In-Jeong Kim

Lymphocyte Activation Gene-3 (CD223) Regulates the Size of the Expanding T Cell Population Following Antigen Activation In Vivo

Lymphocyte activation gene-3 (LAG-3) is a CD4-related, activation-induced cell surface molecule that binds to MHC class II with high affinity. In this study, we used four experimental systems to reevaluate previous suggestions that LAG-3−/− mice had no T cell defect. First, LAG-3−/− T cells exhibited a delay in cell cycle arrest following in vivo stimulation with the superantigen staphylococcal enterotoxin B resulting in increased T cell expansion and splenomegaly. Second, increased T cell expansion was also observed in adoptive recipients of LAG-3−/− OT-II TCR transgenic T cells following in vivo Ag stimulation. Third, infection of LAG-3−/− mice with Sendai virus resulted in increased numbers of memory CD4+ and CD8+ T cells. Fourth, CD4+ T cells exhibited a delayed expansion in LAG-3−/− mice infected with murine gammaherpesvirus. In summary, these data suggest that LAG-3 negatively regulates T cell expansion and controls the size of the memory T cell pool.

γ-Herpesvirus Latency Is Preferentially Maintained in Splenic Germinal Center and Memory B Cells

The γ-herpesviruses are oncogenic B cell lymphotrophic viruses that establish life-long latency in the host. Murine γ-herpesvirus 68 (MHV-68) infection of mice represents a unique system for analyzing γ-herpesvirus latency in splenic B cells at different stages of infection. After intranasal infection with MHV-68 we analyzed the establishment of latency 14 days after infection, and the maintenance of latency 3 months after infection in different purified subpopulations of B cells in the spleen. The data show that MHV-68 latency is mainly established in germinal center B cells and that long-term latency is preferentially maintained in two different subsets of isotype-switched B cells, germinal center and memory B cells. Cell cycle analysis indicates that MHV-68 is located in both cycling and resting isotype-switched B cells. Analysis of viral gene expression showed that both lytic and latent viral transcripts were differentially expressed in germinal center and memory B cells during long-term latency. Together, these observations suggested that γ-herpesviruses exploit the B cell life cycle in the spleen.

Antibody-Mediated Control of Persistent γ-Herpesvirus Infection

The human γ-herpesviruses, EBV and Kaposi’s sarcoma-associated herpesvirus, establish life-long latency and can reactivate in immunocompromised individuals. T cells play an important role in controlling persistent EBV infection, whereas a role for humoral immunity is less clear. The murine γ-herpesvirus-68 has biological and structural similarities to the human γ-herpesviruses, and provides an important in vivo experimental model for dissecting mechanisms of immune control. In the current studies, CD28−/− mice were used to address the role of Abs in control of persistent murine γ-herpesvirus-68 infection. Lytic infection was controlled in the lungs of CD28−/− mice, and latency was maintained in B cells at normal frequencies. Although class-switched virus-specific Abs were initially generated in the absence of germinal centers, titers and viral neutralizing activity rapidly waned. T cell depletion in CD28−/− mice with compromised Ab responses, but not in control mice with intact Ab responses, resulted in significant recrudescence from latency, both in the spleen and the lung. Recrudescence could be prevented by passive transfer of immune serum. These data directly demonstrate an important contribution of humoral immunity to control of γ-herpesvirus latency, and have significant implications for clinical intervention.

Differential γ-Herpesvirus Distribution in Distinct Anatomical Locations and Cell Subsets During Persistent Infection in Mice

Murine γ-herpesvirus 68 (MHV-68) provides an important experimental model for analyzing γ-herpesvirus latent infection. After intranasal infection with MHV-68, we analyzed the distribution of the virus in different anatomical locations and purified populations of cells. Our data show that long-term latency is maintained in a variety of anatomical locations and cell populations with different frequencies. Importantly, we demonstrate that although latency in the lung is established in a variety of cell subsets, long-term latency in the lung is only maintained in B cells. In contrast, splenic latency is maintained in macrophages and dendritic cells, as well as in B cells. In blood, isotype-switched B cells constitute the major viral reservoir. These results show that the cell subsets in which latency is established vary within different anatomical sites. Finally, we demonstrate that long-term latency is accompanied by a low level of infectious virus in lung and spleen. These data have important implications for understanding the establishment and maintenance of latency by γ2-herpesviruses.

Murid Herpesvirus 4 Strain 68 M2 Protein Is a B-Cell-Associated Antigen Important for Latency but Not Lymphocytosis

ABSTRACT This work describes analyses of the function of the murid herpesvirus 4 strain 68 (MHV-68) M2 gene. A frameshift mutation was made in the M2 open reading frame that caused premature termination of translation of M2 after amino acid residue 90. The M2 mutant showed no defect in productive replication in vitro or in lungs after infection of mice. Likewise, the characteristic transient increase in spleen cell number, Vβ4 T-cell-receptor-positive CD8+ T-cell mononucleosis, and establishment of latency were unaffected. However, the M2 mutant virus was defective in its ability to cause the transient sharp rise in latently infected cells normally seen in the spleen after infection of mice. We also demonstrate that expression of M2 is restricted to B cells in the spleen and that M2 encodes a 30-kDa protein localizing predominantly in the cytoplasm and plasma membrane of B cells.

Maintenance of Long Term γ-Herpesvirus B Cell Latency Is Dependent on CD40-Mediated Development of Memory B Cells

It has been proposed that the γ-herpesviruses maintain lifelong latency in B cells by gaining entry into the memory B cell pool and taking advantage of host mechanisms for maintaining these cells. We directly tested this hypothesis by kinetically monitoring viral latency in CD40+ and CD40− B cells from CD40+CD40− mixed bone marrow chimera mice after infection with a murine γ-herpesvirus, MHV-68. CD40+ B cells selectively entered germinal centers and differentiated into memory B cells. Importantly, latency was progressively lost in the CD40− B cells and preferentially maintained in the long-lived, isotype-switched CD40+ B cells. These data directly demonstrate viral exploitation of the normal B cell differentiation pathway to maintain latency.

Vaccination Against Murine γ-Herpesvirus Infection

The γ-herpesviruses establish life-long latency in the host and are important human pathogens. T cells play a major role in controlling the initial acute infection and subsequently maintaining the virus in a quiescent state. However, the nature of the T-cell response to γ-herpesvirus infection and the requirements for effective vaccination are poorly understood. The recent development of a murine γ-herpesvirus (murine herpesvirus-68 [MHV-68]) has made it possible to analyze T-cell responses and test vaccination strategies in a small animal model. Intranasal infection with MHV-68 induces an acute infection in the lung and the subsequent establishment of long-term latency, which is associated with splenomegaly and an infectious mononucleosis-like syndrome. Here we review the T-cell response to different phases of the infection and the impact of vaccination against either lytic-cycle, or latency-associated T-cell epitopes.

T Cell Reactivity during Infectious Mononucleosis and Persistent Gammaherpesvirus Infection in Mice

Intranasal infection of mice with murine gammaherpesvirus 68 causes a dramatic increase in numbers of activated CD8+ T cells in the blood, analogous in many respects to EBV-induced infectious mononucleosis in humans. In the mouse model, this lymphocytosis has two distinct components: an early, conventional virus-specific CD8+ T cell response, and a later response characterized by a dramatic increase among CD8+ T cells that bear Vβ4+ TCRs. We previously demonstrated that Vβ4+CD8+ T cells recognize an uncharacterized ligand expressed on latently infected B cells in an MHC-independent manner. The frequency of Vβ4+CD8+ T cells increases dramatically following the peak of viral latency in the spleen. In the current studies, we show that elevated Vβ4+CD8+ T cell levels are sustained long-term in persistently infected mice, apparently a consequence of continued ligand expression. In addition, we show that Vβ4+CD8+ T cells can acquire effector functions, including cytotoxicity and the capacity to secrete IFN-γ, although they have an atypical activation profile compared with well-characterized CD8+ T cells specific for conventional viral epitopes. The characteristics of Vβ4+CD8+ T cells (potential effector function, stimulation by latently infected B cells, and kinetics of expansion) suggested that this dominant T cell response plays a key role in the immune control of latent virus. However, Ab depletion and adoptive transfer studies show that Vβ4+CD8+ T cells are not essential for this function. This murine model of infection may provide insight into the role of unusual populations of activated T cells associated with persistent viral infections.

Factors Controlling Levels of CD8+ T-Cell Lymphocytosis Associated with Murine γ-Herpesvirus Infection

Intranasal infection of mice with murine gamma-herpesvirus 68 (MHV-68) elicits a striking CD8+ T-cell lymphocytosis following the establishment of latency, which includes a marked increased frequency of Vbeta4+ CD8+ T cells. The Vbeta4+ CD8+ T cells do not recognize a conventional viral peptide, but are stimulated by an uncharacterized ligand expressed on latently infected, activated B cells. The selective expansion of Vbeta4+ CD8+ T cells after MHV-68 infection is observed in all mouse strains examined, although the fold-increase varies widely, ranging from less than twofold to greater than 10-fold. The factors controlling the variation are currently undefined. In the current study, CD8+ T cell activation and Vbeta4+ CD8+ T-cell frequencies were analyzed in 18 inbred strains of mice. The data show that the magnitude of the Vbeta4+ CD8+ T-cell response correlates with the degree of CD8+ T cell-activation, and that both major histocompatibility complex (MHC) and non-MHC genes contribute to the magnitude of the activation. Furthermore, the magnitude of the response does not reflect major differences in susceptibility to viral infection and/or corresponding differences in the acute response. Rather the degree of Vbeta4+ CD8+ T cell activation may be determined by differences in levels of expression of the stimulatory ligand at the peak of latency.

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 γHV68 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.