Fig 2: Enteric glial MHC-II expression affects T-lymphocyte activation. (A) Schematic showing enteric glia expressing MHC-II when stimulated with IFNγ and LPS. MHC-II molecules interact with T-lymphocytes and cause changes in activation state as measured by CD69 expression. (B) Representative image of GFAP (grey) and CD4 (red) immunofluorescent labeling in a colon cross-section showing the close proximity of CD4-expressing T-lymphocytes with GFAP expressing enteric glia in the myenteric plexus. Scale bar: 50 μm. (C) Representative flow cytometric contour plots of mesenteric lymph nodes labeled with CD4 and CD69 in glialMHC2KO (knockout [KO]) and wild-type (WT) littermates pretreated with IFNγ and then challenged with LPS or saline control. The proportion of total gated cells in each quadrant is expressed as a percentage. (D) Levels of activated CD4+ cells quantified as a percentage of total CD4+ cells that are CD4+CD69+ (n = 8–10 mice). (E) Representative contour plots of mesenteric lymph nodes labeled with CD8 and CD69. Proportions of gated cells in each quadrant are expressed as a percentage. (F) Proportion of total CD8+ T-lymphocytes expressing CD69 quantified in each treatment condition (n = 3–4 mice). Two-way analysis of variance with Tukey-corrected multiple comparisons; ∗P < .05, ∗∗∗P < .001.

Fig 3: Loaded NPs activate endogenous TRP2-tetramer+CD8+ TILs in vivo, leading to suppression of tumor growth, prolongation of overall survival, and establishment of protective memory in B16F10 melanoma. (A) The average tumor growth curve of B16F10 melanoma-bearing mice (n = 10) who received three treatments of PBS or NPs on days 5, 11, and 17. Data were analyzed by two-way ANOVA. ****P < 0.0001. (B) The percentage survival of B16F10 melanoma-bearing mice (n = 13 to 15) after three treatments was monitored over time. Data were analyzed by log-rank (Mantel–Cox) test. ****P < 0.0001. (C) NP drives endogenous TRP2-tetramer+CD8+ T cell response strongly biased toward effector memory (TEM: CD44hiCD62LloCD8+ T) populations (flow plot [Left]; quantification [Right]) on day 17 (n = 5 to 7). (D) The representative flow cytometry plot (Left) and the corresponding quantification (Right) of surface activation marker staining (CD44 and CD69) of TRP2-tetramer+CD8+ T cells from freshly isolated B16F10 melanoma on day 17 (n = 5 to 7). (E) The representative flow cytometry plot (Left) and the corresponding quantification (Right) of PD-1 staining of TRP2-tetramer+CD8+ T cells from freshly isolated B16F10 melanoma on day 17 (n = 4 to 5). In Fig. C–E, all the data are means ± SEM. Data were analyzed by unpaired t test with Welch's correction. *P < 0.05; **P < 0.01; ***P < 0.001. (F) The representative flow cytometry plot (Left) and the corresponding quantification (Right) of IFN-γ–producing TRP2-specific CD8+ T cells after ex vivo stimulation of TRP2 peptide on day 17 (n = 4 to 6). (G) The representative flow cytometry plot (Left) and the corresponding quantification (Right) showing that NP increases the frequency of IFN-γ–producing CD8+ T cells in total CD8+ T cell population in B16F10 melanoma after ex vivo stimulation of cell stimulation mixture on day 17 (n = 8 to 10). In panels F and G, all the data are means ± SEM. Data were analyzed by Mann-Whitney test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: Effect of anti-CD103 and TGF-ß neutralizing mAb on tumour growth and PD-1 blockade therapy in B16F10E-KO tumours.a Neutralizing anti-CD103 mAb inhibits the effect of anti-PD-1. Growth of B16F10E-KO tumours, treated with anti-PD-1 and then receiving anti-CD103 blocking mAb (i.t.) or isotype control. Tumour volumes are given as means ± SEM of six mice/group (*p = 0.043). Data are from two independent experiments out of two. b Cytotoxic activity of CD8+ TIL isolated from B16F10E-KO tumours (n = 3) treated with anti-PD-1 (i.p.) plus isotype control (i.t.) or anti-PD-1 plus anti-CD103 (i.t.). Cytotoxicity against B16F10E-KO tumour cells was determined by a conventional 51Cr release assay at indicated E:T ratios. Data are from one independent experiment out of two (*p = 0.030). c Effects of anti-TGF-ß and anti-CD103 neutralizing mAb on tumour growth. Mice were engrafted with B16F10E-KO and then treated with anti-TGF-ß, anti-CD103, or isotype control (i.t.). Tumour volumes are given as means ± SEM of six mice/group. Right, tumour weights of B16F10E-KO (n = 6) recovered at day 17 (*p = 0.024). d Left, absolute cell counts of total TIL and right, CD8+ T cells infiltrating B16F10E-KO tumours treated with anti-TGF-ß, anti-CD103, or isotype control (n = 6). e Effects of anti-TGF-ß neutralizing mAb on PD-1 blockade. Growth of B16F10E-KO tumours, treated with anti-PD-1 and then received anti-TGF-ß blocking mAb (i.t.) or isotype control. Tumour volumes are given as means ± SEM of six mice/group (p = 0.196). f Left, absolute counts of total TIL in B16F10E-KO tumours treated with anti-PD-1 plus anti-TGF-ß or anti-PD-1 plus isotype control (p = 0.418). Middle panel, absolute counts of CD8+ T cells (p = 0.343) and right, percentages of CD69+CD103+ (*p = 0.020) among CD8+ T cells from B16F10E-KO tumours treated with anti-PD-1 mAb (i.p.) plus anti-TGF-ß (i.t.) or anti-PD-1 plus isotype control (n = 6). Each symbol represents an individual tumour; horizontal lines correspond to mean ± SEM (c right, d, f). Data were calculated with unpaired t-test (a-c, e, f) and one-way ANOVA with Tukey’s correction (c right, d.). Source data are provided as a Source Data file.

Fig 5: Effects of BAC on the infiltration of CD4+ T cells in the conjunctiva and CLNs. (A) Representative image of CD4+ T cell staining in the conjunctiva. Scale bars: 50 µm. (B) Statistical analysis of the number of CD4+ T cells in the conjunctiva. The data are shown as the mean ± SD. **P < 0.01 (n = 5). (C) Representative FCM plots of CD4+CD69+ T cells. (D) Representative image of IFN-γ+CD4+ and IL-17+CD4+ T cells in the conjunctiva. Scale bars: 50 µm. (E) Representative FCM plots of IFN-γ+CD4+ and IL-17+CD4+ T cells.