Human CD4+ iNKT cell adoptive immunotherapy induces anti-tumour responses against CD1d-negative EBV-driven B lymphoma.

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that are uniquely suitable as off-the-shelf cellular immunotherapies due to their lack of alloreactivity. Two major subpopulations of human iNKT cells have been delineated, a CD4- subset that has a TH1/cytolytic profile, and a CD4+ subset that appears polyfunctional and can produce both regulatory and immunostimulatory cytokines. Whether these two subsets differ in anti-tumour effects is not known. Using live cell imaging, we found that CD4- iNKT cells limited growth of CD1d+ Epstein-Barr virus (EBV)-infected B-lymphoblastoid spheroids in vitro, whereas CD4+ iNKT cells showed little or no direct anti-tumour activity. However, the effects of the two subsets were reversed when we tested them as adoptive immunotherapies in vivo using a xenograft model of EBV-driven human B cell lymphoma. We found that EBV-infected B cells down-regulated CD1d in vivo, and administering CD4- iNKT cells had no discernable impact on tumour mass. In contrast, xenotransplanted mice bearing lymphomas showed rapid reduction in tumour mass after administering CD4+ iNKT cells. Immunotherapeutic CD4+ iNKT cells trafficked to both spleen and tumour and were associated with subsequently enhanced responses of xenotransplanted human T cells against EBV. CD4+ iNKT cells also had adjuvant-like effects on monocyte-derived DCs and promoted antigen-dependent responses of human T cells in vitro. These results show that allogeneic CD4+ iNKT cellular immunotherapy leads to marked anti-tumour activity through indirect pathways that do not require tumour cell CD1d expression and that are associated with enhanced activity of antigen-specific T cells.

Human CD4+ memory phenotype T cells use mitochondrial metabolism to generate sensitive IFN-γ responses.

The transition of naive T lymphocytes into antigenically activated effector cells is associated with a metabolic shift from oxidative phosphorylation to aerobic glycolysis. This shift facilitates production of the key anti-tumor cytokine interferon (IFN)-γ; however, an associated loss of mitochondrial efficiency in effector T cells ultimately limits anti-tumor immunity. Memory phenotype (MP) T cells are a newly recognized subset that arises through homeostatic activation signals following hematopoietic transplantation. We show here that human CD4+ MP cell differentiation is associated with increased glycolytic and oxidative metabolic activity, but MP cells retain less compromised mitochondria compared to effector CD4+ T cells, and their IFN-γ response is less dependent on glucose and more reliant on glutamine. MP cells also produced IFN-γ more efficiently in response to weak T cell receptor (TCR) agonism than effectors and mediated stronger responses to transformed B cells. MP cells may thus be particularly well suited to carry out sustained immunosurveillance against neoplastic cells.

Analysis of Butyrophilin-Mediated Activation of γδ T Cells from Human Spleen.

There is considerable interest in therapeutically engaging human γδ T cells. However, due to the unique TCRs of human γδ T cells, studies from animal models have provided limited directly applicable insights, and human γδ T cells from key immunological tissues remain poorly characterized. In this study, we investigated γδ T cells from human spleen tissue. Compared to blood, where Vδ2+Vγ9+ T cells are the dominant subset, splenic γδ T cells included a variety of TCR types, with Vδ1+ T cells typically being the most frequent. Intracellular cytokine staining revealed that IFN-γ was produced by a substantial fraction of splenic γδ T cells, IL-17A by a small fraction, and IL-4 was minimal. Primary splenic γδ T cells frequently expressed NKG2D (NK group 2 member D) and CD16, whereas expression of DNAM-1 (DNAX accessory molecule 1), CD28, PD-1, TIGIT, and CD94 varied according to subset, and there was generally little expression of natural cytotoxicity receptors, TIM-3, LAG-3, or killer Ig-like receptors. In vitro expansion was associated with marked changes in expression of these activating and inhibitory receptors. Analysis of functional responses of spleen-derived Vδ2+Vγ9+, Vδ1+Vγ9+, and Vδ1+Vγ9- T cell lines to recombinant butyrophilin BTN2A1 and BTN3A1 demonstrated that both Vδ2+Vγ9+ and Vδ1+Vγ9+ T cells were capable of responding to the extracellular domain of BTN2A1, whereas the addition of BTN3A1 only markedly enhanced the responses of Vδ2+Vγ9+ T cells. Conversely, Vδ1+Vγ9+ T cells appeared more responsive than Vδ2+Vγ9+ T cells to TCR-independent NKG2D stimulation. Thus, despite shared recognition of BTN2A1, differential effects of BTN3A1 and coreceptors may segregate target cell responses of Vδ2+Vγ9+ and Vδ1+Vγ9+ T cells.

Inflammatory CD4/CD8 double-positive human T cells arise from reactive CD8 T cells and are sufficient to mediate GVHD pathology.

An important paradigm in allogeneic hematopoietic cell transplantations (allo-HCTs) is the prevention of graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) activity of donor T cells. From an observational clinical study of adult allo-HCT recipients, we identified a CD4+/CD8+ double-positive T cell (DPT) population, not present in starting grafts, whose presence was predictive of ≥ grade 2 GVHD. Using an established xenogeneic transplant model, we reveal that the DPT population develops from antigen-stimulated CD8 T cells, which become transcriptionally, metabolically, and phenotypically distinct from single-positive CD4 and CD8 T cells. Isolated DPTs were sufficient to mediate xeno-GVHD pathology when retransplanted into naïve mice but provided no survival benefit when mice were challenged with a human B-ALL cell line. Overall, this study reveals human DPTs as a T cell population directly involved with GVHD pathology.

Harnessing invariant natural killer T cells to control pathological inflammation.

Invariant natural killer T (iNKT) cells are innate T cells that are recognized for their potent immune modulatory functions. Over the last three decades, research in murine models and human observational studies have revealed that iNKT cells can act to limit inflammatory pathology in a variety of settings. Since iNKT cells are multi-functional and can promote inflammation in some contexts, understanding the mechanistic basis for their anti-inflammatory effects is critical for effectively harnessing them for clinical use. Two contrasting mechanisms have emerged to explain the anti-inflammatory activity of iNKT cells: that they drive suppressive pathways mediated by other regulatory cells, and that they may cytolytically eliminate antigen presenting cells that promote excessive inflammatory responses. How these activities are controlled and separated from their pro-inflammatory functions remains a central question. Murine iNKT cells can be divided into four functional lineages that have either pro-inflammatory (NKT1, NKT17) or anti-inflammatory (NKT2, NKT10) cytokine profiles. However, in humans these subsets are not clearly evident, and instead most iNKT cells that are CD4+ appear oriented towards polyfunctional (TH0) cytokine production, while CD4- iNKT cells appear more predisposed towards cytolytic activity. Additionally, structurally distinct antigens have been shown to induce TH1- or TH2-biased responses by iNKT cells in murine models, but human iNKT cells may respond to differing levels of TCR stimulation in a way that does not neatly separate TH1 and TH2 cytokine production. We discuss the implications of these differences for translational efforts focused on the anti-inflammatory activity of iNKT cells.

SEC is an antiangiogenic virulence factor that promotes Staphylococcus aureus endocarditis independent of superantigen activity.

The superantigen staphylococcal enterotoxin C (SEC) is critical for Staphylococcus aureus infective endocarditis (SAIE) in rabbits. Superantigenicity, its hallmark function, was proposed to be a major underlying mechanism driving SAIE but was not directly tested. With the use of S. aureus MW2 expressing SEC toxoids, we show that superantigenicity does not sufficiently account for vegetation growth, myocardial inflammation, and acute kidney injury in the rabbit model of native valve SAIE. These results highlight the critical contribution of an alternative function of superantigens to SAIE. In support of this, we provide evidence that SEC exerts antiangiogenic effects by inhibiting branching microvessel formation in an ex vivo rabbit aortic ring model and by inhibiting endothelial cell expression of one of the most potent mediators of angiogenesis, VEGF-A. SEC's ability to interfere with tissue revascularization and remodeling after injury serves as a mechanism to promote SAIE and its life-threatening systemic pathologies.

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells.

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.

iNKT cells coordinate immune pathways to enable engraftment in nonconditioned hosts.

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that interact with key antigen-presenting cells to modulate adaptive T-cell responses in ways that can either promote protective immunity, or limit pathological immune activation. Understanding the immunological networks engaged by iNKT cells to mediate these opposing functions is a key pre-requisite to effectively using iNKT cells for therapeutic applications. Using a human umbilical cord blood xenotransplantation model, we show here that co-transplanted allogeneic CD4+ iNKT cells interact with monocytes and T cells in the graft to coordinate pro-hematopoietic and immunoregulatory pathways. The nexus of iNKT cells, monocytes, and cord blood T cells led to the release of cytokines (IL-3, GM-CSF) that enhance hematopoietic stem and progenitor cell activity, and concurrently induced PGE2-mediated suppression of T-cell inflammatory responses that limit hematopoietic stem and progenitor cell engraftment. This resulted in successful long-term hematopoietic engraftment without pretransplant conditioning, including multi-lineage human chimerism and colonization of the spleen by antibody-producing human B cells. These results highlight the potential for using iNKT cellular immunotherapy to improve rates of hematopoietic engraftment independently of pretransplant conditioning.

Different Human Immune Lineage Compositions Are Generated in Non-Conditioned NBSGW Mice Depending on HSPC Source.

NBSGW mice are highly immunodeficient and carry a hypomorphic mutation in the c-kit gene, providing a host environment that supports robust human hematopoietic expansion without pre-conditioning. These mice thus provide a model to investigate human hematopoietic engraftment in the absence of conditioning-associated damage. We compared transplantation of human CD34+ HSPCs purified from three different sources: umbilical cord blood, adult bone marrow, and adult G-CSF mobilized peripheral blood. HSPCs from mobilized peripheral blood were significantly more efficient (as a function of starting HSPC dose) than either cord blood or bone marrow HSPCs at generating high levels of human chimerism in the murine blood and bone marrow by 12 weeks post-transplantation. While T cells do not develop in this model due to thymic atrophy, all three HSPC sources generated a human compartment that included B lymphocytic, myeloid, and granulocytic lineages. However, the proportions of these lineages varied significantly according to HSPC source. Mobilized blood HSPCs produced a strikingly higher proportion of granulocyte lineage cells (~35% as compared to ~5%), whereas bone marrow HSPC output was dominated by B lymphocytic cells, and cord blood HSPC output was enriched for myeloid lineages. Following transplantation, all three HSPC sources showed a shift in the CD34+ subset towards CD45RA+ progenitors along with a complete loss of the CD45RA-CD49f+ long-term HSC subpopulation, suggesting this model promotes mainly short-term HSC activity. Mice transplanted with cord blood HSPCs maintained a diversified human immune compartment for at least 36 weeks after the primary transplant, although mice given adult bone marrow HSPCs had lost diversity and contained only myeloid cells by this time point. Finally, to assess the impact of non-HSPCs on transplantation outcome, we also tested mice transplanted with total or T cell-depleted adult bone marrow mononuclear cells. Total bone marrow mononuclear cell transplants produced significantly lower human chimerism compared to purified HSPCs, and T-depletion rescued B cell levels but not other lineages. Together these results reveal marked differences in engraftment efficiency and lineage commitment according to HSPC source and suggest that T cells and other non-HSPC populations affect lineage output even in the absence of conditioning-associated inflammation.

Early T Cell Activation Metrics Predict Graft-versus-Host Disease in a Humanized Mouse Model of Hematopoietic Stem Cell Transplantation.

Acute graft-versus-host disease (GVHD) is a frequent complication of hematopoietic transplantation, yet patient risk stratification remains difficult, and prognostic biomarkers to guide early clinical interventions are lacking. We developed an approach to evaluate the potential of human T cells from hematopoietic grafts to produce GVHD. Nonconditioned NBSGW mice transplanted with titrated doses of human bone marrow developed GVHD that was characterized by widespread lymphocyte infiltration and organ pathology. Interestingly, GVHD was not an inevitable outcome in our system and was influenced by transplant dose, inflammatory status of the host, and type of graft. Mice that went on to develop GVHD showed signs of rapid proliferation in the human T cell population during the first 1-3 wk posttransplant and had elevated human IFN-γ in plasma that correlated negatively with the expansion of the human hematopoietic compartment. Furthermore, these early T cell activation metrics were predictive of GVHD onset 3-6 wk before phenotypic pathology. These results reveal an early window of susceptibility for pathological T cell activation following hematopoietic transplantation that is not simply determined by transient inflammation resulting from conditioning-associated damage and show that T cell parameters during this window can serve as prognostic biomarkers for risk of later GVHD development.

An EBNA3A-Mutated Epstein-Barr Virus Retains the Capacity for Lymphomagenesis in a Cord Blood-Humanized Mouse Model.

Epstein-Barr virus (EBV) causes B cell lymphomas and transforms B cells in vitro The EBV protein EBNA3A collaborates with EBNA3C to repress p16 expression and is required for efficient transformation in vitro An EBNA3A deletion mutant EBV strain was recently reported to establish latency in humanized mice but not cause tumors. Here, we compare the phenotypes of an EBNA3A mutant EBV (Δ3A) and wild-type (WT) EBV in a cord blood-humanized (CBH) mouse model. The hypomorphic Δ3A mutant, in which a stop codon is inserted downstream from the first ATG and the open reading frame is disrupted by a 1-bp insertion, expresses very small amounts of EBNA3A using an alternative ATG at residue 15. Δ3A caused B cell lymphomas at rates similar to their induction by WT EBV but with delayed onset. Δ3A and WT tumors expressed equivalent levels of EBNA2 and p16, but Δ3A tumors in some cases had reduced LMP1. Like the WT EBV tumors, Δ3A lymphomas were oligoclonal/monoclonal, with typically one dominant IGHV gene being expressed. Transcriptome sequencing (RNA-seq) analysis revealed small but consistent gene expression differences involving multiple cellular genes in the WT EBV- versus Δ3A-infected tumors and increased expression of genes associated with T cells, suggesting increased T cell infiltration of tumors. Consistent with an impact of EBNA3A on immune function, we found that the expression of CLEC2D, a receptor that has previously been shown to influence responses of T and NK cells, was markedly diminished in cells infected with EBNA3A mutant virus. Together, these studies suggest that EBNA3A contributes to efficient EBV-induced lymphomagenesis in CBH mice.IMPORTANCE The EBV protein EBNA3A is expressed in latently infected B cells and is important for efficient EBV-induced transformation of B cells in vitro In this study, we used a cord blood-humanized mouse model to compare the phenotypes of an EBNA3A hypomorph mutant virus (Δ3A) and wild-type EBV. The Δ3A virus caused lymphomas with delayed onset compared to the onset of those caused by WT EBV, although the tumors occurred at a similar rate. The WT EBV and EBNA3A mutant tumors expressed similar levels of the EBV protein EBNA2 and cellular protein p16, but in some cases, Δ3A tumors had less LMP1. Our analysis suggested that Δ3A-infected tumors have elevated T cell infiltrates and decreased expression of the CLEC2D receptor, which may point to potential novel roles of EBNA3A in T cell and NK cell responses to EBV-infected tumors.

B cells infected with Type 2 Epstein-Barr virus (EBV) have increased NFATc1/NFATc2 activity and enhanced lytic gene expression in comparison to Type 1 EBV infection.

Humans are infected with two distinct strains (Type 1 (T1) and Type 2 (T2)) of Epstein-Barr virus (EBV) that differ substantially in their EBNA2 and EBNA 3A/B/C latency genes and the ability to transform B cells in vitro. While most T1 EBV strains contain the "prototype" form of the BZLF1 immediate-early promoter ("Zp-P"), all T2 strains contain the "Zp-V3" variant, which contains an NFAT binding motif and is activated much more strongly by B-cell receptor signalling. Whether B cells infected with T2 EBV are more lytic than cells infected with T1 EBV is unknown. Here we show that B cells infected with T2 EBV strains (AG876 and BL5) have much more lytic protein expression compared to B cells infected with T1 EBV strains (M81, Akata, and Mutu) in both a cord blood-humanized (CBH) mouse model and EBV-transformed lymphoblastoid cell lines (LCLs). Although T2 LCLs grow more slowly than T1 LCLs, both EBV types induce B-cell lymphomas in CBH mice. T1 EBV strains (M81 and Akata) containing Zp-V3 are less lytic than T2 EBV strains, suggesting that Zp-V3 is not sufficient to confer a lytic phenotype. Instead, we find that T2 LCLs express much higher levels of activated NFATc1 and NFATc2, and that cyclosporine (an NFAT inhibitor) and knockdown of NFATc2 attenuate constitutive lytic infection in T2 LCLs. Both NFATc1 and NFATc2 induce lytic EBV gene expression when combined with activated CAMKIV (which is activated by calcium signaling and activates MEF2D) in Burkitt Akata cells. Together, these results suggest that B cells infected with T2 EBV are more lytic due to increased activity of the cellular NFATc1/c2 transcription factors in addition to the universal presence of the Zp-V3 form of BZLF1 promoter.

Mucosal-Associated Invariant T Cells in Tumors of Epithelial Origin.

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes that circulate in blood and also reside in mucosal tissues. Blood MAIT cells are typically highly Th1-polarized, while those in mucosal tissues include both Th1- and Th17-polarized subsets. MAIT cells mount cytokine and cytolytic responses as a result of T cell receptor (TCR)-mediated recognition of microbially derived metabolites of riboflavin (vitamin B2) presented by the MR1 antigen-presenting molecule. Additionally, MAIT cells can be activated by inflammatory cytokines produced by antigen-presenting cells (APCs) that have been exposed to pathogen-associated molecular patterns (PAMPs). Since the antigenic metabolites of riboflavin recognized by MAIT cells are produced by many microorganisms, including pathogens as well as non-pathogenic colonists, the inflammatory state of the tissue may be a key feature that determines the nature of MAIT cell responses. Under normal conditions where inflammatory cytokines are not produced, MAIT cell responses to microbial metabolites may simply serve to help maintain a healthy balance between epithelial cells and microbial colonists. In contrast, in situations where inflammatory cytokines are produced (e.g., pathogenic infection or damage to epithelial tissue), MAIT cell responses may be more potently pro-inflammatory. Since chronic inflammation and microbial drivers are associated with tumorigenesis and also trigger MAIT cell responses, the nexus of MAIT cells, local microbiomes, and epithelial cells may play an important role in epithelial carcinogenesis. This chapter reviews current information about MAIT cells and epithelial tumors, where the balance of evidence suggests that enrichment of Th17-polarized MAIT cells at tumor sites associates with poor patient prognosis. Studying the role of MAIT cells and their interactions with resident microbes offers a novel view of the biology of epithelial tumor progression and may ultimately lead to new approaches to target MAIT cells clinically.

Loss of Chondroitin Sulfate Modification Causes Inflammation and Neurodegeneration in skt Mice.

One major aspect of the aging process is the onset of chronic, low-grade inflammation that is highly associated with age-related diseases. The molecular mechanisms that regulate these processes have not been fully elucidated. We have identified a spontaneous mutant mouse line, small with kinky tail (skt), that exhibits accelerated aging and age-related disease phenotypes including increased inflammation in the brain and retina, enhanced age-dependent retinal abnormalities including photoreceptor cell degeneration, neurodegeneration in the hippocampus, and reduced lifespan. By positional cloning, we identified a deletion in chondroitin sulfate synthase 1 (Chsy1) that is responsible for these phenotypes in skt mice. CHSY1 is a member of the chondroitin N-acetylgalactosaminyltransferase family that plays critical roles in the biosynthesis of chondroitin sulfate, a glycosaminoglycan (GAG) that is attached to the core protein to form the chondroitin sulfate proteoglycan (CSPG). Consistent with this function, the Chsy1 mutation dramatically decreases chondroitin sulfate GAGs in the retina and hippocampus. In addition, macrophage and neutrophil populations appear significantly altered in the bone marrow and spleen of skt mice, suggesting an important role for CHSY1 in the functioning of these immune cell types. Thus, our study reveals a previously unidentified impact of CHSY1 in the retina and hippocampus. Specifically, chondroitin sulfate (CS) modification of proteins by CHSY1 appears critical for proper regulation of immune cells of the myeloid lineage and for maintaining the integrity of neuronal tissues, since a defect in this gene results in increased inflammation and abnormal phenotypes associated with age-related diseases.

Expansion and Adoptive Transfer of Human Vδ2+ T Cells to Assess Antitumor Effects In Vivo.

Recent clinical trials have yielded promising results suggesting that γδ T cell62-based immunotherapies can be effective against hematological malignancies. Human T cells expressing Vγ9Vδ2+ receptors are particularly attractive candidates for this application, since they can be readily expanded in vitro in large quantities for adoptive transfer and do not require HLA-matching of donors and recipients. While it is well established that Vγ9Vδ2+ T cells are potently cytolytic against many human cancers and it has been shown that they can control transplanted human tumors in xenogeneic model systems, little is known about the parameters that determine the antitumor efficacy of adoptively transferred Vγ9Vδ2+ T cells in physiologically relevant scenarios. In particular, it may be important to separate their immunosurveillance functions from those employed in the context of an established tumor. Moreover, it is critical to understand how the presence of an immunosuppressive environment, such as one where tumor-infiltrating T cells are held in check by inhibitory ligands, affects the functions of Vγ9Vδ2+ T cells. This chapter describes how to establish Epstein-Barr virus (EBV) infection of human umbilical cord blood mononuclear cells (CBMCs) within immunodeficient mice, so as to drive the in vivo formation of human B cell lymphomas that contain an immunosuppressive environment. Details are provided on how to expand human Vγ9Vδ2+ T cells from peripheral blood mononuclear cells (PBMCs), administer them to the mice, and evaluate tumors and other tissues.

Mucosal associated invariant T cells from human breast ducts mediate a Th17-skewed response to bacterially exposed breast carcinoma cells.

BACKGROUND: Antimicrobial 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. METHODS: Epithelial 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. RESULTS: We 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 Vß 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. CONCLUSIONS: These 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.

LFA-1 Ligation by High-Density ICAM-1 Is Sufficient To Activate IFN-γ Release by Innate T Lymphocytes.

By binding to its ligand ICAM-1, LFA-1 is known to mediate both adhesion and costimulatory signaling for T cell activation. The constitutively high LFA-1 cell surface expression of invariant NKT (iNKT) cells has been shown to be responsible for their distinctive tissue homing and residency within ICAM-rich endothelial vessels. However, the functional impact of LFA-1 on the activation of iNKT cells and other innate T lymphocyte subsets has remained largely unexplored. In particular, it is not clear whether LFA-1 contributes to innate-like pathways of T cell activation, such as IFN-γ secretion in response to IL-12. Using a recombinant ICAM-1-Fc fusion protein to stimulate human iNKT cells in the absence of APCs, we show that LFA-1 engagement enhances their IL-12-driven IFN-γ production. Surprisingly, exposure to high densities of ICAM-1 was also sufficient to activate iNKT cell cytokine secretion independently of IL-12 and associated JAK/STAT signaling. LFA-1 engagement induced elevated cytoplasmic Ca2+ and rapid ERK phosphorylation in iNKT cells, and the resulting IFN-γ secretion was dependent on both of these pathways. Analysis of freshly isolated human PBMC samples revealed that a fraction of lymphocytes that showed elevated LFA-1 cell surface expression produced IFN-γ in response to plate-bound ICAM-1-Fc. A majority of the responding cells were T cells, with the remainder NK cells. The responding T cells included iNKT cells, MAIT cells, and Vδ2+ γδ T cells. These results delineate a novel integrin-mediated pathway of IFN-γ secretion that is a shared feature of innate lymphocytes.

An EBNA3C-deleted Epstein-Barr virus (EBV) mutant causes B-cell lymphomas with delayed onset in a cord blood-humanized mouse model.

EBV causes human B-cell lymphomas and transforms B cells in vitro. EBNA3C, an EBV protein expressed in latently-infected cells, is required for EBV transformation of B cells in vitro. While EBNA3C undoubtedly plays a key role in allowing EBV to successfully infect B cells, many EBV+ lymphomas do not express this protein, suggesting that cellular mutations and/or signaling pathways may obviate the need for EBNA3C in vivo under certain conditions. EBNA3C collaborates with EBNA3A to repress expression of the CDKN2A-encoded tumor suppressors, p16 and p14, and EBNA3C-deleted EBV transforms B cells containing a p16 germline mutation in vitro. Here we have examined the phenotype of an EBNAC-deleted virus (Δ3C EBV) in a cord blood-humanized mouse model (CBH). We found that the Δ3C virus induced fewer lymphomas (occurring with a delayed onset) in comparison to the wild-type (WT) control virus, although a subset (10/26) of Δ3C-infected CBH mice eventually developed invasive diffuse large B cell lymphomas with type III latency. Both WT and Δ3C viruses induced B-cell lymphomas with restricted B-cell populations and heterogeneous T-cell infiltration. In comparison to WT-infected tumors, Δ3C-infected tumors had greatly increased p16 levels, and RNA-seq analysis revealed a decrease in E2F target gene expression. However, we found that Δ3C-infected tumors expressed c-Myc and cyclin E at similar levels compared to WT-infected tumors, allowing cells to at least partially bypass p16-mediated cell cycle inhibition. The anti-apoptotic proteins, BCL2 and IRF4, were expressed in Δ3C-infected tumors, likely helping cells avoid c-Myc-induced apoptosis. Unexpectedly, Δ3C-infected tumors had increased T-cell infiltration, increased expression of T-cell chemokines (CCL5, CCL20 and CCL22) and enhanced type I interferon response in comparison to WT tumors. Together, these results reveal that EBNA3C contributes to, but is not essential for, EBV-induced lymphomagenesis in CBH mice, and suggest potentially important immunologic roles of EBNA3C in vivo.

Modeling Human Antitumor Responses In Vivo Using Umbilical Cord Blood-Engrafted Mice.

Mice engrafted with human immune cells offer powerful in vivo model systems to investigate molecular and cellular processes of tumorigenesis, as well as to test therapeutic approaches to treat the resulting cancer. The use of umbilical cord blood mononuclear cells as a source of human immune cells for engraftment is technically straightforward, and provides T lymphocytes and autologous antigen-presenting cells (including B cells, monocytes, and DCs) that bear cognate antigen presenting molecules. By using a human-specific oncogenic virus, such as Epstein-Barr virus, de novo neoplastic transformation of the human B cells can be induced in vivo in a manner that models progressive stages of tumorigenesis from nascent neoplasia to the establishment of vascularized tumor masses with an immunosuppressive environment. Moreover, since tumorigenesis occurs in the presence of autologous T cells, this type of system can be used to investigate how T cells become suppressed during tumorigenesis, and how immunotherapies counteract immunosuppression. This minireview will provide a brief overview of the use of human umbilical cord blood transplanted into immunodeficient murine hosts to model antitumor responses.