Fig 2: Re-FMT modulates the tumor immune microenvironment in subcutaneous colon cancer. (A) Schematic showing the experimental design and schedule of the subcutaneous tumor grafted mice. (B) Flow cytometry detection of immune cell recruitment to the tumor microenvironment. (C) Representative graphs of CD69 expression in CD4+ T cells and positive statistics. Representative graphs of CD69 (D), Granzyme B (E), and PD-1 (F) expression in CD8+ T cells and positive statistics. (G) Representative map of T cell infiltration at the tumor margin. The numbers of CD4+ T cells (H) and CD8+ T cells (I) at the tumor margin were counted. (J) Representative image of T cell infiltration in the tumor parenchyma. Counts of CD4+ T cells (K) and CD8+ T cells (L) in the tumor parenchyma. n =6 mice per group. Data represent mean ± SEM., the P value was determined by a Mann-Whitney t test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: Aged CAR-T cells are unable to maintain stem-like properties.(a) Proportion of CD8+ Tn (CD44−CD62L+), Tcm (CD44+CD62L+) and Tem (CD44+CD62L−) cells in the spleens of young and old mice. On the right, representative CD44/CD62L dot plot (n = 5 biologically independent samples). (b) Proportion of Tcm cells (CD44+CD62L+) upon activation of young and old naïve CD8+ T cells and expansion under effector-like (IL-2) or memory-like (IL-7 IL-15) conditions (n = 10 biologically independent samples). (c) Mean fluorescence intensity (MFI) of TCF-1 within young and old Tcm populations expanded from naïve CD8+ T cells with IL-7 and IL-15 (n = 4 biologically independent samples). (d) Killing capacity against B16-HER2 tumor cells of young and old CAR-T cells expanded with IL-2 (n = 3 biologically independent samples). (e) Proportion of IFNγ+ and TNFα+ young and old CAR-T cells expanded with IL-2 upon rechallenge with B16-HER2 tumor cells (n = 4 biologically independent samples). (f) Proportion of IFNγ+ and TNFα+ young and old exhausted CAR-T cells generated upon chronic stimulation in vitro (n = 3 biologically independent samples). (g) MFI of IFNγ and TNFα within IFNγ+ and TNFα+ populations, respectively, of young and old exhausted CAR-T cells generated upon chronic stimulation in vitro (n = 3 biologically independent samples). (h-l) Levels of the exhaustion markers PD-1, TOX, TCF-1, LAG3 and TIM3 in young and old exhausted CAR-T cells (n = 3 biologically independent samples). (m) Growth of B16-HER2 tumors after ACT of young and old CAR-T cells expanded with IL-2 (n = 6 mice per group). (n) CAR-T cells (CD45.1+) generated from young or old mice were adoptively transferred into young or old recipient mice bearing B16-HER2 tumors. Number of CD45.1+ CD8+ T cells were analyzed in the spleens 20 days post-ACT (n = 9 Y > Y, n = 5 O > Y, n = 8 Y > O, n = 8 O > O). Data are represented as mean values +/- SEM. Statistical analysis was performed using two-way ANOVA (A-C), unpaired t-test (E, G and M) or one-way ANOVA with multiple comparisons (F, H-I and N), as appropiate. ns = p > 0.05. Source data

Fig 4: Impaired T-dependent immune response in Sam68 KO mice against SRBC. A. Scheme showing the experimental timeline for T-dependent SRBC immunization. B. Flow cytometric analysis of B cell activation and GC reaction in SRBC treated WT and Sam68 KO mice 10 days post immunization. C. Histogram showing expression of GL7 on the activated (CD95+ CD38−) B cells in the spleen of SRBC treated WT and KO mice. D. Quantification of CD38− CD95+ activated B cells in the spleen of SRBC treated WT and KO mice. Bars represent mean ± SEM. Statistical analysis- unpaired t-test. E. Quantification of anti-SRBC IgG1, IgG2b and IgG3 in the serum of WT (black) and Sam68 KO (red) mice at day10 after immunization by flow cytometry. Statistical analysis: Two-way ANOVA, multiple comparison test (n = 5–6, two independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001. F. Flow cytometric analysis of T-follicular helper (TfH, CD4+ CXCR5+ PD1+) cells in the spleen of WT and Sam68 KO mice 10 days after SRBC immunization. G. Histogram showing the expression of BCL6 in the TfH cells in WT and Sam68 KO mice. H-I. Quantification of CD4+ T cells (H) and TfH cells (I) in the spleen of immunized WT and Sam68 KO mice. Bars represent mean ± SEM. Statistical analysis- unpaired t-test

Fig 5: rhIL-7-hyFc treatment improves T cell function in the immunosuppressive GBM microenvironment.A Multiplex cytokine array of tumor-infiltrating CD3+ T cells collected from rhIL-7-hyFc and ZIKV-treated brains on day 21 compared to those from ZIKV treatment alone (ZIKV+diluent n = 2, ZIKV + IL7 n = 2). B Representative flow cytometry histograms of MHC I and MHC II expression on SB28 cells. C Quantification of C SB28 and D CT2A tumor cell MHC I expression when co-cultured with CD3+ TILs from rhIL-7-hyFc and ZIKV-treated mice, as well as in the setting of IFN-γ blocking antibody (each data point represents one experimental replicate, n = 3). Percentage of E PD-1+, F TIM-3+, and G LAG-3+ CD8+ T cells in the brain at day 17 and 21 (each data point represents one individual mouse; PBS+diluent n = 9, PBS + IL7 n = 10, ZIKV+diluent n = 9, ZIKV + IL7 n = 10). H Absolute number of circulating CD3+, CD4+, and CD8+ T cells following rhIL-7-hyFc and ZIKV treatment (each data point represents one individual mouse). I Percentage of PD-1+, TIM-3+, and LAG-3+ CD8+ T cells in circulation at day 21 (each data point represents one individual mouse; PBS+diluent n = 8, PBS + IL7 n = 9, ZIKV+diluent n = 8, ZIKV + IL7 n = 10). Bar columns represent the mean, and error bars represent the standard deviation. Statistical differences were determined by (C–I) one-way ANOVA test with Tukey’s posttest (*P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001). Source data and exact p-values are provided as a Source Data file. ZIKV = Zika virus, IL-7 = rhIL-7-hyFc.