Biomedical subjects

Ann B Hill

Publications and source records attributed to Ann B Hill.

14 recordsLinked to original sources

PubMed2006

Coordinated function of murine cytomegalovirus genes completely inhibits CTL lysis.

Murine CMV (MCMV) encodes three viral genes that interfere with Ag presentation (VIPRs) to CD8 T cells, m04, m06, and m152. Because the functional impact of these genes during normal infection of C57BL/6 mice is surprisingly modest, we wanted to determine whether the VIPRs are equally effective against the entire spectrum of H-2(b)-restricted CD8 T cell epitopes. We also wanted to understand how the VIPRs interact at a functional level. To address these questions, we used a panel of MCMV mutants lacking each VIPR in all possible combinations, and CTL specific for 15 H-2(b)-restricted MCMV epitopes. Only expression of all three MCMV VIPRs completely inhibited killing by CTL specific for all 15 epitopes, but removal of any one VIPR enabled lysis by at least some CTL. The dominant interaction between the VIPRs was cooperation: m06 increased the inhibition of lysis achieved by either m152 or m04. However, for 1 of 15 epitopes m04 functionally antagonized m152. There was little differential impact of any of the VIPRs on K(b) vs D(b), but a surprising degree of differential impact of the three VIPRs for different epitopes. These epitope-specific differences did not correlate with functional avidity, or with timing of VIPR expression in relation to Ag expression in the virus replication cycle. Although questions remain about the molecular mechanism and in vivo role of these genes, we conclude that the coordinated function of MCMV's three VIPRs results in a powerful inhibition of lysis of infected cells by CD8 T cells.

Animals↗

PubMed2006

Four distinct patterns of memory CD8 T cell responses to chronic murine cytomegalovirus infection.

CMVs are beta 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 (CD62L(low), IL-7Ralpha(-), IL-15Rbeta(-)) 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.

3T3 Cells↗

PubMed2006

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-2(b)-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.

Acute Disease↗

PubMed2006

Cellular and molecular requirements for association of the murine cytomegalovirus protein m4/gp34 with major histocompatibility complex class I molecules.

The murine cytomegalovirus (MCMV) protein m4/gp34 is unique among known viral genes that target the major histocompatibility complex (MHC) class I pathway of antigen presentation in the following two ways: it is found in association with class I MHC molecules at the cell surface, and it inhibits antigen presentation without reducing cell surface class I levels. The current study was undertaken to define more clearly the structural and cellular requirements for m4/gp34 association with the MHC class I molecule K(b). We first assessed the role of the peptide-loading complex in m4/gp34-K(b) association, using cell lines lacking TAP, tapasin, or beta(2)m. m4/gp34-K(b) complexes formed in the absence of TAP or tapasin, although not as efficiently as in wild-type cells. The expression of full-length and truncation mutants of m4/gp34 in a gutless adenovirus vector revealed that the transmembrane region of m4/gp34 was required for efficient association with the K(b) heavy chain. However, the peptide-loading complex was not absolutely required for the association, since m4/gp34 readily formed complexes with K(b) in detergent lysates. The addition of K(b)-binding peptide to the detergent lysates facilitated but was not essential for the formation of the complexes. The ease of complex formation in detergent lysates contrasted with the small fractions of m4/gp34 and K(b) that form complexes in infected cells, suggesting that the endoplasmic reticulum (ER) environment restricts access of m4/gp34 to K(b). Finally, although m4/gp34-K(b) complexes could form when m4 was carried either by MCMV or by the adenovirus vector, they were only efficiently exported from the ER in MCMV-infected cells, suggesting that MCMV provides additional factors needed for transport of the complexes.

Animals↗

PubMed2006

Murine cytomegalovirus interference with antigen presentation contributes to the inability of CD8 T cells to control virus in the salivary gland.

Compared to other organs, murine cytomegalovirus (MCMV) replication in the salivary gland is uniquely resistant to CD8 T-cell control. The contribution of viral genes that interfere with antigen presentation (VIPRs) to this resistance was assessed using a mutant lacking MCMV's known VIPRs. Salivary gland titers of the VIPR-deficient virus were at least 10-fold lower than those of the wild type during the persistent phase of infection; the defect was reversed by depleting CD8 T cells. Thus, VIPRs contribute to CD8 T cells' inability to control virus in the salivary gland.

Animals↗

PubMed2005

Viral interference with antigen presentation to CD8+ T cells: lessons from cytomegalovirus.

Cytomegaloviruses (CMV), in common with other Herpesviruses, establish lifelong persistence in their hosts. These highly host-specific viruses each encode viral genes that interfere with antigen presentation to CD8+ T cells, although the molecular mechanisms by which this end is achieved differ for human and murine CMVs. In each case, there has been a presumption that these genes are necessary for virus persistence in the host. However, recent data in the murine model casts doubt on that presumption. Here, we review the molecular mechanisms of interference with the class I pathway, and the published data regarding functional significance, with a focus on the murine model.

Alleles↗

PubMed2004

Murine cytomegalovirus interference with antigen presentation has little effect on the size or the effector memory phenotype of the CD8 T cell response.

As with most herpesviruses, CMVs encode viral genes that inhibit Ag presentation to CD8 T cells (VIPRs). VIPR function has been assumed to be essential for CMV to establish its characteristic lifetime infection of its host. We compared infection of C57BL/6 mice with wild-type murine CMV (MCMV) and a virus lacking each of MCMV's three known VIPRs: m4, m6, and m152. During acute infection, there was very little difference between the two viruses with respect to the kinetics of viral replication and clearance, or in the size and kinetics of the virus-specific CD8 T cell response. During chronic infection, a large, effector memory, virus-specific CD8 T cell population (CD8(low)CD62L(-)CD11c(+)NKG2A(+)) was maintained in both infections; the size and phenotype of the CD8 T cell response to both viruses was remarkably similar. The characteristic effector memory phenotype of the CD8 T cells suggested that both wild-type and Deltam4+m6+m152 virus continued to present Ag to CD8 T cells during the chronic phase of infection. During the chronic phase of infection, MCMV cannot be isolated from immunocompetent mice. However, upon immunosuppression, both Deltam4+m6+m152 and wild-type virus could be reactivated from mice infected for 6 wk. Thus, restoring the ability of CD8 T cells to detect MCMV had little apparent effect on the course of MCMV infection and on the CD8 T cell response to it. These results challenge the notion that VIPR function is necessary for CMV persistence in the host.

Animals↗

PubMed2004

4-1BB and OX40 stimulation enhance CD8 and CD4 T-cell responses to a DNA prime, poxvirus boost vaccine.

4-1BB (CD137) is a tumour necrosis factor receptor (TNFR) family member, expressed primarily on CD8 T cells after activation. Signalling through 4-1BB has been reported to enhance CD8 T-cell expansion and to protect activated CD8 T cells from death, resulting in an enlarged memory population. Although stimulating 4-1BB has been shown to significantly improve the immune response to weak immunogens such as tumours, little is known about its effect on the CD8 T-cell response to a powerful viral vector such as vaccinia. To test 4-1BB's ability to improve the murine CD8 T cell response to a DNA prime, poxvirus boost vaccine, similar to those used for human immunodeficiency virus and simian immunodeficiency virus vaccines, we administered 4-1BB agonist antibody at the time of the poxvirus boost. 4-1BB stimulation increased the number of functional memory CD8 T cells by two- to fourfold. However, we saw a similar enhancement at the peak of the response and in the memory phase, thus we found no evidence in the context of virus infection that 4-1BB stimulation could increase the percentage of CD8 T cells that survive the acute activation phase to become memory cells. OX40 (CD134) is an analogous TNFR family member expressed primarily on activated CD4 T cells. OX40 stimulation increased the number of antigen-specific CD4 T cells approximately threefold. Stimulating both 4-1BB and OX40 enhanced the CD8 T-cell response more than 4-1BB alone. Thus stimulating these receptors can improve the response to a powerful virus vector, and may be useful in vaccine development.

Animals↗

PubMed2003

Spermine compaction is an efficient and economical method of producing vaccination-grade DNA.

Plasmid DNA inoculations can induce both humoral and cellular immunity, and this technique is now being employed in developing vaccination regimens for a large number of applications. DNA vaccination studies require the preparation of large amounts of purified plasmid DNA with low endotoxin contamination, and the cost burden for multiple injections, multiple animal or large animal studies is significant. We recently reported that selective compaction with spermine can be used to purify large quantities of DNA. We wanted to determine whether this method would produce DNA suitable for vaccination. Endotoxin levels for spermine-compacted DNA were 0.3+/-0.01 endotoxin units (EU)/microg, well within the accepted range (less than 3 EU/microg) for in vivo use. When injected intramuscularly into mice, column-purified and spermine-compacted DNA induced an equivalent antigen-specific CD8+ T-cell response. The labor and time involved in purifying 5 mg of DNA by each method were similar, but the cost of spermine-compacted DNA was only 20% of the cost of column-purified DNA. We conclude that spermine compaction is an efficient and economical method for preparing vaccination-grade DNA.

Animals↗

PubMed2003

Effective inhibition of K(b)- and D(b)-restricted antigen presentation in primary macrophages by murine cytomegalovirus.

Macrophages play an important role in murine cytomegalovirus (MCMV) infection in vivo, both in disseminating infection and in harboring latent virus. MCMV encodes three immune evasion genes (m4, m6, and m152) that interfere with the ability of cytotoxic T cells (CTL) to detect virus-infected fibroblasts, but the efficacy of immune evasion in macrophages has been controversial. Here we show that MCMV immune evasion genes function in H-2(b) primary bone marrow macrophages (BMMphi) in the same way that they do in fibroblasts. Metabolic labeling experiments showed that class I is retained in the endoplasmic reticulum by MCMV infection and associates with m4/gp34 to a similar extent in fibroblasts and BMMphi. We tested a series of K(b)- and D(b)-restricted CTL clones specific for MCMV early genes against a panel of MCMV wild-type virus and mutants lacking m152, m4, or m6. MCMV immune evasion genes effectively inhibited antigen presentation. m152 appeared sufficient to abolish D(b)-restricted presentation in infected macrophages, as has been previously observed in infected fibroblasts. However, for inhibition of recognition of infected macrophages by K(b)-restricted CTL, m4, m6, and m152 were all required. The contribution of m4 to inhibition of recognition appeared much more important in macrophages than in fibroblasts. Thus, MCMV immune evasion genes function effectively in primary macrophages to prevent CTL recognition of early antigens and show the same pattern of major histocompatibility complex class I allele discrimination as is seen in fibroblasts. Furthermore, for inhibition of K(b)-restricted presentation, a strong synergistic effect was noted among m152, m4, and m6.

Animals↗

PubMed2002

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 D(b)-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 MCMVDelta(m)152 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.

Adjuvants, Immunologic↗

PubMed2002

Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors.

Natural killer (NK) cells express inhibitory receptors for major histocompatibility complex (MHC) class I antigens, preventing attack against healthy cells. Mouse cytomegalovirus (MCMV) encodes an MHC-like protein (m157) that binds to an inhibitory NK cell receptor in certain MCMV-susceptible mice. In MCMV-resistant mice, this viral protein engages a related activating receptor (Ly49H) and confers host protection. These activating and inhibitory receptors are highly homologous, suggesting the possibility that one evolved from the other in response to selective pressure imposed by the pathogen.

3T3 Cells↗

PubMed2002

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.

Animals↗

PubMed2002

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.

Animals↗