Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma

The successful use of expanded tumor-infiltrating lymphocytes (TIL) in adoptive TIL therapies has been reported, but the effects of the TIL expansion, immunophenotype, function, and T cell receptor (TCR) repertoire of the infused products relative to the tumor microenvironment (TME) are not well understood. In this study, we analyzed the tumor samples (n = 58) from treatment-naïve patients with renal cell carcinoma (RCC), “pre-rapidly expanded” TILs (pre-REP TIL, n = 15) and “rapidly expanded” TILs (REP TIL, n = 25) according to a clinical-grade TIL production protocol, with single-cell RNA (scRNA)+TCRαβ-seq (TCRαβ sequencing), TCRβ-sequencing (TCRβ-seq), and flow cytometry. REP TILs encompassed a greater abundance of CD4+ than CD8+ T cells, with increased LAG-3 and low PD-1 expressions in both CD4+ and CD8+ T cell compartments compared with the pre-REP TIL and tumor T cells. The REP protocol preferentially expanded small clones of the CD4+ phenotype (CD4, IL7R, KLRB1) in the TME, indicating that the largest exhausted T cell clones in the tumor do not expand during the expansion protocol. In addition, by generating a catalog of RCC-associated TCR motifs from >1,000 scRNA+TCRαβ-seq and TCRβ-seq RCC, healthy and other cancer sample cohorts, we quantified the RCC-associated TCRs from the expansion protocol. Unlike the low-remaining amount of anti-viral TCRs throughout the expansion, the quantity of the RCC-associated TCRs was high in the tumors and pre-REP TILs but decreased in the REP TILs. Our results provide an in-depth understanding of the origin, phenotype, and TCR specificity of RCC TIL products, paving the way for a more rationalized production of TILs. Significance: TILs are a heterogenous group of immune cells that recognize and attack the tumor, thus are utilized in various clinical trials. In our study, we explored the TILs in patients with kidney cancer by expanding the TILs using a clinical-grade protocol, as well as observed their characteristics and ability to recognize the tumor using in-depth experimental and computational tools.

[1]  W. V. van Harten,et al.  Tumor-Infiltrating Lymphocyte Therapy or Ipilimumab in Advanced Melanoma. , 2022, The New England journal of medicine.

[2]  H. Lähdesmäki,et al.  Evolution and modulation of antigen-specific T cell responses in melanoma patients , 2022, Nature Communications.

[3]  F. Luciani,et al.  VDJdb in the pandemic era: a compendium of T cell receptors specific for SARS-CoV-2 , 2022, Nature Methods.

[4]  H. Lähdesmäki,et al.  Single-cell characterization of leukemic and non-leukemic immune repertoires in CD8+ T-cell large granular lymphocytic leukemia , 2022, Nature Communications.

[5]  Kumiko Goto,et al.  The impact of CCR8+ regulatory T cells on cytotoxic T cell function in human lung cancer , 2022, Scientific Reports.

[6]  Y. Doki,et al.  CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory , 2022, Proceedings of the National Academy of Sciences.

[7]  S. Mustjoki,et al.  T and NK cell abundance defines two distinct subgroups of renal cell carcinoma , 2022, Oncoimmunology.

[8]  Xueda Hu,et al.  Pan-cancer single-cell landscape of tumor-infiltrating T cells , 2021, Science.

[9]  R. Bhatt,et al.  176 Successful generation of tumor-infiltrating lymphocyte (TIL) product from renal cell carcinoma (RCC) tumors for adoptive cell therapy , 2021, Journal for ImmunoTherapy of Cancer.

[10]  S. Tavaré,et al.  Determinants of anti-PD-1 response and resistance in clear cell renal cell carcinoma , 2021, Cancer cell.

[11]  M. Ornstein,et al.  Clinical Review on the Management of Metastatic Renal Cell Carcinoma. , 2021, JCO oncology practice.

[12]  Christopher C. Griffith,et al.  Functional HPV-specific PD-1+ stem-like CD8 T cells in head and neck cancer , 2021, Nature.

[13]  Xiaochen Bo,et al.  clusterProfiler 4.0: A universal enrichment tool for interpreting omics data , 2021, Innovation.

[14]  J. Larkin,et al.  Lifileucel, a Tumor-Infiltrating Lymphocyte Therapy, in Metastatic Melanoma , 2021, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[15]  C. Leslie,et al.  Single-cell sequencing links multiregional immune landscapes and tissue-resident T cells in ccRCC to tumor topology and therapy efficacy. , 2021, Cancer cell.

[16]  H. Lähdesmäki,et al.  Somatic mutations in lymphocytes in patients with immune-mediated aplastic anemia , 2021, Leukemia.

[17]  H. Lähdesmäki,et al.  Predicting recognition between T cell receptors and epitopes with TCRGP , 2021, PLoS Comput. Biol..

[18]  Andrew P. Voigt,et al.  Mapping the immune environment in clear cell renal carcinoma by single-cell genomics , 2021, Communications biology.

[19]  E. V. Van Allen,et al.  Beyond conventional immune-checkpoint inhibition — novel immunotherapies for renal cell carcinoma , 2021, Nature Reviews Clinical Oncology.

[20]  M. V. van Loenen,et al.  T cells expanded from renal cell carcinoma display tumor-specific CD137 expression but lack significant IFN-γ, TNF-α or IL-2 production , 2021, Oncoimmunology.

[21]  Z. Szallasi,et al.  Qualitative Analysis of Tumor-Infiltrating Lymphocytes across Human Tumor Types Reveals a Higher Proportion of Bystander CD8+ T Cells in Non-Melanoma Cancers Compared to Melanoma , 2020, Cancers.

[22]  David Chisanga,et al.  Early precursor T cells establish and propagate T cell exhaustion in chronic infection , 2020, Nature Immunology.

[23]  Guideng Li,et al.  T cell antigen discovery , 2020, Nature Methods.

[24]  S. Mustjoki,et al.  Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease , 2020, Nature Communications.

[25]  R. Offringa,et al.  The Outcome of Ex Vivo TIL Expansion Is Highly Influenced by Spatial Heterogeneity of the Tumor T-Cell Repertoire and Differences in Intrinsic In Vitro Growth Capacity between T-Cell Clones , 2020, Clinical Cancer Research.

[26]  Mark M. Davis,et al.  Analyzing the M. tuberculosis immune response by T cell receptor clustering with GLIPH2 and genome-wide antigen screening , 2020, Nature Biotechnology.

[27]  M. Atkins,et al.  Checkpoint inhibitor immunotherapy in kidney cancer , 2020, Nature Reviews Urology.

[28]  Shannon K. Boi,et al.  Stem, Effector, and Hybrid States of Memory CD8+ T Cells. , 2019, Trends in immunology.

[29]  A. Kamphorst,et al.  An intra-tumoral niche maintains and differentiates stem-like CD8 T cells , 2019, Nature.

[30]  G. Freeman,et al.  Proliferating Transitory T Cells with an Effector-like Transcriptional Signature Emerge from PD-1+ Stem-like CD8+ T Cells during Chronic Infection. , 2019, Immunity.

[31]  Andrew K. Sewell,et al.  VDJdb in 2019: database extension, new analysis infrastructure and a T-cell receptor motif compendium , 2019, Nucleic Acids Res..

[32]  C. Kwak,et al.  Retrospective Multicenter Long-Term Follow-up Analysis of Prognostic Risk Factors for Recurrence-Free, Metastasis-Free, Cancer-Specific, and Overall Survival After Curative Nephrectomy in Non-metastatic Renal Cell Carcinoma , 2019, Front. Oncol..

[33]  J. Verbsky,et al.  Heterogeneity of human bone marrow and blood natural killer cells defined by single-cell transcriptome , 2019, Nature Communications.

[34]  K. Bensalah,et al.  Updated European Association of Urology Guidelines on Renal Cell Carcinoma: Immune Checkpoint Inhibition Is the New Backbone in First-line Treatment of Metastatic Clear-cell Renal Cell Carcinoma. , 2019, European urology.

[35]  M. Delorenzi,et al.  TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection , 2019, Nature.

[36]  S. Berger,et al.  TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion , 2019, Nature.

[37]  G. Naik,et al.  Prognostic impact of immune gene expression signature and tumor infiltrating immune cells in localized clear cell renal cell carcinoma , 2019, Journal of Immunotherapy for Cancer.

[38]  X. Ren,et al.  Prognostic Value of the Neo-Immunoscore in Renal Cell Carcinoma , 2019, Front. Oncol..

[39]  E. Wherry,et al.  CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. , 2019, Annual review of immunology.

[40]  F. Hodi,et al.  Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade , 2019, Nature Immunology.

[41]  Aviv Regev,et al.  Checkpoint Blockade Immunotherapy Induces Dynamic Changes in PD‐1−CD8+ Tumor‐Infiltrating T Cells , 2019, Immunity.

[42]  Daniel E. Speiser,et al.  Intratumoral Tcf1+PD‐1+CD8+ T Cells with Stem‐like Properties Promote Tumor Control in Response to Vaccination and Checkpoint Blockade Immunotherapy , 2019, Immunity.

[43]  Lai Guan Ng,et al.  Dimensionality reduction for visualizing single-cell data using UMAP , 2018, Nature Biotechnology.

[44]  M. Nykter,et al.  Immunogenomic landscape of hematological malignancies , 2018, bioRxiv.

[45]  J. Haanen,et al.  Adoptive cellular therapies: the current landscape , 2018, Virchows Archiv.

[46]  Christoph Hafemeister,et al.  Comprehensive integration of single cell data , 2018, bioRxiv.

[47]  Paul J. Hoover,et al.  Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma , 2018, Cell.

[48]  Francesco Ferrari,et al.  High-dimensional single cell analysis identifies stem-like cytotoxic CD8+ T cells infiltrating human tumors , 2018, The Journal of experimental medicine.

[49]  M. Donia,et al.  T cells isolated from patients with checkpoint inhibitor-resistant melanoma are functional and can mediate tumor regression , 2018, Annals of oncology : official journal of the European Society for Medical Oncology.

[50]  J. Schachter,et al.  Establishment of adoptive cell therapy with tumor infiltrating lymphocytes for non-small cell lung cancer patients , 2018, Cancer Immunology, Immunotherapy.

[51]  Steven J. M. Jones,et al.  The Immune Landscape of Cancer , 2018, Immunity.

[52]  B. Seliger,et al.  T-cell Responses in the Microenvironment of Primary Renal Cell Carcinoma—Implications for Adoptive Cell Therapy , 2018, Cancer Immunology Research.

[53]  Yufeng Shen,et al.  Human Tissue-Resident Memory T Cells Are Defined by Core Transcriptional and Functional Signatures in Lymphoid and Mucosal Sites. , 2017, Cell reports.

[54]  Laurence Zitvogel,et al.  The immune contexture in cancer prognosis and treatment , 2017, Nature Reviews Clinical Oncology.

[55]  S. Mustjoki,et al.  Somatic mutations in clonally expanded cytotoxic T lymphocytes in patients with newly diagnosed rheumatoid arthritis , 2017, Nature Communications.

[56]  A. K. Srivastava,et al.  Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two-stage, single-arm, phase 2 study. , 2017, The Lancet. Oncology.

[57]  S. Lira,et al.  CCR8+FOXp3+ Treg cells as master drivers of immune regulation , 2017, Proceedings of the National Academy of Sciences.

[58]  William S. DeWitt,et al.  Immunosequencing identifies signatures of cytomegalovirus exposure history and HLA-mediated effects on the T cell repertoire , 2017, Nature Genetics.

[59]  Ludmila V. Danilova,et al.  Tumor immune microenvironment characterization in clear cell renal cell carcinoma identifies prognostic and immunotherapeutically relevant messenger RNA signatures , 2016, Genome Biology.

[60]  Charles H. Yoon,et al.  Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq , 2016, Science.

[61]  A. Ravaud,et al.  Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma. , 2015, The New England journal of medicine.

[62]  Mikhail Pogorelyy,et al.  VDJtools: Unifying Post-analysis of T Cell Receptor Repertoires , 2015, PLoS Comput. Biol..

[63]  A. Broeks,et al.  Sunitinib pretreatment improves tumor-infiltrating lymphocyte expansion by reduction in intratumoral content of myeloid-derived suppressor cells in human renal cell carcinoma , 2015, Cancer Immunology, Immunotherapy.

[64]  S. Rosenberg,et al.  Adoptive cell transfer as personalized immunotherapy for human cancer , 2015, Science.

[65]  D. Gilham,et al.  Efficient and reproducible generation of tumour-infiltrating lymphocytes for renal cell carcinoma , 2015, British Journal of Cancer.

[66]  B. Shalmon,et al.  Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Patients with Metastatic Melanoma: Intent-to-Treat Analysis and Efficacy after Failure to Prior Immunotherapies , 2013, Clinical Cancer Research.

[67]  M. Donia,et al.  Characterization and Comparison of ‘Standard’ and ‘Young’ Tumour‐Infiltrating Lymphocytes for Adoptive Cell Therapy at a Danish Translational Research Institution , 2012, Scandinavian journal of immunology.

[68]  S. Steinberg,et al.  Durable Complete Responses in Heavily Pretreated Patients with Metastatic Melanoma Using T-Cell Transfer Immunotherapy , 2011, Clinical Cancer Research.

[69]  B. Shalmon,et al.  Establishment and Large-scale Expansion of Minimally cultured “Young” Tumor Infiltrating Lymphocytes for Adoptive Transfer Therapy , 2011, Journal of immunotherapy.

[70]  Abigail Wacher,et al.  Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells. , 2009, Blood.

[71]  S. Steinberg,et al.  High‐dose interleukin‐2 for the treatment of metastatic renal cell carcinoma , 2008, Cancer.

[72]  J. Cheville,et al.  An outcome prediction model for patients with clear cell renal cell carcinoma treated with radical nephrectomy based on tumor stage, size, grade and necrosis: the SSIGN score. , 2002, The Journal of urology.

[73]  H Nagura,et al.  Proliferative activity of intratumoral CD8(+) T-lymphocytes as a prognostic factor in human renal cell carcinoma: clinicopathologic demonstration of antitumor immunity. , 2001, Cancer research.

[74]  Nicholas L. Bormann,et al.  scRepertoire: An R-based toolkit for single-cell immune receptor analysis. , 2020, F1000Research.

[75]  J. Larkin,et al.  Combined Nivolumab and Ipilimumab or Monotherapy in Previously Untreated Melanoma , 2017 .