CD45 is a receptor-like protein tyrosine phosphatase highly expressed on all nucleated hematopoietic cells. We previously generated mice containing a point mutation in the juxtamembrane wedge of CD45. Demonstrating the critical negative regulatory function of the wedge, the CD45 E613R mutation led to a lymphoproliferative disorder (LPD) and a lupus-like autoimmune syndrome. Here we show the central role of B cells in this phenotype. Genetic elimination of B cells, but not T cells, ablates the LPD. In contrast to CD45-deficient B cells, the E613R mutation generates hyperresponsive B cells. Comparison of CD45-deficient and CD45 E613R mice reveals dichotomous effects of these mutations on B cell development. Together, the results support a role for CD45 as a rheostat, with both positive and negative regulatory functions, that fine-tunes the signal transduction threshold at multiple checkpoints in B cell development.

The cooperative activity of protein tyrosine kinases and phosphatases plays a central role in regulation of T cell receptor (TCR) signal strength. Perturbing this balance, and thus the threshold for TCR signals, has profound impacts on T cell development and function. We previously generated mice containing a point mutation in the juxtamembrane wedge of the receptor-like protein tyrosine phosphatase CD45. Demonstrating the critical negative regulatory function of the wedge, the CD45 E613R (WEDGE) mutation led to a lymphoproliferative disorder (LPD) and a lupus-like autoimmune syndrome. Using genetic, cellular, and biochemical approaches, we now demonstrate that the CD45 wedge influences T cell development and function. Consistent with increased TCR signal strength, WEDGE mice have augmented positive selection and enhanced sensitivity to the CD4-mediated disease experimental autoimmune encephalitis (EAE). These correspond with hyperresponsive calcium and pERK responses to TCR stimulation in thymocytes, but surprisingly, not in peripheral T cells, where these responses are actually depressed. Together, the data support a role for the CD45 wedge in regulation of T cell responses in vivo and suggest that its effects depend on cellular context. autoimmunity ͉ tyrosine phosphatase ͉ tyrosine kinase ͉ thymocyte development T cell receptor (TCR) signal strength is influenced by the integration of multiple inputs including affinity for antigen, presence and activity of costimulatory molecules, and duration of the interaction with antigen-presenting cells (APCs) (1). Alterations in TCR signal strength impact many aspects of T cell biology including CD4 versus CD8 lineage commitment (1), effector and memory cell generation (2), and autoimmunity (3, 4). Currently, the factors defining signal strength and its functional outcome are incompletely understood.CD45, a receptor-like protein tyrosine phosphatase (RPTP) expressed on all nucleated hematopoietic cells, plays a critical positive regulatory role in antigen receptor signaling. Its absence in both mice and humans results in severe combined immunodeficiency (5, 6). CD45 mediates its effects, at least in part, by modulating the activation state of Src family protein tyrosine kinases (SFKs) (5, 7). Phosphorylation of the SFK C-terminal tyrosine by Csk inhibits the kinase while autophosphorylation of the catalytic domain tyrosine results in full activity. CD45 opposes Csk by dephosphorylating the negative regulatory tyrosine, generating a pool of primed SFKs capable of rapid activation upon receptor stimulation. In some cell types, CD45 can also dephosphorylate the catalytic tyrosine and negatively regulate SFKs.Regulation of CD45 itself is complex. Alternative splicing of the extracellular domain, regulated in a cell-and activation-specific manner, generates multiple isoforms differing in size and charge (5). Interestingly, CD45 polymorphisms influencing its alternative splicing, and thus isoform expression, are associated with several human autoimmune diseases (8). We initi...

The autoimmune disease systemic lupus erythematosus (SLE) has a complex environmental and multi-factorial genetic basis. Genome wide association studies have recently identified numerous disease-associated polymorphisms, but it remains unclear in which cells and during which step of pathogenesis specific polymorphisms interact to cause disease. Using a mouse model in which the same activating mutation (CD45E613R) causes distinct genetic background-dependent disease phenotypes, we perform a screen for genetic modifiers of autoreactivity between anti-nuclear antibody (ANA)-resistant CD45E613R.B6 and ANA-permissive CD45E613R.BALB/c mice. Within a novel autoreactivity-associated locus on chromosome 9, we identify a putative modifier, TLR9. Validating a role for TLR9 in modifying autoreactivity in the context of the CD45E613R mutation, manipulation of TLR9 gene dosage eliminates ANA in CD45E613R.BALB/c, but confoundingly permits ANA in CD45E613R.B6. We demonstrate that sensitivity to ANA is modulated by strength of TLR9 signal, since stronger TLR9B6 signals, but not weaker TLR9BALB/c signals, negatively regulate CD45E613R B cell development during competitive reconstitution at the central tolerance checkpoint. Our results identify a novel autoreactivity-associated locus and validate Tlr9 as a candidate gene within the locus. We further demonstrate a novel role for TLR9 signal strength in central tolerance, providing insight into the interplay of disease-associated polymorphisms at a discrete step of SLE pathogenesis.

The dynamic regulation of protein tyrosine phosphorylation represents a powerful control point for integration of environmental signals into cellular responses. By modulating the activity of Src family protein tyrosine kinases, the receptor-like protein tyrosine phosphatase CD45 plays a critical role in regulating phosphotyrosine levels in hematopoietic cells. We previously generated mice containing a single point mutation (denoted CD45E613R) in the juxtamembrane wedge of CD45. Demonstrating the critical negative regulatory function of the wedge, in a mixed 129/Sv-C57Bl/6 (B6) genetic background, the CD45E613R mutation led to T and B cell activation, a lymphoproliferative disorder (LPD) characterized by splenomegaly and lymphadenopathy, and a lupus-like syndrome with autoantibody production and glomerulonephritis (GN). To determine which components of this phenotype were due to the direct effects of the wedge mutation and which were the consequence of genetic modifiers known to segregate between different murine strains, we backcrossed the CD45E613R mice at least 9 generations onto B6, 129/Sv, and BALB/c genetic backgrounds. Here, we use cellular and biochemical approaches to demonstrate that the CD45E613R mutation leads to cell autonomous hyperresponsiveness in B, T, and myeloid cells in each genetic background. However, the phenotypic consequence of this hyperresponsiveness is sensitive to strain-specific genetic modifiers. Despite similar signaling defects at a biochemical level, evaluation of cohorts of F9 CD45E613R and control mice (n=20 mice/genotype/strain) indicate that the LPD and autoimmune phenotypes can be genetically separated in homogenous inbred strains. For example, F9 B6 CD45E613R mice develop a very mild LPD and no evidence of autoimmunity (autoantibodies or GN) when examined serially to 18 months of age. In contrast, 100% of F9 BALB/c CD45E613R mice develop autoantibodies by 12 weeks of age. Despite autoantibody production, these animals fail to develop end-organ damage with age. The phenotype of F9 129/Sv CD45E613R mice is also modest with all animals developing a mild LPD with age and only 15% of the animals developing autoantibodies and GN. Confirming the influence of genetic modifiers in the CD45E613R phenotype, generation of F1 B6-129 mice recapitulated the original phenotype by developing a serve LPD, elevated white blood cell counts, production of autoantibodies, and GN. Disease was also more pronounced in B6-BALB/c F1 mice. To begin to identify modifying loci, 262 F2 CD45E613R B6-BALB mice were generated and characterized at 4 months of age. Isolated autoantibody production was observed in 15.6%, isolated LPD in 7.7%, and both LPD and autoantibodies in 6.5% of F2 mice. Analysis of 94 CD45E613R N2 backcross (B6-BALB/c F1 backcrossed to BALB/c) progeny revealed autoantibody in 64.9% of mice. Quantitative trait loci (QTL) mapping using 358 SNP markers to differentiate B6, BALB/c, and 129/Sv strains is being utilized to identify loci that either enhance or prevent lymphoproliferation and autoimmunity in the context of cell-intrinsic lymphocyte hyperactivity conferred by the CD45E613R mutation.