Genetic dissection of systemic lupus erythematosus pathogenesis: partial functional complementation between Sle1 and Sle3/5 demonstrates requirement for intracellular coexpression for full phenotypic expression of lupus

Sle1 on chromosome 1 and Sle3/5 on chromosome 7 are two of the most critical lupus susceptibility loci of the New Zealand Black/White-derived NZM2410 mouse strain. In contrast to C57BL/6 mice congenic for either Sle1 (B6.Sle1) or Sle3/5 (B6.Sle3/5), strains that express only a modest lupus-related phenotype, the bicongenic B6.Sle1.Sle3/5 strain has a robust phenotype, suggesting a critical role for epistatic interactions in lupus pathogenesis. Mixed chimera experiments indicated that the two loci are functionally expressed by different cell populations and predicted that phenotypic expression of the phenotypic features of the B6.Sle1.Sle3/5 strain could be fully reproduced with a combination of B6.Sle1 and B6.Sle3/5 bone marrow. Contrary to our expectations, there was only a partial functional complementation in these mixed chimeras. Spleen enlargement, CD4:CD8 ratio elevation, and epitope spreading of autoantibodies were fully developed in B6+B6.Sle1.Sle3/5 but not in B6.Sle1+B6.Sle3/5 mixed chimeras. This study is the first to present evidence that the pathways mediated by two critical lupus susceptibility loci derived from the New Zealand White strain must be integrated intracellularly for epistatic interactions to occur. Our mixed chimera approach continues to provide novel insights into the functional genetic pathways underlying this important murine model of systemic autoimmunity.     

IL-6 produced by dendritic cells from lupus-prone mice inhibits CD4+CD25+ T cell regulatory functions

The B6.Sle1.Sle2.Sle3 triple congenic mouse (B6.TC) is a model of lupus coexpressing the three major NZM2410-derived susceptibility loci on a C57BL/6 background. B6.TC mice produce high titers of antinuclear nephrogenic autoantibodies and a highly penetrant glomerulonephritis. Previous studies have shown the Sle1 locus is associated with a reduced number of regulatory T cells (Treg) and that Sle3 results in intrinsic defects of myeloid cells that hyperactivate T cells. In this report, we show that B6.TC dendritic cells (DCs) accumulate in lymphoid organs and present a defective maturation process, in which bone marrow-derived, plasmacytoid, and myeloid DCs express a significantly lower level of CD80, CD86, and MHC class II. B6.TC DCs also induce a higher level of proliferation in CD4(+) T cells than B6 DCs, and B6.TC DCs block the suppressive activity of Treg. B6.TC DCs overproduce IL-6, which is necessary for the blockade of Treg activity, as shown by the effect of anti-IL-6 neutralizing Ab in the suppression assays. The overproduction of IL-6 by DCs and the blockade of Treg activity maps to Sle1, which therefore not only confers a reduced number of Treg but also blocks their ability to regulate autoreactive T cells. Taken together, these results provide a genetic and mechanistic evidence for systemic autoimmunity resulting from an impaired regulatory T cell compartment in both number and function and for Sle1-expressing DCs playing a major role in the latter defect though their production of IL-6.     

Murine lupus susceptibility locus Sle1c2 mediates CD4+ T cell activation and maps to estrogen-related receptor γ

Sle1c is a sublocus of the NZM2410-derived Sle1 major lupus susceptibility locus. We have shown previously that Sle1c contributes to lupus pathogenesis by conferring increased CD4(+) T cell activation and increased susceptibility to chronic graft-versus-host disease (cGVHD), which mapped to the centromeric portion of the locus. In this study, we have refined the centromeric sublocus to a 675-kb interval, termed Sle1c2. Mice from recombinant congenic strains expressing Sle1c2 exhibited increased CD4(+) T cell intrinsic activation and cGVHD susceptibility, similar to mice with the parental Sle1c. In addition, B6.Sle1c2 mice displayed a robust expansion of IFN-γ-expressing T cells. NZB complementation studies showed that Sle1c2 expression exacerbated B cell activation, autoantibody production, and renal pathology, verifying that Sle1c2 contributes to lupus pathogenesis. The Sle1c2 interval contains two genes, only one of which, Esrrg, is expressed in T cells. B6.Sle1c2 CD4(+) T cells expressed less Esrrg than B6 CD4(+) T cells, and Esrrg expression was correlated negatively with CD4(+) T cell activation. Esrrg encodes an orphan nuclear receptor that regulates oxidative metabolism and mitochondrial functions. In accordance with reduced Esrrg expression, B6.Sle1c2 CD4(+) T cells present reduced mitochondrial mass and altered mitochondrial functions as well as altered metabolic pathway utilization when compared with B6 CD4(+) T cells. Taken together, we propose Esrrg as a novel lupus susceptibility gene regulating CD4(+) T cell function through their mitochondrial metabolism.     

Genetic dissection of Sle pathogenesis: Sle3 on murine chromosome 7 impacts T cell activation, differentiation, and cell death.

Polyclonal, generalized T cell defects, as well as Ag-specific Th clones, are likely to contribute to pathology in murine lupus, but the genetic bases for these mechanisms remain unknown. Mapping studies indicate that loci on chromosomes 1 (Sle1), 4 (Sle2), 7 (Sle3), and 17 (Sle4) confer disease susceptibility in the NZM2410 lupus strain. B6.NZMc7 mice are C57BL/6 (B6) mice congenic for the NZM2410-derived chromosome 7 susceptibility interval, bearing Sle3. Compared with B6 controls, B6.NZMc7 mice exhibit elevated CD4:CD8 ratios (2.0 vs 1.34 in 1- to 3-mo-old spleens); an age-dependent accumulation of activated CD4+ T cells (33.4% vs 21.9% in 9- to 12-mo-old spleens); a more diffuse splenic architecture; and a stronger immune response to T-dependent, but not T-independent, Ags. In vitro, Sle3-bearing T cells show stronger proliferation, increased expansion of CD4+ T cells, and reduced apoptosis (with or without anti-Fas) following stimulation with anti-CD3. With age, the B cells in this strain acquire an activated phenotype. Thus, the NZM2410 allele of Sle3 appears to impact generalized T cell activation, and this may be causally related to the low grade, polyclonal serum autoantibodies seen in this strain. Epistatic interactions with other loci may be required to transform this relatively benign phenotype into overt autoimmunity, as seen in the NZM2410 strain.     

The major murine systemic lupus erythematosus susceptibility locus Sle1 results in abnormal functions of both B and T cells

Sle1 is a major susceptibility locus in the NZM2410 murine model of systemic lupus erythematosus. When isolated on a C57BL/6 background in the B6.Sle1 congenic strain, Sle1 results in the production of high levels of anti-chromatin IgG Abs, histone-specific T cells, and increased B and T cell activation. We have shown by mixed bone marrow chimeras with allotypic markers that Sle1 is expressed in B cells. Using the same technique, we now show that it is also expressed in T cells. To assess whether Sle1 results in intrinsic defects in B or T cells, we have bred the muMT and Tcralpha(-/-) mutations onto B6.Sle1 resulting in the absence of circulating B cells and alphabeta T cells in B6.Sle1.muMT and B6.Sle1.Tcralpha(-/-), respectively. The immune phenotypes in these two strains were compared with that of B6.Sle1 and B6.muMT or B6.Tcralpha(-/-). Sle1-expressing B cells broke tolerance to chromatin in the absence of T cells, as shown by high levels of anti-ssDNA IgM Abs in B6.Sle1.Tcralpha(-/-) mice, and had an increased expression of activation markers. Conversely, increased expression of activation markers and increased cytokine production were observed in Sle1-expressing T cells in the absence of B cells in B6.Sle1.muMT mice. However, the production of IgG antinuclear Abs required the presence of both T and B cells. These experiments showed that Sle1 expression results in both B and T cells intrinsic defects and demonstrate that the documented involvement of each cell compartment in the production of anti-chromatin Abs corresponds to genetic defects rather than bystander effects.     

Differential control of autoantibodies and lymphoproliferation by Fas ligand expression on CD4+ and CD8+ T cells in vivo.

We have previously shown that the gld autoimmune syndrome is suppressed in lethally irradiated gld mice reconstituted with a mixture of normal and gld bone marrow (BM). Furthermore, in vivo depletion of normal Thy-1+ cells restores lymphoproliferation and autoantibody production in such chimeras, suggesting that T cells bearing Fas ligand are responsible for correcting the gld defect. In this study, mixed-BM chimeras lacking either normal CD4+ (B6CD4KO-B6gld) or normal CD8+ T cells (B6CD8KO-B6gld) were generated to determine the contribution of the normal T cell subsets to disease suppression. Lymphoproliferation was completely suppressed in B6CD4KO-B6gld chimeras but only modestly in B6CD8KO-B6gld chimeras. On the other hand, both types of mixed-BM chimeras had incomplete effects on the suppression of serum autoantibodies when compared with B6gld reconstituted with isologous BM. These results suggest that both T cell subsets provide Fas ligand to suppress immune cells responsible for autoantibody production; however, CD8+ T cells are mainly responsible for preventing lymphoproliferation.     

Genetic dissection of the murine lupus susceptibility locus Sle2: contributions to increased peritoneal B-1a cells and lupus nephritis map to different loci

Lupus pathogenesis in the NZM2410 mouse model results from the expression of multiple interacting susceptibility loci. Sle2 on chromosome 4 was significantly linked to glomerulonephritis in a linkage analysis of a NZM2410 x B6 cross. Yet, Sle2 expression alone on a C57BL/6 background did not result in any clinical manifestation, but in an abnormal B cell development, including the accumulation of B-1a cells in the peritoneal cavity and spleen. Analysis of B6.Sle2 congenic recombinants showed that at least three independent loci, New Zealand White-derived Sle2a and Sle2b, and New Zealand Black-derived Sle2c, contribute to an elevated number of B-1a cells, with Sle2c contribution being the strongest of the three. To determine the contribution of these three Sle2 loci to lupus pathogenesis, we used a mapping by genetic interaction strategy, in which we bred them to B6.Sle1.Sle3 mice. We then compared the phenotypes of these triple congenic mice with that of previously characterized B6.Sle1.Sle2.Sle3, which express the entire Sle2 interval in combination with Sle1 and Sle3. Sle2a and Sle2b, but not Sle2c, contributed significantly to lupus pathogenesis in terms of survival rate, lymphocytic expansion, and kidney pathology. These results show that the Sle2 locus contains several loci affecting B cell development, with only the two NZW-derived loci having the least effect of B-1a cell accumulation significantly contributing to lupus pathogenesis.     

Murine lupus susceptibility locus Sle1a controls regulatory T cell number and function through multiple mechanisms

The Sle1 locus is a key determinant of lupus susceptibility in the NZM2410 mouse model. Within Sle1, we have previously shown that Sle1a expression enhances activation levels and effector functions of CD4(+) T cells and reduces the size of the CD4(+)CD25(+)Foxp3(+) regulatory T cell subset, leading to the production of autoreactive T cells that provide help to chromatin-specific B cells. In this study, we show that Sle1a CD4(+) T cells express high levels of ICOS, which is consistent with their increased ability to help autoreactive B cells. Furthermore, Sle1a CD4(+)CD25(+) T cells express low levels of Foxp3. Mixed bone marrow chimeras demonstrated that these phenotypes require Sle1a to be expressed in the affected CD4(+) T cells. Expression of other markers generally associated with regulatory T cells (Tregs) was similar regardless of Sle1a expression in Foxp3(+) cells. This result, along with in vitro and in vivo suppression studies, suggests that Sle1a controls the number of Tregs rather than their function on a per cell basis. Both in vitro and in vivo suppression assays also showed that Sle1a expression induced effector T cells to be resistant to Treg suppression, as well as dendritic cells to overproduce IL-6, which inhibits Treg suppression. Overall, these results show that Sle1a controls both Treg number and function by multiple mechanisms, directly on the Tregs themselves and indirectly through the response of effector T cells and the regulatory role of dendritic cells.     

Characterization of reciprocal Lmb1-4 interval MRL-Faslpr and C57BL/6-Faslpr congenic mice reveals significant effects from Lmb3

Susceptibility to severe lupus in MRL-Fas(lpr) mice requires not only the lpr mutation but also other predisposing genes. Using (MRL-Fas(lpr) x B6-Fas(lpr))F2 (where B6 represents C57BL/6) intercrosses that utilize the highly susceptible MRL and poorly susceptible B6 backgrounds, we previously mapped CFA-enhanced systemic lupus-like autoimmunity to four loci, named Lmb1-4, on chromosomes 4, 5, 7, and 10. In the current study, we generated and analyzed reciprocal interval congenic mice for susceptibility to CFA-enhanced autoimmunity at all four Lmb loci. Although all loci had at least a slight effect on lymphoproliferation, only Lmb3 demonstrated a major effect on lymphoproliferation and anti-chromatin Ab levels. Further characterization of Lmb3, primarily by comparing MRL-Fas(lpr) with MRL.B6-Lmb3 Fas(lpr) congenic mice, revealed that it also played a significant role in spontaneous lupus, modifying lymphoproliferation, IgG and autoantibody levels, kidney disease, and survival. The less susceptible B6 Lmb3 locus was associated with a marked reduction in numbers of CD4(+) and double-negative (CD4(-)CD8(-)) T cells, particularly in lymph nodes, as well as reduced T cell proliferation and enhanced T cell apoptosis, both in vivo and in vitro. IFN-gamma-producing CD4(+) T cells were also reduced in MRL.B6-Lmb3 Fas(lpr) mice. Further mapping using subinterval congenic mice placed Lmb3 in the telomeric portion of chromosome 7. Thus, Lmb3, primarily through its effects on CD4(+) and double-negative T cells, appears to be a highly penetrant lupus-modifying locus. Identification of the underlying genetic alteration responsible for this quantitative trait locus should provide new insights into lupus-modifying genes.     

The centromeric region of chromosome 7 from MRL mice (Lmb3) is an epistatic modifier of Fas for autoimmune disease expression

Lupus is a prototypic systemic autoimmune disease that has a significant genetic component in its etiology. Several genome-wide screens have identified multiple loci that contribute to disease susceptibility in lupus-prone mice, including the Fas-deficient MRL/Fas(lpr) strain, with each locus contributing in a threshold liability manner. The centromeric region of chromosome 7 was identified as a lupus susceptibility locus in MRL/Fas(lpr) mice as Lmb3. This locus was backcrossed onto the resistant C57BL/6 (B6) background, in the presence or absence of Fas, resulting in the generation of B6.MRLc7 congenic animals. Detailed analysis of these animals showed that Lmb3 enhances and accelerates several characteristics of lupus, including autoantibody production, kidney disease, and T cell activation, as well as accumulation of CD4(-)CD8(-) double-negative T cells, the latter a feature of Fas-deficient mice. These effects appeared to be dependent on the interaction between Lmb3 and Fas deficiency, as Lmb3 on the B6/+(Fas-lpr) background did not augment any of the lupus traits measured. These findings confirm the role of Lmb3 in lupus susceptibility, as a modifier of Fas(lpr) phenotype, and illustrate the importance of epistatic interaction between genetic loci in the etiology of lupus. Furthermore, they suggest that the genetic lesion(s) in MRLc7 is probably different from those in NZMc7 (Sle3/5), despite a significant overlap of these two intervals.     

Genetic dissection of spontaneous autoimmunity driven by 129-derived chromosome 1 Loci when expressed on C57BL/6 mice

Extensive evidence indicates that genetic predisposition is a central element in susceptibility to systemic lupus erythematosus both in humans and animals. We have previously shown that a congenic line carrying a 129-derived chromosome 1 interval on the C57BL/6 background developed humoral autoimmunity. To further dissect the contribution to autoimmunity of this 129 interval, we have created six subcongenic strains carrying fractions of the original 129 region and analyzed their serological and cellular phenotypes. At 1 year of age the congenic strain carrying a 129 interval between the microsatellites D1Mit15 (87.9 cM) and D1Mit115 (99.7 cM) (B6.129chr1b) had high levels of autoantibodies, while all the other congenic lines were not significantly different from the C57BL/6 controls. The B6.129chr1b strain displayed only mild proliferative glomerulonephritis despite high levels of IgG and C3 deposited in the kidneys. FACS analysis of the spleens revealed that the B6.129chr1b mice had a marked increase in the percentage of activated T cells associated with a significant reduction in the proportion of CD4(+)CD25(high) regulatory T cells. Moreover, this analysis showed a significantly reduced percentage of marginal zone B cells that preceded autoantibody production. Interestingly the 129chr1b-expressing bone marrow-derived macrophages displayed an impaired uptake of apoptotic cells in vitro. Collectively, our data indicate that the 129chr1b segment when recombined on the C57BL/6 genomic background is sufficient to induce loss of tolerance to nuclear Ags. These findings have important implication for the interpretation of the autoimmune phenotype associated with gene-targeted models.     

CD86 (B7-2) can function to drive MHC-restricted antigen-specific CTL responses in vivo

Activation of T cells requires both TCR-specific ligation by direct contact with peptide Ag-MHC complexes and coligation of the B7 family of ligands through CD28/CTLA-4 on the T cell surface. We recently reported that coadministration of CD86 cDNA along with DNA encoding HIV-1 Ags i.m. dramatically increased Ag-specific CTL responses. We investigated whether the bone marrow-derived professional APCs or muscle cells were responsible for the enhancement of CTL responses following CD86 coadministration. Accordingly, we analyzed CTL induction in bone marrow chimeras. These chimeras are capable of generating functional viral-specific CTLs against vaccinia virus and therefore represent a useful model system to study APC/T cell function in vivo. In vaccinated chimeras, we observed that only CD86 + Ag + MHC class I results in 1) detectable CTLs following in vitro restimulation, 2) detectable direct CTLs, 3) enhanced IFN-gamma production in an Ag-specific manner, and 4) dramatic tissue invasion of T cells. These results support that CD86 plays a central role in CTL induction in vivo, enabling non-bone marrow-derived cells to prime CTLs, a property previously associated solely with bone marrow-derived APCs.     

Genetic determination of T cell help in loss of tolerance to nuclear antigens

Sle1 is a major lupus susceptibility locus in NZM2410 lupus model that is associated with a loss of tolerance to nuclear Ags. At least three genes, Sle1a, Sle1b, and Sle1c contribute to Sle1, and their relative role in lupus pathogenesis is unknown. We show here that Sle1-expressing CD4(+) T cells present an activated phenotype associated with increased proliferation and cytokine production. In addition, Sle1 CD4(+) T cells provide help to anti-chromatin B cells to produce anti-nuclear antibodies, whether or not these B cells express Sle1. The Sle1a locus alone accounts for all these Sle1 phenotypes, implying that a specific genetic defect in Sle1a is necessary and sufficient to produce autoreactive T cells. However, Sle1c induces intermediate T cell activation and only provides help to Sle1-expressing anti-chromatin-producing B cells, demonstrating the synergic interactions between Sle1c T and Sle1 B cells. Moreover, Sle1a and Sle1c were associated with a significantly reduced level of CD4(+)CD25(+) regulatory T cells that precedes autoantibody production, suggesting a causal relationship with the generation of autoreactive T cells. Our study identifies for the first time that a specific genetic defect is responsible for lupus pathogenesis by inducing autoreactive T cells to break self-tolerance and that this genetic defect is also associated with a decreased number of regulatory T cells.     

Epistatic suppression of systemic lupus erythematosus: fine mapping of Sles1 to less than 1 mb

Sle is a susceptibility locus for systemic autoimmunity derived from the lupus-prone NZM2410 mouse. The New Zealand White-derived suppressive modifier Sles1 was identified as a specific modifier of Sle1 and prevents the development of IgG anti-chromatin autoantibodies mediated by Sle1 on the C57BL/6 (B6) background. Fine mapping of Sles1 with truncated congenic intervals localizes it to a approximately 956-kb segment of mouse chromosome 17. Sles1 completely abrogates the development of activated T and B cell populations in B6.Sle1. Despite this suppression of the Sle1-mediated cell surface activation phenotypes, B6.Sle1 Sles1 splenic B cells still exhibit intrinsic ERK phosphorylation. Classic genetic complementation tests using the nonautoimmmune 129/SvJ mouse suggests that this strain possesses a Sles1 allele complementary to that of New Zealand White, as evidenced by the lack of glomerulonephritis, splenomegaly, and antinuclear autoantibody production seen in (129 x B6.Sle1 Sles1)F(1)s. These findings localize and characterize the suppressive properties of Sles1 and implicate 129 as a useful strain for aiding in the identification of this elusive epistatic modifier gene.     

The granulocyte colony stimulating factor pathway regulates autoantibody production in a murine induced model of systemic lupus erythematosus

Introduction

An NZB-derived genetic locus (Sle2c2) that suppresses autoantibody production in a mouse model of induced systemic lupus erythematosus contains a polymorphism in the gene encoding the G-CSF receptor. This study was designed to test the hypothesis that the Sle2c2 suppression is associated with an impaired G-CSF receptor function that can be overcome by exogenous G-CSF.

Methods

Leukocytes from B6.Sle2c2 and B6 congenic mice, which carry a different allele of the G-CSF receptor, were compared for their responses to G-CSF. Autoantibody production was induced with the chronic graft-versus-host-disease (cGVHD) model by adoptive transfer of B6.bm12 splenocytes. Different treatment regimens varying the amount and frequency of G-CSF (Neulasta^®) or carrier control were tested on cGVHD outcomes. Autoantibody production, immune cell activation, and reactive oxygen species (ROS) production were compared between the two strains with the various treatments. In addition, the effect of G-CSF treatment was examined on the production autoantibodies in the B6.Sle1.Sle2.Sle3 (B6.TC) spontaneous model of lupus.

Results

B6.Sle2c2 and B6 leukocytes responded differently to G-CSF. G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment. G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils. In contrast, the expression of G-CSF responsive genes indicated a higher G-CSF receptor signaling in B6.Sle2c2 cells. G-CSF treatment restored the ability of B6.Sle2c2 mice to produce autoantibodies in a dose-dependent manner upon cGVHD induction, which correlated with restored CD4⁺ T cells activation, as well as dendritic cell and granulocyte expansion. Steady-state ROS production was higher in B6.Sle2c2 than in B6 mice. cGVHD induction resulted in a larger increase in ROS production in B6 than in B6.Sle2c2 mice, and this difference was eliminated with G-CSF treatment. Finally, a low dose G-CSF treatment accelerated the production of anti-dsDNA IgG in young B6.TC mice.

Conclusion

The different in vivo and in vitro responses of B6.Sle2c2 leukocytes are consistent with the mutation in the G-CSFR having functional consequences. The elimination of Sle2c2 suppression of autoantibody production by exogenous G-CSF indicates that Sle2c2 corresponds to a loss of function of G-CSF receptor. This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus. Overall, these results suggest that the G-CSF pathway regulates the production of autoantibodies in murine models of lupus.     

Mechanisms of peritoneal B-1a cells accumulation induced by murine lupus susceptibility locus Sle2

The abundance of B-1a cells found in the peritoneal cavity of mice is under genetic control. The lupus-prone mouse New Zealand Black and New Zealand White (NZB x NZW)F(1) and its derivative NZM2410 are among the strains with the highest numbers of peritoneal B1-a cells. We have previously identified an NZM2410 genetic locus, Sle2, which is associated with the production of large numbers of B-1a cells. In this paper, we examined the mechanisms responsible for this phenotype by comparing congenic C57BL/6 mice with or without Sle2. Fetal livers generated more B-1a cells in B6.Sle2 mice, providing them with a greater starting number of B-1a cells early in life. Sle2-expressing B1-a cells proliferated significantly more in vivo than their B6 counterparts, and reciprocal adoptive transfers showed that this phenotype is intrinsic to Sle2 peritoneal B cells. The rate of apoptosis detected was significantly lower in B6.Sle2 peritoneal cavity B-1a cells than in B6, with or without exogenous B cell receptor cross-linking. Increased proliferation and decreased apoptosis did not affect Sle2 peritoneal B-2 cells. In addition, a significant number of peritoneal cavity B-1a cells were recovered in lethally irradiated B6.Sle2 mice reconstituted with B6.Igh(a) bone marrow, showing radiation resistance in Sle2 B-1a cells or its precursors. Finally, B6.Sle2 adult bone marrow and spleen were a significant source of peritoneal B-1a cells when transferred into B6.Rag2(-/-) mice. This suggests that peritoneal B-1a cells are replenished throughout the animal life span in B6.Sle2 mice. These results show that Sle2 regulates the size of the B-1a cell compartment at multiple developmental checkpoints.     

Genetic control of anti-Sm autoantibody production in NOD congenic mice narrowed to the Idd9.3 region

Anti-Smith (anti-Sm) autoantibodies are directed to proteins in the small-nuclear ribonucleoprotein (snRNP) family and are considered specific for systemic lupus erythematosus (SLE) in both humans and mice. We previously established that NOD.c3c4 mice, carrying B6 and B10 congenic segments from chromosomes 3 to 4 on an nonobese diabetic (NOD) background, and NOD.Idd9R28 mice, carrying a B10 segment on c4 alone, developed significant penetrance of anti-Sm antibody production. Here we determine autoantibody incidence in additional NOD.Idd9 congenic strains and use a congenic mapping approach to narrow the interval necessary for enhanced autoantibody production to a ∼5.6-Mb region containing insulin-dependent diabetes (Idd)9.3. The Idd9.3 interval contains the candidate molecule cluster of differentiation (CD)137, which is a member of the tumor necrosis factor (TNF) receptor superfamily, functions as an inducible costimulator of T cells, and controls T–B interactions. The NOD and B10 CD137 alleles have sequence polymorphisms and different functional effects on T cells; the NOD CD137 allele mediates weaker T cell proliferative responses and decreased interleukin (IL)-2 production after CD137-mediated costimulation. Our work establishes CD137 as a candidate gene for control of autoantibody production in NOD.Idd9.3 congenic mice.     

NK cells play a critical role in the regulation of class I-deficient hemopoietic stem cell engraftment: evidence for NK tolerance correlates with receptor editing

The role that NK cells play in the rejection of hemopoietic stem cell (HSC) and tolerance induction has remained controversial. In this study, we examined whether NK cells play a direct role in the rejection of HSC. Purified HSC from MHC class II-deficient mice engrafted readily in congenic mice, while HSC from class I-deficient donors (beta(2)-microglobulin(-/-) (beta(2)m(-/-))) failed to engraft. Recipient mice lacking CD8(+), CD4(+), or T cells also rejected HSC from class I-deficient donors, pointing directly to NK cells as the effector in rejection of HSC. Recipients, deficient in or depleted of NK cells, engrafted readily with beta(2)m(-/-) HSC. Expression of the activating Ly-49D and inhibitory Ly-49G2 receptors on recipient NK cells was significantly decreased in these beta(2)m(-/-)-->B6 chimeras, and the proportion of donor NK cells expressing Ly-49D was also significantly decreased. Notably, beta(2)m(-/-) chimeras accepted beta(2)m(-/-) HSC in second transplants, demonstrating that NK cells in the chimeras had been tolerized to beta(2)m(-/-). Taken together, our data demonstrate that NK cells play a direct role in the regulation of HSC engraftment, and down-regulation and/or deletion of specific NK subsets in mixed chimeras can contribute to the induction of NK cell tolerance in vivo. Moreover, our data show that bone marrow-derived elements significantly contribute to NK cell development and tolerance.     

Expression of CD80/86 on B cells is essential for autoreactive T cell activation and the development of arthritis

Depletion of B cells in rheumatoid arthritis is therapeutically efficacious. Yet, the mechanism by which B cells participate in the inflammatory process is unclear. We previously demonstrated that Ag-specific B cells have two important functions in the development of arthritis in a murine model of rheumatoid arthritis, proteoglycan (PG)-induced arthritis (PGIA). PG-specific B cells function as autoantibody-producing cells and as APCs that activate PG-specific T cells. Moreover, the costimulatory molecule CD86 is up-regulated on PG-specific B cells in response to stimulation with PG. To address the requirement for CD80/CD86 expression on B cells in the development of PGIA, we generated mixed bone marrow chimeras in which CD80/CD86 is specifically deleted on B cells and not on other APC populations. Chimeras with a specific deficiency in CD80/CD86 expression on B cells are resistant to the induction of PGIA. The concentration of PG-specific autoantibody is similar in mice sufficient or deficient for CD80/86-expressing B cells, which indicates that resistance to PGIA is not due to the suppression of PG-specific autoantibody production. CD80/86-deficient B cells failed to effectively activate PG-specific autoreactive T cells as indicated by the failure of T cells from PG-immunized CD80/86-deficient B cell chimeras to transfer arthritis into SCID mice. In vitro secondary recall responses to PG are also dependent on CD80/86-expressing B cells. These results demonstrate that a CD80/86:CD28 costimulatory interaction between B cells and T cells is required for autoreactive T cell activation and the induction of arthritis but not for B cell autoantibody production.