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Background: HIV-1-specific CD8+ T cells are required for immune suppression of HIV-1 replication and elimination of the associated viral reservoirs. However, effective induction of functional HIV-1-specific CD8+ T cells from naïve cells remains problematic in the setting of human vaccine trials. In this study, we investigated priming of functional HIV-1-specific CD8+ T cells from naïve cells. Methods: HIV-1-specific CD8+ T cells were primed from naïve T cells of HIV-1-seronegative individuals using TLR4 ligand LPS or STING ligand 3′3′-cGAMP in vitro. We established HIV-1-specific CD8+ T cell lines from primed T cells and then investigated functional properties of these cells. Findings: HIV-1-specific CD8+ T cells primed with LPS failed to suppress HIV-1. In contrast, 3′3′-cGAMP effectively primed HIV-1-specific CD8+ T cells with strong ability to suppress HIV-1. 3′3′-cGAMP-primed T cells had higher expression levels of perforin and granzyme B than LPS-primed ones. The expression levels of granzyme B and perforin and viral suppression ability of 3′3′-cGAMP-primed T cells were positively correlated with the production level of type I IFN from PBMCs stimulated with 3′3′-cGAMP. Interpretation: The present study demonstrates the potential of 3′3′-cGAMP to induce HIV-1-specific CD8+ T cells with strong effector function from naïve cells via a strong type I IFN production and suggests that this STING ligand may be useful for AIDS vaccine and cure treatment. Keywords: HIV-1, CD8+ T cells, Priming, STING ligand, Naïve T cell

Because of the enormous complexity and breadth of the overall HIV-specific CD8(+) T-cell response, invaluable information regarding important aspects of T-cell efficacy against HIV can be sourced from studies performed on individual clonotypes. Data gathered from ex vivo and in vitro analyses of T-cell responses and viral evolution bring us one step closer towards deciphering the correlates of protection against HIV. HIV-responsive CD8(+) T-cell populations are characterized by specific clonotypic immunodominance patterns and public TCRs. The TCR endows T-cells with two key features, important for the effective control of HIV: avidity and crossreactivity. While TCR avidity is a major determinant of CD8(+) T-cell functional efficacy against the virus, crossreactivity towards wildtype and mutant viral epitopes is crucial for adaptation to HIV evolution. The properties of CD4(+) T-cell responses in HIV controllers appear also to be shaped by high avidity public TCR clonotypes. The molecular nature of the TCR, together with the clonotypic composition of the HIV-specific T-cell response, emerge as major determinants of anti-viral efficacy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Flow cytometry with fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) tetramers has transformed the study of antigen-specific T-cells by enabling their visualization, enumeration, phenotypic characterization and isolation from ex vivo samples. Here, we demonstrate that the reversible protein kinase inhibitor (PKI) dasatinib improves the staining intensity of human (CD8+ and CD4+) and murine T-cells without concomitant increases in background staining. Dasatinib enhances the capture of cognate pMHC tetramers from solution and produces higher intensity staining at lower pMHC concentrations. Furthermore, dasatinib reduces pMHC tetramer-induced cell death and substantially lowers the T-cell receptor (TCR)/pMHC interaction affinity threshold required for cell staining. Accordingly, dasatinib permits the identification of T-cells with very low affinity TCR/pMHC interactions, such as those that typically predominate in tumour-specific responses and autoimmune conditions that are not amenable to detection by current technology.

The development of soluble recombinant peptide-major histocompatibility complex class I (pMHCI) molecules conjugated in multimeric form to fluorescent labels has enabled the physical quantification and characterization of antigen-specific CD8(+) T cell populations by flow cytometry. Several factors determine the binding threshold that enables visualization of cognate CD8(+) T cells with these reagents; these include the affinity of the T cell receptor (TCR) for pMHCI antigen. Here, we show that multimers constructed from peptide-human leukocyte antigen (pHLA) A0201 monomers engineered in the heavy chain alpha2 domain to enhance CD8 binding (K(D) approximately 85 microM) without impacting the TCR binding platform can detect cognate CD8(+) T cells bearing low affinity TCRs that are not visible with the corresponding wildtype pHLA A0201 multimeric complexes. Mechanistically, this effect is mediated by a disproportionate enhancement of the TCR/pMHCI association rate. In direct ex vivo applications, these coreceptor-enhanced multimers exhibit faithful cognate binding properties; concomitant increases in background staining within the non-cognate CD8(+) T cell population can be resolved phenotypically using polychromatic flow cytometry as a mixture of naïve and memory cells. These findings provide the first validation of a novel approach to the physical detection of low avidity antigen-specific CD8(+) T cell populations; such coreceptor-enhanced multimeric reagents are likely to be useful in a multitude of settings for the detection of auto-immune, tumor-specific and cross-reactive CD8(+) T cells.