Using these doses, a dose-dependent
suppression of the response was observed with 125 mg/kg reducing the response to background levels (Fig. 1a,b). In the DNFB-induced model, CTLA-4-Ig inhibited the ear swelling in a dose-dependent manner and 25 mg/kg virtually inhibited the response completely (Fig. 1c,d). Taken together, these results show that CTLA-4-Ig mediates a dose-dependent immune suppression in both models and that the DNFB-induced model was responsive to lower doses of CTLA-4-Ig than the oxazolone-induced model. Three weeks after the first sensitization and challenge, mice were resensitized Epigenetic Reader Domain inhibitor and rechallenged with DNFB or oxazolone, respectively, without any further treatment with CTLA-4-Ig. As shown in Fig. 2a, mice in the DNFB-induced model dosed previously with 25 mg/kg still exhibited a significantly reduced ear-swelling response compared to the hIgG1 control group. In the oxazolone-induced model,
the highest dose also exerted a suppressive effect 3 weeks after administration (Fig. 2b). Exposure analysis of circulating levels of CTLA-4-Ig 3 and 21 days after administration (Fig. 2c,d) were performed subsequently. Figure 2c shows serum levels 3 days after administration and clearly revealed detectable levels of CTLA-4-Ig. However, after 21 days the levels of CTLA-4-Ig in the serum samples were below the detection level of the assay (<0·43 μg/ml), suggesting that no or very low levels of CTLA-4-Ig were present in the serum (Fig. 2d). Based on this, we conclude that MK-8669 nmr treatment with CTLA-4-Ig results in a sustained suppression of the ear-swelling response in both models independent of the presence of detectable, Janus kinase (JAK) circulating levels of CTLA-4-Ig in the serum. To investigate the mechanism by which CTLA-4-Ig exerts its suppressive function in greater detail, cells isolated from the inguinal lymph node
draining the area of sensitized skin were stained for activation markers and analysed by flow cytometry 24 h post-sensitization (Fig. 3). CTLA-4-Ig treatment led to a reduced number of CD8+ and CD4+ T cells in the draining lymph node (Fig. 3a,b, right). This reduction was due to an overall lower number of cells in the lymph nodes, as the percentages of CD4+ and CD8+ T cells of CD45+ live cells were similar between the CTLA-4-Ig-treated and the isotype-treated group (Fig. 3a,b, left). Because inflammation in this model is dependent on CD8+ T cells , we investigated this cell population in greater detail. Figure 3c,d shows that CD8+ T cells in the draining lymph node have a less activated phenotype after CTLA-4-Ig treatment, as the number and percentage of CD44+CD62L–CD8+ T cells and CD69+CD8+ T cells were reduced significantly in the CTLA-4-Ig-treated mice compared to the control group.