Autoantibody specificity was also assayed by indirect immunofluorescence staining of Hep2A cells and revealed a homogeneous nuclear pattern, consistent with the original observation in mixed B6/129 background CD45W/W mice (Figure 4A)

Autoantibody specificity was also assayed by indirect immunofluorescence staining of Hep2A cells and revealed a homogeneous nuclear pattern, consistent with the original observation in mixed B6/129 background CD45W/W mice (Figure 4A). even potent susceptibility loci must interact with one another to account for this phenomenon. How then can this complex process be effectively modeled and studied? encodes Lyp, a hematopoietic phosphatase the mouse homolog of which is Pep (PEST-domain enriched tyrosine phosphatase) (8, 9). Pep/Lyp negatively regulates TCR signaling by dephosphorylating the activating tyrosine of the Src-family kinase (SFK) Lck (10, 11). SFKs are critical mediators of signal transduction by ITAM-bearing immunoreceptors such as the TCR (12). Pep is cytoplasmic but is brought into proximity of its target, coreceptor-associated Lck, at the plasma membrane via its constitutive association with the cytoplasmic tyrosine kinase Csk (10, 13). Csk phosphorylates the inhibitory tyrosine of the SFKs and is itself a potent negative regulator of TCR signaling (14). Csk is recruited to Harmaline the membrane by the transmembrane adaptor PAG, which is phosphorylated under basal conditions (15, 16). Following TCR ligation, PAG is rapidly dephosphorylated, releasing Csk from the membrane (16). Pep and Csk interact functionally as well as physically to cooperatively inhibit TCR signaling (10, 11). This cooperative inhibition has been shown to depend upon the C-terminal proline rich sequence (PRS) of Pep and the SH3 domain of Csk (10). The PRS of Pep Rabbit Polyclonal to OR2T2 (613C621 PPPLPEacts as an autoimmune susceptibility locus, and the genetic contexts in which this might occur remain unclear. Given the significance of for human autoimmunity, we sought to establish systems in which we could model and study the risk allele in the context of Csk and in the context of a permissive genetic background. To this end, Harmaline we studied co-expression of Csk Harmaline and Pep as well as Lyp variants in Jurkat cells. To model the genetic cooperation of with other autoimmune susceptibility loci, we took advantage of CD45 E613R mice in which hyper-responsive B cells, characteristic of human and murine SLE, drive a lupus-like disease only on certain genetic backgrounds. CD45 is a receptor-like tyrosine phosphatase expressed at high levels on all nucleated hematopoietic cells (23). CD45 deficiency in humans and mice Harmaline leads to SCID phenotypes, revealing a crucial positive role for CD45 in immunoreceptor signaling and lymphocyte development (24C27). Polymorphisms in CD45 which influence regulated splicing are associated with autoimmune disease in humans (28). CD45 dephosphorylates the negative regulatory tyrosine of SFKs, thereby priming cells to respond to signals through immunoreceptors (23). This positive regulatory role is normally counterbalanced by the cooperating module of Csk/Pep. Our lab previously identified a critical membrane proximal residue (E613) in the cytoplasmic domain of CD45 which mediates dimerization-induced inhibition of phosphatase activity. Mutation of this site to an arginine ablates such inhibition (29, 30). Further support for the critical regulatory role of this residue was provided by the E613R `Wedge’ (CD45w/w) knockin mouse which developed lymphoproliferation, polyclonal lymphocyte activation, autoantibodies, and immune complex glomerulonephritis, a phenotype reminiscent of Harmaline human SLE (31). Genetic deletion of B cells revealed that the lymphoproliferation was B cell driven (32). At the cellular level, B cells were extremely hyper-responsive to B cell receptor signaling, a characteristic shared with spontaneous and engineered mouse models of lupus as well as B cells from patients with SLE (32C35). However, further backcrossing of the original knockin revealed a remarkable background dependence of the disease phenotype (Hermiston et al., manuscript in preparation). B cell hyper-responsiveness was noted on all backgrounds, but the original disease phenotype was recapitulated only on the B6/129 F1 background. Indeed, CD45 E613R mice on the pure B6 background develop no autoantibodies or end-organ disease. Therefore, CD45 E613R effectively functions as a murine autoimmune susceptibility allele in which lupus-like B cells cooperate with other factors. This result suggested that the CD45W/W B6 mouse might provide a suitable background against which to study candidate genetic modifiers such as polymorphism, we undertook in vitro studies of the risk allele in the Jurkat human.