T follicular helper cells (T _FH cells) are important regulators of antigen-specific B cell responses. The B cell chemoattractant CXCL13 has recently been linked with T _FH cell infiltration and improved survival in human cancer. Although human T _FH cells can produce CXCL13, their immune functions are currently unknown. This study presents data from human breast cancer, advocating a role for tumor-infiltrating CXCL13-producing (CXCR5 ^– ) T _FH cells, here named T _FH X13 cells, in promoting local memory B cell differentiation. T _FH X13 cells potentially trigger tertiary lymphoid structure formation and thereby generate germinal center B cell responses at the tumor site. Follicular DCs are not potent CXCL13 producers in breast tumor tissues. We used the T _FH cell markers PD-1 and ICOS to identify distinct effector and regulatory CD4 ⁺ T cell subpopulations in breast tumors. T _FH X13 cells are an important component of the PD-1 ^hi ICOS ^int effector subpopulation and coexpanded with PD-1 ^int ICOS ^hi FOXP3 ^hi Tregs. IL2 deprivation induces CXCL13 expression in vitro with a synergistic effect from TGFβ1, providing insight into T _FH X13 cell differentiation in response to Treg accumulation, similar to conventional T _FH cell responses. Our data suggest that human T _FH X13 cell differentiation may be a key factor in converting Treg-mediated immune suppression to de novo activation of adaptive antitumor humoral responses in the chronic inflammatory breast cancer microenvironment.

We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4⁺ Tfh TIL, CD8⁺ TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1^hiICOS^int phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67⁺ TIL-B in active germinal centers) and cytotoxic (GZMB⁺CD8⁺ and GZMB⁺CD68⁺ TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25⁺CXCR5⁺GARP⁺FOXP3⁺ phenotype and a demethylated FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-β–dependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8⁺ TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade.

There is an exponentially growing interest in targeting immune checkpoint molecules in breast cancer (BC), particularly in the triple-negative subtype where unmet treatment needs remain. This study was designed to analyze the expression, localization, and prognostic role of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, and TIM3 in primary BC. Gene expression analysis using the METABRIC microarray dataset found that all six immune checkpoint molecules are highly expressed in basal-like and HER2-enriched compared to the other BC molecular subtypes. Flow cytometric analysis of fresh tissue homogenates from untreated primary tumors show that PD-1 is principally expressed on CD4⁺ or CD8⁺ T cells and CTLA-4 is expressed on CD4⁺ T cells. The global proportion of PD-L1⁺, PD-L2⁺, LAG3⁺, and TIM3⁺ tumor-infiltrating lymphocytes (TIL) was low and detectable in only a small number of tumors. Immunohistochemically staining fixed tissues from the same tumors was employed to score TIL and tertiary lymphoid structures (TLS). PD-L1⁺, PD-L2⁺, LAG3⁺, and TIM3⁺ cells were detected in some TLS in a pattern that resembles secondary lymphoid organs. This observation suggests that TLS are important sites of immune activation and regulation, particularly in tumors with extensive baseline immune infiltration. Significantly improved overall survival was correlated with PD-1 expression in the HER2-enriched and PD-L1 or CTLA-4 expression in basal-like BC. PD-1 and CTLA-4 proteins were most frequently detected on TIL, which supports the correlations observed between their gene expression and improved long-term outcome in basal-like and HER2-enriched BC. PD-L1 expression by tumor or immune cells is uncommon in BC. Overall, the data presented here distinguish PD-1 as a marker of T cell activity in both the T and B cell areas of BC associated TLS. We found that immune checkpoint molecule expression parallels the extent of TIL and TLS, although there is a noteworthy amount of heterogeneity between tumors even within the same molecular subtype. These data indicate that assessing the levels of immune checkpoint molecule expression in an individual patient has important implications for the success of therapeutically targeting them in BC.

The programmed cell death 1 (PD-1) pathway is an important regulator of immune responses in peripheral tissues, including abnormal situations such as the tumor microenvironment. This pathway is currently the principal target for immunotherapeutic compounds designed to block immune checkpoint pathways, with these drugs improving clinical outcomes in a number of solid and hematological tumors. Medical oncology is experiencing an immune revolution that has scientists and clinicians looking at alternative, non-redundant inhibitory pathways also involved in regulating immune responses in cancer. A variety of targets have emerged for combinatorial approaches in immune checkpoint blockade. The main purpose of this narrative review is to summarize the biological role of lymphocyte activation gene 3 (LAG3), an emerging targetable inhibitory immune checkpoint molecule. We briefly discuss its role in infection, autoimmune disease and cancer, with a more detailed analysis of current data on LAG3 expression in breast cancer. Current clinical trials testing soluble LAG3 immunoglobulin and LAG3 antagonists are also presented in this work.