Sandeep K. Tripathy

Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors.

The use of replication–defective adenoviruses (RDAd) for human gene therapy has been limited by host immune responses that result in transient recombinant gene expression in vivo. It remained unclear whether these immune responses were directed predominantly against viral proteins or, alternatively, against foreign transgene–encoded proteins. In this report, we have compared the stability of recombinant gene expression in adult immunocompetent mice following intramuscular (i.m.) injection with identical RDAd encoding self (murine) or foreign (human) erythropoietin. Our results demonstrate that immune responses directed against foreign transgene–encoded proteins are the major determinants of the stability of gene expression following i.m. injection of RDAd. Moreover, we demonstrate long–term recombinant gene expression in immunocompetent animals following a single i.m. injection of RDAd encoding a self protein. These findings are important for the design of future preclinical and clinical gene therapy trials.

Continuous engagement of a self-specific activation receptor induces NK cell tolerance

Natural killer (NK) cell tolerance mechanisms are incompletely understood. One possibility is that they possess self-specific activation receptors that result in hyporesponsiveness unless modulated by self–major histocompatability complex (MHC)–specific inhibitory receptors. As putative self-specific activation receptors have not been well characterized, we studied a transgenic C57BL/6 mouse that ubiquitously expresses m157 (m157-Tg), which is the murine cytomegalovirus (MCMV)–encoded ligand for the Ly49H NK cell activation receptor. The transgenic mice were more susceptible to MCMV infection and were unable to reject m157-Tg bone marrow, suggesting defects in Ly49H+ NK cells. There was a reversible hyporesponsiveness of Ly49H+ NK cells that extended to Ly49H-independent stimuli. Continuous Ly49H–m157 interaction was necessary for the functional defects. Interestingly, functional defects occurred when mature wild-type NK cells were adoptively transferred to m157-Tg mice, suggesting that mature NK cells may acquire hyporesponsiveness. Importantly, NK cell tolerance caused by Ly49H–m157 interaction was similar in NK cells regardless of expression of Ly49C, an inhibitory receptor specific for a self-MHC allele in C57BL/6 mice. Thus, engagement of self-specific activation receptors in vivo induces an NK cell tolerance effect that is not affected by self-MHC–specific inhibitory receptors.

Muscle-based gene therapy: realistic possibilities for the future

The past five years have witnessed tremendous growth in the field of gene therapy, with pre-clinical and clinical gene therapy trials for diseases as diverse as cancer, AIDS and atherosclerosis. These studies have utilized many different vectors and target organs in order to achieve therapeutic effects. In this review, we examine the rationale for using skeletal muscle as a target tissue for gene therapy, discuss the wide array of vectors that have been used for muscle-based gene therapy, summarize the disease-targets that have been approached using these techniques, and discuss some of the obstacles that remain to be overcome en route to successful muscle-based human gene therapy.

Expression of m157, a Murine Cytomegalovirus-Encoded Putative Major Histocompatibility Class I (MHC-I)-Like Protein, Is Independent of Viral Regulation of Host MHC-I

ABSTRACT A murine cytomegalovirus (MCMV)-encoded protein, m157, has a putative major histocompatibility complex class I (MHC-I) structure and is recognized by the Ly49H NK cell activation receptor. Using a monoclonal antibody against m157, in this study we directly demonstrated that m157 is a cell surface-expressed glycophosphatidylinositol-anchored protein with early viral gene kinetics. Beta-2 microglobulin and TAP1 (transporter associated with antigen processing 1) were not required for its expression. MCMV-encoded proteins that down-regulate MHC-I did not affect the expression of m157. Thus, m157 is expressed on infected cells in a manner independent of viral regulation of host MHC-I.

Activation Receptor-Induced Tolerance of Mature NK Cells In Vivo Requires Signaling through the Receptor and Is Reversible

NK cell responses are determined by signals received through activating and inhibitory cell surface receptors. Ly49H is an NK cell-specific activating receptor that accounts for the genetic resistance to murine CMV (MCMV). The Ly49H receptor has been shown to interact with two adaptor proteins (DAP12 and DAP10). In the context of MCMV infection, interaction of m157 (the MCMV-encoded ligand for Ly49H) with Ly49H results in activation of Ly49H-expressing NK cells. Chronic exposure of Ly49H with m157, however, induces tolerance in these same cells. The mechanism of this tolerance remains poorly understood. Using a transgenic mouse model, we demonstrate that induction of tolerance in Ly49H+ NK cells by chronic exposure to m157, in vivo, requires signaling through the Ly49H adaptor protein DAP12, but not the DAP10 adaptor protein. Furthermore, mature Ly49H-expressing NK cells from wild-type mice can acquire a tolerant phenotype by 24 h posttransfer into a transgenic C57BL/6 mouse that expresses m157. The tolerant phenotype can be reversed, in vivo, if tolerant NK cells are transferred to mice that do not express the m157 protein. Thus, continuous activating receptor engagement can induce a transient tolerance in mature NK cells in vivo. These observations provide new insight into how activating receptor engagement shapes NK cell function and has important implications in how NK cells respond to tumors and during chronic viral infection.

Acquisition of Activation Receptor Ligand by Trogocytosis Renders NK Cells Hyporesponsive

Because NK cells secrete cytotoxic granules and cytokines that can destroy surrounding cells and help shape the subsequent immune response, they must be kept under tight control. Several mechanisms, at different levels, are in place to control NK cell function. In this study, we describe a novel mechanism regulating NK cell function in which NK cells acquire ligands for activating receptors from target cells by trogocytosis, rendering the NK cells hyporesponsive. In this model, murine NK cells acquire m157, the murine CMV–encoded ligand for the Ly49H-activating receptor, from target cells both in vitro and in vivo. Although acquisition of m157 requires cell-to-cell contact, it does not require the expression of the Ly49H receptor by the NK cell. Acquired m157 protein is expressed on the NK cell surface with a glycosylphosphatidylinisotol linkage and interacts with the Ly49H receptor expressed on the NK cell. This interaction results in blocking the Ly49H receptor that prevents the NK cells from recognizing m157-expressing targets and continuous engagement of the Ly49H-activating receptor, which results in the hyporesponsiveness of the Ly49H+ NK cell to stimulation through other activating receptors. Thus, NK cell acquisition of a ligand for an activation receptor by trogocytosis renders them hyporesponsive. This mechanism, by which mature NK cell function can be altered, has important implications in regard to how NK cells respond to tumors in specific microenvironments as well as the use of expanded NK cells in treating various malignancies.

Viral infection transiently reverses activation receptor‐mediated NK cell hyporesponsiveness in an MHC class I‐independent mechanism

Continuous engagement of the Ly49H activating receptor with its ligand (m157) in a transgenic mouse expressing m157 (m157‐Tg) results in hyporesponsiveness of Ly49H+ NK cells. The same interaction, during murine cytomegalovirus (MCMV) infection, leads to activation of Ly49H+ NK cells. MCMV infection results in decreased MHC class I (MHC‐I) expression on the infected cell as well as inflammatory responses, both of which do not take place in the uninfected m157‐Tg mouse, potentially allowing for activation of NK cells in the context of MCMV infection. In this study, we demonstrated that viral infection transiently reverses activation receptor‐mediated NK cell hyporesponsiveness in an MHC‐I‐independent mechanism. Furthermore, Ly49H+ NK cells in an MHC‐I‐deficient environment remained hyporesponsive in the context of m157 expression, even when mature WT splenocytes were transferred into m157‐Tg mice in an MHC‐I‐deficient environment. However, the administration of cytokines TNF‐α, IL‐12, and IFN‐β resulted in a partial recovery from activation receptor‐induced hyporesponsiveness. Thus, the release of the aforementioned cytokines during MCMV infection and not the downregulation of MHC‐I expression appears to be responsible for partial resolution of Ly49H receptor‐induced NK cell hyporesponsiveness.

Expression of the inhibitory receptor NKG2A correlates with increased liver and splenic NK cell response to activating receptor engagement: Liver NK cell responses correlate with NKG2A levels

Natural killer (NK) cells play a critical role in the innate immune response to viruses and tumors, and comprise a large proportion of the hepatic lymphocyte population. They must remain tolerant to non‐pathogenic antigens while protecting the host from harmful agents. Herein, we investigate how the NK cell response to activation receptor engagement is altered in the liver.