49P - CXCL13-Producing Follicular Helper T Cells in Human Breast Cancer

Abstract

Background: Our previous work detected PD-1hi CD200hi follicular helper T cells infiltrating breast tumors (BC Tfh TIL) and associated them with good clinical outcomes. Tfh TILs are the principal cellular source of CXCL13, a strong chemoattractant for CXCR5-expressing cells (B cells and some T cells including Tfh cells). CXCL13 was also the best single gene for survival prediction in HER2+ BC. The aim of this study is to fully characterize CXCL13-producing Tfh TILs and understand their anti-tumor functions. Methods: Ten color flow cytometry was used to analyze human CD4+ TIL in fresh breast tumor specimens and compare Tfh cells with those from tonsils. A retrospective analysis of FFPE samples (N = 80) was assessed for CXCL13 and other Tfh marker gene expression by qPCR. Results: We found that PD-1, CD200 and TIGIT, but not CXCR5 (a hallmark surface receptor for tonsillar Tfh cells) are the most specific surface markers for CXCL13-secreting CD4+ TIL. However, some CXCR5hi CXCL13+ cells are also detected in rare tumors with exceptionally high infiltrates. Interestingly, PD-1hi CD200hi (principally CXCR5neg) TIL abundance is directly proportional to the number of germinal center (GC) B cells, suggesting they play a functional role in B cell maturation. Additionally, PD-1 in association with ICOS revealed three distinct subpopulations in CD4+ TIL. CXCL13-producing cells are enriched in the PD-1hiICOSmed group, FoxP3+ cells (activated and regulatory T cells) are principally in the PD-1medICOShi population, while the PD-1loICOSlo are unactivated cells. qPCR data confirmed CXCL13's value for predicting survival with PDCD1 (PD-1), TIGIT and ICOS also showing predictive potential. Re-examination of our gene expression data found that interferon g (IFNG) mRNA is inversely correlated with surface CXCR5 in cells exposed to tumor supernatant, suggesting a relationship between CXCR5 downregulation and the immunosuppressive potential of the tumor microenvironment. Conclusion: Using novel combinations of surface markers we have identified distinct subpopulations of CD4+ TILs, which although phenotypically different from their tonsillar counterparts may also promote local B cell maturation thereby contributing to the clinical benefit associated with CXCL13 gene expression. Disclosure: All authors have declared no conflicts of interest.