Association of class II with invariant chain (ii) can have several profound effects on class II biosynthesis and function. First, in the endoplasmic reticulum (ER), Ii protects the class II peptide binding site from associating with peptides and facilitates class II assembly, folding, and egress from the ERA Second, Ii enhances the localization of class II into proteolytic, endosomal compartments. Third, the alternatively spliced p41 form of Ii is required for most Ii-dependent antigen presentation events. Fourth, the chondroitin sulfate form of Ii (Ii-CS) can function at the cell surface as a T cell interaction molecule. Finally, T cell development and/or maturation is defective in mice lacking expression of Ii. The overall goals of the studies outlined in this proposal are to understand the intracellular transport pathways that are followed by class II associated with various forms of Ii, how any differences in transport reflect on class II function, and how these differences modulate the specific functions associated with subtypes of class II-positive cells in vivo. We will pursue these goals through three Specific Aims:
Specific Aim 1. Define the role of invariant chain cytosolic tail and transmembrane region in intracellular targeting. The invariant chain cytosolic tail has several signals that regulate endosomal sorting and retention, but the mechanisms that mediate these events are not understood. Studies are proposed to analyze the nature of the Ii degradation signal that we have recently identified within aa 18-29 of the Ii cytosolic tail and to identify cytosolic proteins that interact with the Ii cytosolic tail and mediate endosomal localization.
Specific Aim 2. Determine the intracellular events that regulate the biosynthesis of the chondroitin sulfate form of Ii. Only a small subset of Ii is modified by the addition of chondroitin sulfate (CS) and this form can function at the cell surface by facilitating interaction of antigen presenting cells (APC) with T cells. We will examine the factors that regulate the amount of Ii that gets modified with CS and how these complexes bypass endosomal targeting and degradation and arrive in association with class II at the cell surface.
Specific Aim 3. Analysis of Ii function in T cell development and in various populations of antigen presenting cells by reconstitution of Ii- negative mice. Mice that lack expression of Ii (Ii-o) have profound defects in class II expression and function diminished positive and negative selection of CD4+ T cells, and an altered phenotype of CD4+ T cells in the periphery. We have established a system to dissect the specific functions of Ii associated with these defects by reconstituting Ii-o mice with transgenes that express various forms of Ii. Our preliminary data indicate that even extremely low level of Ii, that do not appear to have any structural effects on class II, can restore CD4 T cell selection events. Experiments are proposed to determine the basis for this effect of Ii and to examine the function of the alternative spliced Ii gene products in various antigen presenting cell populations.
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