Epitope-specific in vivo protection against cytomegalovirus disease by CD8 T cells in the murine model of preemptive immunotherapy

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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...

Section: Cd8 T Cells Recognize Infected Cells By Interaction Of Theirmentioning

confidence: 93%

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

Lemmermann

Gergely

Böhm

et al. 2010

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“…Custom peptide synthesis to a purity of Ͼ80% was performed by Jerini Peptide Technologies (Berlin, Germany). An IE1 peptide (168-YPHFMPTNL-176)-specific, polyclonal cytolytic T-lymphocyte (CTL) line (IE1-CTLL) was generated and tested for purity, broadness of TCR V␤-chain usage, and functional avidity as described previously (8,48).…”

Section: Procedures Of Infectionmentioning

confidence: 99%

The Immune Evasion Paradox: Immunoevasins of Murine Cytomegalovirus Enhance Priming of CD8 T Cells by Preventing Negative Feedback Regulation

Böhm

Simon

Podlech

et al. 2008

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Cytomegaloviruses express glycoproteins that interfere with antigen presentation to CD8 T cells. Although the molecular modes of action of these "immunoevasins" differ between cytomegalovirus species, the convergent biological outcome is an inhibition of the recognition of infected cells. In murine cytomegalovirus, m152/gp40 retains peptide-loaded major histocompatibility complex class I molecules in a cis-Golgi compartment, m06/gp48 mediates their vesicular sorting for lysosomal degradation, and m04/gp34, although not an immunoevasin in its own right, appears to assist in the concerted action of all three molecules. Using the L d -restricted IE1 epitope YPHFMPTNL in the BALB/c mouse model as a paradigm, we provide here an explanation for the paradox that immunoevasins enhance CD8 T-cell priming although they inhibit peptide presentation in infected cells. Adaptive immune responses are initiated in the regional lymph node (RLN) draining the site of pathogen exposure. In particular for antigens that are not virion components, the magnitude of viral gene expression providing the antigens is likely a critical parameter in priming efficacy. We have therefore focused on the events in the RLN and have related priming to intranodal viral gene expression. We show that immunoevasins enhance priming by downmodulating an early CD8 T-cell-mediated "negative feedback" control of the infection in the cortical region of the RLN, thus supporting the model that immunoevasins improve antigen supply for indirect priming by uninfected antigen-presenting cells. As an important consequence, these findings predict that deletion of immunoevasin genes in a replicative vaccine virus is not a favorable option but may, rather, be counterproductive.

“…Assay for antigenic peptide presentation. The ELISPOT assay was used to detect the presentation of endogenously processed antigenic IE1 peptide 168-Y PHFMPTNL-176 in infected cells on the basis of gamma interferon secretion by sensitized cytolytic T lymphocytes (CTL) of an IE1 epitope-specific cell line (IE1-CTLL) characterized previously (5,60). The peptide-presenting stimulator cells were BALB/c (H-2 d ) MEF infected with the respective recombinant viruses under conditions of selective and enhanced expression of IE-phase proteins (see above).…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

“…Several recent articles have reviewed what is known today of CMV enhancers and their cognate MIE genes and proteins (4, 12, 51-53, 71, 80, 81, 85). IE1 proteins of both human CMV (hCMV) and murine CMV (mCMV), in addition to their key regulatory roles in viral-gene expression, play major roles in the antiviral immune response (5,9,11,26,37,43,60,65,67,82; reviewed in references 34 and 64). As shown by Sylwester and colleagues (82), MIE genes of hCMV contribute to HLA polymorphic immunity to a degree above the prediction made by their genomic sequence coverage, thus indicating a nonstochastic mechanism favoring a high quantitative representation of MIE-specific T cells in memory T-cell pools.…”

mentioning

confidence: 99%

Transactivation of Cellular Genes Involved in Nucleotide Metabolism by the Regulatory IE1 Protein of Murine Cytomegalovirus Is Not Critical for Viral Replicative Fitness in Quiescent Cells and Host Tissues

Wilhelmi

Simon

Podlech

et al. 2008

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Despite its high coding capacity, murine CMV (mCMV) does not encode functional enzymes for nucleotide biosynthesis. It thus depends on cellular enzymes, such as ribonucleotide reductase (RNR) and thymidylate synthase (TS), to be supplied with deoxynucleoside triphosphates (dNTPs) for its DNA replication. Viral transactivation of these cellular genes in quiescent cells of host tissues is therefore a parameter of viral fitness relevant to pathogenicity. Previous work has shown that the IE1, but not the IE3, protein of mCMV transactivates RNR and TS gene promoters and has revealed an in vivo attenuation of the mutant virus mCMV-ΔIE1. It was attractive to propose the hypothesis that lack of transactivation by IE1 and a resulting deficiency in the supply of dNTPs are the reasons for growth attenuation. Here, we have tested this hypothesis with the mutant virus mCMV-IE1-Y165C expressing an IE1 protein that selectively fails to transactivate RNR and TS in quiescent cells upon transfection while maintaining the capacity to disperse repressive nuclear domains (ND10). Our results confirm in vivo attenuation of mCMV-ΔIE1, as indicated by a longer doubling time in host organs, whereas mCMV-IE1-Y165C replicated like mCMV-WT and the revertant virus mCMV-IE1-C165Y. Notably, the mutant virus transactivated RNR and TS upon infection of quiescent cells, thus indicating that IE1 is not the only viral transactivator involved. We conclude that transactivation of cellular genes of dNTP biosynthesis is ensured by redundancy and that attenuation of mCMV-ΔIE1 results from the loss of other critical functions of IE1, with its function in the dispersal of ND10 being a promising candidate.