We have further demonstrated complete dependence of the anergic phenotype on PD-1, as depletion of PD-1 with siRNA duplexes at late times results in a total reversal of anergy
We have further demonstrated complete dependence of the anergic phenotype on PD-1, as depletion of PD-1 with siRNA duplexes at late times results in a total reversal of anergy. mediated by PD-1 is wholly IL-2 dependent, and likewise intrinsic to the tolerized cells. 1. Introduction The immunosuppressive brokers used in current transplantation protocols are known to increase the risk of contamination and neoplasia due to their nonspecific dampening of the immune response [1C3]. One alternative to generalized suppression of the immune system is usually a more tailored approach that seeks to induce a state of selective peripheral tolerance specifically to transplanted grafts [4C7]. Inducing an allo-specific tolerant state would permit the introduction of an organ graft into an otherwise fully competent immune environment, capable of immune surveillance and pathogen eradication. The mechanisms that generate peripheral transplantation tolerance are not SNX-5422 Mesylate yet fully elucidated, but it is known that grafts are rejected as the result of both acute and chronic immune activation [8], processes that involve numerous immune mechanisms [9C11]. It is well accepted that CD4+ T lymphocytes are central to the rejection of allografts and that they are also necessary for the successful induction of tolerance [4, 12]. A number of immune processes discovered in animal models that are posited to result in immunological tolerance include clonal deletion, suppression of reactive lymphocyte subsets by regulatory T cells, and T cell anergy [13, 14]. CD4+ T lymphocytes require two signals for optimal activation and production of IL-2, which drive entry into the cell cycle and subsequent clonal expansion [15, 16]. Signal 1 SNX-5422 Mesylate is usually delivered through the TCR upon encounter with antigen. When signal 1 is usually delivered in the absence of a costimulatory signal, known as signal 2, the levels of IL-2 produced are not sufficient to drive clonal expansion. Instead, the T cell acquires a phenotype characterized by antigen unresponsiveness defined as clonal anergy [17]. After a T cell is usually rendered anergic, it is unable to produce IL-2 or proliferate, even when provided a signal through the TCR in the presence of costimulation. Early studies exhibited that anergy is an active phenotype that requires protein synthesis and can be prevented by treatment with cycloheximide and cyclosporine A [18]. These findings suggest that anergy is established through a TCR-dependent signal transduction pathway. The search for factors that participate in this putative anergy pathway is usually ongoing. A number of genes that are upregulated early in the course of anergy induction in T cells have been identified. These include the transcription factor Egr-2 [19, 20] and the E3-ubiquitin ligases Cbl-b [21, 22] and GRAIL [23, 24]. Recent studies have shown that the products of these genes are each necessary SNX-5422 Mesylate for establishing the anergic phenotype. We have previously shown that Egr-2 is necessary for the induction of anergy, but does not appear to have a role in maintaining unresponsiveness once the anergic phenotype is established [19]. The aims of this study were to identify genes that are differentially expressed during the maintenance phase of anergy and to determine whether they contribute to the anergic phenotype. We show that PD-1, a known unfavorable costimulatory receptor [25], is usually upregulated in anergic cells for at least five days after anergy induction, and that depletion of PD-1 protein levels with RNAi at this time results in complete, IL-2-dependent reversal of the anergic phenotype. We further show that, at this late time point, the effect of PD-1 depletion is usually specific to anergic cells, as treatment of fully costimulated cells with siRNA directed against PD-1 does not increase antigen responsiveness. 2. Methods 2.1 Mice B10.BR (models. In the A.E7 model, PD-1 expression remains high for at least five to seven days, during the period at which the cells are hyporesponsive to restimulation with antigen. In contrast, fully stimulated A.E7 T cells that initially received both signal 1 and signal 2 downregulate PD-1 levels by this time point, and proliferate in response to antigen. We have further exhibited complete dependence of the anergic phenotype on PD-1, as depletion of PD-1 with siRNA duplexes at late times results in a total reversal of anergy. The same treatment had no effect on fully activated A.E7 T cells, suggesting that in the A.E7 model this optimally stimulated population is not susceptible to unfavorable regulation by this pathway. This is likely explained by the fact that, prior to restimulation, expression levels of PD-1 mRNA and protein are at or below even SNX-5422 Mesylate the background levels seen in control unstimulated cells (Physique 2) and that due to this low expression, they exhibit maximal proliferation that cannot be further enhanced. A hallmark of anergic T cells is usually a block in the signal transduction pathway responsible for IL-2 upregulation [17], and previous studies have exhibited a relationship between Rabbit Polyclonal to FOXD3 PD-1 engagement and decreased IL-2 production [30]. To determine the effect of PD-1 depletion on IL-2 signaling in the A.E7 model, we included a neutralizing IL-2 monoclonal antibody in the anergic cultures stimulated with PCC. We found that, neutralization.