Data shown are representative examples of three independent animals per group

Data shown are representative examples of three independent animals per group. In order to explore whether CD25 blockade would have comparable effects around the activation and expansion of high-frequency donor-reactive T cell responses in vivo, we performed an experiment in which CFSE-labeled cells from a B6 animal were injected into an irradiated VU 0357121 fully allogeneic BALB/c responder. that this IL-2 pathway plays an important role in the costimulation blockade-resistant response in murine models of transplantation [16], and previous work from Wells and colleagues suggested that CD28 blockade altered expression of CD25 following antigenic activation [17]. An additional modifying factor of both programmed T cell growth and the relative efficacy of costimulation blockade-based treatment in transplantation is the initial precursor frequency of the responding donor-reactive T cell populace [18; 19; 20]. We have previously shown that na?ve CD4+ and CD8+ T cell precursor frequency plays a critical role in determining the quantity and VU 0357121 quality of the donor-reactive T cell response following transplantation, and thus in mediating costimulation blockade-resistant rejection [18; 19; 20]. Specifically, we reported that high frequency populations of na?ve graft-specific CD8+ T cells expanded and differentiated into competent effectors even in the presence of costimulation blockade, thus precipitating graft rejection [18]. In contrast, low-frequency populations of na?ve graft-specific CD8+ T cells failed to significantly expand in the presence of costimulation blockade and did not differentiate into high quality effectors that were capable of rejecting a skin graft. These studies exhibited that high-frequency na?ve T cell populations may obviate the requirement for costimulation during priming and play a significant role in mediating costimulation blockade-resistant allograft rejection. In this study we addressed the ability of blockade of the CD28 pathway to impact expression of the IL-2 receptor alpha chain (CD25) during T cell activation under conditions where the initial anti-donor frequency is usually either high or low. Measuring the magnitude and kinetics of this effect, we found that blockade of the CD28 pathway resulted in division-dependent downregulation of CD25. Due to decreased numbers of cell divisions in cells stimulated at an initial high frequency, CD25 expression levels were managed on a subset of cells within this populace, suggesting that these cells may be responsible for mediating costimulation blockade-resistant rejection system in which na?ve monoclonal CD8+ TCR transgenic T cells (OT-I) were stimulated with cognate peptide antigen in the presence or absence of blockade of the CD28 pathway through CTLA-4 Ig, blockade of the CD40/CD154 pathway using anti-CD154 (MR-1), or a combination of the two. activation with cognate OVA peptide resulted in the generation VU 0357121 of activated CD8+ Thy1.1+ antigen-specific effector T cells, which expressed CD69, granzyme B, and CD107 around the cell surface following incubation with OVA peptide-loaded splenocytes cells (data not shown). These data show that this antigen-specific T cells were activated following in vitro activation with OVA peptide. As shown in Physique 1, effector T cells receiving antigen activation also exhibited dramatic upregulation of CD25 by 24 hours post-stimulation, whereas those T cells not receiving antigenic activation did not upregulate CD25. However, results from the different treatment conditions revealed that in the presence of CD28 blockade, activated T cells first upregulated (at 24 VU 0357121 hours) and then rapidly downregulated their CD25 expression by 48 hours post activation. Antigen-specific CD8+ T cells stimulated in the presence of CTLA-4 Ig continued to further down-regulate this molecule with increasing time, such that by 96 hours post-stimulation it experienced returned to baseline levels much like unstimulated controls. In contrast, antigen-specific CD8+ T cells in untreated samples maintained a high level of CD25 expression even to 96 hours post-stimulation. CD40/CD154 blockade-treated T cells exhibited a delayed reduction in CD25 expression, such that expression was much like untreated controls at 48 and 72 hours but was significantly decreased at 96 hours post-transplant. Cultures treated with a combination of the two treatments did not exhibit a synergistic or additive reduction in CD25 expression levels, but LEG8 antibody instead mimicked the magnitude and kinetics of downregulation observed in samples treated with CTLA-4 Ig alone. Open in a separate window Physique 1 Blockade of CD28 signaling through CTLA-4 Ig results in downregulation of CD25 on.