Abstract 4689: Immune functions and regulation of follicular helper CD4+CXCR5+T cells in human breast cancer
Authors: Langouo Mireille , Noël Gregory , Eynden Gert Van den , Wind Alexandre De , Garaud Soizic , Silva Pushpamali De , Solinas Cinzia , Boisson Anais , Naveaux Celine , Duvillier Hugues , Craciun Ligia , Larsimont Denis , Piccart-Gebhart Martine , Willard-Gallo Karen 
Published: August 2018

Abstract
Introduction: High levels of tumor infiltrating lymphocytes (TIL) have been associated with good clinical outcomes in patients with HER2-positive (HER2+) and triple-negative (TN) breast cancer (BC) . Recently, we demonstrated that 60% of BC TIL are organized in tertiary lymphoid structures (TLS) located in the stroma. We further identified a CXCL13-producing CD4+ T follicular helper cell (Tfh) subpopulation and demonstrated that this chemokine, important for TLS formation, is associated with positive clinical outcomes in BC. The aim of the present study was to investigate how conventional CD4+ Tfh cells, expressing the CXCL13 receptor CXCR5, contribute to immune function and regulation in BC-associated TLS.
Methods: We prospectively collected fresh primary BC tissues and prepared enzyme-free homogenates to prepare tumor supernatant and TIL for flow cytometric analysis and sorting. Matching formalin-fixed paraffin-embedded (FFPE) were used for subsequent spatial analysis by dual IHC and confocal microscopy.
Results: Flow cytometric analyses show that ~15% CD4+, ~13% CD8+ and &gt;95% B cell TIL express CXCR5 while confocal microscopy reveals that these CXCR5+ TIL subpopulations co-localize in BC TLS and their presence is tightly correlated. BC Tfh TIL (compared to activated tonsillar Tfh) have a memory phenotype and express ICOS and PD-1 suggesting they are activated, but BCL6 is undetectable and CXCL13 positive cells are rare in only some tumors. RNA analysis detected high expression of IL-21, IL-10 and CXCL13 in Tfh TIL. Investigation into the role of Tfh cells in TLS functionality was accomplished via an in vitro assay where Tfh TIL were activated with allogeneic splenic B cells. These experiments reveal that only ICOS+PD-1+Tfh TIL from TN/HER2+ BC are capable of inducing IgM and IgG secretion by B cells. A comparison of ICOS+PD-1+ with ICOS-PD-1- Tfh TIL confirms an activated, functional nature for the former characterized by high levels of IL-21, IL-10 and CXCL13 mRNA expression. We also found specialized follicular regulatory T cells (Tfr), expressing CXCR5, CD25, demethylated Foxp3 and GARP, localized in TLS. To understand the regulation of Tfh function(s) by Tfr we quantified immunoglobulins in the primary tumor supernatant, finding a correlation between the Tfh/Tfr ratio (fresh tissues) and IgG and IgM production (supernatant) in TN/HER2+ BC. These data suggest that the balance between effector and regulatory Tfh influences B cell differentiation in TLS.
Conclusions: We show that it is possible to isolate functional Tfh TIL from fresh BC tissues, demonstrate their activation and functional state and link a Tfr presence with negative regulation of TLS function. These data begin to shed light on anti-tumor immune responses occurring in TLS at the tumor site, whose functional activities may have important treatment implications, particularly for immunotherapy in BC.
Citation Format: Mireille Langouo, Gregory Noël, Gert Van den Eynden, Alexandre De Wind, Soizic Garaud, Pushpamali De Silva, Cinzia Solinas, Anais Boisson, Celine Naveaux, Hugues Duvillier, Ligia Craciun, Denis Larsimont, Martine Piccart-Gebhart, Karen Willard-Gallo. Immune functions and regulation of follicular helper CD4+CXCR5+T cells in human breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4689.