Article Text

Download PDFPDF

968 PD-1hiFoxp3-CD4+ tumor-infiltrating T-cell lineage commitment impacts the immunotherapy outcome
  1. Levi Mark Mangarin1,
  2. Stephen Martis2,
  3. Andrea Orlando1,
  4. Sara Schad3,
  5. Jedd D Wolchok1,
  6. Benjamin Greenbaum2,
  7. Taha Merghoub1 and
  8. Roberta Zappasodi1,3
  1. 1Weill Cornell Medicine, New York, NY, USA
  2. 2Memorial Sloan Kettering Cancer Center, New York, NY, USA
  3. 3Weill Cornell Graduate School of Medical Sciences, New York, NY, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


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-1hiFoxp3-CD4+ T cells (4PD1hi) 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 4PD1hiup-regulate T-follicular-helper-cell(Tfh)-related genes.1 4PD1hi cells suppressing immunotherapy responses were recently described in mouse sarcoma models; however, in this setting, 4PD1hi did not over-express Tfh genes.5 Here, we sought to deconvolve the lineage commitment of 4PD1hi tumor-infiltrating lymphocytes (TILs) in relationship with their immune function and impact on ICB outcome.

Methods Single-cell RNA-sequencing (scRNAseq) was performed in 4PD1hi, 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 patients6–9 were used to extract 4PD1hi 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 4PD1hi, 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 4PD1hi cells from tumor-bearing mice polarize toward Tfh, Tregs toward a canonical Treg phenotype, and 4Dneg toward Th1. Conversely, tumor-derived 4PD1hi 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 4PD1hi cells over-expressed Tfh-related genes. This was less clear in 4PD1hi from mixed NSCLC samples, encompassing primary tumors and different metastatic sites.9 However, 4PD1hi from ICB-non-responder patients in this study9 displayed the greatest Tfh-signature scores. Consistently, 4PD1hi TILs from ICB-non-responders in the other NSCLC and melanoma datasets up-regulated Tfh-related genes. To test 4PD1hi 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, 4PD1hi TILs did not substantially decrease. In this setting, 4PD1hi TILs lost suppressive function, down-regulated Pdcd1 and Il10, and up-regulated Ifng, suggesting a Th1 phenotypic switch.

Conclusions These results indicate that 4PD1hi TILs are heterogeneous and their Tfh/Th1 polarization influences immunotherapy responses possibly in a tumor-tissue dependent way. In melanoma, 4PD1hi 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.


  1. Zappasodi R, Budhu S, Hellmann MD, Postow MA, Senbabaoglu Y, Manne S, et al. Non-conventional Inhibitory CD4(+)Foxp3(-)PD-1(hi) T Cells as a Biomarker of Immune Checkpoint Blockade Activity. Cancer Cell. 2018;33(6):1017–32 e7.

  2. Zheng H, Liu X, Zhang J, Rice SJ, Wagman M, Kong Y, et al. Expression of PD-1 on CD4 + T cells in peripheral blood associates with poor clinical outcome in non-small cell lung cancer. Oncotarget. 2016;7(35).

  3. Arrieta O, Montes-Servín E, Hernandez-Martinez J-M, Cardona AF, Casas-Ruiz E, Crispín JC, et al. Expression of PD-1/PD-L1 and PD-L2 in peripheral T-cells from non-small cell lung cancer patients. Oncotarget. 2017;8(60).

  4. Duchemann B, Naigeon M, Auclin E, Ferrara R, Cassard L, Jouniaux JM, et al. CD8(+)PD-1(+) to CD4(+)PD-1(+) ratio (PERLS) is associated with prognosis of patients with advanced NSCLC treated with PD-(L)1 blockers. J Immunother Cancer. 2022;10(2).

  5. Hussein S, Kelly M, Yuang S, Samuel A, Ton S, Yik Andy Y, et al. 578 CD8-targeted IL-2 drives potent anti-tumor efficacy and promotes action of tumor specific vaccines. Journal for ImmunoTherapy of Cancer. 2021;9(Suppl 2):A607.

  6. Caushi JX, Zhang J, Ji Z, Vaghasia A, Zhang B, Hsiue EH, et al. Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers. Nature. 2021;596(7870):126–32.

  7. Schad SE, Chow A, Mangarin L, Pan H, Zhang J, Ceglia N, et al. Tumor-induced double positive T cells display distinct lineage commitment mechanisms and functions. J Exp Med. 2022;219(6).

  8. Yost KE, Satpathy AT, Wells DK, Qi Y, Wang C, Kageyama R, et al. Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat Med. 2019;25(8):1251–9.

  9. Liu B, Hu X, Feng K, Gao R, Xue Z, Zhang S, et al. Temporal single-cell tracing reveals clonal revival and expansion of precursor exhausted T cells during anti-PD-1 therapy in lung cancer. Nature Cancer. 2022;3(1):108–21.

  10. Ceglia N, Sethna Z, Freeman SS, Uhlitz F, Bojilova V, Rusk N, et al. GeneVector: Identification of transcriptional programs using dense vector representations defined by mutual information. bioRxiv. 2023:2022.04.22.487554.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.