Interaction of Toll-Like Receptors with the Molecular Chaperone Gp96 Is Essential for Its Activation of Cytotoxic T Lymphocyte Response

RESEARCH ARTICLE Interaction of Toll-Like Receptors with the Molecular Chaperone Gp96 Is Essential for Its Activation of Cytotoxic T Lymphocyte Response Weiwei Liu1, Mi Chen1, Xinghui Li1, Bao Zhao1, Junwei Hou1, Huaguo Zheng1, Lipeng Qiu2, Zihai Li3, Songdong Meng1* a11111 1 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, P.R. China, 2 Institute of Life Sciences, Jiangsu University, Zhenjiang, P.R. China, 3 Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, United States of America * OPEN ACCESS Citation: Liu W, Chen M, Li X, Zhao B, Hou J, Zheng H, et al. (2016) Interaction of Toll-Like Receptors with the Molecular Chaperone Gp96 Is Essential for Its Activation of Cytotoxic T Lymphocyte Response. PLoS ONE 11(5): e0155202. doi:10.1371/journal. pone.0155202 Editor: Jean Kanellopoulos, University Paris Sud, FRANCE Received: January 10, 2016 Accepted: April 25, 2016 Published: May 16, 2016 Copyright: © 2016 Liu 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. Abstract The heat shock protein gp96 elicits specific T cell responses to its chaperoned peptides against cancer and infectious diseases in both rodent models and clinical trials. Although gp96-induced innate immunity, via a subset of Toll like receptors (TLRs), and adaptive immunity, through antigen presentation, are both believed to be important for priming potent T cell responses, direct evidence for the role of gp96-mediated TLR activation related to its functional T cell activation is lacking. Here, we report that gp96 containing mutations in its TLR-binding domain failed to activate macrophages, but peptide presentation was unaffected. Moreover, we found that peptide-specific T cell responses, as well as antitumor T cell immunity induced by gp96, are severely impaired when the TLR-binding domain is mutated. These data demonstrate the essential role of the gp96-TLR interaction in priming T cell immunity and provide further molecular basis for the coupling of gp96-mediated innate with adaptive immunity. Data Availability Statement: All relevant data are within the paper. Introduction Funding: This work was supported by a grant from Major State Basic Research Development Program of China (973 Program) (No. 2014CB542602); grants from the National Natural Science Foundation of China (31230026, 81321063, 81471960, 81402840); grants from Natural Science Foundation of Jiangsu Province, China (Grant No. BK20130495); and grants from Shenzhen Science and Technology Innovation Committee (JSGG20140516112337659, CYZZ20130826112642412). The funders had no role As a member of the heat shock protein 90 (HSP90) family, gp96 (glucose-regulated protein 94, GRP94) is one of the most abundant chaperones in the endoplasmic reticulum (ER). Both rodent models and clinical trials have demonstrated that gp96 purified from tumors or complexed with viral antigens in vitro elicits antitumor effects or antigen-specific humoral and CD8+ T cell (CTL) immunity against tumors and viruses [1–3]. The immunogenicity of gp96 is attributed to its ability to activate both the innate and adaptive immune responses. First, together with HSP70 and HSP90 in the cytosol, gp96, TAP (transporter associated with antigen processing) molecules, and calreticulin in the ER are thought to constitute a relay line for antigenic peptide transfer from the cytosol to MHC class I molecules in a concerted PLOS ONE | DOI:10.1371/journal.pone.0155202 May 16, 2016 1 / 14 Gp96 and TLRs Interaction Is Essential for CTL Activation in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. and regulated manner [4, 5]. Under intradermal or subcutaneous immunization, the gp96-antigenic peptide complexes access the draining lymph node and are predominantly internalized by subsets of antigen presenting cells (APCs) through cell surface receptor CD91. Internalized gp96 can effectively present the associated peptides to MHC class I and class II molecules and thus activate specific CD8+ and CD4+ T cell responses [6, 7]. Second, gp96 itself binds to and acts as a master chaperone for Toll-like receptors (TLRs) (e. g., TLR2, TLR4, and TLR9) on APCs, stimulating pro-inflammatory and Th1-type cytokine (TNF-α, IL-1β, and IL-12) secretion [8, 9]. The TLR-2/4-binding domain of gp96 was recently mapped to its C-terminal loop structure [10]. In addition, gp96 also interacts with CD91, which leads to CD91 phosphorylation and activation of NF-κB and p38 MAPK. This allows for the maturation of APCs, releasing cytokines, and priming of T-helper (Th) cells [11]. Both of these events are believed to be important for gp96 to induce robust T cell responses. However, it is still unclear how the gp96-mediated innate immune response via TLRs functionally affects its CTL activation ability through antigen presentation. Our previous studies have shown that gp96 complexed with antigens from tumors or hepatitis B virus (HBV) engages macrophages to cross-present antigens. This allows for activation of specific CTL responses and exhibits significant antitumor or antiviral effects [12–14]. Additionally, consistent with previous studies [8, 10, 15], we also found that gp96 binds to and activates the TLR2/4 pathway in regulatory T cells (Tregs) [16]. While sufficient data exist to demonstrate gp96-mediated innate and adaptive immune functions, direct evidence for the potential role of the gp96-TLRs interaction on T cell activation is lacking. Here, we further dissected the impact of the TLR activation activity of gp96 on its ability to induce T cell responses against cancer and viruses. Materials and Methods Ethics statement Animal studies were carried out according to the guidelines set forth by the Institute of Microbiology, Chinese Academy of Sciences of Research Ethics Committee under the approved protocol numbers PZIMCAS2011001. All animal experiments were performed in strict accordance with institutional guidelines on the handling of laboratory animals. Mice were euthanized when the maximum tumor size (diameter: 2.0 cm) had been reached. Cervical dislocation was applied in the study to minimized animal suffering and distress. The health of the animals was monitored every other day and there was no unexpected deaths. To minimized mice suffering and distress, the only tumor was implanted in the subcutaneous site in the flank on each animal. Cell culture and antibodies The CD8+ T cell hybridoma cell line B3Z, the melanoma cell line B16.F10, and H-2b fibroblast cell line K41 were maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS) (GI (...truncated)