CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

Results: Onset of HIV viremia rapidly induced massive activation of CD8 + T cells from 0.27% ( IQR 0.87 to 0.88 ) pre-infection to 52.7% ( IQR 41.8 to 67.4 ) ( p<0.0001 ), by day14 post onset of plasma viremia. In contrast, Influenza and CMV-specific CD8 + T cells were minimally activated, indicating that bystander activation was not occurring. HIV-specific responses were already detectable prior to peak viremia. Duration to highest frequency of activated T cells and the magnitude of the initial response both had a significant association with lower viral load set point ( time to peak activation, Spearman’s r=0.7, p=0.02; magnitude at peak activation, Spearman’s r=-0.7, p=0.02 ). Stimulation with exogenously infected autologous CD4 + T cells revealed that at least 74% of the early proliferating CD8 + T cell response was HIV-specific with the capacity to degranulate and secrete cytokines. Acute phase HIV-specific CD8 + T cells were prone to apoptosis due to activation induced cell death but this resolved after viral load set point was achieved. Conclusions: Our data provide strong evidence that the initial CD8 + T cell response to HIV is much larger than previously appreciated, but is highly proapoptotic and rapidly declines despite ongoing viremia. The rapidity and magnitude of the earliest response are key factors for successful control, as both inversely correlated with subsequent viral load set point. Overall, these studies suggest that, to facilitate long-term control, the initial CD8 + T cell response must be swift, of high magnitude and sustained. 360 Nonclassical Regulatory HIV-1 – Specific CD8 T Cells in HIV-1 Disease Progression Selena Vigano 2 ; Chun Li 2 ; Jordi J. Negron 2 ; Bruce D.Walker 2 ; Mathias Lichterfeld 1 ; Xu G.Yu 2 1 Massachusetts General Hospital, Boston, MA, US; 2 The Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, US Background: Regulatory T cells may influence HIV-1 disease progression by suppressing immune activation or inhibiting antiviral T cell immune responses. Recently, a distinct population of non-classical regulatory T cells expressing HLA-G was identified. However, the role of HLA-G-expressing T cells specific for HIV-1 is unknown. Methods: 36 HIV-1-specific and 25 CMV/EBV-specific CD8 T cell responses were analyzed in 47 HIV-1 infected patients. 27 patients were chronic progressors with median HIV-1 viral loads of 50,300 copies/ml and median CD4 T cell counts of 456 cells/ul. 20 patients were controllers with viremia below 1000 copies/ml and CD4 T cell counts above 600 cells/ ul. HLA-G expression on HIV-1 specific CD8 T cells was assessed by multimer staining and flow cytometry. Cytokine production of sorted HLA-G + and non-regulatory CD8 T cells was assessed in 12 HIV-1 negative individuals following CD3/CD28 stimulation by Luminex. Results: The frequency of HLA-G + HIV-1 specific ( P =0.0002), but not CMV- or EBV-specific ( P =n.s) CD8 T cells, was increased in controllers when compared to chronic progressors. This increase was mostly driven by HLA-G + CD8 T cells restricted by protective HLA class I alleles ( P <0.004), while no difference between controllers and chronic progressors was observed when CD8 T cells restricted by non-protective alleles were analyzed. The proportion of HLA-G + HIV-1-specific CD8 T cells was directly associated with CD4 T cell counts ( P =0.0002, r=0.57) and inversely with viral loads ( P <0.0001, r=-0.66), while total HIV-1-specific CD8 T cells were not. Of interest, HLA-G + CD8 T cells produced higher amount of IL-10, IL-2, MIP-1 α , MIP-1 β and RANTES than HLA-G-negative CD8 T cells after anti-CD3/amti-CD28 stimulation. Conclusions: These data indicate a potentially protective role of HIV-1-specific HLA-G + regulatory CD8 T cells on HIV-1 disease progression. The production of MIP-1 α , MIP-1 β and RANTES may be pivotal in controlling HIV-1 replication. Further investigations of functional properties of these non-classical regulatory CD8 T cells are necessary in order to better elucidate their role in HIV immunopathogenesis. 361 Nef Plays a Role in the Resistance of SIV-Infected Macrophages to CD8 + T-Cell Suppression Jennifer N. Rainho ; Mauricio A. Martins; Francesc Cunyat; David I.Watkins; Mario Stevenson University of Miami, Miami, FL, US Background: SIV-specific CD8 + T cells kill SIV-infected CD4 + T cells in a MHC Class I (MHC-I) dependent manner. However, they are less efficient at killing SIV-infected macrophages. Since Nef has been shown to down regulate MHC-I molecules and enhance CTL evasion, we examined if Nef played a role in protecting SIV-infected macrophages from killing by freshly sorted SIV-specific CD8 + T cells. Methods: The viral suppression assay (VSA) involves the co-culture of primary SIV-infected CD4 + T lymphocytes or monocyte-derived macrophages (target cells), with enriched, primary unstimulated SIV-specific CD8 + T cells (effector cells) for 24 hrs. Suppression of viral replication was determined by an antigen capture assay for Gag p27 in culture supernatants. Elimination of infected target cells was assessed by flow cytometric quantification of Gag p27 + target cells in the presence and absence of effectors. To explore the role of Nef in CD8 + T cell evasion, target cells were infected with wild type SIVmac239 (239wt- nef ), nef- deleted SIVmac239 (239 Δ nef ), and a Nef point mutant (Y223F– nef ) shown previously to inactivate the ability of Nef to down regulate MHC-I. Our controls included SIV-infected target cells in the absence of effectors and SIV-infected target cells isolated from animals with mismatched MHC-I alleles. Results: Freshly sorted SIV-specific CD8 + T cells eliminated SIV-infected CD4 + T cells, but not SIV-infected macrophages (p=<0.0001). Suppression of viral replication was also observed in cultures of CD4 + T cells, but not in SIV-infected macrophage cultures (p=0.0031.) Interestingly, we observed enhanced killing of macrophages infected with 239 Δ nef (p= 0.0276) compared to macrophages infected with wild type virus. The sensitivity of 239 Δ nef -infected macrophages to CD8 + T-cell-mediated suppression was not fully recapitulated in Y223F– nef- infected macrophages despite the fact that the point mutant was almost as effective as Nef-deleted SIVmac239 in disrupting MHC-I down regulation. Conclusions: SIV-infected macrophages evade CD8 + T cell suppression. Nef may be involved in the resistance of infected macrophages to CD8 + T-cell-mediated suppression. However, this protective effect cannot be fully explained by Nef’s ability to down regulate MHC-I. This study has implications for viral persistence and suggests that macrophages may afford primate lentiviruses some degree of protection from immune surveillance. 362 Defining Efficacious HIV-Specific CTL Responses Using Saporin-Conjugated Tetramers Ellen M. Leitman 1 ; Stuart Sims 2 ; Rebecca P. Payne 1 ; Fabian Chen 3 ; Lynn Riddell 4 ; Soren Buus 5 ; Steven Deeks 6 ; BruceWalker 7 ; Philippa C. Matthews 1 ; Philip J. Goulder 1 1 University of Oxford, Oxford, United Kingdom; 2 ETH Zurich, Basel, Switzerland; 3 Royal Berkshire Hospital, Reading, United Kingdom; 4 Northamptonshire Healthcare NHS Trust, Northampton, United Kingdom; 5 University of Copenhagen, Copenhagen, Denmark; 6 University of California San Francisco, San Francisco, CA, US; 7 Ragon Institute of MGH, MIT and Harvard, Charlestown, MA, US Background: In vitro killing of HIV-infected cells by cytotoxic T lymphocytes (CTL) is a surrogate marker of CTL antiviral efficacy. Tetrameric peptide-MHC complexes (tetramers) enable identification of antigen-specific CD8+ T cells in a mixed population. Due to their rapid internalization by cognate T cells, tetramers are an effective delivery vehicle of any coupled moiety to target specific CTL. Novel tetramers conjugated to saporin (SAP), a potent toxin causing cell death through ribosome-inactivation, have been used in vivo to deplete murine diabetogenic T cells, but no human reports exploiting this technology exist to date. Here, we used toxic tetramers to rapidly eliminate or ‘zap’ human CTL of different specificities in order to compare their anti-HIV potency. Methods: PBMC were treated with HLA-matched SAP-coupled tetramers (tet-SAP) or a control (HLA-matched tet-PE, HLA-mismatched tet-SAP, free SAP) and cell loss was quantified by flow cytometry. In inhibition assays, CD8+ T cells were expanded, treated with tet-SAP or a control and added as effectors to HLA-matched target cells infected with NL4-3-GFP, used as a marker to measure viral inhibition. Results: We show that HLA-matched tet-SAP but not HLA-mismatched tet-SAP or free SAP selectively binds to the surface of antigen-specific CD8+ T cells and is then rapidly internalized by the cognate cells alike a conventional tet-PE of the same specificity. We further show that, by as little as 48h post-tet-SAP treatment, up to 97% of tetramer-specific cells are eliminated from the diverse CTL population (Fig 1A). This elimination is highly targeted pertaining exclusively to the desired specificity. Finally, we compare the abilities

Poster Abstracts

273

CROI 2015