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CD45 Regulates Receptor Thresholds 2.1

CD45 and Lymphocyte Development

The first indication that CD45 has a positive role in regulating signals mediated by antigen receptors came from the investigation of T cell lines and T cell clones that lack CD45 expression. In these contexts it was found that

Cd45 Ectodomain Structure

Fig. 1 Domain structure of the CD45 molecule. The extensive CD45 ectodomain is characterised by three fibronectin type III (FN-III) repeats, a cysteine-rich domain and the three alternatively spliced exons A, B and C at the N-terminus. The ectodomain is heavily glycosylated, mainly N-linked in the FN-III and cysteine-rich regions and O-linked in the A, B and C exon-encoded regions. The CD45R0 isoform lacks the A, B and C exon-encod-ed regions. The protein tyrosine phosphatase (PTPase) signature motif VHCSAGVGRTG is located in the C-terminal portion of domain 1. Domain 2 contains a characteristic 24 amino acid acidic sub-domain and a partial PTPase signature motif, but is not thought to display PTPase activity

Fig. 1 Domain structure of the CD45 molecule. The extensive CD45 ectodomain is characterised by three fibronectin type III (FN-III) repeats, a cysteine-rich domain and the three alternatively spliced exons A, B and C at the N-terminus. The ectodomain is heavily glycosylated, mainly N-linked in the FN-III and cysteine-rich regions and O-linked in the A, B and C exon-encoded regions. The CD45R0 isoform lacks the A, B and C exon-encod-ed regions. The protein tyrosine phosphatase (PTPase) signature motif VHCSAGVGRTG is located in the C-terminal portion of domain 1. Domain 2 contains a characteristic 24 amino acid acidic sub-domain and a partial PTPase signature motif, but is not thought to display PTPase activity stimulation of the T cell antigen receptor (TCR) no longer induced intracellular activating signals, demonstrating that the signal transduction coupling threshold of the receptor was greatly increased in the absence of CD45 (Pingel and Thomas 1989; Koretzky et al. 1990). Later, these findings were extended and confirmed by the generation of three CD45-targeted mouse-lines in which CD45 exons 6, 9 or 12 were deleted to generate mice in which nearly all the CD45 was absent (exon 6) (Kishihara et al. 1993) or was completely absent (exons 9 and 12) (Byth et al. 1996; Mee et al. 1999). The low levels of CD45 that remain expressed on the surface of peripheral T cells in the exon 6-targeted mice (Kong et al. 1995b) may in fact have a marked effect in regulating TCR signalling thresholds: low levels of CD45 (as little as 6%-8% of wild-type levels) are sufficient to restore T cell development and functions (Ogilvy et al. 2003). In mice completely lacking CD45 expression there are striking defects in thymic development so that only about 5% of the normal numbers of mature T cells exit to the periphery (Byth et al.

1996). Three distinct defects occur during development: first, there is a partial defect in the transition from CD25+CD44" to CD25"CD44" cells lacking CD4 and CD8 expression, such that the CD25+CD44" subset accumulates in the absence of CD45; second, there is a marked failure of positive selection of CD4+CD8+ thymocytes, explaining the reduced numbers of mature CD4+ and CD8+ T cells that exit to the periphery (Byth et al. 1996); third, there is a partial defect in negative selection at the CD4+CD8+ stage of differentiation, the extent of the defect depending on the avidity of the selecting antigen (Conroy et al. 1996; Wallace et al. 1997; Mee et al. 1999). The reduced transition from CD25+CD44" to CD25~CD44" cells is readily explained by defects in the signals mediated by the pre-TCR, the immature form of the TCR that is required for this maturation step (Pingel et al. 1999). A further thymic defect that occurs in the absence of CD45 is a marked increase in the basal ap-optosis of the CD4+CD8+ subset: since this population comprises the bulk of the thymus, this survival failure presumably explains much of the 50% reduction in size of the CD45_/_ thymus (Byth et al. 1996). Stimulation of CD45_/_ thymocytes in vitro using a CD3 monoclonal antibody (mAb) has shown that intracellular signals are typically reduced by 50%-80% (Stone et al. 1997). This contrasts with the virtual ablation of TCR signal transduction observed in most CD45-deficient T cell lines. In fact, optimal TCR stimulation can induce calcium signals in CD45_/_ thymocytes to levels comparable with wild-type cells, although the twofold increase in TCR expression on CD45_/" CD4+CD8+ thymocytes (Stone et al. 1997) may compensate for defects in signalling to some extent. Taken together, these findings suggest that only a very potent CD45 inhibitor, with efficacy higher than 90%, would be likely to have any effect on murine thymic differentiation.

B cell development is less affected in the absence of CD45 than T cell development. Whereas the early stages of B cell differentiation appear to be normal, the maturation from IgMhi IgDhi (immunoglobulin-Mhiimmuno-globulin-Dlo; T2) cells into the IgMlo IgDhi phenotype typical of follicular B cells is impaired (Byth et al. 1996). The thresholds for B cell selection events are altered in CD45_/_ mice in a manner analogous to the changes observed in the T lineage. In CD45_/_ mice back-crossed to mice carrying immunoglobulin genes specific for hen egg lysozyme (HEL), the circulating HEL autoantigen which mediates negative selection in wild-type mice instead positively selects HEL-binding B cells, leading to their accumulation as long-lived IgDhi cells (Cyster et al. 1996).

The few patients who have been described to date lacking CD45 expression all display a severe combined immunodeficiency (SCID) (Kung et al. 2000; Tchilian et al. 2001) similar to that noted in the CD45_/_ mice. Overall, therefore, observations from both mouse and human are consistent with the idea that CD45 plays a dominantly positive role during lymphocyte development by increasing the threshold for antigen receptor signal transduction.

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