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Adoptively transferred tumor-specific IL-9-producing cytotoxic CD8+ T cells activate host CD4+ T cells to control tumors with antigen loss

Nature Cancer volume 6, pages 718–735 (2025)Cite this article

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Abstract

Host effector CD4+ T cells emerge as critical mediators for tumor regression but whether they can be activated by adoptively transferred CD8+ T cells remains unknown. We previously reported that adoptive transfer of interleukin 9 (IL-9)-producing cytotoxic CD8+ T (Tc9) cells achieved long-term control of tumor growth. Here, we demonstrate that murine tumor-specific Tc9 cells control the outgrowth of antigen-loss relapsed tumors by recruiting and activating host effector CD4+ T cells. Tc9 cells secreted IL-24 and recruited CCR7-expressing conventional type 2 dendritic cells (cDC2 cells) into tumor-draining lymph nodes to prime host CD4+ T cells against relapsed tumors. Host CD4+ T cell or cDC2 deficiency impaired the ability of Tc9 cells to control relapsed tumor outgrowth. Additionally, intratumoral IL24 expression correlates with cDC2 and CD4+ T cell gene signatures in human cancers and their expression is associated with better patient survival. This study reports a mechanism for activation of tumor-specific CD4+ T cells in vivo.

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Fig. 1: Tumor-specific Tc9 cells control the growth of ALVs.
Fig. 2: Tc9 cells inhibit growth of rechallenged antigenneg tumors.
Fig. 3: Host CD4+ T cells are recruited into chimeric tumors.
Fig. 4: CD4+ T cells are required for control of chimeric tumor growth.
Fig. 5: Tc9 cells recruit cDC2 cells to activate CD4+ T cells.
Fig. 6: cDC2 cells are indispensable for host CD4+ T cell activation.
Fig. 7: IL-24 promotes cDC2 migration to activate host CD4+ T cells.
Fig. 8: IL24, cDC2 cells and effector CD4+ T cell genes in human melanoma.

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Data availability

Microarray, scRNA-seq and scTCR-seq data that support the findings of this study were deposited to the Gene Expression Omnibus under accession codes GSE239842 and GSE240762. Mass cytometry data were deposited to ImmPort under accession code SDY2914. Previously published microarray data that were reanalyzed here are available from the Gene Expression Omnibus udner accession codes GSE77671 (ref. 44) and GSE8401 (ref. 42). For the analysis of genes expressed by cDC2 cells, the GSE77671 dataset was used. For the analysis of persons with melanoma, gene expression and clinical parameters were retrieved from Oncomine (www.oncomine.org) and dataset GSE8401. The human breast cancer data were derived from TCGA Research Network: (https://www.cancer.gov/tcga) using UCSC Xena (https://xenabrowser.net/datapages/)43. The remaining data are available within the article and Supplementary Information or from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by funds from the Houston Methodist Research Institute, awards from the Cancer Prevention and Research Institute of Texas (Recruitment of Established Investigator Award RR180044 and High-Impact/High-Risk Research Award RP210868, to Q.Y.) and grants from the National Institutes of Health National Cancer Institute (R01 CA200539, R01 CA239255, R01 CA282099 and R01 CA2855209, to Q.Y.). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors jointly supervised this work: Liuling Xiao, Qing Yi

Authors and Affiliations

  1. Houston Methodist Neal Cancer Center, Houston Methodist Research Institute, Houston, TX, USA

    Liuling Xiao, Rui Duan, Chuanchao Zhang, Xingzhe Ma, Miao Xian, Qiang Wang, Qi Guo, Wei Xiong, Pan Su, Lingqun Ye, Yabo Li, Ling Zhong, Jianfei Qian, Yong Lu & Qing Yi

  2. First Affiliated Hospital, School of Basic Medicine, Chongqing Medical University, Chongqing, China

    Liuling Xiao

  3. The University of Texas MD Anderson Cancer Center, UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA

    Wendao Liu & Zhongming Zhao

  4. Center for Precision Health, McWilliams School of Biomedical Informatics, The University of Texas Health Science Center at Houston, Houston, TX, USA

    Wendao Liu & Zhongming Zhao

  5. Human Genetics Center, School of Public Health, The University of Texas Health Science Center at Houston, Houston, TX, USA

    Zhongming Zhao

Authors
  1. Liuling Xiao
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Contributions

Q.Y. and L.X. initiated the study, designed the experiments and wrote the manuscript. L.X. performed most experiments and statistical analyses. R.D., C.Z., X.M., P.S., W.X. and Q.G. helped with the animal studies. W.L. and Z.Z. performed the scRNA-seq and scTCR-seq analyses. Q.W. and L.Y. helped with the human bioinformatics analysis. Q.W. helped with the CyTOF bioinformatic analysis. M.X., L.Z., Y. Li, Y. Lu and J.Q. provided reagents and important suggestions.

Corresponding authors

Correspondence to Liuling Xiao or Qing Yi.

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Extended data

Extended Data Fig. 1 Tc9 cells control the outgrowth of natural ALV tumors.

a, MFI of OVA-GFP and OVA (SIINFEKL) on WT B16 and B16OVA cells (n = 3 technical replicates from a single experiment, repeated two times with similar results). b, Western blot detection of OVA in B16 and B16OVA cells (one of two independent experiments). c, Schema of experiments in Fig. 1a. d, Schema of experiments in Fig. 1b. e, Western blot and flow cytometry (n = 3 technical replicates from a single experiment, repeated two times with similar results) detection of gp100 in MC38 and MC38gp100 cells. f, Schema of experiments in Extended Data Fig. 1g. g, PBS, Thy1.1+ Pmel-1 Tc1 or Tc9 cells were transferred into CD45.2+ B6 mice bearing MC38gp100 tumors with the adjuvant treatment, and tumor growth curves were shown (n = 5 mice per group, one of two independent experiments). h. Percentages of OVA-GFPnegative and OVA-GFPpositive CD45− tumor cells on day 10 (n = 3 mice) and day 40 (n = 4 mice) after B16OVA tumor inoculation (one of two independent experiments). i. Percentages of gp100negative and gp100positive CD45− tumor cells on Day 10 and Day 40 after MC38gp100 tumor inoculation (n = 5 mice per group). Data are presented as mean ± SEM. Two-way ANOVA was used to compare antigen percentages in h, i. neg, negative; pos, positive.

Source data

Extended Data Fig. 2 Tc9 cells control the growth of ALV chimeric tumors.

a, Schema of experiments in Fig. 1e. b, Relative Pmel mRNA expression (n = 3 biologically independent samples per group) and gp100 protein level (one of two independent experiments) in WT B16 and B16gp100-kd cells. c, Schema of experiments in Fig. 1f. d, Schema of experiments in Extended Data Fig. 1e. e, Thy1.1+ Pmel-1 Tc1, Tsm, Tcm, or Tc9 cells were transferred into CD45.2+ B6 mice bearing B16 chimeric tumors with the adjuvant treatment, and tumor growth curve were shown (n = 6 mice per group). f, Schema of experiments in Fig. 1g. g, Western blot detection of gp100 protein level in WT B16 and B16gp100-ko cells (one of two independent experiments). h, Schema of experiments in Extended Data Fig. 2i. i, PBS, Thy1.1+ Pmel-1 Tc1 or Tc9 cells were transferred into CD45.2+ B6 mice bearing B16 chimeric (B16 + B16 gp100-ko) tumors with the adjuvant treatment, and tumor growth curve were shown (PBS, n = 3 mice; Tc1, n = 6 mice; Tc9, n = 6 mice). Data are presented as mean ± SEM. Multiple t tests-one per row were used to compare tumor growth in e (day 35), i (day 35); Two-tailed unpaired Student’s t-test was used in b.

Source data

Extended Data Fig. 3 Host CD4 + T cells are enriched in Tc9-treated chimeric tumor.

a, Schema of experiments in Fig. 3a–e. b–g, Thy1.1+ Pmel-1 Tc1 or Tc9 cells were transferred into Thy1.2+ B6 mice bearing B16 chimeric tumor with the adjuvant treatment. Tumor-infiltrating CD45+ cells were isolated for CyTOF analysis. t-SNE analysis of CD3, CD4, CD8 and Thy1.2 expression (b), percentage of CD4+ T cells in CD45+ cells (c), percentage of host Thy1.2+ CD8+ T cells (d), Thy1.2− transferred CD8+ T cells (e) in CD45+ cells, relative median intensity of IFNγ and GZMB in CD4+ T cells (f), percentage of Foxp3+ in CD4+ T cells (g). n = 2 in b-g, pooled from 6 mice. h, i, Thy1.1+ Pmel-1 Tc1 or Tc9 cells were transferred into Thy1.2+ mice bearing MC38 chimeric tumor with the adjuvant treatment, and tumors were analyzed. The number of CD4+ T cells, and IFNγ+ or GZMB+ CD4+ T cells were shown (n = 6 mice per group, one of two independent experiments). j, k, Tumors in mice treated as Fig. 3a were analyzed. MFI of IFNγ+ or GZMB+ and number of host Thy1.1− CD8+ T cells were shown (n = 9 mice per group, pooled from two independent experiments). l, Thy1.1+ Pmel-1 Tc9 cells were transferred into WT or CD8−/− mice bearing B16 chimeric tumors with the adjuvant treatment, and tumor growth curve were shown (n = 5 mice per group). m, n, Tumors in mice treated as Fig. 3a were analyzed. MFI of IFNγ+ or GZMB+ and number of transferred Thy1.1+ CD8+ T cells were shown (n = 9 mice per group, pooled from two independent experiments). o, p, Thy1.1+ Pmel-1 Tc1 or Tc9 cells were transferred into Thy1.2+ B6 mice bearing MC38 chimeric tumor with the adjuvant treatment, and tumors were analyzed. MFI of CD44, CD69 and PD-1 on CD4+ T cells, percentage of ICOS+PD-1low CD4+ T cells in tumors (n = 6 mice per group). Data are presented as mean ± SEM. Two-tailed unpaired Student’s t-tests were used in h–k and m–p. Multiple t tests-one per row were used to compare tumor growth in l (day 40).

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Extended Data Fig. 4 Analysis of host tumor-infiltrating CD4 + T cells.

a, UMAP plot showing the top 3 most highly expanded TCRs in the Th1-like cells from Tc1- and Tc9- treated mice (n = 1pooled from three mice per group). CDR3 amino acid sequences of α/β chains for each TCR are shown. b, Indexes of TCR clonotype diversity for the Th1-like cells from Tc1- and Tc9-treated mice. c, Violin plots of the most differentially regulated TCR genes in the Th1-like cells from Tc1- and Tc9- treated mice. d, Pie charts represent cumulative frequency of TCRα and TCRβ clonotypes. Bar charts illustrate the count of TCR clonotypes with a frequency greater than 1% in the Th1-like cells from mice treated with Tc1 or Tc9 cells. e, Representative IFNγ ELISPOT assay of CD4+ T cells isolated from tumors that were ex vivo stimulated with naive splenocytes pulsed with 5 μg/ml indicated individual peptide (n = 3 biologically independent samples per group). f, Flow cytometry analysis of Vβ5+(reacts with the Vβ 5.1 and Vβ 5.2 TCR), Vβ12+(reacts with the Vβ 12 TCR), Vβ13+ (reacts with the Vβ 13 TCR) clonotypes percentages in tumor-infiltrating CD4+ T cells (n = 5 mice per group, one of two independent experiments). g, IFNγ ELISA analysis of Vβ5+, Vβ12+, Vβ13+ tumor-infiltrating CD4+ T cells reponse to indicated peptide (n = 6 biologically independent samples per group, pooled from two independent experiments). h, Percentages and the numbers of central memory (CM) and effector memory (EM) CD4+ T cells. Tc1, n = 10 mice; Tc9, n = 8 mice. Data are presented as mean ± SEM. Wilcoxon rank sum test were used in c. Two-tailed unpaired Student’s t-tests were used in f, h. One-way ANOVA was used in g.

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Extended Data Fig. 5 Host CD4+ T cell is required for Tc9-mediated tumor restraint.

a, Cytotoxicity of Pmel-1 Thy1.1+ Tc9 cells against target cells was determined by a 6-hour cytotoxicity assay at 10:1 E:T ratio. Target cells were WT B16 cells, relapsed B16 cells from tumors on day 40, B16gp100-kd cells and B16gp100-ko cells (n = 6 biologically independent samples per group, pooled from three independent experiments). b, Schema of experiments in Fig. 4i. c, Schema of experiments in Extended Data Fig. 5d. d, Thy1.1+ Pmel-1 Tc1 cells were transferred into Thy1.2+ B6 mice bearing chimeric (B16 and B16gp100-kd) tumors with the adjuvant treatment, and IgG isotype or αCD4 antibodies were injected on day 20 after tumor cell inoculation. Tumor growth curves of treated mice were shown (n = 5 mice per group). e, Representative plots of CD4+ and CD8+ cell percentages in peripheral blood of WT and Cd4−/− mice. f, g, on day 0, mice (n = 6 for each group) were inoculated s.c. in the right back flank with 0.4 million WT B16 and 0.2 million B16gp100-kd chimeric tumor cells. On day 8, one dose of CTX was given intraperitoneally at 200 mg/kg body weight. On day 9 after tumor inoculation, mice were treated with intravenous injection of 2 million Tc1 or Tc9 cells, followed by i.v. injection of 0.5 million peptide-pulsed bone marrow-derived DCs and 4 doses of rhIL-2 (6 × 105 U). On day 20, mice were injected intraperitoneal every three days with 200μg of InVivoMAb anti mouse NK1.1 or IgG. Schema of experiments in f and tumor growth curve in g. Data are presented as mean ± SEM. One-way ANOVA was used in a; Multiple t tests-one per row were used to compare tumor growth in d (day 40).

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Extended Data Fig. 6 Tc9 ACT increases the infiltration of DCs into tumors.

a, cDC gating strategy in tumors. b, c, Thy1.1+ Pmel-1 Tc1 or Tc9 cells were transferred into Thy1.2+ B6 mice bearing MC38 chimeric tumors with the adjuvant treatment, and tumor tissues were harvested for analysis. Percentages and numbers of cDCs in tumors tissues (b, n = 6 mice per group, one of two independent experiments), number of cDC2 in tumors tissues (c, n = 6 mice per group, one of two independent experiments). d, cDC gating strategy in tdLN. e, f, Thy1.1+ Pmel-1 Tc1 or Tc9 cells were transferred into Thy1.2+ B6 mice bearing MC38 chimeric tumors with the adjuvant treatment, and tdLN were harvested for analysis. Percentages of total migratory cDCs (e, n = 8 mice per group, pooled from two independent experiments) and CD11b+ migratory cDC2 in CD45+ cells in tdLNs (f, n = 8 mice per group, pooled from two independent experiments). g, CD4+ T cell gating strategy in Fig. 5g. h, Schema of experiments in Fig. 6a-f. i, j, Thy1.1+ Pmel-1 Tc9 cells were transferred into Thy1.2+ B6 mice bearing chimeric tumor with the adjuvant treatment, and CCR7 expression on cDC2 cells from tumor tissues (i, n = 4 mice per group, one of two independent experiments) or tdLNs (j, n = 4 mice per group). k, Schema of experiments in Fig. 6g-m. Data are presented as mean ± SEM. Two-tailed unpaired Student’s t-tests were used in b, c, e, f, i, j.

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Extended Data Fig. 7 IL-24 promotes cDC2 migration to activate host CD4+ T cells.

a, b, Tc9 cells were transferred into WT or Il9r−/− B6 mice bearing chimeric tumor with the adjuvant treatment, and tumors were harvested for analysis. Schema of experiments (a), percentages and numbers of cDC2 (b, n = 5 mice per group). c, Pmel-1 T cells were transferred into B6 mice bearing MC38 chimeric tumor with the adjuvant treatment. IL-24 expression in tumor-infiltrating Tc1 or Tc9 cells (n = 6 mice per group, one of two independent experiments). d, cDC2 gating strategy for cultured DC sorting. e, f, Pmel-1 Tc9 cells transduced with vector or Il24 shRNA, IL-24 protein (e) and mRNA expression (f); n = 3 biologically independent samples per group in e, f. g, Schema of experiments in Fig. 7f-l. h, IL-24 expression in tumor-infiltrating vector or Il24 shRNA transduced Tc9 cells (n = 8 mice per group, pooled from two independent experiments). i, Il24 mRNA expression in Pmel-1 Tc1 cells transduced with vector or Il24 shRNA (n = 3 biologically independent samples per group). j-l, Vector- or Il24 shRNA- transduced Tc1 cells were transferred into mice bearing B16 chimeric tumor with the adjuvant treatment, tumors and tdLNs were harvested for analysis. Schema of experiments (j); Number of CD11b+ migratory cDC2 and CD4+ T cells in tdLNs (k); Number of cDC2 and CD4+ T cells in tumors (l); n = 5 mice per group in j–l. m, n, vector- or Il24 shRNA-transduced Tc1 cells and vector-transduced Tc9 cells were transferred into mice bearing B16 chimeric tumor with the adjuvant treatment, tumors were harvested for analysis. The percentage of Thy1.1+ CD8+ T cells (m) and IL-24 concentration (n) in tumors; n = 5 mice per group in m, n. o, Vector- or Il24 shRNA- transduced Tc1 cells were transferred into mice bearing B16 chimeric tumor with the adjuvant treatment, tumor growth curves of treated mice were shown (n = 5 mice per group). Data are presented as mean ± SEM. Unpaired Student’s t-test were used in b,c,e,f,h,i,k,l. One-way ANOVA was used in m,n. Multiple t tests-one per row were used to compare tumor growth in o (day 35).

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Extended Data Fig. 8 IL24, cDC2 and effector CD4 + T cell genes in breast cancer.

a, Prognostic value of IL24 expression for overall survival of breast cancer patients comparing high versus low quartiles (n = 263 per group). b, Correlation of IL24 and cDC2 signature in breast cancer patients (n = 1050). c, d, Heatmap (n = 1050) of ordered, z-transformed expression values of cDC2-specific genes and expression of cDC2 signature genes for top and bottom quartiles (n = 263 per group) in breast cancer patients. e, Prognostic value of cDC2 signature or CD1C expression for overall survival of breast cancer patients comparing high versus low quartiles (n = 263 per group). f, g, Heatmap (n = 1050) of ordered, z-transformed expression values of effector CD4+ T cell-specific genes and expression of effector CD4+ T cell-specific signature genes for top and bottom quartiles (n = 263 per group) in breast cancer patients. h, Prognostic value of effector CD4+ T cell signature expression for overall survival in breast cancer patients comparing high versus low quartiles (n = 263 per group). (i) Correlation of effector CD4+ T cell signature with IL24 or with cDC2 signature in breast cancer patients (n = 1050). Data are presented as mean ± SEM. The log-rank (Mantel–Cox) test was performed to compare survival curves in a, e, h; Linear regression were used in b and i.

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Extended Data Fig. 9 Schematic diagram of the study.

After adoptive T cell transfer, antigen-positive tumor cells lost their primary antigens. Adoptively transferred Tc9 cells not only killed antigen-expressing primary tumors but also secreted high levels of IL-24, which recruited dendritic cells (DCs), particularly cDC2, to the tumor microenvironment. cDC2 then migrated to tumor-draining lymph nodes (tdLNs) in a CCR7-dependent manner, where they activated host Th1-like effector CD4+ T cells. These activated CD4+ T cells subsequently infiltrated tumors and contributed to controlling ALV tumor growth.

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Xiao, L., Duan, R., Liu, W. et al. Adoptively transferred tumor-specific IL-9-producing cytotoxic CD8+ T cells activate host CD4+ T cells to control tumors with antigen loss. Nat Cancer 6, 718–735 (2025). https://doi.org/10.1038/s43018-025-00935-0

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