Sally R. Sarawar

Stimulation via CD40 can substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus

Reactivation of latent herpesviruses is a particular problem in immunocompromised individuals, such as AIDS patients, who lack effective CD4 T helper cell function. An important question is whether residual immune defenses can be mobilized to combat such opportunistic infections, in the absence of CD4 T cells. In the present study, we used a mouse model of opportunistic infection to determine whether stimulation via CD40 could substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus. Treatment with an agonistic antibody to CD40 was highly effective in preventing reactivation of latent murine gammaherpesvirus (MHV-68) in the lungs of CD4 T cell-deficient mice. CD8+ T cells were essential for this effect, whereas virus-specific serum antibody was undetectable and IFN-γ production was unchanged. This demonstration that immunostimulation via CD40 can replace CD4 T cell help in controlling latent virus in vivo has potential implications for the development of novel therapeutic agents to prevent viral reactivation in immunocompromised patients.

Lymphotoxin-alpha-deficient mice can clear a productive infection with murine gammaherpesvirus 68 but fail to develop splenomegaly or lymphocytosis.

ABSTRACT Respiratory challenge with murine gammaherpesvirus 68 (MHV-68) leads to an acute productive infection of the lung and a persistent latent infection in B lymphocytes, epithelia, and macrophages. The virus also induces splenomegaly and an increase in the number of activated CD8 T cells in the circulation. Lymphotoxin- α-deficient (LTα−/−) mice have no lymph nodes and have disrupted splenic architecture. Surprisingly, in spite of the severe defect in secondary lymphoid tissue, LTα−/− mice could clear a productive MHV-68 infection, although with delayed kinetics compared to wild-type mice, and could control latent infection. Cytotoxic T-cell activity was comparable in the lungs and spleens of LTα−/− and wild-type mice. However, splenic gamma interferon responses were substantially reduced in LTα−/− mice. Furthermore, LTα−/− mice failed to develop splenomegaly or lymphocytosis. Although germinal centers were absent, LTα−/− mice were able to class switch and showed significant virus-specific antibody titers. This work demonstrates that organized secondary lymphoid tissue is not an absolute requirement for the generation of immune responses to viral infections.

Protein Kinase C θ Is Not Essential for T-Cell-Mediated Clearance of Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring rodent pathogen with significant homology to human pathogens Epstein-Barr virus and Kaposis sarcoma-associated herpesvirus. T cells are essential for primary clearance of MHV-68 and survival of mice following intranasal infection. Previous reports have suggested that protein kinase C θ (PKCθ) is essential for T-cell activation and cytokine production in vitro. To determine the role of this molecule in vivo during the immune response to a viral infection, PKCθ−/− mice were infected with MHV-68. Despite the essential role of T cells in viral clearance, PKCθ−/− mice survived infection, cleared lytic virus, and maintained effective long-term control of latency. CD8 T-cell expansion, trafficking to the lung, and cytotoxic activity were similar in PKCθ+/+ and PKCθ−/− mice, whereas antiviral antibody and T-helper cell cytokine production were significantly lower in PKCθ−/− mice than in PKCθ+/+ mice. These studies demonstrate a differential requirement for PKCθ in the immune response to MHV-68 and show that PKCθ is not essential for the T-cell activation events leading to viral clearance.

IMMUNOLOGICAL CONTROL OF MURINE GAMMAHERPESVIRUS INFECTION IS INDEPENDENT OF PERFORIN

Perforin-mediated cytotoxic T cell killing has been suggested to be of importance in the control of noncytopathic virus infections, based on studies with lymphocytic choriomeningitis virus (LCMV). We examined the role of perforin in a mouse model of gammaherpesvirus infection using transgenic perforin-deficient mice. Previous work from this laboratory has shown that CD8 T cells are essential for the resolution of the acute lung infection and control of latently infected B cells in murine gamma-herpesvirus 68 infection. The absence of perforin did not significantly affect the kinetics of either the lytic lung infection or the latent spleen infection. Lymphocytes from both perforin-deficient and control mice secreted comparable levels of IFN-gamma, IL-10 and IL-6. In addition, lymphocytes from both strains had similar levels of CD3epsilon-dependent cytotoxic activity in the spleen, draining lymph nodes and bronchoalveolar lavage. These data indicate that the lack of perforin has little affect on the ability of mice to control an experimental gammaherpesvirus infection.

CD28−/− Mice Show Defects in Cellular and Humoral Immunity but Are Able To Control Infection with Murine Gammaherpesvirus 68

ABSTRACT The role of CD28-dependent costimulatory interactions in the development and maintenance of antiviral immune responses was investigated in a mouse model of gammaherpesvirus infection. CD28−/− mice could clear a productive infection with murine gammaherpesvirus 68 (MHV-68), although early lung viral titers were significantly increased. Both CD28−/− and CD28+/+ mice maintained effective long-term control of MHV-68. Gamma interferon responses appeared to develop more slowly in CD28−/− mice, while cytotoxic T-cell activity was similar to that in wild-type mice. Splenomegaly developed normally in CD28−/− mice, whereas virus-specific antibody responses were significantly reduced and aberrant class switching was observed. This work demonstrates that costimulatory interactions involving CD28 are not an absolute requirement for the control of infection with MHV-68.

Cytokines and Costimulatory Molecules in the Immune Response to Murine Gammaherpesvirus-68

Murine gammaherpesvirus 68 (MHV-68) infection of mice provides a useful small animal model for studying gammaherpesvirus pathogenesis and immunity. Recent work has elucidated the cytokine and chemokine profiles during MHV-68 infection and has identified some of the costimulatory interactions that are important for an effective immune response to this virus. Several themes emerge from this work. There is a differential requirement for certain cytokines and costimulatory molecules in the acute and long-term control of MHV-68, and for control of the virus in different anatomical sites. CD4 T cell help is not required for short-term control of MHV-68 in the lung by cytotoxic CD8 T cells, but is essential for effective long-term control. Stimulation via CD40 is an important component of this CD4 T cell help, and interestingly, some of its effects appear to be independent of CD28. MHV-68 infection also increases the expression of several chemokines, which could potentially play important roles in leukocyte trafficking to sites of infection. However, to counter this response, MHV-68 has evolved strategies that enable it to evade or subvert the host chemokine system. Studying the role of cytokines and costimulatory molecules in immunity to MHV-68 may provide useful insights for the development of agents to control gammaherpesviruses that cause human disease.

CD4 T-Cell Help Programs a Change in CD8 T-Cell Function Enabling Effective Long-Term Control of Murine Gammaherpesvirus 68: Role of PD-1-PD-L1 Interactions

ABSTRACT We previously showed that agonistic antibodies to CD40 could substitute for CD4 T-cell help and prevent reactivation of murine gammaherpesvirus 68 (MHV-68) in the lungs of major histocompatibility complex (MHC) class II−/− (CII−/−) mice, which are CD4 T cell deficient. Although CD8 T cells were required for this effect, no change in their activity was detected in vitro. A key question was whether anti-CD40 treatment (or CD4 T-cell help) changed the function of CD8 T cells or another cell type in vivo. To address this question, in the present study, we showed that adoptive transfer of CD8 T cells from virus-infected wild-type mice or anti-CD40-treated CII−/− mice caused a significant reduction in lung viral titers, in contrast to those from control CII−/− mice. Anti-CD40 treatment also greatly prolonged survival of infected CII−/− mice. This confirms that costimulatory signals cause a change in CD8 T cells enabling them to maintain effective long-term control of MHV-68. We investigated the nature of this change and found that expression of the inhibitory receptor PD-1 was significantly increased on CD8 T cells in the lungs of MHV-68-infected CII−/−, CD40−/−, or CD80/86−/− mice, compared with that in wild-type or CD28/CTLA4−/− mice, correlating with the level of viral reactivation. Furthermore, blocking PD-1-PD-L1 interactions significantly reduced viral reactivation in CD4 T-cell-deficient mice. In contrast, the absence of another inhibitory receptor, NKG2A, had no effect. These data suggest that CD4 T-cell help programs a change in CD8 T-cell function mediated by altered PD-1 expression, which enables effective long-term control of MHV-68.

Role of CXCR3 in the Immune Response to Murine Gammaherpesvirus 68

ABSTRACT The chemokine IP-10 (CXCL10) and its cellular receptor CXCR3 are upregulated in the lung during murine gammaherpesvirus 68 (MHV-68) infection. In order to determine the role of the CXCR3 chemokine receptor in the immune response to MHV-68, CXCR3−/− mice were infected with the virus. CXCR3−/− mice showed delayed clearance of replicating MHV-68 from the lungs. This correlated with delayed T-cell recruitment to the lungs and reduced cytolytic activity prior to viral clearance. Splenomegaly and the numbers of latently infected cells per spleen were transiently increased. Ηowever, CXCR3−/− mice showed normal virus-specific antibody titers and effective long-term control of MHV-68 infection.

CD40 Engagement on Dendritic Cells, but Not on B or T Cells, Is Required for Long-Term Control of Murine Gammaherpesvirus 68

ABSTRACT CD4 T cells are not essential for primary clearance of replicating murine gammaherpesvirus 68 (MHV-68) but are required for effective long-term control. The virus reactivates in the lungs of major histocompatibility complex class II-deficient (CII−/−) mice that lack functional CD4 T cells. CD40 ligand (CD40L) is upregulated on activated CD4 T cells, and it is thought that CD40-CD40L interactions are an important component of CD4 T-cell help. Our previous studies have shown that agonistic antibodies to CD40 can substitute for CD4 T-cell function in the long-term control of MHV-68. In the present study, we sought to identify the CD40-positive cell type mediating this effect. To address this question, we adoptively transferred MHV-68 peptide-pulsed CII−/− dendritic cells (DC) that had been treated with an agonistic antibody to CD40 into MHV-68-infected CII−/− recipients. Viral reactivation was significantly lower in mice injected with anti-CD40-treated DC than in those injected with control DC or in mice that did not receive any DC. However, in similar experiments with B cells, anti-CD40 treatment had no effect. We also investigated the requirement for CD40 expression on T cells by adoptive transfer of T cells from CD40+/+ or CD40−/− mice into T-cell-deficient recipients that were subsequently infected with MHV-68. The results showed that CD40 expression on T cells is not necessary for preventing viral reactivation. Taken together, our data suggest that CD40 engagement on DC, but not on T or B cells, is essential for effective long-term control of MHV-68.

Primary Clearance of Murine Gammaherpesvirus 68 by PKCθ−/− CD8 T Cells Is Compromised in the Absence of Help from CD4 T Cells

ABSTRACT CD4 T cells are dispensable for acute control of murine gammaherpesvirus 68 (MHV-68) but are necessary for effective long-term control of the virus by CD8 T cells. In contrast, protein kinase C θ (PKCθ) is not essential for either acute or long-term viral control. However, we found that while either CD4 or CD8 T cells could mediate the clearance of MHV-68 from the lungs of PKCθ+/+ mice, PKCθ−/− mice depleted of either subset failed to clear the virus. These data suggest that there are two alternative pathways for MHV-68 clearance, one dependent on CD4 T cells and the other on PKCθ. Protection mediated by the latter appears to be short-lived. These observations may help to explain the differential requirement for PKCθ in various models of CD8 T-cell activation and differences in the costimulatory requirements for acute and long-term viral control.