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TCF1+PD-1+ tumour-infiltrating lymphocytes predict a favorable response and prolonged survival after immune checkpoint inhibitor therapy for non-small-cell lung cancer

  • Jaemoon Koh
    Affiliations
    Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea

    Laboratory of Immune Regulation in Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea
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  • Sehui Kim
    Affiliations
    Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea

    Department of Pathology, Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea
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  • Yeon Duk Woo
    Affiliations
    Laboratory of Immune Regulation in Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea
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  • Seung Geun Song
    Affiliations
    Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea

    Laboratory of Immune Regulation in Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea
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  • Jeemin Yim
    Affiliations
    Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea
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  • Bogyeong Han
    Affiliations
    Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea
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  • Sojung Lim
    Affiliations
    Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea
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  • Hyun Kyung Ahn
    Affiliations
    Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea

    Cancer Research Institute, Seoul National University, Seoul, Republic of Korea
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  • Seungchan Mun
    Affiliations
    Cancer Research Institute, Seoul National University, Seoul, Republic of Korea

    Interdiscipilinary Program of Cancer Biology, Seoul National University Graduate School, Seoul, Republic of Korea

    Integrated Major in Innovative Medical Science, Seoul National University Graduate School, Republic of Korea
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  • Jung Sun Kim
    Affiliations
    Cancer Research Institute, Seoul National University, Seoul, Republic of Korea

    Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea
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  • Bhumsuk Keam
    Affiliations
    Cancer Research Institute, Seoul National University, Seoul, Republic of Korea

    Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea
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  • Young A Kim
    Affiliations
    Department of Pathology, Seoul Metropolitan Government Boramae Hospital, Seoul, Republic of Korea
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  • Se-Hoon Lee
    Affiliations
    Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea

    Department of Health Sciences and Technology, Samsung Advanced Institute of Health Sciences and Technology, Sungkyunkwan University, Seoul, Republic of Korea
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  • Author Footnotes
    1 Y. K. Jeon and D. H. Chung contributed equally to this work.
    Yoon Kyung Jeon
    Correspondence
    Corresponding author: Department of Pathology, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 03080, Republic of Korea. Fax: +82 2 743 5530.
    Footnotes
    1 Y. K. Jeon and D. H. Chung contributed equally to this work.
    Affiliations
    Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea

    Cancer Research Institute, Seoul National University, Seoul, Republic of Korea

    Interdiscipilinary Program of Cancer Biology, Seoul National University Graduate School, Seoul, Republic of Korea

    Integrated Major in Innovative Medical Science, Seoul National University Graduate School, Republic of Korea
    Search for articles by this author
  • Author Footnotes
    1 Y. K. Jeon and D. H. Chung contributed equally to this work.
    Doo Hyun Chung
    Correspondence
    Corresponding author: Department of Pathology, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 03080, Republic of Korea. Fax: +82 2 743 5530.
    Footnotes
    1 Y. K. Jeon and D. H. Chung contributed equally to this work.
    Affiliations
    Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea

    Laboratory of Immune Regulation in Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea
    Search for articles by this author
  • Author Footnotes
    1 Y. K. Jeon and D. H. Chung contributed equally to this work.
Open AccessPublished:August 13, 2022DOI:https://doi.org/10.1016/j.ejca.2022.07.004

      Highlights

      • Progenitor exhausted T-cell signature is related to a better response to ICI in NSCLC.
      • TCF1+PD-1+ tumour-infiltrating lymphocytes represent progenitor exhausted T cells.
      • High TCF1+PD-1+ TIL levels predict responsiveness to ICI and better survival in NSCLC.
      • TCF1+PD-1+ TIL is a useful predictive biomarker for ICI therapy in NSCLC patients.

      Abstract

      Background

      T-cell factor 1 (TCF1)+Programmed cell death-1 (PD-1)+ tumour-infiltrating lymphocytes (TILs) are a recently defined subset of exhausted T-cells (Texh-cells) that exhibit a progenitor phenotype. They have been associated with a response to immune checkpoint inhibitor (ICI) therapy in murine tumour models and in patients with malignant melanoma. We investigated the significance of TCF1+PD-1+ TILs as a predictive biomarker for ICI therapy response in non-small-cell lung cancer (NSCLC).

      Methods

      Two different cohorts of NSCLC patients treated with ICI targeting the PD-1/PD-L1 pathway were included. RNA-seq was performed using NSCLC tissues obtained from 234 patients prior to immunotherapy (RNA-seq cohort). Double immunostaining of TCF1 and PD-1 and single immunostaining of other immunologic markers were performed in resected tumour tissues from another 116 patients (immunohistochemistry cohort).

      Results

      In the RNA-seq cohort, both Texh-cell and progenitor Texh-cell gene sets were enriched in responders compared with non-responders. Larger Texh-cell fractions and increased progenitor Texh-cell gene sets were significantly associated with better progression-free survival (PFS). In the immunohistochemistry cohort, the TCF1+PD-1+ TIL number and PD-L1 tumour proportion score were significantly higher in responders than in non-responders. A high number of TCF1+PD-1+ TILs was significantly associated with both PFS and overall survival (OS) after ICI therapy, and it independently predicted a better PFS and OS according to multivariate analysis.

      Conclusion

      TCF1+PD-1+ TILs, representing progenitor Texh-cells, predict both better response and survival in NSCLC patients after ICI therapy. Thus, they may be a useful predictive biomarker for ICI therapy in NSCLC.

      Keywords

      1. Introduction

      Immunotherapy based on immune checkpoint inhibitors (ICIs) has become the standard care for lung cancer patients and, in some cases, has replaced conventional treatments as a first-line therapy [
      • Ettinger D.S.
      • Wood D.E.
      • Aisner D.L.
      • Akerley W.
      • Bauman J.R.
      • Bharat A.
      • et al.
      NCCN Guidelines Insights: Non-Small Cell Lung Cancer, version 2.2021.
      ,
      • Herbst R.S.
      • Giaccone G.
      • de Marinis F.
      • Reinmuth N.
      • Vergnenegre A.
      • Barrios C.H.
      • et al.
      Atezolizumab for first-line treatment of PD-L1-selected patients with NSCLC.
      ]. Programmed cell death ligand 1(PD-L1) immunohistochemistry (IHC) is widely used as a companion diagnostic for ICI targeting Programmed cell death-1 (PD-1) and PD-L1 in non-small-cell lung cancer (NSCLC) [
      • Prince E.A.
      • Sanzari J.K.
      • Pandya D.
      • Huron D.
      • Edwards R.
      Analytical concordance of PD-L1 assays utilizing antibodies from FDA-approved diagnostics in advanced cancers: a systematic literature review.
      ]. However, the presence of PD-L1-positive tumour cells is not always correlated with a clinical benefit [
      • Davis A.A.
      • Patel V.G.
      The role of PD-L1 expression as a predictive biomarker: an analysis of all US Food and Drug Administration (FDA) approvals of immune checkpoint inhibitors.
      ,
      • Hirsch L.
      • Zitvogel L.
      • Eggermont A.
      • Marabelle A.
      PD-Loma: a cancer entity with a shared sensitivity to the PD-1/PD-L1 pathway blockade.
      ]. Therefore, the development of a predictive biomarker for ICI therapy would improve the management of NSCLC patients.
      The functional impairment of T-cells in the tumour microenvironment (TME) has been demonstrated in many kinds of cancers [
      • Thommen D.S.
      • Schumacher T.N.
      T cell dysfunction in cancer.
      ,
      • Xia A.
      • Zhang Y.
      • Xu J.
      • Yin T.
      • Lu X.J.
      T cell dysfunction in cancer immunity and immunotherapy.
      ]. Prolonged T-cell activation by cancer antigens leads to the exhaustion of T-cells via the TOX-mediated transcriptional and epigenetic program [
      • Alfei F.
      • Kanev K.
      • Hofmann M.
      • Wu M.
      • Ghoneim H.E.
      • Roelli P.
      • et al.
      TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.
      ,
      • Khan O.
      • Giles J.R.
      • McDonald S.
      • Manne S.
      • Ngiow S.F.
      • Patel K.P.
      • et al.
      TOX transcriptionally and epigenetically programs CD8(+) T cell exhaustion.
      ,
      • Buchholz V.R.
      • Busch D.H.
      Back to the future: effector fate during T cell exhaustion.
      ]. The exhausted T-cells (Texh-cells) express PD-1, a receptor that contributes to the suppression of effector T-cell function upon engagement with its ligand PD-L1 [
      • Ahmadzadeh M.
      • Johnson L.A.
      • Heemskerk B.
      • Wunderlich J.R.
      • Dudley M.E.
      • White D.E.
      • et al.
      Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired.
      ,
      • Baitsch L.
      • Baumgaertner P.
      • Devevre E.
      • Raghav S.K.
      • Legat A.
      • Barba L.
      • et al.
      Exhaustion of tumor-specific CD8(+) T cells in metastases from melanoma patients.
      ]. Recent studies have identified phenotypically and functionally heterogeneous subsets of exhausted CD8 T-cells in chronic viral infections and in murine tumour models [
      • Im S.J.
      • Hashimoto M.
      • Gerner M.Y.
      • Lee J.
      • Kissick H.T.
      • Urger M.C.B.
      • et al.
      Defining CD8(+) T cells that provide the proliferative burst after PD-1 therapy.
      ,
      • Siddiqui I.
      • Schaeuble K.
      • Chennupati V.
      • Marraco S.A.F.
      • Calderon-Copete S.
      • Ferreira D.P.
      • et al.
      Intratumoral Tcf1(+)PD-1(+)CD8(+) T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy.
      ]. Two phenotypically and functionally different subsets have been described: stem-like/or progenitor Texh-cells (progenitor Texh-cells), characterised by the expression of transcription factor TCF1 (encoded by TCF7) and an intermediate level of PD-1 expression; and terminally differentiated Texh-cells, which do not express TCF1 but co-express high levels of PD-1, TIM-3, and other co-inhibitory receptors and effector molecules [
      • Im S.J.
      • Hashimoto M.
      • Gerner M.Y.
      • Lee J.
      • Kissick H.T.
      • Urger M.C.B.
      • et al.
      Defining CD8(+) T cells that provide the proliferative burst after PD-1 therapy.
      ]. Of these two subsets, preclinical models of chronic viral infection and cancer have shown that TCF1+PD-1+ progenitor Texh-cells have stem-like properties, including the ability to expand, self-renew, and differentiate into TCF1PD-1+ T-cells after PD-1 blockade [
      • Siddiqui I.
      • Schaeuble K.
      • Chennupati V.
      • Marraco S.A.F.
      • Calderon-Copete S.
      • Ferreira D.P.
      • et al.
      Intratumoral Tcf1(+)PD-1(+)CD8(+) T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy.
      ,
      • Utzschneider D.T.
      • Charmoy M.
      • Chennupati V.
      • Pousse L.
      • Ferreira D.P.
      • Calderon-Copete S.
      • et al.
      T cell factor 1-expressing memory-like CD8(+) T cells sustain the immune response to chronic viral infections.
      ,
      • Thommen D.S.
      The first shall (be) last: understanding durable T cell responses in immunotherapy.
      ]. Thus, TCF1+PD-1+ progenitor Texh-cells rather than terminal Texh-cells may contribute to the response to ICI therapy [
      • Siddiqui I.
      • Schaeuble K.
      • Chennupati V.
      • Marraco S.A.F.
      • Calderon-Copete S.
      • Ferreira D.P.
      • et al.
      Intratumoral Tcf1(+)PD-1(+)CD8(+) T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy.
      ]. Moreover, TCF1+PD-1+ tumour-infiltrating lymphocytes (TILs) have been observed in the tumour tissues of patients with malignant melanoma, and their presence is associated with responsiveness to ICI therapy [
      • Siddiqui I.
      • Schaeuble K.
      • Chennupati V.
      • Marraco S.A.F.
      • Calderon-Copete S.
      • Ferreira D.P.
      • et al.
      Intratumoral Tcf1(+)PD-1(+)CD8(+) T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy.
      ]. Tumour-infiltrating progenitor CD8 T-cells were also detected in human NSCLC via single-cell sequencing analysis [
      • Brummelman J.
      • Mazza E.M.C.
      • Alvisi G.
      • Colombo F.S.
      • Grilli A.
      • Mikulak J.
      • et al.
      High-dimensional single cell analysis identifies stem-like cytotoxic CD8(+) T cells infiltrating human tumors.
      ]. However, the status of progenitor Texh-cells or TCF1+PD-1+ TILs in patients with NSCLC and their role as a predictive biomarker of ICI therapy are unknown. We address these issues using two different cohorts of NSCLC patients treated with an ICI.

      2. Materials and methods

      2.1 Patients

      Two cohorts of NSCLC patients who received ICI (PD-1 or PD-L1 blockade) therapy were included in this study. In the RNA-seq cohort, tumour tissues obtained prior to ICI therapy from 234 patients (Suppl. Table S1), who were treated at Samsung Medical Center (SMC) and Seoul National University Hospital (SNUH), Seoul, Republic of Korea, were subjected to RNA-seq.
      In the IHC cohort, NSCLC tissues were subjected to histopathological and IHC analyses. Of the 768 NSCLC patients who received ICI therapy at SNUH between 2014 and 2019, 116 patients underwent surgical resection before ICI therapy; whole sections from representative formalin-fixed paraffin-embedded tissue (FFPE) blocks of their resected tumours were subjected to IHC (Suppl. Fig. S1). Treatment response was evaluated based on the Response Evaluation Criteria in Solid Tumours version 1.1, and clinical outcome was defined as detailed in the Supplementary Methods. The study was approved by the Institutional Review Boards of SNUH (H-1905-115-1035) and SMC (SMC-2013-10-112 and SMC-2018-03-130) and all participants provided written informed consent.

      2.2 RNA-seq

      RNA was extracted from fresh or FFPE tumour samples using the AllPrep DNA/RNA mini kit (QIAGEN, Hilden, Germany). The library was prepared using the RNA Access library prep kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions. RNA-seq reads were aligned using STAR 2.5.2b, and gene expression was quantified using RSEM 1.3.0 with batch effect correction. Quality control of mapping was performed using the tools included in Picard and RSeQC.

      2.3 Gene set enrichment analysis

      Gene sets for exhausted CD8 T-cells, progenitor exhausted CD8 T-cells, and terminally differentiated exhausted CD8 T-cells were assembled from previous studies (Suppl. Table S2) [
      • Doering T.A.
      • Crawford A.
      • Angelosanto J.M.
      • Paley M.A.
      • Ziegler C.G.
      • Wherry E.J.
      Network analysis reveals centrally connected genes and pathways involved in CD8+ T cell exhaustion versus memory.
      ,
      • Miller B.C.
      • Sen D.R.
      • Al Abosy R.
      • Bi K.
      • Virkud Y.V.
      • LaFleur M.W.
      • et al.
      Subsets of exhausted CD8(+) T cells differentially mediate tumor control and respond to checkpoint blockade.
      ]. Gene set enrichment analysis (GSEA) was performed using GSEA software (version 3.0) downloaded from the Broad Institute (http://www.broadinstitute.org/gsea).

      2.4 Assessment of progenitor Texh gene signature score

      The progenitor Texh gene signature score in RNA-seq cohort was calculated from the mean gene expression z-scores using the gene sets defined/curated for precursor Texh cell signature in a previous study (Suppl. Table S3) [
      • Connolly K.A.
      • Kuchroo M.
      • Venkat A.
      • Khatun A.
      • Wang J.
      • William I.
      • et al.
      A reservoir of stem-like CD8(+) T cells in the tumor-draining lymph node preserves the ongoing antitumor immune response.
      ].

      2.5 Deconvolution of tumour-infiltrating immune cells

      Deconvolution of the tumour-infiltrating immune cells was performed using RNA-seq data and the web-based tool ImmuCellAI (http://bioinfo.life.hust.edu.cn/web/ImmuCellAI) and the relative proportions of 22 types of immune cells were estimated.

      2.6 Two-colour multiplex chromogenic IHC and single IHC

      Two-colour multiplex chromogenic IHC (double IHC) of TCF1 and PD-1 and single IHC of CD8, CD56, CD163, and Foxp3 were performed using the antibodies listed in Suppl. Table S4 and the Benchmark XT autostainer (Ventana Medical Systems, Tucson, AZ). Whole-slide images were obtained using the Aperio ScanScope slide scanner (Aperio Technologies, Vista, CA). The numbers/mm2 of CD8+, CD56+, Foxp3+, and CD163+ cells were automatically counted using QuPath [
      • Bankhead P.
      • Loughrey M.B.
      • Fernandez J.A.
      • Dombrowski Y.
      • McArt D.G.
      • Dunne P.D.
      • et al.
      QuPath: open source software for digital pathology image analysis.
      ].
      In the double IHC analysis, more than three different high-power fields (HPFs) with the most active signals from a slide were analysed. From these areas, TCF1+, PD-1+, TCF1+PD-1+ TILs (i.e. TILs expressing both TCF1 and PD-1) and TCF1PD-1+ TILs (i.e. TILs expressing PD-1 but not TCF1) were counted; the cell counts are presented as mean numbers per HPF.
      PD-L1 IHC was performed using the PD-L1 IHC 22C3 pharmDx system and the tumour proportion score (TPS) was estimated according to the manufacturer's recommendation (Agilent Technologies, Santa Clara, CA).
      Manual evaluation of IHC was performed by two pathologists (YKJ and JK) blinded to the clinical information.

      2.7 Flow cytometry

      To investigate whether TCF1+PD-1+ TILs are of the progenitor Texh-cell phenotype, matched fresh tumour tissues and FFPE tissue sections from another 15 patients with NSCLC were subject to flow cytometry and double IHC, respectively. Single-cell suspensions were prepared from fresh tumour tissues as described previously [
      • Koh J.
      • Kim H.Y.
      • Lee Y.
      • Park I.K.
      • Kang C.H.
      • Kim Y.T.
      • et al.
      IL23-producing human lung cancer cells promote tumor growth via conversion of innate lymphoid cell 1 (ILC1) into ILC3.
      ], and cells were stained for CD45, CD3, CD8, CXCR5, TIM-3, TCF1, and granzyme β using the antibodies listed in Suppl. Table S5.

      2.8 Quantification of tertiary lymphoid structures

      The numbers of mature and immature tertiary lymphoid structures (TLSs) in a representative tumour area were determined per low-power field (×40) as described in the Supplementary Methods.

      2.9 Statistical analysis

      Statistical analysis was performed using Prism (version 8.0; GraphPad) and SPSS (version 25; IBM Corp.) software as detailed in the Supplementary Methods. Two-sided P-values < 0.05 were considered to indicate statistical significance.

      3. Results

      3.1 Transcriptome analysis of tumour-infiltrating immune cell subsets in NSCLC and the association with patient survival and ICI therapy response

      In the RNA-seq cohort, among the immune subsets (estimated by ImmuCellAI), the proportions of CD8 T-cells and NK cells were significantly higher, and those of CD4 T-cells and regulatory T-cells significantly lower, in patients with partial response (PR) than in those with progressive disease (PD) and/or stable disease (SD) (Fig. 1A). Although the proportion of Texh-cells was highest in patients with SD, it was significantly higher in patients with DCB than in those without DCB (Fig. 1A). The PD-L1 TPS and progenitor Texh gene signature score were significantly higher in patients with PR than in those with PD (Fig. 1B). In the GSEA, both Texh-cell and progenitor Texh-cell gene sets were significantly enriched in the responders to ICI therapy compared with patients with PD (Fig. 1C).
      Fig. 1
      Fig. 1Tumour-infiltrating immune cell subsets and their association with responsiveness to ICI therapy and survival of patients (analysis using RNA-seq data). (A) Proportions of total CD8 T-cells or exhausted CD8 T-cells (upper left), CD4 T-cells, NK-cells, and regulatory T-cells (lower) in ICI-treated NSCLC patients according to treatment response (PD, n = 116; SD, n = 56; PR, n = 62). Proportions of exhausted CD8 T-cells in patients with or without a durable clinical benefit (DCB) after ICI therapy (upper right; no DCB, n = 116; DCB, n = 118). (B) Tumour proportion score (TPS) determined by PD-L1 IHC and progenitor Texh gene signature score determined by RNA-seq in patients according to treatment response. (C) Gene set enrichment analysis of exhausted T-cells and progenitor exhausted T-cells based on the gene signature in patients non-responsive (PD, progressive disease) and responsive (PR, partial response) to ICI therapy. (D) Kaplan–Meier analysis of progression-free survival (PFS) after ICI therapy according to the fraction of CD8 T-cells or exhausted T-cells based on RNA-seq data. (E) Kaplan–Meier analysis of PFS according to PD-L1 TPS or progenitor Texh gene signature score. n.s., not significant. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
      In survival analysis of the RNA-seq cohort, a large Texh-cell subset was correlated with longer progression-free survival (PFS) (P = 0.002, Fig. 1D). Patients with a high PD-L1 TPS (10% cut-off) had a longer PFS than those with a low PD-L1 TPS (P = 0.01) (Fig. 1E). A high progenitor Texh gene signature score was associated with prolonged survival of NSCLC patients treated with ICI (P = 0.038) (Fig. 1E).
      Analysis of publicly available RNA-seq data from patients with NSCLC and melanoma receiving ICI therapy (Supplementary Methods) [
      • Riaz N.
      • Havel J.J.
      • Makarov V.
      • Desrichard A.
      • Urba W.J.
      • Sims J.S.
      • et al.
      Tumor and microenvironment evolution during immunotherapy with nivolumab.
      ] showed that a higher progenitor Texh gene signature score was associated with a better response and survival after ICI therapy (Suppl. Fig. S2A–C). Taken together, these data suggest that progenitor Texh-cells are associated with survival and a response to ICI therapy in NSCLC.

      3.2 Associations among TCF1+PD-1+ progenitor Texh-cells, other immune cell subsets, and clinicopathological features in NSCLC

      To examine the clinicopathological implications of progenitor and terminally differentiated Texh-cells in NSCLC patients treated with ICIs, IHC and histological evaluation were performed using the resected tumour tissues of another cohort including 116 patients treated with ICI (Table 1; Fig. 2A–E). A previous study reported the co-expression of PD-1 and TCF1 as a marker of progenitor exhausted lymphocytes [
      • Miller B.C.
      • Sen D.R.
      • Al Abosy R.
      • Bi K.
      • Virkud Y.V.
      • LaFleur M.W.
      • et al.
      Subsets of exhausted CD8(+) T cells differentially mediate tumor control and respond to checkpoint blockade.
      ]. To confirm that TCF1+PD-1+ TILs detected by double IHC represent progenitor Texh-cells, we performed multicolour flow cytometry and double IHC on NSCLC tissue from 15 patients. In flow cytometry, TCF1highPD-1int CD8 T-cells, which are regarded as progenitor Texh-cells, showed high CXCR5 and low TIM-3 and granzyme β expression levels, suggesting that these cells have the phenotype of progenitor Texh-cells (Suppl. Fig. S3A, B). Furthermore, a significant positive correlation between the number of TCF1+PD-1+ TILs by double IHC and the proportion of TCF1highPD-1int CD8 T-cells of CD3 T-cells was observed (Fig. 2F, G). Based on these findings, TCF1+PD-1+ and TCF1PD-1+ TILs were considered to represent progenitor Texh-cells and terminally differentiated Texh-cells, respectively.
      Table 1Characteristics of patients in the immunohistochemistry cohort.
      CharacteristicsN (% or range)
      AgeMedian (range)63.4 (34–88)
      SexMale93 (80.2)
      Female23 (19.8)
      ECOG PS0–1112 (96.5)
      24 (3.5)
      SmokingNever34 (29.3)
      Ever82 (70.7)
      HistologyADC57 (49.1)
      SqCC32 (27.6)
      Other
      Adenosquamous carcinoma and sarcomatoid carcinoma.
      27 (23.3)
      Tumour sizeMedian (range)3.86 (1–8.6)
      LN metastasisAbsent58 (50.8)
      Present56 (49.2)
      Stage at diagnosisI30
      II31
      III55
      Genetic alterationsEGFR mutation14 (12.3)
      KRAS mutation7 (6.1)
      ALK translocation2 (1.8)
      ImmunotherapyAnti-PD-L1
      Atezolizumab, durvalumab.
      25 (21.6)
      Anti-PD-1
      Nivolumab, pembrolizumab.
      91 (78.4)
      Prior lines of chemotherapy027 (23.3)
      154 (46.5)
      ≥235 (30.2)
      Prior use of targeted therapy
      EGFR tyrosine kinase inhibitors (gefitinib, osimertinib).
      Absent101 (86.8)
      Present15 (13.2)
      Response to ICI therapyComplete response0 (0)
      Partial response40 (34.5)
      Stable disease28 (24.1)
      Progression disease48 (41.4)
      PS, performance status; ADC, adenocarcinoma; SqCC, squamous cell carcinoma.
      a Adenosquamous carcinoma and sarcomatoid carcinoma.
      b Atezolizumab, durvalumab.
      c Nivolumab, pembrolizumab.
      d EGFR tyrosine kinase inhibitors (gefitinib, osimertinib).
      Fig. 2
      Fig. 2Representative images and relationship of immune subsets in the tumour microenvironment (TME), tumour PD-L1 expression, and tertiary lymphoid structures (TLS). (A) Representative image of tumour expression of PD-L1 clone 22C3 as detected by IHC. (B, C) CD8+, Foxp3+, PD-1+, and CD56+ tumour-infiltrating lymphocytes (TILs) (B) and CD163+ cells (C) in the TME. (D) Representative image of double staining of TCF1 (magenta, nuclear staining) and PD-1 (DAB, brown, membrane staining) in lymphocytes. TCF1+PD-1+ TILs expressing both PD-1 and TCF1 (solid arrow) and TCF1PD-1+ TILs expressing only PD-1 (empty arrow) are shown. (A–D: scale bar, 100 μm) (E) Representative image of H&E-stained NSCLC tissue, showing a TLS in the TME. Scale bar, 200 μm. (F) Representative number or fraction of TCF1+PD-1+ TILs according to expression of TCF1 and PD-1 in CD8 TILs in a flow cytometric dot plot (red box) and double IHC of TCF1 and PD-1 (solid arrow). (G) Correlation of the number of TCF1+PD-1+ TILs according to double IHC and TCF1highPD-1int CD8 TILs as determined by flow cytometry of matched tumour samples. (H) Heatmap of immune cell subsets, PD-L1 tumour proportion score (TPS), and TLSs as determined by IHC and H&E staining of tissues from the IHC cohort. Heatmap was generated using the R package complexHeatmap after normalising the values of the variables. (I) PD-L1 TPS; numbers of CD8+, CD56+, Foxp3+, TCF1PD-1+ TILs, and CD163+ cells; and number of TLS according to number of TCF1+PD-1+ TILs (low; ≤1 per HPF, n = 68; high >1 per HPF, n = 48). n.s., not significant. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
      The correlations between the immune subsets and clinicopathologic features are summarised in Table 2. TCF1PD-1+ TIL numbers were higher in tumours ≥3 cm than in those <3 cm in size (P = 0.028). There was no significant difference in the number of TCF1+PD-1+ TILs as a function of the clinicopathological features.
      Table 2Correlations between clinicopathologic factors and immune cell subsets in the tumour microenvironment.
      CharacteristicsTCF1+PD-1+ TILsTCF1PD-1+ TILsCD8+ TILsCD56+ TILsFoxp3+ TILsCD163+ cells
      SexMale2.73 ± 4.4910.83 ± 9.70262.44 ± 160.141.28 ± 1.949.18 ± 19.1630.87 ± 38.45
      Female1.47 ± 3.889.69 ± 12.98228.51 ± 176.030.75 ± 1.165.56 ± 9.7531.28 ± 46.76
      P-value0.2060.7660.3840.2340.3920.966
      Age<652.83 ± 5.0111.75 ± 19.52263.37 ± 153.411.22 ± 1.866.90 ± 14.2729.41 ± 30.94
      ≥652.21 ± 3.629.37 ± 11.92247.59 ± 174.131.12 ± 1.7910.20 ± 20.9132.63 ± 48.21
      P-value0.4610.4340.610.7750.3250.671
      SmokingNever2.19 ± 4.1510.67 ± 15.09225.65 ± 147.681.58 ± 2.687.42 ± 10.7130.44 ± 34.43
      Ever2.66 ± 4.5010.58 ± 16.81267.24 ± 167.991.02 ± 1.368.88 ± 19.8131.15 ± 42.08
      P-value0.6140.980.2280.1450.6980.934
      HistologyADC2.75 ± 4.4610.08 ± 11.91222.91 ± 141.211.19 ± 2.0711.06 ± 23.0627.33 ± 36.62
      SqCC2.56 ± 5.0712.51 ± 22.34263.72 ± 154.471.04 ± 1.205.61 ± 7.6529.11 ± 41.49
      Other1.81 ± 2.658.93 ± 16.64345.70 ± 210.131.37 ± 1.985.45 ± 7.9045.66 ± 45.98
      P-value0.7290.6960.0150.8230.2610.21
      Tumour size<32.19 ± 4.027.17 ± 7.29269.28 ± 13.8231.37 ± 1.8112.52 ± 26.4031.45 ± 47.96
      ≥32.73 ± 4.6112.90 ± 19.57247.87 ± 176.621.06 ± 1.836.08 ± 8.9430.66 ± 34.78
      P-value0.5250.0280.5030.3740.1330.925
      Lymph node metastasisAbsent2.11 ± 4.4412.13 ± 20.57263.28 ± 168.681.15 ± 1.4611.67 ± 23.0231.98 ± 39.54
      Present3.01 ± 4.369.55 ± 10.40243.81 ± 156.261.22 ± 2.165.26 ± 9.0526.06 ± 29.11
      P-value0.2810.4010.5280.8180.0550.37
      EGFR mutationAbsent2.73 ± 4.6910.25 ± 15.77257.35 ± 164.881.11 ± 1.658.56 ± 18.4328.99 ± 34.58
      Present1.81 ± 2.1316.50 ± 20.82244.06 ± 157.091.29 ± 2.639.70 ± 15.5631.03 ± 40.31
      P-value0.4720.1870.7780.7280.8270.841
      ADC, adenocarcinoma; SqCC, squamous cell carcinoma.
      The distributions of PD-L1 TPS, the relative numbers of immune cell subsets, and TLSs in the IHC cohort are presented in Fig. 2H and I. Tumours with high TCF1+PD-1+ TIL levels (>1 per HPF) had a significantly higher PD-L1 TPS and more TCF1PD-1+ TILs compared to tumours with low TCF1+PD-1+ TIL levels (≤1 per HPF) (Fig. 2I and Supple. Fig. S3C). A positive correlation between TCF1+PD-1+ TILs and TCF1PD-1+ TILs is in agreement with their proposed differentiation. However, heatmap (Fig. 2H) showed that there were populations with high levels of TCF1PD-1+ TILs but low levels of TCF1+PD-1+ TILs. These findings suggest that tumours may have a unique progenitor exhausted T-cell repertoire that could not be predicted solely by the status of terminally exhausted T-cells.

      3.3 Relationship between the number of TCF1+PD-1+ TILs and responsiveness to ICI therapy in NSCLC

      In the IHC cohort, the number of TCF1+PD-1+ TILs and the PD-L1 TPS were significantly higher in responders than in non-responders to ICI (Fig. 3A, B). Patients with a DCB after ICI had a significantly higher number of TCF1+PD-1+ TILs and a higher PD-L1 TPS than patients without a DCB (Fig. 3C). By contrast, the number of TCF1PD-1+ TILs was not related to either ICI responsiveness or clinical benefit (Fig. 3A–C). In the group that received anti-PD-1 antibody as ICI therapy (n = 91), a higher PD-L1 TPS and a higher number of TCF1+PD-1+ TILs were significantly related to greater clinical benefit (Suppl. Fig. S4A, B).
      Fig. 3
      Fig. 3Relationship between TCF1+PD-1+ tumour-infiltrating lymphocytes (TILs) and other immune cell subsets and correlations with the ICI treatment response. (A) PD-L1 TPS; numbers of CD8+, CD56+, Foxp3+, PD-1+, TCF1+PD-1+, TCF1PD-1+ TILs, and CD163+ cells; and number of TLS according to response to immunotherapy (PD, n = 48; SD, n = 28; PR, n = 40) (B) PD-L1 TPS; numbers of CD8+, CD56+, Foxp3+, PD-1+, TCF1+PD-1+, TCF1PD-1+ TILs, and CD163+ cells; and number of TLS according to response to immunotherapy (non-responder, n = 76; responder, n = 40). (C) PD-L1 TPS; numbers of CD8+, CD56+, Foxp3+, PD-1+, TCF1+PD-1+, TCF1PD-1+ TILs, and CD163+ cells; and number of TLS according to durable clinical benefit (DCB) (no DCB, n = 48; DCB, n = 68). n.s., not significant. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

      3.4 Prognostic significance and the role of a predictive biomarker for TCF1+PD-1+ TILs in patients with NSCLC receiving ICI therapy

      In a survival analysis of the IHC cohort, the PD-L1 TPS (1% cut-off), TLS, and numbers of CD8+, CD56+, and TCF1PD-1+ TILs were not significantly related to PFS or overall survival (OS) (Fig. 4A–C). By contrast, large numbers of PD-1+ and TCF1+PD-1+ TILs were significantly related to a prolonged PFS and OS; the statistical significance was greater for TCF1+PD-1+ TILs (P < 0.001) than PD-1+ TILs (P = 0.01) for both PFS and OS (Fig. 4C, D). When the patients were categorised into three groups according to the number of TCF1+PD-1+ TILs, an increasing number of TCF1+PD-1+ TILs was correlated with better PFS and OS (Fig. 4D). By contrast, an analysis of the recurrence-free survival of patients after surgical resection of the primary tumour showed that the number of TCF1+PD-1+ TILs was not a significant prognostic indicator (Fig. 4E). In the group that received anti-PD-1 antibody, a large number of TCF1+PD-1+ TILs was significantly related to prolonged PFS and OS (Suppl. Fig. S4C).
      Fig. 4
      Fig. 4An increased number of TCF1+PD-1+ tumour-infiltrating lymphocytes (TILs) predicts longer survival after ICI therapy in patients with NSCLC. (A) Kaplan–Meier analysis of progression-free survival (PFS) and overall survival (OS) in NSCLC patients treated with ICI according to a PD-L1 TPS 1% cut-off (TPS <1, n = 59; TPS ≥1, n = 57). (B) PFS and OS according to number of CD8+ TILs (low, ≤200, n = 50; high, >200, n = 66), CD56+ TILs (low, ≤1, n = 71; high, >1, n = 45), and TLS (low, ≤1, n = 75; high, >1, n = 41). (C) PFS and OS according to number of PD-1+ TILs (low, ≤8, n = 54; high, >8, n = 62) and TCF1PD-1+ TILs (low, ≤5, n = 71; high, >5, n = 45). (D) PFS and OS in patients with a low (≤1 per HPF, n = 64) or high (>1 per HPF, n = 52) number of TCF1+PD-1+ TILs (left) and stratified into three groups based on the number of TCF1+PD-1+ TILs (low, <1, n = 48; intermediate, 1–5, n = 51; high, >5, n = 17) (right). (E) Kaplan–Meier curve for postoperative recurrence-free survival of NSCLC patients who underwent surgery before ICI treatment based on the number of TCF1+PD-1+ TILs (low, ≤1; high, >1). Survival difference was analysed using the Kaplan–Meier method and log-rank test. (F) Multivariate Cox regression analysis of PFS and OS after ICI therapy. For each variable, the estimated hazard ratio and 95% confidence interval are shown.
      In a multivariate analysis, a large number of TCF1+PD-1+ TILs was an independent predictor of improved PFS (P < 0.001) and OS (P = 0.007) after ICI therapy in patients with NSCLC (Fig. 4F).

      4. Discussion

      The clinical implications of progenitor Texh-cells as a predictive biomarker for ICI therapy in patients are unclear. This is the first study to explore TCF1+PD-1+ TILs as a surrogate marker of progenitor Texh-cells in human NSCLC. Our results showed that the number of TCF1+PD-1+ TILs predicts improvements in both the therapy responsiveness and survival of patients with NSCLC after ICI therapy.
      The transcription factor TCF1 plays an important role in follicular helper T-cell (Tfh) differentiation, memory CD8 T-cell formation, and sustained cytotoxicity against pathogens and tumours [
      • Raghu D.
      • Xue H.H.
      • Mielke L.A.
      Control of lymphocyte fate, infection, and tumor immunity by TCF-1.
      ]. It was also recently identified as a key transcription factor in maintaining progenitor Texh-cell populations and in effectively reinvigorating the anti-tumour immune response after ICI therapy [
      • Thommen D.S.
      The first shall (be) last: understanding durable T cell responses in immunotherapy.
      ]. Our study consisted of a retrospective analysis of the potential effect of Texh-cells on the clinical outcome after ICI therapy, determined using two cohorts of NSCLC patients evaluated by RNA-seq or IHC. Our results demonstrated that gene sets for progenitor Texh-cells were enriched in responders to ICI and large numbers of TCF1+PD-1+ TILs were identified as an independent predictor of prolonged survival in patients with NSCLC after ICI therapy.
      Several studies have reported that TCF1+ T-cells form aggregates in the TME and are topologically related to TLSs [
      • Vanhersecke L.
      • Brunet M.
      • Guegan J.P.
      • Rey C.
      • Bougouin A.
      • Cousin S.
      • et al.
      Mature tertiary lymphoid structures predict immune checkpoint inhibitor efficacy in solid tumors independently of PD-L1 expression.
      ,
      • Jansen C.S.
      • Prokhnevska N.
      • Master V.A.
      • Sanda M.G.
      • Carlisle J.W.
      • Bilen M.A.
      • et al.
      An intra-tumoral niche maintains and differentiates stem-like CD8 T cells.
      ]. TLS presence was associated with a better prognosis after ICI therapy in malignant melanoma [
      • Cabrita R.
      • Lauss M.
      • Sanna A.
      • Donia M.
      • Larsen M.S.
      • Mitra S.
      • et al.
      Tertiary lymphoid structures improve immunotherapy and survival in melanoma (vol 577, 561, 2020).
      ,
      • Helmink B.A.
      • Reddy S.M.
      • Gao J.J.
      • Zhang S.J.
      • Basar R.
      • Thakur R.
      • et al.
      B cells and tertiary lymphoid structures promote immunotherapy response.
      ]. However, a significant positive correlation between TCF1+PD-1+ TIL and TLS numbers was not observed in this study, although TCF1+PD-1+ TILs were frequently located in immature TLSs characterised by lymphoid aggregates. In addition, the number of TLSs had no prognostic significance in NSCLC patients treated with an ICI. These results suggest that TCF1+PD-1+ TILs rather than TLSs might be a better predictor of clinical outcome after ICI therapy in NSCLC.
      The prognostic implication of PD-1+ TILs in cancer patients undergoing ICI treatment is controversial [
      • Mazzaschi G.
      • Madeddu D.
      • Falco A.
      • Bocchialini G.
      • Goldoni M.
      • Sogni F.
      • et al.
      Low PD-1 expression in cytotoxic CD8(+) tumor-infiltrating lymphocytes confers an immune-privileged tissue microenvironment in NSCLC with a prognostic and predictive value.
      ,
      • Mazzaschi G.
      • Minari R.
      • Zecca A.
      • Cavazzoni A.
      • Ferri V.
      • Mori C.
      • et al.
      Soluble PD-L1 and circulating CD8+PD-1+ and NK cells enclose a prognostic and predictive immune effector score in immunotherapy treated NSCLC patients.
      ]. In this study, we evaluated the number of PD-1+ TILs selectively in the immune-inflamed tumour area rather than the whole tumour and identified a predictive role for PD-1+ TILs in ICI therapy. However, the prognostic significance of TCF1+PD-1+ TILs was greater than PD-1+ TILs. These findings suggest the heterogeneity of PD-1+ TILs in the TME, as reported previously [
      • Kim C.G.
      • Kim G.
      • Kim K.H.
      • Park S.
      • Shin S.
      • Yeo D.
      • et al.
      Distinct exhaustion features of T lymphocytes shape the tumor-immune microenvironment with therapeutic implication in patients with non-small-cell lung cancer.
      ,
      • Ma J.
      • Zheng B.
      • Goswami S.
      • Meng L.
      • Zhang D.
      • Cao C.
      • et al.
      PD1(Hi) CD8(+) T cells correlate with exhausted signature and poor clinical outcome in hepatocellular carcinoma.
      ,
      • Thommen D.S.
      • Koelzer V.H.
      • Herzig P.
      • Roller A.
      • Trefny M.
      • Dimeloe S.
      • et al.
      A transcriptionally and functionally distinct PD-1(+) CD8(+) T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade.
      ], and emphasise the need for optimisation of PD-1+ TIL evaluation in the TME as a predictive biomarker for ICI.
      Notably, the number of TCF1+PD-1+ TILs was not related to tumour recurrence or progression after initial surgery and IHC was performed using surgically resected tumour tissues obtained 3 months to 16 years (median, 3 years) before ICI therapy. Our findings thus identify TCF1+PD-1+ TILs as a specific biomarker that predicts the response to ICI rather than as a nonspecific prognostic biomarker in patients with NSCLC. They also suggest that the TCF1+PD-1+ lymphocyte-mediated anti-tumour immune response is initiated at an early time point in tumourigenesis, such that it is established and imprinted to the tumour at a considerable level by the time of surgery. Progenitor Texh-cells are thought to infiltrate the TME and to reside or circulate in tumour-draining lymph nodes and peripheral blood [
      • Pauken K.E.
      • Shahid O.
      • Lagattuta K.A.
      • Mahuron K.M.
      • Luber J.M.
      • Lowe M.M.
      • et al.
      Single-cell analyses characterize circulating anti-tumor CD8(+) T cells in mice and humans and identify markers for their enrichment.
      ,
      • Dammeijer F.
      • van Gulijk M.
      • Mulder E.E.
      • Lukkes M.
      • Klaase L.
      • van den Bosch T.
      • et al.
      The PD-1/PD-L1-checkpoint restrains T cell immunity in tumor-draining lymph nodes.
      ,
      • Buchwald Z.S.
      • Nasti T.H.
      • Lee J.
      Tumor-draining lymph node is important for a robust abscopal effect stimulated by radiotherapy (vol 8, e000867, 2020).
      ]. Therefore, each tumour might have a unique progenitor Texh-cell repertoire, which could be estimated at the time of surgical resection or in surgical specimens, if available, to predict the efficacy of ICI therapy.
      This study had a few limitations. First, the utility of TCF1+PD-1+ TILs as a predictive biomarker may be limited in assays using small biopsies, because they are present in only low numbers in the TME. Nonetheless, they were identifiable in surgical resection specimens using double IHC. Second, whether TCF1+PD-1+ TILs represent bona fide progenitor Texh-cells is debatable. TCF1 is expressed in various types of T-cells, including naïve T-cells, memory T-cells, and Tfh-cells [
      • Raghu D.
      • Xue H.H.
      • Mielke L.A.
      Control of lymphocyte fate, infection, and tumor immunity by TCF-1.
      ,
      • Xing S.J.
      • Gai K.X.
      • Li X.
      • Shao P.
      • Zeng Z.H.
      • Zhao X.D.
      • et al.
      Tcf1 and Lef1 are required for the immunosuppressive function of regulatory T cells.
      ]. Among these T-cell subsets, Tfh-cells co-express PD-1 (bright) and TCF1 in secondary B-cell follicles, which uncommon in the TME of NSCLC, and most double-stained cells in this study did not have an intense PD-1 signal [
      • Chen Y.
      • Yu M.
      • Zheng Y.
      • Fu G.
      • Xin G.
      • Zhu W.
      • et al.
      CXCR5(+)PD-1(+) follicular helper CD8 T cells control B cell tolerance.
      ]. Furthermore, we observed a significant positive correlation between the number of TCF1+PD-1+ TILs as detected by IHC and the proportion of PD-1intTCF1high CD8 T-cells as determined by flow cytometry. Therefore, double IHC for TCF1 and PD-1 might be a useful approach for identifying progenitor Texh-cells using FFPE tissue.

      5. Conclusions

      In summary, using two cohorts of NSCLC patients treated with ICI therapy, we demonstrated that TCF1+PD-1+ TILs in the TME serve as a specific predictive biomarker of the response to ICI therapy in patients with NSCLC.

      Authors' contributions

      Conceptualisation: JK and YKJ. Methodology: JK, SK, YDW, SSG, JY, BH, SL, HKA, SM and SHL. Formal analysis: JK, YAK and YKJ. Resources: JK, JSK, BK and SHL. Investigation: JK, SK and YDW. Writing, review, and/or revision of the manuscript: JK, YKJ and DHC. Funding acquisition: DHC, JK and SHL. Supervision: YKJ and DHC.
      All authors read and approved the final manuscript.

      Data availability statement

      The data generated in this study are available upon request from the corresponding author.

      Conflict of interest statement

      The authors declare no conflicts of interest.

      Acknowledgements

      This work was supported by the Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (grant number 2020R1A4A1017515) and the NRF grant funded by the Korea government (Ministry of Science and ICT) (grant number 2020R1F1A1070825, 2020R1A2C3006535), and the Post-Genome Technology Development Program (Business model development driven by clinico-genomic database for precision immuno-oncology) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea) (grant number 10067758).

      Appendix A. Supplementary data

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