Tumor-infiltrating CD39+CD8+ T cells determine poor prognosis and immune evasion in clear cell renal cell carcinoma patients

Abstract

Purpose: Tumor microenvironment is important in the progression of clear cell renal cell carcinoma (ccRCC), and its prognostic value is still unclear. Recent reports demonstrated tumor-infiltrating CD39⁺CD8⁺ T cells are abundant, but their function remains obscure. We aim to assess clinical value of CD39⁺CD8⁺ T cells and seek a potential therapeutic target in ccRCC.

Experimental design: We immunohistochemically evaluated clinical value of CD39⁺CD8⁺ T cells in a retrospective Zhongshan Hospital cohort of 243 ccRCC patients. Fresh tumor samples (n = 48), non-tumor tissues and peripheral blood for flow cytometry analyses were collected to analyze immune cell functions from Zhongshan Hospital. The survival benefit of tyrosine kinase inhibitors (TKIs) in this subpopulation was evaluated. Kaplan-Meier analysis and COX regression model were applied for survival analyses. Bioinformatics analysis performed in TCGA KIRC cohort and the scRNA-seq cohort.

Results: We found that accumulation of CD39⁺CD8⁺ T cells indicated poor prognosis (p < 0.0001) and indicated therapeutic benefit of TKIs therapy (p = 0.015). CD39⁺CD8⁺ T cells showed decreased TNF-α and IFN-γ with elevated PD-1 and TIM-3 expression. Further analysis of tumor-infiltrating immune cell landscape in the ccRCC revealed the positive correlation between CD39⁺CD8⁺ T cells and Tregs (p = 0.037) and M2-polarized macrophages (p < 0.0001). Finally, inhibition of CD39 partially restores the anti-tumor function of CD8⁺ T cells.

Conclusions: High CD39⁺CD8⁺ T cells indicated poor prognosis in ccRCC, due to impaired anti-tumor function of CD39⁺CD8⁺ T cells and indicated therapeutic benefit of TKIs therapy.

Keywords: CD39; CD8+ T cell; Clear cell renal cell carcinoma; Prognosis; Tyrosine kinase inhibitors.

Fig. 1

Accumulation of CD39⁺CD8⁺ T cells in renal cell carcinoma associated with disease progression. a, b The representative flow cytometry and statistics analysis of CD39 expression on CD8⁺ T cells in matched PBMCs, non-tumor tissues and tumor from patients with ccRCC (n = 10). ^✱✱✱✱p < 0.0001 by paired t test. c Representative images of the immunofluorescence staining with CD39 (red), CD8 (green),DAPI (blue) and Merge (double positive) on ccRCC tissues. Scans were imaged at 200 magnification. d, e The percentage of CD39 expressed on CD8⁺ T cells is more in tumor than in non-tumor tissues. ^✱✱✱✱p < 0.0001 by Student’ s t test. f The percentage of CD39⁺CD8⁺ T cells was elevated as the TNM stage progressed. ^✱p < 0.05 by Chi square test. B = blood, N = non-tumor, T = tumor

Fig. 2

CD39⁺CD8⁺ T cells predict sunitinib therapeutic response. Kaplan–Meier curve of overall survival (OS) and recurrence-free survival (RFS) of 243 non-metastatic ccRCC patients according to the CD8⁺ T cells expression (a, d), and percentage of CD39⁺CD8⁺ cells (b, e) and to the combination of both markers (c, f). Log-rank tests were used to derive p values for comparisons between groups. Multivariate Cox analysis identified the independent prognostic factors for OS and RFS in ccRCC patients (g, i). ROC analysis for predictive accuracy of SSIGN alone and combined with CD39 expression level for 10-year OS (h) and RFS (j). HR, hazard radio; CI, confidence interval

Fig. 3

The proliferation of CD39⁺CD8⁺ T cells is repressed along with loss of function. a, b Representative flow cytometry plots for TNF-α, IFN-γ, GZMB and PRF-1 in CD39⁻ and CD39⁺ cells, with quantification below for 26 patients. c Correlation assessed by Pearson’s correlation analysis and linear regression analysis between percentage of CD39⁺CD8⁺ T cells and the frequencies of four effector markers in ccRCC patients based on the results of flow cytometry. d, e Representative FACS figures and statistics analysis of Ki-67⁺ cells frequencies in ccRCC tissue from patients with CD39⁻ and CD39⁺. f Correlation between expression of ENTPD-1 and effector/exhausted markers through scRNA-seq. Bar plots show mean ± SD in panel B. ns: no significance, ^✱p < 0.05; ^✱✱p < 0.01 by paired t test and Spearman’s test

Fig. 4

PD-1 and Tim-3 are increased in the CD39⁺CD8⁺ T cells, and POM-1 can recover partial function of CD39⁺CD8⁺ T cells. a, b Expression of immune checkpoint in CD39⁻CD8⁺ and CD39⁺CD8⁺ T cells (n = 26, FMO was used for the control). c Frequencies of TNF-α⁺CD8⁺ T cells and IFN-γ⁺CD8⁺ T cells from patients (n = 12) after treated POM-1 or DMSO. Expression of PD-1 and Tim-3 from patients (n = 12) after treated POM-1 or DMSO. Bar plots show mean ± SD. ns: no significance, ^✱p < 0.05; ^✱✱p < 0.01; ^✱✱✱p < 0.001 and ^✱✱✱✱p < 0.0001 by paired t test. Low: percentage of CD39⁺CD8⁺ T cells low; high: percentage of CD39⁺CD8⁺ T cells high

Fig. 5

Infiltration of CD39⁺CD8⁺ T cells in the tumor microenvironment is accompanied by Tregs and M2. a Heat map displaying scaled expression of various immune cell types between high/low percentage of CD39⁺CD8⁺ T cells. b–f Comparison of the various immune cell types between high/low percentage of CD39⁺CD8⁺ T cells. ns: no significance, ^✱p < 0.05 and ^✱✱p < 0.01 by Mann–Whitney U test; B cell = B cells; CD4 = CD4⁺ T cells; Th1 = type 1 T helper cells; Th2 = type 2 T helper cells; CD8 = CD8⁺ T cells; DC = dendritic cells; M1 = M1 macrophages; M2 = M2 macrophages; Mast = mast cells; Neut = neutrophils; NK = nature killer cells; Treg = regulatory T cells

Fig. 6

CD39⁺CD8⁺ T cells predict sunitinib therapeutic response. a, b TKI treatment responses (n = 85) in the group of CD8⁺ and the percentage of CD39⁺CD8⁺ cells