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Latency reversal agents (LRA) variably induce HIV re-expression in CD4 T cells but reservoirs are not cleared. Whether HIV epitope presentation is similar between latency reversal and initial infection of CD4 T cells is unknown yet crucial to define immune responses able to detect HIV-infected CD4 T cells after latency reversal. HIV peptides displayed by MHC comes from the intracellular degradation of proteins by proteasomes and post-proteasomal peptidases but the impact of LRAs on antigen processing is not known. Here we show that HDAC inhibitors (HDCAi) reduced cytosolic proteolytic activities while PKC agonists (PKCa) increased them to a lesser extent than that induced by TCR activation. During the cytosolic degradation of long HIV peptides in LRA-treated CD4 T cells extracts, HDACi and PKCa modulated degradation patterns of peptides and altered the production of HIV epitopes in often opposite ways. Beyond known HIV epitopes, HDACi narrowed the coverage of HIV antigenic fragments by 8-11aa degradation peptides while PKCa broadened it. LRAs altered HIV infection kinetics and modulated CD8 T cell activation in an epitope- and time-dependent manner. Interestingly the efficiency of endogenous epitope processing and presentation to CD8 T cells was increased by PKCa Ingenol at early time points despite low levels of antigens. LRA-induced modulations of antigen processing should be considered and exploited to enhance and broaden HIV peptide presentation by CD4 T cells and to improve immune recognition after latency reversal. This property of LRAs, if confirmed with other antigens, might be exploited to improve immune detection of diseased cells beyond HIV., Author summary Latently HIV-infected CD4 T cells persist and remain invisible to the immune system. Strategies to flush out HIV reservoirs propose to re-express HIV with latency reversal agents (LRAs), leading to CD4 T cell death or clearance by HIV-specific immune responses. LRAs tested so far variably induced HIV re-expression but did not eliminate reservoirs. The activation of HIV-specific immune responses is triggered by HIV peptides displayed by infected cells after HIV intracellular degradation. Whether HIV antigens are similarly degraded and displayed by CD4 T cells after latency reversal or during initial infection is unknown. We showed that LRAs altered the activities of the degradation machinery and changed the degradation patterns of HIV into peptides. LRA-treated HIV-infected CD4 T cells were variably recognized by immune cells in a time- and peptide-dependent manner. Some LRAs increased the efficiency of HIV peptide presentation despite low levels of HIV antigens inside CD4 T cells. The modulation of HIV peptide presentation by current or future LRAs should be accounted for and exploited to improve HIV peptide presentation and enhance immune detection of HIV-infected CD4 T cells after latency reversal.

CD8 T cells provide limited protection against Mycobacterium tuberculosis (Mtb) infection in the mouse model. As Mtb causes chronic infection in mice and humans, we hypothesize that Mtb impairs T cell responses as an immune evasion strategy. TB10.4 is an immunodominant antigen in people, nonhuman primates, and mice, which is encoded by the esxH gene. In C57BL/6 mice, 30–50% of pulmonary CD8 T cells recognize the TB10.44−11 epitope. However, TB10.4-specific CD8 T cells fail to recognize Mtb-infected macrophages. We speculate that Mtb elicits immunodominant CD8 T cell responses to antigens that are inefficiently presented by infected cells, thereby focusing CD8 T cells on nonprotective antigens. Here, we leverage naturally occurring polymorphisms in esxH, which frequently occur in lineage 1 strains, to test this “decoy hypothesis”. Using the clinical isolate 667, which contains an EsxHA10T polymorphism, we observe a drastic change in the hierarchy of CD8 T cells. Using isogenic Erd.EsxHA10T and Erd.EsxHWT strains, we prove that this polymorphism alters the hierarchy of immunodominant CD8 T cell responses. Our data are best explained by immunodomination, a mechanism by which competition for APC leads to dominant responses suppressing subdominant responses. These results were surprising as the variant epitope can bind to H2-Kb and is recognized by TB10.4-specific CD8 T cells. The dramatic change in TB10.4-specific CD8 responses resulted from increased proteolytic degradation of A10T variant, which destroyed the TB10.44-11epitope. Importantly, this polymorphism affected T cell priming and recognition of infected cells. These data support a model in which nonprotective CD8 T cells become immunodominant and suppress subdominant responses. Thus, polymorphisms between clinical Mtb strains, and BCG or H37Rv sequence-based vaccines could lead to a mismatch between T cells that are primed by vaccines and the epitopes presented by infected cells. Reprograming host immune responses should be considered in the future design of vaccines., Author summary An important question for vaccine developers is the relative potency of CD4 vs. CD8 T cells against Mtb, as strategies differ for eliciting these different T cell subsets. Despite robust antigen-specific pulmonary CD8 T cell responses, CD4 T cells mediate more protection than CD8 T cells in the murine model. Most CD8 T cells recognize a single antigen, TB10.4, which is encoded by the esxH gene. Based on finding that TB10.44−11-specific CD8 T cells poorly recognize Mtb-infected macrophages, we hypothesized that Mtb evades detection by CD8 T cells and focuses the CD8 T cell response on non-protective antigen. We termed these antigens “decoy antigens.” To test this hypothesis, we took advantage of a natural variant of the esxH gene, which contains an A10T polymorphism within the TB10.44−11 epitope. This polymorphism drastically alters the hierarchy of CD8 T cell responses elicited by Mtb. These data suggest that immunodomination by the TB10.4 epitope acts to suppress subdominant CD8 T cell responses to other Mtb antigens, impairing the CD8 T cell response to other Mtb antigens, some of which might be presented by Mtb-infected macrophages and be targets of protective immunity. Importantly, this single amino acid polymorphism, which does not significantly alter MHC-binding or T cell recognition, alters the half-life of the epitope and consequently, has a profound effect on CD8 T cell priming and recognition of infected cells. These data also provide a mechanism that could be exploited to manipulate the hierarchy of immunodominant responses.

Iron–sulfur cluster coordination was probed in the [FeFe]-hydrogenase H cluster maturation scaffold HydF. Putative Cys thiol and His imidazole ligation identified through multiple sequence alignments and structural studies were subjected to amino acid substitution and the variants were biochemically characterized. The results implicate a role for C304, C353, C356, and H306 of Clostridium acetobutylicum HydF in FeS cluster binding. Individual ligand substitutions affect both [4Fe–4S] and [2Fe–2S] cluster coordination suggesting shared coordination or cluster interconversion. Substitutions at C353 and H306 appear to preferentially impact the presence of the [2Fe–2S] cluster complement of the resulting variants of HydF. The results implicate a potential role for these residues in biosynthesis specifically and potential in bridging the [4Fe–4S] cluster to 2Fe subcluster biosynthetic intermediates. Structured summary of protein interactions HydF and HydF bind by molecular sieving (View interaction: 1 , 2 )