Augmenting antitumor efficacy of Th17-derived Th1 cells through IFN-γ-induced type I interferon response network via IRF7

Abstract

The importance of CD4⁺ T cells in cancer immunotherapy has gained increasing recognition. Particularly, a specific subset of CD4⁺ T cells coexpressing the T helper type 1 (Th1) and Th17 markers has demonstrated remarkable antitumor potential. However, the underlying mechanisms governing the differentiation of these cells and their subsequent antitumor responses remain incompletely understood. Single-cell RNA sequencing (scRNA-seq) data reanalysis demonstrated the presence of Th₁₇1 cells within tumors. Subsequent trajectory analysis found that these Th₁₇1 cells are initially primed under Th17 conditions and then converted into IFN-γ-producing cells. Following the in vivo differentiation trajectory of Th₁₇1 cells, we successfully established in vitro Th₁₇1 cell culture. Transcriptomic profiling has unveiled a substantial resemblance between in vitro-generated Th₁₇1 cells and their tumor-infiltrating counterparts. Th₁₇1 cells exhibit more potent antitumor responses than Th1 or Th17 cells. Additionally, Th₁₇1chimeric antigen receptor T (CAR-T) cells eradicate solid tumors more efficiently. Importantly, Th₁₇1 cells display an early exhaustion phenotype while retaining stemness. Mechanistically, Th₁₇1 cells migrate faster and accumulate more in tumors in an extracellular matrix protein 1 (ECM1)-dependent manner. Furthermore, we show that IFN-γ up-regulated IRF7 to promote the type I interferon response network and ECM1 expression but decreased the exhaustion status in Th₁₇1 cells. Taken together, our findings position Th₁₇1 cells as a great candidate for improving targeted immunotherapies in solid malignancies.

Keywords: T helper cells; adoptive cell transfer; exhaustion; tumor immunotherapy; type I interferon pathway.

Fig. 1.

IL-17⁺IFN-γ⁺ cells are present in tumor microenvironment and positively correlated with patient survival. (A) Feature plot shows coexpression of IL-17 and IFN-γ in tumor-infiltrating CD4⁺ T cells. (B) Annotation of cell types for distinct groups. (C) Marker specificity for Th1 and Th17 cells shown in a dot plot. (D) GSVA of pathway alterations in Th₁₇1 cells compared to other cell types. (E) Pseudotemporal analysis showing evolutionary trajectory of tumor-infiltrating CD4⁺ T Cells. (F) Kaplan–Meier survival analysis and ROC curves showing prognostic performance of Th1, Th17, and Th₁₇1 signatures across cancers. AUC (Area Under the ROC Curve). (G) Percentage of GFP⁺ Th17 cells from CD45.1⁺ OTII IL-17 reporter mice before and after sorting. (H and I) Flow cytometry analysis of IFN-γ and IL-17A in CD45.1⁺ OTII IL-17 reporter Th17 cells 5 d posttransfer in both the spleen and tumor from MB49-OVA tumor-bearing mice. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001.

Fig. 2.

Th₁₇1 cells mimic the intratumor IL-17⁺IFN-γ⁺ subsets. (A) A schematic diagram depicts the experimental design for Th₁₇1 cell differentiation in vitro. (B) Flow cytometry showing IL-17 and IFN-γ expression in cells from first and second differentiation rounds on days 3 and 9. (C) Comparison of intratumoral scRNA-seq data of IL-17⁺IFN-γ⁺ subsets (9) with our bulk RNA-seq transcriptional profiles of Th1, Th17, and Th₁₇1 cells differentiated in vitro by universal Th1 and Th17 markers. (D) UMAP of in vivo Th₁₇1 cells from pan-cancer patients and the reflection of in vitro-differentiated Th₁₇1 cells by the 41 genes set. (E) Kaplan–Meier survival analysis based on high or low expression of the 41-gene Th₁₇1 signature. Experiments shown in B were performed with three biological replicates. Data are presented as mean ± SEM. ****P < 0.0001.

Fig. 3.

Th₁₇1 cells possess the best antitumor effect. (A and B) C57BL/6 mice were intravenously injected with B16-OVA cells and CD45.1⁺ OTII Th1, Th17, Th₁17, and Th₁₇1 cells (cultured for 10 d in vitro). Lung images show tumor burden in different treatment groups (A) and tumor weight measurements (B) (n = 4). (C–H) Mice with subcutaneous B16-OVA tumors were treated with CD45.1⁺ OTII Th1, Th17, or Th₁₇1 cells on day 5. (C) Tumor growth curves are shown (n = 6 to 10). Percentages of donor T cells in blood (D and E) and tumors (F), endogenous CD8⁺ T cells in blood (G) on indicated days. (H) Tumor growth in the B16-OVA model treated with isotype or CD8 antibody (n = 4 to 6). (I–M) CAR-T cell treatment in tumor model. Tumor growth (I) and survival (J) curves for Th17 and Th₁₇1 CAR-T cells (n = 8 to 10). PD-1 expression in CAR-T cells in vitro and in vivo (K), CAR-T cells in blood (L), and tumor (M). Data are from independent experiments (A, C, and H) and single experiment (H). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4.

Th₁₇1 cells exhibit early exhausted phenotype and better stemness. (A) Heatmap comparing exhaustion stage-specific genes (SI Appendix, Table S3) in RNA-seq data from in vitro-differentiated Th1, Th17, and Th₁₇1 cells. (B) Diagram of T cell exhaustion progression across four stages. (C and D) Flow cytometry analysis of CD69 and Ly108 expression on in vitro-differentiated Th1, Th17, and Th₁₇1 cells on day 10. (E and F) Exhaustion stages of transferred Th1, Th17, and Th₁₇1 cells in the blood of B16-OVA tumor-bearing mice on days 5, 10, and 15 posttransfer (n = 3). (G and H) CD69 and Ly108 expression on transferred cells in the spleen on day 10. (I) CD28, PD-1, and LAG-3 expression on in vitro-differentiated cells on day 11. (J) Heatmap showing stemness marker expression in Th1, Th17, and Th₁₇1 cells. (K) qPCR analysis of Il2rg, Myb, and Sell expression. Data are representative of at least two independent experiments except RNA-seq data. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.

Th₁₇1 cells migrate better in an ECM1-dependent manner. (A) CBN plot illustrating gene pathway relationships in Th₁₇1 cells. (B) GSEA of scRNA-seq data (Fig. 1) showing cellular characteristics. (C and D) Flow cytometry analysis of migrated T cells in transwell assays with B16 tumor cells after 12 h. (E) Volcano plot from RNA-seq comparing gene expression between Th₁₇1 and Th1 cells. (F) qPCR analysis of Ecm1 expression in Th1, Th17, and Th₁₇1 cells. (G) Western blot of ECM1 expression in differentiated cells on day 9. (H and I) Migration assay comparing ECM1 KO and WT Th₁₇1 cells. (J and K) Tumor growth and survival in MB49-CD19 tumor-bearing mice treated with ECM1 KO or WT Th₁₇1 CAR-T cells (n = 7 to 11). (L) Tumor infiltration of CAR-T cells on day 7 posttransfer (n = 3). (M) GFP⁺ CAR-T cells in blood on day 14. Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 6.

IRF7-mediated type I IFN response network accounts for the early exhausted feature and ECM1 upregulation in Th₁₇1 cells. (A) Changes in pathways (Th₁₇1 vs. Th1 and Th₁₇1 vs. Th17) analyzed by GSVA of bulk RNA-seq data. (B) Heatmap of type I interferon response genes in Th1, Th17, and Th₁₇1 cells. (C) PPI network of up-regulated genes in Th₁₇1 cells. (D) IRF7 protein expression in differentiated cells (day 9). (E and F) Ly108 and CD69 staining in control and IRF7 knockdown Th₁₇1 cells. (G) Predicted IRF7 binding site on Ecm1 gene. (H) ChIP analysis of the binding of IRF7 to the Ecm1 gene in Th₁₇1 cells. Ecm1 mRNA expression (I) and migration assay (J) for the Th₁₇1 cells after Irf7 knockdown. Experiments were performed with at least three biological replicates with at least two independent experiments except bioinformatics data. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 7.

IFN-γ endows Th₁₇1 cells a unique type I IFN response–related transcriptional pattern by up-regulating IRF7. (A and B) Relative mRNA expression of Tbx21 and Ifng in in vitro-differentiated Th1, Th17, and Th₁₇1 cells. (C) GSEA of the pathway receptor signaling pathway via JAK-STAT utilizing bulk RNA-seq results from Th1, Th17, and Th₁₇1 cells. (D) Western blot analysis of STAT1 expression and phosphorylation on day 9. (E and F) mRNA expression of Irf7 and Isg15 under control, IFN-γ blockade (αIFN-γ), IFNAR1 blockade (αIFANR1), and combined IFN-γ/IFNAR1 blockade. (G) Western blot analysis of p-STAT1, IRF7, and ECM1 in Th₁₇1 cells with or without IFN-γ blockade. (H–J) Exhaustion status and migration ability of Th₁₇1 cells with or without IFN-γ blockade. (K–N) Effects of STAT1 inhibitor on exhaustion status, migration ability, and expression of p-STAT1, IRF7, and ECM1 in Th₁₇1 cells. (O) Flow cytometry analysis of IFN-γ⁺, IL-17A⁺, and IFN-γ⁺IL-17A⁺ T cells in the blood of healthy donors and bladder cancer patients. (P and Q) IRF7 expression and mean fluorescence intensity (MFI) in IFN-γ⁺, IL-17A⁺, and IFN-γ⁺IL-17A⁺ T cells. Data are presented as mean ± SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001.