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. 2016 Sep;6(9):1022-35.
doi: 10.1158/2159-8290.CD-15-1412. Epub 2016 Jun 13.

Autocrine Complement Inhibits IL10-Dependent T-cell-Mediated Antitumor Immunity to Promote Tumor Progression

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Free PMC article

Autocrine Complement Inhibits IL10-Dependent T-cell-Mediated Antitumor Immunity to Promote Tumor Progression

Yu Wang et al. Cancer Discov. .
Free PMC article

Abstract

In contrast to its inhibitory effects on many cells, IL10 activates CD8(+) tumor-infiltrating lymphocytes (TIL) and enhances their antitumor activity. However, CD8(+) TILs do not routinely express IL10, as autocrine complement C3 inhibits IL10 production through complement receptors C3aR and C5aR. CD8(+) TILs from C3-deficient mice, however, express IL10 and exhibit enhanced effector function. C3-deficient mice are resistant to tumor development in a T-cell- and IL10-dependent manner; human TILs expanded with IL2 plus IL10 increase the killing of primary tumors in vitro compared with IL2-treated TILs. Complement-mediated inhibition of antitumor immunity is independent of the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) immune checkpoint pathway. Our findings suggest that complement receptors C3aR and C5aR expressed on CD8(+) TILs represent a novel class of immune checkpoints that could be targeted for tumor immunotherapy. Moreover, incorporation of IL10 in the expansion of TILs and in gene-engineered T cells for adoptive cell therapy enhances their antitumor efficacy.

Significance: Our data suggest novel strategies to enhance immunotherapies: a combined blockade of complement signaling by antagonists to C3aR, C5aR, and anti-PD-1 to enhance anti-PD-1 efficacy; a targeted IL10 delivery to CD8(+) TILs using anti-PD-1-IL10 or anti-CTLA4-IL10 fusion proteins; and the addition of IL10 in TIL expansion for adoptive cellular therapy. Cancer Discov; 6(9); 1022-35. ©2016 AACR.See related commentary by Peng et al., p. 953This article is highlighted in the In This Issue feature, p. 932.

Figures

Figure 1
Figure 1
Regulation of IL-10 expression in CD8+ T cells by complement. A, heat map of the differentially-expressed genes in IL-10+ and IL-10 CD8+ T cells. B, pathway analysis of differentially expressed genes as shown in (A). Shown are the top 10 pathways that are highly enriched in IL-10+CD8+ T cells. C and D, mRNA expression of complement (C) and complement receptors (D) in IL-10+CD8+ (GFP+) and IL-10CD8+ (GFP) T cells. Plots show relative expression levels of mRNAs for each indicated gene based on gene chip data. Shown are the mean ± SEM from data deposited by Trandem et al (Reference 26). E, expression of IL-10 in CD8+ TILs from WT and C3−/− mice. IL-10 reporter (Tiger) mice were crossed with C3−/− mice and inoculated with B16 melanoma cells. TILs were analyzed by flow cytometry from day 12 to 13. The percentages of IL-10+CD8+ TILs from six C3−/− Tiger mice are shown in the right panel. Error bars indicate SEM. Significance was determined in all panels by Student’s t test (*p≤0.05, **p≤0.01, ***p≤0.001).
Figure 2
Figure 2
Suppression of T cell-mediated antitumor immunity by complement. A-C, melanoma development in C3−/− mice. B16F10 melanoma cells (2×105/mouse) were subcutaneously (s.c.) inoculated into WT and C3−/− mice. Tumor growth was monitored daily starting from day 7. Shown are tumor volume, size, and weight in these mice (n=9 mice per group). D-F, breast cancer development in C3−/− mice. E0771 breast cancer cells (1×106/mouse) were s.c. inoculated into WT and C3−/− mice. Tumor growth was monitored every other day starting from day 7. Shown are tumor volume, size, and weight in these mice (n=9 mice per group). G and H, phenotypes of CD8+ TILs from C3−/− mice. WT and C3−/− mice were s.c. inoculated with B16F10 cells (2×105/mouse). Total, IFNγ–, and TNFα-producing CD8+ TILs were analyzed by flow cytometry (n=5 mice per group) at day 12 after tumor inoculation. I, B16F10 tumor development in WT, C3−/−, TCRα−/−, and C3−/− TCRα−/− mice (n=6 mice per group). All experiments shown are representative of at least three independent experiments. Bars and error bars indicate mean ± SEM. Significance was determined in all panels by Student’s t test (ns, p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001).
Figure 3
Figure 3
Non-CD8+ T cell responses in tumor-bearing mice. B16F10 melanoma cells (2×105/mouse) were s.c. inoculated into WT and C3−/− mice. The draining lymph nodes (dLNs) and tumors were treated with collagenase and DNase to generate a single-cell suspension. Leukocytes were pre-gated on CD45+ cells. A, CD11b and GR1 expression in leukocytes from tumor-infiltrating leukocytes (TILs) were analyzed by flow cytometry (n=4 mice per group). B, regulatory CD4+ T cell population in leukocytes from TILs was analyzed by flow cytometry using CD4 and Foxp3 as markers (n=4 mice per group). C, NK population in leukocytes from TILs was analyzed by flow cytometry using NK1.1 as a marker (n=4 mice per group). Experiments shown are representative of three independent experiments. Bars and error bars indicate mean ± SEM. Significance was determined in all panels by Student’s t test (ns, p>0.05).
Figure 4
Figure 4
Essential role for IL-10 in the antitumor response in C3−/− mice. A-C, melanoma development in C3−/− mice. B16F10 melanoma cells (2×105/mouse) were s.c. inoculated into WT, Il10−/−, C3−/−, and Il10−/−C3−/− mice. Tumor growth was monitored daily starting from day 7. Shown are tumor volume, size, and weight in these mice (n=8 mice per group). D-F, breast cancer development in C3−/− mice. E0771 breast cancer cells (1×106/mouse) were s.c. inoculated into WT, C3−/−, and Il10−/−C3−/− mice. Tumor growth was monitored every other day starting from day 7. Shown are tumor volume, size, and weight in these mice (n=8 mice per group). All experiments shown are representative of three independent experiments. Bars and error bars indicate mean ± SEM. Significance was determined by Student’s t test in panels (A) and (D), and by ANOVA in panels (C) and (F) (ns, p>0.05, *p≤0.05, **p≤0.01).
Figure 5
Figure 5
IL-10 enhances the function of TILs from cancer patients. A, cell number of in vitro-expanded TILs from lung cancer patients. TILs were isolated and cultured in the presence of 6000 U/ml rIL-2, 100 U/ml rIL-10, or 6000 U/ml rIL-2 plus 100 U/ml rIL-10. The numbers of live TILs counted are shown (y-axis). The inserted panel shows the ratio of TILs from two types of culture from 3 patients. B, killing activity of in vitro-expanded TILs. The expanded TILs in (A) were activated by anti-CD3/CD28 antibodies for 24 hrs and tested for their ability to kill autologous primary tumor cells at an E:T ratio of 20:1. The killing activity was measured at 15 minute intervals by Impendance assay. C-D, IFNγ and TNFα expression in CD8+ TILs expanded in vitro. TILs from lung cancers were expanded in complete culture medium with 6000 U/ml rIL-2 alone or combined with 100 U/ml rIL-10 for 20 days. IFNγ and TNFα expression in CD8+ TILs were analyzed by flow cytometry. A-D show results representative of three to six patients. E, heat map of the differentially-expressed genes in IL-10-treated human lung tumor CD8+ TILs. F-J, mRNA expression of different pathways as indicated in IL-10/IL-2-treated human CD8+ TILs. Plotted are relative expression levels of mRNAs compared to those from IL-2-treated cells for each indicated gene based on gene chip data. Shown are the mean ± SEM. Significance was determined by ANOVA in panels (F-J) *p≤0.05, **p≤0.01.
Figure 6
Figure 6
Suppression of IL-10 production by autocrine C3. A, schematic of T cell transfer to C3−/−TCR−/− mice and tumor development. B, melanoma development in chimeric mice. B16F10 melanoma cells were inoculated into T cell-reconstituted C3−/−TCR−/− mice and tumor development was monitored daily (n=5 mice per group). C, FACS profiles of C3aR and C5aR expression on CD8+ T cells from dLNs of naïve mice. D, FACS profiles of C3aR and C5aR expression on CD8+ T cells from dLNs and melanomas. B16F10 melanoma cells (2×105/mouse) were s.c. inoculated into WT mice, and TILs were isolated at day 13 and analyzed by flow cytometry. E, FACS profiles of C3aR and C5aR expression on CD8+ T cells from breast cancer. E0771 cells (1×106/mouse) were s.c. inoculated into WT mice. The expression of C3aR and C5aR on CD8+ TILs was analyzed at day 19 by flow cytometry. F, summary of results from (D) and (E). G, FACS profiles of C3aR and C5aR expression on CD8+ T cells from PBMCs and TILs from liver cancer (n=5). H, effect of carboxypeptidase N (CPN) expression in tumor cells on IL-10 production in CD8+ TILs. Control and CPN-expressing B16F10 cells (4×105/mouse) were s.c. inoculated into WT Tiger mice. IL-10-reporter eGFP expression in CD8+ TILs was assayed at day 13. Right panel shows the percentages of IL-10+CD8+ TILs from 5 mice. I, effect of C3aR and C5aR antagonists on IL-10 expression in CD8+ TILs. B16 tumor-bearing WT Tiger mice were treated with control or C3aR and C5aR antagonists (n=3 per group). J, effect of C3aR and C5aR antagonists on IL-10 expression in in vitro-activated CD8+ T cells (n=4). K, effect of complement signaling blockade on breast cancer development. E0771 breast cancer cells (1×106/mouse) were s.c. inoculated into WT, C3−/−, and Il10−/− mice. WT and Il10−/− mice were treated with C3aR and C5aR antagonists or control solution every 12 hrs starting from day 9 after tumor implantation. Tumor volume was monitored every other day (n=8 mice per group). (B and K) show results representative of three independent experiments. Bars and error bars indicate mean ± SEM. Significance was determined by Student’s t test in panel (B) and by ANOVA in panels (J) and (K) (ns, p>0.05, **p≤0.01, ***p≤0.001).
Figure 7
Figure 7
Complement inhibits antitumor immunity through a PD-1-independent pathway. A, B16F10 melanoma cells (2×105/mouse) were s.c. inoculated into WT and C3−/− mice. Expression levels of PD-1 on CD8+ TILs were analyzed by flow cytometry at day 12 after tumor implantation. B, expression level of PD-1 on CD8+ T cells activated with anti-CD3/CD28 antibodies in the presence of IL-10. T cells from LNs of naïve mice were activated by incubation with 3μg/ml anti-CD3/CD28 antibodies for 48 hrs with or without 500 U/ml IL-10 and analyzed for PD-1 expression. C-D, PD-L1 expression on tumor cells developed in WT and C3−/− mice. B16F10 melanoma cells (2×105/mouse) or E0771 breast cancer cells (1×106/mouse) were s.c. inoculated into WT and C3−/− mice. PD-L1 expression in CD45 B16 tumor cells (C) and CD45 E0771 cells (D) were measured by flow cytometry at day 12 and day 19, respectively. E, PD-L1 expression in tumor cells after IL-10 stimulation. B16F10 cells were cultured with or without 500 U/ml IL-10 for 5 days. PD-L1 expression was analyzed by flow cytometry. F, PD-L1 expression on gene-engineered B16F10 cells. B16F10 cells were transduced with control virus or sg-RNA-targeting PD-L1 virus and selected with puromycin to generate a stable PD-L1-silenced polyclonal cell line. G, IL-10 expression in CD8+ TILs from PD-L1-silenced B16F10 tumors. Control or PD-L1-silenced B16F10 melanoma cells (4×105/mouse) were s.c. inoculated into C3−/− and C3−/− Tiger mice. GFP expression in CD3+CD8+ TILs was analyzed by flow cytometry. Right panel shows the percentages of IL-10+CD8+ TILs from 4 individual mice. H, effect of blockade of PD-1/PD-L1 and complement signaling pathways on tumor development. Control or PD-L1-silenced B16F10 melanoma cells (4×105/mouse) were s.c. inoculated into WT and C3−/− mice. Tumor development was monitored every day starting from day 7 after implantation (n=7, 7, 9, and 8 mice respectively). I, B16F10 melanoma cells (5×104/mouse) were s.c. inoculated into WT mice. Mice were randomized into 4 groups 6 days after implantation. Each group of mice received control antibody, anti-PD-1 antibody, C3aR and C5aR antagonists, or anti-PD-1 anitbody plus C3aR and C5aR antagonists. (n=8 mice per group). (A-F) Solid gray color indicates isotype control staining. (A-E) Data represent a pool of 6-8 mice in each group. Significance was determined in panels (H) and (I) by Student’s t test.* p≤0.05, **p≤0.01.

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