Although recent evidence demonstrates heterogeneity among CD8+ T cells during chronic infection, developmental relationships and mechanisms underlying their fate decisions remain incompletely understood. Using single-cell RNA and TCR sequencing, we traced the clonal expansion and differentiation of CD8+ T cells during chronic LCMV infection. We identified immense clonal and phenotypic diversity, including a subset termed intermediate cells. Trajectory analyses and infection models showed intermediate cells arise from progenitor cells before bifurcating into terminal effector and exhausted subsets. Genetic ablation experiments identified that type I IFN drives exhaustion through an IRF7-dependent mechanism, possibly through an IFN-stimulated subset bridging progenitor and exhausted cells. Conversely, Zeb2 was critical for generating effector cells. Intriguingly, some T cell clones exhibited lineage bias. Mechanistically, we identified that TCR avidity correlates with an exhausted fate, whereas SHP-1 selectively restricts low-avidity effector cell accumulation. Thus, our work elucidates novel mechanisms underlying CD8+ T cell fate determination during persistent infection and suggests two potential pathways leading to exhaustion.

Gammaherpesviruses are ubiquitous pathogens that are associated with B cell lymphomas. In the early stages of chronic infection, these viruses infect naive B cells and subsequently usurp the B cell differentiation process through the germinal center response to ensure latent infection of long-lived memory B cells. A unique feature of early gammaherpesvirus chronic infection is a robust differentiation of irrelevant, virus-nonspecific B cells with reactivities against self-antigens and antigens of other species. In contrast, protective, virus-specific humoral responses do not reach peak levels until a much later time. While several host factors are known to either promote or selectively restrict gammaherpesvirus-driven germinal center response, viral mechanisms that contribute to the irrelevant B cell response have not been defined. In this report we show that the expression and the enzymatic activity of the gammaherpesvirus-encoded conserved protein kinase selectively facilitates the irrelevant, but not virus-specific, B cell responses. Further, we show that lack of interleukin-1 (IL-1) receptor attenuates gammaherpesvirus-driven B cell differentiation and viral reactivation. Because germinal center B cells are thought to be the target of malignant transformation during gammaherpesvirus-driven lymphomagenesis, identification of host and viral factors that promote germinal center responses during gammaherpesvirus infection may offer an insight into the mechanism of gammaherpesvirus pathogenesis. IMPORTANCE Gammaherpesviruses are ubiquitous cancer-associated pathogens that usurp the B cell differentiation process to establish life-long latent infection in memory B cells. A unique feature of early gammaherpesvirus infection is the robust increase in differentiation of B cells that are not specific for viral antigens and instead encode antibodies that react with self-antigens and antigens of other species. Viral mechanisms that are involved in driving such irrelevant B cell differentiation are not known. Here, we show that gammaherpesvirus-encoded conserved protein kinase and host IL-1 signaling promote irrelevant B cell responses and gammaherpesvirusdriven germinal center responses, with the latter thought to be the target of viral transformation.

Gammaherpesviruses are ubiquitous viruses that establish lifelong infections. Importantly, these viruses are associated with numerous cancers and lymphoproliferative diseases. While risk factors for developing gammaherpesvirus-driven cancers are poorly understood, it is clear that elevated viral reactivation from latency often precedes oncogenesis. Here, we demonstrate that the liver X receptor alpha isoform (LXRα) restricts gammaherpesvirus reactivation in an anatomic-site-specific manner. We have previously demonstrated that deficiency of both LXR isoforms (α and β) leads to an increase in fatty acid and cholesterol synthesis in primary macrophage cultures, with a corresponding increase in gammaherpesvirus replication. Interestingly, expression of fatty acid synthesis genes was not derepressed in LXRα-deficient hosts, indicating that the antiviral effects of LXRα are independent of lipogenesis. Additionally, the critical host defenses against gammaherpesvirus reactivation, virus-specific CD8+ T cells and interferon (IFN) signaling, remained intact in the absence of LXRα. Remarkably, using a murine gammaherpesvirus 68 (MHV68) reporter virus, we discovered that LXRα expression dictates the cellular tropism of MHV68 in the peritoneal cavity. Specifically, LXRα−/− mice exhibit reduced latency within the peritoneal B cell compartment and elevated latency within F4/80+ cells. Thus, LXRα restricts gammaherpesvirus reactivation through a novel mechanism that is independent of the known CD8+ T cell-based antiviral responses or changes in lipid synthesis and likely involves changes in the tropism of MHV68 in the peritoneal cavity. IMPORTANCE Liver X receptors (LXRs) are nuclear receptors that mediate cholesterol and fatty acid homeostasis. Importantly, as ligand-activated transcription factors, LXRs represent potential targets for the treatment of hypercholesterolemia and atherosclerosis. Here, we demonstrate that LXRα, one of the two LXR isoforms, restricts reactivation of latent gammaherpesvirus from peritoneal cells. As gammaherpesviruses are ubiquitous oncogenic agents, LXRs may represent a targetable host factor for the treatment of poorly controlled gammaherpesvirus infection and associated lymphomagenesis.

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Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections and are associated with a variety of malignancies, including lymphomas. Interferon Regulatory Factor 7 (IRF-7) is an innate immune transcription factor that restricts acute replication of diverse viruses, including mouse gammaherpesvirus 68 (MHV68). Importantly, very little is known about the role of IRF-7 during chronic virus infections. In this study we demonstrate that IRF-7 attenuates chronic infection by restricting establishment of gammaherpesvirus latency in the peritoneal cavity and, to a lesser extent, viral reactivation in the spleen. Despite the classical role of IRF-7 as a stimulator of type I interferon (IFN) transcription, there were no global effects on the expression of IFN-induced genes (ISGs) in the absence of IRF-7, with only a few ISGs showing attenuated expression in IRF-7 deficient peritoneal cells. Further, IRF-7 expression was dispensable for the induction of virus-specific CD8 T cell response. In contrast, IRF-7 expression restricted latent gammaherpesvirus infection in the peritoneal cavity under conditions when the viral latent reservoir is predominantly hosted by peritoneal B cells. This report is the first demonstration of the antiviral role of IRF-7 during the chronic stage of gammaherpesvirus infection. Importance The innate immune system of the host is critical for the restriction of acute viral infections. In contrast, the role of the innate immune network during chronic herpesvirus infection remains poorly defined. Interferon Regulatory Factor 7 (IRF-7) is a transcription factor with many target genes, including type I interferons (IFNs). In this study we show that the antiviral role of IRF-7 continues into the chronic phase of gammaherpesvirus infection, wherein IRF-7 restricts the establishment of viral latency and viral reactivation. This study is to our knowledge the first to define the role of IRF-7 in chronic virus infection.

Gammaherpesviruses are ubiquitous pathogens that are associated with cancers, including B cell lymphomas. These viruses are unique in that they infect naive B cells and subsequently drive a robust polyclonal germinal center response in order to amplify the latent reservoir and to establish lifelong infection in memory B cells. The gammaherpesvirus-driven germinal center response in combination with robust infection of germinal center B cells is thought to precipitate lymphomagenesis. Importantly, host and viral factors that selectively affect the gammaherpesvirus-driven germinal center response remain poorly understood. Global deficiency of antiviral tumor-suppressive interferon regulatory factor 1 (IRF-1) selectively promotes the murine gammaherpesvirus 68 (MHV68)-driven germinal center response and expansion of the viral latent reservoir. To determine the extent to which antiviral effects of IRF-1 are B cell intrinsic, we generated mice with conditional IRF-1 deficiency. Surprisingly, B cell-specific IRF-1 deficiency attenuated the establishment of chronic infection and the germinal center response, indicating that MHV68 may, in a B cell-intrinsic manner, usurp IRF-1 to promote the germinal center response and expansion of the latent reservoir. Further, we found that B cell-specific IRF-1 deficiency led to reduced levels of active tyrosine phosphatase SHP1, which plays a B cell-intrinsic proviral function during MHV68 infection. Finally, results of this study indicate that the antiviral functions of IRF-1 unveiled in MHV68-infected mice with global IRF-1 deficiency are mediated via IRF-1 expression by non-B cell populations. IMPORTANCE Gammaherpesviruses establish lifelong infection in over 95% of all adults and are associated with B cell lymphomas. The virus’s manipulation of the germinal center response and B cell differentiation to establish lifelong infection is thought to also precipitate malignant transformation, through a mechanism that remains poorly understood. The host transcription factor IRF-1, a well-established tumor suppressor, selectively attenuates MHV68-driven germinal center response, a phenotype that we originally hypothesized to occur in a B cell-intrinsic manner. In contrast, in testing, B cell-intrinsic IRF-1 expression promoted the MHV68-driven germinal center response and the establishment of chronic infection. Our report highlights the underappreciated multifaceted role of IRF-1 in MHV68 infection and pathogenesis.

Gammaherpesviruses establish lifelong infection and are associated with a variety of cancers, including B cell lymphomas. These viruses manipulate the B cell differentiation process to establish lifelong infection in memory B cells. Specifically, gammaherpesviruses infect naive B cells and promote entry of both infected and uninfected naive B cells into germinal centers, where the virus usurps rapid proliferation of germinal center B cells to exponentially increase its cellular latent reservoir. In addition to facilitating the establishment of latent infection, germinal center B cells are thought to be the target of viral transformation. In this study, we have uncovered a novel proviral role of host interleukin 17A (IL-17A), a well-established antibacterial and antifungal factor. Loss of IL-17A signaling attenuated the establishment of chronic gammaherpesvirus infection and gammaherpesvirus-driven germinal center response in a route of inoculation-dependent manner. Further, IL-17A treatment directly supported gammaherpesvirus reactivation and de novo lytic infection. This study is the first demonstration of a multifaceted proviral role of IL-17 signaling. IMPORTANCE Gammaherpesviruses establish lifelong infections in a majority of humans and are associated with B cell lymphomas. IL-17A is a host cytokine that plays a well-established role in the clearance of bacterial and fungal infections; however, the role of IL-17A in viral infections is poorly understood. In this study, we show that IL-17A signaling promoted the establishment of chronic gammaherpesvirus infection following the mucosal route of infection, viral lytic replication, and reactivation from latency. Thus, our study unveils a novel proviral role of IL-17A signaling in gammaherpesvirus infection.

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