Biomarkers of tumor-reactive CD4⁺ and CD8⁺ TILs associate with improved prognosis in endometrial cancer

Background: Despite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear.

Methods: To better understand the role of TILs in EC, we analyzed the phenotypic traits of CD8⁺ and CD4⁺ EC^-resident T cells from 47 primary tumors by high-dimensional flow cytometry. In addition, CD8⁺ and CD4⁺ TIL subpopulations were isolated based on the differential expression of programmed cell death protein-1 (PD-1) (negative, dim and high) and CD39 (positive or negative) by fluorescence activated cell sorting (FACS), expanded in vitro, and screened for autologous tumor recognition. We further investigated whether phenotypic markers preferentially expressed on CD8⁺ and CD4⁺ tumor-reactive TIL subsets were associated with the four distinct molecular subtypes of EC, tumor mutational burden and patient survival.

Results: We found that CD8⁺TILs expressing high levels of PD-1 (PD-1hi) co-expressed CD39, TIM-3, HLA-DR and CXCL13, as compared with TILs lacking or displaying intermediate levels of PD-1 expression (PD-1^- and PD-1^dim, respectively). Autologous tumor reactivity of sorted and in vitro expanded CD8+ TILs demonstrated that the CD8⁺PD-1^dimCD39⁺ and PD-1^hiCD39⁺ T cell subsets both contained tumor-reactive TILs and that a higher level of PD-1 expression was associated with increased CD39 and a superior frequency of tumor reactivity. With respect to CD4⁺ T conventional (Tconv) TILs, co-expression of inhibitory and activation markers was more apparent on PD-1^hi compared with PD-1^- or PD-1^dim T cells, and in fact, it was the CD4⁺PD-1^hi subpopulation that accumulated the antitumor T cells irrespective of CD39 expression. Most importantly, detection of CD8⁺PD-1^hiCD39+ and CD4⁺PD-1^hi tumor-reactive T-cell subsets, but also markers specifically expressed by these subpopulations of TILs, that is, PD-1^hi, CD39, CXCL13 and CD103 by CD8⁺ TILs and PD-1^hi and CXCL13 by CD4⁺ Tconv TILs, correlated with prolonged survival of patients with EC.

Conclusions: Our results demonstrate that EC are frequently infiltrated by tumor-reactive TILs, and that expression of PD-1^hi and CD39 or PD-1^hi can be used to select and expand CD8⁺ and CD4⁺ tumor-reactive TILs, respectively. In addition, biomarkers preferentially expressed on tumor-reactive TILs, rather than the frequency of CD3⁺, CD8⁺ and CD4⁺ lymphocytes, hold prognostic value suggesting their protective role in antitumor immunity.