Our data here show that this TLR3 deficiency does not impede CD8 T cell expansion, but its absence may even be beneficial for CD8 effector differentiation in response to poly I:C. specific roles of how dsRNA receptors shape CD8 T cell responses, which should be considered as poly I:C is usually authenticated as a SSR240612 therapeutic adjuvant used in vaccines. and is associated with food poisoning outbreaks, toxic shock, SSR240612 and recently respiratory diseases (19, 20). SEA crosslinks MHC II on antigen presenting cells with the T cell receptor V1, V3, V10, V11 or V17 chains on T cells (21). Thus, following immunization with SEA endogenous CD8 and CD4 T cell expansion and effector differentiation is usually incredibly robust. Here, it is exhibited that poly I:C preferentially induced CD8 V3 T cell expansion over CD4. Secondly, although TLR3 pathway deficiency did not significantly alter the magnitude of CD8 T cell expansion, effector differentiation was actually enhanced in the absence of TLR3. To better understand this counterintuitive result, cells from TLR3?/? mice were analyzed against wild type for cytokine SSR240612 output in response to PAMPs. The TLR3 impartial pathway induced high amounts of the immunosuppressive cytokine IL-10 in response to CpG but not in response to poly I:C, while wild type cells responded well to each PAMP. Although IL-10 may suppress effector differentiation (22), we postulated that IFN and cell killing potential was fundamentally dependent upon the presence of innate cytokines not the absence of immunosuppressive ones. Thus, we exhibited that CD8 effector differentiation was completely dependent upon TLR3-impartial production of IFN/. These data suggest that efficacious therapeutic use of poly I:C requires careful consideration in targeting the desired dsRNA receptor pathway. Materials and Methods Mice and reagents C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and NCI-Frederick (Frederick, MD). TRIF-deficient mice around the C57BL/6 background were purchased from the Jackson Laboratory (Strain name: C57BL/6J-enterotoxin A (SEA) was purchased from Toxin Technology Inc. (Sarasota, FL). Poly SSR240612 I:C was purchased from Invivogen (San Diego, CA) and Alexis Biochemicals (Axxora LLC, San Diego, CA). CpG was purchased from The Midland Certified Reagent Co. (Midland, TX). LPS, derived from culture. For experiments involving liver and lung lymphocytes, animals were Rabbit Polyclonal to Cofilin first perfused with PBS made up of heparin (Sigma-Aldrich) at 75U/ml. Livers were crushed through cell strainers and the cell suspension was partitioned on 35% Percoll (Sigma-Aldrich) to obtain lymphocytes. Remaining red blood cells in the samples were lysed with Gey’s solution. Lungs were cut into smaller pieces, incubated in BSS made up of 1.3mM EDTA (pH 7) at 37C for 30 min, followed by digestion with collagenase: was performed for all those data shown. Error bars indicate standard error of mean. Results Poly I:C enhances T cell expansion in vivo in a TLR3- and TRIF-independent manner enterotoxin A is usually a well characterized pathogenic protein and that we utilized to study endogenous T cell expansion in TLR3?/? mice. SEA activates endogenous T cells that express V3 T cell receptor (TCR) but not those that express V6. In wild type mice, poly I:C increased the frequency of V3+ T cells within the CD8 population by approximately 3-fold compared to SEA immunization alone (Fig. 1A). The dose of poly I:C was based on titration studies (data not shown). We hypothesized that since poly I:C was administered in a soluble form but not in complex with any transfecting reagent, it would be detected by endocytosis. Consequently, its adjuvant effects should be mediated through the TLR3 pathway in the endosomal compartment. We predicted that poly I:C would fail to enhance CD8 T cell expansion in TLR3?/? and TRIF-deficient mice; however, the expansion of CD8+V3+ T cells was not impaired in response to poly I:C (Fig. 1A). Likewise, total numbers of CD8+V3+ cells in the spleen of knockout mice were increased by poly I:C immunization. (data not shown). The frequency of V6+ control T cells within the CD8 population was not increased by poly I:C treatment (Fig. 1A, left), showing that in this model the effects of poly I:C can only be detected with SEA-activated T cells. In comparison to CD8 T cells, poly I:C had a less dramatic but nonetheless significant effect on CD4 T cell expansion. After poly I:C treatment, the frequency of V3+ within the CD4 population was increased by 40% in wild type mice, no significant increase in.
