Fig 2: Characterization of macrophages and T cells.(A, B, C) Representative plots for the identification of the macrophage population (A) and of their two subtypes, M1-like (B) and M2-like (C) macrophages versus days after implantation. (D, E) Percentage of macrophages (D) and macrophage subtypes (E) versus days after implantation. (F, G) T cells (CD3) identification strategy (F) for the CD4+ and CD8+ subpopulations (G). (H, I) Percentage of T cells among viable cells (H) and T cells subpopulations (I) among all T cells, both versus days after implantation. (J, K, L) Representative plots for identifying T regulatory cells (CD4+CD127-, J, CD25+, K) among T cells, versus days after implantation. (L). Data shown as mean ± SEM. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

Fig 3: Measurement of 6AzGal uptake ex vivo and in vivo.a Workflow for multicolor phenotyping of cells receiving 6AzGal ex vivo and in vivo. For ex vivo experiments, cells isolated from tissues were incubated with 6AzGal, stained with antibodies and a viability dye, labeled with BDP-DBCO, and analyzed by flow cytometry. For in vivo experiments, 6AzGal was injected into mice, followed by cell isolation, multiple labeling, and flow cytometric analysis. b Ex vivo 6AzGal uptake in splenocytes. Cells isolated from mouse spleens were treated with 6AzGal, labeled with anti-CD marker antibodies, FVD780, and BDP-DBCO, and analyzed by flow cytometry to quantify 6AzGal labeling in B and T cells (gating strategy shown in Fig. S4b). c Increased 6AzGal uptake in ex vivo-activated T cells. CD3/CD28-stimulated CD4+ T cells were analyzed by flow cytometry to quantify CD69 expression and 6AzGal labeling (gating strategy shown in Fig. S4f). Their correlation was evaluated by the order of BDP-DBCO MFI in subdivided populations (in right panel, one dot represents the MFI of 1 × 103 cells). d In vivo 6AzGal uptake in mouse tissues. After 6AzGal injection into mice, cells isolated from the indicated tissues were labeled with BDP-DBCO and analyzed by flow cytometry (Fig. S5a). e In vivo 6AzGal uptake in splenocytes. Splenocytes isolated from 6AzGal-injected mice were subjected to flow cytometric quantification of 6AzGal labeling (Fig. S5d). f Competitive inhibition of in vivo 6AzGal uptake by D-glucose supplementation. Splenocytes isolated from mice cotreated with 6AzGal and D-glucose were subjected to flow cytometric quantification of 6AzGal labeling. g Increased 6AzGal uptake in inflammation models. After 6AzGal injection into LPS-treated mice, splenocytes were isolated and subjected to flow cytometric quantification of 6AzGal labeling (Fig. S6b). h Blocking 6AzGal uptake in tumors by the GLUT inhibitor. Nude mice with subcutaneous K562 xenografts were coinjected with 6AzGal and WZB-117, and subjected to flow cytometric quantification of 6AzGal labeling (Fig. S6e). Bar graphs represent means ± SEM. P values were determined by the t test.

Fig 4: Heat hyperalgesia in mice deficient for Penk in regulatory T cell (Treg).(A) Withdrawal latency of wild-type (WT) and LOX mice before (baseline) and after administration of Tamoxifen (TMX). Each dot corresponds to the mean latency response (in seconds) of four measurements taken before TMX administration (baseline) and four measurements taken from day 7 onwards (TMX). Statistical modeling was performed using a non-parametric unpaired Mann–Whitney t-test with multiple corrections. The results shown in this figure are cumulative from two independent experiments with a total of 44 mice (26 WT and 18 LOX). Each dot is a mouse. (B) Representative flow cytometry contour plot of CD25 and Foxp3 staining on pad skin CD45+CD3+CD4+ cells from WT or LOX mice 17 days after TMX gavage. (C) Quantification of the frequency of Treg (Foxp3+CD25+) among CD4+ T cells in WT and LOX mice pad as shown in (B). The indicated p value was determined by a Mann–Whitney test. Each dot is a mouse from a single experiment. (D) Immunofluorescence staining of Calcitonin Gene-Related Peptide (CGRP) neurons (red), Foxp3-GFP cells (green), and 4'6-diamidino-2-phenylindole (DAPI) (blue) of footpad skin section of a female LOX mouse (scale bar represents 50 µm). The right panel is the magnification of the area indicated on the left panel.

Fig 5: miR-155 represses Ship-1, indirectly inhibiting Tcf-1 and enhancing anti-tumor immunity.(A–D, F, and G) From WT or 155-KO mice challenged with MC38 cells, frequency of intratumoral Tcf1+Ship-1+CD8+ T cells (A and B) and CM CD44+CD62L− (C), eff CD44+CD62L− (D), Ccr7+Cx3cr1− (F), and Cx3cr1+ (G) subsets.(E and H) Histogram and MFI of Ship-1 in Cxcr6+Tcf1−Ship-1+ CD8+ T cells; n = 10 per group; t test with Welsh’s correction.(I–M) p-Akt+ cells (I), frequency of p-Akt+Ship-1+, and MFI of p-Akt and Ship-1 in vitro anti-CD3/CD28 activated CD8+ T cells (J) and in Ccr7+Cx3cr1− (K), Cx3cr1+ (L), and Cxcr6+ (M) CD8+ T cell subsets; n = 3 per group; t test with Welsh’s correction.(N and O) Ki-67+ CD8+ T cell frequency from tumor (N) or tdLN (O); n= 8–9 per group from WT or miR-155 TKO challenged with MC38 cells; t test with Welsh’s correction.(P and Q) Tumor growth (P) and mass (Q) in TCRβKO mice challenged with MC38-OVA cells and given 2E5 OT-1-Cas9 (blue), OT1-Cas9-miR-155KO (orange), or OT1-Cas9-miR-155/Ship-1KO (green) CD8+ T cells. One-way ANOVA with multiple comparisons of means of each group; n= 6–7; Grubbs’ outlier test Alpha = 0.01. Frequency of intratumoral CD8+ T cells (R); n= 5–7; Brown-Forsythe and Welch ANOVA test with multiple comparisons of means of each group. Grubbs’ outlier test Alpha = 0.05; *p > 0.05, **p > 0.01, ***p > 0.001, ****p > 0.0001. Bars represent mean; error bars represent SEM.See also Figure S5.