Background In contrast to PD-1⁺CD8⁺ T cells, PD-1⁺CD4⁺ T cells and their impact in tumor progression and immunotherapy response remain relatively unexplored. We previously reported that PD-1^hiFoxp3^-CD4⁺ T cells (4PD1^hi) from melanoma-bearing mice and patients with melanoma or non-small cell lung cancer (NSCLC) suppress T-cell function and correlate with unfavorable outcomes upon immune checkpoint blockade (ICB) therapy.¹ CD4⁺PD-1⁺ T cells were also found to correlate with poor prognosis in other NSCLC patient cohorts.^2–4 We showed that 4PD1^hiup-regulate T-follicular-helper-cell(Tfh)-related genes.¹ 4PD1^hi cells suppressing immunotherapy responses were recently described in mouse sarcoma models; however, in this setting, 4PD1^hi did not over-express Tfh genes.⁵ Here, we sought to deconvolve the lineage commitment of 4PD1^hi tumor-infiltrating lymphocytes (TILs) in relationship with their immune function and impact on ICB outcome.

Methods Single-cell RNA-sequencing (scRNAseq) was performed in 4PD1^hi, PD-1^-Foxp3^-CD4⁺ (4Dneg), and Foxp3⁺CD4⁺ T cells (Tregs) FACS-sorted from ICB-treated B16F10-melanoma bearing Foxp3-GFP mice. scRNAseq datasets of TILs from ICB-treated cancer patients^6–9 were used to extract 4PD1^hi and analyze their profiles. Tfh-deficient SAP knock-out (KO) and CD4KO:CXCR5KO mixed bone marrow (BM) transplanted RAGKO and control mice were implanted with B16F10, treated with ICB, and 4PD1^hi, 4Dneg, and Tregs were quantified by flow cytometry and FACS-sorted for functional analyses.

Results Using prior-knowledge-based signatures and mutual-information-based cell-type classification,¹⁰ we found that spleen-derived 4PD1^hi cells from tumor-bearing mice polarize toward Tfh, Tregs toward a canonical Treg phenotype, and 4Dneg toward Th1. Conversely, tumor-derived 4PD1^hi were not significantly skewed toward these phenotypes but gained in Th1 polarization after an effective anti-CTLA-4 treatment. In human primary melanoma,⁷ NSCLC,⁶ and squamous/basal cell carcinoma,⁸ 4PD1^hi cells over-expressed Tfh-related genes. This was less clear in 4PD1^hi from mixed NSCLC samples, encompassing primary tumors and different metastatic sites.⁹ However, 4PD1^hi from ICB-non-responder patients in this study⁹ displayed the greatest Tfh-signature scores. Consistently, 4PD1^hi TILs from ICB-non-responders in the other NSCLC and melanoma datasets up-regulated Tfh-related genes. To test 4PD1^hi TIL Tfh polarization in ICB response, we used SAPKO and CD4KO:CXCR5KO BM chimera mice. Both Tfh-deficient models showed better tumor responses to a suboptimal anti-CTLA-4 treatment¹; however, 4PD1^hi TILs did not substantially decrease. In this setting, 4PD1^hi TILs lost suppressive function, down-regulated Pdcd1 and Il10, and up-regulated Ifng, suggesting a Th1 phenotypic switch.

Conclusions These results indicate that 4PD1^hi TILs are heterogeneous and their Tfh/Th1 polarization influences immunotherapy responses possibly in a tumor-tissue dependent way. In melanoma, 4PD1^hi TIL Tfh polarization drives immunosuppression and ICB resistance.

Acknowledgements This study was supported in part by the Parker Institute for Cancer Immunotherapy. S. M. and A.O. contributed equally to this work.

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