Article Figures & Data
Figures
- Figure 1.
IL35 is upregulated in CD4+ T cells and B cells in PDA. A, Representative flow cytometry plots of CD4+ and CD19+ cells isolated from the spleens of WT or KC mice at 4 months of age. Cells were processed for intracellular staining with anti-p35 and anti-Ebi3. Percentage of p35+Ebi3+CD4+ T cells and p35+Ebi3+CD19+ B cells is indicated. B, Percentage of IL35+ splenic T cells, B cells, or myeloid cells in WT (n = 12) or splenic and intratumoral IL35+ T cells, B cells, or myeloid cells KC mice (n = 12). C, Percentage of IL35+ splenic T cells, B cells, or myeloid cells in WT or splenic and intratumoral IL35+ splenic T cells, B cells, or myeloid cells in mice orthotopically injected with KPC 4662 cells and collected 3 weeks after tumor cell injection. D, Representative flow cytometry plots of intratumoral B conventional (Bcon; CD19+CD21+CD5−CD1d−) and Bregs (CD19+CD21hiCD5+CD1dhi) from KC mice at 4 months of age or mice orthotopically injected with KPC cells and collected 3 weeks after tumor cell injection. Cells were analyzed by staining with anti-p35 antibody. Percentage of p35+ Bcon or Bregs is indicated. E, Representative flow cytometry plots of intratumoral CD4+Foxp3− T cells and CD4+Foxp3+ Tregs from KC mice at 4 months of age. Cells were analyzed by staining with anti-p35 antibody. Percentage of p35+ CD4+Foxp3− or Tregs is indicated. F, Representative flow cytometry plots of intratumoral CD4+Foxp3− T cells and Tregs from mice orthotopically injected with KPC cells and collected 3 weeks after tumor cell injection. Cells were analyzed by staining with anti-p35 antibody. Percentage of p35+ CD4+Foxp3− or Tregs is indicated. error bars, SEM. p-values were calculated using Student t test (unpaired, two-tailed); NS, not significant; *, P < 0.05; ***, P < 0.001. Data represent 3 independent experiments.
- Figure 2.
IL35 regulation of tumor growth is accompanied by suppression of CD4+ effector T-cell activity and expansion of Tregs. A, Representative images of pancreatic tumors from WT, Il10−/−, p35−/−, and Ebi3−/− mice orthotopically injected with KPC 4662 cells and collected at 3 weeks after injection of tumor cells. B, Quantification of tumor weights from WT, Il10−/−, p35−/−, and Ebi3−/− (N = 12; n = 3/group) mice. C, Quantification of tumor weights from WT (n = 9), Il10−/− (n = 9), and p35−/− (n = 9) mice orthotopically injected with KPC 2173 cells and collected 3 weeks after tumor cell injection. D, Representative flow cytometry plots of gating strategy for identifying CD4+ T cells and frequency of intratumoral CD4+CD25− effector T cells isolated from WT, Il10−/−, p35−/−, and Ebi3−/− mice orthotopically injected with KPC 4662 cells and collected 3 weeks after injection of tumor cells. Percentage of CD45+CD4+ cells is indicated. E, Quantification of the frequency of CD45+CD4+ T cells from WT, Il10−/−, p35−/−, and Ebi3−/− (n = 3/group) mice. F, Representative flow-cytometric plots of CD4+IFNγ+ and CD4+TNFα+ T cells isolated from WT, Il10−/−, p35−/−, and Ebi3−/− mice orthotopically injected with KPC 4662 cells and collected 3 weeks after injection of tumor cells. G, Quantification of the frequency of CD4+IFNγ+ T cells (left graph, % of CD4+ T cells) and CD4+TNFα+ T cells (right graph, % of CD4+ T cells) from WT, Il10−/−, p35−/−, and Ebi3−/− (n = 3/group) mice. H, Representative flow-cytometric plots of intratumoral CD4+Foxp3+ Tregs isolated from WT, Il10−/−, p35−/−, and Ebi3−/− mice. I, Quantification of the frequency of CD4+Foxp3+ (% of CD4+ T cells) from WT, Il10−/−, p35−/−, and Ebi3−/− (n = 3/group) mice. J, Mean CD4+ effector T cell to Treg ratio was calculated based on the percentage of 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 < 0.05; **, P < 0.01; ***, P < 0.001. Data represent 3–4 independent experiments.
- Figure 3.
IL27 signaling does not confer immunosuppression in PDA. A, Representative images of pancreatic tumors from WT, Il27ra−/−, and p35−/− mice orthotopically injected with KPC 4662 cells and collected at 3 weeks after injection of tumor cells. B, Quantification of tumor weights from WT, Il27ra−/−, and p35−/− (n = 3/group) mice. C, Representative flow-cytometric plots of intratumoral CD4+IFNγ+, CD4+TNFα+, and CD4+Foxp3+ T cells isolated from WT, Il27ra−/−, and p35−/− mice. D, Quantification of the frequency of intratumoral CD4+IFNγ+ T cells (left graph, % of CD4+ T cells), CD4+TNFα+ T cells (middle graph, % of CD4+ T cells), or CD4+Foxp3+ regulatory T cells (right graph, % of CD4+ T cells) from WT, Il27ra−/−, and p35−/− (n = 3/group) mice. E, Quantification of the frequency of intratumoral CD45+CD8+ T cells from WT, Il27ra−/−, and p35−/− (n = 3/group) mice determined by flow cytometry. F, Representative gating strategy for sorting CD8+ T cells and flow cytometry plots of intratumoral CD8+IFNγ+ T cells isolated from WT, Il27ra−/−, and p35−/− mice. Percentage of CD8+ T cells is indicated. G, Quantification of the frequency of intratumoral CD8+IFNγ+ T cells (% of CD8+ T cells) from WT, Il27ra−/−, and p35−/− (n = 3/group) mice. 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 3 independent experiments.
- Figure 4.
IL35 suppresses cytotoxic CD8+ T-cell infiltration and activity in PDA. A, Immunohistochemical detection of CD8+ T cells by DAB in tumor tissues from WT, Il10−/−, p35−/−, and Ebi3−/− mice orthotopically injected with KPC 4662 cells and collected at 3 weeks after injection of tumor cells. Scale bars, 100 μm. B, Quantification of the frequency of tumor-infiltrating CD45+CD8+ T cells from WT, Il10−/−, p35−/−, and Ebi3−/− (n = 3/group) mice determined by flow cytometry. C, Mean CD8+ cytotoxic T cell to Treg ratio was calculated based on the percentage of positive lymphocyte population determined by flow cytometry. D, Representative flow cytometry plots of intratumoral CD8+IFNγ+ T cells isolated from WT, Il10−/−, p35−/−, and Ebi3−/− mice. Percentage of CD8+ T cells is indicated. E, Quantification of frequency of CD8+IFNγ+ T cells (% of CD8+ T cells) from WT, Il10−/−, p35−/−, and Ebi3−/− (n = 12) mice. F, Representative immunofluorescence staining. Top, Ebi3 (red) and CD20 (green); bottom, Ebi3 (red) and CD4 (green) in samples of human PDA. Scale bars, 25 μm. G, Number of CD8+ T cells in tumor cell nests as a function of low versus high numbers of Ebi3+ immune cells. Each point is the number of tumor cell adjacent CD8+ T cells per 10× FOV. Data were derived from counting 3 to 6 FOV per tumor sample (n = 11 tumor samples). 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 3–4 independent experiments.
- Figure 5.
Deficiency in IL35 potentiates efficacy of anti–PD-1 checkpoint blockade. A, Schematic of the antibody treatment regimen. Anti-CD8 or control IgG (red arrows) was administered for 3 days prior to tumor cell injection and then twice weekly on days 3, 5, 9, 11, 15, and 17. Administration of anti–PD-1 (green arrows) was initiated on day 7 after tumors reached approximately 4 mm in diameter (blue circle). Two more doses of anti–PD-1 were administered on days 9 and 11. Mice were sacrificed 3 weeks after tumor cell injection. B, Quantification of tumor weights from WT and p35−/− (n = 3/group) mice orthotopically injected with KPC 4662 cells and treated with antibodies as described in A. C, Representative flow-cytometric plots of intratumoral CD4+IFNγ+, CD4+TNFα+, and CD4+Foxp3+ T cells isolated from WT and p35−/− mice. Percentage of CD4+ T cells is indicated. D, Quantification of the frequency of intratumoral CD4+TNFα+ T cells (% of CD4+ T cells) from WT and p35−/− (n = 3/group) mice. E, Quantification of the frequency of intratumoral CD4+IFNγ+ T cells (% of CD4+ T cells) from WT and p35−/− (n = 3/group) mice. F, Quantification of the frequency of intratumoral CD4+Foxp3+ regulatory T cells (% of CD4+ T cells) from WT and p35−/− (n = 9) mice. H, Quantification of the frequency of tumor-infiltrating CD45+CD8+ T cells by flow cytometry from WT and p35−/− (n = 3/group) mice. I, Representative flow-cytometric plots of CD8+IFNγ+ T cells isolated from WT and p35−/− mice. Percentage of CD8+ T cells is indicated. J, Quantification of the frequency of CD8+IFNγ+ T cells (% of CD8 T cells) from WT and p35−/− (n = 3/group) mice. Error bars, SEM. P values were calculated using the Student t test (unpaired, two-tailed); NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data represent 3 independent experiments.
Additional Files
Supplementary Data
- Supplementary Figure 1 - This figure demonstrates that host deficiency for IL-35 does not affect myeloid cell frequency
- Supplementary Figure 2 - This figure demonstrates that infiltrating immune cells contain low amounts of cytokines IL12 and IL27
- Supplementary Figure 3 - This figure demonstrates that deletion of IL12 subunit p40 does not activate anti-tumor immune response
- Supplementary Figure 4 - This Figure demonstrates examples of p35+ and Ebi3+ B cells and T cells in primary human PDAC tumors, as well as gives examples of CD8 T cell infltrated versus depleted regions in tumors
- Supplementary Figure 5 - This Figure documents successful depletion of CD8 T cells from mice, and shows results of tumor growth studies on mice with IL35 deficiency and treated with anti-PD1
Volume 6, Issue 9
