The Co-Stimulatory Effects of MyD88-Dependent Toll-Like Receptor Signaling on Activation of Murine γδ T Cells

et al. (2014) The Co-Stimulatory Effects of MyD88-Dependent Toll-Like Receptor Signaling on Activation of Murine cd T Cells. PLoS ONE 9(9): e108156. doi:10.1371/journal.pone.0108156 The Co-Stimulatory Effects of MyD88-Dependent Toll- Like Receptor Signaling on Activation of Murine cd T Cells Jinping Zhang 0 Jia Wang 0 Lan Pang 0 Guorui Xie 0 Thomas Welte 0 Vandana Saxena 0 Jason Wicker 0 Brian Mann 0 Lynn Soong 0 Alan Barrett 0 Willi Born 0 Rebecca O'Brien 0 Tian Wang 0 Dong-Yan Jin, University of Hong Kong, Hong kong 0 1 Department of Microbiology & Immunology, University of Texas Medical Branch , Galveston, Texas , United States of America, 2 Department of Pathology, University of Texas Medical Branch , Galveston, Texas , United States of America, 3 Integrated Department of Immunology, National Jewish Health Center , Denver, Colorado , United States of America cd T cells express several different toll-like receptor (TLR)s. The role of MyD88- dependent TLR signaling in TCR activation of murine cd T cells is incompletely defined. Here, we report that Pam3CSK4 (PAM, TLR2 agonist) and CL097 (TLR7 agonist), but not lipopolysaccharide (TLR4 agonist), increased CD69 expression and Th1-type cytokine production upon anti-CD3 stimulation of cd T cells from young adult mice (6-to 10-week-old). However, these agonists alone did not induce cd T cell activation. Additionally, we noted that neither PAM nor CL097 synergized with anti-CD3 in inducing CD69 expression on cd T cells of aged mice (21-to 22-month-old). Compared to young cd T cells, PAM and CL097 increased Th-1 type cytokine production with a lower magnitude from anti-CD3- stimulated, aged cd T cells. Vc1+ and Vc4+ cells are two subpopulations of splenic cd T cells. PAM had similar effects in anti-CD3-activated control and Vc4+ subset- depleted cd T cells; whereas CL097 induced more IFN-c production from Vc4+ subset-depleted cd T cells than from the control group. Finally, we studied the role of MyD88-dependent TLRs in cd T cell activation during West Nile virus (WNV) infection. cd T cell, in particular, Vc1+ subset expansion was significantly reduced in both MyD88- and TLR7- deficient mice. Treatment with TLR7 agonist induced more Vc1+ cell expansion in wild-type mice during WNV infection. In summary, these results suggest that MyD88dependent TLRs provide co-stimulatory signals during TCR activation of cd T cells and these have differential effects on distinct subsets. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by a NIH grant to T.W. (R01AI072060). G.X. was supported by a fellowship from the Sealy Center for Vaccine Development at UTMB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Co-author Tian Wang is currently serving as an academic editor for PLOS ONE. This does not alter the authors adherence to PLOS ONE Editorial policies and criteria. . These authors contribute equally to this work. cd T cells are a minority of CD3+ T cells in lymphoid tissue and blood of humans and rodents, but are well represented at epithelial and mucosal sites [1]. They can rapidly proliferate after parasitic, bacterial, and viral infections and produce inflammatory cytokines, such as IFN-c and TNF-a [26]. These cells lack major histocompatibility complex (MHC) restriction and have the potential capacity to respond to antigens without a requirement for conventional antigen processing [7,8]. Unlike ab T cells, there are few antigens recognized by cd T cell receptor [9]. Human Vd2 T cells recognize small bacterial phosphoantigens, alkylamines and synthetic aminobisphosphonates; whereas Vd1 T cells recognize stress-inducible MHC-related molecules-MICA/B and other ligands [10,11]. The class I molecules, including class Ib, and CD1d, are ligands for some murine cd T cells [1214]. In addition, both murine and human cd T cells recognize the algae protein phycoerythrin [15]. Taken together, these unique features suggest that cd T cells play a role in innate immunity during microbial infection. However, the underlying immune mechanisms of cd T cell activation are not clearly understood. Toll-like receptors (TLRs) play a fundamental role in host innate immunity by mounting a rapid and potent inflammatory response to pathogen infection by their recognition of conserved structural patterns in diverse microbial molecules. They are expressed by a wide range of cells, including both immune cells and non-immune cells. The core TLR signaling pathway (except for TLR3) utilizes myeloid differentiation factor 88 (MyD88) as the primary adaptor [1618]. Mouse and human cd T cells express TLR2, TLR3, TLR4 and TLR7/8 [1923]. Some studies suggest TLR-mediated signaling pathways can indirectly activate cd T cells, mainly via cross- talk between these cells and dendritic cells (DCs) [2427]. For human cd T cells, TLR ligands are also known to co-stimulate TCR-activation. For example, TLR2, TLR3 and TLR5 ligands induced higher cytokine production and increased expression of cell surface activation markers [2830]. However, the direct effect of TLR ligands on activation of murine cd T cells is not clearly defined. In this study, we investigated the role of MyD88-dependent TLRs in activating murine cd T cells. Materials and Methods Mice 6-to 10-week-old control C57BL/6 (B6), TLR4 deficient (TLR42/2) mice and 21-to 22-month-old B6 mice were purchased from Jackson Laboratories (Bar Harbor, ME) and the National Institute of Aging (Bethesda, MD), respectively. MyD882/2 mice were bred to the B6 background by backcrossing for 10 successive generations [31,32]. TLR72/2 mice (B66129 F2 background) were obtained from Regeneron Inc. (Tarrytown, NY) and bred to the B6 background by backcrossing for 7 successive generations [33,34]. Groups were age- and sex-matched for each experiment and housed under identical conditions. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were approved by the Animal Care and Use Committee at the University of Texas Medical Branch (Permit #0902011). Stimulation of cd T cells with anti-CD3 with or without TLR agonists cd T cells were purified from the pooled spleens of 35 mice by using a TCRc/d+ T Cell Isolation Kit according to the manufacturers instructions (Miltenyi Biotec, Auburn, CA). The purity of cd T cells was examined by staining with streptavidin-PE and anti-CD3 FITC. cd T cells (16105 cells/well) were cultured for 2 days at 37uC in RPMI-1640 medium (Invitrogen, Carlsbad, CA) in 96-well plates coated with 5 mg/ml anti-CD3 (eBioscience, San Diego, CA) in the presence of 1 mg/ml of Pam3CSK4 (PAM, Invivogen, San Di (...truncated)