UCSF

Systemic lupus erythematosus (SLE) is a highly heterogeneous and prevalent autoimmune disease. The development of anti-nuclear autoantibodies (ANAs) is a hallmark of SLE and many patients will go on to develop glomerulonephritis (GN), a potentially fatal destruction of the kidneys. In the CD45E613R murine model of SLE, a single point mutation in the juxtamembrane wedge region of the protein tyrosine phosphatase CD45, results in hyperactivated immunoreceptor tyrosine-based activation motif (ITAM) signaling. Interestingly, despite the mutation functioning biochemically identically, different strains expressing the mutation result in different phenotypic consequences. CD45E613R B6 and 129 mice develop normally, while CD45E613R B6-129 F1 mice develop double stranded (ds)DNA autoantibodies, lymphoproliferative disease (LPD) and GN. CD45E613R BALB/c mice develop dsDNA autoantibodies, slight LPD, and no GN. This indicated genetic modifiers on these backgrounds modulated disease phenotype, and a single nucleotide polymorphism (SNP) screen performed between B6 and BALB/c mice for the development of ANAs identified two susceptibility loci. One locus, termed Wedge Associated Modifier (Wam) 1 on the BALB/c background identified tlr9 as the putative modifier gene. The second locus identified on the B6 background corresponded to the MHC H2 region.

Here we examine the role of these two susceptibility regions in the CD45E613R BALB/c and B6 models of SLE. Previous studies from our lab identified two alleles of TLR9, the B6 (TLR9B6) and BALB/c (TLR9Ba) alleles, hypothesized to result in the signaling difference observed between the two backgrounds. In CD45E613R BALB/c TLR9-/- mice, B cell expression of CD45E613R and TLR9 is required for ANA development. Interestingly, CD45E613R B6 TLR9-/- mice acquired ANA development, suggesting the two alleles are regulating ANA and B cell tolerance in opposing ways. In Chapter II, we developed TLR9 congenic mice on the B6 and BALB/c backgrounds with the CD45E613R polymorphisms to determine if signaling difference and autoantibody development observed in intact mice were intrinsic to the TLR9 alleles. We show here that the TLR9Ba allele positively regulates autoreactive B cell development and is required for the development of ANAs, GN, and results in hyperresponsive B cell activation upon stimulation resistant CD45E613R B6 mice. Furthermore, the TLR9B6 allele negatively regulates ANA production, resulting in tolerance in susceptible CD45E613R BALB/c mice. The TLR9 alleles also dictate the signaling differences, such that the TLR9B6 allele results in hyperresponsive signaling and the TLR9Ba allele results in hyporesponsive signaling, regardless of genetic background, potentiating the differences in disease phenotypes. Together, these data demonstrate the dual role two allelic variants of TLR9 plays in regulating B cell tolerance.

In Chapter III, we determine the role of the B6 susceptibility loci, MHC H2b (MHCB6), in the development of pathogenesis in the CD45E613R BALB/c model. We hypothesized the protective endogenous BALB/c MHC allele was preventing development of the severe GN observed in the CD45E613R B6-129 F1 model. In order to test this, we developed congenic CD45E613R BALB/c mice congenic for the MHC H2 region. CD45E613R BALB/c MHCB6 mice developed accelerated autoantibody production compared to CD45E613R BALB/c mice and eventually GN upon aging. B cells from CD45E613R BALB/c MHCB6 mice became dysregulated during development and were hyperactivated upon stimulation compared to CD45E613R BALB/c B cells. Further investigation revealed the B6 MHC H2b allele resulted in increased MHC class II expression on the surface of B cells compared to the BALB/c MHC H2d allele, but not other cell populations prior to disease onset. These data demonstrate one mechanism by which MHC susceptibility alleles identified in autoimmune patient populations are required for disease progression from autoreactivity to autoimmunity. Taken together with the TLR9 studies, we hypothesize the MHCB6 allele, in cooperation with the TLR9Ba allele and the sensitizing CD45E613R mutation results in the severe progression of autoimmunity in the CD45E613R B6 TLR9Ba model.