Abstract A40: The balance between activated follicular helper T cells and follicular regulatory T cells within tertiary lymphoid structures guides antitumor immune responses in human breast cancer
Authors: Noël Grégory , Langouo Mireille , Garaud Soizic , Boisson Anaïs , Duvillier Hugues , Van den Eynden Gert , Larsimont Denis , Willard-Gallo Karen 
Published: June 2021

Abstract
Tumor-infiltrating lymphocytes (TIL) have been associated with good clinical outcomes in numerous large clinical trials for HER2-positive (HER2+) and triple-negative (TN) breast cancer (BC). Our previous work in human demonstrated that TIL can organize in tertiary lymphoid structures (TLS) with the B-cell chemoattractant, CXCL13, playing a key role in their formation as well as having a strong association with positive clinical outcomes. The present study investigated how TLS functionally contribute to immune responses in human BC. We prospectively collected fresh primary human BC tissues and prepared enzyme-free homogenates to produce TIL suspensions and tumor supernatants for flow cytometry and cytokine/chemokine/immunoglobulin (Ig) quantification, respectively. Matching formalin-fixed, paraffin-embedded tumor tissues were used for spatial analysis by dual immunohistochemistry and immunofluorescent confocal microscopy. We show that CD3+CD4+, CD3+CD8+ and CD19+ TIL in human BC contain subpopulations expressing CXCR5 (the CXCL13 receptor) and are co-localized in TLS. An in vitro functional assay activating Tfh cells with allogeneic B cells from human spleen evaluated the functionality of human BC Tfh TIL (CD45+CD3+CD4+CXCR5+). These experiments reveal that ICOS+PD-1hi Tfh TIL from TN and HER2+, but not luminal, BC possess helper activities for B cell IgG production. This observation is supported by a correlation between ICOS+PD-1hi Tfh TIL densities and IgG concentrations in primary human BC supernatants. A comparison of ICOS+PD-1hi and ICOS-PD-1- Tfh TIL confirms the activated, functional nature of these specialized TIL, which are characterized by high IL-21, IL-10 and CXCL13 mRNA expression. High IFNγ mRNA expression was detected in ICOS+PD-1hi Tfh TIL, suggesting their functional Th1 orientation. We also identified follicular regulatory T cells (Tfr), located in TLS that express CXCR5, CD25, demethylated Foxp3 and GARP, a marker of TGFβ activity. Tfr regulation of Tfh functions were evaluated by determining the ratio between functional Tfh TIL and GARP+ Tfr TIL. This analysis reveals a strong correlation between the Tfh/Tfr ratio and IgG production in primary tumor supernatants from TN and HER2+ human BC. In addition, strong correlations were observed with IgA and IgM production in HER2+ human BC. Our data show that Tfh TIL in human BC are major contributors to TLS activity, with the ratio between functional Tfh TIL and activated Tfr TIL directly influencing Ig production by B cells. Effective antitumor immunity in BC is therefore not only characterized by TIL density and organization but also by the balance between effector and regulatory TIL. These findings constitute important elements to consider when choosing patients for immunotherapy.
Citation Format: Grégory Noël, Mireille Langouo, Soizic Garaud, Anaïs Boisson, Hugues Duvillier, Gert Van den Eynden, Denis Larsimont, Karen Willard-Gallo. The balance between activated follicular helper T cells and follicular regulatory T cells within tertiary lymphoid structures guides antitumor immune responses in human breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A40.