The goal of the proposed research project is to gain a better understanding of how the expression of HLA class II proteins is regulated at the transcriptional level. Cell lines from patients with a form of severe combined immunodeficiency disease and mutant human B lymphoblastoid cell lines exist which fail to express HLA class II proteins because they lack different functional transactivating factors. These class II negative cell lines fall into four complementation groups, corresponding to four defective gene products required for the expression of HLA class II proteins. The identity of these gene products remains unknown. The specific objective of the proposed project is to identify the transactivating factor defects responsible for the failure of class II gene transcription in the immunodeficient and mutant lines. Success in identifying the genetic defects in the class II negative cells would further our understanding of the transcriptional regulation of HLA class II proteins as well as suggest a site to target gene therapy for correcting the underlying defect in one of the forms of severe combined immunodeficiency disease. In addition, knowledge of how HLA class II genes are turned off would be applicable to transplantation and the treatment of autoimmune diseases in which minimizing the expression of class II proteins at the gene level might allow for the use of less immunosuppressive agents and hence a reduction in the complications associated with available immunosuppressants. The experimental approach to be taken will be to complement class II negative cell lines by electroporetic transfection of the cells with a cDNA library derived from a parent B lymphoblastoid cell line that expresses class II proteins normally. The library will utilize a vector with a CMV promoter for high expression of cDNA, EBV ori P sequence for stable episomal replication of transfected cDNA, hygromycin resistance as a selectable marker for transfected cells, and a class II promoter dependent-methotrexate resistance for selection of complemented class II negative cells. The described vector has been constructed and preliminary data indicates that it can be used to distinguish class II expressing cells from class II negative cells. Complementation of class II negative cells will be confirmed by looking for surface expression of class II using a fluorescently labelled monoclonal antibody specific for one of the class II proteins and by hybridization of mRNA on a northern blot to a radiolabelled class II cDNA. Episomal vector will be isolated from methotrexate resistant, presumably complemented, class II expressing cells in a Hirt supernatant and amplified by transformation of E. coli. Recovered vector will be transfected again into class II negative cells to confirm complementation and maximize selection of complementing cDNA. The cDNA of the vector recovered from the second round of transfection will be sequenced and compared to known gene sequences. Confirmation of the identified cDNA being the genetic defect in a particular class II negative line will come from sequencing the corresponding cellular gene using techniques of PCR. This approach should allow for identification of the transactivating factors missing in the class II negative cells regardless of whether or not they are DNA binding proteins.
| Muczynski, K A; Anderson, S K; Pious, D (1998) Discoordinate surface expression of IFN-gamma-induced HLA class II proteins in nonprofessional antigen-presenting cells with absence of DM and class II colocalization. J Immunol 160:3207-16 |
