Inhibitory effects of cytomegalovirus proteins US2 and US11 point to contributions from direct priming and cross-priming in induction of vaccinia virus-specific CD8(+) T cells

The extent to which naive CD8(+) CTLs (T(CD8)(+)) are primed by APCs presenting endogenous Ags (direct priming) or Ags acquired from other infected cells (cross-priming) is a critical topic in basic and applied immunology. To examine the contribution of direct priming in the induction of VV-specific T(CD8)(+), we generated recombinant vaccinia viruses that express human CMV proteins (US2 and US11) that induce the destruction of newly synthesized MHC class I molecules. Expression of US2 or US11 was associated with a 24-63% decrease in numbers of primary or secondary VV-specific T(CD8)(+) responding to i.p. infection. Using HPLC-isolated peptides from VV-infected cells, we show that US2 and US11 selectively inhibit T(CD8)(+) responses to a subset of immunogenic VV determinants. Moreover, VV-US2 and lysates from VV-infected histoincompatible cells elicit T(CD8)(+) specific for a similar subset of VV determinants. These findings indicate that US2 and US11 can function in vivo to interfere with the activation of virus-specific T(CD8)(+). Furthermore, they suggest that 1) both cross-priming and direct priming contribute significantly to the generation of VV-specific T(CD8)(+), 2) the sets of immunogenic vaccinia virus determinants generated by cross-priming and direct priming are not completely overlapping, and 3) cross-priming overrides the effects of cis-acting viral interference with the class I Ag presentation pathway.     

Vaccinia virus infection induces dendritic cell maturation but inhibits antigen presentation by MHC class II

Vaccinia virus (VV) infection is known to inhibit dendritic cells (DC) functions in vitro. Paradoxically, VV is also highly immunogenic and thus has been used as a vaccine. In the present study, we investigated the effects of an in vivo VV infection on DC function by focusing on early innate immunity. Our data indicated that DC are activated upon in vivo VV infection of mice. Splenic DC from VV-infected mice expressed elevated levels of MHC class I and co-stimulatory molecules on their cell surface and exhibited the enhanced potential to produce cytokines upon LPS stimulation. DC from VV-infected mice also expressed a high level of interferon-beta. However, a VV infection resulted in the down-regulation of MHC class II expression and the impairment of antigen presentation to CD4 T cells by DC. Thus, during the early stage of a VV infection, although DC are impaired in some of the critical antigen presentation functions, they can promote innate immune defenses against viral infection.     

Acquisition of MHC:Peptide Complexes by Dendritic Cells Contributes to the Generation of Antiviral CD8+ T Cell Immunity In Vivo

There is an increasing body of evidence suggesting that the transfer of preformed MHC class I:peptide complexes between a virus-infected cell and an uninfected APC, termed cross-dressing, represents an important mechanism of Ag presentation to CD8(+) T cells in host defense. However, although it has been shown that memory CD8(+) T cells can be activated by uninfected dendritic cells (DCs) cross-dressed by Ag from virus-infected parenchymal cells, it is unknown whether conditions exist during virus infection in which naive CD8(+) T cells are primed and differentiate to cytolytic effectors through cross-dressing, and indeed which DC subset would be responsible. In this study, we determine whether the transfer of MHC class I:peptide complexes between infected and uninfected murine DC plays a role in CD8(+) T cell priming to viral Ags in vivo. We show that MHC class I:peptide complexes from peptide-pulsed or virus-infected DCs are indeed acquired by splenic CD8α(-) DCs in vivo. Furthermore, the acquired MHC class I:peptide complexes are functional in that they induced Ag-specific CD8(+) T cell effectors with cytolytic function. As CD8α(-) DCs are poor cross-presenters, this may represent the main mechanism by which CD8α(-) DCs present exogenously encountered Ag to CD8(+) T cells. The sharing of Ag as preformed MHC class I:peptide complexes between infected and uninfected DCs without the restraints of Ag processing may have evolved to accurately amplify the response and also engage multiple DC subsets critical in the generation of strong antiviral immunity.     

A tumor-associated glycoprotein that blocks MHC class II-dependent antigen presentation by dendritic cells

Tumors evade immune surveillance despite the frequent expression of tumor-associated Ags (TAA). Tumor cells escape recognition by CD8(+) T cells through several mechanisms, including down-regulation of MHC class I molecules and associated Ag-processing machinery. However, although it is well accepted that optimal anti-tumor immune responses require tumor-reactive CD4(+) T cells, few studies have addressed how tumor cells evade CD4(+) T cell recognition. In this study, we show that a common TAA, GA733-2, and its murine orthologue, mouse epithelial glycoprotein (mEGP), function in blocking MHC class II-restricted Ag presentation by dendritic cells. GA733-2 is a common TAA that is expressed normally at low levels by some epithelial tissues and a subset of dendritic cells, but at high levels on colon, breast, lung, and some nonepithelial tumors. We show that ectopic expression of mEGP or GA733-2, respectively, in dendritic cells derived from murine bone marrow or human monocytes results in a dose-dependent inability to stimulate proliferation of Ag-specific or alloreactive CD4(+) T cells. Dendritic cells exposed to cell debris from tumors expressing mEGP are similarly compromised. Furthermore, mice immunized with dendritic cells expressing mEGP from a recombinant adenovirus vector exhibited a muted anti-adenovirus immune response. The inhibitory effect of mEGP was not due to down-regulation of functional MHC class II molecules or active suppression of T cells, and did not extend to T cell responses to superantigen. These results demonstrate a novel mechanism by which tumors may evade CD4(+) T cell-dependent immune responses through expression of a TAA.     

Human cytotoxic CD4+ T cells recognize HLA-DR1-restricted epitopes on vaccinia virus proteins A24R and D1R conserved among poxviruses

We previously demonstrated that vaccinia virus (VV)-specific CD4(+) cytolytic T cells can persist for >50 years after immunization against smallpox in the absence of re-exposure to VV. Nevertheless, there have been few studies focusing on CD4(+) T cell responses to smallpox vaccination. To ensure successful vaccination, a candidate vaccine should contain immunodominant CD4(+) T cell epitopes as well as CD8(+) T and B cell epitopes. In the present study, we established cytotoxic CD4(+) T cell lines from VV-immune donors, which recognize epitopes in VV proteins D1R and A24R in association with HLA-DR1 Ags. Comparisons of sequences between different members of the poxvirus family show that both epitopes are completely conserved among VV, variola viruses, and most mammalian poxviruses, including monkeypox, cowpox, and ectromelia. The CD4(+) T cell lines lysed VV-infected, Ag- and peptide-pulsed targets, and the lysis was inhibited by concanamycin A. We also detected these peptide-specific cytolytic and IFN-gamma-producing CD4(+) T cells in short-term bulk cultures of PBMC from each of the three VV-immune donors tested. These are the first VV-specific CD4(+) T cell epitopes identified in humans restricted by one of the most common MHC class II molecules, HLA-DR1, and this information may be useful in analyzing CD4(+) T cell responses to pre-existing or new generation VV vaccines against smallpox.     

Human CD8+ T cells recognize the 60-kDa cysteine-rich outer membrane protein from Chlamydia trachomatis

The intracellular bacterial pathogen Chlamydia is sequestered from the host cell cytoplasm by remaining within an inclusion body during its replication cycle. Nevertheless, CD8(+) T cells recognizing Chlamydia Ags in the context of MHC class I molecules are primed during infection. We have recently described derivation of Chlamydia-specific human CD8(+) T cells by using infected dendritic cells as a surrogate system to reflect Chlamydia-specific CD8(+) T cell responses in vivo. These CD8(+) T cell clones recognize chlamydial Ags processed via the conventional class Ia processing pathway, as assessed by treatment of infected APC with lactacystin and brefeldin A, suggesting that the Ags are translocated from the chlamydial inclusion into the host cell cytosol. In this study, outer membrane protein 2 (OmcB) was identified as the Ag recognized by one of these Chlamydia-specific human CD8(+) T cells, and we defined the HLA*A0101-restricted epitope from this Ag. CD8(+) T cell responses to this epitope were present at high frequencies in the peripheral blood of both of two HLA*A0101 donors tested. In vitro chlamydial growth was completely inhibited by the OmcB-specific CD8(+) T cell clone independently of lytic mechanisms. OmcB is a 60-kDa protein that has been postulated to be associated with the Chlamydia outer membrane complex. The subcellular localization of OmcB to the cytosol of infected cells, as determined by conventional MHC class I Ag processing and presentation, suggests the possibility of an additional, cytosolic-associated function for this protein.     

Infection of APC by human cytomegalovirus controlled through recognition of endogenous nuclear immediate early protein 1 by specific CD4(+) T lymphocytes

Infections by human CMV are controlled by cellular immune responses. Professional APC such as monocytes and macrophages can be infected in vivo and are considered as a reservoir of virus. However, CMV-specific CD4(+) responses against infected APC have not been reported. To develop a model of CD4-infected APC interaction, we have transfected the U373MG astrocytoma cell line with the class II transactivator (CIITA). Confocal microscopy experiments showed that U373MG-CIITA cells expressed markers characteristic of APC. Functional assays demonstrated that infected U373MG-CIITA APC processed and presented both exogenous and endogenously neosynthesized nuclear immediate early (IE) protein 1 through the MHC class II pathway. More importantly, endogenous presentation of IE1 by infected APC lead to efficient control of CMV infection as revealed by decreased viral titer. Thus, these results describe the endogenous presentation of a nuclear viral protein by the MHC class II pathway and suggest that IE1-specific CD4(+) T cells may play an important role in CMV infection by directly acting against infected APC.     

Human cytomegalovirus disrupts constitutive MHC class II expression

CD8(+) and CD4(+) T lymphocytes are important in controlling human CMV (HCMV) infection, but the virus has evolved protean mechanisms to inhibit MHC-based Ag presentation and escape T lymphocyte immunosurveillance. Herein, the interaction of HCMV with the MHC class II Ag presentation pathway was investigated in cells stably transfected with class II transactivator. Flow cytometry experiments demonstrate that HCMV infection decreases cell-surface MHC class II expression. HCMV down-regulates MHC class II surface expression without a significant effect on class II RNA or steady-state protein levels. SDS-stability and confocal microscopy experiments demonstrate normal levels of steady-state peptide-loaded class II molecules in infected cells and that class II molecules reach late endosomal and HLA-DM positive peptide-loading compartments. However, MHC class II positive vesicles are retained in an abnormal perinuclear distribution. Finally, experiments with a mutant HCMV strain demonstrate that this novel mechanism of decreased MHC class II expression is not mediated by one of the known HCMV immunomodulatory genes. These defects in MHC class II expression combined with previously identified CMV strategies for decreasing MHC class I expression enables infected cells to evade T lymphocyte immunosurveillance.     

Emergence of CD8+ T cells expressing NK cell receptors in influenza A virus-infected mice

Both innate and adaptive immune responses play an important role in the recovery of the host from viral infections. In the present report, a subset of cells coexpressing CD8 and NKR-P1C (NK1.1) was found in the lungs of mice infected with influenza A virus. These cells were detected at low numbers in the lungs of uninfected mice, but represented up to 10% of the total CD8(+) T cell population at day 10 postinfection. Almost all of the CD8(+)NK1.1(+) cells were CD8alphabeta(+)CD3(+)TCRalphabeta(+) and a proportion of these cells also expressed the NK cell-associated Ly49 receptors. Interestingly, up to 30% of these cells were virus-specific T cells as determined by MHC class I tetramer staining and by intracellular staining of IFN-gamma after viral peptide stimulation. Moreover, these cells were distinct from conventional NKT cells as they were also found at increased numbers in influenza-infected CD1(-/-) mice. These results demonstrate that a significant proportion of CD8(+) T cells acquire NK1.1 and other NK cell-associated molecules, and suggests that these receptors may possibly regulate CD8(+) T cell effector functions during viral infection.     

Priming of CTLs by lymphocytic choriomeningitis virus depends on dendritic cells

Appropriate activation of naive CD8(+) T cells depends on the coordinated interaction of these cells with professional APC that present antigenic peptides in the context of MHC class I molecules. It is accepted that dendritic cells (DC) are efficient in activating naive T cells and are unique in their capacity to prime CD8(+) T cell responses against exogenous cell-associated Ags. Nevertheless, it is unclear whether epitopes, derived from endogenously synthesized proteins and presented by MHC class I molecules on the surface of other APC including B cells and macrophages, can activate naive CD8(+) T cells in vivo. By infecting transgenic CD11c-DTR/GFP mice that allow conditional depletion of DC with lymphocytic choriomeningitis virus (LCMV), which infects all types of APC and elicits a vigorous CTL response, we unambiguously show that priming of LCMV-specific CD8(+) T cells is crucially dependent on DC, despite ample presence of LCMV-infected macrophages and B cells in secondary lymphoid organs.     

Cellular and humoral immunity against vaccinia virus infection of mice

Despite the widespread use of vaccinia virus (VV) as a vector for other Ags and as the smallpox vaccine, there is little information available about the protective components of the immune response following VV infection. In this study, protection against wild-type VV was evaluated in mice with respect to the relative contributions of CD8(+) T cells vs that of CD4(+) T cells and Ab. C57BL/6 mice primed with the Western Reserve strain of VV mount significant IgM and IgG Ab responses, specific cytotoxic T cell responses, IFN-gamma responses in CD4(+) and CD8(+) T cells, and effectively clear the virus. This protection was abrogated by in vivo depletion of CD4(+) T cells or B cells in IgH(-/-) mice, but was not sensitive to CD8(+) T cell depletion alone. However, a role for CD8(+) T cells in primary protection was demonstrated in MHC class II(-/-) mice, where depleting CD8(+) T cells lead to increase severity of disease. Unlike control MHC class II(-/-) mice, the group depleted of CD8(+) T cells developed skin lesions on the tail and feet and had adrenal necrosis. Adoptive transfer experiments also show CD8(+) T cells can mediate protective memory. These results collectively show that both CD4(+) and CD8(+) T cell-mediated immunity can contribute to protection against VV infection. However, CD4(+) T cell-dependent anti-virus Ab production plays a more important role in clearing virus following acute infection, while in the absence of Ab, CD8(+) T cells can contribute to protection against disease.     

Expression of herpes simplex virus ICP47 and human cytomegalovirus US11 prevents recognition of transgene products by CD8(+) cytotoxic T lymphocytes

The in vivo persistence of gene-modified cells may be limited by the development of a host immune response to vector-encoded proteins. Herpesviruses evade cytotoxic T-lymphocyte (CTL) recognition by expressing genes which interfere selectively with presentation of viral antigens by class I major histocompatibility complex (MHC) molecules. Here, we studied the use of retroviral vectors encoding herpes simplex virus ICP47, human cytomegalovirus (HCMV) US3, or HCMV US11 to decrease presentation of viral proteins and transgene products to CD8(+) CTL. Human fibroblasts and T cells transduced to express the ICP47, US3, or US11 genes alone exhibited a decrease in cell surface class I MHC expression. The combination of ICP47 and US11 rendered fibroblasts negative for surface class I MHC and allowed a class I MHC-low population of T cells to be sorted by flow cytometry. Fibroblasts and T cells expressing both ICP47 and US11 were protected from CTL-mediated lysis and failed to stimulate specific memory T-cell responses to transgene products in vitro. Our findings suggest that expression of immunoregulatory viral gene products could be a potential strategy to prolong transgene expression in vivo.     

RNA based vaccines

The recognition that CD8(+) T-cell mediated Th1 immune responses were necessary to produce immunity to intracellular and transformed self pathogens led to intense interest in the delivery of nucleic acids, DNA, or RNA encoding candidate antigens, as vaccines. Antigen presenting cells (APC) encounter most protein and vaccine immunogens as extracellular proteins and, thus, present them on major histocompatibility complex (MHC) class II molecules leading to the activation of CD4(+) T cells. Protein antigens encoded by nucleic acids delivered to dendritic cell (DC) are produced inside the cell and, thus, can stimulate MHC class I mediated activation of CD8(+) T-cell immune responses. Unfortunately, DCs are not readily transfected with DNA (Akbari et al., 1999) resulting in the requirement for high concentrations of DNA and repeated immunizations to achieved immune responses. RNA, on the other hand, is readily taken up and expressed by DC, making it an alternative vaccine candidate. In this article, we will discuss immune responses developed, interactions between APC and RNA that activate and dictate DC activation, and preliminary studies using RNA in vivo and in vitro to develop protective immunity.     

Varicella-zoster virus retains major histocompatibility complex class I proteins in the Golgi compartment of infected cells

We sought to examine the effects of varicella-zoster virus (VZV) infection on the expression of major histocompatibility complex class I (MHC I) molecules by human fibroblasts and T lymphocytes. By flow cytometry, VZV infection reduced the cell surface expression of MHC I molecules on fibroblasts significantly, yet the expression of transferrin receptor was not affected. Importantly, when human fetal thymus/liver implants in SCID-hu mice were inoculated with VZV, cell surface MHC I expression was downregulated specifically on VZV-infected human CD3+ T lymphocytes, a prominent target that sustains VZV viremia. The stage in the MHC I assembly process that was disrupted by VZV in fibroblasts was examined in pulse-chase and immunoprecipitation experiments in the presence of endoglycosidase H. MHC I complexes continued to be assembled in VZV-infected cells and were not retained in the endoplasmic reticulum. In contrast, immunofluorescence and confocal microscopy showed that VZV infection resulted in an accumulation of MHC I molecules which colocalized to the Golgi compartment. Inhibition of late viral gene expression by treatment of infected fibroblasts with phosphonoacetic acid did not influence the modulation of MHC I expression, nor did transfection of cells with plasmids expressing immediate early viral proteins. However, cells transfected with a plasmid carrying the early gene ORF66 did result in a significant downregulation of MHC I expression, suggesting that this gene encodes a protein with an immunomodulatory function. Thus, VZV downregulates MHC I expression by impairing the transport of MHC I molecules from the Golgi compartment to the cell surface; this effect may enable the virus to evade CD8+ T-cell immune recognition during VZV pathogenesis, including the critical phase of T-lymphocyte-associated viremia.     

Mutations in a dominant Nef epitope of simian immunodeficiency virus diminish TCR:epitope peptide affinity but not epitope peptide:MHC class I binding

Viruses like HIV and SIV escape from containment by CD8(+) T lymphocytes through generating mutations that interfere with epitope peptide:MHC class I binding. However, mutations in some viral epitopes are selected for that have no impact on this binding. We explored the mechanism underlying the evolution of such epitopes by studying CD8(+) T lymphocyte recognition of a dominant Nef epitope of SIVmac251 in infected Mamu-A*02(+) rhesus monkeys. Clonal analysis of the p199RY-specific CD8(+) T lymphocyte repertoire in these monkeys indicated that identical T cell clones were capable of recognizing wild-type (WT) and mutant epitope sequences. However, we found that the functional avidity of these CD8(+) T lymphocytes for the mutant peptide:Mamu-A*02 complex was diminished. Using surface plasmon resonance to measure the binding affinity of the p199RY-specific TCR repertoire for WT and mutant p199RY peptide:Mamu-A*02 monomeric complexes, we found that the mutant p199RY peptide:Mamu-A*02 complexes had a lower affinity for TCRs purified from CD8(+) T lymphocytes than did the WT p199RY peptide:Mamu-A*02 complexes. These studies demonstrated that differences in TCR affinity for peptide:MHC class I ligands can alter functional p199RY-specific CD8(+) T lymphocyte responses to mutated epitopes, decreasing the capacity of these cells to contain SIVmac251 replication.     

Redox regulation facilitates optimal peptide selection by MHC class I during antigen processing

Activated CD8(+) T cells discriminate infected and tumor cells from normal self by recognizing MHC class I-bound peptides on the surface of antigen-presenting cells. The mechanism by which MHC class I molecules select optimal peptides against a background of prevailing suboptimal peptides and in a considerably proteolytic ER environment remained unknown. Here, we identify protein disulfide isomerase (PDI), an enzyme critical to the formation of correct disulfide bonds in proteins, as a component of the peptide-loading complex. We show that PDI stabilizes a peptide-receptive site by regulating the oxidation state of the disulfide bond in the MHC peptide-binding groove, a function that is essential for selecting optimal peptides. Furthermore, we demonstrate that human cytomegalovirus US3 protein inhibits CD8(+) T cell recognition by mediating PDI degradation, verifying the functional relevance of PDI-catalyzed peptide editing in controlling intracellular pathogens. These results establish a link between thiol-based redox regulation and antigen processing.     

Gag-specific CD8+ T lymphocytes recognize infected cells before AIDS-virus integration and viral protein expression

CD8(+) T cells are a key focus of vaccine development efforts for HIV. However, there is no clear consensus as to which of the nine HIV proteins should be used for vaccination. The early proteins Tat, Rev, and Nef may be better CD8(+) T cell targets than the late-expressed structural proteins Gag, Pol, and Env. In this study, we show that Gag-specific CD8(+) T cells recognize infected CD4(+) T lymphocytes as early as 2 h postinfection, before proviral DNA integration, viral protein synthesis, and Nef-mediated MHC class I down-regulation. Additionally, the number of Gag epitopes recognized by CD8(+) T cells was significantly associated with lower viremia (p = 0.0017) in SIV-infected rhesus macaques. These results suggest that HIV vaccines should focus CD8(+) T cell responses on Gag.     

Cutting edge: efficient MHC class I cross-presentation during early vaccinia infection requires the transfer of proteasomal intermediates between antigen donor and presenting cells

Priming of CD8(+) T cells requires presentation of short peptides bound to MHC class I molecules of professional APCs. Cross-presentation is a mechanism whereby professional APC present on their own MHC class I molecules peptides derived from degradation of Ags synthesized by other Ag "donor cells." The mechanism of cross-presentation is poorly understood, and the nature of the transferred Ag is unknown. In this report, we demonstrate that the bulk of a cross-presented Ag transferred from donor cells recently infected with vaccinia virus are proteasomal products that are susceptible to peptidases within the donor cell cytosol and not full-length proteins or mature epitopes either free or bound to chaperones.