Progressive natural killer cell dysfunction in advanced-stage clear-cell renal cell carcinoma and association with clinical outcomes

Abstract

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.

Keywords: NK cells; immunogenomics; renal cell carcinoma; single-cell transcriptomics.

Figure 1

A cluster of non-cytotoxic bright-like NK cells is enriched in advanced ccRCC. (A) Schematic of single-cell transcriptomic profiling of NK cells subsetted from an scRNA-seq dataset of immune cells in advancing disease stages of ccRCC and adjacent normal tissue. (B) Relative proportions of NK cells compared with total immune cell populations (PTPRC/CD45+ cell clusters) in normal (non-malignant adjacent) kidney, localized ccRCC tumors (stage I-III), and advanced RCC (stage IV) samples. P values displayed for Wilcoxon rank sum test for pair-wise comparison between the different stages of ccRCC (green and red) and control (orange). (C) Dot plot of canonical NK gene markers consisting of markers for bright NK cells and dim NK cells, including annotation of re-clustered NK populations based on the markers. (D) UMAP representation of sub-clustered NK cell populations, displaying substantial heterogeneity (five clusters). (E) UMAP representations of NK cells in normal kidney, localized RCC, and advanced RCC. In metastatic RCC, cluster 0 is highlighted. (F) Box plot of the relative proportion of NK subpopulations (as a proportion of total NK cells), normalized within the sample across disease stages and control with sample count normal = 13, localized = 8, advanced = 4. For box plots, hinges are 25th-75th percentiles; central lines are medians, whiskers are highest and lowest values no greater than 1.5× interquartile range, and dots are outliers. ccRCC, clear-cell renal cell carcinoma; NK, natural killer; scRNA-seq, single-cell RNA sequencing; UMAP, uniform manifold approximation and projection.

Figure 2

NK cells in advanced ccRCC are depleted of cytotoxic genes and express markers associated with tissue residency. (A) Volcano plot of differentially expressed genes in C0.Bright-like compared with all other NK populations. In red are genes significantly enriched in the C0.Bright-like population (q value < 0.05; log₂ fold change >1; q value is the Bonferroni-corrected P value). In blue are genes significantly depleted in the C0.Bright-like population. Select genes displayed are involved in cytotoxicity and tissue residency. (B and C) Heatmap of selected significantly (B) up-regulated or (C) downregulated in the C0.Bright-like cluster compared with all other NK populations. (D) Characteristics of the C0.Bright-like NK population. (Left) UMAP feature plots show module scores of defined biologically relevant gene sets smoothed over 634 neighbor cells (see ‘Materials and methods’ section). Violin plots for smoothed module scores are shown in all NK subpopulations (middle) and across RCC stages (right). ccRCC, clear-cell renal cell carcinoma; NK, natural killer; RCC, renal cell carcinoma; UMAP, uniform manifold approximation and projection.

Figure 3

C0.Bright-like NK cells are associated with worse overall survival in external clinical datasets. (A) Box plots showing significantly higher signature score of C0.Bright-like population in advanced RCC compared with normal and localized RCC in the external The Cancer Genome Atlas ccRCC cohort (TCGA KIRC; two-sided Wilcoxon rank sum test for pair-wise comparison). (B) Overall survival for TCGA KIRC cohort based on high gene signature for C0.Bright-like (≥median) versus low signature expression (high versus low threshold at the median; log-rank test). Higher signature for the C0 cluster indicates a lower survival. (C) PFS for CheckMate 025 cohort patients within the nivolumab treatment group based on high gene signature for C0.Bright-like (≥median) versus low signature expression, showing no significant difference in PFS. Log-rank test for significance. (D) PFS for CheckMate 025 cohort patients within the everolimus treatment group based on high gene signature for C0.Bright-like (≥median) versus low signature expression, showing no significant difference in PFS. Log-rank test for significance. (E) Overall survival for all CheckMate 025 cohort patients based on high gene signature for C0.Bright-like (≥median) versus low signature expression, showing worse overall survival for patients with a high signature score. Log-rank test for significance. PFS, progression-free survival; RCC, renal cell carcinoma; TCGA, The Cancer Genome Atlas; UMAP, uniform manifold approximation and projection.

Figure 4

CD49a+CD9+ NK cells isolated from metastatic tumors lack antitumor functional activity. (A) Schematic describing in vitro assays to assess the function of NK cells isolated from ccRCC patients with metastatic disease. (B) Proportions of NK cells isolated from the tumor (sorted live, CD56+ cells) based on CD49a and CD9 expression. (C) Flow cytometry gating strategy. (D) Graph showing the percentage of CD49a+CD9+ and CD49a−CD9− NK cells that are IFN-γ positive after 6-h co-culture with K562 target cells. (E) Graph showing the percentage of CD49a+CD9+ or CD49a−CD9− NK cells that are CD107a+ after co-culture for 6 h with K562 target cells. n = 5 renal cell carcinoma cases. ∗P < 0.05 one-tailed paired t-test. ccRCC, clear-cell renal cell carcinoma; IFN-γ, interferon-γ; NK, natural killer.