Ann B. Hill

T cells and viral persistence: lessons from diverse infections

Persistent virus infections create specific problems for their hosts. Although the dynamics of immune responses after acute infection are well studied and very consistent, especially in mouse models, the patterns of responses noted during persistent infection are more complex and differ depending on the infection. In particular, CD8+ T cell responses differ widely in quantity and quality. In this review we examine these diverse responses and ask how they may arise; in particular, we discuss the function of antigen re-encounter and the CD4+ T cell responses to and the escape strategies of specific viruses. We focus on studies of four main human pathogens, cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus and hepatitis C virus, and their animal models.

Stable binding of the herpes simplex virus ICP47 protein to the peptide binding site of TAP.

The herpes simplex virus (HSV) ICP47 protein inhibits the MHC class I antigen presentation pathway by inhibiting the transporter associated with antigen presentation (TAP) which translocates peptides across the endoplasmic reticulum membrane. At present, ICP47 is the only inhibitor of TAP. Here, we show that ICP47 produced in bacteria can block human, but not mouse, TAP, and that heat denaturation of ICP47 has no effect on its ability to block TAP. ICP47 inhibited peptide binding to TAP without affecting ATP binding, consistent with previous observations that the peptide binding and ATP binding sites of TAP are distinct. ICP47 bound to TAP with a higher affinity (KD approximately 5 × 10(‐8) M) than did peptides, and ICP47 did not dissociate from TAP. ICP47 was not transported by TAP and remained sensitive to proteases added from the cytosolic surface of the membrane. Peptides acted as competitive inhibitors of ICP47 binding to TAP, and this inhibition required a 100‐ to 1000‐fold molar excess of peptide. These results demonstrate that ICP47 binds to a site which includes the peptide binding domain of TAP and remains bound to this site in a stable fashion.

Viral interference with antigen presentation.

CD8+ T cells play an important role in immunity to viruses. Just how important these cells are is demonstrated by the evolution of viral strategies for blocking the generation or display of peptide–major histocompatibility complex class I complexes on the surfaces of virus-infected cells. Here, we focus on viral interference with antigen presentation; in particular we consider the importance (and difficulty) of establishing the evolutionary significance (that is, the ability to enhance viral transmission) of viral gene products that interfere with antigen presentation in vitro.

Assembly and antigen-presenting function of MHC class I molecules in cells lacking the ER chaperone calreticulin

MHC class I molecules expressed in a calreticulin-deficient cell line (K42) assembled with beta 2-microglobulin (beta2-m) normally, but their subsequent loading with optimal peptides was defective. Suboptimally loaded class I molecules were released into the secretory pathway. This occurred despite the ability of newly synthesized class I to interact with the transporter associated with antigen processing (TAP) loading complex. The efficiency of peptide loading was reduced by 50%-80%, and impaired T cell recognition was observed for three out of four antigens tested. The peptide-loading function was specific to calreticulin, since the defect in K42 could be rectified by transfection with calreticulin but not a soluble form of calnexin, which shares its lectin-like activity.

Memory Inflation During Chronic Viral Infection is Maintained by Continuous Production of Short-Lived Functional T Cells

During persistent murine cytomegalovirus (MCMV) infection, the T cell response is maintained at extremely high intensity for the life of the host. These cells closely resemble human CMV-specific cells, which compose a major component of the peripheral T cell compartment in most people. Despite a phenotype that suggests extensive antigen-driven differentiation, MCMV-specific T cells remain functional and respond vigorously to viral challenge. We hypothesized that a low rate of antigen-driven proliferation would account for the maintenance of this population. Instead, we found that most of these cells divided only sporadically in chronically infected hosts and had a short half-life in circulation. The overall population was supported, at least in part, by memory T cells primed early in infection, as well as by recruitment of naive T cells at late times. Thus, these data show that memory inflation is maintained by a continuous replacement of short-lived, functional cells during chronic MCMV infection.

A mouse cytomegalovirus glycoprotein, gp34, forms a complex with folded class I MHC molecules in the ER which is not retained but is transported to the cell surface

Murine cytomegalovirus (MCMV) interferes with antigen presentation by means of retaining major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER). Here we identify and characterize an MCMV‐encoded glycoprotein, gp34, which tightly associates with properly conformed MHC class I molecules in the ER. Gp34 is synthesized in large quantities during MCMV infection and it leaves the ER only in association with MHC class I complexes. Many but not all class I molecules are retained in the ER during the early phase of MCMV infection, and we observe an inverse correlation between amounts of gp34 synthesized during the course of infection and class I retention. An MCMV deletion mutant lacking several genes, including the gene encoding gp34, shows increased class I retention. Thus, MCMV gp34 may counteract class I retention, perhaps to decrease susceptibility of infected cells to recognition by natural killer cells.

Four Distinct Patterns of Memory CD8 T Cell Responses to Chronic Murine Cytomegalovirus Infection

CMVs are β herpesviruses that establish lifelong latent infection of their hosts. Acute infection of C57BL/6 mice with murine CMV elicits a very broad CD8 T cell response, comprising at least 24 epitopes from 18 viral proteins. In contrast, we show here that the CD8 T cell response in chronically infected mice was dominated by only five epitopes. Altogether, four distinct CD8 T cell kinetic patterns were evident. Responses to some epitopes, including M45, which dominates the acute response, contracted sharply after day 7 and developed into stable long-term memory. The response to m139 underwent rapid expansion and contraction, followed by a phase of memory inflation, whereas the response to an M38 epitope did not display any contraction phase. Finally, responses against two epitopes encoded by the immediate early gene IE3 were readily detectable in chronically infected mice but near the limit of detection during acute infection. CD8 T cells specific for the noninflationary M45 epitope displayed a classic central memory phenotype, re-expressing the lymph node homing receptor CD62L and homeostatic cytokine receptors for IL-7 and IL-15, and produced low levels of IL-2. Responses to two inflationary epitopes, m139 and IE3, retained an effector memory surface phenotype (CD62Llow, IL-7Rα−, IL-15Rβ−) and were unable to produce IL-2. We suggest that immunological choices are superimposed on altered viral gene expression profiles to determine immunodominance during chronic murine CMV infection.

Genome-Wide Analysis Reveals a Highly Diverse CD8 T Cell Response to Murine Cytomegalovirus

Human CMV establishes a lifelong latent infection in the majority of people worldwide. Although most infections are asymptomatic, immunocompetent hosts devote an extraordinary amount of immune resources to virus control. To increase our understanding of CMV immunobiology in an animal model, we used a genomic approach to comprehensively map the C57BL/6 CD8 T cell response to murine CMV (MCMV). Responses to 27 viral proteins were detectable directly ex vivo, the most diverse CD8 T cell response yet described within an individual animal. Twenty-four peptide epitopes were mapped from 18 Ags, which together account for most of the MCMV-specific response. Most Ags were from genes expressed at early times, after viral genes that interfere with Ag presentation are expressed, consistent with the hypothesis that the CD8 T cell response to MCMV is largely driven by cross-presented Ag. Titration of peptide epitopes in a direct ex vivo intracellular cytokine staining assay revealed a wide range of functional avidities, with no obvious correlation between functional avidity and the strength of the response. The immunodominance hierarchy varied only slightly between mice and between experiments. However, H-2b-expressing mice with different genetic backgrounds responded preferentially to different epitopes, indicating that non-MHC-encoded factors contribute to immunodominance in the CD8 T cell response to MCMV.

The murine cytomegalovirus immunomodulatory gene m152 prevents recognition of infected cells by M45-specific CTL but does not alter the immunodominance of the M45-specific CD8 T cell response in vivo.

Although in vitro studies have shown that herpesviruses, including murine CMV (MCMV), encode genes that interfere with the MHC class I pathway, their effects on the CTL response in vivo is unclear. We identified a Db-restricted CTL epitope from MCMV M45 by screening an MCMV genomic library using CTL clones isolated from mice infected with MCMV lacking m152. Because m152 severely inhibits CTL recognition of M45 in vitro, we questioned whether an M45-specific response would be generated in mice infected with wild-type MCMV expressing m152. Mice infected with wild-type MCMV or MCMVΔm152 made similar responses to the M45 Ag. Moreover, we saw no skewing of the proportion of M45-specific CD8 T cells within the total MCMV-specific response after infection with MCMV with m152. Despite the profound effect m152 has on presentation of M45 in vitro, it does not affect the immunodominance of M45 in the CTL response in vivo.

Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response

Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-Dk and the MCMV m04 protein. Using H2 chimeras between H2-Db and -Dk, we demonstrate that the H2-Dk peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Δm04) virus infection. MA/My mice, which express Ly49P and H2-Dk, are resistant to MCMV; however, infection with Δm04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Δm04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatability complex class I–restricted recognition of virally infected cells by an activating NK cell receptor.