CD4 T cells specific for a latency-associated γ-herpesvirus epitope are polyfunctional and cytotoxic.

The oncogenic γ-herpesviruses EBV and Kaposi sarcoma-associated herpesvirus are ubiquitous human pathogens that establish lifelong latent infections maintained by intermittent viral reactivation and reinfection. Effector CD4 T cells are critical for control of viral latency and in immune therapies for virus-associated tumors. In this study, we exploited γHV68 infection of mice to enhance our understanding of the CD4 T cell response during γ-herpesvirus infection. Using a consensus prediction approach, we identified 16 new CD4 epitope-specific responses that arise during lytic infection. An additional epitope encoded by the M2 protein induced uniquely latency-associated CD4 T cells, which were not detected at the peak of lytic infection but only during latency and were not induced postinfection with a latency-deficient virus. M2-specific CD4 T cells were selectively cytotoxic, produced multiple antiviral cytokines, and sustained IL-2 production. Identification of latency-associated cytolytic CD4 T cells will aid in dissecting mechanisms of CD4 immune control of γ-herpesvirus latency and the development of therapeutic approaches to control viral reactivation and pathology.

Promotion of a subdominant CD8 T cell response during murine gammaherpesvirus 68 infection in the absence of CD4 T cell help.

CD8 and CD4 T cells are each critically important for immune control of murine gammaherpesvirus 68 (γHV68) infection. In immunocompetent mice, acute γHV68 infection results in lifelong latency, but in the absence of CD4 T cell help, mice succumb to viral recrudescence and disease. However, the requirements for CD4 T cell help in the generation and maintenance of antiviral CD8 T cell responses are incompletely understood, and it is unclear whether there are epitope-specific differences in the requirement of CD8 T cells for CD4 help. In this report, we characterized the CD8 T cell response to γHV68 in major histocompatibility complex (MHC) class II(-/-) mice, which lack CD4 T cells, or after antibody-mediated depletion of CD4 T cells. All antiviral CD8 T cells exhibited marked upregulation of surface expression of the inhibitory receptor programmed death-1 (PD-1), but surprisingly, while the immunodominant memory response appeared to be functionally impaired, helpless CD8 T cells of a subdominant specificity had increased numbers and enhanced functionality. Thus, we demonstrate differential requirements for CD4 help in the antiviral CD8 T cell response to a latent gammaherpesvirus. Importance: γHV68 is a mouse pathogen closely related to the oncogenic human γHVs, which infect a majority of the world's population. Reactivation of these viruses from latency can lead to complications, disease, and even death. CD4 T cells are required for complete immune control of long-term infection, in part by providing key signals to dendritic cells that in turn instruct optimal antiviral CD8 T cell responses. We have investigated multiple virus-specific CD8 T cell responses during infection and identified a subdominant CD8 T cell response that is numerically and functionally enhanced in the absence of CD4 T cell help. This occurs in spite of high surface expression of an inhibitory receptor and in contrast to the immunodominant response, which is impaired. Our data suggest that signals from CD4 T cells are important in maintaining the CD8 T cell hierarchy during γHV infections.

Gammaherpesvirus latency induces antibody-associated thrombocytopenia in mice.

Human herpesviruses establish lifelong latency. Viral recrudescence can lead to the development of cancers, immunoproliferative disorders, transplantation complications, and thrombocytopenia. Although platelet-specific autoantibodies have been reported in patients infected with the Epstein-Barr virus (EBV), the mechanisms by which thrombocytopenia is induced remain unclear, as do the relative contributions of lytic viral replication and latent viral gene expression. The human gammaherpesviruses are tightly restricted in their ability to infect other mammals, so they are difficult to study in live animal models. Here we show that infection of mice with murine gammaherpesvirus-68 (γHV68), a rodent-specific pathogen closely related to EBV, induces the production of platelet-binding antibodies and causes thrombocytopenia. Infection of antibody-deficient mice does not lead to thrombocytopenia, indicating the platelet decrease is mediated by antibody. Additionally, infection with a latency-null recombinant γHV68 does not induce thrombocytopenia, suggesting factors associated with viral latency drive the infection-induced antibody-mediated thrombocytopenia. These studies describe an important animal model of gammaherpesvirus-induced autoimmune thrombocytopenia and demonstrate that this pathology is mediated by antibody and dependent on viral latency. This model will allow studies of the underlying mechanisms of disease progression and the testing of therapeutic strategies for the alleviation of virus-induced thrombocytopenia.

γ-Herpesvirus reactivation differentially stimulates epitope-specific CD8 T cell responses.

The γ-herpesviruses are characterized by their ability to establish lifelong latency. Subsequent immune suppression leads to viral reactivation from latency and the onset of a variety of pathologies, including lymphoproliferative disease and cancers. CD8 T cells play a key role in preventing reactivation of latent virus. Therefore, to develop effective therapeutic immune strategies, it is essential to understand the maintenance of CD8 T cell responses during latency. Because the γ-herpesviruses are highly species-specific and mice cannot be infected with the human pathogens, EBV or Kaposi's sarcoma-associated herpesvirus, we have used a natural rodent γ-herpesvirus experimental infection model, γ-herpesvirus-68. In this report, we show that during long-term latent infection, naive CD8 T cells are recruited into the ongoing immune response in an epitope-specific manner. When virus reactivation is induced in vivo, the recruitment of CD8 T cells for some, but not all, epitopes is enhanced. The variation in recruitment is not due to differences in epitope presentation. We also show that CD8 T cells that are newly stimulated during reactivation are functionally impaired compared with acutely stimulated cells in terms of cytokine production. Thus, our results demonstrate unexpected complexity in the response of CD8 T cells specific for different viral epitopes that were stimulated during acute infection, quiescent latency, and reactivation.

Importance of antibody in virus infection and vaccine-mediated protection by a latency-deficient recombinant murine γ-herpesvirus-68.

The human γ-herpesviruses EBV and Kaposi's sarcoma-associated herpesvirus establish lifelong latent infections, can reactivate in immunocompromised individuals, and are associated with the development of malignancies. Murine γ-herpesvirus-68 (γHV68), a rodent pathogen related to EBV and Kaposi's sarcoma-associated herpesvirus, provides an important model to dissect mechanisms of immune control and investigate vaccine strategies. Infection of mice with γHV68 elicits robust antiviral immunity, and long-term protection from γHV68 reactivation requires both cellular and humoral immune responses. Vaccination of mice with AC-replication and transcription activator (RTA), a highly lytic latency-null recombinant γHV68, results in complete protection from wild-type γHV68 infection that lasts for at least 10 mo. In this report, we examine the immune correlates of AC-RTA-mediated protection and show that sterilizing immunity requires both T cells and Ab. Importantly, Ab was also critical for mitigating viral infection in the brain, and in the absence of Ab-mediated control, amplification of the AC-RTA virus in the brain resulted in fatality. Our results highlight important considerations in the development of vaccination strategies based on live-attenuated viruses.

Cutting edge: activation of virus-specific CD4 T cells throughout γ-herpesvirus latency.

CD4 T cells are essential for immune control of γ-herpesvirus latency. We previously identified a murine MHC class II-restricted epitope in γ-herpesvirus-68 gp150 (gp150(67-83)I-A(b)) that elicits CD4 T cells that are maintained throughout long-term infection. However, it is unknown whether naive cells can be recruited into the antiviral CD4 T cell pool during latency. In this study, we generate a mouse transgenic for a gp150-specific TCR and show epitope-specific activation of transgenic CD4 T cells during acute and latent infections. Furthermore, although only dendritic cells can stimulate virus-specific CD8 T cells during latency, we show that both dendritic cells and B cells stimulate transgenic CD4 T cells. These studies demonstrate that naive CD4 T cells specific for a viral glycoprotein can be stimulated throughout infection, even during quiescent latency, suggesting that CD4 T cell memory is maintained in part by the continual recruitment of naive cells.

De novo infection of B cells during murine gammaherpesvirus 68 latency.

The mechanisms by which gammaherpesviruses maintain latency are unclear. Here we used a murine gammaherpesvirus model to show that previously uninfected B cells in immunocompetent mice can acquire virus during latency. In vivo depletion of T cells allowed viral reactivation, as measured by increased viral loads, but not enhanced transfer of virus to new cells. In the absence of both immune T cells and antibody following the transfer of latently infected cells into naïve animals, there was robust infection of new B cells. These data confirm that both T cells and antibody contribute to the control of gammaherpesvirus latency, reactivation, and spread.

Inflammatory chemokine receptors regulate CD8(+) T cell contraction and memory generation following infection.

The development of T cell memory from naive precursors is influenced by molecular cues received during T cell activation and differentiation. In this study, we describe a novel role for the chemokine receptors CCR5 and CXCR3 in regulating effector CD8(+) T cell contraction and memory generation after influenza virus infection. We find that Ccr5(-/-) Cxcr3(-/-) cells show markedly decreased contraction after viral clearance, leading to the establishment of massive numbers of memory CD8(+) T cells. Ccr5(-/-) Cxcr3(-/-) cells show reduced expression of CD69 in the lung during the peak of infection, which coincides with differential localization and the rapid appearance of memory precursor cells. Analysis of single chemokine receptor-deficient cells revealed that CXCR3 is primarily responsible for this phenotype, although there is also a role for CCR5 in the enhancement of T cell memory. The phenotype could be reversed by adding exogenous antigen, resulting in the activation and contraction of Ccr5(-/-) Cxcr3(-/-) cells. Similar results were observed during chronic Mycobacterium tuberculosis infection. Together, the data support a model of memory CD8(+) T cell generation in which the chemokine-directed localization of T cells within infected tissues regulates antigen encounter and controls the extent of CD8(+) T cell activation and differentiation, which ultimately regulates effector versus memory cell fate decisions.

Differential impact of ageing on cellular and humoral immunity to a persistent murine gamma-herpesvirus.

BACKGROUND: Oncogenic gamma-herpesviruses establish life-long infections in their hosts and control of these latent infections is dependent on continual immune surveillance. Immune function declines with age, raising the possibility that immune control of gamma-herpesvirus infection becomes compromised with increasing age, allowing viral reactivation and/or increased latent load, both of which are associated with the development of malignancies. RESULTS: In this study, we use the experimental mouse gamma-herpesvirus model, gammaHV68, to investigate viral immunity in aged mice. We found no evidence of viral recrudescence or increased latent load in aged latently-infected mice, suggesting that effective immune control of gamma-herpesvirus infection remains intact with ageing. As both cellular and humoral immunity have been implicated in host control of gammaHV68 latency, we independently examined the impact of ageing on gammaHV68-specific CD8 T cell function and antibody responses. Virus-specific CD8 T cell numbers and cytolytic function were not profoundly diminished with age. In contrast, whereas ELISA titers of virus-specific IgG were maintained over time, there was a progressive decline in neutralizing activity. In addition, although aged mice were able to control de novo acute infection with only slightly delayed viral clearance, serum titers of neutralizing antibody were reduced in aged mice as compared to young mice. CONCLUSION: Although there is no obvious loss of immune control of latent virus, these data indicate that ageing has differential impacts on anti-viral cellular and humoral immune protection during persistent gammaHV68 infection. This observation has potential relevance for understanding gamma-herpesvirus immune control during disease-associated or therapeutic immunosuppression.

gamma-Herpesvirus-induced protection against bacterial infection is transient.

Herpesviruses are widely disseminated in the population and establish lifelong latency, which is associated with a variety of pathological consequences. A recent report showed that mice latently infected with either murine gamma-herpesvirus-68 (gammaHV68) or murine cytomegalovirus (mCMV), mouse pathogens genetically similar to the human herpesviruses, Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and cytomegalovirus, had enhanced resistance to subsequent bacterial infection, suggesting protective as well as deleterious effects of latency. Here we confirm that latent gammaHV68 infection confers protection against subsequent infection with Listeria monocytogenes. However, the effect is transient, lasting only a few months.

Perturbation of B cell activation in SLAM-associated protein-deficient mice is associated with changes in gammaherpesvirus latency reservoirs.

Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP)) interactions with SLAM family proteins play important roles in immune function. SAP-deficient mice have defective B cell function, including impairment of germinal center formation, production of class-switched Ig, and development of memory B cells. B cells are the major reservoir of latency for both EBV and the homologous murine gammaherpesvirus, gammaherpesvirus 68. There is a strong association between the B cell life cycle and viral latency in that the virus preferentially establishes latency in activated germinal center B cells, which provides access to memory B cells, a major reservoir of long-term latency. In the current studies, we have analyzed the establishment and maintenance of gammaHV68 latency in wild-type and SAP-deficient mice. The results show that, despite SAP-associated defects in germinal center and memory B cell formation, latency was established and maintained in memory B cells at comparable frequencies to wild-type mice, although the paucity of memory B cells translated into a 10-fold reduction in latent load. Furthermore, there were defects in normal latency reservoirs within the germinal center cells and IgD(+)"naive" B cells in SAP-deficient mice, showing a profound effect of the SAP mutation on latency reservoirs.