CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

222 IL-15 DRIVES THE GENERATION AND SURVIVAL OF SENESCENT CD8 T CELLS IN HIV INFECTION Stephen R. Morris 1 , Bonnie Chen 2 , Soumya Panigrahi 2 , Scott F Sieg 2 , Souheil A Younes 2 , Sara Gianella 3 , Michael M. Lederman 2 , Michael L. Freeman 2 1 Louis Stokes Cleveland VA Medical Center, Cleveland, OH, USA, 2 Case Western Reserve University, Cleveland, OH, USA, 3 University of California San Diego, La Jolla, CA, USA Background: There is a significant expansion of activated CD8 T cells in HIV infected people, even when virus is controlled with antiretroviral therapy (ART). This expansion leads to an inversion of the CD4/CD8 ratio, and a low ratio is linked to increased co-morbidities, including increased cardiovascular disease (CVD) risk. Many of the expanded CD8 T cells express the fractalkine receptor (CX3CR1) and contain cytolytic granules, suggesting these cells can traffic to sites of endothelial cell dysfunction and contribute to CVD. CX3CR1+ CD8 T cells are highly enriched for senescent cells calling into question how these poorly replicative cells are maintained long-term. Here we tested the role of the inflammatory cytokine IL-15 in promoting the formation and survival of senescent CD8 T cells. Methods: Peripheral blood CD8 T cells were isolated from ART-treated HIV- infected individuals (n=14) or HIV-uninfected controls (n=9) and analyzed by surface and intracellular flow cytometry. In some experiments, cells were treated in culture with recombinant IL-2 (100U/mL), recombinant IL-15 (20ng/ mL), or anti-CD3 (5ng/mL) and anti-CD28 (5μg/mL) for 1, 2, 4, or 7 days. Results: Principal component and t-stochastic neighbor embedding analyses reveal that CX3CR1+ CD8 T cells are a fundamentally distinct population of memory CD8 T cells characterized by elevated expression of CD57, a marker of immune senescence. To determine what factors contribute to the persistence and expansion of a poorly replicative T cell population, we sorted subpopulations of CX3CR1+ CD8 T cells and stimulated with IL-2, IL-15, or via T cell receptor (TCR) ligation. IL-2 and IL-15, but not TCR signals, supported CD57+ CD8 T cell viability, and IL-15 and TCR signals, but not IL-2, promoted proliferation of CD57+ CD8 T cells. Although both IL-15 and TCR signals upregulated c-myc expression and oxidative phosphorylation, only IL-15 also induced expression of the pro-survival factor Bcl-2. Additionally, IL-15 upregulated CD57 expression on sorted CX3CR1+CD57- CD8 T cells. Conclusion: IL-15 induced the generation, proliferation, and survival of CX3CR1+CD57+ CD8 T cells. This effect was consistent with an upregulation of c-myc, mitochondrial activity, and Bcl-2 expression. As elevated IL-15 has been demonstrated in chronic untreated HIV infection, and in HIV-uninfected individuals with CVD, our data suggest that the proinflammatory environment in HIV disease contributes to the generation of and maintenance of senescent cytotoxic CD8 T cells. 223 REGULATION OF B CELLS BY IGG3 IN HIV-INFECTED INDIVIDUALS Christine Youn 1 , Lela Kardava 1 , Haewon Sohn 2 , James W. Austin 1 , Wei Wang 1 , Clarisa M. Buckner 1 , Marissa Hand 1 , Kathleen Gittens 3 , Richard Kwan 3 , Michael Sneller 1 , Tae-Wook Chun 1 , Susan K. Pierce 2 , Susan Moir 1 1 NIAID, Bethesda, MD, USA, 2 NIAID, Rockville, MD, USA, 3 NIH, Bethesda, MD, USA Background: The role of B cells in HIV immunopathology has been actively studied over the past few decades. Early antibody responses to vaccination or natural infection, including HIV, often involve IgG3. In chronic HIV viremia, we recently observed abnormalities involving the binding of soluble IgG3 to IgM- expressing B cells. Here, we describe a regulatory role for soluble IgG3 B-cell binding in chronic HIV-infection. Methods: Flow cytometry was performed to identify and sort B-cell populations. Biochemical and molecular analyses were performed to identify soluble and surface-expressed immunoglobulins (Ig). IgG3 complexes (IgG3-C) were isolated from serum by PEG precipitation. Imaging cytometry and total internal reflection fluorescence (TIRF) microscopy were used for colocalization analyses. Calcium influx and phosphorylation assays were performed for functional analyses. Results: Peripheral blood B cells were isolated from 108 HIV-infected individuals. Frequencies of IgG3+IgM+ B cells were found to vary by disease status, being highest in infected individuals with chronic HIV viremia while completely absent in HIV-negative individuals. IgG3+IgM+ B cells were also restricted to certain B-cell subsets, namely tissue-like memory (TLM) cells and to a lesser extent, naïve cells. Trypsin treatment and mixed light chain pattern suggested that soluble IgG3 was bound to the surface of IgM B-cell receptor (BCR) expressing cells. Transcriptional analyses confirmed that IgG3+IgM+ B cells exclusively expressed IGHMmRNA. Imaging cytometry and TIRF

microscopy revealed a significant colocalization between IgM and IgG3 on TLM B cells. TIRF analyses demonstrated that in the absence of IgG3, IgM was evenly distributed on the cell surface. In contrast, when IgG3 bound to the cells, IgM-BCR was highly clustered, consistent with antigen-induced polarization, and highly colocalized with IgG3. PEG precipitation of Ig complexes from serum of individuals with high- but not low-intensity IgG3+IgM+ B cells led to binding of IgG3-C to B cells of HIV-negative individuals. TIRF microscopy, as well as anti- CD32 treatment, demonstrated involvement of CD32b in the binding of soluble IgG3. Lastly, IgG3+IgM+ TLM B cells responded poorly to BCR stimulation, as observed by calcium signaling and phosphorylation of downstream substrates. Conclusion: Our study provides a new functional role of IgG3 in dampening the B-cell response in HIV-infected individuals through its strong association with IgM-expressing TLM B cells. 224LB RESISTANCE OF HIV-INFECTED MACROPHAGES TO CTL KILLING DRIVES IMMUNE ACTIVATION Kiera L. Clayton 1 , David R. Collins 2 , Joshua Lengieza 1 , Farokh Dotiwala 3 , Judy Lieberman 3 , Bruce D. Walker 1 1 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 2 Massachusetts General Hospital, Boston, MA, USA, 3 Boston Children’s Hospital, Boston, MA, USA Background: CD4 T lymphocytes are the principal target of HIV infection, but macrophages can also become infected and contribute to viral pathogenesis. CD8 cytotoxic T lymphocytes (CTLs) control virus levels during acute and chronic stages of HIV infection and reduce HIV disease progression. Most studies have focused on CTL control of infected CD4 T cells with less focus on infected macrophages. Recent work suggests that SIV-infected macrophages are relatively resistant to CTL-mediated killing, but the mechanism behind their differential susceptibility is unknown. Thus, the objective of this work was to characterize the interactions between CTLs and HIV-infected targets, both CD4 T cells and macrophages, to delineate immunoevasion mechanisms of macrophage resistance to CTL-mediated elimination. Methods: Monocytes were matured into macrophages while CD4 T cells were activated to permit infection with HIV. Flow cytometry-based elimination assays and HIV Gag p24 ELISA-based suppression assays were used to asses the susceptibility of autologous HIV-infected targets to CTL-mediated killing. Flow cytometry and ELISA-based recognition assays were used to characterize the CTL degranulation and cytokine response to the targets. Imaging flow cytometry was used to assess effector-target conjugates while cytokine-bead arrays characterized pro-inflammatory chemokines released by macrophages. Results: We demonstrate that macrophages exhibit delayed CTL-mediated killing as compared to CD4 T cells (p<0.0001), resulting in inefficient HIV suppression (p = 0.0005). Mechanistic studies reveal that delayed killing of macrophages is caspase-3- and granzyme B-dependent, whereas rapid killing of CD4 T cells is caspase-independent and does not require granzyme B. Moreover, impaired killing of macrophages is associated with prolonged effector-target contact time (p=0.0022) and greater CTL IFN-γ expression (p = 0.0047), inducing macrophage production of pro-inflammatory chemokines that trigger recruitment of monocytes and T cells. Conclusion: These results suggest that inefficient CTL-mediated killing of macrophages may contribute to reservoir persistence and chronic inflammation in HIV infection. An improved understanding of the precise mechanisms underlying the observed resistance to target cell killing and resulting hypersecretion of pro-inflammatory cytokines and chemokines will be necessary to develop approaches capable of efficiently eliminating infected macrophages and hampering chronic inflammation. 225 SPECIFIC CTFH FREQUENCY CORRELATES WITH MEMORY B CELL RESPONSES IN HIV CONTROLLERS Lisa Chakrabarti 1 , Mathieu Claireaux 1 , Moran Galperin 1 , Daniela Benati 1 , Alexandre Nouel 1 , Madhura Mukhopadhyay 1 , Pierre de Truchis 2 , David Zucman 3 , Samia Hendou 4 , Faroudy Boufassa 4 , Olivier Lambotte 2 1 Institute Pasteur, Paris, France, 2 AP–HP, Paris, France, 3 Hopital Foch, Suresnes, France, 4 INSERM, Le Kremlin-Bicetre, France Background: Follicular helper T cells (Tfh) play an essential role in the affinity maturation of the antibody response by providing help to B cells. To determine whether this CD4+ T cell subset may contribute to the spontaneous control of HIV infection, we analyzed the phenotype and function of circulating Tfh (cTfh) in patients from the ANRS CO21 CODEX cohort who naturally controlled HIV-1

Poster Abstracts

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CROI 2018