CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

266 LYMPH NODE HIV-SPECIFIC CXCR5+ CTLs ARE ASSOCIATED WITH ENHANCED VIRAL CONTROL Zaza Ndhlovu 1 , Funsho Ogunshola 2 , Omolara Baiyegunhi 2 , Nikoshia Mewalal 2 , Amber Moodley 2 , Krista Dong 1 , Thumbi Ndung’u 2 , Bruce D. Walker 1 1 Ragon Inst of MGH, MIT and Harvard, Cambridge, MA, USA, 2 Univ of KwaZulu Natal, Durban, South Africa Background: HIV replication occurs in follicular CD4+ T helper cells localized within germinal centers (GC) of secondary lymphoid follicles, and exclusion of CTL within GC is thought to contribute to the maintenance of the viral reservoir. Recent studies have identified a CD8+ T cell population termed follicular CD8+ T cells that expresses the chemokine receptor CXCR5, which is associated with trafficking into GCs, which secrete its ligand CXCL13. However, the frequency, precise localization and antiviral function of HIV-specific CXCR5+ CD8+ T cells within lymph nodes have not been determined. We investigated the frequency, function and localization of HIV-specific CXCR5+ CD8+ T cells in lymph nodes (LN) and peripheral blood (PB) during treated and untreated chronic clade C HIV-1 infection. Methods: Biopsied LN and paired PB samples from 5 chronic untreated, 2 treated in chronic phase and 2 subjects treated during hyperacute HIV infection were analyzed. MHC class I tetramers and ICS assays were used to characterize HIV-specific responses. Immunohistochemistry (IHC) staining for Gag p24 was used to identify the location of HIV infected cells. IHC was also used to locate CXCR5+ CD8+ T cells within LN. Results: Comparative analysis of HIV-specific CD8+ T cells of the same tetramer specificity in LN and paired PB illustrated phenotypic and functional dissimilarities. Notably, in contrast to PB responses, LN cells were significantly more activated (CD38+HLA-DR+), more exhausted (PD-1 high) and secreted less cytokines upon ex vivo stimulation with mitogens (SEB). IHC revealed limited overlap between Gag p24+ CD4+ T cells and CD8+ T cells. The frequencies of CXCR5+ tetramer+ CD8+ T cells were very low in PB averaging about 0.9% (IQR 0.0:3.2), but were readily detectable in LMC samples by flowcytometry as well as in LN tissues by IHC. More importantly the frequency of CXCR5+ tetramer+ CD8+ T cells in LN inversely correlated with plasma viral load (Spearman’s r=−0.8; p=0.01). Conclusion: Taken together our results indicate that HIV-specific CD8+ T cells in LN are functionally more impaired than PB responses. Within LN, CXCR5+ tetramer+ CD8+ T cells may contribute to enhanced virus control. Therefore, efforts aimed at redirecting HIV-specific CD8+ T cells into GC though induction of CXCR5 expression may contribute to enhanced virus suppression. 267 RESIDENT MEMORY CD8+ T CELLS FORM THE FRONT-LINE DEFENSE IN HIV-INFECTED LYMPH NODES Marcus Buggert 1 , Lalit Beura 2 , Son Nguyen 1 , David Canaday 3 , Ali Naji 4 , Constantinos Petrovas 5 , Gustavo Reyes-Terán 6 , Steven G. Deeks 7 , David Masopust 2 , Michael R. Betts 1 1 Univ of Pennsylvania, Philadelphia, PA, USA, 2 Univ of Minnesota, Minneapolis, MN, USA, 3 Case Western Reserve Univ, Cleveland, OH, USA, 4 Hosp of the Univ of Pennsylvania, Philadelphia, PA, USA, 5 NIH, Bethesda, MD, USA, 6 Rsr Cntr in Infectious Diseases, Mexico City, Mexico, 7 Univ of California San Francisco, San Francisco, CA, USA Background: CD8+ T cells are key players in HIV control and future cure efforts. Current knowledge of HIV-specific CD8+ T cells relies almost entirely on circulating cells, although HIV is primarily a disease of lymphoid tissue. Accumulating evidence indicates that non-circulating ‘resident memory’ (TRM) CD8+ T cell responses are critical for pathogen control in tissues; however, whether CD8+ TRMs are found in lymphoid tissue is uncertain. Conceptually, most lymphoid memory CD8+ T cells are thought to recirculate, but such data are based on human blood and hygienic mouse models. Methods: Human peripheral blood and lymphoid tissues frommultiple locations were collected from healthy controls and HIV-infected subjects, including ART-, ART+, and elite controllers from different sites in North and Central America. Peripheral blood and lymph nodes were also compared between laboratory mice living in ultra-hygienic specific- pathogen free (SPF) environment and “dirty” mice that had more physiologic infectious experience. Multi-parametric flow cytometry, histo-cytometry, gene expression, and tetramer analysis were used to assess the localization, phenotype, and transcriptional profile of CD8+ T cells. Data were analyzed in FlowJo, GraphPad Prism, and R Studio. Results: We here provide evidence that a majority of memory CD8+ T cells in lymphoid tissue of healthy humans bear a TRM phenotype, with high expression of CD69 and variable levels of CD103. Furthermore, “dirty” mice exhibited substantially more of the TRM phenotype within lymph nodes than SPF mice. Lymphoid TRMs are detectable in HIV-infected lymph nodes using histo-cytometry and most CD8+ T cells with B cell follicle homing potential (CXCR5+) have a TRM phenotype. Importantly, the majority of HIV-specific CD8+ T cells in lymph nodes are TRMs and show transcriptional and phenotypic characteristics that are distinct from peripheral blood HIV-specific CD8+ T cells. Finally, we find that HLA- B57/27+ elite controllers demonstrate high magnitudes of HIV-specific TRMs in lymph nodes that selectively target immunodominant epitopes within Gag. Conclusion: We identify that HIV-specific TRMs are the front-line defense in HIV-infected lymph nodes. TRMs do not share the same phenotypic and transcriptional characteristics with circulating HIV-specific CD8+ T cells that have classically been studied in the context of HIV pathogenesis. Elicitation of functional and high numbers of lymphoid CD8+ TRMs should be a priority for any HIV vaccine or eradication strategy. 268 HIV-1 ESCAPE FROM CD8+ CYTOTOXIC T-LYMPHOCYTES DEFINED AT CLONAL RESOLUTION Background: A small number of HIV-infected individuals are able to effectively suppress HIV replication without antiretroviral therapy. Certain “protective” HLA types correlate with this ability to suppress HIV replication, indicating that the CD8+ cytotoxic T-lymphocyte (CTL) response in these individuals is responsible for the efficient containment of HIV. However, the mechanism behind CTL mediated control is unclear and has been suggested to be a result of either increased ability of the CTLs to recognize escape mutants or due to the ability of protective HLA types to present conserved viral epitopes. In this project we define all possible single and double amino acid mutations that the HIV can assume to escape CTL targeting, and the effect of these mutations on relative viral fitness within two HIV-1 immunodominant epitopes: SL9 (Gag 77-85) and KF11 (Gag 162-172) presented by the non-protective HLA A*02 and the protective HLA B*5701, respectively. Methods: Plasmid libraries encoding full length HIV with all possible single and double amino acid mutations within the SL9 and KF11 epitope were synthesized. Live virus was created from this library and passaged in HIV permissive cells in the absence or presence of epitope-specific CTLs. The passaged virus was deep sequenced to identify viable variants and variants that could escape CTL recognition. Results: 48 variants within SL9 and 33 within KF11 maintained reasonable relative fitness. KF11 specific CTLs recognized 13-16 of these variants while SL9 specific CTLs recognized 7-24 variants. 2 KF11 and 11 SL9 variants were at least as fit as wild type. KF11 specific CTLs recognized all of these most fit variants, while SL9 specific CTLs recognized only 0-45% of the most fit variants. Conclusion: CTLs targeting SL9 and KF11 do not differ in their ability to cross recognize escape variants. However, HIV is far less tolerant of mutations within KF11 than SL9, giving the virus fewer options for escape at this epitope. Furthermore, since KF11 specific CTLs can recognize all of the most fit KF11 variants (while SL9 specific CTLs cannot), HIV must sacrifice replicative capacity to escape CTL recognition of KF11. Our findings indicate that the beneficial effect of protective HLA types derives from the ability of these HLAs to present highly conserved regions of the virus, and from the ability of CTLs targeting these epitopes to cross recognize only the few fit variants that exist within such regions. 269 INVESTIGATING THE ROLE OF TCR STRUCTURE IN FUNCTIONAL ACTIVITY OF HIV-SPECIFIC CTLs Nina C. Flerin 1 , Tynisha Glover 1 , Huabiao Chen 2 , Jianhua Zheng 1 , Harris Goldstein 1 1 Albert Einstein Coll of Med, Bronx, NY, USA, 2 Ragon Inst of Massachusetts General Hosp, Cambridge, MA, USA Background: Lentiviral vectors expressing effective HIV-specific TCR clonotypes can transform naïve CD8+ T cells into potent HIV-specific CTLs, a possible gene therapy approach to increase HIV-specific immunity. To optimize this approach, it is crucial to identify TCR clonotypes which confer the most potent anti-HIV activity. CTLs with superior antiviral efficacy, termed effective CTLs, are well-represented in HIV-1 controllers but are rare or absent in HIV-1 progessors. However, the contributory role of the HIV-specific CTL TCR clonotype on the potency of its antiviral activity is unclear; this compromises our ability to identify which HIV-1 specific TCR α and β chain genes most effectively convert primary CD8+ T cells into potent HIV-specific CTLs. Aleksandr Gorin , Yushen Du, Ren Sun, Otto Yang Univ of California Los Angeles, Los Angeles, CA, USA

Poster and Themed Discussion Abstracts

CROI 2017 106