For LPS, staining was detected with N-Histofine simple stain MAX PO (M Nichirei) and ImmPACT DAB peroxidase substrate, samples counterstained with hematoxylin QS and mounted with VectaMount permanent mounting medium (all Vector Labs). Images were acquired using FV10-ASW software. Slides were mounted with Prolong Gold with DAPI (Invitrogen) and imaged at room temperature on a FluoView FV1000 confocal microscope (Olympus) using a 40×/1.30 Oil UPlan FLN objective lens. Controls for peroxidase blocking were included in all experiments. Samples were reblocked with hydrogen peroxide and sodium azide between each stain. Tyramide signal amplification, with TSA-plus Cy5, C圓, and FITC reagents (PerkinElmer), was used to visualize staining of MDR-1, CD8, and CD3, respectively. For immunofluorescent staining, samples were stained sequentially, initially for MDR-1 (detected with peroxidase-conjugated donkey anti–mouse IgG secondary (Jackson ImmunoResearch Laboratories), and then for CD3 and CD8 (detected sequentially with peroxidase-conjugated donkey anti–rabbit IgG (Jackson ImmunoResearch Laboratories), and peroxidase-conjugated goat anti–mouse IgG1 (Invitrogen) secondaries. Primary antibodies included anti–MDR-1 (5A12.2, mouse IgG2b, Merck Millipore), anti-CD3 (F7.2.38, mouse IgG1, Dako) anti-CD8 (rabbit polyclonal, Abcam), anti-lipopolysaccharide (LPS) core (WN1 222-5, mouse IgG2A, Hycult Biotech), and isotype-matched controls. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide and 0.13% sodium azide (both Sigma-Aldrich), and sections blocked with 0.5% blocking reagent (Perkin Elmer). Heat-induced antigen retrieval was performed using a pressure cooker (The Retriever, Electron Microscopy Sciences) and R-Buffer A (lipolysaccharide) or B (MDR-1, CD3, CD8 Electron Microscopy Sciences). Immunohistochemistry was performed on 5-μm thick sections of formalin-fixed, paraffin-embedded tissues. For proliferation assays, PBMCs were stained with CellTrace Violet (Invitrogen) as per the manufacturer's instructions. Dead cell were excluded with Near-IR Dead Cell Stain (Invitrogen). This loss may impact mucosal defense and could be important in susceptibility to specific opportunistic infections in HIV.Īll antibodies were from BD Bioscience unless otherwise indicated. We provide evidence that CD161 ++/MAIT cells are not preferentially infected but may be depleted through diverse mechanisms including accumulation in tissues and activation-induced cell death. We show that the CD161 ++/MAIT cell population is significantly decreased in early HIV infection and fails to recover despite otherwise successful treatment. We analyzed the frequency and function of CD161 ++/MAIT cells in peripheral blood and tissue from patients with early stage or chronic-stage HIV infection. In adults they dominantly express the semi-invariant TCR Vα7.2, the canonical feature of mucosal associated invariant T (MAIT) cells and have been recently implicated in host defense against pathogens. CD161 ++CD8 + T cells are a tissue-infiltrating population that produce IL17A, IL22, IFNγ, and TNFα, cytokines important in mucosal immunity. HIV infection is associated with immune dysfunction, perturbation of immune-cell subsets and opportunistic infections.
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