327, p<0.05, MS: r2=0.446, p<0.05). Of interest, the Treg-mediated inhibition of Tconv proliferation also positively correlated with IL-7Rα-MFIs on Tconv (HC: r2=0.175, p<0.05 MS: r2=0.587, p<0.01; Fig. 3), suggesting that IL-7Rα expression by affecting

frequencies of circulating RTE-Treg also interferes with Treg function. Proliferative responses of stimulated and unstimulated Tconv were comparable in samples obtained from MS patients and healthy donors. We measured sIL-7Rα in plasma specimens obtained from MS patients (n=20, 12 with active disease, 8 in remission) and age- and sex-matched control inidividuals (n=17) using an in-house ELISA protocol and IL-7 levels with a conventional ELISA Kit as described in the Materials and methods section. We found an inverse correlation between IL-7 plasma levels and IL-7Rα-MFIs on total Tconv in patients with MS (IL-7: HC: r2=0.142, p=0.103; MS: r2=0.252, p=0.027;

Fig. 4B). Concentrations of both IL-7 and sIL-7Rα were AG-014699 manufacturer elevated in 20 patient-derived samples as compared to 17 HC-derived PF-02341066 solubility dmso samples, which was statistically significant for IL-7 only (IL-7 [pg/mL]: HC 5.1±1.5, MS 11.2±5.9, p=0.050; sIL-7Rα [ng/mL]: HC 107.5±40.6, MS 145.0±53.7, p=0.161; Fig. 4A). Enhanced IL-7 and sIL-7Rα plasma levels were detectable in both patients with active and inactive disease. TSLP and TSLPR-expressing MDCs were previously shown to be critically involved in thymic Treg development 13. Therefore, we analyzed surface expression levels of TSLPR on circulating MDCs in blood samples obtained from MS patients (n=12, 8

with active disease, 4 in remission) and age- and sex-matched normal donors (n=11). TSLPR-MFIs were significantly lower on patient-derived MDCs (HC 96.0±15.9, MS 59.6±17.4; p<0.01; Fig. 5) and did not differ between RRMS patients with active or stable Isoconazole disease. In addition, expression levels of IL-7Rα and TSLPR strongly correlated in both study cohorts (MS: r2=0.57, p<0.05; HC: r2=0.61, p<0.05; not depicted). It was previously shown, that 10–30% of peripheral T cells and up to 99% of human Treg express two distinct TCR-Vα chains 21. Here, in both study cohorts (HC: n=33, MS: n=56) approximately a quarter of total Tconv harbored TCRs with dual specificity (HC: 31.9±14.0%, MS: 29.6±18.2%, p=0.47). In contrast, 85.6±17.1% of control-derived, but only 55.8±31.2% of patient-derived Treg expressed two TCR-Vα chains (p<0.01; Fig. 6A). Overall, there was a strong correlation of IL-7Rα-MFIs of Tconv and TSLPR-MFIs on MDCs with the amount of dual TCR specific Treg in both patients and control donors (IL-7Rα: HC: r2=0.247, p=0.011; MS: r2=0.355, p=0.008, Fig. 6B; TSLPR: HC: r2=0.214, p=0.031; MS: r2=0.333, p=0.016; not depicted). Screening for rs6897932-SNP 15–18 associated with MS, type 1 diabetes and chronic inflammatory arthropathies 19 was performed by SNAP-shot PCR.