Fig 2: Immunohistochemical staining assays to measure the infiltration of T cells in EMT-6 tumors. a–d The presentative images of tumor-infiltrating CD3+ cells in the tumor periphery and the tumor center. Scale bars, 250 µm or 50 µm. e The quantitative analyses for the number of tumor-infiltrating CD3+ cells, and the proportion of CD3+ area was used. f The quantitative analysis for the infiltration depth. The infiltration depth of CD3+ cells was calculated by the mean nearest distance of all CD3+ cells to the tumor border. The mean nearest distance was scaled by the distance between the corresponding tumor border to tumor center. g, h The presentative images of tumor-infiltrating CD4+ and CD8+ cells in the tumor periphery and the tumor center. Scale bars, 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 denote the significant difference relative to YM101 treatment. a-TGF-ß: anti-TGF-ß, a-PD-L1: anti-PD-L1

Fig 3: Effect of Spry2 deletion on T cells.(A) CD4+ T cells from Tamoxifen-treated Spry2f/f (Spry2+/+) (n = 2) and ERT2-Cre:Spry2f/f (Spry2-/-) mice (n = 3) analyzed by western blot to confirm Tamoxifen-inducible deletion of Spry2. (B) Real-time PCR analysis of basal Spry2 mRNA expression in splenic CD4+ T cells from Spry2+/+ and Spry2-/- mice (n = 3). (C, D) Representative dot plot and statistical analysis of splenic CD4+ and CD8+ T cells from Spry2+/+ and Spry2-/- mice (n = 3). (E, F) Representative dot plot and statistical analysis of SP CD4+, CD8+, DP and DN thymocytes from Spry2+/+ and Spry2-/- mice (n = 3). (G) Histogram of Bcl2 expression in splenic CD4+ and CD8+ T cells from Spry2+/+ and Spry2-/- mice (n = 3). (H) Western blot showing CC-3 levels in splenic CD4+ T cells from Spry2+/+ and Spry2-/- mice (n = 3). (I, J) Annexin-V staining analysis of unstimulated or anti-CD3/CD28-stimulated CD4+ T cells from Spry2+/+ and Spry2-/- mice (n = 3). (K) Proliferation analysis of CFSE-labeled CD4+ T cells from Spry2+/+ and Spry2-/- mice after 72 h culture (n = 3) (L) Histogram of surface TCR-ß expression in CD4+ T cells from Spry2+/+ and Spry2-/- mice. Values represent mean ± SEM. ** p < 0.005 by unpaired Student t test. All the data of this figure can be found in the S1 and S2 Data files. Bcl2, B cell lymphoma 2; CC-3, Cleaved Caspase-3; CFSE, carboxyfluorescein succinimidyl ester; DN, double-negative; DP, double-positive; SP, single-positive; Spry2, Sprouty2; TCR-ß, T cell receptor beta.

Fig 4: IGFBP5 increased the percentage of Treg cells and decreased the percentage of Th17 cells. Flow cytometry and RT-qPCR were used to detect the effect of IGFBP5 on T cell subsets in vitro. (A, C) Flow cytometry showed that the percentage of CD4+ IL-17A+ Th17 cells in the CD3/CD28 activated T cells decreased after the treatment of recombinant IGFBP5. (B, D) Flow cytometry showed that the percentage of CD4+ CD25+ Treg cells in the CD3/CD28 activated T cells increased after IGFBP5 treatment. (E, G) Flow cytometry showed that the percentage of CD4+ IFN-?+ Th1 cells in the CD3/CD28 activated T cells decreased slightly after the treatment of recombinant IGFBP5. (F, H) Flow cytometry showed that the percentage of CD4+ IL-4+ Th2 cells in the CD3/CD28 activated T cells increased slightly after IGFBP5 treatment. (I, J) RT-qPCR showed that the expression of ROR?t was significantly reduced and the expression of Foxp3 was markedly increased in IGFBP5 group. (K, L) RT-qPCR showed that the expression of T-bet was slightly reduced and there was little effect on the expression of Gata3 after the treatment of IGFBP5. (M-Q) The relative mRNA levels of Ki-67 and activation-related CD25, CD44, CD69 and IL-2. Student’s t-test was used to determine statistical significance. The data are presented as the mean ± SEM (n=3). The experiment was repeated three times independently. *p < 0.05, **p < 0.01; ns, not significant.

Fig 5: Siglec-1 binding to Tregs depends on Treg activation status.( A) Freshly isolated CD4 T cells were induced to become Tregs and analysed for expression of Siglec-1 ligands using pre-complexed Siglec-1-Fc (Sn-Fc). Compared to the negative control non-binding mutant, Siglec-1-R97A-Fc (SnR97A-Fc), the induced and activated Tregs showed strong Siglec-1 binding. The binding was lost when the cells were rested in IL-2 and TGF-ß for four days, but was fully recovered when the cells were reactivated with anti-CD3 mAb for three days (upper panels). The freshly induced Tregs continued to show strong Siglec-1 binding when kept under activating conditions in the presence of anti-CD3 mAb, but binding was lost when anti-CD3 mAb was withdrawn for three days (lower panels). The whole set of experiments was performed twice with similar results. Analysis of Siglec-1 binding to resting and activated Tregs was repeated more than 20 times and in each case binding was much higher to the activated cells. ( B) Siglec-1 binding to Tregs depends on the T cell receptor signal strength. Freshly induced Tregs were rested for four days and then stimulated using different concentrations of anti-CD3 mAb and analysed for Siglec-1 binding at 24, 48 and 72 hours. Binding was both dose- and time-dependent. Similar results were observed in two independent experiments.