The goals of this research project for the next five years are a continuing analysis of the structural characteristics of class II MHC molecules which govern their interactions with peptides and their role in determining immune responsiveness to specific peptide epitopes from foreign and self molecules.
The specific aims are: 1) Completion of site specific mutations in the I-Ab s chain to introduce the first and/or third hypervariable region sequences from I-Abu into I-Ab s. These mutant constructs will be introduced into both cell lines and into mice, and the functional effects of these mutations on antigen presentation and immune responsiveness will be determined in in vitro and in vivo studies. A subsidiary part of this project will lead to the production of I-Au transgenic mice. These mice will be crossed with NZB mice to test the hypothesis that the MHC linked genetic factor contributed by the NZW strain to the NZB x NZW lupus nephritis model is the I-Au molecule. 2) Expression of phosphoinositol (P.I.) linked I-Aa u /I-Abu heterodimers in CHO cells. This willpermit isolation of soluble I-Au molecules through the action of phosolipae C. These molecules which appear to be in large part empty will be used to study the interaction of MBP Ac1-11 and its analogs with I-Au to determine the ability of isolated I-Aau and I-Ab u peptides to bind MBP Ac1-11, and to attempt to produce large amounts of the I-Au in which most or all of the molecules are binding the same peptide, namely MBP Ac1-11. Attempts will then be made in collaboration with the department of structural biology at Stanford to determine whether these molecules are capable of forming crystals. 3) Production of a series of peptides based on the MBP Ac1-11 sequence in which all the residues except positions 5, 6 and 10 or 2, 5, 6 and 10 are replaced by L-alanine. The ability of thisprototype peptide to bind to I-Au will then be tested. If, as predicted, this peptide binds, a series of related peptides will be synthesized in which the L-alanines are replaced by D-alanine in a sequence proceeding from the N terminus, a second sequence preceding from the C terminus and a third sequence in which the D-alanine will replace L-alanine at every third alanine residue. These peptides should permit determination of whether the MBP 1-11 peptide binds as a alpaheliy or as an extended peptide, and should also permit a determination of the extent to which the I-Au binding site distinguishes chirality of a peptide prior to binding. 4) Transgenic mice will be produced which express a construct consisting of the metallothionein promoter with the invariant chain genomic coding sequences. This construct should permit high level expression of the invariant chain. These mice will be crossed with the I-Abk and I-Aa k /I-Ab k transgenic mice to rescue their defect in B cell development. If this experiment is successful, it will indicate that one of the main functions of the invariant chain is to facilitate assembly and transport of the I-A molecules to the surface. 5) To further test the function of the invariant chain in assembly, transport and function of the I-A and I-E molecules, we will produce transgenic mice homozygous for inactivation of the invariant chain gene by homologous recombination in an embryonal stem cell line, followed by introduction of these cells into blastoysts. These mice should fail to express the invariant chain and are expected to have defects in assembly, transport and cell surface expression of class II MHC molecules. 6) To facilitate functional testing of HLA-DQ and DR molecules expressed in transgenic mice, transgenic mice will be produced will be produced using the human CD4 gene. These transgenic mice will then be crossed with transgenic lines expressing HLA-DR4 or HLA DQw2, 6, 7or 8. In the presence of human CD4, these HLA DR and DQ molecules should function in antigen presentation in positive and negative selection in the thymus, thus permitting careful analysis of peptide epitopes presented to T cells by these molecules.
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