Efficient repression of endogenous major histocompatibility complex class II expression through dominant negative CIITA mutants isolated by a functional selection strategy

Major histocompatibility complex class II (MHC-II) molecules present peptide antigens to CD4-positive T cells and are of critical importance for the immune response. The MHC-II transactivator CIITA is essential for all aspects of MHC-II gene expression examined so far and thus constitutes a master regulator of MHC-II expression. In this study, we generated and analyzed mutant CIITA molecules which are able to suppress endogenous MHC-II expression in a dominant negative manner for both constitutive and inducible MHC-II expression. Dominant negative CIITA mutants were generated via specific restriction sites and by functional selection from a library of random N-terminal CIITA deletions. This functional selection strategy was very effective, leading to strong dominant negative CIITA mutants in which the N-terminal acidic and proline/serine/threonine-rich regions were completely deleted. Dominant negative activity is dependent on an intact C terminus. Efficient repression of endogenous MHC-II mRNA levels was quantified by RNase protection analysis. The quantitative effects of various dominant negative CIITA mutants on mRNA expression levels of the different MHC II isotypes are very similar. The optimized dominant negative CIITA mutants isolated by functional selection should be useful for in vivo repression of MHC-II expression.