Epstein-Barr virus (EBV) infection causes B cell lymphomas in humanized mouse models and contributes to a variety of different types of human lymphomas. T cells directed against viral antigens play a critical role in controlling EBV infection, and EBV-positive lymphomas are particularly common in immunocompromised hosts. We previously showed that EBV induces B cell lymphomas with high frequency in a cord blood-humanized mouse model in which EBV-infected human cord blood is injected intraperitoneally into NOD/LtSz-scid/IL2Rγnull (NSG) mice. Since our former studies showed that it is possible for T cells to control the tumors in another NSG mouse model engrafted with both human fetal CD34+ cells and human thymus and liver, here we investigated whether monoclonal antibodies that block the T cell inhibitory receptors, PD-1 and CTLA-4, enhance the ability of cord blood T cells to control the outgrowth of EBV-induced lymphomas in the cord-blood humanized mouse model. We demonstrate that EBV-infected lymphoma cells in this model express both the PD-L1 and PD-L2 inhibitory ligands for the PD-1 receptor, and that T cells express the PD-1 and CTLA-4 receptors. Furthermore, we show that the combination of CTLA-4 and PD-1 blockade strikingly reduces the size of lymphomas induced by a lytic EBV strain (M81) in this model, and that this anti-tumor effect requires T cells. PD-1/CTLA-4 blockade markedly increases EBV-specific T cell responses, and is associated with enhanced tumor infiltration by CD4+ and CD8+ T cells. In addition, PD-1/CTLA-4 blockade decreases the number of both latently, and lytically, EBV-infected B cells. These results indicate that PD-1/CTLA-4 blockade enhances the ability of cord blood T cells to control outgrowth of EBV-induced lymphomas, and suggest that PD-1/CTLA-4 blockade might be useful for treating certain EBV-induced diseases in humans.

A hallmark of T cell activation in vitro and in vivo is the clustering of T cells with each other via interaction of the LFA-1 integrin with ICAM-1. The functional significance of these homotypic aggregates in regulating T cell function remains unknown. We used an APC-free in vitro activation system to demonstrate that stimulation of purified naïve CD8 T cells results in enhanced expression of ICAM-1 on T cells that is sustained by the inflammatory cytokine IL-12 and associated with robust T cell aggregates. ICAM-1 deficient CD8 T cells proliferate normally but demonstrate a striking failure to aggregate. Interestingly, loss of ICAM-1 expression results in elevated levels of interferon-γ (IFN-γ) and Granzyme B, as well as enhanced cytotoxicity. Similar results were obtained when anti-LFA-1 antibody was used to block the clustering of wild-type T cells. ICAM-1 ligation is not required for IFN-γ regulation, as clustering of ICAM-1 deficient CD8 T cells with wild-type T cells reduces IFN-γ expression. Analysis using a fluorescent reporter that monitors TCR signal strength indicates that T cell clustering limits T cell exposure to antigen during activation. Furthermore, T cell clustering promotes the upregulation of the CTLA-4 inhibitory receptor and the downregulation of eomesodermin, which controls effector molecule expression. Activation of ICAM-1 deficient CD8 T cells in vivo results in an enhanced percentage of KLRG-1+ T cells indicative of short-lived effectors. These results suggest that T cell clustering represents a mechanism that allows continued proliferation but regulates T cell effector function and differentiation.

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Developing strategies to enhance cancer prevention is a paramount goal, particularly given recent concerns about surgical treatment of pre-invasive states such as ductal carcinoma in situ. Promoting effective immunosurveillance by leukocytes that scan for nascent neoplastic transformations represents a potential means to achieve this goal. Since most breast cancers arise within the ductal epithelium, enhancing protective immunosurveillance will likely necessitate targeting one or more of the distinctive lymphocyte types found in these sites under normal conditions. Here, we have characterized the intraepithelial lymphocyte compartment of non-cancerous human breast tissue and identified a subset of T lymphocytes that can be pharmacologically targeted to enhance their responses to breast cancer cells. Specifically, Vδ2+ γδ T cells were consistently present in preparations of mammary ductal epithelial organoids and they proliferated in response to zoledronic acid, an aminobisphosphonate drug. Vδ2+ T cells from breast ductal organoids produced the anti-tumor cytokine IFN-γ and efficiently killed bisphosphonate-pulsed breast carcinoma cells. These findings demonstrate the potential for exploiting the ability of Vδ2+ γδ T cells to respond to FDA-approved bisphosphonate drugs as a novel immunotherapeutic approach to inhibit the outgrowth of breast cancers.

BackgroundAntimicrobial T cells play key roles in the disease progression of cancers arising in mucosal epithelial tissues, such as the colon. However, little is known about microbe-reactive T cells within human breast ducts and whether these impact breast carcinogenesis.MethodsEpithelial ducts were isolated from primary human breast tissue samples, and the associated T lymphocytes were characterized using flow cytometric analysis. Functional assays were performed to determine T-cell cytokine secretion in response to bacterially treated human breast carcinoma cells.ResultsWe show that human breast epithelial ducts contain mucosal associated invariant T (MAIT) cells, an innate T-cell population that recognizes specific bacterial metabolites presented by nonclassical MR1 antigen-presenting molecules. The MAIT cell population from breast ducts resembled that of peripheral blood in its innate lymphocyte phenotype (i.e., CD161, PLZF, and interleukin [IL]-18 receptor coexpression), but the breast duct MAIT cell population had a distinct T-cell receptor Vβ use profile and was markedly enriched for IL-17-producing cells compared with blood MAIT cells. Breast carcinoma cells that had been exposed to Escherichia coli activated MAIT cells in an MR1-dependent manner. However, whereas phorbol 12-myristate 13-acetate/ionomycin stimulation induced the production of both interferon-γ and IL-17 by breast duct MAIT cells, bacterially exposed breast carcinoma cells elicited a strongly IL-17-biased response. Breast carcinoma cells also showed upregulated expression of natural killer group 2 member D (NKG2D) ligands compared with primary breast epithelial cells, and the NKG2D receptor contributed to MAIT cell activation by the carcinoma cells.ConclusionsThese results demonstrate that MAIT cells from human breast ducts mediate a selective T-helper 17 cell response to human breast carcinoma cells that were exposed to E. coli. Thus, cues from the breast microbiome and the expression of stress-associated ligands by neoplastic breast duct epithelial cells may shape MAIT cell responses during breast carcinogenesis.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-1036-5) contains supplementary material, which is available to authorized users.