CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

Results: LN were evaluated from 6 HIV+men receiving ART for a median of 20 years (range, 7-29 years) with documented plasma HIV RNA <20 copies/ mL at the time of screening. For comparison, LN sections from 8 HIV+men not receiving ART, and 6 HIV seronegative men (SN) were also evaluated. As expected, frequencies of Tregs in the tissue cross sections were significantly higher in untreated HIV+ compared to SN men (medians, 56 vs 21 cells/mm 2 ; p=0.008), and a similar trend was seen for TFR within follicles (medians, 59 versus 22 cells/mm 2 ; p=0.10). In ART treated HIV+, Tregs (median, 36 cells/ mm 2 ) and TFR (median, 76 cells/mm 2 ) did not differ significantly from either group. Tregs and TFR from ART-treated HIV+ demonstrated a clear dichotomy; 3 subjects had elevated levels similar to what was seen in untreated HIV+, and 3 had low levels of TFR similar to SN. HIV RNA+ cells were detected in LN of all ART-treated HIV+ (range, 0.05 to 0.5 cells/mm 2 ) and were significantly fewer than those detected in untreated HIV+ (range, 0.2 to 2.6 cells/mm 2 ; p=0.04). Higher concentrations of HIV RNA+ cells were found in the LN follicles of all ART treated individuals (median, 0.4 cells/mm 2 ) compared to extrafollicular regions (median, 0.2 cells/mm 2 ) with median F:EF ratio of 1.8 (range, 1.5 to 3.2). Frequencies of both Tregs and TFR in LN of ART-treated individuals correlated directly with frequencies of HIV RNA+ cells (r=0.88; p=0.03 for both). Conclusion: Persistent expansion of Tregs including TFR is seen in LN of some ART-treated individuals with plasma virus suppression and is related to tissue HIV RNA expression. Expansions in TFR may contribute to impairments in humoral immunity seen in some ART-treated individuals. 198 DENDRITIC CELLS CROSSTALK WITH FOLLICULAR CD8+ T-CELLS IN HIV- INFECTED LYMPH NODES Beatriz Dominguez-Molina 1 , Sara Ferrando-Martinez 2 , Rebeca S de Pablo- Bernal 1 , Fernando D. Docobo 3 , Manuel Leal 1 , Richard A. Koup 2 , Ezequiel Ruiz-Mateos 1 1 Institute of Biomedicine of Seville, Sevilla, Spain, 2 Vaccine Research Center, NIAID, Bethesda, MD, USA, 3 Hospital Universitario Virgen del Rocio, Sevilla, Spain Background: Plasmacytoid dendritic cells (pDCs) have been related with HIV spontaneous control. However, a deregulation mainly consisting in an aberrant IFN-I production lead to an inflammatory environment that could enhance HIV pathogenesis. It has been communicated that HIV provokes alteration in phenotype and location of CD8 T-cells within lymph nodes (LN). In a mouse model, a cooperative activity between pDCs and XCR1 dendritic cells (DCs) in order to effectively antigen cross-priming to CD8+T cells has been reported. However, the phenotype, function of pDCs and its interaction with other cell types in lymphoid tissues in relation with HIV-disease outcomes remain largely unknown. Methods: Seven inguinal LN samples prior to antiretroviral onset were obtained from HIV-infected patients. PBMCs were obtained at the same time point of LN biopsies. A comprehensive analysis of DCs, pDCs and T-cells was performed by deep immunophenotyping using multiparametric flow cytometry. Results: pDCs levels were inversely correlated with viral load (VL) both in PBMCs and LNs (r=-0.893; p=0.007 and r=-0.9; p=0.037, respectively). Alternatively, VL positively correlated with exhausted pDCs in LN, assessed by PDL-1 expression (r=0.829; p=0.042). Indeed, we found a strong positive correlation between the frequency of pDCs in PBMCs and pDCs in LN (r>0.9, p=0.016). Interestingly, associations between pDCs survival and CD141 mDCs and frequency of follicular CD8 (fCD8) T-cells within LN were presented. The more frequency of CD141 mDCs was present in LN, the less pDCs exhibiting an early stage of apoptosis and the less frequency of fCD8 T-cells were present (r=-0.829, p=0.042 and r=- 0.886, p=0.019, respectively). Furthermore, we found a strong correlation between the percentage of pDCs expressing pDL1 and the levels of follicular CD8+T cells PD1+ (r=0.943, p=0.005). Of note, this correlation was not present neither between the levels of pDCs PD1+ and non-follicular CD8+T cell levels nor with other myeloid dendritic cells expressing PDL1. Conclusion: We explored a pDCs crosstalk with CD141+mDC and fCD8+T-cells, and its relation with fCD8 T-cell exhaustion in LNs of HIV-infected patients. This pathway may be a drug target that may enhance HIV-specific response within LNs, allowing the development of HIV curative strategies.

199 LONGITUDINAL DYNAMICS OF FOLLICULAR CD4+ T CELLS IN ACUTE SIV INFECTION Leticia Kuri Cervantes 1 , Claire Deleage 2 , Emily Roberts 1 , Heidi M. Gunzelman 1 , Diane G. Carnathan 3 , Guido Silvestri 3 , Michael R. Betts 1 1 University of Pennsylvania, Philadelphia, PA, USA, 2 Frederick National Laboratory for Cancer Research, Frederick, MD, USA, 3 Emory University, Atlanta, GA, USA Background: Follicular T helper CD4+ (Tfh) cells play a critical role in germinal center (GC) formation and B cell maturation. GCs in lymph nodes (LN), particularly within Tfh cells, are sites for preferential SIV infection and replication. Changes in Tfh phenotype and functions in early acute SIV infection may be a major determinant in the development effective antibody-mediated control of SIV infection. Methods: Eighteen rhesus macaques were intravenously infected with SIVmac251 and underwent staggered necropsy during acute and chronic infection. Tfh cells from surface LN (sLN), mesenteric LN (mLN) and spleen were immunophenotyped. We further examined mLN to quantify and localize viral RNA (vRNA) using immunohistochemistry, and performed gene expression and pathway enrichment analyses on sorted Tfh cells from LNs in resting and stimulated conditions. Results: The frequency of Tfh cells decreased at day 10 post-infection (p.i.) and partially rebounded after 20 days in all tissues. Using principal component analyses we found similar phenotypic profiles in Tfh frommLN and sLN; in contrast, Tfh isolated from the spleen clustered separately after 10 days p.i. Although plasma viremia (pVL) peaked day 10 p.i., vRNA in mLNs was detectable as early as day 5 p.i. within follicles and the T cell zone. While pVL decreased after 20 days, tissue vRNA was increased until 90 days p.i. but was not preferentially found within the follicles during this early period. Very early following infection, transcriptional profiling of Tfh-related genes showed profound modulation of cytokine production and inflammatory pathways. Moreover, we observed a decrease in Tfh responsiveness to stimulation as early as day 5 p.i. This functional ability was partially recovered after 20 days p.i. irrespective of the increasing vRNA found the tissue. tSNE analyses showed independent clustering pre- and post-infection, and Tfh cells from day 90 p.i. had the most similar profile to pre-infection suggesting a partial recovery in responsiveness in later stages of infection. Conclusion: SIV infection has a profound effect in Tfh frequencies, phenotypic and genetic profiles across tissues since acute infection. This effect suggests a temporal decrease in Tfh ability to provide B cell help during early stages of infection associated with high levels of viremia in blood and tissues, that may directly impact or delay the early induction of SIV-specific antibody production. 200LB CYTOLYTIC HIV-SPECIFIC CD8+ T CELLS DO NOT RECIRCULATE THROUGH TISSUES Marcus Buggert 1 , Son Nguyen 2 , Sam Darko 3 , Amy Ransier 3 , Gustavo Reyes- Terán 4 , Daniel Douek 3 , Yoav Dori 5 , Ian Frank 2 , Max G. Itkin 5 , Michael R. Betts 2 1 Karolinska Institute, Stockholm, Sweden, 2 University of Pennsylvania, Philadelphia, PA, USA, 3 Vaccine Research Center, NIAID, Bethesda, MD, USA, 4 National Institute of Respiratory Diseases, Mexico City, Mexico, 5 Children’s Hospital of Philadelphia, Philadelphia, PA, USA Background: Cytolytic effector memory HIV- and SIV-specific CD8+ T cells are key correlates for natural and vaccine-induced viral control. While assumed, it remains unknown if these cells leave the blood to access HIV reservoirs in lymphoid and peripheral tissues. To directly address this question, we present for the first time a spatial map of the tissue egressing (recirculating) immune system by sampling blood, lymph nodes (LNs) and thoracic duct lymph (TDL) in HIV-seronegative and seropositive individuals. Methods: We isolated LNs and matched human blood and TDL mononuclear cells through thoracic duct cannulation of HIV-seronegative and seropositive individuals on and off antiretroviral therapy. Functional and phenotypic assays on total and HIV+ lymphocytes were performed by flow cytometry and transcriptional data was collected through RNAseq using the SMARTseq2 platform. The results were analyzed using RStudio, FlowJo, and GraphPad Prism. Results: Through transcriptional, functional and phenotypic analysis, we show that expression of cytolytic molecules by effector memory CD8+ T cells are almost entirely confined to blood, while their phenotypic counterparts in the thoracic duct, and many tissues, represent non-cytolytic T cells with a higher regenerative capacity. Unlike their blood counterparts, HIV- and CMV-specific CD8+ T cells in TDL and LNs generally lack cytolytic molecule expression and killing ability. We further demonstrate that those HIV-specific CD8+ T cells

Poster Abstracts

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CROI 2019