Immune Evasion Proteins of Murine Cytomegalovirus Preferentially Affect Cell Surface Display of Recently Generated Peptide Presentation Complexes

Abstract

For recognition of infected cells by CD8 T cells, antigenic peptides are presented at the cell surface, bound to major histocompatibility complex class I (MHC-I) molecules. Downmodulation of cell surface MHC-I molecules is regarded as a hallmark function of cytomegalovirus-encoded immunoevasins. The molecular mechanisms by which immunoevasins interfere with the MHC-I pathway suggest, however, that this downmodulation may be secondary to an interruption of turnover replenishment and that hindrance of the vesicular transport of recently generated peptide-MHC (pMHC) complexes to the cell surface is the actual function of immunoevasins. Here we have used the model of murine cytomegalovirus (mCMV) infection to provide experimental evidence for this hypothesis. To quantitate pMHC complexes at the cell surface after infection in the presence and absence of immunoevasins, we generated the recombinant viruses mCMV-SIINFEKL and mCMV-⌬m06m152-SIINFEKL, respectively, expressing the K b -presented peptide SIINFEKL with early-phase kinetics in place of an immunodominant peptide of the viral carrier protein gp36.5/m164. The data revealed ϳ10,000 K b molecules presenting SIINFEKL in the absence of immunoevasins, which is an occupancy of ϳ10% of all cell surface K b molecules, whereas immunoevasins reduced this number to almost the detection limit. To selectively evaluate their effect on preexisting pMHC complexes, cells were exogenously loaded with SIINFEKL peptide shortly after infection with mCMV-SIINFEKA, in which endogenous presentation is prevented by an L174A mutation of the C-terminal MHC-I anchor residue. The data suggest that pMHC complexes present at the cell surface in advance of immunoevasin gene expression are downmodulated due to constitutive turnover in the absence of resupply. CD8 T cells recognize infected cells by interaction of theirT-cell receptor (TCR) with a cell surface presentation complex composed of a cognate antigenic peptide bound to a presenting allelic form of a major histocompatibility complex class I (MHC-I) glycoprotein (77,85,97,98). The number of such "peptide receptors" per cell has been estimated to be on the order of 10 5 to 10 6 for each MHC-I allomorph (for a review, see reference 82). Viral antigenic peptides are generated within infected cells by proteolytic processing of viral proteins, usually in the proteasome, and associate with nascent MHC-I proteins in the endoplasmic reticulum (ER) before the peptide-MHC (pMHC) complexes travel to the cell surface with the cellular vesicular flow (for reviews, see references 13, 87, 92, and 93). CD8 T cells have long been known to protect against cytomegalovirus (CMV) infection and disease in animal models (60, 72; reviewed in references 33 and 36) and in humans (9,61,67,75,76). As shown only recently in the murine CMV (mCMV) model of infection of immunocompromised mice by adoptive transfer of epitope-specific CD8 T cells, antiviral protection against CMV is indeed TCR mediated and epitope dependent. Specifically, memory cells purified by TCR-bas...

Antigen presentation under the influence of ‘immune evasion’ proteins and its modulation by interferon-gamma: implications for immunotherapy of cytomegalovirus infection with antiviral CD8 T cells

“…Notably, m04, which so far did not show a convincing inhibitory function, appears to help m06 and m152 in downmodulating MHC-I cell surface expression (Fig. 1b, Target cell conditioning by IFN-c almost levels the differences in antigen presentation imposed by vRAPs As we have shown previously, downmodulation of total cell surface MHC-I expression by the combined action of vRAPs m06 and m152 results primarily from blocking the trafficking of recently loaded pMHC-I complexes to the cell surface, leading to a lack of resupply of MHC-I molecules in the otherwise constitutive MHC-I turnover [54] (see also the accompanying review article by Lemmermann et al in this issue of MMI). This predicts the patterns of vRAP function in cell surface presentation of defined pMHC-I complexes and their recognition by cognate CD8 T cells follow the patterns seen for total MHC-I cell surface expression (which actually includes MHC-I molecules loaded with self-peptides or peptides from unrelated antigens).…”

“…Response of all CD8 T cells of a polyclonal cell population is thus a threshold phenomenon that indicates nothing more than presentation of pMHC-I complexes sufficient for sensitizing all CD8 T cells characterized by a particular functional avidity (and TCR structural avidity) distribution, whereas the actual number of pMHC-I complexes on the cell surface of the stimulating infected cells may still differ widely in the absence or presence of vRAPs. Evidence in support of this possibility has been provided by a direct quantitation of pMHC-I (SIINFEKL-K b ) complexes with an antibody specific for this particular complex [54] (see also the accompanying review by Lemmermann et al in this issue of MMI). Functional evidence is also provided by taking into account the intensity of CD8 T-cell sensitization that is indicated by the amounts of IFN-c secreted during the ELISpot assay, which are reflected by spot sizes.…”

Nodular Inflammatory Foci Are Sites of T Cell Priming and Control of Murine Cytomegalovirus Infection in the Neonatal Lung

“…Both viruses encode the SIINFEKL peptide but MCMV-3DΔvRAP lacks the “ v iral r egulator of a ntigen p resentation” genes encoding for the MHC class I evasion proteins gp48/m06 and gp40/m152 [53], [54]. In contrast to MCMV-3D, this mutant is therefore expected to lack the ability to interfere with MHC class I peptide surface expression as was recently shown for a related virus mutant [55]. We speculated that MHC class I bound SIINFEKL peptide presentation on MCMV-3DΔvRAP-infected cells would allow direct recognition of infected cells by OTI T cells and decreased luciferase activity in organs of these animals would be an in vivo indicator for the activity of cytotoxic T lymphocytes (CTL).…”

The Epstein-Barr Virus-Encoded BILF1 Protein Modulates Immune Recognition of Endogenously Processed Antigen by Targeting Major Histocompatibility Complex Class I Molecules Trafficking on both the Exocytic and Endocytic Pathways

“…Lemmermann et al (18) showed that the immune evasion proteins from murine cytomegalovirus preferentially affect cell surface display of recently generated peptide presentation complexes. These immunoevasins only slowly downmodulate preexisting cell surface MHC-I/peptide complexes, which is a passive mechanism due to constitutive turnover in the absence of resupply.…”