CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

2014; Salabert N. et al., Eur. J. Immunol., 2015). The development of these immunization strategies in humans requires a better understanding of early immune events driven by targeted LCs. We investigated the effects of αLC/HIV-Env on the differentiation of naïve CD4+ T-cells. Methods: Anti-human Langerin recombinant human IgG4 antibody fused with HIV-Env gp140 at the C-terminus of the H-chain were produced in CHO cells. Purified cord-blood CD34+ progenitor cells were differentiated into LCs (Caux C. et al., Blood, 1997), incubated with αLC/HIV-Env, and co-cultured eight days with autologous naïve CD4+ T-cells. T-cells differentiation was assessed by flow cytometry. Results: We show that: i) αLC/HIV-Env candidate vaccine specifically target skin or vaginal explant LC (CD1ahi/CD207+) as demonstrated by FACS and immunohisto-staining; ii) In vitro CD34-derived LCs exhibited a phenotype similar to ex vivo isolated LCs from human skin; iii) In vitro derived LCs incubated with αLC/HIV-Env induced the differentiation of co-cultured naïve CD4+ T-cells into Tfh-like cells (CXCR5+PD-1+Bcl-6+) significantly as compared to culture conditions with control HuFc-IgG4 or aCD40 fusion antibodies; iv) In the same culture conditions, monocyte-derived DCs and BDCA1+ primary DCs did not promote this differentiation of CD4+ T cells. Conclusion: These results revealed that LC activated through CD207 promotes the differentiation of Tfh cells. Gene expression and cytokine profiles of vaccine-targeted LC and differentiated Tfh cells are ongoing. These data support the development of novel DC targeting vaccine approaches. 263 HIV-1-INFECTION–ASSOCIATED CHANGES IN THE HUMAN CD4+ T-CELL PROTEOME Johannes Nemeth 1 , Valentina Vongrad 2 , Karin Metzner 2 , Victoria P. Strouvelle 2 , Rainer Weber 2 , Patrick Pedrioli 3 , Ruedi Aebersold 3 , Huldrych F. Günthard 2 , Ben Collins 3 1 Cntr for Infectious Disease Rsr, Seattle, WA, USA, 2 Univ of Zürich, Zürich, Switzerland, 3 ETH Zürich, Zürich, Switzerland Background: Host directed therapies against HIV-1 are supposed to be critical for long term containment of the HIV-1 pandemic but remain elusive. Since HIV-1 infects and manipulates important effectors of both the innate and adaptive immune system, identifying modulations of the host cell systems in humans during HIV-1 infection may be crucial for the development of immune based therapies. Methods: Here, we measured the changes of the proteome in human CD4+ T cells upon HIV-1 infection, both in vitro and in vivo. To our best knowledge, this is the first attempt to measure the proteome of CD4+ T cells in HIV infected humans. In an exploratory study, a bi-phasic SWATH-MS approach was used to measure the proteome of primary CD4+ T cells infected with HIV-1 in vitro as well as CD4+ T cells from HIV-1 infected patients with paired samples on and off antiretroviral treatment. Results: In the in vitro experiment, 1742 host cell proteins and 5 HIV-1 proteins were measured, with 121 proteins changing significantly during the time course. Changes in the proteome peaked 24 hours after infection, concomitantly with significant HIV-1 protein production. In sorted CD4+ T cells from clinical samples, 940 proteins were detected consistently, 174 of which were considered to be significantly different between viraemic patients and patients undergoing successful treatment. The proteome of in vitro infected CD4+ T cells was modulated on multiple functional levels, including TLR-4 signalling and the type 1 interferon signalling pathway. Likewise, perturbations in the type 1 interferon signalling pathway were recapitulated in CD4+ T cells from patients. Conclusion: SWATH-MS is capable of detecting significant changes on different functional levels of the proteome human CD4+ T cells to a yet unaccomplished depth. Exploring the perturbations in the proteome associated with HIV-1 infection may help to identify new targets for immune based interventions. 264 CHARACTERIZATION OF PREEXISTING HIV-SPECIFIC CD4 T CELLS IN UNINFECTED INDIVIDUALS Audrey Daigneault 1 , Nathalie Brassard 2 , Amy E. Baxter 1 , Lucie Barblu 1 , Colin Havenar-Daughton 3 , Roxanne Charlebois 2 , Jean-Pierre Routy 4 , Shane Crotty 3 , Daniel E. Kaufmann 1 1 Cntr de Rsr du Cntr Hosp de l’Univ de Montréal, 2 La Jolla Inst for Allergy and Immunol, La Jolla, CA, USA, 4 McGill Univ Hlth Cntr, Glen site, Montréal, Canada Background: CD4 T cells (Thelper, TH) play a key role in antiviral immunity. The magnitude of HIV-specific TH responses generated by infection or vaccination is highly variable. Previous studies suggest that antiviral TH responses can be generated prior to pathogen exposure by cross-reactivity with other microorganisms, and may shape TH responses upon infection or immunization. However, the functionality of such pre-existing HIV-specific TH in HIV-negative subjects is unknown. Methods: We investigated HIV-specific TH responses in HIV-uninfected donors (UD, n=18) and compared them to those of HIV-infected subjects (HI, n=53). We measured TH proliferative responses to HIV antigen (Ag) peptide pools using CFSE assays and grew HIV-specific TH cell lines (CL). We determined ex vivo responses firstly by ICS and secondly by co-upregulation of activation-induced markers (AIM) after Ag stimulation: i) CD69 and CD40L; or ii) CD25 and Ox40. Results: We identified a high prevalence of HIV-specific proliferative TH responses in UD; 33% had a robust CFSE response to one or more HIV Ags (Gag, Env, Nef or Pol; net >1% and >2x No Ag background). Gag was less immunodominant in UD than HI: the strongest response was against Gag in 33% of UD vs. 68% of HI (p=0.013, Fisher exact test). While ICS for Th0/1 cytokines (IFNγ, IL2, TNF) and CD40L failed to identify HIV-specific TH in PBMCs directly ex vivo, the same ICS assay on Gag- and gp41-specific CL derived from UD (n=12) showed that these responses were functional and dominated by TNF and CD40L, but produced little IFNγ. In contrast to ICS, AIM assays detected HIV-specific TH from UD without pre-expansion: net HIV-specific TH frequencies (cutoff: >2x No Ag value) ranged from 0.15 to 0.79% for CD69/CD40L and 0.3 to 1.60% for CD25/Ox40. Compared to total TH, HIV-specific Ox40+CD25+ TH were enriched in central memory (median 62% vs. 36%) and T follicular helper cells (median 20% vs. 14%), and preferentially expressed CXCR3 (median 46% vs. 35%). There was a direct correlation between the magnitudes of HIV-specific TH measured by the AIM and CFSE assays (n=20; CD69/CD40L: p=0.01, r=0.55; Ox40/ CD25: p=0.01, r=0.56; Spearman). Conclusion: These results demonstrate that HIV-specific TH cells exist in a substantial proportion of UD, can target diverse HIV Ags and are detectable by functional assays including CFSE and AIM. These cross-reactive CD4 T cells could impact the development of virus-specific TH responses upon acute HIV infection and influence vaccine-induced immunity. 265LB TFH CELLS FUNCTIONAL PROFILE DRIVES ABNORMAL B CELL MATURATION IN HIV INFECTION Giuseppe Pantaleo 1 , Alessandra Noto 1 , Andrew McDavid 2 , Phu Van 2 , Agostino Riva 3 , Guy Cavet 4 , Song Ding 5 , Raphael Gottardo 2 , Craig Fenwick 1 1 Univ Hosp of Lausanne, Lausanne, Switzerland, 2 FRCHC, Seattle, WA, USA, 3 Luigi Sacco Univ Hosp, Milan, Italy, Milano, Italy, 4 Atreca, Redwood City, CA, USA, 5 Eurovacc Foundation, Amsterdam, Netherlands Background: The mechanisms underlying abnormalities in B cell maturation and antibody response in HIV-1 infection have been only partially elucidated. The large majority of the studies have been performed in blood B cells while limited information is available on B cell populations in lymph nodes and on the association between the functional profile of T follicular helper (Tfh) cells and the maturation of B cell populations in lymph nodes. Methods: In the present study, we have investigated the phenotype and function of T follicular helper cells (Tfh) and B cells in lymph nodes of healthy and HIV-1 infected individuals using cutting-edge technologies such as mass cytometry and single cell gene expression. Lymph node biopsies were obtained from 21 HIV-1 infected viremic individuals naive to antiretroviral therapy and 18 healthy HIV negative individuals undergoing surgery for vascular pathologies or herniorrhaphy. Lymph node mononuclear cells (LNMC) were stimulated with phorbol-12-myristate-13-acetate (PMA) and ionomycin for 6 hours and stained with a unique panel of 32 markers primarily defining memory CD4 T cell and B cell populations including antibodies measuring differentiation, trafficking receptors and function. Results: In HIV-1 infected individuals, we demonstrate a significant increase (2-3 fold) in the Tfh cells defined by the CXCR3+T-bet+ phenotype and IFN-γ production along with a significant decrease (2-3 fold) in Tfh cells expressing CCR4, CCR6 and producing IL-4. The CXCR3, T-bet and IFN-γ signatures of Tfh cells in HIV infection were strongly associated with the appearance of memory B cells expressing CXCR3 and T-bet, which accounted for 30% of memory B cells. CXCR3+ B cells had decreased expression of CXCR4 and CXCR5 and antibody production by CXCR3+ cells was different both quantitatively, e.g. higher producer cells, and qualitatively, e.g. lower levels of hyper somatic mutations, as compared to CXCR3- B cells. We then identified IFN-γ as the causative factor inducing the differentiation of the CXCR3+T-bet+memory B cells and as a strong suppressive factor of antibody production. The identification of Th1-like Tfh cells and IFN-γ as the main mechanisms affecting B cell maturation and antibody production in HIV infection will provide novel insights into strategies to develop optimal antibody responses in HIV infection and following vaccination.

Poster and Themed Discussion Abstracts

CROI 2017 105