The clinical relevance of tumor infiltrating lymphocytes (TIL) in breast cancer (BC) is now broadly accepted; however, the relationship between protective immunity, observed in some patients, and critical features of lymphoid subset composition and organization are largely unknown. Our recent work revealed that tertiary lymphoid structures (TLS), principally composed of T and B cells, present in the peri-tumoral regions of BC are associated with increased TIL infiltration in the tumor bed and good clinical outcomes in both the neo-adjuvant and adjuvant settings. To gain insight into the immune components linked with a significant TIL and TLS presence, we initiated a prospective study of T and B cell TIL. Breast tissues from tumors (current n=483) and matched non-adjacent non-tumor tissues (NANT) as well as normal tissue from mammary reductions (n=49) were systematically immunophenotyped by flow cytometry on the day of surgery. Primary tumor supernatants derived from the tissue homogenates (non-enzymatic) and plasma from all patients were stored for cytokine/chemokine and immunoglobulin analysis. TIL organization and spatial distribution was analyzed on paraffin sections using immunohistochemistry (IHC) and immunofluorescence (IF). The fresh tissue analyses revealed that TIL distribution in BC forms a continuum. The infiltration levels detected in normal tissue and NANT were used to define cutoffs that discriminate between TIL positive (TILpos) and negative (TILneg) tumors. TILneg tumors are defined as those with a TIL density <99th percentile of the lymphocyte density in normal breast tissue. TILpos tumors include TIL-high (TILhi) where TIL density is >99th percentile of lymphocyte density in NANT and TIL-intermediate (TILint). Applying these thresholds to the TIL infiltration levels identified approximately 25% of tumors as TILneg with infiltrating lymphocytes similar to normal breast tissue. The TILpos tumors (75%) are subdivided into 36% TILint and 39% TILhi tumors. TILpos tumors are characterized by an increase in CD4+ T cells and CD19+/CD20+ B cells. The median CD4/CD8 ratio was >1 in TILpos compared to <1 in TILneg tumors and NANT. CD4+ and CD8+ T cells were predominantly CD45RO+ memory cells with a significant proportion expressing PD-1. All stages of B cell differentiation were detected in the infiltrate; however, memory B cells were significantly increased in BC (50%) compared to normal tissues (<15%) with the highest levels seen in TILhi tumors. An increase in centroblasts and centrocytes, the germinal center (GC) B cells, was associated with Tfh cells, both resident in peri-tumoral TLS. Their presence was positively correlated with antibody-secreting cells, suggesting local humoral immune responses are generated in TLS, which is supported by the increased immunoglobulin levels detected in tumor supernatants compared to NANT/normal tissue supernatants. Closer examination of Tfh cells infiltrating BC indicates that CXCL13-producing Tfh TIL vary from Tfh cells in secondary lymphoid organs such as tonsils. Tfh TIL express the highest levels of some GC Tfh cell markers (PD-1, CD200 and TIGIT) but intermediate levels of others (ICOS and BCL6) and are essentially negative for CXCR5. Closer examination of Tfh cells in the tumor bed revealed that they directly contact CD20+ B cells, IgG+ and IgA+ plasma cells as well as CXCL13–CD8+ TIL. Taken together, our data suggest that active cross-talk between adaptive immune cells in the tumor microenvironment may be key to establishing immunological memory sufficiently robust to control residual BC over the long term. These data further signify that TLS and/or the presence of GC-associated Tfh and B cells may be important biomarkers that identify effective anti-tumor immunity, which can ultimately be used to independently grade therapeutic responses.

Citation Format: Soizic Garaud, Laurence Buisseret, Chunyan Gu-Trantien, Gert Van den Eynden, Alexandre de Wind, Edoardo Migliori, Roberto Salgado, Denis Larsimont, Martine Piccart, Karen Willard-Gallo. Tertiary lymphoid structures: Predictors of effective antitumor immunity in human breast cancer? [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A124.