Nicholas R. Schindler

Abstract Immune checkpoint inhibitors targeting the PD-1 pathway have transformed treatment of advanced renal cell carcinoma (RCC), but mechanisms underlying the therapeutic response remain largely unknown. In this study, we perform transcriptomic analysis on RCC biospecimens from 102 patients enrolled in a phase II clinical trial of first-line nivolumab (NCT03117309) to investigate determinants of response to anti–PD-1 monotherapy. Through bulk analysis, we identify an enrichment of genes associated with tertiary lymphoid structures (TLS) in responding patients. Using single-cell transcriptomics and external cohort validation, we identify a population of tissue-resident (ZNF683+SLAMF7+) exhausted CD8+ T cells enriched in patients with poor clinical outcomes. Integrating these findings, we find tumors with high TLS and low tissue-resident exhausted CD8+ T cells that have superior clinical outcomes with nivolumab. Altogether, these analyses contribute to a growing understanding of how the tumor microenvironment drives immune checkpoint inhibitor resistance and propose possible therapeutic targets to rationally overcome resistance to anti–PD-1 monotherapy. Significance: We describe a paradigm wherein combined high TLS and low tissue-resident exhausted CD8+ T cells are required for optimal response to PD-1 blockade in RCC. This analysis identifies key determinants of response to PD-1 blockade in advanced RCC and suggests avenues for future immune modulation through rational combination therapy strategies.

BACKGROUND: Natural killer (NK) cells are important contributors to antitumor immunity in clear-cell renal cell carcinoma (ccRCC). However, their phenotype, function, and association with clinical outcomes in ccRCC remain poorly understood. MATERIALS AND METHODS: We analyzed single-cell RNA sequencing data from 13 primary tumors, 1 localized tumor extension, and 1 metastasis from ccRCC patients at different clinical stages. For each primary tumor specimen, paired normal kidneys were also analyzed. Differential gene expression analysis was carried out to investigate NK cell phenotypes and to derive a gene expression signature. Gene signatures from NK cell subclusters of interest were used to interrogate bulk transcriptomic datasets and expression with clinical outcomes. Finally, tumor-infiltrating NK cell function (cytokine production and cytotoxicity) was assessed by isolation of live NK cells from ccRCC tissue, co-culture with K562 target cells, and measurement of cytokine production (interferon-γ) and cytotoxicity (CD107a) markers by flow cytometry. RESULTS: Single-cell transcriptomic data were analyzed from 13 patients with ccRCC (tumor/normal kidney), resulting in 21 139 NK cells. Clustering analysis revealed six NK cell subsets. Bright-like NK cells were significantly enriched in advanced ccRCC compared with localized ccRCC and normal kidney, expressed markers of tissue residency (ZNF683/Hobit, ITGA1/CD49a, CD9, ITGAE/CD103), and had decreased expression of cytotoxicity genes (GZMB/Granzyme-B, PRF1/perforin). In independent cohorts (The Cancer Genome Atlas ccRCC cohort, CheckMate 025), a gene expression score representing this dysfunctional NK cell phenotype was enriched in advanced ccRCC and was associated with worse overall survival. Functional interrogation of tumor-infiltrating NK cells from ccRCC confirmed that tumor-resident CD49a+CD9+ NK cells had impaired cytotoxicity compared with CD49a-CD9- NK cells. CONCLUSIONS: A dysfunctional, tumor-resident NK cell phenotype was enriched among patients with metastatic disease and associated with worse survival in patients with advanced ccRCC across multiple patient cohorts. Restoration of NK cell function (via cytokine stimulation or NK cell engineering) could provide a novel avenue for therapeutic intervention against ccRCC.

PURPOSE While immune checkpoint inhibition (ICI) has transformed the management of many advanced renal cell carcinomas (RCCs), the determinants of effective antitumor immunity for chromophobe RCC (ChRCC) and renal oncocytic tumors remain an unmet clinical and scientific need. METHODS Single-cell transcriptomic and T-cell receptor profiling was performed on tumor and adjacent normal tissue of patients with ChRCC and renal oncocytic neoplasms. Using machine learning, the cellular origin of renal oncocytic neoplasms was evaluated, with analysis of associated oncogenic pathways. Using immunohistochemistry, immune infiltration was analyzed in renal oncocytic neoplasms in comparison with clear cell RCC (ccRCC). Immune checkpoint expression, clonal expansion, and tumor specificity were compared between ChRCC and ccRCC. Using the International Metastatic RCC Database Consortium data set, clinical outcomes of patients with metastatic ChRCC (mChRCC) treated with first-line systemic regimens were compared with those of patients with ccRCC. RESULTS We validated α-intercalated cells as the cellular origin of renal oncocytic neoplasms. We identified a downregulation of HLA class I molecules with enrichment of potentially targetable pathways including mammalian target of rapamycin and ferroptosis in ChRCC. The tumor microenvironment of ChRCC showed markedly decreased immune infiltration, with a pronounced depletion in tumor-infiltrating CD8 + T cells. ChRCC-infiltrating CD8 + T cells demonstrated lower immune checkpoint expression, diminished clonal expansion, and decreased tumor specificity. Clinical analysis identified poor survival outcomes selectively among patients with mChRCC treated with immune-based therapies. CONCLUSION Immunogenomic analysis of ChRCC revealed profound depletion of T cells, with an immune phenotype marked by a lack of expression of immune checkpoints and poor tumor specificity, suggesting that the few T cells in these tumor types are likely nonspecific bystanders. This immune-cold environment hinders an effective response to immunotherapy and underscores the need for ChRCC-tailored treatments designed to improve tumor-specific T-cell infiltration into the microenvironment.

<div>Abstract<p>Immune checkpoint inhibitors targeting the PD-1 pathway have transformed treatment of advanced renal cell carcinoma (RCC), but mechanisms underlying the therapeutic response remain largely unknown. In this study, we perform transcriptomic analysis on RCC biospecimens from 102 patients enrolled in a phase II clinical trial of first-line nivolumab (NCT03117309) to investigate determinants of response to anti–PD-1 monotherapy. Through bulk analysis, we identify an enrichment of genes associated with tertiary lymphoid structures (TLS) in responding patients. Using single-cell transcriptomics and external cohort validation, we identify a population of tissue-resident (<i>ZNF683</i><sup>+</sup><i>SLAMF7</i><sup>+</sup>) exhausted CD8<sup>+</sup> T cells enriched in patients with poor clinical outcomes. Integrating these findings, we find tumors with high TLS and low tissue-resident exhausted CD8<sup>+</sup> T cells that have superior clinical outcomes with nivolumab. Altogether, these analyses contribute to a growing understanding of how the tumor microenvironment drives immune checkpoint inhibitor resistance and propose possible therapeutic targets to rationally overcome resistance to anti–PD-1 monotherapy.</p>Significance:<p>We describe a paradigm wherein combined high TLS and low tissue-resident exhausted CD8<sup>+</sup> T cells are required for optimal response to PD-1 blockade in RCC. This analysis identifies key determinants of response to PD-1 blockade in advanced RCC and suggests avenues for future immune modulation through rational combination therapy strategies.</p></div>