7, 9, and 11; or with monotherapy or combination therapy and vaccine <t>(gp100/anti-CD40)</t> on day 7. Blood was analyzed by flow cytometry on day 14. ( B – D ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET-1 mice on day 0. Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28 along with mono- or combination therapy (50 µg aOX40 or control rat IgG on days 28 and 29 and 200 µg aCTLA-4 on days 28, 30, and 32). Splenocytes were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict ( B ) the total number of OT-I + cells; the total number of Ki-67 + , granzyme B + (GrzB), KLRG-1 + , CD62L + , and CD25 + OT-I; ( C and D ) and the percentage ( C ) and total number ( D ) of IFNγ-, TNFα-, and IL-2--producing OT-I cells. Data are shown for one representative mouse ( C ) or for all mice in each cohort for donor OT-I cells ( B and D ). Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group). * P
    Anti Cd40, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    341G2 h2 Mediates Super-agonistic Activity In Vivo (A) OTI cells (1 × 10 5 ) were adoptively transferred into hCD40Tg mice 1 day before treatment with 30 μg anti-CD40 mAbs as indicated. Mice were bled on day 5 and SIINFEKL + cells were expressed as a percentage of total CD8 + T cells. Means ± SEM, n = 5, data representative of three experiments. Each dot represents one mouse. Two-tailed, non-paired Student’s t test, ∗ p

    Journal: Cancer Cell

    Article Title: Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity

    doi: 10.1016/j.ccell.2020.04.013

    Figure Lengend Snippet: 341G2 h2 Mediates Super-agonistic Activity In Vivo (A) OTI cells (1 × 10 5 ) were adoptively transferred into hCD40Tg mice 1 day before treatment with 30 μg anti-CD40 mAbs as indicated. Mice were bled on day 5 and SIINFEKL + cells were expressed as a percentage of total CD8 + T cells. Means ± SEM, n = 5, data representative of three experiments. Each dot represents one mouse. Two-tailed, non-paired Student’s t test, ∗ p

    Article Snippet: For monotherapy, mice were treated with 30 μg anti-CD40 mAb starting on day 5 every 3 days for 3 doses.

    Techniques: Activity Assay, In Vivo, Mouse Assay, Two Tailed Test

    341G2 h2 Potently Activates Dendritic Cells (A) hCD40Tg and hCD40Tg/FcγRnull mice received 30 μg anti-CD40 mAbs intravenously and spleens were harvested on day 2. Expression levels of CD80 and CD86 on splenic CD11c + CD8 + DEC205 + DCs were analyzed by flow cytometry. Histograms representative of six to seven mice from two experiments. (B) Experiments same as in (A). MFI values for CD80 and CD86 are quantified, and the frequency of CD11c + CD8 + DEC205 + DC expressed as the percentage of CD11c + cells. Means ± SEM, n = 6–7, data pooled from two experiments. Each dot represents one mouse. Two-tailed, non-paired Student’s t test, ∗ p

    Journal: Cancer Cell

    Article Title: Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity

    doi: 10.1016/j.ccell.2020.04.013

    Figure Lengend Snippet: 341G2 h2 Potently Activates Dendritic Cells (A) hCD40Tg and hCD40Tg/FcγRnull mice received 30 μg anti-CD40 mAbs intravenously and spleens were harvested on day 2. Expression levels of CD80 and CD86 on splenic CD11c + CD8 + DEC205 + DCs were analyzed by flow cytometry. Histograms representative of six to seven mice from two experiments. (B) Experiments same as in (A). MFI values for CD80 and CD86 are quantified, and the frequency of CD11c + CD8 + DEC205 + DC expressed as the percentage of CD11c + cells. Means ± SEM, n = 6–7, data pooled from two experiments. Each dot represents one mouse. Two-tailed, non-paired Student’s t test, ∗ p

    Article Snippet: For monotherapy, mice were treated with 30 μg anti-CD40 mAb starting on day 5 every 3 days for 3 doses.

    Techniques: Mouse Assay, Expressing, Flow Cytometry, Two Tailed Test

    341G2 hIgG1 (h1) and hIgG4 (h4) Suppress Immune Function In Vitro and In Vivo (A) Ramos cells were incubated with fixed concentration of CD40L and various concentrations of anti-CD40 mAbs. Remaining bound CD40L was detected by anti-FLAG-APC. Means ± SEM, n = 3, data representative of three experiments. (B) Purified hCD40Tg mouse splenic B cells were incubated with 2 μg/mL CD40L in the presence or absence of 5 μg/mL 341G2 h1 and h4 for 2 days. Cell culture images were taken on day 2. Proliferation was measured by 3 H-thymidine incorporation. Means ± SEM, n = 5, data representative of three experiments. Scale bar, 0.5 mm. (C) Purified human B cells were incubated with 2 μg/mL CD40L in the presence or absence of 5 μg/mL 341G2 h1 or h4 for 2 days. Cell culture images were taken on day 2. Proliferation was measured by 3 H-thymidine incorporation. Means ± SEM, n = 3–5, data representative of three donors. Scale bar, 0.5 mm. (D) hCD40Tg mice received 500 μg OVA and 100 μg anti-CD40 mAbs on day 0 and another dose of 100 μg anti-CD40 mAbs on day 3. Mice were bled on day 18 and serum levels of anti-OVA IgG were quantified by ELISA as described in the STAR Methods . Means ± SEM, n = 4–5, data representative of two experiments. Each dot represents one mouse. (E) Mice received the same treatment as in (D). The level of circulating CD19 + B cells in blood on day 2 was quantified by anti-mouse CD19-APC and expressed as the percentage of CD45.2 + cells. Means ± SEM, n = 4–5, data representative of two experiments. Each dot represents one mouse. Two-tailed, non-paired Student’s t test, ∗ p

    Journal: Cancer Cell

    Article Title: Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity

    doi: 10.1016/j.ccell.2020.04.013

    Figure Lengend Snippet: 341G2 hIgG1 (h1) and hIgG4 (h4) Suppress Immune Function In Vitro and In Vivo (A) Ramos cells were incubated with fixed concentration of CD40L and various concentrations of anti-CD40 mAbs. Remaining bound CD40L was detected by anti-FLAG-APC. Means ± SEM, n = 3, data representative of three experiments. (B) Purified hCD40Tg mouse splenic B cells were incubated with 2 μg/mL CD40L in the presence or absence of 5 μg/mL 341G2 h1 and h4 for 2 days. Cell culture images were taken on day 2. Proliferation was measured by 3 H-thymidine incorporation. Means ± SEM, n = 5, data representative of three experiments. Scale bar, 0.5 mm. (C) Purified human B cells were incubated with 2 μg/mL CD40L in the presence or absence of 5 μg/mL 341G2 h1 or h4 for 2 days. Cell culture images were taken on day 2. Proliferation was measured by 3 H-thymidine incorporation. Means ± SEM, n = 3–5, data representative of three donors. Scale bar, 0.5 mm. (D) hCD40Tg mice received 500 μg OVA and 100 μg anti-CD40 mAbs on day 0 and another dose of 100 μg anti-CD40 mAbs on day 3. Mice were bled on day 18 and serum levels of anti-OVA IgG were quantified by ELISA as described in the STAR Methods . Means ± SEM, n = 4–5, data representative of two experiments. Each dot represents one mouse. (E) Mice received the same treatment as in (D). The level of circulating CD19 + B cells in blood on day 2 was quantified by anti-mouse CD19-APC and expressed as the percentage of CD45.2 + cells. Means ± SEM, n = 4–5, data representative of two experiments. Each dot represents one mouse. Two-tailed, non-paired Student’s t test, ∗ p

    Article Snippet: For monotherapy, mice were treated with 30 μg anti-CD40 mAb starting on day 5 every 3 days for 3 doses.

    Techniques: In Vitro, In Vivo, Incubation, Concentration Assay, Purification, Cell Culture, Mouse Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    341G2 h2 Exhibits Antitumor Efficacy in Combination with Peptide Vaccine (A) hCD40Tg mice were inoculated with 1 × 10 5 TC1 tumor cells subcutaneously on day 0 and then were treated with 150 μg peptide in combination with 30 μg anti-CD40 on day 5, or treated with 30 μg anti-CD40 alone on days 5, 8, and 11. Tumor size and survival were assessed, Means ± SEM, n = 5–6, data representative of at least two experiments. The fractions in parentheses indicate the number of tumor-free mice (numerator) out of total number of mice (denominator) in that group at the end of the study. Survival curves were compared by log rank test. ∗ p

    Journal: Cancer Cell

    Article Title: Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity

    doi: 10.1016/j.ccell.2020.04.013

    Figure Lengend Snippet: 341G2 h2 Exhibits Antitumor Efficacy in Combination with Peptide Vaccine (A) hCD40Tg mice were inoculated with 1 × 10 5 TC1 tumor cells subcutaneously on day 0 and then were treated with 150 μg peptide in combination with 30 μg anti-CD40 on day 5, or treated with 30 μg anti-CD40 alone on days 5, 8, and 11. Tumor size and survival were assessed, Means ± SEM, n = 5–6, data representative of at least two experiments. The fractions in parentheses indicate the number of tumor-free mice (numerator) out of total number of mice (denominator) in that group at the end of the study. Survival curves were compared by log rank test. ∗ p

    Article Snippet: For monotherapy, mice were treated with 30 μg anti-CD40 mAb starting on day 5 every 3 days for 3 doses.

    Techniques: Mouse Assay

    Antagonist CFZ533 Is Converted into an Agonist by Isotype Switching to hIgG2 (A) CHO-k1 cells expressing different hCD40 mutants were probed with anti-CD40 mAbs. Bound mAbs were detected by anti-mouse IgG-FITC. (B) Deduced epitope of CFZ533 is shown in red on a color-coded CD40 molecular scaffold (left) and displayed relative to the CD40/CD40L binding interface (right). The structure model is based on PDB: 3QD6 . (C) Jurkat cells stably transfected with human CD40EC-GFP were treated with 10 μg/mL CFZ533 h1 or CFZ533 h2 for 1 h at 37°C. Cells were then fixed, nuclear-stained using DAPI, and imaged using a Leica SP8 confocal microscope. z stack images shown. Blue, nucleus; green, human CD40-GFP. Scale bar, 4 μm. Image representative of at least ten images taken. (D) Jurkat NF-κB GFP reporter cells stably transfected with hCD40 were incubated with various concentrations of anti-CD40 mAbs for 8 h and the level of NF-κB activation was assessed by GFP expression using flow cytometry. Means ± SEM, n = 3, data representative of two experiments. (E) Purified splenic B cells from hCD40Tg mice were incubated with various concentrations of anti-CD40 mAbs for 4 days. Proliferation was measured by 3 H-thymidine incorporation. Means ± SEM, n = 3, data representative of three experiments. (F) Experiments the same as (E), splenic B cells from hCD40Tg/ Fcgr2b −/− mice were used. Means ± SEM, n = 3, data representative of three experiments. (G) 1 × 10 5 OTI cells were adoptively transferred into hCD40Tg mice 1 day before treatment with 100 μg anti-CD40 mAbs as indicated. Mice were bled on day 5 and SIINFEKL + cells were quantified as a percentage of total CD8 + T cells. Means ± SEM, n = 5, each dot represents one mouse, data representative of two experiments. Two-tailed, non-paired Student’s t test, ∗ p

    Journal: Cancer Cell

    Article Title: Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity

    doi: 10.1016/j.ccell.2020.04.013

    Figure Lengend Snippet: Antagonist CFZ533 Is Converted into an Agonist by Isotype Switching to hIgG2 (A) CHO-k1 cells expressing different hCD40 mutants were probed with anti-CD40 mAbs. Bound mAbs were detected by anti-mouse IgG-FITC. (B) Deduced epitope of CFZ533 is shown in red on a color-coded CD40 molecular scaffold (left) and displayed relative to the CD40/CD40L binding interface (right). The structure model is based on PDB: 3QD6 . (C) Jurkat cells stably transfected with human CD40EC-GFP were treated with 10 μg/mL CFZ533 h1 or CFZ533 h2 for 1 h at 37°C. Cells were then fixed, nuclear-stained using DAPI, and imaged using a Leica SP8 confocal microscope. z stack images shown. Blue, nucleus; green, human CD40-GFP. Scale bar, 4 μm. Image representative of at least ten images taken. (D) Jurkat NF-κB GFP reporter cells stably transfected with hCD40 were incubated with various concentrations of anti-CD40 mAbs for 8 h and the level of NF-κB activation was assessed by GFP expression using flow cytometry. Means ± SEM, n = 3, data representative of two experiments. (E) Purified splenic B cells from hCD40Tg mice were incubated with various concentrations of anti-CD40 mAbs for 4 days. Proliferation was measured by 3 H-thymidine incorporation. Means ± SEM, n = 3, data representative of three experiments. (F) Experiments the same as (E), splenic B cells from hCD40Tg/ Fcgr2b −/− mice were used. Means ± SEM, n = 3, data representative of three experiments. (G) 1 × 10 5 OTI cells were adoptively transferred into hCD40Tg mice 1 day before treatment with 100 μg anti-CD40 mAbs as indicated. Mice were bled on day 5 and SIINFEKL + cells were quantified as a percentage of total CD8 + T cells. Means ± SEM, n = 5, each dot represents one mouse, data representative of two experiments. Two-tailed, non-paired Student’s t test, ∗ p

    Article Snippet: For monotherapy, mice were treated with 30 μg anti-CD40 mAb starting on day 5 every 3 days for 3 doses.

    Techniques: Expressing, Binding Assay, Stable Transfection, Transfection, Staining, Microscopy, Incubation, Activation Assay, Flow Cytometry, Purification, Mouse Assay, Two Tailed Test

    341G2 hIgG2 (h2) Is a Super-agonist In Vitro (A) Purified hCD40Tg mouse splenic B cells were incubated with various concentrations of 341G2 h1, h2, and h4 for 2 days. Cell culture images were taken on day 2. Proliferation was measured by 3 H-thymidine incorporation. Means ± SEM, n = 3, data representative of three experiments. Scale bar, 0.5 mm. (B) Purified human B cell proliferation assay performed the same as in (A). Means ± SEM, n = 3, data representative of three donors. Scale bar, 0.5 mm. (C) Purified hCD40Tg mouse splenic B cells were incubated with anti-CD40 mAbs for various periods of time as indicated above each plot. Proliferation was measured by 3 H-thymidine incorporation. Means ± SEM, n = 3, data representative of three experiments. (D) Purified hCD40Tg mouse splenic B cells were incubated with 2 μg/mL clinical anti-CD40 mAbs for 3 days as indicated above each plot. Proliferation was measured by 3 H-thymidine incorporation. Means ± SEM, n = 4, data representative of three experiments. (E) Jurkat cells stably transfected with human CD40EC-GFP were treated with 10 μg/mL anti-CD40 mAbs as indicated for 1 h at 37°C. Cells were then fixed, nuclear-stained using DAPI, and imaged using a Leica SP8 confocal microscope. z stack images shown. Blue, nucleus; green, human CD40-GFP. Scale bar, 4 μm. Image representative of at least ten images taken. See also Figure S1 .

    Journal: Cancer Cell

    Article Title: Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity

    doi: 10.1016/j.ccell.2020.04.013

    Figure Lengend Snippet: 341G2 hIgG2 (h2) Is a Super-agonist In Vitro (A) Purified hCD40Tg mouse splenic B cells were incubated with various concentrations of 341G2 h1, h2, and h4 for 2 days. Cell culture images were taken on day 2. Proliferation was measured by 3 H-thymidine incorporation. Means ± SEM, n = 3, data representative of three experiments. Scale bar, 0.5 mm. (B) Purified human B cell proliferation assay performed the same as in (A). Means ± SEM, n = 3, data representative of three donors. Scale bar, 0.5 mm. (C) Purified hCD40Tg mouse splenic B cells were incubated with anti-CD40 mAbs for various periods of time as indicated above each plot. Proliferation was measured by 3 H-thymidine incorporation. Means ± SEM, n = 3, data representative of three experiments. (D) Purified hCD40Tg mouse splenic B cells were incubated with 2 μg/mL clinical anti-CD40 mAbs for 3 days as indicated above each plot. Proliferation was measured by 3 H-thymidine incorporation. Means ± SEM, n = 4, data representative of three experiments. (E) Jurkat cells stably transfected with human CD40EC-GFP were treated with 10 μg/mL anti-CD40 mAbs as indicated for 1 h at 37°C. Cells were then fixed, nuclear-stained using DAPI, and imaged using a Leica SP8 confocal microscope. z stack images shown. Blue, nucleus; green, human CD40-GFP. Scale bar, 4 μm. Image representative of at least ten images taken. See also Figure S1 .

    Article Snippet: For monotherapy, mice were treated with 30 μg anti-CD40 mAb starting on day 5 every 3 days for 3 doses.

    Techniques: In Vitro, Purification, Incubation, Cell Culture, Proliferation Assay, Stable Transfection, Transfection, Staining, Microscopy

    Antagonist Anti-CD40 mAb 341G2 Binds CRD1 and CRD2 Domains at the CD40/CD40L Interface (A) CHO-k1 cells expressing full-length hCD40 or domain-truncated variants encompassing CRD2-4 or CRD3-4 were incubated with 10 μg/mL anti-CD40 mAb Lob 7/6, ChiLob 7/4, or 341G2. Bound anti-CD40 mAb was detected by anti-human Fc-PE. (B) His-tagged recombinant soluble proteins corresponding to the full-length extracellular domain (EC) of hCD40 or its truncated variants were analyzed by western blotting with the mAb indicated above each panel used for detection. Image represents a composite of multiple blots. (C) CHO-k1 cells expressing different hCD40 mutants were probed with anti-CD40 mAbs. Bound mAbs was detected by anti-mouse IgG-FITC. (D) His-tagged soluble hCD40 mutants were captured for 30 s using anti-His mAbs immobilized on a CM5 chip, and Lob 7/6 and 341G2 were injected at 1,000, 333, 111, 37, 12.4, and 4.1 nM using a Biacore T100 instrument. The association and dissociation phases lasted 180 and 300 s, respectively. (E) Structural prediction of 341G2 Fab-hCD40 by SEC-SAXS followed by homology modeling and docking analysis. 341G2 Fab homology modeled as described in the methods shown in magenta cartoon representation. CD40 EC represented as CRD colored sticks. ChiLob 7/4 shown as cyan cartoon overlaid with CD40 (PDB: 6FAX ). CD40L shown as a gray surface (PDB: 3QD6 ) shows overlap with proposed 341G2 Fab position. CD40 residues involved in 341G2 interaction highlighted as red sticks.

    Journal: Cancer Cell

    Article Title: Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity

    doi: 10.1016/j.ccell.2020.04.013

    Figure Lengend Snippet: Antagonist Anti-CD40 mAb 341G2 Binds CRD1 and CRD2 Domains at the CD40/CD40L Interface (A) CHO-k1 cells expressing full-length hCD40 or domain-truncated variants encompassing CRD2-4 or CRD3-4 were incubated with 10 μg/mL anti-CD40 mAb Lob 7/6, ChiLob 7/4, or 341G2. Bound anti-CD40 mAb was detected by anti-human Fc-PE. (B) His-tagged recombinant soluble proteins corresponding to the full-length extracellular domain (EC) of hCD40 or its truncated variants were analyzed by western blotting with the mAb indicated above each panel used for detection. Image represents a composite of multiple blots. (C) CHO-k1 cells expressing different hCD40 mutants were probed with anti-CD40 mAbs. Bound mAbs was detected by anti-mouse IgG-FITC. (D) His-tagged soluble hCD40 mutants were captured for 30 s using anti-His mAbs immobilized on a CM5 chip, and Lob 7/6 and 341G2 were injected at 1,000, 333, 111, 37, 12.4, and 4.1 nM using a Biacore T100 instrument. The association and dissociation phases lasted 180 and 300 s, respectively. (E) Structural prediction of 341G2 Fab-hCD40 by SEC-SAXS followed by homology modeling and docking analysis. 341G2 Fab homology modeled as described in the methods shown in magenta cartoon representation. CD40 EC represented as CRD colored sticks. ChiLob 7/4 shown as cyan cartoon overlaid with CD40 (PDB: 6FAX ). CD40L shown as a gray surface (PDB: 3QD6 ) shows overlap with proposed 341G2 Fab position. CD40 residues involved in 341G2 interaction highlighted as red sticks.

    Article Snippet: For monotherapy, mice were treated with 30 μg anti-CD40 mAb starting on day 5 every 3 days for 3 doses.

    Techniques: Expressing, Incubation, Recombinant, Western Blot, Chromatin Immunoprecipitation, Injection

    341G2 h2 Exhibits Antitumor Efficacy and Potentiates Adoptive T Cell Therapy (A) hCD40Tg mice were inoculated with 5 × 10 5 MC38 tumor cells subcutaneously. On day 6, when the tumor became palpable, mice were treated with 30 μg anti-CD40 mAbs and again 3 days later. Tumor size and survival were assessed, n = 11–14, data pooled from two experiments. The fractions in parentheses indicate the number of tumor-free mice (numerator) out of the total number of mice (denominator) in that group at the end of the study. Survival curves were compared by log rank test. ∗ p

    Journal: Cancer Cell

    Article Title: Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity

    doi: 10.1016/j.ccell.2020.04.013

    Figure Lengend Snippet: 341G2 h2 Exhibits Antitumor Efficacy and Potentiates Adoptive T Cell Therapy (A) hCD40Tg mice were inoculated with 5 × 10 5 MC38 tumor cells subcutaneously. On day 6, when the tumor became palpable, mice were treated with 30 μg anti-CD40 mAbs and again 3 days later. Tumor size and survival were assessed, n = 11–14, data pooled from two experiments. The fractions in parentheses indicate the number of tumor-free mice (numerator) out of the total number of mice (denominator) in that group at the end of the study. Survival curves were compared by log rank test. ∗ p

    Article Snippet: For monotherapy, mice were treated with 30 μg anti-CD40 mAb starting on day 5 every 3 days for 3 doses.

    Techniques: Mouse Assay

    Combination therapy with vaccination reverses CD8 T-cell anergy and restores T-cell function in a spontaneous adenocarcinoma model. ( A ) Naive Pmel TCR transgenic CD8 T cells were purified by negative selection and adoptively transferred on day 6 into B16F10 tumor-bearing wild-type C57BL/6 mice. Mice were treated with IgG or aOX40 on days 7 and 11; with aCTLA-4 on days 7, 9, and 11; or with monotherapy or combination therapy and vaccine (gp100/anti-CD40) on day 7. Blood was analyzed by flow cytometry on day 14. ( B – D ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET-1 mice on day 0. Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28 along with mono- or combination therapy (50 µg aOX40 or control rat IgG on days 28 and 29 and 200 µg aCTLA-4 on days 28, 30, and 32). Splenocytes were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict ( B ) the total number of OT-I + cells; the total number of Ki-67 + , granzyme B + (GrzB), KLRG-1 + , CD62L + , and CD25 + OT-I; ( C and D ) and the percentage ( C ) and total number ( D ) of IFNγ-, TNFα-, and IL-2--producing OT-I cells. Data are shown for one representative mouse ( C ) or for all mice in each cohort for donor OT-I cells ( B and D ). Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group). * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice

    doi: 10.1073/pnas.1510518113

    Figure Lengend Snippet: Combination therapy with vaccination reverses CD8 T-cell anergy and restores T-cell function in a spontaneous adenocarcinoma model. ( A ) Naive Pmel TCR transgenic CD8 T cells were purified by negative selection and adoptively transferred on day 6 into B16F10 tumor-bearing wild-type C57BL/6 mice. Mice were treated with IgG or aOX40 on days 7 and 11; with aCTLA-4 on days 7, 9, and 11; or with monotherapy or combination therapy and vaccine (gp100/anti-CD40) on day 7. Blood was analyzed by flow cytometry on day 14. ( B – D ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET-1 mice on day 0. Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28 along with mono- or combination therapy (50 µg aOX40 or control rat IgG on days 28 and 29 and 200 µg aCTLA-4 on days 28, 30, and 32). Splenocytes were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict ( B ) the total number of OT-I + cells; the total number of Ki-67 + , granzyme B + (GrzB), KLRG-1 + , CD62L + , and CD25 + OT-I; ( C and D ) and the percentage ( C ) and total number ( D ) of IFNγ-, TNFα-, and IL-2--producing OT-I cells. Data are shown for one representative mouse ( C ) or for all mice in each cohort for donor OT-I cells ( B and D ). Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group). * P

    Article Snippet: Murine anti–DEC-205–OVA and anti–DEC-205/HER2 mAbs were provided by Celldex Therapeutics, Inc., and 5 μg were injected s.c. along with 50 μg anti-CD40 (clone FGK4.5; BioXCell) or 50 μg poly(I:C) (InvivoGen) as an adjuvant.

    Techniques: Cell Function Assay, Transgenic Assay, Purification, Selection, Mouse Assay, Flow Cytometry, Cytometry

    ( A and C ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET mice (day 0). Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28, along with mono- or combination therapy: 50 µg aOX40 or control rat IgG on days 28 and 29, and 200 µg aCTLA-4 on days 28, 30, and 3. ( A ) Model of treatment. ( B ) Naive OT-I CD8 T cells (2.5 × 10 6 ) were purified by negative selection and adoptively transferred into TRAMP/POET or wild-type C57BL/6 mice. Three days later, lymph nodes were processed, and OX40 and CTLA-4 expression was analyzed by flow cytometry on donor OT-1 cells. For CTLA-4 expression on donor OT-1 cells, the unstained control is shown in gray; the wild-type control is shown as a solid black line; TRAMP/POET mice are shown as a purple line. For OX40 expression ( Middle and Right ), images represent OT-I + (Thy1.1 + ) CD8 T cells versus OX40 expression; the numbers shown are the percentage of OX40 + OT-I cells. ( C ) Splenocytes and prostate-draining lymph nodes (pooled for each group) were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice

    doi: 10.1073/pnas.1510518113

    Figure Lengend Snippet: ( A and C ) Naive OT-I CD8 T cells (5 × 10 5 ) were purified by negative selection and adoptively transferred into TRAMP/POET mice (day 0). Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28, along with mono- or combination therapy: 50 µg aOX40 or control rat IgG on days 28 and 29, and 200 µg aCTLA-4 on days 28, 30, and 3. ( A ) Model of treatment. ( B ) Naive OT-I CD8 T cells (2.5 × 10 6 ) were purified by negative selection and adoptively transferred into TRAMP/POET or wild-type C57BL/6 mice. Three days later, lymph nodes were processed, and OX40 and CTLA-4 expression was analyzed by flow cytometry on donor OT-1 cells. For CTLA-4 expression on donor OT-1 cells, the unstained control is shown in gray; the wild-type control is shown as a solid black line; TRAMP/POET mice are shown as a purple line. For OX40 expression ( Middle and Right ), images represent OT-I + (Thy1.1 + ) CD8 T cells versus OX40 expression; the numbers shown are the percentage of OX40 + OT-I cells. ( C ) Splenocytes and prostate-draining lymph nodes (pooled for each group) were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 + Thy1.1 + events. Graphs depict the mean ± SEM for one of three independent experiments ( n = 4 or 5 per group).

    Article Snippet: Murine anti–DEC-205–OVA and anti–DEC-205/HER2 mAbs were provided by Celldex Therapeutics, Inc., and 5 μg were injected s.c. along with 50 μg anti-CD40 (clone FGK4.5; BioXCell) or 50 μg poly(I:C) (InvivoGen) as an adjuvant.

    Techniques: Purification, Selection, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    Diminished CD8 T cell responses to infection in mice lacking SOCS1 in DC a) Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl littermates were primed with 1×10 5 cfu ΔactA QV, 5μg anti-DEC205-OVA with 25 μg anti-CD40 or 1×10 6 pfu VV-OVA. Spleens were harvested 7 days post priming and IFNγ + SIINFEKL-specific CD8 T cells determined by using ICS. i) Representative flow-cytometry plots of IFNγ expression in response to SIINFEKL peptide. Quantitation of ii) the percent and iii) absolute numbers of IFNγ + SIINFEKL-specific CD8 T cells per spleen. b) Quantitation of the percent of IFNγ + i) B8R 20–27 -specific or ii) A42R 88–96 -specific CD8 T cells in spleens from mice immunized with ΔactA QV. c) Percent IFNγ + LLO 190-201 –specific CD4 T cells 7 days post immunization with ΔactA QV. d) OVA-specific CD90.1 + OT1 CD8 T cells were isolated from OT1 transgenic mice and 10,000 cells per mouse adoptively transferred to Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice. The following day mice were immunized with 1×10 5 cfu ΔactA QV as above, spleens harvested 7 days later, cells stained and CD90.1 + (OT1) CD8 T cells analyzed. i) Representative flow-cytometry plot of CD90.1 + CD8 T cell expansion in Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice. ii) The percent and iii) total number of OT1 cells per mouse. e) CD8 T cells from Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice were purified and transferred to Rag1 -/- mice. 24hr later mice were immunized with 1×10 5 cfu ΔactA QV. Spleens were harvested 7 post infection and splenocytes stimulated in vitro with the indicated peptide. i) Percent and ii) absolute numbers of antigen-specific CD8 T cells as well as iii) percent of LLO 190-201 CD4 T cell responses are shown. Each symbol represents one mouse, n=3-5 mice per group. Data represents the mean ± SEM of each group. The displayed experiments are representative of 3 to 5 independent repeats. Statistics calculated by Student's t test; ns = no significant differences observed between the groups analyzed; ** = p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Amplifying IFNγ signaling in DC by CD11c-specific loss of SOCS1 increases innate immunity to infection while decreasing adaptive immunity

    doi: 10.4049/jimmunol.1700909

    Figure Lengend Snippet: Diminished CD8 T cell responses to infection in mice lacking SOCS1 in DC a) Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl littermates were primed with 1×10 5 cfu ΔactA QV, 5μg anti-DEC205-OVA with 25 μg anti-CD40 or 1×10 6 pfu VV-OVA. Spleens were harvested 7 days post priming and IFNγ + SIINFEKL-specific CD8 T cells determined by using ICS. i) Representative flow-cytometry plots of IFNγ expression in response to SIINFEKL peptide. Quantitation of ii) the percent and iii) absolute numbers of IFNγ + SIINFEKL-specific CD8 T cells per spleen. b) Quantitation of the percent of IFNγ + i) B8R 20–27 -specific or ii) A42R 88–96 -specific CD8 T cells in spleens from mice immunized with ΔactA QV. c) Percent IFNγ + LLO 190-201 –specific CD4 T cells 7 days post immunization with ΔactA QV. d) OVA-specific CD90.1 + OT1 CD8 T cells were isolated from OT1 transgenic mice and 10,000 cells per mouse adoptively transferred to Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice. The following day mice were immunized with 1×10 5 cfu ΔactA QV as above, spleens harvested 7 days later, cells stained and CD90.1 + (OT1) CD8 T cells analyzed. i) Representative flow-cytometry plot of CD90.1 + CD8 T cell expansion in Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice. ii) The percent and iii) total number of OT1 cells per mouse. e) CD8 T cells from Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice were purified and transferred to Rag1 -/- mice. 24hr later mice were immunized with 1×10 5 cfu ΔactA QV. Spleens were harvested 7 post infection and splenocytes stimulated in vitro with the indicated peptide. i) Percent and ii) absolute numbers of antigen-specific CD8 T cells as well as iii) percent of LLO 190-201 CD4 T cell responses are shown. Each symbol represents one mouse, n=3-5 mice per group. Data represents the mean ± SEM of each group. The displayed experiments are representative of 3 to 5 independent repeats. Statistics calculated by Student's t test; ns = no significant differences observed between the groups analyzed; ** = p

    Article Snippet: Cre- SOCS1fl/fl and Cre+ SOCS1fl/fl littermates made equivalent responses to vaccination with anti-DEC205-OVA and anti-CD40 , suggesting that DC function is intact when antigen is delivered via this route.

    Techniques: Infection, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Quantitation Assay, Isolation, Transgenic Assay, Staining, Purification, In Vitro

    Increased innate response and TipDC activity in mice lacking SOCS1 in DC a) i) Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice were immunized with anti-DEC205-OVA and anti-CD40 to generate an equivalent priming event then boosted 21 days later with 1×10 5 cfu ΔactA QV. ii) Spleens were harvested 5 days post-boost and IFNγ + SIINFEKL-specific CD8 T cells determined by using ICS. b) Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice were left untreated or primed with 1×10 5 cfu ΔactA QV and challenged 21 days later with 1×10 5 cfu wt L. monocytogenes . Three days later i) spleens and ii) livers were harvested, homogenized, plated on BHI plates and cfu numbers determined after overnight incubation at 37°C. c) Spleens from naïve mice were harvested, stained and analyzed by flow-cytometry. i) Summary of the absolute number of CD11b + Ly6C high cells and ii) the absolute number of Ly6G + Ly6C int cells gated in the population. d) Mice were left untreated (0hr) or immunized with 1×10 4 cfu wt, spleens removed at 24hr and 48hr post-infection, dissociated, stained and iNOS synthesis determined by ICS. i) Representative expression of iNOS and MHCII in CD3 - CD19 - CD11b + Ly6C high monocytes over time, and ii) summary of the number of iNOS + MHCII + Tip-DC/spleen. Each symbol represents one mouse. Data represents the mean ± SEM of each group. Results shown are representative of three independent experiments. Statistics calculated by Student's t test; ns = no significant differences observed between the groups analyzed; * = p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Amplifying IFNγ signaling in DC by CD11c-specific loss of SOCS1 increases innate immunity to infection while decreasing adaptive immunity

    doi: 10.4049/jimmunol.1700909

    Figure Lengend Snippet: Increased innate response and TipDC activity in mice lacking SOCS1 in DC a) i) Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice were immunized with anti-DEC205-OVA and anti-CD40 to generate an equivalent priming event then boosted 21 days later with 1×10 5 cfu ΔactA QV. ii) Spleens were harvested 5 days post-boost and IFNγ + SIINFEKL-specific CD8 T cells determined by using ICS. b) Cre - SOCS1 fl/fl and Cre + SOCS1 fl/fl mice were left untreated or primed with 1×10 5 cfu ΔactA QV and challenged 21 days later with 1×10 5 cfu wt L. monocytogenes . Three days later i) spleens and ii) livers were harvested, homogenized, plated on BHI plates and cfu numbers determined after overnight incubation at 37°C. c) Spleens from naïve mice were harvested, stained and analyzed by flow-cytometry. i) Summary of the absolute number of CD11b + Ly6C high cells and ii) the absolute number of Ly6G + Ly6C int cells gated in the population. d) Mice were left untreated (0hr) or immunized with 1×10 4 cfu wt, spleens removed at 24hr and 48hr post-infection, dissociated, stained and iNOS synthesis determined by ICS. i) Representative expression of iNOS and MHCII in CD3 - CD19 - CD11b + Ly6C high monocytes over time, and ii) summary of the number of iNOS + MHCII + Tip-DC/spleen. Each symbol represents one mouse. Data represents the mean ± SEM of each group. Results shown are representative of three independent experiments. Statistics calculated by Student's t test; ns = no significant differences observed between the groups analyzed; * = p

    Article Snippet: Cre- SOCS1fl/fl and Cre+ SOCS1fl/fl littermates made equivalent responses to vaccination with anti-DEC205-OVA and anti-CD40 , suggesting that DC function is intact when antigen is delivered via this route.

    Techniques: Activity Assay, Mouse Assay, Incubation, Staining, Flow Cytometry, Cytometry, Infection, Expressing

    Single dose 5Gy SBRT combined with anti-CD40 induces regression of contralateral Panc02 tumors. C57BL/6 mice were inoculated with Panc02 tumors on each flank. Once tumors reached palpable size, the right flank was treated with RT and/or a single dose of anti-CD40 (20 μg) as indicated. (A) Volumes of treated tumors over time, measured by CT. (B) Volumes of contralateral tumors over time, measured by CT. (C) Representative CT imaging of mice at 3 weeks post-treatment. (D) Overall survival. n = 8/group. **** p

    Journal: Frontiers in Immunology

    Article Title: Radiation and Local Anti-CD40 Generate an Effective in situ Vaccine in Preclinical Models of Pancreatic Cancer

    doi: 10.3389/fimmu.2018.02030

    Figure Lengend Snippet: Single dose 5Gy SBRT combined with anti-CD40 induces regression of contralateral Panc02 tumors. C57BL/6 mice were inoculated with Panc02 tumors on each flank. Once tumors reached palpable size, the right flank was treated with RT and/or a single dose of anti-CD40 (20 μg) as indicated. (A) Volumes of treated tumors over time, measured by CT. (B) Volumes of contralateral tumors over time, measured by CT. (C) Representative CT imaging of mice at 3 weeks post-treatment. (D) Overall survival. n = 8/group. **** p

    Article Snippet: Monoclonal anti-CD40 (clone FGK, BioXcell) was injected intratumorally into the treated tumors of relevant mice.

    Techniques: Mouse Assay, Imaging

    Multiple fractions of image guided SBRT delivers radiation precisely to pancreatic tumors, but fails to achieve an abscopal effect. (A) A Small Animal Radiation Research Platform (SARRP) was used for RT. Image guided RT was given to the left tumor only. (B,C) Dosimetry showing the CT view of a mouse during image guided RT and dosimetry showing RT distribution in treated and contralateral tumors, as well as surrounding normal tissue. (D) C57BL/6 mice were inoculated with Panc02 tumors on each flank. Once tumors were palpable, mice were treated on one flank with no SBRT, 10Gy on three consecutive days, or 5Gy on 6 consecutive days. Tumor growth on each side was measured. n = 5 mice/group. (E) Mice were treated as in (D) , except that anti-CD40 was administered (10 μg, intratumoral) with the first and last dose of SBRT, or two injections 5 days apart in mice receiving no SBRT. n = 5 mice/group. Error bars are SD.

    Journal: Frontiers in Immunology

    Article Title: Radiation and Local Anti-CD40 Generate an Effective in situ Vaccine in Preclinical Models of Pancreatic Cancer

    doi: 10.3389/fimmu.2018.02030

    Figure Lengend Snippet: Multiple fractions of image guided SBRT delivers radiation precisely to pancreatic tumors, but fails to achieve an abscopal effect. (A) A Small Animal Radiation Research Platform (SARRP) was used for RT. Image guided RT was given to the left tumor only. (B,C) Dosimetry showing the CT view of a mouse during image guided RT and dosimetry showing RT distribution in treated and contralateral tumors, as well as surrounding normal tissue. (D) C57BL/6 mice were inoculated with Panc02 tumors on each flank. Once tumors were palpable, mice were treated on one flank with no SBRT, 10Gy on three consecutive days, or 5Gy on 6 consecutive days. Tumor growth on each side was measured. n = 5 mice/group. (E) Mice were treated as in (D) , except that anti-CD40 was administered (10 μg, intratumoral) with the first and last dose of SBRT, or two injections 5 days apart in mice receiving no SBRT. n = 5 mice/group. Error bars are SD.

    Article Snippet: Monoclonal anti-CD40 (clone FGK, BioXcell) was injected intratumorally into the treated tumors of relevant mice.

    Techniques: Mouse Assay

    Combination 10Gy RT and anti-CD40 induces regression of contralateral KPC tumors, but TGFβ blockade counteracts the abscopal effect of anti-CD40. C57BL/6 mice were inoculated on each flank with 150,000 KPC cells. Once tumors reached palpable size (11–14 days post-implantation), mice were treated with 10Gy SBRT, anti-CD40 (20 μg once, intratumoral), both RT and anti-CD40, or PBS control. (A) Volume of treated side tumors over time. (B) Volume of contralateral tumors over time. (C) Overall survival. (D–F) C57BL/6 mice were treated as in (A–C) , except anti-TGFβ (200μg intraperitoneal every 3 days starting at the time of SBRT) was included where indicated. n = 5/group. Error bars are SEM.

    Journal: Frontiers in Immunology

    Article Title: Radiation and Local Anti-CD40 Generate an Effective in situ Vaccine in Preclinical Models of Pancreatic Cancer

    doi: 10.3389/fimmu.2018.02030

    Figure Lengend Snippet: Combination 10Gy RT and anti-CD40 induces regression of contralateral KPC tumors, but TGFβ blockade counteracts the abscopal effect of anti-CD40. C57BL/6 mice were inoculated on each flank with 150,000 KPC cells. Once tumors reached palpable size (11–14 days post-implantation), mice were treated with 10Gy SBRT, anti-CD40 (20 μg once, intratumoral), both RT and anti-CD40, or PBS control. (A) Volume of treated side tumors over time. (B) Volume of contralateral tumors over time. (C) Overall survival. (D–F) C57BL/6 mice were treated as in (A–C) , except anti-TGFβ (200μg intraperitoneal every 3 days starting at the time of SBRT) was included where indicated. n = 5/group. Error bars are SEM.

    Article Snippet: Monoclonal anti-CD40 (clone FGK, BioXcell) was injected intratumorally into the treated tumors of relevant mice.

    Techniques: Mouse Assay