Fig 2: IL-33 enhanced DNT cell function by promoting its expansion.A Flowchart showing suppression test method in vivo. B Representative flow cytometric results showing the proliferation of adoptively transferred CD4+ T cells, as examined by CFSE in the spleen. C Statistical analysis of the percentage of proliferating CD3+ T cells, CD4+ T cells and CD8+ T cells in spleen and lymph nodes in each group. Representative flow cytometric results of apoptosis in adoptively transferred CD4+ T (D) and DNT cells (F) in the spleen. E, G Statistical analysis of the apoptosis rates in the spleen and lymph nodes in each group. H Representative flow cytometric results of Bcl-2 expression in adoptively transferred DNT cells in the spleen. I Quantification of Bcl-2 expression levels in spleen and lymph nodes in each group. Data in C and E were analyzed using One-way ANOVA post hoc Holm-Sidak’s test. Data in G and I were analyzed using unpaired Student’s t test with or without Welch’s correction. The data are represented as the mean ± SD, n = 5/group. *p < 0.05, **p < 0.01.

Fig 3: IL-33 regulated the survival of DNT cells.Converted DNT cells were stimulated with or without IL-33 for 24 h and 48 h in vitro, and representative flow cytometry plots and statistical analysis of ST2 (A), cleaved caspase-3 (B), Annexin V (C), and Ki67 expression (D) levels were performed (n = 4/group). E Flowchart showing the adoptive transfer mode. B6D2F1 mice were received a total of 3 × 106 CD45.1 positive DNT cells by tail vein injection. At the same time, IL-33 was administrated daily by intraperitoneal injection for 3 days. Representative flow cytometry plots and statistical analysis of cleaved caspase-3 (F), Annexin V (G), Ki67 (H), and BrdU (I) expression in CD45.1 positive DNT cells (n = 5/group). Data in A–D were analyzed using repeated measures One-way ANOVA post hoc Turkey’s test. Data in F–I were analyzed using unpaired Student’s t test without Welch’s correction (equal variances). Data are represented as the mean ± SD. *P < 0.05, **P < 0.01, ns no significance.

Fig 4: IL-33 promoted DNT cells survival via the NF-κB signaling pathway.A The data were presented as a heatmap exhibiting differential gene expression related to IL-33 signaling. B Genes mRNA expression was confirmed by real-time PCR after IL-33 stimulation (n = 4/group). C GSEA of a NFKB_Q6_01 gene set in DNT cells stimulated with IL-33 compared with control DNT cells. D Relative mRNA expression of NF-κB signaling pathway-related genes (n = 4/group). E Flow cytometric and statistical analysis of P50 protein expression, and the phosphorylation of IKKα/β and IκBα protein expression in DNT cells with or without IL-33 stimulation for 48 h (n = 4/group). Representative flow cytometry plots (F) and statistical analysis (G) of cleaved caspase-3+, Annexin V+ and Ki67+ DNT cells percentages after incubation with an NF-κB signaling inhibitor (BAY 11-7082) with or without IL-33 stimulation for 48 h (n = 4 or 5/group). Representative flow cytometry plots (H) and statistical analysis (I) of the Bcl-2+, Bcl-xl+, and Survivin+ DNT cells percentages after incubation with an NF-κB signaling inhibitor (BAY 11-7082) with or without IL-33 for 48 h (n = 4/group). Data in B, D and E were analyzed using unpaired Student’s t test with or without Welch’s correction depending on the F-test results. Data in G and I were analyzed using repeated measures One-way ANOVA post hoc Turkey’s test. The data are represented as the mean ± SD. *p < 0.05, **p < 0.01. See also Fig. S5.

Fig 5: DNT cells therapy combined with IL-33 could further ameliorate ConA-induced liver injury.A Flowchart showing ConA, DNT cells and IL-33 administration in each group. B Serum ALT was measured in each group. C Representative H&E staining of the liver. D Liver pathological scores in each group. E Relative mRNA levels of Ifng, Tnfa, Il6, Il17a, and T-bet in liver tissues. F Representative histogram plots showing the quantification of Annexin V, Ki67 and Bcl-2 in CD45.2+ DNT cells in the liver and spleen in ConA+DNT and ConA+DNT+IL-33 groups. G Representative histogram plots and quantification of IKKα/β and IκBα phosphorylation and P50 protein expression in CD45.2+ DNT cells in the two groups. Data in B and D were analyzed using One-way ANOVA post hoc Tukey’s test. Data in E were analyzed using One-way ANOVA post Holm-Sidak’s test. Data in F and G were analyzed using unpaired Student’s t test without Welch’s correction (equal variances). The data are represented as the mean ± SD, n = 4 or 5/group. *p < 0.05, **p < 0.01.