Abstract 3853: The anti-tumor immune responses by active and quiescent tertiary lymphoid structures to breast cancer
Authors: De Silva Pushpamali , Garaud Soizic , Solinas Cinzia , Noël Grégory , Fontsa Mireille Langouo , Boisson Anaïs , de Wind Alexandre , Jose Vinu , Van den Eynden Gert , Thomas Noemie , Duvillier Hugues , Naveaux Céline , Craciun Ligia , Bron Dominique , Piccart-Gebhart Martine , Larsimont Denis , Willard-Gallo Karen 
Published: September 2020

Abstract
There is a growing interest in active immune responses generated by tertiary lymphoid structures (TLS) arising in solid tumors; however, their clinical impact in breast cancer (BC) remains unclear. Several studies show that transcription factors contribute to TLS formation via their regulation of cytokine and chemokine production. The Forkhead box (FOX) protein 1 (FOXP1) has been shown to play critical roles in regulating immune cells, including our recent work revealing its effects on TIL migration. These data lead us to further investigate FOXP1 expression in tumor infiltrating lymphocytes (TIL) and TLS. We identify two types of TLS based on FOXP1 expression: 1) those that contain a germinal center (GC+) and those that do not (GC-). Comparative analysis of FOXP1 expression in secondary lymphoid organs, including more immune active tonsils (many GC) and less immune active spleens (primarily without GC) confirm differences in FOXP1 expression associated with GC. In BC, TLS containing tumors were more frequently GC- than GC+ (n=49), with triple-negative tumors having higher numbers of GC+ TLS compared to luminal or HER2+ tumors. Immunofluorescence and multiplex immunohistochemistry was used to closely examine the GC+ and GC- TLS, finding an immune active profile in the former, characterized by T follicular helper cells (PD1+CD4+ T), mature dendritic cells (CD21+ and CD23+), actively proliferating (Ki67+) B cells undergoing immunoglobulin (Ig) class switch recombination (AID+) and a plasma cell presence (CD138+). Analysis of Ig's in the primary tumor supernatants revealed that BC with ≥1 GC+ TLS (n=20) were characterized by increases in total Ig, IgG1, IgG2 and IgA, reflecting active humoral immunity, compared to BC containing only GC- TLS (n=29). Gene expression analysis of individual micro-dissected TLS demonstrated upregulation of Th1, Th2 and Tfh immune genes in the GC+ compared to the GC- TLS, suggesting the former also sustain cell-mediated immune responses. Immune infiltrates in tumors with ≥1 GC+ TLS are specifically characterized by high global TIL, CD3+, CD4+ or CD8+ T cell TIL and CD20+ TIL-B (n=29). Analysis of BC TIL spatial distribution identified increased stromal TIL (all subpopulations) while intratumoral TIL increases were predominantly CD3+ and CD8+ T cell TIL in tumors with GC+ TLS. Overall, our data indicate that GC+ TLS house active immune responses in BC while GC- TLS are quiescent.
Citation Format: Pushpamali De Silva, Soizic Garaud, Cinzia Solinas, Grégory Noël, Mireille Langouo Fontsa, Anaïs Boisson, Alexandre de Wind, Vinu Jose, Gert Van den Eynden, Noemie Thomas, Hugues Duvillier, Céline Naveaux, Ligia Craciun, Dominique Bron, Martine Piccart-Gebhart, Denis Larsimont, Karen Willard-Gallo. The anti-tumor immune responses by active and quiescent tertiary lymphoid structures to breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3853.