The Activation State of CD4 T Cells Alters Cellular Peptidase Activities, HIV Antigen Processing, and MHC Class I Presentation in a Sequence-Dependent Manner

Abstract

CD4 T cell activation is critical to the initiation of adaptive immunity. CD4 T cells are also the main targets of HIV infection, and their activation status contributes to the maintenance and outcome of infection. Although the role of activation in the differentiation and proliferation of CD4 T cells is well studied, its impact on the processing and MHC class I (MHC-I) presentation of epitopes and immune recognition by CD8 T cells are not investigated. In this study, we show that the expression and hydrolytic activities of cellular peptidases are increased upon TCR-dependent and MHC-peptide activation of primary CD4 T cells from healthy or HIV-infected persons. Changes in peptidase activities altered the degradation patterns of HIV Ags analyzed by mass spectrometry, modifying the amount of MHC-I epitopes produced, the antigenicity of the degradation products, and the coverage of Ags by degradation peptides presentable by MHC-I. The computational analysis of 2237 degradation peptides generated during the degradation of various HIV-antigenic fragments in CD4 T cells identified cleavage sites that were predictably enhanced, reduced, or unchanged upon cellular activation. Epitope processing and presentation by CD4 T cells may be modulated by the activation state of cells in a sequence-dependent manner. Accordingly, cellular activation modified endogenous Ag processing and presentation and killing of HIV-infected CD4 T cells by CD8 T cells in a way that mirrored differences in in vitro epitope processing. The clearance of HIV-infected cells may rely on different immune responses according to activation state during HIV infection.

Latency reversal agents modulate HIV antigen processing and presentation to CD8 T cells

“…In contrast PKCa significantly increased aminopeptidase and proteasomal activities (2.05-to 4.9-fold increase depending on the activities) but to a lesser extent than TCR activation. Our previous work indicated that the increase in peptidase activities following aCD3/CD28-stimulation is positively correlated with cellular activation of primary CD4 T cells [37]. We assessed the expression of activation markers CD25 and CD69 at the surface of primary CD4 T cells following LRA-treatment ( Fig 1D).…”

“…We measured with a fluorescence-based enzymatic assay the hydrolytic activities of the proteasome (chymotryptic, tryptic-like and caspase-like activities) and the post-proteasomal aminopeptidase activities involved in MHC-I presentation in live CD4 T cells of healthy donors at 6, 24 and 48 hours post-treatment [37] (Fig 1). Peptidase activities significantly changed after a 48 hours treatment ( Fig 1A).…”

“…First, we compared the degradation of a HIV-1 Gag 35mer containing 6 MHC-I-restricted HIV epitopes, p24-10-35m MVHQAISPRTLNAWVKVVEEKAFSPEVIPMFAALS, in matched cellular extracts of primary CD4 T cells treated for 48 hours with DMSO, aCD3/CD28, Bryostatin, Ingenol, Panobinostat or Disulfiram (Fig 2A). Degradation peptides generated at various time points were purified, identified and quantified by mass spectrometry as in [37]. We compared the degradation patterns of the 35-mer by quantifying the relative amount of peptides starting or ending at each aa.…”

“…Peptides displayed by MHC-I to CD8 T or NK cells derive from the intracellular degradation of proteins by the antigen processing machinery, including proteasomes and post-proteasomal peptidases in the cytosol and endoplasmic reticulum (ER) where LRA-induced HIV antigens may be found [31]. The expression and hydrolytic activities of the antigen processing machineries are modulated by various stimuli such as cytokines [32], viral infection [33], TLR ligands [34][35][36], TCR-induced cellular activation [37], oxidative stress [38] or enzyme inhibitors [39][40][41][42][43]. We previously showed that TCR-mediated cellular activation enhanced peptidase activities, modulated protein degradation patterns into peptides and CTL recognition [37].…”

“…The expression and hydrolytic activities of the antigen processing machineries are modulated by various stimuli such as cytokines [32], viral infection [33], TLR ligands [34][35][36], TCR-induced cellular activation [37], oxidative stress [38] or enzyme inhibitors [39][40][41][42][43]. We previously showed that TCR-mediated cellular activation enhanced peptidase activities, modulated protein degradation patterns into peptides and CTL recognition [37]. The metabolic environment for HIV protein expression and degradation differs drastically between productive infection in activated CD4 T cells [44,45] and latency reversal in differentiated resting CD4 T cells [46,47], but the consequences on the degradation machinery and epitope production are not known.…”