Identification of HLA-B upstream elements required for induction by IFN- and by CIITA.

(A) YHHH cells were transfected with CAT reporter vectors and cultured with (+) or without (−) IFN-α as described in “Materials and methods.” Transfections were performed in parallel and the CAT activity of the HLA-B vector in the presence of IFN-α was defined as 100 U. For this and subsequent figures, error bars represent SD on the mean from at least 3 independent transfections. “C” is the promoterless CAT vector control and “B” is the HLA-B reporter, with 500 bp of sequence upstream of the start codon of HLA-B57.28 Truncated (Tr) versions with 200 bp of HLA-B upstream sequence include the conserved Rel site in ENH-A, which was left intact, or mutated (Tr-mE). (B) Cotransfections with the HLA-B reporters were performed with 1 μg of a control (−) or CIITA (+) vector, and induced activity of the wild-type reporter was defined as 100 U. (C) The IFN-α inducibility of the full length HLA-BCAT vector, “B,” was compared with versions that had been mutated in the IRE, X1, R1, α, or IC elements (Figure 1B). (D) The HLA-B mutant reporter series was tested with the CIITA vector as described in panel B. (E) HeLa cells were transfected with the HLA-B reporter series −/+ CIITA as described for YHHH cells.