Somatic cell hybridization of human T- and B- lymphoblastoid cell lines (LCL) induces the expression of silent HLA genes encoded by the T-LCL partner. By using as fusion partners regulatory mutant B-LCL lacking class I or class II antigens on the cell surface, a minimum of two trans-acting genes regulating class I antigen expression and two trans-acting genes regulating class II antigen expression have been identified. Three of these genes act by regulating levels of specific mRNA. The fourth facilitates the assembly of HLA class I glycoprotein-beta2 microglobulin dimers. The objectives of this proposal are to characterize these regulatory genes, and other HLA regulatory genes which may be identified in additional mutant B-LCL. Genes will be isolated by molecular cloning techniques. They will be identified by subtractive hybridization approaches, or by secondary mutagenesis, using retroviral insertion, of hybrids of T-LCL with mutant B-LCL. Restoration of class I or class II antigen expression in the appropriate negative variant cell line upon transfection of a cloned gene will provide conclusive evidence of its regulatory function. Identification and structural characterization of genes which mediate the pre- and post- translational control of HLA antigen expression may ultimately contribute to our understanding of the regulation of in vivo immune responses.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI015775-12
Application #
3126424
Study Section
Immunobiology Study Section (IMB)
Project Start
1979-05-01
Project End
1991-07-31
Budget Start
1990-07-01
Budget End
1991-07-31
Support Year
12
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Duke University
Department
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Anderson, K S; Cresswell, P (1994) A role for calnexin (IP90) in the assembly of class II MHC molecules. EMBO J 13:675-82
Anderson, K S; Alexander, J; Wei, M et al. (1993) Intracellular transport of class I MHC molecules in antigen processing mutant cell lines. J Immunol 151:3407-19
Crumpacker, D B; Alexander, J; Cresswell, P et al. (1992) Role of endogenous peptides in murine allogenic cytotoxic T cell responses assessed using transfectants of the antigen-processing mutant 174xCEM.T2. J Immunol 148:3004-11
Click, E M; Anderson, K S; Androlewicz, M J et al. (1992) Transport and expression of class I MHC glycoproteins in an antigen-processing mutant cell line. Cold Spring Harb Symp Quant Biol 57:571-7
Storkus, W J; Salter, R D; Cresswell, P et al. (1992) Peptide-induced modulation of target cell sensitivity to natural killing. J Immunol 149:1185-90
Storkus, W J; Salter, R D; Alexander, J et al. (1991) Class I-induced resistance to natural killing: identification of nonpermissive residues in HLA-A2. Proc Natl Acad Sci U S A 88:5989-92
Koppelman, B; Cresswell, P (1990) Rapid nonlysosomal degradation of assembled HLA class II glycoproteins incorporating a mutant DR alpha-chain. J Immunol 145:2730-6
Alexander, J; Payne, J A; Shigekawa, B et al. (1990) The transport of class I major histocompatibility complex antigens is determined by sequences in the alpha 1 and alpha 2 protein domains. Immunogenetics 31:169-78
Liegler, T; Alexander, J; Cresswell, P et al. (1990) An analysis of insulin receptor expression and binding on MHC class I positive and negative human lymphoblastoid cells. J Immunol 145:1788-93
Storkus, W J; Alexander, J; Payne, J A et al. (1989) The alpha 1/alpha 2 domains of class I HLA molecules confer resistance to natural killing. J Immunol 143:3853-7

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