Fig 2: Enforced expression of CXXC4 leads to suppression of gastric cancer cells proliferation and the activation of T cells by inhibiting the expression of CDK18. A, The expression of CDK18 in gastric cancer and adjacent normal tissues measured by RT‐qPCR, n = 37, *P < .05 tumour tissues vs adjacent normal tissues. B, The expression of CDK18 in GES‐1 and the gastric cancer cell line SGC7901 assessed by RT‐qPCR, n = 3, *P < .05 SGC7091 cell line vs GES‐1 cell line. C, The expression of CDK18 in gastric cancer cell line SGC7901 normalized to β‐actin measured by Western blot analysis, n = 3, *P < .05 oe‐MIR100HG vs oe‐NC. D, The expression of CDK18 in gastric cancer cell line SGC7901 normalized to β‐actin assessed by Western blot analysis, n = 3. E, The expression of CXXC4, MIR100HG and CDK18 measured by RT‐qPCR, n = 3. F, The proliferation of SGC7901 cells detected by CCK‐8 assay, n = 3. G, The proliferation ability and the proportion of activated IFN‐γ+ T cells of CD3+ detected by flow cytometry. H, The cytokine IFN‐γ secreted by T cells measured by ELISA, n = 3, *P < .05 oe‐CXXC4 + oe‐NC vs oe‐NC, # P < .05 oe‐CXXC4 + oe‐CDK18 vs oe‐CXXC4 + oe‐NC. Measurement data were expressed as mean ± SD. Comparisons between tumour and adjacent normal tissues in panel A were analysed using paired t test. The data conforming to normal distribution and homogeneous variance between two groups in panels B and C were analysed by unpaired t test. Comparisons among multiple groups in panels D, E, G and H were analysed using the one‐way ANOVA, followed by Tukey's post hoc test. The data at different time points in panel F were analysed by the repeated measures ANOVA followed by Bonferroni's post hoc test. The experiment was repeated three times independently

Fig 3: 6-AN, a small-molecule inhibitor of 6PGD, recapitulates the phenotype and function of 6PGD-/- T cells(A–F) Naive CD8+ T cells (CD3+CD8+CD44lowCD62Lhigh) were isolated from spleens of 6PGDfl/fl mice and were stimulated in vitro with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL) in the presence of DMSO or 6-AN (10 µM) for 4 days. Expression of CD44 and CD62L (A) and CD69 (B) was assessed by flow cytometry. IFN-? production was assessed by flow cytometry (C) and ELISA (D). Expression of granzyme B was assessed by flow cytometry (E and F).(G) CTL activity was assessed as described in STAR methods. Results are representative of three independent experiments with n = 4 per experiment.(H–L) CD8+ T cells were isolated from OTI/6PGDfl/fl mice and were stimulated in vitro with aCD3 + aCD28 mAbs and IL-2 (20 U/mL) for 4 days in the presence of 6-AN or DMSO followed by adoptive transfer to congenic (CD45.1+) mice bearing EG7 lymphoma tumor, as indicated in the schema (H). Tumor growth was evaluated every 2 days, and tumor volume was calculated (I). Results are representative of two independent experiments with n = 12 mice per group. The fraction of OTI+ CTLs (CD45.2+) in TILs (J) (p < 0.05) and their capacity to produce IFN-? in the tumor microenvironment after treatment with 6-AN or DMSO (K) were assessed by flow cytometry (p < 0.05). No difference in IFN-? production by CD45.2- host CD8+ TILs was observed between recipients of 6-AN-treated and DMSO-treated CD45.2+ antigen-specific CD8+ T cells (p > 0.05) (L). Error bars represent ± SEM. **p < 0.01; ***p < 0.001.

Fig 4: Analysis of skin-infiltrating T cells in IMQ-treated K14-IκBζ–KO mice.All analyses were performed after 7 days of IMQ treatment. (A) Flow cytometry analysis of T cell subsets in the ears of IMQ-treated Ctrl and K14-KO mice. T cell subsets were detected as CD45+ and CD3+, αβTCR+, or γδTCR+ cells. Single data points derive from 2 ears. Shown is the mean of 4–12 mice per group ± SEM. (B) Gene expression analysis of Il22 and Il17a in skin tissue of untreated and IMQ-treated control and K14-KO mice, normalized to the reference gene Actin. n = 4–14 ± SEM. (C) Determination of the percentage of IL-17A– and IL-22–producing αβ and γδ T cells in IMQ-treated control and K14-KO mice. After fixation and permeabilization, cells were gated as in A, except for an additional gating on either IL-17A+ or IL-22+ cells. n = 3 ± SEM. (D) Gene expression analysis of Il1b and Il23a in untreated and IMQ-treated mice, similar as in B. (E) mRNA levels of Ccl2, Ccl17, and Ccl20 in untreated mice, similar as in B. (F) mRNA levels of Ccr2, Ccr4, and Ccr6 in the skin of untreated control and K14-KO mice, similar as in B. n = 5–14 ± SEM. P values were calculated using 2-tailed Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig 5: Exosomes derived from M1 macrophages promote the immune response of T cells. Mice are treated with PBS or M1 macrophage-derived exosomes. (A) Immunohistochemistry of CD3+ T infiltration in the tumors of mice (400×). (B) Flow cytometry of INF-γ+ T cells in CD3+ T cells (n = 5). (C) ELISA of IL-2, TNF-α and INF-γ expression in the supernatant of T cells (n = 5). (D) Flow cytometry of PD-1+ T cell proliferation in GC cells treated with M1 macrophage-derived exosomes and anti-PD-L1 after co-cultured with PD-1+ or PD-1– T cells. (E) Flow cytometry of PD-1– T cell proliferation in GC cells treated with M1 macrophage-derived exosomes and anti-PD-L1 after co-cultured with PD-1– T cells or PD-1– T cells. *p < 0.05 compared to mice treated with PBS as well as in comparison to the GC cells without any treatment; #p < 0.05 compared with GC cells treated with M1 macrophage-secreted exosomes. Measurement data representative of three independently conducted experiments expressed as mean ± standard deviation. Comparisons between two groups analyzed by non-paired t test as well as comparisons among multiple groups are analyzed by one-way ANOVA. Post hoc test was conducted using Tukey’s test.