Fig 1: Tumor-infiltrating Treg cells are regulated by CD300a.Tumor tissues were harvested 3 weeks after B16 melanoma inoculation. (A) Representative flow cytometry plots of Treg cells in the tumor and draining lymph node (LN) (left). Numbers adjacent to outlined areas indicate the percentage of Foxp3+ (Treg) CD4+ cells. The frequencies of Foxp3+ cells among CD4+ T cells in both wild-type (WT, n = 7) and Cd300a-/- mice (n = 8) are shown (right). (B) Fluorescence microscopy of tumor sections from Foxp3-eGFP WT (n = 4) and Cd300a-/- (n = 7) mice, stained with an anti-GFP monoclonal antibody (green) and the DNA-binding dye 4',6-diamidino-2-phenylindole (DAPI; left). The number of Foxp3+ cells was quantified from for low-power fields (LPF) (right). White arrow shows Foxp3-positive cells. Scale bar, 200 µm. (C) Flow cytometric analysis of the frequencies of Foxp3+ cells among CD4+ T cells in the tumor (n = 11 in each group) and draining lymph node (n = 8 in each group) in Cd300afl/fl and Cd300afl/fl;ItgaxCre mice. Flow cytometric analysis of Ki67 (D) and CTLA-4 (E) expressions of Treg cells in the tumor in Cd300afl/fl (n = 9 in D, n = 3 in E) and Cd300afl/fl;ItgaxCre mice (n = 10 in D, n = 4 in E). Representative histogram (left), frequency (right), (D), and mean fluorescent intensity (MFI, right, E). (F and G) Tumor growth curve of WT (control mAb, n = 7; anti-CD25 mAb, n = 5), Cd300a-/- (control mAb, n = 8; anti-CD25 mAb, n = 6), Cd300afl/fl (control mAb, n = 4; anti-CD25 mAb, n = 3), and Cd300afl/fl;ItgaxCre (control mAb, n = 3; anti-CD25 mAb, n = 5) mice that were treated with an anti-CD25 mAb or a control antibody three times (days -6, -3, and 0) and then inoculated with B16 melanoma cells. (H) Representative histogram of IFN-? production from tumor-infiltrating T cells after phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation (left). The proportion of IFN-?+ cells is shown (right) (n = 6 in each group). (I–K) Flow cytometric analysis of the expressions of programmed cell death-1(PD-1) (I), PD-1 and Tim3 (J), and CTLA-4 (K) in CD8+ T cells in the tumor of Cd300afl/fl (n = 4 in I, n = 3 in J and K) and Cd300afl/fl;ItgaxCre (n = 6 in I, n = 5 in J, n = 4 in K) mice. Representative histogram (left, I and K) or dot plots (left, J) and MFI (I and K, right) or frequency (J, right). Data are given as means ± standard error of the means (SEMs). N.S.: not significant. *p < 0.05, **p < 0.01, and ***p < 0.001. p values were obtained by using a two-way analysis of variance (ANOVA) followed by Bonferroni’s post-test (A, C, and H) and the Student’s t-test (B, D, E, and I–K). Data were pooled from two (B, H, and F) or three (A, C–E, G, and I–K) independent experiments. Figure 2—source data 1.Source data for Figure 2A-K.