The Phenotypic Consequences of an Activating Mutation in the Juxtamembrane Wedge of CD45 Is Sensitive to Genetic Modifiers.

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.