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.

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.

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.

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.

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.

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.

The 2.5 Å structure of CD1c in complex with a mycobacterial lipid reveals an open groove ideally suited for diverse antigen presentation.

CD1 molecules function to present lipid-based antigens to T cells. Here we present the crystal structure of CD1c at 2.5 Å resolution, in complex with the pathogenic Mycobacterium tuberculosis antigen mannosyl-ß1-phosphomycoketide (MPM). CD1c accommodated MPM's methylated alkyl chain exclusively in the A' pocket, aided by a unique exit portal underneath the α1 helix. Most striking was an open F' pocket architecture lacking the closed cavity structure of other CD1 molecules, reminiscent of peptide binding grooves of classical major histocompatibility complex molecules. This feature, combined with tryptophan-fluorescence quenching during loading of a dodecameric lipopeptide antigen, provides a compelling model by which both the lipid and peptide moieties of the lipopeptide are involved in CD1c presentation of lipopeptides.

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.

PD-1/CTLA-4 Blockade Inhibits Epstein-Barr Virus-Induced Lymphoma Growth in a Cord Blood Humanized-Mouse Model.

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.

Human Invariant NKT Cells Induce IL-1ß Secretion by Peripheral Blood Monocytes via a P2X7-Independent Pathway.

The cytokine IL-1ß plays a central role in inflammatory responses that are initiated by microbial challenges, as well as in those that are due to endogenous processes (often called sterile inflammation). IL-1ß secretion that occurs independently of microbial stimulation is typically associated with the presence of endogenous alarmins, such as extracellular ATP (an indicator of cytopathic damage). In this study, we show that IL-2-activated human invariant NKT (iNKT) cells stimulate the secretion of IL-1ß protein by human peripheral blood monocytes in a manner that requires neither the presence of microbial compounds nor signaling through the extracellular ATP receptor P2X7 Monocyte IL-1ß production was specifically induced by iNKT cells, because similarly activated polyclonal autologous T cells did not have this effect. Secretion of IL-1ß protein occurred rapidly (within 3-4 h) and required cell contact between the iNKT cells and monocytes. Similar to IL-1ß production induced by TLR stimulation, the iNKT-induced pathway appeared to entail a two-step process involving NF-κB signaling and IL1B gene transcription, as well as assembly of the NLRP3 inflammasome and activation of caspase-1. However, in contrast to the classical inflammasome-mediated pathway of IL-1ß production, activation of monocytes via P2X7 was dispensable for iNKT-induced IL-1ß secretion, and potassium efflux was not required. Moreover, the iNKT-induced effect involved caspase-8 activity, yet it induced little monocyte death. These results suggest that IL-2-activated human iNKT cells induce monocytes to produce IL-1ß through a distinctive pathway that does not require the presence of microbial danger signals or alarmins associated with cytopathic damage.

Are human iNKT cells keeping tabs on lipidome perturbations triggered by oxidative stress in the blood?

The central paradigm of conventional MHC-restricted T cells is that they respond specifically to foreign peptides, while displaying tolerance to self-antigens. In contrast, it is now becoming clear that a number of innate-like T cell subsets-CD1-restricted T cells, Vγ9Vδ2 T cells, and MAIT cells-may operate by different rules: rather than focusing on the recognition of specific foreign antigens, these T cells all appear to respond to alterations to lipid-related pathways. By monitoring perturbations to the "lipidome," these T cells may be able to spring into action to deal with physiological situations that are of self as well as microbial origin. iNKT cells are a prime example of this type of lipidome-reactive T cell. As a result of their activation by self lyso-phospholipid species that are generated downstream of blood lipid oxidation, human iNKT cells in the vasculature may respond sensitively to a variety of oxidative stresses. Some of the cytokines produced by activated iNKT cells have angiogenic effects (e.g., GM-CSF, IL-8), whereas others (e.g., IFN-γ) are pro-inflammatory factors that can propagate vascular pathology by influencing the functions of macrophages and dendritic cells. Consistent with this, evidence is accumulating that iNKT cells contribute to atherosclerosis, which is one of the most common inflammatory pathologies, and one that is integrally related to characteristics of the lipidome.

Transcriptional regulation of the human TNFSF11 gene in T cells via a cell type-selective set of distal enhancers.

In addition to osteoblast lineage cells, the TNF-like factor receptor activator of NF-κB ligand (RANKL) is expressed in both B and T cells and may play a role in bone resorption. Rankl gene (Tnfsf11) expression in mouse T cells is mediated through multiple distal elements marked by increased transcription factor occupancy, histone tail acetylation, and RNA polymerase II recruitment. Little is known, however, of the regulation of human TNFSF11 in T cells. Accordingly, we examined the consequence of T cell activation on the expression of this factor both in Jurkat cells and in primary human T cells. We then explored the mechanism of this regulation by scanning over 400 kb of DNA surrounding the TNFSF11 locus for regulatory enhancers using ChIP-chip analysis. Histone H3/H4 acetylation enrichment identified putative regulatory regions located between -170 and -220 kb upstream of the human TNFSF11 TSS that we designated the human T cell control region (hTCCR). This region showed high sequence conservation with the mouse TCCR. Inhibition of MEK1/2 by U0126 resulted in decreased RANKL expression suggesting that stimulation through MEK1/2 was a prerequisite. ChIP-chip analysis also revealed that c-FOS was recruited to the hTCCR as well. Importantly, both the human TNFSF11 D5a/b (RLD5a/b) enhancer and segments of the hTCCR mediated robust inducible reporter activity following TCR activation. Finally, SNPs implicated in diseases characterized by dysregulated BMD co-localized to the hTCCR region. We conclude that the hTCCR region contains a cell-selective set of enhancers that plays an integral role in the transcriptional regulation of the TNFSF11 gene in human 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.

Mice engrafted with human fetal thymic tissue and hematopoietic stem cells develop pathology resembling chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a significant roadblock to long-term hematopoietic stem cell (HSC) transplantation success. Effective treatments for cGVHD have been difficult to develop, in part because of a paucity of animal models that recapitulate the multiorgan pathologies observed in clinical cGVHD. Here we present an analysis of the pathology that occurs in immunodeficient mice engrafted with human fetal HSCs and implanted with fragments of human fetal thymus and liver. Starting at time points generally later than 100 days post-transplantation, the mice developed signs of illness, including multiorgan cellular infiltrates containing human T cells, B cells, and macrophages; fibrosis in sites such as lungs and liver; and thickened skin with alopecia. Experimental manipulations that delayed or reduced the efficiency of the HSC engraftment did not affect the timing or progression of disease manifestations, suggesting that pathology in this model is driven more by factors associated with the engrafted human thymic organoid. Disease progression was typically accompanied by extensive fibrosis and degradation of the thymic organoid, and there was an inverse correlation of disease severity with the frequency of FoxP3(+) thymocytes. Hence, the human thymic tissue may contribute T cells with pathogenic potential, but the generation of regulatory T cells in the thymic organoid may help to control these cells before pathology resembling cGVHD eventually develops. This model thus provides a new system to investigate disease pathophysiology relating to human thymic events and to evaluate treatment strategies to combat multiorgan fibrotic pathology produced by human immune cells.

An Epstein-Barr Virus (EBV) mutant with enhanced BZLF1 expression causes lymphomas with abortive lytic EBV infection in a humanized mouse model.

Immunosuppressed patients are at risk for developing Epstein-Barr Virus (EBV)-positive lymphomas that express the major EBV oncoprotein, LMP1. Although increasing evidence suggests that a small number of lytically infected cells may promote EBV-positive lymphomas, the impact of enhanced lytic gene expression on the ability of EBV to induce lymphomas is unclear. Here we have used immune-deficient mice, engrafted with human fetal hematopoietic stem cells and thymus and liver tissue, to compare lymphoma formation following infection with wild-type (WT) EBV versus infection with a "superlytic" (SL) mutant with enhanced BZLF1 (Z) expression. The same proportions (2/6) of the WT and SL virus-infected animals developed B-cell lymphomas by day 60 postinfection; the remainder of the animals had persistent tumor-free viral latency. In contrast, all WT and SL virus-infected animals treated with the OKT3 anti-CD3 antibody (which inhibits T-cell function) developed lymphomas by day 29. Lymphomas in OKT3-treated animals (in contrast to lymphomas in the untreated animals) contained many LMP1-expressing cells. The SL virus-infected lymphomas in both OKT3-treated and untreated animals contained many more Z-expressing cells (up to 30%) than the WT virus-infected lymphomas, but did not express late viral proteins and thus had an abortive lytic form of EBV infection. LMP1 and BMRF1 (an early lytic viral protein) were never coexpressed in the same cell, suggesting that LMP1 expression is incompatible with lytic viral reactivation. These results show that the SL mutant induces an "abortive" lytic infection in humanized mice that is compatible with continued cell growth and at least partially resistant to T-cell killing.

Expression of CD1c enhances human invariant NKT cell activation by α-GalCer.

Invariant natural killer T (iNKT) cells are innate T lymphocytes that specifically recognize α-linked glycosphingolipids (α-GSLs) as antigens presented by CD1d molecules. Activating iNKT cells by administering α-GSLs improves disease outcomes in murine cancer models and, thus, there is great interest in the clinical potential of these lipids for treating human cancers. However, humans possess several other CD1 isoforms that are not present in mice and it is not clear whether these CD1 molecules, which also bind lipids, affect human iNKT cell responses. We demonstrate here that CD1c, which is co-expressed with CD1d on blood dendritic cells and on a fraction of B cells, is able to present α-galactosylceramide (α-GalCer) as a weak agonist to human iNKT cells, and that the presence of CD1c synergistically enhances α-GalCerdependent activation of iNKT cells by CD1d. Primary human B cells expressing CD1c induced stronger iNKT cell responses to α-GalCer than the CD1c- subset, and an antibody against CD1c inhibited iNKT cell cytokine secretion. These results suggest that therapeutic activation of human iNKT cells by α-GSLs will be driven preferentially by CD1c+ cell types. Thus, B cell neoplasias that co-express CD1c and CD1d may be particularly susceptible to α-GSL therapy, and cancer vaccines using α-GSLs as adjuvants may be most effective when presented by CD1c+ antigen-presenting cells.

A live imaging cell motility screen identifies prostaglandin E2 as a T cell stop signal antagonist.

The T cell migration stop signal is a central step in T cell activation and inflammation; however, its regulatory mechanisms remain largely unknown. Using a live-cell, imaging-based, high-throughput screen, we identified the PG, PGE(2), as a T cell stop signal antagonist. Src kinase inhibitors, microtubule inhibitors, and PGE(2) prevented the T cell stop signal, and impaired T cell-APC conjugation and T cell proliferation induced by primary human allogeneic dendritic cells. However, Src inhibition, but not PGE(2) or microtubule inhibition, impaired TCR-induced ZAP-70 signaling, demonstrating that T cell stop signal antagonists can function either upstream or downstream of proximal TCR signaling. Moreover, we found that PGE(2) abrogated TCR-induced activation of the small GTPase Rap1, suggesting that PGE(2) may modulate T cell adhesion and stopping through Rap1. These results identify a novel role for PGs in preventing T cell stop signals and limiting T cell activation induced by dendritic 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.

A new model of Epstein-Barr virus infection reveals an important role for early lytic viral protein expression in the development of lymphomas.

Epstein-Barr virus (EBV) infects cells in latent or lytic forms, but the role of lytic infection in EBV-induced lymphomas is unclear. Here, we have used a new humanized mouse model, in which both human fetal CD34(+) hematopoietic stem cells and thymus/liver tissue are transplanted, to compare EBV pathogenesis and lymphoma formation following infection with a lytic replication-defective BZLF1-deleted (Z-KO) virus or a lytically active BZLF1(+) control. Both the control and Z-KO viruses established long-term viral latency in all infected animals. The infection appeared well controlled in some animals, but others eventually developed CD20(+) diffuse large B cell lymphomas (DLBCL). Animals infected with the control virus developed tumors more frequently than Z-KO virus-infected animals. Specific immune responses against EBV-infected B cells were generated in mice infected with either the control virus or the Z-KO virus. In both cases, forms of viral latency (type I and type IIB) were observed that are less immunogenic than the highly transforming form (type III) commonly found in tumors of immunocompromised hosts, suggesting that immune pressure contributed to the outcome of the infection. These results point to an important role for lytic EBV infection in the development of B cell lymphomas in the context of an active host immune response.