Interaction of the B cell surface antigen CD4O with its ligand (CD4OL) expressed on activated T cells plays a critical role in T-B cell collaboration, particularly in immunoglobulin (Ig) isotype switching. Mutations in the CD4OL in patients with X-linked Hyper IgM syndrome and disruption of the CD4O or CD4OL genes in mice result in deficient Ig isotype switching. The unique capacity of CD4O to trigger isotype switching in B cells suggests that a novel signaling pathway must be utilized by CD4O. We have recently identified a 26 kD protein associated on the B cell surface with human CD4O. The sequence of three internal peptides derived from p26 indicate that p26 is a novel protein. We have evidence to suggest that p26 may be used to transduce signals following ligation of CD4O. We propose to characterize p26, analyze its interaction with CD4O and dissect the individual role of p26 and CD4O in immunoglobulin isotype switching using transgenic mice with disrupted CD4O and p26 genes.
In Aim 1 we propose to clone and characterize p26 by a) isolation and sequencing of human and mouse p26 cDN, b) generation of antibodies to p26 and detection of p26 and of p26 associated proteins and c) studying the expression of p26 during B cell development.
In Aim II we propose to analyze the interaction between CD4O and p26. We will a) examine the association of CD4O and p26 in cells cotransfected with CD4O and p26 cDNA and b) determine the role of CD4O in p26 surface expression by studying p26 expression on B cells from CD4O-/- mice.
In Aim III we will perform a functional analysis of signaling via p26 by a) examining signaling via anti-p26 antibodies and b) determining the role of CD4O in p26 signaling using B cells from CD4O-/- mice and B cells expressing a CD8:p26 chimeric molecule.
In Aim I V we will examine the role of p26 in CD4O signaling by constructing p26 deficient mice and assessing CD4O signaling in these mice. Given the central role of CD4O in Ig isotype switching and in B cell presentation of antigen to T cells, a detailed understanding of the mechanisms of CD4O signalling is critical for devising therapeutic strategies aimed at preventing isotype switching and/or inducing tolerance to antigens presented by B cells. These strategies are applicable to the treatment of allergic and autoimmune diseases and to the prevention of allograft rejection.
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