Tumor-infiltrating lymphocytes (TIL) have been associated with good clinical outcomes in HER2-positive (HER2+) and triple-negative (TN) breast cancer (BC) patients. Recently, we demonstrated that in 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 (Tfh) cell subpopulation that is associated with positive clinical outcomes in BC.

We aimed to investigate how CD4+Tfh cells, expressing the CXCL13 receptor CXCR5, contribute to immune response in BC TLS.

Prospectively collected fresh primary BC tissues were dissociated without enzymes to separate tumor supernatants and TIL and used for Immunoglobulin (lg)/cytokines quantification and flow cytometric analysis/sorting. Matching formalin-fixed paraffin-embedded (FFPE) were used for spatial analysis.

In our BC cohort, around 15%CD4+TIL, 13% CD8+TIL and >95% B cell TIL express CXCR5 while confocal microscopy reveals that they colocalized in BC TLS. in some BC, Tfh TIL (as activated tonsillar Tfh) express ICOS and PD-1 suggesting they are activated. RNA analysis on Tfh TIL detected high expression of IL-21, INFg, and CXCL13. In an in vitro assay where Tfh TIL were activated with splenic B cells revealed that only ICOS+PD-1+Tfh TIL from TN/HER2+ BC are capable of inducing Ig secretion by B cells. mRNA analysis of sorted ICOS+PD-1+Tfh TIL confirms an activated, functional nature for the former with high levels of IL-21, INFg, and CXCL13. Moreover, we found that CXCR5+CD8 TIL expressing ICOS and PD-1 expressed high level of CCL4, FASL, granzyme B and IFNy. Multiplex IHC analyses confirm the presence of activated CD8+ TIL in close contact with functional Tfh and B TIL. A high correlation between the presence of functional Tfh with activated CD8 TIL and with Ig secreted in the NANT from TN/HER2+ BC was observed.

We describe two distinct subpopulations of Tfh TIL. The CXCR5 TfhX13 producing CXCL13 that may recruit the CXCR5+Tfh TIL in TLS. Functional Tfh TIL probesto guide humoral and cytotoxic antitumor immunity in TLS. These data shed light on anti-tumor immune responses taking place in the TLS, whose functional activities may have important treatment implications, particularly for immunotherapy in BC.