The Cis-Cis Interaction Model

There is an extensive literature describing the association of CD45 with other proteins in cis-cis interactions at the cell surface (the earlier literature is reviewed in Alexander 1997). Reconstitution of a CD45-deficient murine cell line with CD45 isoforms suggested that the CD45R0 isoform promoted greater IL-2 secretion upon engagement of the TCR with the cognate MHC-peptide as compared to other isoforms (Novak et al. 1994). Co-capping experiments in these cells revealed preferential CD4-CD45R0 association (Leitenberg et al. 1996). However, more detailed capping and co-immu-noprecipitation studies indicated a basal association of CD45R0 with the TCR independent of CD4 expression and suggested that co-capping of CD4 with CD45R0 was mediated by this prior CD45R0-TCR association (Leitenberg et al. 1999). Nevertheless, CD4-CD45 association has also been described in primary CD4+ T cells (Mittler et al. 1991; Bonnard et al. 1997). In a further study in which fluorescence resonance energy transfer (FRET) analysis was used to investigate cis-cis interactions, different CD45 isoforms were transfected into a CD45-deficient T cell line expressing the TCR, CD4

and CD8. The results suggested that CD45R0 preferentially associated with CD4 and CD8 relative to the CD45RBC and CD45RABC isoforms (Dornan et al. 2002). The CD45R0-CD4 association correlated with up-regulated protein tyrosine phosphorylation events under both basal and TCR-stimulated conditions. These studies suggest a model in which the juxtaposition of CD45 PTPase activity in relation to its CD4- or CD8-associated p56lck kinase substrate promotes the action of the kinase, thereby up-regulating TCR-stimu-lated intracellular phosphorylation events.

Attractive as such a model may be, one problem with such cell line studies is that the glycosylation status of CD45 isoforms and their associating glycoproteins may not be the same as in primary cells, resulting in associations that do not accurately reflect those found under more physiological conditions. To address this question, CD45_/_ mice have been reconstituted with specific CD45 isoforms. In one study it was shown that either CD45R0 or CD45RABC transgenes, driven by a proximal Ick promoter, restored T cell numbers to near normal in lymph nodes, while no restoration of T cell populations in the spleen was found (Kozieradzki et al. 1997). In contrast, the Vav promoter has more recently been utilised to generate several lines of transgenic mice expressing either CD45R0 or CD45RB on the CD45_/_ background in all haematopoietic lineages (Ogilvy et al. 2003). When expressed at levels comparable with the total CD45 expression found in hemizygous CD45+/_ mice, thymic development was restored to apparently normal levels and reconstitution of mature T cells in both the lymph nodes and spleen was observed. In fact expression of CD45R0 at a level only 6%-8% of the total wild-type CD45 expression level was sufficient to restore thymic development to levels equivalent to wild-type. Mice expressing either the CD45R0 or CD45RB isoforms at comparable levels were equally efficient in their ability to mount T-dependent antigenic responses. Interestingly, however, neither isoform at this level of expression was able to restore effective B cell maturation, nor BCR-stimulated proliferation, showing that CD45 isoforms play unique roles which differ between the T and B lineages.

Overall, therefore, there is considerable support, based largely on cell line studies, for the cis-cis interaction model, but the model remains to be confirmed as being relevant to the function of CD45 isoforms in the whole animal context.

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