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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., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Genomic variation in the untranslated region (UTR) has been shown to influence HLA class I expression level and associate with disease outcomes. Sequencing of the 3'UTR of common HLA-A alleles indicated the presence of two polyadenylation signals (PAS). The proximal PAS is conserved, whereas the distal PAS is disrupted within certain alleles by sequence variants. Using 3'RACE, we confirmed expression of two distinct forms of the HLA-A 3'UTR based on use of either the proximal or the distal PAS, which differ in length by 100 bp. Specific HLA-A alleles varied in the usage of the proximal versus distal PAS, with some alleles using only the proximal PAS, and others using both the proximal and distal PAS to differing degrees. We show that the short and the long 3'UTR produced similar mRNA expression levels. However, the long 3'UTR conferred lower luciferase activity as compared with the short form, indicating translation inhibition of the long 3'UTR. RNA affinity pull-down followed by mass spectrometry analysis as well as RNA coimmunoprecipitation indicated differential binding of Syncrip to the long versus short 3'UTR. Depletion of Syncrip by small interfering RNA increased surface expression of an HLA-A allotype that uses primarily the long 3'UTR, whereas an allotype expressing only the short form was unaffected. Furthermore, specific blocking of the proximal 3'UTR reduced surface expression without decreasing mRNA expression. These data demonstrate HLA-A allele-specific variation in PAS usage, which modulates their cell surface expression posttranscriptionally., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Immune recognition by T cells relies on the presentation of pathogen-derived peptides by infected cells, but the persistence of chronic infections calls for new approaches to modulate immune recognition. Ag cross-presentation, the process by which pathogen Ags are internalized, degraded, and presented by MHC class I, is crucial to prime CD8 T cell responses. The original degradation of Ags is performed by pH-dependent endolysosomal cathepsins. In this article, we show that HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate cathepsin activities in human APCs, dendritic cells and macrophages, and CD4 T cells, three cell subsets infected by HIV. Two HIV PIs acted in two complementary ways on cathepsin hydrolytic activities: directly on cathepsins and indirectly on their regulators by inhibiting Akt kinase activities, reducing NADPH oxidase 2 activation, and lowering phagolysosomal reactive oxygen species production and pH, which led to enhanced cathepsin activities. HIV PIs modified endolysosomal degradation and epitope production of proteins from HIV and other pathogens in a sequence-dependent manner. They altered cross-presentation of Ags by dendritic cells to epitope-specific T cells and T cell-mediated killing. HIV PI-induced modulation of Ag processing partly changed the MHC self-peptidome displayed by primary human cells. This first identification, to our knowledge, of prescription drugs modifying the regulation of cathepsin activities and the MHC-peptidome may provide an alternate therapeutic approach to modulate immune recognition in immune disease beyond HIV., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Increased IFN-α production contributes to the pathogenesis of infectious and autoimmune diseases. Plasmacytoid dendritic cells (pDCs) from females produce more IFN-α upon TLR7 stimulation than pDCs from males, yet the mechanisms underlying this difference remain unclear. In this article, we show that basal levels of IFN regulatory factor (IRF) 5 in pDCs were significantly higher in females compared with males and positively correlated with the percentage of IFN-α-secreting pDCs. Delivery of recombinant IRF5 protein into human primary pDCs increased TLR7-mediated IFN-α secretion. In mice, genetic ablation of the estrogen receptor 1 (Esr1) gene in the hematopoietic compartment or DC lineage reduced Irf5 mRNA expression in pDCs and IFN-α production. IRF5 mRNA levels furthermore correlated with ESR1 mRNA levels in human pDCs, consistent with IRF5 regulation at the transcriptional level by ESR1. Taken together, these data demonstrate a critical mechanism by which sex differences in basal pDC IRF5 expression lead to higher IFN-α production upon TLR7 stimulation in females and provide novel targets for the modulation of immune responses and inflammation., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Dendritic cells (DCs), macrophages (MPs), and monocytes are permissive to HIV. Whether they similarly process and present HIV epitopes to HIV-specific CD8 T cells is unknown despite the critical role of peptide processing and presentation for recognition and clearance of infected cells. Cytosolic peptidases degrade endogenous proteins originating from self or pathogens, exogenous Ags preprocessed in endolysosomes, thus shaping the peptidome available for endoplasmic reticulum translocation, trimming, and MHC-I presentation. In this study, we compared the capacity of DCs, MPs, and monocyte cytosolic extracts to produce epitope precursors and epitopes. We showed differences in the proteolytic activities and expression levels of cytosolic proteases between monocyte-derived DCs and MPs and upon maturation with LPS, R848, and CL097, with mature MPs having the highest activities. Using cytosol as a source of proteases to degrade epitope-containing HIV peptides, we showed by mass spectrometry that the degradation patterns of long peptides and the kinetics and amount of antigenic peptides produced differed among DCs, MPs, and monocytes. Additionally, variable intracellular stability of HIV peptides prior to loading onto MHC may accentuate the differences in epitope availability for presentation by MHC-I between these subsets. Differences in peptide degradation led to 2- to 25-fold differences in the CTL responses elicited by the degradation peptides generated in DCs, MPs, and monocytes. Differences in Ag-processing activities between these subsets might lead to variations in the timing and efficiency of recognition of HIV-infected cells by CTLs and contribute to the unequal capacity of HIV-specific CTLs to control viral load., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Ag processing by intracellular proteases and peptidases and epitope presentation are critical for recognition of pathogen-infected cells by CD8+ T lymphocytes. First-generation HIV protease inhibitors (PIs) alter proteasome activity, but the effect of first- or second-generation PIs on other cellular peptidases, the underlying mechanism, and impact on Ag processing and epitope presentation to CTL are still unknown. In this article, we demonstrate that several HIV PIs altered not only proteasome but also aminopeptidase activities in PBMCs. Using an in vitro degradation assay involving PBMC cytosolic extracts, we showed that PIs altered the degradation patterns of oligopeptides and peptide production in a sequence-specific manner, enhancing the cleavage of certain residues and reducing others. PIs affected the sensitivity of peptides to intracellular degradation, and altered the kinetics and amount of HIV epitopes produced intracellularly. Accordingly, the endogenous degradation of incoming virions in the presence of PIs led to variations in CTL-mediated killing of HIV-infected cells. By altering host protease activities and the degradation patterns of proteins in a sequence-specific manner, HIV PIs may diversify peptides available for MHC class I presentation to CTL, alter the patterns of CTL responses, and provide a complementary approach to current therapies for the CTL-mediated clearance of abnormal cells in infection, cancer, or other immune disease.

Viruses evade immune detection partly through immune-associated mutations. Analyses of HIV sequences derived from infected individuals have identified numerous examples of HLA-associated mutations within or adjacent to T cell epitopes, but the potential impact of most mutations on epitope production and presentation remains unclear. The multistep breakdown of proteins into epitopes includes trimming of N-extended peptides into epitopes by aminopeptidases before loading onto MHC class I molecules. Definition of sequence signatures that modulate epitope production would lead to a better understanding of factors driving viral evolution and immune escape at the population level. In this study, we identified cytosolic aminopeptidases cleavage preferences in primary cells and its impact on HIV Ag degradation into epitopes in primary human cell extracts by mass spectrometry and on epitope presentation to CTL. We observed a hierarchy of preferred amino acid cleavage by cytosolic aminopeptidases. We demonstrated that flanking mutations producing more or less cleavable motifs can increase or decrease epitope production and presentation by up to 14-fold. We found that the efficiency of epitope production correlates with cleavability of flanking residues. These in vitro findings were supported by in vivo population-level analyses of clinically derived viral sequences from 1134 antiretroviral-naive HIV-infected individuals: HLA-associated mutations immune pressures drove the selection of residues that are less cleavable by aminopeptidases predominantly at N-flanking sites, leading to reduced epitope production and immune recognition. These results underscore an important and widespread role of Ag processing mutations in HIV immune escape and identify molecular mechanisms underlying impaired epitope presentation.