IFN-γ induces fas expression in normal human CD34⁺ cells. (A) RT-PCR analysis of RNA obtained from cells exposed for 6 hours to various doses of IFN-γ are shown. Fas is expressed constitutively and mRNA increases slightly with all doses of IFN-γ tested except the highest which suppressed fas RNA. (B) Fas immunoblot: CD34⁺ cells were incubated in the absence (lane 1) or presence (lane 2) of IFN-γ for 6 hours. Each lane was loaded with 50 μg of protein from whole cell lysates. (C) Flow cytometry: Flow cytometric analysis of surface fas expression on CD34⁺ cells purified from normal BM. The horizontal axis illustrates linear fluorescence intensity, and the vertical axis indicates absolute cell number. The dotted line represents CD34⁺ cells cultured without IFN-γ exposed to the isotypic control antibody, the dot-dashed line shows fas expression in CD34⁺ cells cultured in the absence of IFN-γ. The shaded histogram indicates fas expression by CD34⁺ cells exposed to 100 ng/mL IFN-γ for 18 hours. Treatment of cells with 1,000 ng/mL IFN-γ yielded an identical histogram. (D) IFN-γ primes the fas pathway in human CD34⁺ cells. Results of one representative experiment (of three separate experiments) is shown. Human CD34⁺ cells exposed to various doses of IFN-γ for 60 minutes followed by exposure to antihuman fas monoclonal antibodies (“anti-fas,” ▪), or isotype control antibodies (“control,” □), for 3 hours were suspended in methylcellulose medium containing the same dose of IFN-γ. CFU-GM (not shown) and BFU-E (mean ± SD of triplicate plates) were counted on day 14 of culture. In the absence of IFN (control) BFU-E were 154 ± 14/5,000 CD34⁺ cells. Although IFN-γ suppresses clonal growth of normal BFU-E, the addition of anti-fas augments clonal inhibition by IFN-γ, particularly at IFN levels above 0.5 ng/mL.