Subramanya Hegde

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

ABSTRACT 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.

Autoreactive natural killer T cells: promoting immune protection and immune tolerance through varied interactions with myeloid antigen-presenting cells

Natural killer T (NKT) cells are innate T lymphocytes that are restricted by CD1d antigen‐presenting molecules and recognize lipids and glycolipids as antigens. NKT cells have attracted attention for their potent immunoregulatory effects. Like other types of regulatory lymphocytes, a high proportion of NKT cells appear to be autoreactive to self antigens. Thus, as myeloid antigen‐presenting cells (APCs) such as monocytes, dendritic cells (DCs) and myeloid‐derived suppressor cells (MDSCs) constitutively express CD1d, NKT cells are able to interact with these APCs not only during times of immune activation but also in immunologically quiescent periods. The interactions of NKT cells with myeloid APCs can have either pro‐inflammatory or tolerizing outcomes, and a central question is how the ensuing response is determined. Here we bring together published results from a variety of model systems to highlight three critical factors that influence the outcome of the NKT–APC interaction: (i) the strength of the antigenic signal delivered to the NKT cell, as determined by antigen abundance and/or T‐cell receptor (TCR) affinity; (ii) the presence or absence of cytokines that costimulate NKT cells [e.g. interleukin (IL)‐12, IL‐18 and interferon (IFN)‐α]; (iii) APC intrinsic factors such as differentiation state (e.g. monocyte versus DC) and Toll‐like receptor (TLR) stimulation. Together with recent findings that demonstrate new links between NKT cell activation and endogenous lipid metabolism, these results outline a picture in which the functions of NKT cells are closely attuned to the existing biological context. Thus, NKT cells may actively promote tolerance until a critical level of danger signals arises, at which point they switch to activating pro‐inflammatory immune responses.

NKT cells direct monocytes into a DC differentiation pathway

Monocytes can differentiate into macrophags or dendritic cells (DCs). The processes that promote their differentiation along one pathway rather than the other remain unknown. NKT cells are regulatory T cells that respond functionally to self and foreign antigens presented by CD1d molecules. Hence, in addition to contributing to antimicrobial responses, they may carry out autoreactively activated functions when there is no infectious challenge. However, the immunological consequences of NKT cell autoreactivity remain poorly understood. We show here that human NKT cells direct monocytes to differentiate into immature DCs. The ability to induce monocyte differentiation was CD1d‐dependent and appeared specific to NKT cells. Addition of exogenous antigens or costimulation from IL‐2 was not required but could enhance the effect. DC differentiation was a result of NKT cell secretion of GM‐CSF and IL‐13, cytokines that were produced by the NKT cells upon autoreactive activation by monocytes. NKT cells within PBMC samples produced GM‐CSF and IL‐13 upon exposure to autologous monocytes directly ex vivo, providing evidence that such NKT cell‐autoreactive responses can occur in vivo. These results show that when NKT cells are activated by autologous monocytes, they are capable of providing factors that specifically direct monocyte differentiation into immature DCs. Thus, autoreactively activated NKT cells may contribute to the maintenance of the immature DC population, and microbial infection or inflammatory conditions that activate NKT cells further could stimulate them to promote an increased rate of DC differentiation.

Human invariant natural killer T cells acquire transient innate responsiveness via histone H4 acetylation induced by weak TCR stimulation

Invariant NKT cells (iNKT cells) are innate T lymphocytes that are thought to play an important role in producing an early burst of IFN-γ that promotes successful tumor immunosurveillance and antimicrobial immunity. The cellular activation processes underlying innate IFN-γ production remain poorly understood. We show here that weak T cell receptor (TCR) stimulation that does not directly activate iNKT cell IFN-γ messenger RNA transcription nevertheless induces histone H4 acetylation at specific regions near the IFNG gene locus. This renders the iNKT cells able to produce IFN-γ in an innate manner (i.e., not requiring concurrent TCR stimulation) upon exposure to IL-12 and IL-18. The iNKT cells retain the capacity for innate activation for hours to days after the initial weak TCR stimulation, although their innate responsiveness gradually declines as a function of histone deacetylation. These results explain how iNKT cells are able to mediate rapid innate IFN-γ secretion in a manner that does not require them to undergo permanent T(H1) differentiation. Moreover, our results also indicate that iNKT cell motility is maintained during activation by IL-12 and IL-18. Therefore, iNKT cells activated through this pathway can continue to migrate and may thus disseminate the IFN-γ that they produce, which may amplify its impact.

Human NKT cells promote monocyte differentiation into suppressive myeloid antigen-presenting cells

NKT cells have been shown to promote peripheral tolerance in a number of model systems, yet the processes by which they exert their regulatory effects remain poorly understood. Here, we show that soluble factors secreted by human NKT cells instruct human peripheral blood monocytes to differentiate into myeloid APCs that have suppressive properties. NKT‐instructed monocytes acquired a cell surface phenotype resembling myeloid DCs. However, whereas control DCs that were generated by culturing monocytes with recombinant GM‐CSF and IL‐4 had a proinflammatory phenotype characterized by the production of IL‐12 with little IL‐10, NKT‐instructed APCs showed the opposite cytokine production profile of high IL‐10 with little or no IL‐12. The control DCs efficiently stimulated peripheral blood T cell IFN‐γ secretion and proliferation, whereas NKT‐instructed APCs silenced these T cell responses. Exposure to NKT cell factors had a dominant effect on the functional properties of the DCs, since DCs differentiated by recombinant GM‐CSF and IL‐4 in the presence of NKT cell factors inhibited T cell responses. To confirm their noninflammatory effects, NKT‐instructed APCs were tested in an in vivo assay that depends on the activation of antigen‐specific human T cells. Control DCs promoted substantial tissue inflammation; however, despite a marked neutrophilic infiltrate, there was little edema in the presence of NKT‐instructed APCs, suggesting the inflammatory cascade was held in check. These results point to a novel pathway initiated by NKT cells that can contribute to the regulation of human antigen‐specific Th1 responses.

Analysis of the CD1 Antigen Presenting System in Humanized SCID Mice

CD1 molecules are glycoproteins that present lipids and glycolipids for recognition by T cells. CD1-dependent immune activation has been implicated in a wide range of immune responses, however, our understanding of the role of this pathway in human disease remains limited because of species differences between humans and other mammals: whereas humans express five different CD1 gene products (CD1a, CD1b, CD1c, CD1d, and CD1e), muroid rodents express only one CD1 isoform (CD1d). Here we report that immune deficient mice engrafted with human fetal thymus, liver, and CD34+ hematopoietic stem cells develop a functional human CD1 compartment. CD1a, b, c, and d isoforms were highly expressed by human thymocytes, and CD1a+ cells with a dendritic morphology were present in the thymic medulla. CD1+ cells were also detected in spleen, liver, and lungs. APCs from spleen and liver were capable of presenting bacterial glycolipids to human CD1-restricted T cells. ELISpot analyses of splenocytes demonstrated the presence of CD1-reactive IFN-γ producing cells. CD1d tetramer staining directly identified human iNKT cells in spleen and liver samples from engrafted mice, and injection of the glycolipid antigen α-GalCer resulted in rapid elevation of human IFN-γ and IL-4 levels in the blood indicating that the human iNKT cells are biologically active in vivo. Together, these results demonstrate that the human CD1 system is present and functionally competent in this humanized mouse model. Thus, this system provides a new opportunity to study the role of CD1-related immune activation in infections to human-specific pathogens.

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, PGE2, as a T cell stop signal antagonist. Src kinase inhibitors, microtubule inhibitors, and PGE2 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 PGE2 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 PGE2 abrogated TCR-induced activation of the small GTPase Rap1, suggesting that PGE2 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.

Human NKT cells direct the differentiation of myeloid APCs that regulate T cell responses via expression of programmed cell death ligands

NKT cells are innate lymphocytes that can recognize self or foreign lipids presented by CD1d molecules. NKT cells have been shown to inhibit the development of autoimmunity in murine model systems, however, the pathways by which they foster immune tolerance remain poorly understood. Here we show that autoreactive human NKT cells stimulate monocytes to differentiate into myeloid APCs that have a regulatory phenotype characterized by poor conjugate formation with T cells. The NKT cell instructed myeloid APCs show elevated expression of the inhibitory ligand PD-L2, and blocking PD-L1 and PD-L2 during interactions of the APCs with T cells results in improved cluster formation and significantly increased T cell proliferative responses. The elevated expression of PD-L molecules on NKT-instructed APCs appears to result from exposure to extracellular ATP that is produced during NKT-monocyte interactions, and blocking purinergic signaling during monocyte differentiation results in APCs that form clusters with T cells and stimulate their proliferation. Finally, we show that human monocytes and NKT cells that are injected into immunodeficient mice co-localize together in spleen and liver, and after 3 days in vivo in the presence of NKT cells a fraction of the myeloid cells have upregulated markers associated with differentiation into professional APCs. These results suggest that autoreactive human NKT cells may promote tolerance by inducing the differentiation of regulatory myeloid APCs that limit T cell proliferation through expression of PD-L molecules.

Natural killer T cells: innate lymphocytes positioned as a bridge between acute and chronic inflammation?

Natural killer T cells are an innate population of T lymphocytes that recognize antigens derived from host lipids and glycolipids. In this review, we focus on how these unique T cells are positioned to influence both acute and chronic inflammatory processes through their early recruitment to sites of inflammation, interactions with myeloid antigen presenting cells, and recognition of lipids associated with inflammation.

Differential requirement for P2X7R function in IL-17 dependent vs. IL-17 independent cellular immune responses.

IL17‐dependent autoimmunity to collagen type V (Col V) has been associated with lung transplant obliterative bronchiolitis. Unlike the T helper 1 (Th1)‐dependent immune responses to Tetanus Toxoid (TT), the Th17 response to Col V in lung transplant patients and its Th1/17 variant observed in coronary artery disease patients requires IL‐1β, tumor necrosis factor α and CD14+ cells. Given the involvement of the P2X7 receptor (P2X7R) in monocyte IL‐1β responses, we investigated its role in Th17‐, Th1/17‐ and Th1‐mediated proinflammatory responses. Transfer of antigen‐pulsed peripheral blood mononucleated cells (PBMCs) from Col V‐reactive patients into SCID mouse footpads along with P2X7R antagonists revealed a selective inhibition of Col V‐, but not TT‐specific swelling responses. P2X7R inhibitors blocked IL‐1β induction from monocytes, including both Col V‐α1 peptide‐induced (T‐dependent), as well as native Col V‐induced (T‐independent) responses. Significantly higher P2X7R expression was found on CXCR3negCCR4+/6+ CD4+ [Th17] versus CXCR3+CCR4/6neg CD4+ [Th1] subsets in PBMCs, suggesting that the paradigm of selective dependence on P2X7R might extend beyond Col V autoimmunity. Indeed, P2X7R inhibitors suppressed not only anti‐Col V, but also Th1/17‐mediated alloimmunity, in a heart transplant patient without affecting anti‐viral Epstein–Barr virus responses. These results suggest that agents targeting the P2X7R might effectively treat Th17‐related transplant pathologies, while maintaining Th1‐immunity to infection.