Tumor infiltrating lymphocytes are enriched for highly suppressive CD4+Foxp3+regulatory T cells compared to peripheral blood: a flow cytometric analysis of two distinct cancer types

Abstract Characterizing TIL composition allows monitoring of immunotherapies and can be a prognostic factor for clinical outcome. Here we analyze the phenotype and function of lymphocytes collected from peripheral blood and tumor infiltrating lymphocytes, immediately following resection, from 15 patients with colorectal cancer liver metastases (CRLM) and 21 ovarian cancer primary tumors (OVC). Samples were analyzed using a multi-color flow panel and by qPCR for cytokine production. The percentage of different lymphocyte subpopulations was similar in both patients. Highly suppressive Tregs expressing HLA-DR were significantly increased in TIL compared to PBL in CRLM: 69.4% vs 31.7% (p=0.0002) and in OVC: 74.8% versus 38.6% (p<0.0001). Enhanced expression of CD39, CTLA-4 and Helios on the surface of the Treg, confirmed that TIL are enriched for a highly suppressive subpopulation. The cytokine profile showed that IL-6, is uniquely detected in CD4+ T cells in TIL samples. Both TIL populations also contained a significantly higher percentage of activated CD8+ T cells (HLA-DR+/CD38+) compared to PBL (CRLM: 30.2% vs 7.7%, (p=0.0012), OVC 57.3% vs 11.9%, (p<0.0001)). This study demonstrates that multi-color flow of fresh tumor samples is an effective method to understand the activation state of TIL and may reveal subpopulations not observed in fixed tissue samples. TIL composition were remarkably similar and enriched for highly suppressive Tregs and activated CD8+ T cells.