+ Exposure to Apoptotic Activated CD4 T Cells Induces Maturation and APOBEC3G- Mediated Inhibition of HIV-1 Infection in Dendritic Cells Venkatramanan Mohanram., Ulrika Johansson . , Annette E. Sko¨ ld, Joshua Fink, Sushil Kumar Pathak, Barbro Ma¨ kitalo, Lilian Walther-Jallow", Anna-Lena Spetz*" Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden Abstract Dendritic cells (DCs) are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4+ T cells (ApoInf) or apoptotic uninfected activated CD4+ T cells (ApoAct) induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4+ T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1a, MIP-1b, MCP-1, and TNF-a; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-a using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4+ T cells (ApoRest). Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection. Citation: Mohanram V, Johansson U, Sko¨ ld AE, Fink J, Kumar Pathak S, et al. (2011) Exposure to Apoptotic Activated CD4+ T Cells Induces Maturation and APOBEC3G- Mediated Inhibition of HIV-1 Infection in Dendritic Cells. PLoS ONE 6(6): e21171. doi:10.1371/journal.pone.0021171 Editor: Roberto F. Speck, University Hospital Zurich, Switzerland Received December 10, 2010; Accepted May 21, 2011; Published June 16, 2011 Copyright: 2011 Mohanram et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the Swedish Research Council, Swedish International Development Cooperation Agency/Department for Research Cooperation, Hedlunds Foundation, SLL 20050348, European Commission; EC-FP6-018953 Auto/AlloCell, EC-FP6-037611 EUROPRISE, EC-FP7- 242135 CHAARM and Swedish Foundation for Strategic Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: A.-L. Spetz and L. Walther-Jallow have affiliation with Avaris AB. This does not alter the authors’ adherence to the PLoS ONE policies on sharing data and materials. * E-mail: anna-lena.spetz@ki.se . These authors contributed equally to this work. " These authors also contributed equally to this work. Introduction DCs are professional antigen-presenting cells that play a central role in connecting innate and adaptive immunity[1]. DCs residing in peripheral tissues engulf both foreign microbial material and dying cells of the host. Maturation signals are, however, required for DCs to undergo phenotypic and functional changes to acquire a fully competent antigen-presenting capacity. Maturation of DCs is a process that includes a transient increased capacity for antigen uptake; migration to draining lymph nodes; and simultaneous upregulation of molecules including chemokine receptors, major histocompatibility complex (MHC) class I and II molecules, and co-stimulatory molecules [1,2]. DC maturation is triggered by CD40 ligand, danger associated molecular pattern released from certain dying cells and by pro-inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), IL-1b, IL-6, and interferon-a (IFN-a) [3]. In addition, infected apoptotic cells may provide a DC maturation signal through interactions with pattern recognition receptors including members of the Toll-like receptor (TLR) family [4–6]. DCs are located in mucosal and lymphoid tissues and because of their localization in the mucosa, are likely to be among the first cells to encounter invading HIV particles [7]. It has been shown that DCs can be infected by HIV and support viral repli- cation, suggesting that HIV-infected DCs may be a viral reservoir in vivo [8]. Experiments using human cervical explants models showed that emigrating DCs had captured HIV and were capable of transmitting the virus to CD4+ T cells in trans [9]. In vitro experiments further support a role for DCs in transmitting HIV to CD4+ T cells which was demonstrated to occur both in cis and trans [7]. DC maturation suppresses HIV infection through multifaceted mechanisms that involve decreased viral fusion [10], a block of reverse transcription [11] and a post-integration restriction that has been proposed to exist at the transcriptional level [3]. There are several described intrinsic factors that act at discrete steps in the viral life cycle to mediate suppression of HIV replication in DCs. These include chemokines and their receptors, which play a central role in HIV infection and disease progression. HIV uses PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21171