Portable flanking sequences modulate CTL epitope processing
Sylvie Le Gall, … , Pamela Stamegna, Bruce D. Walker
Sylvie Le Gall, … , Pamela Stamegna, Bruce D. Walker
Published November 1, 2007
Citation Information: J Clin Invest. 2007;117(11):3563-3575. https://doi.org/10.1172/JCI32047.
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Technical Advance

Portable flanking sequences modulate CTL epitope processing

Abstract

Peptide presentation is critical for immune recognition of pathogen-infected cells by CD8+ T lymphocytes. Although a limited number of immunodominant peptide epitopes are consistently observed in diseases such as HIV-1 infection, the relationship between immunodominance and antigen processing in humans is largely unknown. Here, we have demonstrated that endogenous processing and presentation of a human immunodominant HIV-1 epitope is more efficient than that of a subdominant epitope. Furthermore, we have shown that the regions flanking the immunodominant epitope constitute a portable motif that increases the production and antigenicity of otherwise subdominant epitopes. We used a novel in vitro degradation assay involving cytosolic extracts as well as endogenous intracellular processing assays to examine 2 well-characterized HIV-1 Gag overlapping epitopes presented by the same HLA class I allele, one of which is consistently immunodominant and the other subdominant in infected persons. The kinetics and products of degradation of HIV-1 Gag favored the production of peptides encompassing the immunodominant epitope and destruction of the subdominant one. Notably, cytosolic digestion experiments revealed flanking residues proximal to the immunodominant epitope that increased the production and antigenicity of otherwise subdominant epitopes. Furthermore, specific point mutations in these portable flanking sequences modulated the production and antigenicity of epitopes. Such portable epitope processing determinants provide what we believe is a novel approach to optimizing CTL responses elicited by vaccine vectors.

Authors

Sylvie Le Gall, Pamela Stamegna, Bruce D. Walker

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Figure 5

Point mutations in the N terminal portable sequences alter epitope production.

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Point mutations in the N terminal portable sequences alter epitope produ...
(A) The in vitro degradation of 8 nmol of peptides DOM-ATK9-DOM, DOMe-ATK9-DOM (where flanking I is mutated to E), and sub-ATK9-sub was performed as described in the Figure 4 legend. The amount of ATK9 produced at time 60 was analyzed by mass spectrometry and RP-HPLC. 100% corresponds to the amount of ATK9 produced in 1 hour during DOM-ATK9-DOM degradation (lower left panel). The digestion products from DOM-ATK9-DOM (DD, filled squares), DOMe-ATK9-DOM (DeD, open squares), and sub-ATK9-sub (ss, triangles) were used at 0.05 μg/ml to pulse 51Cr-labeled HLA-A3+ B cells used as targets in a 51Cr release assay using ATK9-specific CTLs. (B) Similar to A except that the 3 peptides were sub-ATK9-sub, subI-ATK9-sub (where flanking E is replaced by I), and DOM-ATK9-sub. 100% corresponds to the amount of ATK9 produced in 1 hour during sub-ATK9-sub degradation. The digestion products from DOM-ATK9-sub (Ds, diamonds), subI-ATK9-sub (sIs, open triangles), and sub-ATK9-sub (ss, triangles) were used in a 51Cr release assay as in A. (C) Similar to A except that the 3 peptides were WT-ATK9-WT, DOM-ATK9-DOM, and DOM+1-ATK9- DOM+1 (Gag flanking sequences shifted by 1 residue). 100% corresponds to the amount of ATK9 produced in 1 hour during WT-ATK9-WT degradation. The digestion products from WT-ATK9-WT (WT, Xs), DOM-ATK9-DOM (DD, squares), and DOM+1-ATK9-DOM+1 (DD+1, circles) were used in a 51Cr release assay as in A. Data are the average of 3 experiments performed with extracts from 3 healthy donors.