Systematic analysis of CD39, CD103, CD137, and PD-1 as biomarkers for naturally occurring tumor antigen-specific TILs

Abstract

The detection of tumor-specific T cells in solid tumors is integral to interrogate endogenous antitumor responses and to advance downstream therapeutic applications. Multiple biomarkers are reported to identify endogenous tumor-specific tumor-infiltrating lymphocytes (TILs), namely CD137, PD-1, CD103, and CD39; however, a direct comparison of these molecules has yet to be performed. We evaluated these biomarkers in primary human ovarian tumor samples using single-cell mass cytometry to compare their relative phenotypic profiles, and examined their response to autologous tumor cells ex vivo. PD-1⁺ , CD103⁺ , and CD39⁺ TILs all contain a CD137⁺ cell subset, while CD137⁺ TILs highly co-express the aforementioned markers. CD137⁺ TILs exhibit the highest expression of cytotoxic effector molecules compared to PD-1⁺ , CD103⁺ , or CD39⁺ TILs. Removal of CD137⁺ cells from PD-1⁺ , CD103⁺ , or CD39⁺ TILs diminish their IFN-γ secretion in response to autologous tumor cell stimulation, while CD137⁺ TILs maintain high HLA-dependent IFN-γ secretion. CD137⁺ TILs exhibited an exhausted phenotype but with CD28 co-expression, suggesting possible receptiveness to reinvigoration via immune checkpoint blockade. Together, our findings demonstrate that the antitumor abilities of PD-1⁺ , CD103⁺ , and CD39⁺ TILs are mainly derived from a subset of CD137-expressing TILs, implicating CD137 as a more selective biomarker for naturally occurring tumor-specific TILs.

Keywords: CD103; CD137; CD39; PD-1; tumor-infiltrating lymphocytes.

Figure 1.

A small population of patient TILs positive for activation, tumor-specific markers also co-express effector molecules. CyTOF was performed on human ovarian tumor digests and analyzed using metaPhenoGraph.Experiment was repeated twice for a total of 5 samples (A) Metaclustering analysis identified 7 metaPhenoGraph metaclusters for ovarian cancer patient CD3⁺CD45⁺ TILs (n = 5). (B) Bar plot represents metacluster frequency per patient. Error bars represent the 95% confidence interval (n = 5). (C) metaPhenoGraph cluster results are represented as a heatmap, showing expression of Ki67, CD4, CD8, and effector markers (n = 5). (D) metaPhenoGraph heatmap displaying activation/tumor-specific markers (n = 5).

Figure 2.

TILs positive for CD137 have enhanced expression of effector molecules compared to other markers indicative of tumor-specificity. CyTOF analysis was performed on human ovarian tumor digests and analyzed via viSNE (A) or traditional biaxial gating (B). (A) viSNE plots of tumor-specific markers used in the literature (CD137, CD103, PD-1, CD39), activation markers (CD25, CD69), a TNFR family member (OX40), and effector molecules IFN-γ and TNF-α. CD4 and CD8 TILs are represented as well. Experiment was conducted twice for a total of 5 samples. (B) Plots comparing frequency of effector molecule expression between activation and markers of tumor-specificity (n= 15).Experiment was independently performed four times, with the exception of CD39 where the experiment was repeated three times. The Student’s two-tailed, paired t-test was run to determine statistical significance. NS represents a P-value >0.050, (*) represents a P-value ≤ 0.050, (**) represents a P-value <0.01, (***) represents a P-value < 0.001, (****) represents a P-value < 0.0001, boxplots depict the median, quartiles, with whiskers representing the min and max, error bars are 95% confidence interval.

Figure 3.

CD137⁺ TILs are rare and highly co-express PD-1, CD103, and CD39. Human ovarian tumor digests were interrogated using CyTOF and analyzed using traditional biaxial gating. Experiment was conducted a total of four times, with the exception of CD39 which was performed three times independently.(A) Frequency of activation and tumor-specific markers within CD3⁺CD45⁺ TILs (n= 15).(B) Co-expression patterns of CD137⁺, PD-1⁺, CD103⁺, and CD39⁺ TILs (n = 15 with the exception of CD39⁺ TIL co-expression for PD-1, n = 10). (C) Schematic of CD137, PD-1, CD103, and CD39 predicted T cell expression according to phenotypic findings. The Student’s two-tailed, paired t-test was run to determine statistical significance. NS represents a P-value > 0.050, (*) represents a P-value ≤ 0.050, (**) represents a P-value < 0.01, (***) represents a P-value < 0.001, (****) represents a P-value < 0.0001, error bars are 95% CI with center values representing the mean.

Figure 4.

Removal of CD137⁺ T cells decreases effector molecule production in other biomarker subsets. (A and B) CyTOF analysis was run on human ovarian tumor digests and analyzed via biaxial gating. (C) TIL subsets were co-cultured with autologous tumor cells and supernatants were analyzed after 24 h by LEGENDplax. (A) Expression of IFN-γ and (B) Granzyme B within CD137⁺ and CD137⁻ subpopulation within tumor-specific and activation markers (n = 15). Experiment was conducted four times, for a total of total of 15 samples, with the exception of the CD39 plot where the experiment was repeated three times for a total of 15 samples. Representative gating is shown in Supporting Information Figure 2C. (C) IFN-γ secretion following autologous tumor co-culture in three different ovarian patient samples. Blue dashed and blue number on the y-axis indicates the lowest sensitivity of the assay according to the standard curve. Assay flow setup is represented in Supporting Information Figure 2D. Experimented was conducted three times, with one patient ran at a time. The Student’s two-tailed, paired t-test was run to determine statistical significance. NS represents a P-value > 0.050, (*) represents a P-value ≤ 0.050, (**) represents a P-value < 0.01, (***) represents a P-value < 0.001, (****) represents a P-value < 0.0001, error bars are 95% confidence interval with center values representing the mean.

Figure 5.

Both CD4⁺ and CD8⁺ TILs express markers indicative of antitumor reactivity. Human ovarian tumor digests were interrogated by CyTOF and analyzed via biaxial gating. Experiment was independently run four times, with the exception for CD39 which was repeated three times. (A) Frequency of CD4⁺ and CD8⁺ TILs in live CD3⁺CD45⁺ TILs (n= 19).(B) Frequency of tumor-specific markers within CD4⁺ and CD8⁺ TILs. Sample size per group: CD137 (n = 19), CD39 (n = 14), CD103, PD-1, CD25, CD69, and OX40 (n = 18). (C) Frequency of effector molecules within CD4⁺ and CD8⁺ TILs. The Student’s two-tailed, paired t-test was run to determine statistical significance. NS represents a P-value > 0.050, (*) represents a P-value ≤ 0.050, (**) represents a P-value < 0.01, (***) represents a P-value < 0.001, (****) represents a P-value < 0.0001, error bars are 95% confidence interval with center values representing the mean.

Figure 6.

CD137⁺ TILs within the ovarian cancer tumor microenvironment have a phenotype indicative of exhaustion. CyTOF analysis was performed on human ovarian tumor digests and analyzed via biaxial gating. (A) metaPhenoGraph heatmap (same samples and setup as in Figure 1) displaying markers mainly of activation, co-stimulation, and exhaustion. Experiment was conducted twice (n = 5). (B) Frequencies of exhaustion markers TIGIT, EOMES, and CD39 in CD137⁺ versus CD137⁻ TILs (n = 11). Experiment was repeated three times. (C) Representative gating of EOMES^hiT–bet^lo and EOMES^hi T-bet^lo in CD137⁺ and CD137⁻ live CD3⁺CD45⁺ TILs. (D) EOMES^hiT–bet^lo and EOMES^hi T-bet^lo frequencies in CD137⁺ and CD137⁻ TILs (n = 7). Experiment was performed independently twice. The Student’s two-tailed, paired t-test was run to determine statistical significance. NS represents a P-value > 0.050, (*) represents a P-value ≤ 0.050, (**) represents a P-value <0.01, (***) represents a P-value < 0.001, (****) represents a P-value < 0.0001, error bars are 95% confidence interval with center values representing the mean.