Variable Processing and Cross-presentation of HIV by Dendritic Cells and Macrophages Shapes CTL Immunodominance and Immune Escape

Abstract

Dendritic cells (DCs) and macrophages (Møs) internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8+ T cells (CTL). However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation.

Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses

“…While some cleavage sites were shared among all three cell types, some, such as the cleavage at P position 9 in 293T, appeared uniquely in one cell subset and increased in abundance over time. The cell type-specific degradation patterns are in accordance with our previous findings (19,28,34). While some peptides such as AQ14 (ASLRSLFGSDPSSQ) were produced in extracts from all three cell types and displayed by all of them, others such as RQ11 (RSLFGSDPSSQ) were produced by all subsets but displayed by only one or two cell types, showing that the presentation of these peptides is limited not by cell type-specific production but rather by the HLAs of the cells.…”

“…The degradation was stopped with 2.5 l of 100% formic acid, and peptide fragments were purified by the use of 5% trichloroacetic acid (TCA) precipitation. Degradation peptides generated in 2 to 3 independent degradation experiments were identified by in-house LC/MS-MS as previously described (20,21,28).…”

HIV Protease Inhibitor–Induced Cathepsin Modulation Alters Antigen Processing and Cross-Presentation

“…The degradation was stopped with 2.5μL of 100% formic acid and peptide fragments were purified by 5% trichloroacetic acid precipitation. Degradation peptides were identified by in-house MS as previously described (8, 19). …”

“…Proteolytic activity measurement was performed as we previously described in (8, 19, 25). Briefly, 5x104 DCs, Møs or CD4T cells were pretreated with indicated PIs and omnicathepsin, cathepsin S, cathepsin D or cathepsin E activities were measured using Z-FR-AMC, Z-VVR-AMC, Ac-RGFFP-AMC or MOCAc-GSPAFLAK-Dnp-R specific fluorogenic substrates respectively.…”

“…Upon phagocytosis, phagosomes fuse with early and late endosomes and with lysosomes, thus acquiring progressively both the acidification machinery (mainly the V-ATPase complex) and lysosomal proteases (18). Acidification results in the activation of cathepsins with optimal proteolytic activities between pH 4-6 (18, 19). The activation and recruitment of NADPH oxidase 2 (NOX2) at the phagosomal membrane generates reactive oxygen species (ROS), increasing pH and limiting cathepsin activities and antigen degradation in DCs (20, 21).…”