Figure 2

Tyk-2 inhibited T cell proliferation in asthma.

(a). and (b). CFSE labelled spleen CD4⁺ T cells from naïve and asthmatic Tyk-2^(−/−) mice were analyzed for their proliferation (n = 4–5; M = Mitosis: M1: p = 0,0000, M2: p = 0,0000, M3: p = 0,0001, M4: p = 0,0005, M5: p = 0,0459). (c–e). CD4⁺T cells were isolated from the lungs of naïve and OVA-treated wild type and Tyk-2^(−/−) mice and cultured for 24 h with α-CD3 and α-CD28 antibodies and supernatants were analyzed by ELISA for IL-6 ((c): n = 2–3; p = 0.0014, p = 0,0053, p = 0,0052), IL-21 ((d): n = 2–3; p = 0.0015), TGF-β ((e): n = 3)). (f). TGF-β levels in the supernatants of total lung cells (n = 3). (g). CD4⁺ T cells were isolated from the lungs of naïve and OVA-treated wild type and Tyk-2^(−/−) mice and cultured for 24 h with α-CD3 and α-CD28 antibodies and supernatants were analyzed by ELISA for IL-4 (n = 2–4; p = 0,019 p = 0,005 p = 0,001 p = 0,0046). (h). Total lung cells from wild type and Tyk-2^−/− mice were incubated with antibodies against CD4 and pSTAT3 (n = 4–5; p = 0.002) and analyzed by flow cytometry. (i). Total lung RNA was analyzed after cDNA synthesis by qPCR for the expression of Stat5 mRNA (n = 3). (j). Western blot analysis was performed on total lung protein extracts from OVA-treated mice. The membrane was probed with antibodies against SOCS3 and β-Actin as normalization control (n = 5; p = 0.00003).