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The importance of CD4+ follicular helper T cells and tertiary lymphoid structures in the anti-tumor immune response to breast cancer

par Migliori, Edoardo
Président du jury Le Moine, Alain
Promoteur Piccart-Gebhart, Martine
Co-Promoteur Willard-Gallo, Karen
Publication Non publié, 2017-10-03

Thèse de doctorat

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Résumé :

Breast cancer (BC) is the most common cancer in women. It is a highly heterogeneous disease in terms of histology, therapeutic response and patient outcomes. Early and accurate detection of breast cancer is crucial as the patient prognosis varies greatly depending on the diagnosis of the disease. Patient outcomes have been linked to the presence of tumor infiltrating lymphocytes (TIL) in solid tumors. In human BC, higher TIL infiltration is associated with a better prognosis and also predicts relevant responses to pre-operative chemotherapy. TIL are primarily composed of T cells, albeit around 20% of BC patients (pts) show significant B cell infiltration, and can organize in tertiary lymphoid structures (TLS) located in the peritumoral stroma, which are associated with survival in HER2+ and triple negative BC patients. Further, these studies revealed that CD4+ follicular helper T (Tfh) cells producing CXCL13 were specifically associated with peritumoral TLS. CXCL13 is an important B cell chemoattractant whose function is to recruit B cells to the germinal center (GC) in secondary lymphoid organs and TLS, where they can mature and differentiate into memory or antibody-producing B cells. The principal objective of this thesis project was to investigate the role of CXCL13 and Tfh cells play in the development and/or maintenance of GC-like structures in BC-associated TLS.Further understanding of the factors that promote TLS formation in vivo could provide important insight for treatment decisions in BC. CXCL13 expression was originally identified as an important signal associated with TLS that was predictive for patient outcomes. We investigated factors capable of inducing CXCL13 expression in CD4+ T cells isolated from peripheral blood, using flow cytometry analysis. Treatment with TGFβ1 alone, or together with several cytokines (IL12, IL21, and in particular IL2 blockade), increased CXCL13 expression in activated CD4+ T cells. Similar to our characterization of Tfh TIL in fresh tumor tissues, these CXCL13-producing CD4+ T cells were CXCR5 negative and expressed the Tfh markers PD-1 and ICOS. The positive correlation, in treated cells and fresh TIL, between CXCL13-producing CD4+ T cells and FoxP3-expressing regulatory CD4+ T cells, and the diminished chemokine production upon depletion of the latter population, suggest a possible positive relationship between regulatory CD4+ T cells and CXCL13-producing CD4+ T cells.We then derived a GC-associated B cell gene signature for integration in our previously published Tfh cell gene signature, including CXCL13 gene. The combined GC gene signature was tested for its ability to sensitively detect BC-associated TLS using a qRT-PCR-based assay on two different cohorts, a primary BC set (n=83) and a retrospective series (n=52) of formalin-fixed paraffin-embedded (FFPE) BC tissues. These data revealed a correlation between gene signature expression and the extent of TLS scored by trained pathologists on dual-immunohistochemistry stained (CD3+CD20 for T and B cells, respectively) FFPE tissue sections. In addition, the high GC signature expression predicted better overall and disease-free survival of BC pts in our retrospective BC cohort, as well as in public microarray data.This thesis research has demonstrated that CXCL13-producing CD4+ T cells lacking CXCR5 differentiate and exert their function in IL-2-limited but TGF-β1-rich conditions. Furthermore, we developed a GC-associated gene signature able to detect TLS in BC and predict BC pts better survival.