Journal: bioRxiv

Article Title: Diet Modifies Colonic Microbiota and CD4 + T Cell Repertoire to Trigger Flares in a Novel Model of Colitis Induced by IL-23

doi: 10.1101/262634

Figure Lengend Snippet: IL-23 Expression by CX 3 CR1 + Cells Induces Colonic and Cecal Inflammation. ( A ) Tamoxifen (TAM) in diet 2019 (green) was fed to R23FR and FR mice during the indicated times. After each cycle of TAM treatment, animals were switched to our mouse facility diet 5053 (gray). ( B ) Representative H&E-stained cecum and colon sections of R23FR mice at different time points. Scale bars, 100 μm. ( C ) Fecal lipocalin-2 levels in the stools of R23FR and FR mice were measured by ELISA (n= 5-15 per group per time point). ( D ) Histological scores of the colon and cecum of R23FR and FR mice at different time points (n= 5-15 per group per time point). Error bars represent mean ± SEM.

Article Snippet: R23FR mice after TAM treatment for 49 days were injected intraperitoneally (i.p.) with 2μg recombinant mouse IL-23 (R&D System) per mouse.

Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Diet Modifies Colonic Microbiota and CD4 + T Cell Repertoire to Trigger Flares in a Novel Model of Colitis Induced by IL-23

doi: 10.1101/262634

Figure Lengend Snippet: IL-23-induced CD4 + T Cells Drive Inflammation in Adoptively Transferred Mice. ( A ) Experimental setup for adoptive transfer of pooled CD4 + T cells from cecum and mLN of R23FR or FR mice into Rag -/- mice. (B) Representative H&E staining (left) and histological scores (right) of the colon and cecum of Rag -/- mice that received R23FR and FR mice CD4 + T cells fed with different diets. *** p<0.001; by nonparametric Mann-Whitney test. (C) Experimental setup for adoptive transfer of CD4 + T cells from large intestine or colonic mLN (cMLN) of R23FR mice into Rag -/- mice fed with different diets. (D) Representative H&E stained sections (left) and histological scores (right) of the colon and cecum of Rag -/- mice that received mLN or intestinal CD4 + T cells fed with different diets. *p<0.05, *** p<0.001; by nonparametric Mann-Whitney test.

Article Snippet: R23FR mice after TAM treatment for 49 days were injected intraperitoneally (i.p.) with 2μg recombinant mouse IL-23 (R&D System) per mouse.

Techniques: Adoptive Transfer Assay, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vasoactive intestinal peptide confers anticipatory mucosal immunity by regulating ILC3 activity

doi: 10.1101/729400

Figure Lengend Snippet: a - c , Mixed bone marrow chimeras were generated by reconstituting CD45.1 +/+ lethally irradiated recipients with F1 (CD45.1 × CD45.2, CD45.1 + CD45.2 + ) wild-type and CD45.2 +/+ Bmal1 ΔIL-7R bone marrow. a , Representative contour plots of frequency of different lymphocyte subsets isolated in the small intestine of mixed bone marrow chimeras. Plots show live CD45.1 + CD45.2 + cells for wild-type (F1) mice (upper panels) or CD45.2 +/+ cells for Bmal1 -deficient cells (lower panels). b , Frequency of different lymphocyte subsets derived from wild-type (red) and Bmal1 -deficient (blue) donor bone marrow isolated from the small intestine of chimeric mice. c , Frequency of wild-type (CD45.1 + CD45.2 + , red) and Bmal1 -deficient (CD45.2 +/+ , blue) IL-22-producing ILC3 isolated from the small intestine of chimeric mice. a - c Data are representive of 2 independent experiments ( a ) and show the mean ± s.e.m. for one experiment ( n = 6-8 mice/experiment) ( b , c ). d , 4 × 10 3 ILC3 were purified from the small intestine of wild-type (red) or Bmal1 ΔIL7R (blue) mice and cultivated 16 hours in presence of recombinant IL-7 (10ng/mL) to determine constitutive IL-22 expression, or IL-7 together with IL-23 (10 ng/mL) to elicit maximal IL-22 cytokine expression. IL-22 production was determined in culture supernatants by ELISA. e-f , Frequency of IL-5 and IL-22-producing ( e ) lung ILC2, and ( f ) small intestinal ILC3 at T4 and T16 isolated from wild-type and Bmal1 ΔIL7R mice. Left panel shows the constitutive expression of cytokines; right panel shows the cytokine expression after cultivation with IL-7 (10 ng/mL) and IL-23 (10 ng/mL). e , f Data show the mean ± s.e.m. of one of two similar experiments ( n = 4 mice for each genotype per time point per experiment).

Article Snippet: For longer term cultures, media was supplemented with 10 ng/mL recombinant human IL-7 (R&D Systems)(a survival factor), with or without 10ng/mL recombinant murine IL-23 (R&D Systems).

Techniques: Generated, Irradiation, Isolation, Derivative Assay, Purification, Recombinant, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Vasoactive intestinal peptide confers anticipatory mucosal immunity by regulating ILC3 activity

doi: 10.1101/729400

Figure Lengend Snippet: a , Food consumption of mice was determined at the indicated time points. Data show the mean ± s.e.m for one of two similar experiments ( n = 12 mice per experiment). b , Schematic representation of the time-restricted feeding regime used in c and d . Mice were given food only during the day (day food) or the night (night food) for seven days. All mice were analysed at T4 on day 8. c,d , Analyses of IL-5-expressing ILC2 ( c ) and IL-22-expressing ILC3 ( d ) from the small intestine, mesenteric LN and lung of mice fed only during the day (white) or only during the night (blue) as determined by flow cytometric analyses of intracellular cytokine. Representiative flow cytometric histograms of cytokine expression (left panels) and the frequency of constitutively producing IL-5 + or IL-22 + ILC2 and ILC3, respectively (right panels), showing individual mice (circles) together with the mean ± s.e.m. pooled from two independent experiments (gut and mLN: n = 6, 6 mice for each condition/experiment; lung: n = 4, 6 mice for each condition/experiment). e , Mice were treated with combination antibiotics (ampicillin, streptomycin and colistin) for two weeks before analysis of constitutive IL-22 production by intracellular staining. Data show the mean ± s.e.m ( n =6 mice per experimental condition). f , g , Expression of IL-5-expressing ILC2 ( f ) and IL-22-expressing ILC3 ( g ) from the small intestine and mesenteric LN from mice fed (green) or fasted for 16 hours (white). f , g Data show the mean ± s.e.m. of ( n = 6 mice per group per condition) and show one of two similar experiments. h , Gut ILC3 (7.5 × 10 3 ) were purified from fed (green) or fasted for 14 h (white) and cultivated 16 hours in presence of IL-7 (10 ng/mL) alone (constitutive expression) and IL-7 and IL-23 (10 ng/mL). IL-22 secretion was determined in culture supernatants by ELISA. Data show the response of cells pooled from 2 independent experiments with 3 mice per well for each condition in each experiment.

Article Snippet: For longer term cultures, media was supplemented with 10 ng/mL recombinant human IL-7 (R&D Systems)(a survival factor), with or without 10ng/mL recombinant murine IL-23 (R&D Systems).

Techniques: Expressing, Staining, Purification, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Vasoactive intestinal peptide confers anticipatory mucosal immunity by regulating ILC3 activity

doi: 10.1101/729400

Figure Lengend Snippet: a , Intracellular expression of IL-22 detected in ILC3 after four hours of incubation with IL-7 and IL-23 (10 ng/mL) in presence of vehicle (DMSO), VIP (1 μM) or Dibutyryl cyclic-AMP (10 μM). b , IL-22 secretion by purified ILC3 isolated from the small intestine and cultivated for 16 hours with IL-7 (10 ng/mL) or IL-7 and IL-23 (10 ng/mL) in presence of vehicle (DMSO) or VIP (1 μM). IL-22 was measured in the supernatant by ELISA. Data are a pool of three independent experiments (mean ± s.e.m) from a total of six experiments. c , d , Titration effect of VIP ( c ) and Dibutyryl cyclic-AMP ( d ) on the production of IL-22 by intestinal ILC3. Purified ILC3 were stimulated with IL-7 alone or IL-7 and IL-23 (10 ng/mL) and different concentrations of either VIP ( c ) or dibutyryl cyclic-AMP ( d ). ( e ) IL-22 production of ILC3 cultured with IL-7 (10 ng/mL) or IL-7 and IL-23 (10 ng/mL) in the presence of VIP (100mM) or VIPR2 agonist BAY55-9837 (100mM). f, IL-22 production of ILC3 cultured with IL-7 and IL-23 (10 ng/mL) in the presence of VIP (100mM) or VIP (100mM) with the VIPR2 antagonist PG99-465 (1μM) for 16 hours. e , f , Data are pooled from two independent experiments, each dot represents a pool of two mice (mean ± s.e.m).

Article Snippet: For longer term cultures, media was supplemented with 10 ng/mL recombinant human IL-7 (R&D Systems)(a survival factor), with or without 10ng/mL recombinant murine IL-23 (R&D Systems).

Techniques: Expressing, Incubation, Purification, Isolation, Enzyme-linked Immunosorbent Assay, Titration, Cell Culture

Journal: bioRxiv

Article Title: Vasoactive intestinal peptide confers anticipatory mucosal immunity by regulating ILC3 activity

doi: 10.1101/729400

Figure Lengend Snippet: a , Mice were injected i.p. with 2 nM VIP or PBS 2h (T2) before sacrifice (T4). Constitutive expression of IL-5 by ILC2, and IL-22 and IL-17 by ILC3 from the small intestine was determined by flow cytometry. Data show the mean ± s.e.m. of one of two similar experiments ( n = 5-6 mice per time point per experiment). b , Intracellular expression of ILC3 from the small intestine of wild-type or Vipr2 −/− mice after 4 hours of incubation with IL-7. Data show the mean ± s.e.m. of one of two similar experiments ( n = 8 mice per time point per experiment). c , 4.5 × 10 3 ILC3 purified from small intestine of wild-type or Vipr2 −/− were cultivated with IL-7 (10 ng/mL) alone or IL-7 and IL-23 (10 ng/mL) in presence of vehicle or VIP (1 μM) for 16 hours. IL-22 was measured in the supernatant by ELISA. Data represent a pool of 2 independent experiments and each dot represents a pool of two mice (mean ± s.e.m).

Article Snippet: For longer term cultures, media was supplemented with 10 ng/mL recombinant human IL-7 (R&D Systems)(a survival factor), with or without 10ng/mL recombinant murine IL-23 (R&D Systems).

Techniques: Injection, Expressing, Flow Cytometry, Incubation, Purification, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: Allograft dendritic cell p40 homodimers activate donor-reactive memory CD8 + T cells

doi: 10.1172/jci.insight.96940

Figure Lengend Snippet: (A) Groups of A/J mice (n = 4–8/group) received cardiac allografts subjected to 0.5 or 8 hours of CIS from either WT C57BL/6 (WTB6) or B6.IL12p40–/– donors. The indicated recipients of WT allografts were treated with 200 μg anti–IL-12 p40 mAb i.p. on days 0 and 1. All recipients were injected with 100 μg BrdU i.p. on days 0 and 1 after transplant. The following day, allografts were harvested and digested, and cell aliquots were stained with antibody and analyzed by flow cytometry to assess total numbers and proliferation of infiltrating memory CD4+ and CD8+ T cells. *P < 0.05; **P < 0.01, as determined by the Mann-Whitney nonparametric test. (B) Groups of A/J mice (n = 4/group) received cardiac allografts subjected to 0.5 or 8 hours of CIS from WT C57BL/6 or B6.IL12p35–/– donors. All recipients were injected with 100 μg BrdU i.p. on days 0 and 1. On day 2 after transplant, allografts were harvested and digested, and cell aliquots were stained with antibody and analyzed by flow cytometry to assess total numbers and proliferation of infiltrating memory CD4+ and CD8+ T cells. *P < 0.05 as determined by the Mann-Whitney nonparametric test. (C) Groups of C57BL/6 mice (n = 5–6/group) received C57BL/6 cardiac isografts or A/J cardiac allografts subjected to 0.5 or 8 hours of CIS. Grafts were harvested on day 2 after transplant, total RNA was isolated, and expression of the indicated cytokine mRNA was tested by qPCR. Results shown indicate relative expression vs. expression in naive hearts of A/J mice. **P < 0.01, as determined by the Mann-Whitney nonparametric test.

Article Snippet: Quantification of IL-12 heterodimer and p40 homodimer protein was performed on heart iso- and allograft lysates, and serum was collected at 48 hours after transplant using Mouse IL-12 p70 and Mouse p40 Quantikine ELISA Kits, respectively, from R&D Systems.

Techniques: Injection, Staining, Flow Cytometry, MANN-WHITNEY, Isolation, Expressing

Journal: JCI Insight

Article Title: Allograft dendritic cell p40 homodimers activate donor-reactive memory CD8 + T cells

doi: 10.1172/jci.insight.96940

Figure Lengend Snippet: (A) Groups of C57BL/6 mice (n = 5/group) received A/J cardiac allografts or C57BL/6 isografts subjected to 0.5 or 8 hours of CIS. The indicated recipients of 8 hours of CIS allografts were treated with 200 μg control rat IgG or anti-CD4 mAb on days –3, –2, and –1 prior to transplant. On day 2 after transplant, serum and graft lysates were prepared and quantities of p40 homodimers and IL-12 p70 heterodimers were tested by ELISA. *P < 0.05, as determined by the Mann-Whitney nonparametric test. (B) Groups of C57BL/6 mice (n = 5/group) received A/J cardiac allografts subjected to 0.5 or 8 hours of CIS and the indicated recipients were injected with 200 μg recombinant p40 homodimers i.p. on days 0 and 1. All recipients were injected with 100 μg BrdU i.p. on days 0 and 1 after transplant; the next day, allografts were harvested and digested, and cell aliquots were stained with antibody and analyzed by flow cytometry to assess total numbers and numbers of proliferating memory CD8+ T cells infiltrating the grafts. *P < 0.05; **P < 0.01, as determined by the Mann-Whitney nonparametric test. (C) Groups of C57BL/6 mice (n = 4–7/group) received A/J cardiac allografts subjected to 0.5 or 8 hours of CIS, and on days 0 and 1, were treated with 200 μg control rat IgG or 250 μg CTLA-4Ig i.p. with or without 200 μg recombinant p40 homodimers i.p. Allograft survival was monitored by daily abdominal palpation and rejection confirmed by laparotomy. ***P < 0.001 vs. all other groups, as determined using the Log-rank/Mantel-Cox test.

Article Snippet: Quantification of IL-12 heterodimer and p40 homodimer protein was performed on heart iso- and allograft lysates, and serum was collected at 48 hours after transplant using Mouse IL-12 p70 and Mouse p40 Quantikine ELISA Kits, respectively, from R&D Systems.

Techniques: Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Injection, Recombinant, Staining, Flow Cytometry

Journal: Endocrinology

Article Title: Immunotherapy With Apitopes Blocks the Immune Response to TSH Receptor in HLA-DR Transgenic Mice.

doi: 10.1210/en.2018-00306

Figure Lengend Snippet: Figure 2. Validation of adenoviral GD model using MMI and MPred. (A and B) DR3tg mice were immunized with 1010 Ad-TSHR at week 0 and 3 and received vehicle (n = 8) or MMI (n = 8) administered by subcutaneously implanted osmotic pumps. Serum was collected 2 weeks after the first immunization, and anti-TSHR IgG levels and T4 levels were analyzed by ELISA. One-way ANOVA was used to measure overall differences in anti-TSHR IgG levels and T4 levels. Bonferroni post hoc testing was used; significant differences are indicated in the graphs (*P , 0.05; **P , 0.01; ***P , 0.001; ****P , 0.0001). (C–E) DR3tg mice were immunized with 1010 Ad-TSHR at week 0 and 3 and received vehicle treatment (n = 10) or MMI treatment (n = 10). Serum was collected 2 weeks after the first immunization, and anti-TSHR IgG levels were analyzed by ELISA. Thymus weight and total number of splenocytes were determined on termination of the experiment. Each dot represents data from one mouse, and the mean 6 SEM is shown per group. One-way ANOVA and Bonferroni post hoc testing were used; significant differences are indicated in the graphs (*P , 0.05; **P , 0.01; ***P , 0.001; ****P , 0.0001).

Article Snippet: After 48 hours, peptide-induced IL-2 production by the antigenspecific T-cell hybridoma clone was measured by ELISA (mouse IL-2 ELISA kit, R&D Systems).

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay

Journal: Endocrinology

Article Title: Immunotherapy With Apitopes Blocks the Immune Response to TSH Receptor in HLA-DR Transgenic Mice.

doi: 10.1210/en.2018-00306

Figure Lengend Snippet: Figure 3. Prophylactic ATX-GD-59 treatment reduces anti-TSHR antibody levels. (A–C) DR3tg mice were injected subcutaneously in the flank region with 15 pmol, 150 pmol, and 1500 pmol ATX-GD-459 (n = 9) or control treatment (n = 7) on days 215, 213, and 211, followed by three injections of 15 nmol of each peptide (total 45 nmol/dose) of ATX-GD-459 or by PBS control treatment on days 28, 26, and 24 (dose escalation schedule). Mice were injected intramuscularly with 109 Ad-TSHR or Ad-LacZ on two occasions (days 0 and 21). Blood was collected (A) before and (B) 2 and (C) 5 weeks after the first immunization to measure anti-TSHR total IgG levels by ELISA. (D–I) DR3tg mice were injected subcutaneously in the flank region with ATX-GD-59 (n = 11) or control peptide (n = 11) according to the dose escalation schedule, with a 22.5-nmol top dose for each peptide, followed by intramuscular injection of 1010 Ad-TSHR or Ad-LacZ viral particles (n = 7) on week 0 and week 3. Blood was collected (D) before and (E) 2 and (F) 5 weeks after the first immunization to measure anti-TSHR total IgG levels by ELISA. Blood samples of week 2 were used to measure anti-TSHR (G) IgG1, (H) IgG2b, and (I) IgG2c antibodies. Each dot represents data from one mouse, and the group mean 6 SEM is indicated. One-way ANOVA was used to measure overall differences in anti-TSHR IgG levels. Bonferroni post hoc testing was used; significant differences are indicated in the graphs (*P , 0.05; **P , 0.01; ***P , 0.001; ****P , 0.0001).

Article Snippet: After 48 hours, peptide-induced IL-2 production by the antigenspecific T-cell hybridoma clone was measured by ELISA (mouse IL-2 ELISA kit, R&D Systems).

Techniques: Injection, Control, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Influence of hydrocarbon oil structure on adjuvanticity and autoimmunity

doi: 10.1038/s41598-017-15096-z

Figure Lengend Snippet: Effect of adjuvants on ovalbumin-specific recall response and antibody production. ( a – d ) DA rats were injected with ovalbumin together with pristane, pentadecane, phytol, hexadecane, heptadecane, squalane and squalene (n = 8 per group). Draining lymph nodes were harvested 10 days after immunization and restimulated in vitro with ovalbumin. Level of cytokines in the supernatant was determined as a measure of T cell recall response. ( a ) IL-2 was measured after 20 hours, and ( b ) IFN-γ and ( c ) IL-17 were measured after 88 hours of restimulation with ovalbumin. The stimulation index was determined by dividing the level of cytokines produced by the cells cultured in medium with ovalbumin, by the level of cytokines produced by the cells that were cultured in medium without ovalbumin. ( d ) Levels of ovalbumin-specific antibodies IgG2b in the serum was determined by ELISA and shown as arbitrary unit (AU). ( e ) Levels of ovalbumin-specific antibodies IgG2b in the serum in rats injected with ovalbumin together with squalane and squalane, with and without αβ T cell depletion (n = 5–6 per group). Data are shown as mean ± SEM. Statistics were determined with the Mann-Whitney U test. *Denotes p < 0.05, **denotes p < 0.01, ***denotes p < 0.001.

Article Snippet: The level of IL-2 and IL-17 were determined using the Rat IL-2 Duoset ELISA Assay (DY502, R&D Systems) and the Mouse IL-17 DuoSet ELISA Assay (DY421, R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Injection, In Vitro, Produced, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal:

Article Title: Induction of T Helper Type 1 Responses by a Polysaccharide Deacetylase from Cryptococcus neoformans

doi: 10.1128/IAI.71.9.5412-5417.2003

Figure Lengend Snippet: rd25-induced IFN-γ and IL-2 production in cultures of splenocytes from rd25-immunized or C. neoformans-infected mice. Spleens were collected at 7, 14, and 28 days after immunization or infection (see the legend to Fig. ​Fig.1),1), and splenocytes (5 × 106/ml) were stimulated with CneF (10 μg/ml), rd25 (10 μg/ml), or PBS. Cytokine levels were assayed at 24 h (IL-2) or 48 h (IFN-γ) after the addition of the stimuli. Data are expressed as means ± SEM for three experiments, each performed in triplicate. Asterisk, P < 0.05, relative to results with cells from PBS-CFA-injected mice by one-way analysis of variance and the Student-Keuls-Newman test.

Article Snippet: To assess the role of IL-2, groups of d25-immunized and nonimmunized mice were treated with polyclonal goat anti-mouse IL-2 IgG (R&D Systems) or normal goat IgG and challenged with C. neoformans (7.5 × 10 3 CFU).

Techniques: Infection, Injection

Journal:

Article Title: Induction of T Helper Type 1 Responses by a Polysaccharide Deacetylase from Cryptococcus neoformans

doi: 10.1128/IAI.71.9.5412-5417.2003

Figure Lengend Snippet: rd25-induced IFN-γ and IL-2 production in cultures of CD4+ or CD8+ cells from C. neoformans-infected mice. Spleens were collected at 14 days after infection, and CD4+ or CD8+ T cells (5 × 106/ml), separated by means of a panning procedure (16), were cultured in the presence of splenic adherent cells (5 × 105/ml) and CneF (10 μg/ml), d25 (10 μg/ml), or PBS. Cytokine levels were assayed as indicated in the legend to Fig. ​Fig.2.2. Data are expressed as means ± SEM for three experiments, each performed in triplicate. Asterisk, P < 0.05, relative to results with cells from PBS-CFA-injected mice by one-way analysis of variance and the Student-Keuls-Newman test.

Article Snippet: To assess the role of IL-2, groups of d25-immunized and nonimmunized mice were treated with polyclonal goat anti-mouse IL-2 IgG (R&D Systems) or normal goat IgG and challenged with C. neoformans (7.5 × 10 3 CFU).

Techniques: Infection, Cell Culture, Injection

Journal:

Article Title: Induction of T Helper Type 1 Responses by a Polysaccharide Deacetylase from Cryptococcus neoformans

doi: 10.1128/IAI.71.9.5412-5417.2003

Figure Lengend Snippet: Role of IL-12 and TNF-α in the development of cytokine responses to d25. Spleens were collected at 7 and 14 days after immunization with rd25 (100 μg per mouse in CFA), and cultures of splenocytes (5 × 106/ml) were stimulated with rd25 (10 μg/ml). Cytokine levels were assayed at 24 h (IL-2) or 48 h (IFN-γ) after the addition of the stimulus. Experimental groups included immunized mice that were injected i.p. with 0.2 mg of goat polyclonal anti-mouse TNF-α IgG, goat anti-IL-12 IgG, or normal goat IgG at 1 h prior to infection. Data are expressed as means ± SEM for three experiments, each performed in triplicate. Asterisk, significantly (P < 0.05) different from normal IgG control group by one-way analysis of variance and the Student-Keuls-Newman test.

Article Snippet: To assess the role of IL-2, groups of d25-immunized and nonimmunized mice were treated with polyclonal goat anti-mouse IL-2 IgG (R&D Systems) or normal goat IgG and challenged with C. neoformans (7.5 × 10 3 CFU).

Techniques: Injection, Infection

Journal:

Article Title: Induction of T Helper Type 1 Responses by a Polysaccharide Deacetylase from Cryptococcus neoformans

doi: 10.1128/IAI.71.9.5412-5417.2003

Figure Lengend Snippet: Role of IL-2 and IFN-γ in the protective effects of d25 immunization. Panels A and B show the effects of, respectively, IL-2 blockade and IFN-γ deficiency on C. neoformans-induced lethality in d25-immunized and unimmunized mice. (A) Eight-week-old female BALB/c mice were immunized s.c. with rd25 (100 μg per mouse in CFA) or injected with PBS-CFA. Mice belonging to each of these two subgroups were injected i.p. at the time of immunization and 15 days later with 0.5 mg of goat polyclonal anti-mouse IL-2 IgG or normal goat IgG. All mice were challenged i.v. with 7.5 × 103 viable C. neoformans cells (strain H99) at 1 week after immunization, and lethality was observed daily for 30 days. (B) Eight-week-old female IFN-γ−/− mice on a C57BL/6 background and control WT C57BL/6 mice were immunized with d25 in CFA or injected with PBS-CFA as described above. After 1 week, mice were challenged i.v. with 3 × 104 viable C. neoformans cells (strain H99), and lethality was observed daily for 30 days. Survival data were analyzed with Kaplan-Meier survival plots followed by the log rank test (JMP Software; SAS Institute, Cary, N.C.) on an Apple Macintosh computer.

Article Snippet: To assess the role of IL-2, groups of d25-immunized and nonimmunized mice were treated with polyclonal goat anti-mouse IL-2 IgG (R&D Systems) or normal goat IgG and challenged with C. neoformans (7.5 × 10 3 CFU).

Techniques: Injection, Software

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

Article Title: Anaphylatoxin C5a induces monocyte recruitment and differentiation into dendritic cells by TNF-alpha and prostaglandin E2-dependent mechanisms.

doi: 10.4049/jimmunol.171.5.2631

Figure Lengend Snippet: FIGURE 7. IL-12 and TNF- release from DC ex vivo. Human mono- cytes (2 107) were injected i.v. into SCID mice together with C5a or MIP-1 (10 g each) i.p. Twenty-four hours later, the injection of C5a or MIP-1 was repeated. Another 24 h later, peritoneal cells were harvested and counted in a hemocytometer, and the percentage of HLA-DR human cells was determined by flow cytometry. Absolute numbers of migrated HLA-DR cells were calculated from the percentage of red fluorescent cells and the total peritoneal cell count. HLA-DR cells were cultured at 2 106/ml for 48 h. A, Supernatants of cultured cells were tested by ELISA for IL-12 p40 and TNF- release (n 7). B, IL-12 p70 was mea- sured after culturing cells from C5a-treated mice for 48 h in the absence (n 7) or the presence (n 4) of CD40L/enhancer. The mean SEM are shown. C, CD83 expression on human cells that were obtained from C5a- treated mice and cultured in the absence or the presence of CD40L/en- hancer was analyzed by flow cytometry.

Article Snippet: Secretion of human and murine TNF- , murine IL-1 , murine IL-6, PGE2, human IL-12 p40 (all from R&D Systems, Wiesbaden-Nordenstadt, Germany), and human IL-12 p70 (BD PharMingen, Heidelberg, Germany) in serum or supernatant was measured by ELISA according to the manufacturer’s instructions.

Techniques: Ex Vivo, Injection, Cytometry, Cell Counting, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Nature Immunology

Article Title: IL-23 stabilizes an effector T reg cell program in the tumor microenvironment

doi: 10.1038/s41590-024-01755-7

Figure Lengend Snippet: (a ) Gating strategy to FACS sort γδ T cells and Treg cells from LNs of Foxp3 Cre-YFP Il23r fl/fl and Il23r fl/fl mice for qPCR and example plot showing purity of sorted Treg cells. (b) Bar graphs depicting relative mRNA expression level of Il23r normalized to pol.2 in FACS sorted γδ T cells and Treg cells from steady state LNs of Foxp3 Cre-YFP Il23r fl/fl , Il23r fl/fl and Il23r del/del mice. Data shown from one representative experiment out of two independent experiments with n = 5 (Foxp3 Cre-YFP Il23r fl/fl and IL23R fl/fl mice) or n = 3 (Il23r del/del ) biologically independent samples. Data are displayed as mean +/- SEM. Statistical significance was determined using t-tests. (c) Il23r fl/fl and Foxp3 Cre-YFP Il23r fl/fl mice were inoculated i.d. with B16 tumor cells, inoculated s.c. with YUMMER1.7 tumor cells or inoculated s.c. with MC38 tumor cells. Tumors were analyzed on day 15, 14 or 24 post-inoculation. Pictures depict tumors after harvest. Data from 3 independent experiments with n = 6-10. (d) Kinetics of tumor volume measured by caliper gauge in B16 tumor cell-inoculated i.d. Foxp3 Cre-YFP and Foxp3 Cre-YFP Il23r fl/fl mice. Mean +/- SEM is displayed. Statistical significance was determined using 2-way Anovas. n = 4 biologically independent animals. (e) Kinetics of tumor volume measured by caliper gauge in MC38 tumor cell-inoculated s.c. C57Bl/6 mice i.p. injected either with anti-p19 blocking or isotype antibodies. n = 6 (isotype) and n = 7 (anti p19 antibody) biologically independent animals. Mean +/− SEM is displayed. Statistical significance was determined using 2-way Anovas.

Article Snippet: The mice were randomized to respective treatment groups on day 6 after inoculation and received a total of three injections of 100 μg of anti-p19 (clone G23-8, BioXcell) or isotype control (IgG1, clone MOPC-21, BioXcell) every 72 h intraperitoneally.

Techniques: Expressing, Injection, Blocking Assay

Journal: Nature Immunology

Article Title: IL-23 stabilizes an effector T reg cell program in the tumor microenvironment

doi: 10.1038/s41590-024-01755-7

Figure Lengend Snippet: (a-f) Il23r fl/fl and Foxp3 Cre-YFP Il23r fl/fl mice were inoculated s.c. with MC38 tumor cells (a-c) or inoculated s.c. with YUMMER1.7 tumor cells (d-f) and tumor-infiltrating T cells were analyzed by flow cytometry on day 24 (a-c) or 14 (d-f) post-inoculation. TILs from MC38 tumors were re-stimulated with PMA/Ionomycin prior flow cytometry analysis. Data display 2 independent experiments with n = 6-10. (a, d) UMAP with overlaid FlowSOM clustering (left) (gated on CD45 + TCRβ + and TCRγδ + cells) and heatmap depicting relative marker expression among identified cell clusters (right). (b, e) Violin plots depicting cell numbers of identified T cell clusters per gram tumor. Data are displayed as mean +/- SEM. Statistical significance was determined using two-tailed Mann-Whitney U-tests. (c, f) Dotplots displaying median marker expression in identified T cell clusters comparing Foxp3 Cre-YFP Il23R fl/fl and Il23R fl/fl mice. Color represents log(median expression Foxp3 Cre-YFP Il23R fl/fl / median expression Il23R fl/fl ); that is red means that median expression is decreased in Foxp3 Cre-YFP Il23R fl/fl in comparison to Il23R fl/fl mice; green means that median expression is increased in Foxp3 Cre-YFP Il23R fl/fl mice in comparison to Il23R fl/fl mice. Circle size represent log(p value). Statistically significant changes (p < 0.05) are highlighted with black lines around the circles. Statistical significance was determined using t-tests. (g) C57Bl/6 mice were s.c. inoculated with MC38 tumor cells and tumor-infiltraring T cells were analyzed by flow cytometry on day 14 post-inoculation. Dotplot displaying median marker expression of CD4 + T cell and Treg cell clusters comparing anti-p19 and isotype antibody treated mice. Color represents log(median expression anti-p19/median expression isotype). Circle size represent log(p value). Statistically significant changes (p < 0.05) are highlighted with black lines around the circles. Data display one out of 2 independent experiments with n = 4-7. Statistical significance was determined using t-tests.

Article Snippet: The mice were randomized to respective treatment groups on day 6 after inoculation and received a total of three injections of 100 μg of anti-p19 (clone G23-8, BioXcell) or isotype control (IgG1, clone MOPC-21, BioXcell) every 72 h intraperitoneally.

Techniques: Flow Cytometry, Marker, Expressing, Two Tailed Test, MANN-WHITNEY, Comparison