IL-7 and IL-7Rα.

(A) IL-7 and IL-7Rα RNA expression is normal in patient cells. PCR was performed with IL-7 and IL-7Rα specific primers on cDNA from patient 1 and 3 control EBV cell lines. Control GAPDH products are also shown. (B) IL-7R expression is not altered. Il-7Rα was immunoprecipitated from lysates of control and patient EBV cell lines and human fibroblasts (negative control) and Western blotted with anti–IL-7R antibody. Anti-Cbl immunoprecipitation and Western blot were performed as a positive control. (C) Absence of Jak-3 tyrosine phosphorylation after IL-7 stimulation in patient cells. Patient and control cells were incubated for 10 minutes ± 30 ng/mL rIL-7. Lysates were precipitated with anti–Jak-3 antibody and blotted with antiphosphotyrosine (top panel) and anti–Jak-3 after stripping (bottom panel). (D) COS-7 cells were transfected with expression vectors encoding γc, Jak-3, and either wild-type or mutant IL-7Rα as indicated (top). Cells were incubated ± IL-7 for 10 minutes, lysed, and immunoprecipitated with anti–Jak-3 antibody and subsequently blotted with either antiphosphotyrosine (top) or anti–Jak-3 (bottom) antibodies. (E) Specific binding of¹²⁵I-labeled IL-7 to EBV-transformed B cells derived form patient or healthy donor. IL-7 binding is presented as counts after subtraction of the background binding measured in the presence of a 100-fold excess of unlabeled IL-7.