2011). The data presented herein provide additional evidence for the ability of MTX to suppress serum cytokine levels in RA patients. = 7), NSC 131463 (DAMPA) Moderate (by DAS28-CRP = 13, by DAS28-ESR = 15), and Severe (by DAS28-CRP = 8, by DAS28-ESR = 10). PRT062607 concentration (= 18) or did not receive (No MTX; = 14) stable MTX therapy. The IC50 and 95% confidence interval for each group are shown. Data are represented as mean SEM. (D) RA patients with severe activity as defined by DAS28-ESR scores were separated into two groups based on treatment with MTX. Raw data are shown (= 5 per group) with a curvefit. MTX uniquely restores PRT062607 inhibitory potency in suppression of BCR mediated B-cell activation We next evaluated the effect of stable MTX therapy on the potency of PRT062607 in suppressing BCR-mediated B-cell activation in RA patients. Irrespective NSC 131463 (DAMPA) of the severity of disease activity, the population was separated into two groups; those on stable MTX therapy (= 18) and those not receiving MTX (= 14). Percent inhibition of B-cell activation across a range of PRT062607 concentrations was plotted (Fig. ?(Fig.2C).2C). By comparing the two concentration-effect relationships, we observed that the activity of PRT062607 in MTX-treated patients (IC50 = 224 nmol/L) was similar to that of healthy controls, while for those patients not on MTX the IC50 (385 nmol/L) was higher. The confidence intervals between these two groups were nonoverlapping, and the effect was statistically significant by the Wilcoxon test. Furthermore, it was apparent that complete inhibition (defined as >80%) was more readily achieved by PRT062607 in the MTX-treated patients. Although limited by sample size, the same general observation was made in patients with severe inflammation, separated into two groups (= 5 per group), those receiving MTX and those not. Raw data from this analysis are presented in Figure ?Figure2D.2D. Importantly, when F3 the patient population was grouped-based on prednisone or TNF inhibitor therapy, no impact on the potency of PRT062607 was observed (data not shown), indicating that MTX was unique in its ability to cooperate with PRT062607 to suppress B-cell function. No changes were observed in the percent NSC 131463 (DAMPA) of circulating B cells in the lymphocyte population among the various RA subgroups analyzed in the study (data not shown). Also, BCR/Syk signaling (Fig. S1A) was not affected by disease severity (Fig. S1B) or by MTX (Fig. S1C), suggesting that MTX affected the potency of PRT062607 inhibition of BCR-mediated functional responses by a Syk-independent mechanism. MTX treatment is associated with decreased serum cytokine concentrations MTX controls immune function in part by reducing cytokine burden (Cutolo et al. 2001; Wessels et al. 2008). We therefore utilized fresh frozen serum samples obtained from each of the RA patients to quantify concentrations of various cytokines and other serum markers of disease relevant to RA. As an initial analysis of this data, we sought to confirm the clinical observations and scoring of disease activity by assessing the relationship between disease activity and concentration of the serum proteins. Protein data were separated into three groups, representing remission/mild, moderate, and severe disease based on DAS28 ESR scores, and plotted against concentration on the < 0.05. These were IL2 (= 0.034) and IL17a NSC 131463 (DAMPA) (= 0.027; Fig. ?Fig.4).4). This effect was unique to MTX, as neither prednisone nor TNF inhibitors led to significant reductions in any of the serum proteins measured (data NSC 131463 (DAMPA) not shown). While MTX likely exerts immune modulation by multiple mechanisms, the reduction in IL2 was intriguing because this cytokine lowers the threshold for activation, differentiation, and clonal expansion of both B and T cells. In contrast, IL17 has no known role for directly modulating B-cell function, consistent with the observation that IL17a receptor expression is restricted to T and natural killer cells. Given the reduction in proinflammatory cytokine burden in MTX-treated patients, we predicted that B cells may be less responsive to BCR-mediated cellular activation in RA patients on stable MTX therapy. We tested this by comparing the extent of CD69 upregulation following BCR ligation in whole blood from RA patients untreated or treated.