Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: 4-1BBL + B cells in GBM patients’ peripheral blood and Ag-presenting function. (A) Box plot showing the expression of 4-1BBL in CD20 + CD19 + B cells from newly diagnosed GBM patients’ PBMCs ( n = 90). Box plots express the following values: median (line inside the box), 75th and 25th percentiles (box extremities), and maximum and minimum values (dashes). (B) Histograms representing the intracellular expression of TNFα and IFNγ and surface expression of CD69 and CD86 by 4-1BBL − (black line) and 4-1BBL + (blue line) B cells. (C) Linear regression analysis of 4-1BBL expression by B cells and CD69 expression by CD8 + T cells in newly diagnosed GBM patients’ PBMCs ( n = 68). (D and E) CD8 + T cell costimulation assay using CD8 + T cells activated with anti-CD3 and IL-2 mixed with autologous 4-1BBL − and 4-1BBL + B cells from peripheral blood of three newly diagnosed GBM patients (NU00856, NU01006, and NU00429). CD8 + T cell activation was measured as cellular expansion (D) and expression of intracellular IFNγ and GzmB (E). The experiment was performed in triplicate. (F) B Vax from GL261-OVA mice were pulsed with SIINFEKL (B Vax(SIINFEKL) ) and evaluated for SIINFEKL presentation by MHC class I (H-2K b + SIINFEKL Ab) and the coexpression of MHC class I (H-2K b ) and costimulatory molecules CD86 and 4-1BBL. A representative experiment of three independent experiments is shown. (G and H) B Vax were tested for their ability to uptake Alexa Fluor 488–conjugated OVA (B Vax[OVA] ; G) and present SIINFEKL peptide via MHC class I (H-2K b ; H). Surface transport of the H-2K b + SIINFEKL complex was inhibited using brefeldin A (BFA). A representative experiment of three independent experiments is shown. Scale bars represent 15 µm (fluorescence images) and 50 µm (fluorescence and bright light image). (I) B Naive , B Naive + IFNγ, B Vax , and DCs were pulsed with OVA and tested for their ability to promote OT-I CD8 + T cell activation assessed by cell proliferation (expansion index, x axis) and intracellular expression of GzmB (y axis). The experiment was performed in triplicate. A representative experiment of two independent experiments is shown. (J) OT-I CD8 + T cells cultured with B Naive ; B Vax pulsed with OVA and isotype control, B Vax(OVA) + IC; or MHC class I blocking Ab, B Vax(OVA) + anti-H2K b , and tested for their cellular expansion. A representative experiment of two independent experiments is shown. Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. Statistical significance is depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not statistically significant.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: Expressing, Activation Assay, Fluorescence, Cell Culture, Control, Blocking Assay

Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: 4-1BBL–expressing B cells. (A) 4-1BBL expression levels by CD19 + B cells in the brain, blood, dCLNs, and superficial CLNs of CT2A glioma-bearing mice over time: no tumor (D0), 7, 14, and 21 d after tumor implantation ( n = 4 mice/time point). (B) Murine B cell ability to promote CD8 + T cell activation, measured by cell expansion and expression of intracellular GzmB, was assessed by activated B cells and compared with B cells from 4-1BBL–deficient (4-1BBL KO) mice. B cells were incubated with BAFF, anti-IgM, anti-CD40, and/or recombinant mouse (rm)IFNγ. Representative experiment of a total of three independent experiments performed in triplicate. Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. Histograms are shown as mean ± SD. Statistical significance is depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not statistically significant.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: Expressing, Tumor Implantation, Activation Assay, Incubation, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: B Vax generation. (A) Human B cells from PBMCs were treated with 10 U/ml IFNγ for 24 h. CD86 expression was assessed by flow cytometry. Representative histogram of three independent experiments. (B) Murine B cells from WT C57BL/6 or IFNγR–deficient (IFNγR KO) mice were incubated with 5 μg/ml CD40 activating Ab ± 10 U/ml IFNγ. Expression of CD86 was assessed by flow cytometry. Representative histogram of four independent experiments. (C) Stepwise schema of B Vax generation in vitro. B Vax were produced from 4-1BBL + B cells isolated from tumor-bearing SLOs such as the spleen and deep and superficial CLNs. Cells were then activated with 5 μg/ml CD40 activating Ab and supplemented with 100 nM of B cell survival factor BAFF, which after 24 h allows the up-regulation of CD86, H-2K b , and IFNγ receptor 1 (IFNγRI) compared with only BAFF-treated B cells. The addition of IFNγ for an additional 24 h allowed further up-regulation of CD86 compared with only anti-CD40–treated B cells. Histograms represent mean ± SD of n = 3 mice/treatment. gMFI, geometric mean fluorescence intensity. (D) B Vax overexpress both IA b (MHC class II) and H-2K b compared with only BAFF-treated B cells (B Naive ). Flow cytometry dot plot representative of n = 4 mice/group. (E) B cell subsets (B Naive ± IFNγ and B Vax ) and DCs were tested for their ability to present SIINFEKL peptide to OT-I CD8 + T cells measured by cell proliferation and expression of intracellular GzmB. Representative experiment of three independent experiments performed in triplicate. (F) Same experiment as in E was performed using in addition CD8 + T cells from WT C57BL/6 mice as negative control. Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. Histograms are shown as mean ± SD. Statistical significance is depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not statistically significant.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: Expressing, Flow Cytometry, Incubation, In Vitro, Produced, Isolation, Fluorescence, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: B Vax APC function in vivo. (A) Rag1-deficient mice were challenged intracranially with GL261-OVA. 9 d after, mice received i.v. B Naive or B Vax pulsed with OVA protein. eFluor450-labeled CD8 + T cells from WT C57BL/6 mice were concomitantly injected with B cells ( n = 4 mice/group). 7 d after the cell adoptive transfer, eFluor450 + CD8 + T cells were evaluated by flow cytometry in the tumor-bearing brains and the dCLNs. A representative experiment of two independent experiments is shown. (B) B KO mice were challenged intracranially with GL261-OVA. 9 d after, mice received i.v. B Naive , 4-1BBL − B cells activated with CD40 agonism and IFNγ (B Act ) or B Vax pulsed with OVA protein. A group of B Vax(OVA) mice was pretreated with PTX before injection ( n = 4 mice/group). 7 d after the cell adoptive transfer, SIINFEKL-specific CD8 + T cells were analyzed in the tumor-bearing brains by flow cytometry using SIINFEKL-tetramer. A representative experiment of two independent experiments is shown. (C) B KO mice were challenged intracranially with CT2A cells. 9 d after, mice received i.v. B Naive and B Vax pulsed with CT2A tumor lysates pretreated with or without PTX ( n = 5 mice/group). 7 d after the cell adoptive transfer, CD8 + T cells were evaluated for the intracellular expression of GzmB and IFNγ in the tumor-bearing brain, blood, and dCLNs. A representative experiment of three independent experiments is shown. (D) Rag1-deficient (KO) mice were challenged intracranially with CT2A cells. 9 d after, mice i.v. and concomitantly received both CellTracker red CMPTX B Vax (red) cells and CellTracker green CMFDA-labeled CD8 + T cells (green). B Vax and CD8 + T cell splenic localization was analyzed by fluorescent microscopy. Scale bars represent 100 µm (left image, 20× magnification) and 50 µm (right image, 63× magnification). Images are representative of the spleen and CLN of three mice. For all experiments shown in this figure, differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. Histograms are shown as mean ± SD. Statistical significance is depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not statistically significant.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: In Vivo, Labeling, Injection, Adoptive Transfer Assay, Flow Cytometry, Expressing, Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: 4-1BBL is a key marker for B Vax therapeutic effect. (A) In B cell–deficient mice, B Naive and B Vax were detected in draining dCLNs and the circulation, and B Vax were found in the tumor-bearing brains. Histogram represents mean ± SD of n = 5 mice/group. (B) CD45.1 + B Naive and B Vax were intracranially injected using cannula-guided injections 10 d after tumor implantation. 4 d after, CD45.1 + cells were magnetically isolated from tumors and tested for their ability to activate CD8 + T cells, measured by cell proliferation (expansion index) by flow cytometry. Histogram represents mean ± SD of n = 3 mice/group. (C) B Naive , B Act , and B Vax were tested for their therapeutic effect in CT2A-bearing B cell–deficient (B KO) mice ( n = 9 or 10 mice/group). (D) CT2A-bearing B cell–deficient mice treated with B Vax (pretreated with 4-1BBL–blocking Ab before injection) ± 4-1BBL–blocking Ab (500 μg/mouse × three i.p. injections after B Vax adoptive transfer) were monitored for survival ( n = 10 mice/group). Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. Survival curves were generated via the Kaplan-Meier method and compared by log-rank test, and multiple comparisons were adjusted using Bonferroni method. Statistical significance is depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not statistically significant.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: Marker, Injection, Tumor Implantation, Isolation, Flow Cytometry, Blocking Assay, Adoptive Transfer Assay, Generated

Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: Radiotherapy favors B cell adaption in vivo. (A) CT2A-bearing mice were irradiated (RT), and CD45.1 + B Vax were adoptively transferred i.v. CD45.1 + cells were analyzed by flow cytometry in the spleens, dCLNs, and tumor-bearing brains ( n = 4 mice/group). (B) Irradiated CT2A-bearing mice were bled 36 h and 5 d after RTx ( n = 3 mice/group). Serum BAFF levels were analyzed and compared with nonirradiated (Tumor) and non–tumor-bearing mice serum (No tumor). (C) The same experiment as in A was performed using CD45.1 + B Naive , B Act (4-1BBL − B cells activated with anti-CD40 and IFNγ), or B Vax . CD45.1 + cells were analyzed by flow cytometry in the spleen and dCLN. B cell proliferative status was assessed by the expression of Ki67. A representative animal for each group ( n = 3 or 4 mice/group) is depicted. The experiment was repeated twice independently. (D) Alternatively, a group of mice received B cells pretreated with BAFF-R–blocking Ab. The treatment was also administered to the mice i.v. for 3 consecutive d after B cell transfer. (E) Irradiated CT2A-bearing mice received i.v. vehicle (Mock, black line), CD8 + T cells (gray line), pulsed B Vax (black dashed line), or combined pulsed B Vax + CD8 + T cells (pink dotted line). The experiment was performed using n = 10 mice/group. A representative experiment of three independent experiments is shown. (F) Irradiated CT2A-bearing mice received i.v. vehicle (Mock, black line), CD8 + T cells (gray line), CD8 + T cells i.v. + pulsed DC Vax administered either i.d. (DC Vax(i.d.) ; dashed black line) or i.v. (DC Vax(i.v.) ; blue line), or pulsed B Vax + CD8 + T cells (pink dotted line). The experiment was performed using n = 10 mice/group. A representative experiment of two independent experiments is shown. (G) Irradiated CT2A-bearing mice received i.v. eFluor450-labeled B Vax or DCs. DCs were administered either i.v. (DC (i.v.) ) or i.d. (DC (i.d.) ). 7 d after cell transfer, eFluor450 + cells were analyzed in the dCLN and spleen ( n = 3 or 4 mice/group). A representative experiment of two independent experiments is shown. Differences between two groups were analyzed by Student’s t test. Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. Histograms are shown as mean ± SD. Survival curves were generated via the Kaplan-Meier method and compared by log-rank test, and multiple comparisons were adjusted using the Bonferroni method. Statistical significance is depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not statistically significant.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: In Vivo, Irradiation, Flow Cytometry, Expressing, Blocking Assay, Labeling, Generated

Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: B Vax treatment confers tumor eradication. (A) PD-L1 membrane expression was evaluated in B Vax and B Naive cells. Representative dot plot of four independent experiments. Histograms are shown as mean ± SD. (B) CD8 + T cell infiltration in CT2A tumor in control group (14 d after tumor inoculation, n = 2). Scale bar represents 250 μm. Number of CD8 + T cells per section was assessed in the tumor area (tumor) versus nontumor (brain) area. (C) Immune cell infiltration in LTSs treated with either B Vax and CD8 + T cells (LTS-B Vax +CD8, n = 5) or only CD8 + T cells (LTS-CD8, n = 4) were compared with control CT2A-bearing (Control, n = 3) and Mock brains (No tumor, n = 2). Representative dot plot showing lymphocyte and myeloid cell compartment distribution (CD45 and CD11b expression) and CD8 + and CD4 + T cell distribution. Within the CD4 + T cell compartment, the expression of CD44 and Foxp3 was evaluated. Within the non–T cell compartment (CD8 − CD4 − lymphocytes), the CD19 + B cell compartment–expressing IFNγ and 4-1BBL was evaluated. Values of the populations of interest are shown as mean ± SD. Differences between two groups were analyzed by Student’s t test. Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. GBM patient–derived B Vax promoted anti-tumor CD8 + T cells. (D) Schema of generation of GBM patient–derived B Vax . CD8 + T cell activation and expansion and CD8 + T cell–mediated tumor cell killing assay was performed in autologous settings. (E) Freshly resected tumors from GBM patients were cultured ex vivo as tumor spheroids. Representative picture of adherent cells after 5 d of culture. Scale bars represent 100 μm. SSC, side scatter.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: Membrane, Expressing, Control, Derivative Assay, Activation Assay, Cell Culture, Ex Vivo

Journal: eLife

Article Title: Cadherins regulate nuclear topography and function of developing ocular motor circuitry

doi: 10.7554/elife.56725

Figure Lengend Snippet: Figure 6. Extraocular muscle innervation and neuromuscular junction formation is unperturbed in cdh2DEC larvae. (A–B) Immunostained axon projections of oculomotor and trochlear nerves (a-tubulin) innervating extraocular muscles (myosin heavy chain) at 72 hpf in wild types and cdh2DEC- mCherry. Images are lateral view maximum intensity projections, white arrow indicates the point at which the oculomotor nerve enters the orbit. Boxes show high magnification view of individual branches. Scale bars = 40 mm. (C) Quantification of branch number per genotype for each nerve/branch. There was no significant difference in branch number for any muscle target (ns; SR, p=0.9406; MR, p=0.9451; IR, p=0.8512; IO, p=0.9451; SO, p=0.94512; multiple t-tests, and Holm-Sidak test). Error bars are mean and SD. Both wild-type and cdh2DEC larvae are from three clutches. (D–G’’) Immunostained NMJs composed of presynaptic vesicles (SV2) and acetylcholine receptors (a-bungarotoxin) in wild types (D–E’’) and cdh2DEC (F–G’’) at 4 dpf at the medial rectus (MR) muscle. Scale bars = 20 mm. All images are maximum intensity projections of sub-stacks taken from larger z-stacks. See also Figure 6—figure supplement 1. The online version of this article includes the following source data and figure supplement(s) for figure 6:

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Danio rerio) Tg(Isl1:GFP) Higashijima et al., 2000 RRID:ZFIN_ZDBGENO-070810-2 Genetic reagent (Danio rerio) Tg(isl1:cdh2DEC-mCherry)vc25/Tg(Isl1:GFP) Rebman et al., 2016 Antibody anti-GFP (chicken polyclonal) Abcam RRID:AB_300798 1:500 dilution Antibody anti-RFP (rabbit polyclonal) MBL Life Science RRID:AB_591279 1:200 dilution Antibody Anti-acetylated tubulin (rabbit monoclonal) Cell Signalling Technologies RRID:AB_10544694 1:1000 dilution Antibody Anti-myosin heavy chain (mouse monoclonal) Developmental Studies Hybridoma Bank (DSHB), University of Iowa RRID:AB_528356 1:200 dilution Antibody Anti-SV2A (mouse monoclonal) Developmental Studies Hybridoma Bank (DSHB), University of Iowa RRID:AB_2315387 1:200 dilution Chemical compound, drug Tetramethylrhodamine-a-bungarotoxin conjugate Invitrogen RRID:AB_2313931 1:1000 dilution Commercial assay, kit RNAscope Fluorescent Multiplex Reagent kit ACD Bio Cat# 320850 Software, algorithm FIJI/Image J https://github.com/fiji/fiji RRID:SCR_002285 Software, algorithm Imaris 9 Bitplane RRID:SCR_007370 Software, algorithm MATLAB Mathworks RRID:SCR_001622 Software, algorithm JAGS https://cran.r-project.org/web/ packages/rjags/index.html RRID:SCR_017573 Software, algorithm LabView National Instruments RRID:SCR_014325 Software, algorithm Prism7 GraphPad RRID:SCR_002798 Experimental model and transgenic lines Zebrafish were maintained at 28.5 ̊C on a 14/10 hr light/dark cycle.

Techniques: Muscles

Journal: Vaccines

Article Title: 4-1BBL-Armed Oncolytic Herpes Simplex Virus Exerts Antitumor Effects in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/vaccines12121309

Figure Lengend Snippet: Construction and characterization of the oHSV-4-1BBL virus. ( A ) Schematic diagram of the construction of oHSV-4-1BBL. Two copies of ICP34.5 (neurovirulence factor) were replaced with a gene cassette consisting of the CMV promoter, the GFP or mouse 4-1BBL coding sequence, and SV40 Poly A. In addition, ICP47, which enhances MHC I antigen processing and presentation, was deleted to boost tumor-specific immune responses. ( B ) Expression of 4-1BBL after oHSV-4-1BBL infection in different cells (MOI = 1). After 48 h, the infected cells were collected, blocked with CD16/32 antibody, and stained with PE anti-mouse 4-1BBL antibody, and the expression of 4-1BBL was analyzed by flow cytometry. ( C ) Cytopathic effects caused by viral infection. The cells were infected with the designated virus at an MOI of 1, and an image was taken 24 h post infection. ( D ) Viral replication kinetics. The cells were infected with the indicated virus at an MOI of 0.01, and the titer of the virus at each time point was measured to draw the viral replication kinetics. ( E ) Determination of cell viability after viral infection. Cells were infected with the indicated viruses at three MOIs, and cell viability was monitored at different time points using a CCK-8 kit.

Article Snippet: First, the 4-1BBL mouse coding sequence was amplified by PCR from pCMV3-mTNFSF9 (4-1BBL) (Sino Biological, Beijing, China) and inserted into a plasmid with CMV elements.

Techniques: Virus, Sequencing, Expressing, Infection, Staining, Flow Cytometry, CCK-8 Assay

Journal: Vaccines

Article Title: 4-1BBL-Armed Oncolytic Herpes Simplex Virus Exerts Antitumor Effects in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/vaccines12121309

Figure Lengend Snippet: Therapeutic effects of oHSV and oHSV-4-1BBL in pancreatic cancer models. ( A – C ) Pan02_HVEM model ( n = 6). Mice were inoculated with Pan02-HEVM cells on day 0 and treated with PBS, oHSV, or oHSV-4-1BBL on days 7, 10, and 13 (indicated by arrows). Tumor volumes were measured with a caliper every third day. For each group, the average ( A ) and individual ( B ) tumor volume, as well as the survival curve ( C ), are depicted. ( D – F ) KPC model ( n = 6). Mice were inoculated with KPC cells on day 0 and received treatments on days 4, 7, and 10 (indicated by arrows). Tumor volumes were measured with a caliper every third day. For each group, the average ( D ) and individual ( E ) tumor volume, as well as the survival curve ( F ), are documented. ( G – I ) KPC model ( n = 6), which is similar to the experiment demonstrated in ( D – E ), except that mice were euthanized on day 17. The tumor growth curves ( G ), tumor images ( H ), and tumor weight ( I ) on day 17 post treatment are illustrated. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: First, the 4-1BBL mouse coding sequence was amplified by PCR from pCMV3-mTNFSF9 (4-1BBL) (Sino Biological, Beijing, China) and inserted into a plasmid with CMV elements.

Techniques:

Journal: Vaccines

Article Title: 4-1BBL-Armed Oncolytic Herpes Simplex Virus Exerts Antitumor Effects in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/vaccines12121309

Figure Lengend Snippet: oHSV-4-1BBL treatment promotes T-cell activation and cytotoxicity. ( A ) Classification of KPC tumor model-derived CD8+ TIL cells into distinct exhaustion subsets based on the expression levels of PD-1 and LAG-3. ( B ) Expression of the 4-1BB receptor in different exhausted subsets of CD8+ TIL cells ( n = 16). oHSV-4-1BBL treatment promotes T-cell infiltration. KPC tumor-bearing mice treated according to the protocol described above were sacrificed on day 17; tumors were isolated and prepared as a single-cell suspension and stained with viability dye and corresponding antibodies. Intratumoral CD45+ cells ( C ), CD45+ CD3+ T cells ( D ), CD45+ CD4+ T cells ( E ), CD45+ CD8+ T cells ( F ), CD69+ CD4+ T cells ( G ), CD69+ CD8+ T cells ( H ), IFN-γ+ CD4+ T cells ( I ), IFN-γ+ CD8+ T cells ( J ), and GZMB+ CD8+ T cells ( K ) were analyzed with flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: First, the 4-1BBL mouse coding sequence was amplified by PCR from pCMV3-mTNFSF9 (4-1BBL) (Sino Biological, Beijing, China) and inserted into a plasmid with CMV elements.

Techniques: Activation Assay, Derivative Assay, Expressing, Isolation, Suspension, Staining, Flow Cytometry

Journal: Vaccines

Article Title: 4-1BBL-Armed Oncolytic Herpes Simplex Virus Exerts Antitumor Effects in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/vaccines12121309

Figure Lengend Snippet: Changes in the distribution of CD8+ T cells at each stage of exhaustion after treatment. ( A – D ) oHSV-4-1BBL treatment reduces exhaustive T cells and Treg cells. KPC tumor-bearing mice treated according to the protocol described above were sacrificed on day 17; tumors were isolated, and single-cell suspensions were prepared and stained with corresponding antibodies. CD4+ PD-1+ T cells ( A ), CD8+ PD-1+ T cells ( B ), LAG-3+ CD8+ T cells ( C ), and Treg cells ( D ) were analyzed with flow cytometry. ( E – G ) The changes in CD8+ T cells at each stage of exhaustion within the tumor analyzed by two sets of indicators: CD69 and Ly108 ( E , F ) and PD-1 and LAG-3 ( G ). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: First, the 4-1BBL mouse coding sequence was amplified by PCR from pCMV3-mTNFSF9 (4-1BBL) (Sino Biological, Beijing, China) and inserted into a plasmid with CMV elements.

Techniques: Isolation, Staining, Flow Cytometry

Journal: Vaccines

Article Title: 4-1BBL-Armed Oncolytic Herpes Simplex Virus Exerts Antitumor Effects in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/vaccines12121309

Figure Lengend Snippet: Oncolytic virus combined with PD-1 therapy. ( A ). Regimen of PD-1 antibody combination therapy in the Pan02-HEVM tumor model. (PBS+αPD-1: n = 6, oHSV+αPD-1: n = 7, oHSV-4-1BBL+αPD-1, n = 7). Tumor volumes were measured with a caliper every third day. The tumor growth curves of the average ( B ) and individual mice ( C ) for each group are shown. ( D ) Survival curves of Pan02_HVEM tumor-bearing mice. *** p < 0.001.

Article Snippet: First, the 4-1BBL mouse coding sequence was amplified by PCR from pCMV3-mTNFSF9 (4-1BBL) (Sino Biological, Beijing, China) and inserted into a plasmid with CMV elements.

Techniques: Virus