In addition to their antigen presenting function, MHC class II molecules transduce signals that regulate the function of immune cells. We have shown that MHC class II molecules serve as receptors for bacterial superantigens such as TSST-1 and that engagement of MHC class II molecules by TSST-1 induces IL-1 and TNF-alpha synthesis in monocytes and delivers proliferation and differentiation signals to B cells. MHC class II signalling involved activation of src type tyrosine kinases (hck and fgr in monocytes and lyn in B cells), activation of phospholipase C-gamma, Ca+2 fluxes, activation of protein kinase C and of the nuclear transcription factors NF-kappaB and AP-1. An important consequence of MHC class II signalling is the induction of expression of B7, the counterreceptor of the costimulatory T cell molecules CD28/CTLA4. There is evidence to suggest that the cytoplasmic domains of murine MHC class II molecules is important for antigen presentation and B7 expression. We propose to carry out a detailed structure/function analysis of the signalling properties of human MHC class II molecules, and to define the mechanisms of class II mediated activation of B7 expression in the course of cognate T-B cell interactions.
In Aim 1, we will examine the role of the cytoplasmic domain of MHC class II molecules in signalling. We will transfect truncated DRalpha and DRbeta chains, chimeric CD8/DRalpha and CD8/DRbeta, chains, and mutated DRalpha and DRbeta molecules into class II- B cell lines and analyze antigen presentation, homotypic adhesion, protein tyrosine phosphorylation and B7 expression in these cells We will use, as required, a similar approach to examine the role of the transmembrane domains and of the extracellular domains DRalpha and DRbeta in signalling.
In Aim 2, we propose to identify MHC class II-associated proteins. We will overexpress the DRalpha and DRbeta cytoplasmic domains as fusion proteins and use them to identify associated proteins by immunoprecipitation, Western blotting and screening of expression libraries.
In Aim 3, we will investigate the mechanisms of transcriptional activation of B7 by MHC Class II ligands. We will also dissect the interplay between MHC class II and CD4O mediated signals in the induction of B7 expression in the course of cognate T-B cell interactions which involve interaction between TCR and class II/peptide complexes as well as interaction between the B cell antigen CD4O and its ligand on activated T cells. In these experiments we will make use of CD4O ligand deficient alloreactive T cells from patients with the HyperIgM syndrome and we will use B cells from class II knockout and CD4O knockout mice to define the relative contribution of class II and CD4O signals to B7 expression.
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