Human autoimmune diseases arise from environmental challenge in the context of cooperating susceptibility loci, each of which independently confers a small relative risk. Modeling and understanding the mechanisms that underlie such interactions in a tractable genetic system such as the mouse has been challenging.
A polymorphism in the human gene PTPN22
(C1858T / R620W) confers risk for developing multiple autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and type 1 diabetes (1
). Indeed, PTPN22
R620W was the second most significant risk allele identified in two unbiased whole genome scans for rheumatoid arthritis, although the odds ratio associated with carrying the risk allele is quite modest, with a value of less than two (4
). Nevertheless, human autoimmune disease has a striking genetic contribution; RA heritability has been estimated at 60% (7
). Clearly 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
). Pep/Lyp negatively regulates TCR signaling by dephosphorylating the activating tyrosine of the Src-family kinase (SFK) Lck (10
). 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
). Csk phosphorylates the inhibitory tyrosine of the SFKs and is itself a potent negative regulator of TCR signaling (14
). Csk is recruited to the membrane by the transmembrane adaptor PAG, which is phosphorylated under basal conditions (15
). 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
). 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 (613–621 PPPLPER
T), containing the critical R620 residue, is absolutely required for this association and for cooperative inhibition of TCR signaling (13
To complement in vitro over-expression studies, Pep-deficient (Pep−/−
) mice provide in vivo support for a negative regulatory role in TCR signaling (17
). The phenotype is however surprisingly subtle; TCR signaling is enhanced at the CD4+CD8+ (double positive) stage of thymocyte development and in the effector/memory compartment of peripheral T cells, but naïve T cells and B cells appear to have no functional abnormality. While Pep−/−
mice develop an expanded effector/memory T cell compartment and increased numbers of spontaneous germinal centers, no autoantibodies or frank autoimmune disease are observed on the B6 genetic background. One possible explanation for this subtle phenotype is redundancy with the ubiquitously expressed, related phosphatase PTP-PEST (18
The R620W polymorphism partially disrupts the association of Pep/Lyp with Csk (1
). These data are consistent with a critical role for the PRS domain, and suggest that the R620W allele might represent a hypomorphic variant. However, evidence from genetic studies does not clearly identify either a pure recessive or dominant effect of the polymorphism in RA (HET odds ratio 1.98, HOM odds ratio 3.32) (4
). Relatively few in vitro functional studies of the polymorphism have been reported. In a Jurkat overexpression study, the R620W allele of Lyp exerted more potent inhibition of TCR signaling than wild type (19
). Importantly, the polymorphism was not studied in the context of its binding partner Csk to identify its effect on synergistic inhibition of TCR signaling. More recently, another group has studied the functional consequences of the R620W variant directly in primary human T cells from heterozygous and homozygous carriers of the polymorphism. These studies found impaired calcium flux and upregulation of activation markers in response to TCR ligation in peripheral blood memory T and B cells. Notably, naïve T cell function was normal and no significant differences in IL-2 production or proliferation were detectable (20
). Three additional studies in which primary PBMCs from patients with type I diabetes or myasthenia gravis who carry the R620W allele have been subjected to functional assays report mixed results (19
The functional consequences of the R620W polymorphism, the mechanism by which PTPN22 acts as an autoimmune susceptibility locus, and the genetic contexts in which this might occur remain unclear. Given the significance of PTPN22 for human autoimmunity, we sought to establish systems in which we could model and study the risk allele in vitro in the context of Csk and in vivo in the context of a permissive genetic background. To this end, we studied co-expression of Csk and Pep as well as Lyp variants in Jurkat cells. To model the genetic cooperation of PTPN22 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 leads to SCID phenotypes, revealing a crucial positive role for CD45 in immunoreceptor signaling and lymphocyte development (24
). 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
). 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 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 (32
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 PTPN22.
Here we report that misexpression of wild type and R620W Pep and Lyp alleles in the context of Csk in Jurkat cells unmasks R620W as a hypomorphic allele. We took advantage of Pep-deficient mice as a model of the hypomorphic human risk allele, albeit a more extreme allele. We crossed the Pep−/− mice onto the non-autoimmune CD45W/W B6 background in order to model how subtle susceptibility loci might cooperate in human autoimmune disease. We found that these alleles cooperate to break tolerance on the non-autoimmune B6 genetic background. Indeed, double mutant CD45W/W/Pep−/− mice develop lymphadenopathy and splenomegaly, autoantibodies, glomerulonephritis and premature mortality. The cellular phenotype is characterized by polyclonal B and T cell activation as well as early and progressive expansion of the memory T cell compartment. Studies of antigen receptor signal transduction revealed that peripheral B cells in double mutant mice respond like single mutant CD45W/W B cells while peripheral T cells resemble single mutant Pep−/− T cells. In contrast, thymic signaling reflects cooperation between the two mutant alleles. These studies suggest that Pep−/− T cells in the context of a susceptible microenvironment drive CD45W/W B cells to break tolerance.