No abstract

Breast cancer metastasis is known to be extensively promoted by immunosuppression. Here we describe a novel mechanism by which mesenchymal stem cell (MSC)-derived exosomes drive an immunosuppressive program within the breast tumor microenvironment. Initial q-PCR and immunophenotyping of human breast tumor samples, and TCGA data analysis confirmed a positive correlation between infiltrations of MSC and M2 macrophage phenotype, therefore we hypothesized that MSC promote M2 macrophage differentiation in breast tumors. We then performed a series of experiments in vitro and in vivo, and found that MSC-derived exosomes significantly promote differentiation of macrophages into PD-L1 expressing ‘M2-like’ phenotype, while also enhancing malignant progression of breast tumors, in vivo. Additionally, we observed increased invasive potential of tumor cells with higher expression of mesenchymal markers when induced with MSC-derived exosomes. Importantly, our observation of a significantly higher TGF-β production by tumor associated macrophages in exosome-induced tumors, with stronger PD-1 expression by intra-tumoral T cells, suggests TGF-β driven PD-1 upregulation. Blocking of PD-L1 abrogates exosome-induced tumor growth signifying the PD-L1/PD-1 checkpoint pathway is vital to MSC-induced tumor progression. Together, infiltration of MSCs within breast tumors drives the increased invasive potential as well as immune-checkpoint-mediated immunosuppression through inducing PD-L1 expression by tumor associated macrophages and PD-1 expression by T cells. Targeting infiltration of MSCs into the breast tumor therefore is a potential approach to reduce breast tumor metastases and improve efficacy of immunotherapies.

T Follicular Helper cells (TFH) provide both co-stimulation and stimulatory cytokines to B cells to facilitate affinity maturation, class switch recombination, and plasma cell differentiation within the germinal center. However, is not clear how TFH differentiation is regulated. We found that deficiency of the chromatin organizer Satb1 results in increased TFH formation in CD4Cre+Satb1flx/flx mice through up-regulation of the canonical TFH markers ICOS and PD-1 and suppression of Foxp3+PD-1highCXCR5+ T follicular regulatory (TFR) cells as well. Accordingly, CD4Cre+Satb1flx/flx mice, or RAG1−/− mice transferred with Satb1-deficient CD4+ T cells showed a dramatic accumulation of CD4+CXCR5+PD-1high upon ovarian tumor challenge, compared to their Satb1+ counterparts, which was associated with reduced tumor growth. Importantly, intratumoral administration of Satb1-deficient CD4+ T cells re-directed to target ovarian cancer cells through chimeric receptors, but not their Satb1+ counterparts, induce the formation of Tertiary Lymphoid Structures in most tumors. Conclusion Satb1 controls three mechanisms relevant for TFH differentiation and, subsequently, antigen-specific humoral responses; namely, PD- 1 expression, ICOS de-repression and TFR formation. Our results suggest a novel role for Satb1 as a major regulator of TFH differentiation and TLS during tumor formation.

Ovarian carcinoma microenvironmental T cells exert clinically relevant pressure against malignant progression; however current immunotherapies rarely induce ovarian cancer regression. Here we investigate CD277-containing butyrophilin 3A1 (BTN3A1), a poorly investigated immunoregulatory pathway driven by myeloid and tumor cells in ovarian tumor beds. We show that BTN3A1 is overexpressed in ovarian cancer and is associated with a significant survival disadvantage in these patients (n=200). Concomitantly, ectopic expression of BTN3A1 on APCs inhibits αβ T cell proliferation and Th1 cytokine production. Proteomic analyses and binding assays demonstrate that BTN3A1 interacts with the CD45 phosphatase and elements of the TCR. Consequently, TCR ligation in the presence of BTN3A1 inhibits the segregation of CD45 from the immune synapse and blunts downstream signaling by antagonizing the phosphorylation of CD3Zeta, Lck, and Zap70. We developed fully human αCD277 antibodies which rescue αβ T cell proliferation and Th1 cytokine responses, while driving the infiltration of T cells into tumor beds, delaying ovarian tumor progression in novel BTN3A1+ humanized mice and xenograft studies. Paradoxically, αCD277 antibodies promote the activation of γδ T cells by driving a conformational transformation of BTN3A1. Thus, co-transfer of γδ and Ag-specific αβ T cells in the presence of αCD277 antibodies synergize to further impair malignant progression in vivo. Overall, we show that BTN3A1 drives αβ T cell dysfunction in ovarian cancer, while αCD277 antibodies transform this molecule from immunosuppressive to immunostimulatory by rescuing αβ T cells and activating γδ T cells, thus dynamically unleashing T cell-driven antitumor immunity.

Conventional memory T cells classically include central memory T (TCM) cells, residing in lymphoid organs, and effector memory T (TEM) cells, circulating through various tissues. Recently, a novel population of memory T cells has gained interest, namely tissue resident memory T (TRM) cells, which persist in tissues and do not recirculate. It is described that these cells reside in human tumors playing a role in tumor-specific T-cell responses. We found by flow cytometry that between 50%–80% of the CD8+ T cells in human ovarian carcinomas are CD103+CD69+TRM cells. RNA-seq of TRM and their re-circulating counterparts from 7 different human carcinomas showed a very distinctive phenotype, characterized by co-upregulation of effector (GZMB, IFNG) and exhaustion (PD-1, TIM3) markers, along with transcription factors and signaling molecules likely involved in the acquisition of the TRM phenotype. Unexpectedly, we found very little overlap between the TCR repertoire of both populations in multiple tumors, and TRM T cells consistently showed significantly higher clonality. Tumor antigen-specific TRM T cells intratumorally transferred into syngeneic mice were more effective at delaying tumor growth compared with their tumor antigen-specific recirculating counterparts. Finally, both the acquisition and the maintenance of a TRM phenotype within the CD8 T cell compartment at tumor beds are dependent on exposure to tumor cognate antigen. Together, our data indicate that TRM CD8+ T cells, but not their CD103− counterparts, represent tumor antigen-specific effector lymphocytes actively exerting anti-tumor immune pressure in the ovarian cancer microenvironment.