CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

(p=0.0011). Using combinatorial gates, we found a positive correlation between the expression of any of the five activating receptors we measured on CD56 dim CD16 + cells and HIV DNA (OR gate, p=0.037). The diversity of activating receptors was also positively correlated with HIV DNA on both CD56 dim CD16+ and CD56-CD16+CD7+ cells (p=0.034 and 0.046, respectively). Conclusion: We confirmed that higher levels of HIV DNA are associated with the loss of Siglec-7 expression, suggesting possible NK cell dysfunction. We also show that higher HIV DNA levels are associated with a greater frequency of CD62L-expressing NK cells, suggesting that these polyfunctional cells are activated and expand in response to HIV infection. The correlations observed between HIV DNA level and both the frequency and diversity of activating NK cell receptors are likely driven by the level of virus replication. Further studies on the functional capacity of Siglec-7+ and CD62L+ NK cells in relation to the control of HIV infection are warranted. 245 CHARACTERIZATION OF NKG2C+ MEMORY NK CELLS IN SIV INFECTION BY FISH-FLOW CYTOMETRY Daniel Ram , R. Keith Reeves Beth Israel Deaconess Medical Center, Boston, MA, USA Background: Recently, we and others have shown that NK cells have memory-like properties against CMV and SIV infections. While the mechanisms of memory-like responses are unclear, it has been shown in humans that expression of the activating receptor NKG2C is elevated on memory NK cells in response to infection with CMV. The role of NKG2C+ NK cells in experimental SIV infection is unclear as previously it has not been technically possible to distinguish NKG2C from its inhibitory counterpart, NKG2A, due to unfaithful antibody cross-reactivity. Because of the crucial role that macaque models play in modeling HIV (SIV) and CMV (rhCMV) new techniques to functionally and phenotypically characterize NKG2C+ NK memory cells are critical. Methods: Using flow cytometry we phenotyped NK cells from rhesus macaques either chronically infected with SIV (n=8), rhCMV (n=12), or from specific pathogen free (SPF) animals that were negative for both viruses (n=10). Commercial flow cytometry antibodies against rhesus CD3, NKG2A, CD14 and CD20 were used in combination with PrimeFlow (Affymetrix), which combines fluorescence in situ hybridization (FISH) with flow cytometry, allowing for simultaneous detection of NKG2A and NKG2C transcripts (KLRC1 and KLRC2, respectively). Results: Rhesus macaque NK cells were identified using a standard gating strategy (CD3-CD14-CD20-NKG2a/c+) and NK cell frequencies increased in infected relative to SPF animals. This suggests a virus-specific NK cell increase, though, as expected, inhibitory NKG2A could not be distinguished from activating/memory NKG2C expression. Using RNA probes we identified four different populations within NK cells (KLRC1±KLRC2±). Interestingly, frequencies of KLRC1+ NK cells were higher in the SPF animals, while KLRC2+ NK cells were elevated in infected animals. Levels of CD16 (KLRC1-KLRC2+ population) and CD56 (KLRC1±KLRC2+ populations) were also significantly modulated between infected and SPF groups, and CD2 was globally upregulated on NK cells in infected animals. Conclusion: Our data suggests that elevated KLRC2 expression and induction of NKG2C+ NK cells is associated with both rhCMV and SIV infection, suggesting these cells could partially delineate memory responses against these pathogens. We expect that this technical advance from combining flow cytometry and FISH will greatly enhance HIV vaccine and cure-related work utilizing the macaque model. 246 INNATE LYMPHOID CELLS IN ENDOCERVICAL MUCOSA OF HIV INFECTED WOMEN Natasa Strbo , Maria L. Alcaide, Laura Romero, Denisse Garcia, Margaret Fischl University of Miami, Miami, FL, USA Background: Dysbiosis of the vaginal microbiome, referred to as bacterial vaginosis (BV) is associated with increased HIV acquisition and transmission. BV increases local inflammation, and activated CD4+ T cells in the female reproductive tract (FRT). Innate lymphoid cells (ILCs), a novel family of immune effector cells, are key regulators of immune defenses at mucosal surfaces, and crucial for maintaining intact mucosal barriers, but have not been evaluated in the (FRT). ILCs are grouped into ILC1, ILC2, and ILC3, which share functional characteristics with Th1, Th2, and Th17 cells, respectively. The genital mucosa is the initial site of viral replication following vaginal HIV-1 infection. We hypothesized that, BV result in increased HIV acquisition and transmission

via inflammation-induced disruption to epithelial barrier integrity through dysregulation of ILCs. This is the first report comparing distribution and function of ILCs in endocervical mucosa in HIV+ and HIV- individuals. Methods: HIV+ (n=7) and HIV- (n=6) pre-menopausal women participating in the WIHS cohort were recruited. Participants underwent vaginal examination with collection of endocervical cytobrushes and peripheral blood. Frequency and phenotype of ILCs were determined in cervical cytobrush samples and peripheral blood by multicolor flow cytometry. BV was determined by Nugent scoring Results: ILC3 represent the predominant ILCs subset in endocervical intraepithelial cells in HIV- women without BV (BV-). Women with BV (BV+) have lower frequencies of ILC3 than those BV- (BV+ 61 ± 10.01, n=3 vs BV- 77.6 ± 8.647, n=3; p =0.05). In HIV+ women we did not find difference between BV+ and BV- samples, but we found significant decrease of ILC3 in HIV+ women compared to HIV- women (11.2 ± 7.0 n=7 vs 69.6 ± 5.6 n=6; p=0.001). Conclusion: Composition of the endocervical ILC pool differs between women with and without BV, and is altered by HIV status. Our data suggest a link between dysregulated vaginal microbiome and loss of endocervical ILCs and barrier function, thereby allowing for local immune activation. This highlights the important role played by vaginal microbiome on the endocervical innate immune system and suggest that endocervical barrier integrity and ILCs dysregulation are implicated in HIV acquisition in women with BV. 247 HIV-1 VPR SUSTAINS IL-6 PRODUCTION TO PROMOTE HIV-1 REPLICATION IN MACROPHAGES Qi Wang , Lishan Su University of North Carolina Chapel Hill, Chapel Hill, NC, USA Background: Persistent inflammation is a hallmark of HIV-1 pathogenesis, but the mechanism that leads to persistent inflammation remains elusive. As one of the abundant HIV-1 virion-associated proteins, Vpr remains the most enigmatic of HIV-1 accessory proteins. Vpr is efficiently incorporated into virus particles to enhance viral replication in macrophage by unclear mechanisms. In addition, the role of virion-associated Vpr in dysregulating inflammatory cytokines remains unclear during HIV-1 infection. Methods: We utilized different Vpr mutants to determine the proinflammatory cytokines level during HIV-1 infection in primary macrophage. We also utilized a Vpr-deficient HIV-1 and provided Vpr in trans that are packaged into the virions. This system devoids of de novo Vpr production during HIV-1 replication and allows us to exclusively study the role of virion-associated Vpr. Results: We report here that HIV-1 Vpr enhances IL-6 production, correlated with elevated HIV-1 replication in human monocytic cells via a Vpr activity that is independent of its G2 cell cycle arrest activity. We show that Vpr sustains IL-6 production by reducing binding of TET2 and HDACs to the IL-6 promoter during its resolution phase. We further demonstrate that Vpr-enhanced HIV-1 replication in macrophages partially depends on IL-6 signaling. Blocking IL-6 signaling with IL-6 neutralizing antibody or depleting NF-IL6 significantly reduced the ability of Vpr to enhance HIV-1 replication in human primary macrophages. Conclusion: First, we have discovered that HIV-1 Vpr enhances IL-6 production, correlated with elevated HIV-1 replication in monocytic cells via an activity that is independent of its G2 cell cycle arrest activity. Second, we demonstrate that Vpr sustains IL-6 production during its resolution phase by preventing binding of TET2 and HDACs to the IL-6 promoter. Third, we demonstrate that Vpr-enhanced HIV-1 replication in macrophages partially depends on IL-6 signaling. 248 T FOLLICULAR HELPER CELLS ARE MAJOR HIV-2 RESERVOIRS AND SUPPORT PRODUCTIVE INFECTION Ana Godinho-Santos , Ana Antão, Bárbara Tavares, Tiago Ferreira, Ana Serra- Caetano, Paula Matoso, Cheila Rocha, Russell B. Foxall, Ana E. Sousa Instituto de Medicina Molecular, Lisbon, Portugal Background: Life-long antiretroviral treatment is currently required for the 36 million people living with HIV in order to restrain persistent viral reservoirs. Follicular helper T cells (Tfh), a CD4+ T cell subset critical for efficient antibody responses, have been shown to be a main HIV-1 reservoir. HIV-2 represents a unique naturally-occurring model to investigate the role of Tfh in HIV/ AIDS. Despite reservoir establishment, HIV-2-infected patients feature: 1) low to undetectable viremia throughout disease; 2) slow rate of CD4 decline with limited impact on the survival of infected adults; and 3) high titers of neutralizing antibodies (nAbs). Thus, we reasoned that Tfh might be central

Poster Abstracts

86

CROI 2018