B cell–Derived IL35 Drives STAT3-Dependent CD8⁺ T-cell Exclusion in Pancreatic Cancer

STAT3 activation in CD8⁺ T cells is suppressive in vitro and in vivo. A, Representative flow cytometry plots and histograms of IFNγ production and expression of CXCR3, CCR5, phospho(p)-STAT1, pSTAT3, and pSTAT4 in CD3⁺CD8⁺ T cells stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (2 μg/mL) in the presence of OVA (2 μg/mL), rIL35 (50 ng/mL), and/or STA-21 (20 μmol/L) inhibitor. Proportion of CD8⁺ T cells is indicated. B, Quantification of IFNγ production and expression of CXCR3, CCR5, pSTAT1, pSTAT3, and pSTAT4 in CD3⁺CD8⁺ T cells from A. C, Experimental schema used to test the role of Stat3 activation in CD8⁺ T cells. D, Quantification of tumor weight from CD45.1⁺ mice 3 weeks after orthotopic adoptive transfer of tumor-educated CD45.2⁺CD8⁺ T cells pretreated with anti-CD3/CD28 with and without STA-21 CD45.2⁺CD8⁺ T cells and injection with KPC cells (n = 6 mice/group). E, Numbers (no.) of adoptively transferred CD45.2⁺ (left) and endogenous CD45.1⁺ (right) tumor-infiltrating CD3⁺CD8⁺ T cells 3 weeks after orthotopic adoptive transfer to the CD45.1⁺ recipients indicated in D. F, Representative flow cytometry histograms (left) and quantification (right) of pSTAT1, pSTAT3, and pSTAT4 in intratumoral CD45.2⁺CD8⁺ T cells 3 weeks after orthotopic adoptive transfer to CD45.1⁺ recipients indicated in D. G, Representative flow cytometry plots and histograms (left) and quantification (right) of IFNγ production and expression of CXCR3 and CCR5 in intratumoral CD45.2⁺CD8⁺ T cells 3 weeks after orthotopic adoptive transfer to CD45.1⁺ recipients indicated in D. Error bars, SEM; P values were calculated using Student t test (unpaired, two-tailed); NS, not significant. **, P < 0.01; ***, P < 0.001. Data represent three independent experiments.

B cell–derived IL35 promotes tumor growth and mediates suppression of IFNγ and chemotactic receptors CXCR3 and CCR5 in CD8⁺ T cells. A, Experimental schema used to generate tumor-bearing mixed bone marrow chimeras containing B cell–specific deletion of p35 (B^p35−/−) and corresponding control B^WT mice. B, Quantification of tumor weight from B^WT and B^p35−/− mice 3 weeks post-orthotopic injection with KPC4662 cells (n = 6 mice/group). C, Representative flow cytometry histograms (left) and quantification (right) of intracellular p35 expression in intratumoral and intrasplenic CD19⁺CD21^hiCD5⁺CD1d^hi Bregs from mice in B. Proportion of p35⁺ Bregs is indicated. D, Representative flow cytometry histograms (left) and quantification (right) of intratumoral CD45⁺CD3⁺CD4⁺CD25⁻ effector T cells in B^WT and B^p35−/− mice from B. E, Representative flow cytometry plots (left) and quantification (right) of intracellular IFNγ (isotype; rat IgG1, κ) and TNFα (isotype; rat IgG1, κ) expression by CD3⁺CD4⁺ intratumoral T cells from mice in B. Proportion of CD4⁺ T cells is indicated. F, Representative flow cytometry histograms (left) and quantification (right) of intratumoral CD3⁺CD4⁺Foxp3⁺ Tregs from mice in B. Proportion of CD4⁺ T cells is indicated. G, Representative flow cytometry histograms (left) and quantification (right) of intracellular p35 (isotype; rat IgG2a, ĸ) and IL10 (isotype; rat IgG2b, κ) expression by intratumoral CD3⁺CD4⁺ T cells from mice in B. H, Quantification of frequency of tumor-infiltrating CD45⁺CD3⁺CD8⁺ T cells from B^WT and B^p35−/− mice from B determined by flow cytometry. I, Representative flow cytometry plots (left) and quantification (right) of IFNγ (isotype; rat IgG1, κ) in intratumoral CD45⁺CD3⁺CD8⁺ T cells from mice in B. J, Ratio of mean CD3⁺CD4⁺CD25⁻ effector T cells (Teff) to Tregs (left) and ratio of mean CD3⁺CD8⁺ cytotoxic T cells to Tregs (right) were calculated on the basis of the percent-positive lymphocyte population determined by flow cytometry. Error bars, SEM; P values were calculated using Student t test (unpaired, two-tailed); NS, not significant. **, P < 0.01; ***, P < 0.001. Data represent three independent experiments.

Production of IL35 by B cells impedes anti–PD-1 therapy. A, Experimental schema used to generate tumor-bearing mice that contain B cell–specific deletion of Ebi3 (B^Ebi3−/−) and corresponding control B^Ebi3+/− mice. B, Quantification of tumor weight from anti–PD-1–treated (200 μg) and IgG-treated (200 μg) B^Ebi3+/− and B^Ebi3−/− mice 3 weeks after orthotopic injection with KPC cells (n = 6 mice/group). C, Quantification of tumor growth by ultrasound from B^Ebi3+/− and B^Ebi3−/− mice from B. D, Quantification of frequency of tumor-infiltrating CD45⁺CD3⁺CD8⁺ T cells from B^Ebi3+/− and B^Ebi3−/− mice from B determined by flow cytometry. E, Representative flow cytometry plots of intratumoral CD45⁺CD3⁺CD8⁺ T cells stained for IFNγ (isotype; rat IgG1, κ) from mice in B. F, Quantification of intratumoral CD8⁺IFNγ⁺ T cells from all mice in B. G, Representative flow cytometry histograms (left) and quantification (right) of expression of CXCR3, CCR5, phospho(p)-STAT1, pSTAT3, and pSTAT4 in intratumoral CD3⁺CD8⁺ T cells isolated from B^Ebi3+/− and B^Ebi3−/− mice from B. H, Representative flow cytometry plots and histograms (left) and quantification (right) of IFNγ production and expression of CXCR3, CCR5, pSTAT1, pSTAT3, and pSTAT4 in CD3⁺CD8⁺ T cells 48 hours after coculture with Bregs in 1:1 ratio derived from the indicated mice. Experiment were done in triplicate with 6 mice per group. Error bars, SEM; P values were calculated using Student t test (unpaired, two-tailed); NS, not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data represent three independent experiments.

IL35 blockade relieves immunosuppression of CD8⁺ T cells and synergizes with anti–PD-1. A, Schematic of the antibody treatment regimen. Anti-IL35 (200 μg first dose, followed by 100 μg/week) or control IgG antibody was administered in therapeutic schedule (1 week after tumor cell injection on days 7, 11, and 15). Administration of anti–PD-1 (200 μg) was initiated on day 7 after tumors reached approximately 4 to 5 mm in diameter. Two more doses of anti–PD-1 were administered on days 9 and 11. Mice were sacrificed 3 weeks after tumor cell injection or assessed for survival. B, Quantification of tumor weight from WT mice treated with therapeutic anti-IL35, anti–PD-1, or combination (as in A) 3 weeks after orthotopic injection with KPC cells (n = 6 mice/group). C, Quantification of tumor growth by ultrasound from WT mice in B. D, Survival plot of orthotopically injected WT mice from B treated with the indicated therapeutic antibodies. E, Quantification of tumor growth by ultrasound from spontaneous KPC mice treated with therapeutic anti-IL35, anti–PD-1, or combination as in A. Treatment was initiated when tumor measuring approximately 5 mm was visualized by ultrasound (n = 6 mice/group). Data represent three independent experiments. F, Quantification of frequency of orthotopic tumor-infiltrating CD45⁺CD3⁺CD8⁺ T cells from mice in B. G, Representative flow cytometry plots of intracellular IFNγ in intratumoral CD45⁺CD3⁺CD8⁺ T cells from the mice in B. Proportion of CD8⁺ T cells is indicated. H, Quantification of intratumoral CD8⁺IFNγ⁺ T cells from the mice in B. I, Quantification of CXCR3, CCR5, pSTAT3, and pSTAT4 expression in intratumoral CD3⁺CD8⁺ T cells from mice in B. Error bars, SEM; P values were calculated using Student t test (unpaired, two-tailed); NS, not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data represent three independent experiments.