Fig 2: Lung tissue and serum levels of IL-17 are suppressed following abatacept treatment and negatively correlated with blood Breg and Treg levels.(A) Lung tissue gene expression level of IL-17 following treatment with the abatacept IN and IP. (B-D) Correlation between percentage of IL-135+IL-10+ producing Bregs, LAG3+ cTregs, or LAG3+ uTregs in the lung and lung level of IL-17. (E) Serum level of IL-17 following treatment with the abatacept IN or IP. (F-H) Correlation between percentage of IL-135+IL-10+ producing Bregs, LAG3+ cTregs, or LAG3+ uTregs in the lung and serum level of IL-17. Lung tissues of 5 mice and PBMCs of 10 mice were used. Statistical test; Pearson’s correlation coefficient with a one-sided test for significance (P<0.05 significant).

Fig 3: Compensatory upregulation of immune checkpoints in murine OVC after blockade of single checkpoint pathway. (A, B) Elevated expression of other checkpoints in tumor-bearing PD-1KO or LAG-3KO mice. The levels of LAG-3, CTLA-4, and TIM-3 are elevated in the TALs from the tumor-bearing PD-1KO mice, whereas PD-1, TIM-3, and 2B4 are increased in tumor-bearing LAG-3KO mice. Tumor implantation was performed as described in Fig. 1. TALs and TILs (data not shown) were collected from tumor-bearing C57BL6 (wild-type), PD-1KO, and LAG-3KO mice (five animals per group) at early time points and stained for the antibody against the indicated checkpoints. Data are representative of two independent experiments. (C, D) Individual blockade with immune checkpoint antibody anti-PD-1, or anti-LAG-3, or anti-CTLA-4 in the wild-type mice results in elevated expression of the other immune inhibitory checkpoints. Ten days after tumor implantation, mice were treated intraperitoneally with antibody (200 µg per mouse) every other day for four times. T cells were analyzed 7 d after the end of the treatment. Data were analyzed using GraphPad Prism 6. Error bars represent SD. Statistical significance was determined by Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001. Data were obtained from five animals and are representative of three independent experiments.

Fig 4: Complement C1q binding protein (C1QBP) knockdown impacts tumor progression. 2 × 105 MC38 cells were injected subcutaneously into the flanks of C1QBP+/+ and C1QBP+/- mice (five mice per group). A, Representative bioluminescence images of MC38 xenograft tumors in C1QBP+/+ and C1QBP+/- mice after 21 d of injection. B, Tumor tissues were collected from C1QBP+/+ and C1QBP+/- mice after 21 d of injection. C, The bar graphs show the alterations of tumor volume and weight from C1QBP+/+ and C1QBP+/- mice after 21 d of tumor injection. D, Ki67 (proliferation marker) and Puma (anti–apoptotic marker) expressed in tumor tissues from C1QBP+/+ and C1QBP+/- mice were assessed by immunohistochemistry. Scale bars, 10 µm (200×), 5 µm (400×). E, The relative percentages of tumor-infiltrating CD4+ and CD8+ T cells from C1QBP+/+ and C1QBP+/- mice were assessed by flow cytometry. F, The anti–CD4 and anti–CD8 antibodies were used to detect T cell tumor infiltration in C1QBP+/+ and C1QBP+/- mice by immunohistochemistry. Scale bars, 10 µm (200×), 5 µm (400×). G, Exhaustion markers (PD1, Tim-3, LAG3) expressed in CD4+ and CD8+ T cells from C1QBP+/+ and C1QBP+/- mice were assessed by flow cytometry. The bar graphs show the means ± SD of three independent experiments (n = 3); ***P < .001, **P < .01, *P < .05

Fig 5: Effect of chronic treatment with ATX-MS-1467 on the splenic iTreg population of (DR2 × Ob1)F1 mice. For a–c, the spleens were harvested and fresh cells stained as described in the methods section. a, b are representative plots showing the populations of LAG3+CD49b+ cells (CD3+CD8-B220-) from total CD4+ lymphocytes, for which the averages are shown in c. For d–g, the harvested splenocytes were stimulated in vitro for 3 h in the presence of PMA, ionomycin and monensin, to allow intracellular accumulation and subsequent staining of IL-10. The contributions of Foxp3- and Foxp3+ cells to the total IL 10-producing CD4+ lymphocytes is shown in d and e, which are representative dot plots of the groups of mice treated with HLAbp (n = 8) or ATX-MS-1467 (n = 8), respectively. The averaged data are shown in f and g. * and ** indicate p < 0.05 and < 0.01, respectively, by unpaired t test with Welch’s correction versus the HLAbp group (n = 8). Foxp3 forkhead box p3, HLAbp human leukocyte antigen-binding peptide, IL interleukin, iTreg induced regulatory T cells, PMA phorbol myristate acetate