Study of Antigen-Processing Steps Reveals Preferences Explaining Differential Biological Outcomes of Two HLA-A2-Restricted Immunodominant Epitopes from Human Immunodeficiency Virus Type 1

Abstract

Cytotoxic T-lymphocyte (CTL) responses directed to different human immunodeficiency virus (HIV) epitopesvary in their protective efficacy. In particular, HIV-infected cells are much more sensitive to lysis by anti-Gag/ p17(77-85)/HLA-A2 than to that by anti-polymerase/RT(476-484)/HLA-A2 CTL, because of a higher density of p17(77-85) complexes. This report describes multiple processing steps favoring the generation of p17(77-85) complexes: (i) the exact COOH-terminal cleavage of epitopes by cellular proteases occurred faster and more frequently for p17(77-85) than for RT(476-484), and (ii) the binding efficiency of the transporter associated with antigen processing was greater for p17(77-85) precursors than for the RT(476-484) epitope. Surprisingly, these peptides, which differed markedly in their antigenicity, displayed qualitatively and quantitatively similar immunogenicity, suggesting differences in the mechanisms governing these phenomena. Here, we discuss the mechanisms responsible for such differences.The mechanisms of presentation of intracellular antigens by major histocompatibility class I (MHC-I) molecules are well documented, and several of these steps have been described previously (28). First, antigenic proteins are degraded in peptides by different cytosolic complexes, the most highly implicated and frequently described being the proteasome. Second, peptides are translocated by the transporter associated with antigen-processing (TAP) into the endoplasmic reticulum (ER), where they bind newly synthesized empty MHC-I molecules. Third, stable peptide/MHC-I complexes are exported via the Golgi apparatus to the cell surface, where they can be recognized by CD8 ϩ T cells expressing a specific T-cell receptor. However, the steps in antigen processing which determine a dominant T-cell response are still poorly understood. Elucidating the mechanisms that determine the antigenicity of epitopes for MHC-I presentation is critical for defining targets for peptide-designed vaccines or immunotherapies that limit viral spread.In the present work, we investigated the processing of two HLA-A2-restricted epitopes, p17(77-85), from the Gag/p17 matrix protein, and RT(476-484), from the Pol/reverse transcriptase (RT) complex, which are naturally presented by human immunodeficiency virus (HIV)-infected cells (35). Previous studies have shown that p17(77-85)-specific cytotoxic T-lymphocyte (CTL) clones are more efficient than those specific for RT(476-484) in lysing infected target cells, because of a better presentation of p17 epitope (35,38). To understand the molecular basis of the dominance of p17(77-85) over , a detailed analysis of antigen presentation was performed. First, the kinetics and efficiency of the degradation by enriched proteasome fractions of protein fragments encompassing the epitopes were compared. Binding assays of the digestion products to TAP and of the optimal epitopes to HLA-A2 molecules were then performed. The avidities of spontaneously induced CD8 ϩ T cells against each epitope in HIV-infected ...

Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients

“…Cleavage products were separated by RP-HPLC (PerkinElmer, Norwalk, CT). Chromatograms were recorded at 214 nm (36). Fractions containing cleavage products corresponding to HPLC peaks and regions between peaks were lyophilized before mass spectrometry analysis or functional tests.…”

“…The IFN-␥ ELISPOT assay was performed as described in ref. 36. PBMCs were suspended in complete medium supplemented with 10% FCS (PAN Biotech, Aidenbach, Germany), plated in triplicate at 3 ϫ 10 5 cells per well and incubated overnight in the presence of 10 g͞ml peptide and 25 units͞ml human IL-2.…”

Portable flanking sequences modulate CTL epitope processing

“…50). Poorly processed epitopes or epitopes with a low affinity for transporter associated with antigen processing (TAP) will be less likely to enter the ER and be loaded onto the MHC (29,34,46). The importance of antigen-processing efficiency on epitope hierarchy is indirectly demonstrated by the effect of mutations impairing epitope production and presentation.…”

“…In addition to the above factors, the efficiency of antigen processing has been shown to influence viral epitope hierarchy in animal models (29,(45)(46)(47)(48)(49). The processing of epitopes from ubiquitinylated proteins or from defective ribosomal translation products relies on sequential cleavages, mainly by the proteasome and other amino- or endopeptidases in the cytosol and in the ER (reviewed in ref.…”

De l’immunologie fondamentale au développement de la vaccinologie

“…On a montré notamment que les conditions modulant la libération d'épitopes d'intérêt et l'effet de molécules accessoires sur l'assemblage CMH/peptide agissaient sur le profil de restriction HLA au niveau des cellules présentatrices d'antigè-nes et sur la nature des lymphocytes T engendrés. Pour comprendre les mécanismes d'induction des réponses T par les lipopeptides, la dégradation in vitro des fragments d'antigènes du VIH inclus dans les préparations vaccinales par des systèmes enzymatiques cellulaires de référence a été réalisée et la possibilité de produire des épitopes d'intérêt a été mise en évidence [19,20]. Dans la même optique, le devenir des lipopeptides au niveau de la cellule dendritique responsable de la stimulation primaire des lymphocytes T a été étudié [21].…”