MHC CLASS I PEPTIDE CHARACTERIZATION FROM HIV

3.1
TITLE: MHC CLASS I PEPTIDE CHARACTERIZATION FROM HIV–INFECTED CELLS

Heather D. Hickman,¹ Mary E. Ellexson–Turner,¹ Angela D. Luis,¹ Wilfried Bardet,¹ Kenneth W. Jackson,² William H. Hildebrand.¹

¹Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; ²Warren Medical Research Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK

Immunity to viruses and intracellular pathogens is determined in part by the presentation of peptides by MHC class I molecules to immune effector cells such as CTLs. Recognition of the antigenic peptide as foreign or virus–derived leads to destruction of infected cells. The characterization of peptide epitopes unique to infected cells will guide viral vaccine architects and provide insights into host–pathogen interactions. In order to characterize changes in epitope presentation that result from infection with HIV, we have developed an approach to identify class I peptides from infected cells. Class I ligands from HIV–infected and –uninfected cells are directly compared, and unique peptide–epitopes are identified. Briefly, we transfect CD4+ T cells to secrete a soluble class I molecule, in particular A*0201 or B*0702. Transfected cells are cultured in hollow–fiber bioreactors as either uninfected cells or after infection with HIV. Soluble–HLA containing supernatant is collected as harvest, affinity purified, and the peptides released using acid–denaturation. Peptides are separated using reverse–phase HPLC and then mapped on a Q–STAR Q–TOF mass spectrometer. Maps are compared to identify peptides which are presented only or in higher proportion on infected cells. These peptides are then sequenced using tandem mass spectrometry. Analysis of peptides upregulated or presented solely on HIV–infected cells has yielded HIV–derived epitopes and numerous peptides derived from self–proteins. For example, one such endogenous peptide, AARPATSTL, derives from the eukaryotic translation initiation factor 4G (eIF4G). Western blot and real–time PCR data confirm the degradation of eIF4G after HIV infection. Thus, a large number of endogenous proteins are presented as class I epitopes on infected cells. The immune response to these neo–antigens is unknown, but it is interesting to speculate that virus–induced changes in the class I presentation of endogenous proteins may contribute to virus diversion of T–cell recognition or virally–induced autoimmune disease.