Direct visualization of the dynamics of antigen presentation in human cells infected with cytomegalovirus revealed by antibodies mimicking TCR specificity

Abstract

There are no direct means to study class I MHC presentation in human normal or diseased cells. Using CMV-infected human cells and applying novel mAb that mimic T-cell receptor specificity directed toward the immunogenic epitope of the viral pp65 protein presented on HLA-A2 molecules, we directly imaged the dynamics of Ag presentation in infected cells. We demonstrate that following infection large intracellular pools of HLA-A2/pp65 complexes are localized to the Golgi. These HLA-A2/pp65 pools account for the majority of total HLA-A2 molecules in infected cells. Interestingly, these large pools are sequestered inside infected cells and only a small portion of them are exported to the cell surface. Virus-induced class I MHC down-regulation did not affect the intracellular pool of HLA-A2/ pp65 complexes. Our data also suggest that proteasome function influences the release of class I complexes to the membrane. We present herein a new and direct molecular tool to study the dynamics of viral Ag presentation that may further elucidate the balance between immune response versus viral escape.Key words: Ab . Ag presentation/processing . MHC . Virology Supporting Information available online IntroductionImmunity to CMV is complex and involves humoral and cellmediated responses [1][2][3][4][5]. Studies showed that both NK cells and CTL are of primary importance in the prevention of recurrence [6][7][8]. Many gene products participate in generating the CTL response, but the high frequencies of the viral protein pp65 found in subsequent studies showed that this protein is the chief target of the CTL-mediated immune response [9,10]. Cytosolic proteins, usually synthesized in the cells, such as these viral proteins, enter the class I MHC pathway of Ag presentation. The proteasome is a cytoplasmic multiprotein complex that proteolytically degrades ubiquitinated cytoplasmic proteins and probably generates a large portion of the peptides destined for display by class I MHC molecules. Peptides are delivered from the cytoplasm to the ER by the TAP molecules. Newly formed class I MHC dimmers in the ER associate with and bind peptides delivered by the TAP. Peptide binding stabilizes class I MHC molecules and permits their movement out of the ER, through the Golgi, to the cell surface. This pathway ensures that any cell synthesizing viral proteins can 1552be marked for recognition and killing by CD8 1 CTL [11,12]. Among all the pp65 peptides, CMV-specific CTL activity in HLA-A2-positive individuals was found to be mainly directed to the peptide pp65 495-503 NLVPMVATV [13].Characterization of class I MHC/peptide presentation is essential for studying the relationships between immunity and viral escape. However, there are no direct means available to study class I MHC/peptide presentation; for example, study of viralderived peptide/MHC complexes on the surface and inside cells during the course of infection. mAb with peptide-specific, MHCrestricted recognition patterns, termed TCR-like Ab, are currently the only tool for such studies. ...

MHC class I antigen processing distinguishes endogenous antigens based on their translation from cellular vs. viral mRNA

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