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.