Abstract P5-04-12: Functional CXCR5+CD4+ follicular helper T cells in breast cancer associated tertiary lymphoid structures signal active immune responses at the tumor site
Authors: Willard-Gallo Karen , Noel Gregory , Fontsa Mireille Langouo , Garaud Soizic , de Wind Alexandre , Van den Eynden Gert , Salgado Roberto , Boisson Anais , Naveaux Celine , Duvillier Hugues , Craciun Ligia , Piccart-Gebhart Martine , Larsimont Denis 
Published: March 2020

Abstract
Background: The association between tumor infiltrating lymphocytes (TIL) and positive clinical outcomes in breast cancer (BC) is now commonly recognized. Previous work from our laboratory demonstrated that TIL can organize in tertiary lymphoid structures (TLS) in BC-associated stroma. We further showed that CXCL13, produced by specialized CD4+ T follicular helper (TfhX13) cell, is an important TLS chemoattractant and associated with positive clinical outcomes. The current study investigated how immune cell functionally and regulation in TLS contributes to immune responses in BC.
Methodology: We prospectively collected fresh primary BC tissues and prepared enzyme-free homogenates to produce TIL suspensions and tumor supernatants for flow cytometry and cytokine/chemokine/immunoglobulin (Ig) analysis, respectively. Matching formalin-fixed paraffin-embedded tumor tissues were analyzed using dual immunohistochemistry (IHC) and immunofluorescence (IF) confocal microscopy or multiplex IHC.
Results: We show that CXCR5, the CXCL13 receptor, is expressed on subpopulations of CD4+ (Tfh) and CD8+ T cell TIL as well as the majority of TIL-B, with all CXCR5+ TIL co-localizing in TLS. The functional activities of Tfh TIL, evaluated using an in vitro assay with allogeneic human splenic B cells, reveals that PD-1hiICOS+ Tfh TIL (in some triple negative and HER2+ but not luminal BC) can provide help to TIL-B for Ig production. This observation is strengthened by additional data showing: 1) a correlation between functional (PD-1hiICOS+) Tfh TIL densities and IgG concentrations in primary BC supernatants; 2) a strong correlation between PD-1+ Tfh TIL and Ki67+ TIL-B in BC-associated TLS; and 3) cell-to-cell contact between Tfh TIL and TIL-B in TLS with active germinal centers. PD-1hiICOS+ (functional) and PD-1lo/intICOS− (non-functional) Tfh TIL were sorted for mRNA analysis with functional Tfh TIL expressing higher levels of IL-21, IFNγ and CXCL13. Higher IFNγ expression by PD-1hiICOS+ Tfh TIL suggests their functional Th1 orientation. CXCR5+ T follicular regulatory (Tfr) TIL were also detected in TLS and shown to express, CD25, demethylated FOXP3, and GARP, a marker of active TGFβ. Analyzing the ratio between Tfh and GARP+ Tfr TIL revealed that when the balance favors Tfh TIL, IgG production is increased. This Tfh/Tfr ratio was also correlated with activated CXCR5+CD8+ TIL in TLS. Multiplex IHC identified the positioning of CXCR5+ TIL subpopulations and demonstrated there are important cell-to-cell contacts between these subpopulations in active TLS.
Conclusions: Our data show that Tfh TIL subpopulations play major roles in the functionality of BC-associated TLS. The balance between functionally active and regulatory CXCR5+ Tfh TIL together with active CXCR5+CD8+ TIL and CXCR5+ TIL-B appears to regulate immune activities in the tumor microenvironment. Tumors with functional Tfh TIL are linked to a more robust stromal and intratumoral infiltrate together with a ratio of active TLS &gt;1. Thus, while TIL density scores provide important primary insight on immune activity in BC, their organization and subpopulation balances may offer key information for fine-tuning treatment selection options, particularly for immune-based therapies.
Citation Format: Karen Willard-Gallo, Gregory Noel, Mireille Langouo Fontsa, Soizic Garaud, Alexandre de Wind, Gert Van den Eynden, Roberto Salgado, Anais Boisson, Celine Naveaux, Hugues Duvillier, Ligia Craciun, Martine Piccart-Gebhart, Denis Larsimont. Functional CXCR5+CD4+ follicular helper T cells in breast cancer associated tertiary lymphoid structures signal active immune responses at the tumor site [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-12.