Background: CD8+ tumor-infiltrating lymphocytes (TIL) are associated with survival in ovarian cancer. A second subpopulation of TIL – defined by FoxP3 expression – has been reported to inhibit tumor immunity, resulting in decreased patient survival. However, recent studies by our lab and others have challenged this paradigm by showing that FoxP3+ T cells, in some patient cohorts, are associated with favorable prognosis.
Hypothesis: FoxP3 expression encompasses heterogeneous populations of effector and regulatory T cells that vary in proportion between patients and provide differential prognostication.
Methodology: We used multi-parameter flow cytometry to assess CD25, CD28, CD39, CD45RO, CD103, CD127, CTLA-4, CCR4, CCR7, GITR, Helios, ICOS, IFN-gamma, and PD-1 in CD3+CD4+CD8-FoxP3+ TIL in 12 patients with high-grade serous ovarian cancer (HGSC). CD4 and CD8 expression within FoxP3+ TIL was assessed by four channel multi-parameter immuno-histochemistry (mIHC). CD8-FoxP3+ TIL were assessed for CD25 and CD39 expression by mIHC. Survival analysis will be assessed via Kaplan-Meier plots, log-rank tests and Cox regression analyses.
Results: Consistent with prior reports, flow cytometry revealed that FoxP3 was expressed predominantly by CD4+ TIL. All CD4+FoxP3+ cells also expressed CD28, CD45RO, ICOS and CTLA-4, while they lacked expression of CD103 and IFN-gamma. However, we observed highly varied expression of regulatory T cell markers, including CD25, CD39, and GITR. Furthermore, on average, 4% (range 0-25%) of FoxP3+ TIL were CD8+. TCR spectratyping revealed similar TCR Vβ usage patterns between FoxP3- and FoxP3+ TIL. Although mIHC confirmed the variable expression of CD39 in CD8-FoxP3+ TIL, the expression of CD39 by a portion of HGSC tumors eliminated our ability to assess the clinical significance of this subset. The clinical significance of CD25 expression in CD8-FoxP3+ TIL is currently being analyzed in 245 cases of HGSC by mIHC.
Conclusions: FoxP3+ TIL demonstrate profound phenotypic heterogeneity within and between HGSC patients. FoxP3- and FoxP3+ TIL appear to be clonally related, suggesting their distinct phenotypes reflect microenvironmental influences rather than developmental origin or antigen specificity. The variable expression of CD25 and CD39 may be of importance in defining clinically important FoxP3+ TIL subsets. Further study of FoxP3+ TIL subpopulations in HGSC is required to better understand their contributions to tumor immunity and patient survival.
Citation Format: Ronald J. deLeeuw, Sara E. Kost, Brad H. Nelson. Clinical significance of FOXP3+ T cell heterogeneity in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B88.