The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheung, Y.-H.
Right arrow Articles by Wither, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheung, Y.-H.
Right arrow Articles by Wither, J. E.
The Journal of Immunology, 2005, 175: 8154-8164.
Copyright © 2005 by The American Association of Immunologists

Functional Interplay between Intrinsic B and T Cell Defects Leads to Amplification of Autoimmune Disease in New Zealand Black Chromosome 1 Congenic Mice1

Yui-Ho Cheung*,{dagger}, Nan-Hua Chang*, Yong-Chun Cai*, Gabriel Bonventi*, Ralph MacLeod* and Joan E. Wither2,*,{dagger},{ddagger}

* Arthritis Centre of Excellence, Toronto Western Research Institute, Toronto, Ontario, Canada; {dagger} Department of Immunology, University of Toronto, Toronto, Ontario, Canada; and {ddagger} Department of Medicine, University Health Network, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Genetic loci on New Zealand Black (NZB) chromosome 1 play an important role in the development of lupus-like autoimmune disease. We have shown previously that C57BL/6 mice with an introgressed NZB chromosome 1 interval extending from ~35 to 106 cM have significantly more severe autoimmunity than mice with a shorter interval extending from ~82 to 106 cM. Comparison of the cellular phenotype in these mice revealed that both mouse strains had evidence of increased T cell activation; however, activation was more pronounced in mice with the longer interval. Mice with the longer interval also had increased B cell activation, leading us to hypothesize that there were at least two independent lupus susceptibility loci on chromosome 1. In this study, we have used mixed hemopoietic radiation chimeras to demonstrate that autoimmunity in these mice arises from intrinsic B and T cell functional defects. We further show that a T cell defect, localized to the shorter interval, leads to spontaneous activation of T cells specific for nucleosome histone components. Despite activation of self-reactive T cells in mixed chimeric mice, only chromosome 1 congenic B cells produce anti-nuclear Abs and undergo class switching, indicating impaired B cell tolerance mechanisms. In mice with the longer chromosome 1 interval, an additional susceptibility locus exacerbates autoimmune disease by producing a positive feedback loop between T and B cell activation. Thus, T and B cell defects act in concert to produce and amplify the autoimmune phenotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemiclupus erythematosus (SLE)3 is a multisystem autoimmune disease of unknown etiology that mainly affects women of childbearing age. One of the hallmarks of this disease is the loss of tolerance to self-Ags, particularly nuclear Ags (1). This loss of tolerance leads to production of autoantibodies directed against nuclear Ags, such as chromatin, and results in the formation of immune complexes. Deposition of immune complexes in the glomeruli, skin, and other organs induces tissue damage resulting in the manifestations of disease, which include skin rash, arthritis, and glomerulonephritis (2). Although the genes responsible for development of SLE in humans have not been clearly identified, there is convincing evidence that SLE has a strong, but complex, genetic basis (3).

Identification of the immune abnormalities that lead to the development of SLE has been greatly aided by the study of lupus-prone mouse strains. New Zealand Black (NZB) mice develop a lupus-like autoimmune disease characterized by production of anti-ssDNA and anti-RBC Abs leading to hemolytic anemia (4). Mapping studies have revealed that susceptibility to lupus in NZB mice is polygenic with multiple genes contributing to the generation of high affinity autoantibodies, nephritis, and mortality in various crosses (5, 6, 7, 8, 9, 10, 11, 12, 13). In particular, genetic loci on NZB chromosome 1 have been shown in multiple crosses to play a prominent role in disease susceptibility (5, 7, 9, 10, 12, 13). Indeed, in one cross between NZB and B6.H2z mice, a region on distal NZB chromosome 1 was found, in combination with H2z, to confer >90% of the susceptibility to nephritis and autoantibody production (9, 10).

The presence of a lupus susceptibility gene(s) on distal NZB chromosome 1 was confirmed by Rozzo et al. (14), who showed that B6 congenic mice with an NZB interval extending from ~79 to 109 cM, produced IgG anti-nuclear Abs. Three candidate genes have been proposed to contribute to the development of autoimmunity in this interval: Ifi202 (14), Fcgr2b (15, 16), and the SLAM/CD2 cluster (17). However, work by ourselves and others (12, 13) suggested that it was unlikely that this interval contained all of the lupus susceptibility genes located on NZB chromosome 1. For example, Kono et al. (5) showed linkage between a NZB region around 67 cM and anti-chromatin Ab production and splenomegaly in a (NZB x New Zealand White (NZW))F2 intercross. In our mapping study of (B6 x NZB)F2 intercross mice, IgG anti-ssDNA Ab production was linked to a broad region on NZB chromosome 1 with 2 shallow lod score peaks at 63 and 92 cM, and a similar area of broad linkage was demonstrated for several B cell activation phenotypes, including increased expression of B7.1, B7.2, ICAM-1, and CD44 (13). Indeed, the peak lod scores for IgG anti-ssDNA Ab production, ICAM-1, and CD44 were all located in the 63–76 cM region.

To investigate the presence of additional susceptibility genes on NZB chromosome 1, we generated congenic mice with NZB chromosome 1 intervals of various lengths and compared their cellular and autoimmune phenotype (18). We found that mice with a congenic interval extending from ~35 to 106 cM had a significantly more severe autoimmune phenotype than mice with an interval extending from ~82 to 106 cM, confirming the presence of at least two independent lupus susceptibility loci on NZB chromosome 1. Mice with the shorter interval (labeled B6.NZBc1S in this article) had evidence of increased T cell activation with an increased proportion of memory T cells, whereas mice with the longer interval (B6.NZBc1L) had more robust T cell activation and an increased proportion of activated B cells. The increased T cell activation in B6.NZBc1L mice, as compared with B6.NZBc1S mice, was B cell dependent, leading us to hypothesize that the genetic locus located within the shorter interval predominantly affected T cell function while that located in the longer interval altered B cell function.

In this study, we have further investigated the nature and location of the immune abnormalities in these mice. By generating mixed hemopoietic radiation chimeras, we show that autoimmunity in B6.NZBc1L mice results from intrinsic B and T cell defects. We further show that the susceptibility locus located within the shorter interval leads to spontaneous activation of T cells with specificity for nucleosome histone components. Despite the presence of activated self-reactive T cells in mixed chimeric mice, tolerance in B6-derived B cells is sufficient to prevent autoantibody production because only congenic B cells produce anti-nuclear Abs. Thus, in mice with the shorter interval T and B cell defects must act together to produce the autoimmune phenotype. In mice with the longer NZB interval, autoimmunity is exacerbated by the presence of an additional susceptibility locus that leads to enhanced T and B cell activation through generation of a positive feedback loop. The data provide new insights into the nature of the immune defects that produce autoimmunity in NZB chromosome 1 congenic mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 (B6), C57BL/6.Thy1aIgHaGpia (denoted here as B6.Thy1aIgHa), and B6.129S2-Tcratm1Mom (B6.TCR{alpha}–/–) breeders were purchased from The Jackson Laboratory. NZB mice were purchased from Harlan Sprague Dawley. Congenic mice were produced by backcrossing mice with NZB chromosome 1 intervals onto the B6 background, using the speed congenic technique. Mice were typed at each successive generation using polymorphic microsatellite markers that discriminate between NZB and B6 DNA. Markers were spaced at ~20 cM intervals throughout the genome, except for regions containing lupus susceptibility genes where more densely spaced markers were used. Fully backcrossed mice were obtained in six to seven generations and intercrossed to produce congenic mice that were homozygous for NZB chromosome 1 intervals. Mice used in this study, denoted B6.NZBc1L and B6.NZBc1S for simplicity, correspond to the B6.NZBc1 (35–106) and B6.NZBc1 (85–106) mice published previously (18). B6.NZBc1L and B6.NZBc1S mice have introgressed NZB intervals extending from between D1Mit21 (32.8 cM) and D1Mit303 (34.8 cM) or D1Mit396 (79 cM) and D1Mit159 (81.6 cM) to between D1Mit223 (106 cM) and D1Mit210 (109 cM). The borders of these intervals have been refined from our previous publication by identification and testing of additional polymorphic primers. B6.NZBc1L and B6.NZBc1S mice expressing transgenes encoding IgM/IgD H and L chains specific for hen egg white lysozyme (anti-HEL Ig transgenic (Tg), MD4; Ref.19) were produced by backcrossing the anti-HEL Ig transgene from B6 anti-HEL Ig Tg mice (purchased from The Jackson Laboratory). Offspring were genotyped by PCR, using primers specific for the V region of the Ig H chain. B6.NZBc1L.TCR{alpha}–/– mice were produced by backcrossing the gene deletion onto the B6.NZBc1L background and then intercrossing to produce knockout mice. TCR{alpha} knockout mice were genotyped by PCR, using neo- and TCR{alpha}-specific primers. The mice were housed in microisolators in the animal facility at the Toronto Western Hospital and were specific-pathogen free. All of the mice used in this study were female.

Generation of bone marrow reconstituted and mixed chimeric mice

For bone marrow reconstitution experiments, 6- to 8-wk-old female B6.Thy1aIgHa recipient mice were put on sterilized acid water at least 1 wk before reconstitution. On the day of cell transfer, recipient mice were given two doses of gamma radiation (525 rad) separated by 2 h and injected, through the tail vein, with 1 x 107 T cell-depleted bone marrow cells from B6 or B6.NZBc1L mice. For mixed chimeras, 6- to 8-wk-old female B6 mice were treated, as above, and injected with a mixture of T cell-depleted bone marrow from B6.Thy1aIgHa mice and B6 or B6.NZBc1L mice at a ratio of 3:1 or 1:1. Recipients were sacrificed and analyzed 4 or 7 mo later.

Flow cytometry analysis

RBC-depleted splenocytes (5 x 105) were incubated with 10 µg/ml mouse IgG (Sigma-Aldrich) for 15 min to block FcRs and stained with various combinations of directly conjugated mAbs. Following washing, allophycocyanin-conjugated streptavidin (BD Pharmingen) was used to reveal biotin-conjugated Ab staining. Dead cells were excluded by staining with 0.6 µg/ml propidium iodide (Sigma-Aldrich). Flow cytometry of the stained cells was performed using a dual laser FACSCalibur (BD Biosciences) and analyzed using CellQuest (BD Biosciences) software. Live cells were gated on the basis of propidium iodide uptake and scattering characteristics with 10,000 events being acquired for each sample. The following directly conjugated mAbs were purchased from BD Pharmingen: biotin anti-CD11c (N418), biotin anti-CD11b (Mac1), biotin anti-CD4 (L3T4), biotin anti-CD8 (53-6.7), biotin anti-CD62L (MEL-14), biotin anti-B220 (RA3-6B2), biotin anti-Thy1.1 (HIS51), biotin anti-Thy1.2 (53-2.1), biotin anti-IgMa (DS-1), biotin anti-IgMb (AF6-78), biotin anti-IgG2aa (8.3), biotin anti-IgG2ab (5.7), PE anti-B7.1 (16-10A1), PE anti-B7.2 (GL1), PE anti-CD3 (145-2C11), PE anti-CD69 (H1.2F3), PE anti-CD44 (IM7), PE anti-NK1.1 (PK136), PE anti-CD4 (H129.19), FITC anti-CD3 (145-2C11), FITC anti-CD4 (L3T4), FITC anti-CD8 (53-6.7), FITC anti-CD21/CD35 (7G6), FITC anti-CD25 (7D4), FITC anti-B220 (RA3-6B2), FITC anti-TCR{beta} (H57-597), and FITC anti-TCR{delta}{gamma} (GL3). FITC anti-CD62L was purchased from Cedarlane Laboratories. Biotinylated HEL was prepared using an EZ-Link Sulfo-NHS-LC Biotinylation kit (Pierce). All isotype controls, with the exception of hamster IgG controls (BD Pharmingen), were purchased from Cedarlane Laboratories.

APC isolation

Single-cell suspensions of splenocytes were isolated from 2- to 3-mo-old B6 mice by pressing through a nylon mesh. RBCs were removed by lysis in Geys’ solution. The cell suspension was then incubated with purified anti-Thy1.2 (HO-13-4) and anti-CD4 (GK1.5) mAb followed by guinea pig complement (Cedarlane Laboratories) to remove T cells. Following lysis, the resultant cell population (<1% CD4+ T cell contamination) was resuspended in RPMI 1640 containing 0.5% normal mouse serum (NMS).

CD4+ T cell isolation

Single-cell suspensions of RBC-depleted splenocytes were suspended in 2% FBS/PBS and incubated with biotinylated anti-B220, -I-Ad, -CD8, -Mac1, -CD11c, and -CD24 Abs on ice for 30 min. Excess Abs were removed by washing, and the cells were then resuspended in 2% FBS/PBS at a concentration of 100 x 106 cells/ml. Streptavidin-conjugated Dynabeads (M-280; Dynal Biotech) were washed with 2% FBS/PBS to remove azide and resuspended at 160 x 106 beads/ml in PBS. An equal volume of washed Dynabeads was added to the cell suspension and incubated at room temperature for 1 h. CD4+ T cells were then purified by negative selection with a magnet and resuspended in 0.5% NMS/RPMI 1640. The resultant cell population contained >80% CD4+ T cells.

T cell proliferation and cytokine assays

T cell-depleted splenic APCs, isolated from B6 mice, were irradiated with 2000 rad and then incubated for 1 h at 37°C with medium alone or containing 1 µg/ml bovine total histones, H1, H2A, H2B, H3, or H4 (Roche). Excess Ag was removed by washing with PBS, and the cells were resuspended in 0.5% NMS/RPMI 1640. Ag-pulsed APCs were cultured at 0.5 x 106 cells/well together with purified CD4+ T cells (0.5 x 106 per well) for 48 h at 37°C in 96-well flat-bottom plates. Anti-CD3 (5 µg/ml) or Con A (5 µg/ml) were added to control wells at the time of plating. Proliferation was measured by [3H]thymidine incorporation after a 16-h pulse with 1 µCi/well. Uptake of [3H]thymidine was quantified by a scintillation counter and expressed as mean counts per minute ± SD of triplicate wells. For each Ag condition, a stimulation index was calculated by dividing the mean counts per minute in the presence of Ag by the mean counts per minute in the absence of Ag.

IL-4 and IFN-{gamma} levels in tissue culture supernatants were measured at 48 h. Anti-IL-4 and -IFN-{gamma} capture Abs, biotinylated-anti-IL-4 and -IFN-{gamma} detection Abs, and rIL-4 and IFN-{gamma} were purchased from BD Pharmingen. Assays were performed as per the manufacturer’s recommendations. The concentration of cytokine in each supernatant was calculated from a standard curve with log-log plot of absorbance vs concentration of recombinant cytokine preparation.

Measurement of Ab production

Levels of IgM, IgMa, IgMb, IgG, IgG2a, IgG2aa, and IgG2ab anti-chromatin and -ssDNA Abs in the sera were measured by ELISA. ssDNA was prepared by boiling dsDNA (isolated from calf thymus DNA) for 10 min and quick cooling on ice. H1-stripped chromatin was prepared from chicken RBCs as described previously (20). ELISA plates were coated overnight with either ssDNA (20 µg/ml) or chromatin (8 µg/ml) in PBS, washed with 0.05% Tween 20/PBS, and blocked with 2% BSA/PBS for 1 h at room temperature. After washing, serum samples were diluted 1/100 in 2% BSA/0.05% Tween 20/PBS, added to ELISA plates, and incubated for 1 h at room temperature. The presence of bound Abs was detected by adding alkaline-phosphatase-conjugated anti-IgM, -IgG, or -IgG2a (Caltag Laboratories) as secondary reagents. For allotype-specific ELISAs, biotinylated anti-IgG2aa and -IgG2ab, or -IgMa and -IgMb mAb (BD Pharmingen) were used and detected with alkaline-phosphatase-conjugated streptavidin (BD Pharmingen). To control for differences in the ability of the allotype-specific Abs to detect bound Ab, control sera were run on each assay that were of "a" (MRL/lpr/lpr) or "b" (B6.NZBc1L) allotype, and the results for allotype-specific assays were normalized to those seen with assays using anti-IgG2a or -IgM Abs that detected both allotypes.

Immunofluorescence staining of tissue sections

Spleens were snap frozen in OCT compound (Sakura Finetek) at the time of sacrifice. Cryostat sections (5 µm) were fixed in acetone, washed with PBS, and blocked with 5% normal goat serum/PBS. Sections were stained with biotinylated peanut lectin (agglutinin) (PNA) (Sigma-Aldrich) with or without FITC anti-CD21 to detect germinal centers. For allotype-specific Ab staining of germinal centers, sections were stained with biotinylated anti-IgMa, -IgMb, -IgG2aa, or -IgG2ab mAb together with FITC anti-CD21. Biotin staining was revealed using rhodamine-conjugated streptavidin as a secondary reagent (Molecular Probes). Stained sections were mounted with Mowiol (Calbiochem) and tissue fluorescence visualized using a Zeiss Axioplan 2 imaging microscope (Zeiss). Digital images were obtained using the manufacturer’s imaging system. Germinal centers were scored as positive for IgMa, IgMb, IgG2aa, and IgG2ab cells, when one or more cells could be clearly localized to the PNA+ region of the germinal center using CD21 staining to align serial sections. Cells that stained with anti-allotype Ab but were CD21bright, and thus likely to be follicular dendritic cells, were excluded from the analysis.

Statistics

Comparisons of differences between groups of mice were performed using a Mann-Whitney U nonparametric test with the exception of IFN-{gamma} levels where a Wilcoxon two-sample test was used. Values of p < 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Reconstitution of B6 mice with bone marrow cells from B6.NZBc1L mice transfers the autoimmune phenotype

To determine whether the genetic polymorphism(s) leading to autoimmunity in B6.NZBc1L mice affect bone marrow-derived immune cell populations, bone marrow cells from B6.NZBc1L (ThylbIgHb) or normal B6 (ThylbIgHb) mice were injected into lethally irradiated B6.ThylaIgHa mice, and the recipients were analyzed 6 mo postreconstitution. In these mice, the majority of T and B cells were derived from the donor bone marrow (%IgMb+B220+ cells > 90%; %Thy1b+CD4+ cells > 90%).

In B6.NZBc1L mice, autoimmunity is characterized by increased production of IgG anti-ssDNA and -chromatin Abs, together with markers of increased T and B cell activation (18). As shown in Table I, 6-mo-old B6.NZBc1L mice analyzed in tandem with reconstituted mice had the same phenotype that we have previously reported for 4-mo-old mice, including splenomegaly, increased proportions of activated (CD69+) and costimulatory molecule expressing B cells, and increased proportions of activated (CD69+) and memory (CD44highCD62Llow) CD4+ cells (18). All of these phenotypes were recapitulated in mice reconstituted with B6.NZBc1L bone marrow. Transfer of B6.NZBc1L bone marrow also resulted in the same altered distribution of peripheral B cell subsets as seen in B6.NZBc1L mice, with increased proportions of CD21lowCD23low and follicular B cells, and decreased proportions of T2 and marginal zone B cells (data not shown). As in our previous study, the increased costimulatory molecule expression was restricted to (for B7.1), or predominantly in (for B7.2 and ICAM-1), the CD21low B cell population (Table I and data not shown).


View this table:
[in this window]
[in a new window]
  		Table I. The autoimmune phenotype in B6.NZBc1L mice can be reproduced by reconstituting lethally irradiated B6.Thy1aIgHa mice with B6.NZBc1L bone marowa

 
Mice reconstituted with B6.NZBc1L bone marrow also had significantly elevated levels of IgG anti-chromatin (OD ± SD; B6.NZBc1L = 0.371 ± 0.261, B6 = 0.006 ± 0.002, p = 0.017) and -ssDNA Abs (OD ± SD; B6.NZBc1L = 0.224 ± 0.159, B6 = 0.018 ± 0.019, p = 0.033), as compared with mice reconstituted with B6 bone marrow. Thus, the immune defects leading to abnormal lymphocyte activation and autoantibody production in NZB chromosome 1 congenic mice affect bone marrow derived cell populations.

B6.NZBc1L mice have intrinsic B and T cell functional defects

To investigate the roles of intrinsic B and T cell functional abnormalities in production of the autoimmune phenotype in NZB chromosome 1 congenic mice, chimeric mice with a mixture of bone marrow from B6.NZBc1L and B6.ThylaIgHa mice were produced. Mice receiving a mixture of bone marrow from normal B6 and B6.ThylaIgHa were used as controls. Bone marrow mixtures were injected into 6- to 8-wk-old lethally irradiated B6 mice at a 3:1 or 1:1 ratio (ThylaIgHa:ThylbIgHb), and the recipients were assessed at 4 (3:1 ratio) or 7 (1:1 ratio) mo after reconstitution. Allotype-specific mAb for Thy1 and IgM was used to discriminate between B6 and B6.NZBc1L T and B cells.

Consistent with previous experiments using bone marrow from New Zealand Mixed (NZM)-derived congenic mouse strains (21, 22), chimeric mice injected with a 3:1 ratio of Thy1aIgHa:Thy1bIgHb bone marrows had similar proportions of Thy1a and Thy1b cells (B6 + B6.Thy1aIgHa, %Thy1a = 10.09 ± 0.84, %Thy1b = 8.106 ± 1.67; B6.NZBc1L + B6.Thy1aIgHa, %Thy1a = 14.14 ± 1.289, %Thy1b = 11.68 ± 2.997) and IgMa and IgMb (B6 + B6.Thy1aIgHa, %IgMa 42.74 ± 4.221, %IgMb 34.73 ± 4.348; B6.NZBc1L + B6.Thy1aIgHa, %IgMa 36.98 ± 3.787, %IgMb 35.85 ± 4.857). Further analysis of these mice revealed that B6.NZBc1L-derived B and T cells retained their abnormal activation phenotype (Fig. 1). In mice with a mixture of B6.NZBc1L and B6.Thy1aIgHa bone marrow, the proportion of Thy1b T cells that were CD69+ and CD44highCD62Llow was significantly increased when compared with Thy1a T cells and compared with Thy1b T cells in mice with a mixture of B6 and B6.Thy1aIgHa bone marrow (Fig. 1, A and B), indicating that the abnormal T cell activation in B6.NZBc1L mice results from an intrinsic T cell defect.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 1. Splenic B and T cell activation in a and b allotype cells from mixed chimeric mice. Freshly isolated splenocytes from 4-mo-old chimeric mice, receiving a mixture of B6 + B6.Thy1aIgHa bone marrow ({blacktriangleup} = a allotype cells, {triangleup} = b allotype cells) or B6.NZBc1L + B6.Thy1aIgHa bone marrow (• = a allotype cells, or {circ} = b allotype cells), were stained with anti-IgMa or -IgMb, anti-CD21, and anti-B7.1, -B7.2, -ICAM-1, or -CD69 Abs to assess B cell activation. T cell activation was investigated by staining with anti-Thy1a or -Thy1b, and anti-CD44 and -CD62L, or anti-CD4 and -CD69, Abs. Shown are the (A) percentage of CD69+ cells within the CD4+ subset, (B) percentage of CD62LlowCD44high cells, (C) percentage of CD69+ cells, (D) percentage of B7.1+ cells within the CD21low subset, (E) MFI for B7.2 staining in the CD21low subset, or (F) MFI for ICAM-1 in the CD21low subset, gating independently on Thy1a or Thy1b (A and B), or IgMa or IgMb (C–F), positive populations. Each symbol represents the determination from an individual mouse. Horizontal lines indicate the mean for each population examined.

 
In contrast to the findings observed for T cells, the proportions of CD69+ IgMa and IgMb B cells in chimeric mice with a mixture of B6.NZBc1L and B6.Thy1aIgHa bone marrow were comparable and significantly elevated when contrasted with their counterparts in mice with a mixture of B6 and B6.Thy1aIgHa bone marrow (Fig. 1C). Nevertheless, costimulatory molecule expression was higher in IgMb than IgMa B cells. In the total B cell population, this achieved statistical significance for B7.2 and ICAM-1 (%B7.1+, IgMa = 16.03 ± 3.23, IgMb = 19.81 ± 4.26, p = NS; mean fluorescent intensity (MFI) B7.2, IgMa = 11.90 ± 1.31, IgMb = 15.38 ± 1.23, p = 0.0002; MFI ICAM-1, IgMa = 206.4 ± 21.91, IgMb = 279.2 ± 67.88, p = 0.015). Staining with anti-CD21 to discriminate between peripheral B cell subsets revealed that costimulatory molecule expression on IgMb cells was significantly elevated as compared with IgMa cells only in the CD21low population (Fig. 1, D–F). Although costimulatory molecule expression was increased somewhat in the CD21int and CD21high populations in mice reconstituted with B6.NZBc1L + B6.Thy1aIgHa bone marrow as compared with mice reconstituted with B6 + B6.Thy1aIgHa bone marrow, this did not differ between IgMa and IgMb B cells. Thus, B6.NZBc1L mice have an intrinsic B cell defect that leads to altered up-regulation of costimulatory molecules in the CD21low population.

We next assessed whether B6 and B6.NZBc1L B cells equivalently differentiate into Ab producing cells. In B6.NZBc1L mice, IgG autoantibodies of the IgG2a, but not IgG1, subclass are produced (data not shown). As shown in Fig. 2A, chimeric mice receiving B6.NZBc1L + B6.Thy1aIgHa bone marrow cells, also had significantly increased levels of IgG2a anti-ssDNA (p < 0.05) and -chromatin (p < 0.005) Abs as compared with B6 controls and B6 + B6.Thy1aIgHa bone marrow chimeric mice. Furthermore, the levels of autoantibodies produced were comparable to those seen in 16-wk-old B6.NZBc1L mice. Characterization of the origin of these Abs using allotype specific Abs revealed that only "b" allotype IgG2a anti-ssDNA (p < 0.05) and -chromatin (p < 0.005) Abs were produced (Fig. 2B). Consistent with our previous results, unmanipulated B6.NZBc1L and chimeric mice receiving B6.NZBc1L + B6.Thy1aIgHa bone marrow cells did not produce significantly increased levels of IgG2a anti-dsDNA Abs (OD ± SD; IgG2a anti-dsDNA, B6 = 0.076 ± 0.053, B6.NZBc1L = 0.135 ± 0.132, p = NS; B6 + B6.Thy1aIgHa = 0.098 ± 0.027, B6.NZBc1L + B6.Thy1aIgHa = 0.237 ± 0.168, p = NS). Nevertheless, "b" allotype IgG2a anti-dsDNA levels were increased slightly in mice reconstituted with B6.NZBc1L + B6.Thy1aIgHa bone marrow cells (OD ± SD; IgG2a anti-dsDNA; "a" allotype, B6 + B6.Thy1aIgHa = 0.053 ± 0.020, B6.NZBc1L + B6.Thy1aIgHa = 0.034 ± 0.014, p = NS; "b" allotype, B6 + B6.Thy1aIgHa = 0.032 ± 0.015, B6.NZBc1L + B6.Thy1aIgHa = 0.150 ± 0.170, p = 0.03). Similar results were obtained for mice analyzed at 7 mo postreconstitution (data not shown).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 2. Autoantibody levels in mixed chimeric mice. Serum samples from (A) 4-mo-old B6 ({blacktriangleup}), B6.NZBc1L (•), or chimeric mice receiving either a mixture of B6 + B6.Thy1aIgHa bone marrow ({triangleup}) or B6.NZBc1L + B6.Thy1aIgHa bone marrow ({circ}) were assayed for the presence of IgG2a anti-ssSNA or chromatin Abs by ELISA. B, Allotype-specific ELISA were used to detect the presence of IgG2aa and IgG2ab anti-ssDNA and anti-chromatin Abs in chimeric mice receiving a mixture of B6 + B6.Thy1aIgHa bone marrow ({blacktriangleup} = IgG2aa and {triangleup} = IgG2ab) or B6.NZBc1L + B6.Thy1aIgHa bone marrow (• = IgG2aa and {circ} = IgG2ab). Each symbol represents the determination from an individual mouse. Horizontal lines indicate the mean for each population examined. Significantly higher titers of "b" allotype anti-ssDNA (p < 0.05) and anti-chromatin (p < 0.005) Abs were detected in mice receiving a combination of B6.NZBc1L + B6.Thy1aIgHa bone marrow.

 
Although B6.NZBc1L mice produce relatively low titers of IgM anti-nuclear Abs, significantly elevated levels of IgMb, but not IgMa, anti-ssDNA and -chromatin Abs could be detected in mice reconstituted with B6.NZBc1L + B6.Thy1aIgHa bone marrow cells (OD ± SD; anti-ssDNA, IgMa = 0.054 ± 0.027, IgMb = 0.562 ± 0.361, p = 0.0002; anti-chromatin, IgMa = 0.038 ± 0.027, IgMb = 0.486 ± 0.49, p = 0.0003). Thus, despite the presence of T cells that can provide help for Ab production in B6.NZBc1L + B6.Thy1aIgHa chimeric mice, only B6.NZBc1L B cells can differentiate into autoantibody-producing cells or undergo class-switching.

We have shown previously that B6.NZBc1L mice have a markedly increased number of germinal centers as compared with B6 and B6.NZBc1S mice (18). This phenotype was shared by B6.NZBc1L + B6.Thy1aIgHa chimeric mice (Fig. 3, A–C), who had a significantly increased number of germinal centers as compared with mice with B6 + B6.Thy1aIgHa bone marrow cells (B6 + B6.Thy1aIgHa = 5.80 ± 2.59, B6.NZBc1L + B6.Thy1aIgHa = 23.50 ± 11.59, p < 0.005). To determine whether B6 and B6.NZBc1L B cells are equivalently recruited into germinal centers, spleen sections were stained with anti-CD21 and PNA to reveal germinal centers and then serial sections stained with anti-CD21 and anti-IgMa or -IgMb mAb to permit identification of B6 and B6.NZBc1L germinal center B cells. A total of 15 PNA+ germinal centers from three different B6 + B6.Thy1aIgHa controls and 30 germinal centers from four different B6.NZBc1L + B6.Thy1aIgHa chimeric mice were scored for the presence of IgMb- and IgMa-expressing cells within the PNA+ population. In B6 + B6.Thy1aIgHa controls, equivalent proportions of germinal centers contained IgMb (11 of 15) and IgMa (11 of 15) cells. In addition, bright staining with anti-IgMa and anti-IgMb mAb could be detected on a subset of the CD21bright cells. This staining was granular in nature suggesting that the anti-IgMa and anti-IgMb mAbs were binding to immune complexes on the surface of the follicular dendritic cells. These data indicate that in normal mice both IgMb- and IgMa-expressing cells are similarly recruited into germinal centers and produce IgM Abs that can complex with germinal center Ags. In contrast, there were significantly more IgMb+ germinal centers (27 of 30) than IgMa+ germinal centers (8 of 30) in B6.NZBc1L + B6.Thy1aIgHa chimeric mice (p < 0.0001 Fisher’s exact test), indicating that B6.NZBc1L B cells are preferentially recruited into autoreactive germinal centers. In support of this concept and consistent with the significantly elevated levels of IgMb, but not IgMa, autoantibodies in B6.NZBc1L + B6.Thy1aIgHa chimeric mice, bright granular staining of CD21high cells was seen only with anti-IgMb mAb (Fig. 3, D–F).



View larger version (136K):
[in this window]
[in a new window]
  		FIGURE 3. Assessment of "a" and "b" allotype B cells in splenic germinal centers. Spleens from (A) B6 + B6.Thy1aIgHa bone marrow or (B) B6.NZBc1L + B6.Thy1aIgHa bone marrow chimeric mice were stained with PNA to delineate germinal centers. C, The PNA staining for the spleen of a representative 4-mo-old B6.NZBc1L mouse for comparison. D–F, A germinal center from a B6.NZBc1L + B6.Thy1aIgHa chimeric mouse. All sections were stained with anti-CD21 to reveal follicular dendritic cells within the germinal center. Shown is one of the few germinal centers with both (E) IgMa and (F) IgMb staining cells within the (D) PNA+ region. Another germinal center from a B6.NZBc1L + B6.Thy1aIgHa chimeric mouse stained with (G) anti-CD21 and PNA to reveal germinal centers. Staining of the same germinal center with anti-IgG2aa (H) and -IgG2ab (I). As shown in these panels, none of the germinal centers stained with anti-IgG2aa, whereas germinal centers with abundant IgG2ab staining cells were seen frequently. Shown in the top right corner of each panel are the stains performed. Magnification: A–C, x2.5; D–F, x40; G–I, x20.

 
Because the majority of PNA+ cells within the germinal centers were IgMa and IgMb negative (Fig. 3, D–F), we stained additional sections with anti-CD21 and anti-IgG2aa or IgG2ab to determine whether the germinal center B cells had undergone class-switching. As shown in Fig. 3, G–I, the majority of germinal centers (60%) contained large numbers of IgG2ab cells, and these were found within both the PNA+ and PNA– populations. In contrast, no IgG2aa cells could be seen in any of the germinal centers of B6.NZBc1L + B6.Thy1aIgHa chimeric mice.

Taken together, these findings indicate that B6.NZBc1L B cells have an intrinsic functional defect that leads to enhanced recruitment of these cells into autoreactive germinal centers and permits class switching of autoreactive B cells.

Increased activation of histone-reactive CD4+ T cells in B6.NZBc1L and B6.NZBc1S mice

B6.NZBc1S and B6.NZBc1L mice produce significantly elevated levels of IgG anti-chromatin Abs (18). Because histone-reactive T cells have been shown to provide support for IgG anti-chromatin Ab production (23, 24), we investigated whether histone-reactive T cells were activated in B6.NZBc1S and B6.NZBc1L mice. To this end, chromosome 1 congenic and control mice were aged to 8 mo, and their splenic CD4+ T cells were purified by negative selection. The purified T cells were then incubated with irradiated T cell-depleted B6 APCs together with various concentrations of purified bovine H1, H2A, H2B, H3, H4 or total histones, and T cell activation quantified by measurement of cytokine secretion and proliferation.

As shown in Fig. 4A, T cells from both B6.NZBc1L and B6.NZBc1S mice demonstrated increased production of IFN-{gamma} as compared with B6 T cells for virtually all of the histones tested. This achieved statistical significance in B6.NZBc1L mice with H1 (p < 0.05) and H3 (p < 0.05) stimulation and in B6.NZBc1S mice with total histone (p < 0.05) and H2A (p < 0.05) stimulation. In contrast, no IL-4 was elaborated following stimulation with any of the histones tested (data not shown). This did not reflect a general inability of these mice to produce IL-4 because IL-4 could be readily detected in the supernatants of wells stimulated with anti-CD3 or Con A, and the levels of these cytokines were comparable in all three strains (data not shown).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 4. Presence of T cells responsive to nucleosome histone components in B6.NZBc1L and B6.NZBc1S mice. Splenic CD4+ T cells, isolated from 8- to 9-mo-old B6 ({blacktriangleup}), B6.NZBc1L (•) and B6.NZBc1S ({blacksquare}) mice, were cultured together with B6 T cell-depleted APCs pulsed with 1 µg/ml of total histones, H1, H2A, H2B, H3, or H4. A, Histone-induced IFN-{gamma} production. IFN-{gamma} was measured in the supernatant of cells cultured for 48 h. Each symbol represents the mean of a triplicate determination from an individual mouse. B, Histone-induced proliferation. Cells were cultured for 48 h and pulsed for the last 16 h with [3H]thymidine. Uptake of [3H]thymidine was quantified by a scintillation counter and expressed as mean counts per minute ± SD of triplicate wells. The stimulation index was calculated by dividing the mean counts per minute in presence of Ag by the mean counts per minute in the absence of Ag (i.e., T cells + APCs only). Each symbol represents the determination from an individual mouse. Horizontal lines indicate the mean for each population examined. Mean background proliferation (T cells + APCs only) was similar for B6, B6.NZBc1L, and B6.NZBc1S T cells.

 
In contrast to the findings for cytokine secretion, significant Ag-specific proliferation following stimulation with histones was seen only for B6.NZBc1L mice, with CD4+ T cells from B6.NZBc1L mice demonstrating significantly increased proliferation in response to stimulation with total histones (p = 0.0005), H2B (p < 0.0001), and H3 (p < 0.05) (Fig. 4B). T cells from B6.NZBc1L mice were not generally more hyperproliferative because these cells proliferated comparably to B6 and B6.NZBc1S cells in response to stimulation with Con A and anti-CD3. Although T cells from 8-mo-old B6.NZBc1S mice did not proliferate in response to histones, proliferation could be detected with T cells from 12-mo-old mice (data not shown). This finding suggests that differences between B6.NZBc1S and B6.NZBc1L mice at 8 mo of age reflect accelerated kinetics of the response in B6.NZBc1L mice rather than qualitative differences. Taken together, the data indicate that the lupus susceptibility locus located within the ~82–106 interval leads to activation of T cells specific for nucleosome histone components and that the susceptibility locus in ~35–82 interval serves to enhance this process.

Self-reactive B cells drive T cell activation in B6.NZBc1L but not B6.NZBc1S mice

We have shown previously that introduction of a nonself reactive Ig transgene recognizing hen egg white lysozyme (Ig Tg) onto the B6.NZBc1L background resulted in normalization of the B cell activation phenotype (18), suggesting that the increased B cell activation in these mice reflects enhanced activation of self-reactive B cells. As a result of this constriction of the B cell repertoire, the proportions of activated (CD69+) and memory (CD44highCD62Llow) CD4+ T cells in B6.NZBc1L Ig Tg mice were significantly reduced, indicating that autoreactive B cells were amplifying T cell activation. However, the proportion of memory T cells in B6.NZBc1L Ig Tg mice was not normalized to levels seen in B6 Ig Tg mice but remained significantly elevated at levels comparable to those seen in B6.NZBc1S non-Tg mice. This suggested that the increased activation of T cells observed in B6.NZBc1S mice might be B cell independent. To address this question, the Ig Tg was backcrossed onto the B6.NZBc1S background.

As in our previous study, >95% of B cells in Ig Tg mice were HEL-specific (data not shown) and the proportion of activated B cells was normalized in B6.NZBc1L and B6.NZBc1S Ig Tg mice to levels seen in control B6 Ig Tg mice (Table II). Consistent with our previous results, the proportions of CD69+ and CD44highCD62Llow CD4+ T cells in B6.NZBc1L mice were markedly reduced by introduction of the Ig Tg but remained elevated as compared with B6 Ig Tg mice at levels seen in non-Tg B6.NZBc1S mice (Fig. 5). In contrast, introduction of the Ig Tg on the B6.NZBc1S background had no impact on T cell activation, with the proportion of memory T cells remaining significantly higher than corresponding B6 Ig Tg controls (Fig. 5). These data indicate that T cell activation in B6.NZBc1L, but not B6.NZBc1S mice, is amplified by the presence of activated self-reactive B cells.


View this table:
[in this window]
[in a new window]
  		Table II. Normalization of the B cell activation phenotype in B6.NZBc1L and B6.NZBc1S mice by introduction of an Ig transgenea

 


View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 5. Impact of an Ig transgene on T cell activation in B6.NZBc1L or B6.NZBc1S congenic mice. Freshly isolated splenocytes from 4-mo-old mice were stained with anti-CD4 together with anti-CD69, or anti-CD62L and -CD44, to determine the proportion of (A) activated CD69+, or (B) memory CD44highCD62Llow, CD4+ T cells in non-Tg (NTg, {blacktriangleup} = B6, • = B6.NZBc1L and {blacksquare} = B6.NZBc1S), and anti-HEL Ig Tg (Tg, {triangleup} = B6, {circ} = B6.NZBc1L and {square} = B6.NZBc1S) mice. Each symbol represents the determination from an individual mouse, with horizontal lines indicating the mean for each population.

 
The increased B cell activation in B6.NZBc1L mice is T cell dependent, indicating the presence of a positive feedback loop

In NZB mice, one of the manifestations of polyclonal B cell activation is increased costimulatory molecule expression on diverse B cell subsets (25). This phenotype is seen in 3- to 4-wk-old mice and is CD40L and T cell independent, suggesting that it arises from an intrinsic B cell defect (J. Wither, N. Chang, C. Loh, G. Bonventi, S. Henrichs, Y. Cai, G. Lajoie, V. Ray, and R. MacLeod, manuscript in preparation; our unpublished observations). In B6.NZBc1L mice, increased costimulatory molecule expression does not develop until ~4 mo of age and, as outlined above, is most marked in the CD21low B cell subset. Because of these differences, we were interested in determining whether the B cell activation phenotype in these mice is similarly T cell-independent. Therefore, we crossed a TCR{alpha}-chain knockout onto the B6.NZBc1L genetic background and assessed B cell costimulatory molecule expression in 4- to 6-mo-old mice using flow cytometry.

Comparison of B cell costimulatory molecule expression on the CD21low B cell population of B6.NZBc1L.TCR{alpha}+/– and B6.NZBc1L.TCR{alpha}–/– mice revealed that B7.1 (p < 0.0001) and B7.2 (p < 0.001) were significantly reduced in knockout mice (Fig. 6). Although there was a trend to decreased ICAM-1 expression, this did not achieve statistical significance. Similar findings were observed for CD69 expression in the total B cell population (%B cells CD69+, B6.NZBc1L.TCR{alpha}+/– = 8.75 ± 3.26, B6.NZBc1L.TCR{alpha}–/– = 6.25 ± 2.57, p < 0.05). Costimulatory molecule expression in the CD21int and CD21high populations was not increased in B6.NZBc1L.TCR{alpha}+/–, as compared with B6.TCR{alpha}+/– mice, and there was no impact of the TCR knockout on this expression (data not shown). The proportion of B cells in peripheral B cell subsets as defined by staining with anti-CD21 and -CD23 was also unaffected by the TCR knockout (data not shown).



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 6. Costimulatory molecule expression on splenic B cells of TCR{alpha}+/+, TCR{alpha}+/–, and TCR{alpha}–/– mice. Freshly isolated splenocytes from 4- to 6-mo-old TCR{alpha}+/+ mice (• = B6, {blacktriangleup} = B6.NZBc1L), TCR{alpha}+/– mice ({circ} = B6, {triangleup} = B6.NZBc1L), and TCR{alpha}–/– mice ({diamond} = B6, {triangledown} = B6.NZBc1L) were stained with anti-B220, anti-CD21, and anti-B7.1 or -B7.2 Abs. The percentage of B7.1+ cells and MFI for B7.2 in the CD21lowB220+ population are shown. Each symbol represents the determination from an individual mouse, with horizontal lines indicating the mean for each population.

 
Despite decreased costimulatory molecule expression in the CD21low subset of B6.NZBc1L.TCR{alpha}–/– mice, B7.1 remained elevated in this subset relative to B6.TCR{alpha}–/– mice (p < 0.0005). It is likely that this residual B cell activation results from activation by the few residual T cells in these mice. Both B6.TCR{alpha}–/– and B6.NZBc1L.TCR{alpha}–/– mice had small populations of T cells that stained with anti-TCR {beta} and {gamma}{delta} mAb, and germinal centers could still be seen in these mice (data not shown). Consistent with the presence of residual T cell help in these mice, B6.NZBc1L.TCR{alpha}–/– mice produced IgG anti-ssDNA Abs (OD ± SD; B6.NZBc1L.TCR{alpha}+/– = 0.525 ± 0.747, B6.NZBc1L.TCR{alpha}–/– = 0.389 ± 0.258, p = NS) and -chromatin Abs (OD ± SD; B6.NZBc1L.TCR{alpha}+/– = 0.360 ± 0.267, B6.NZBc1L.TCR{alpha}–/– = 0.374 ± 0.286, p = NS). In keeping with previous reports (26, 27), B6.TCR{alpha}–/– mice also produced IgG anti-ssDNA Abs (OD ± SD; B6.TCR{alpha}+/– = 0.096 ± 0.062, B6.TCR{alpha}–/– = 0.462 ± 0.492, p < 0.05) and showed a trend to increased IgG anti-chromatin Ab production (OD ± SD; B6.TCR{alpha}+/– = 0.095 ± 0.068, B6.TCR{alpha}–/– = 0.145 ± 0.189, p = NS).

The observation that there was no difference between B6.TCR{alpha}–/– and B6.NZBc1L.TCR{alpha}–/– mice in the proportions of the residual T cell populations or their activation (as indicated by expression of CD69) suggests that the increased B7.1 expression in B6.NZBc1L.TCR{alpha}–/–, as compared with B6.TCR{alpha}–/– B cells, reflects an intrinsic B cell defect that affects the response to T cell help (data not shown). Thus, T cells drive the activation of self-reactive B cells in B6.NZBc1L mice, which in turn amplify T cell activation (as demonstrated in Ig Tg mice), resulting in a positive feedback loop and exacerbation of autoimmune disease in B6.NZBc1L mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Congenic mice are excellent tools for dissecting the complex genetic basis of murine SLE. To define the functional defects associated with the development of autoimmunity on NZB chromosome 1, we generated mice with short and long chromosome intervals. In our previous study, we provided evidence for at least two genetic loci on chromosome 1. One located within the ~82–106 cM interval affecting T cell activation and the second within the ~35- to 82 cM interval that appears to affect B cell activation and leads to enhanced autoantibody production, glomerulonephritis, splenomegaly, and T cell activation (18). In the present study, we have demonstrated that the altered T and B cell activation in these mice results from intrinsic T and B cell functional defects and provide insight into the nature and location of these defects together with their role in the development of autoimmunity.

Although our mixed chimeric experiments used B6.NZBc1L bone marrow cells, our findings permit localization of intrinsic B and T cell defects not only to the B6.NZBc1L interval (~35–106 cM) but also to the B6.NZBc1S interval (~82–106 cM). In B6.NZBc1S mice, autoimmunity is characterized by production of IgG anti-ssDNA and -chromatin Abs. We show that production of these autoantibodies is critically dependent on the presence of an intrinsic B cell defect. Similarly, the increased proportion of CD4+ memory T cells, a phenotype localized to the B6.NZBc1S interval, is restricted to congenic T cells in B6.NZBc1L + B6.Thy1aIgHa mixed chimeric mice. We further characterized this T cell defect by demonstrating that the abnormal T cell activation in B6.NZBc1S mice is B cell independent and associated with priming of IFN-{gamma}-producing T cells with specificity for nucleosome histone components. Thus, in B6.NZBc1S mice, an intrinsic T cell defect that leads to abnormal activation of histone-reactive T cells and an intrinsic B cell defect that permits the abnormal differentiation of autoreactive B cells into Ab-producing cells act together to produce autoimmunity.

Our data also provides insight into how the additional susceptibility allele(s) in B6.NZBc1L mice leads to enhancement of autoimmunity. Using B6.NZBc1L + B6.Thy1aIgHa chimeric mice we show that enhanced up-regulation of costimulatory molecules, a phenotype that we have localized to the ~35–82 interval, reflects an intrinsic B cell defect. We further demonstrate that the up-regulation of costimulatory molecules in B6.NZBc1L B cells is T cell dependent and requires IgR engagement, suggesting that self-reactive B cells in these mice have altered responses to T cell help. In support of this concept, we have found that B6.NZBc1L anti-HEL Ig Tg B cells demonstrate enhanced differentiation to Ab-producing cells following transfer into B6.NZBc1L soluble HEL recipient mice (N.-H. Chang and J. E. Wither, unpublished observations). The more robust T cell activation in B6.NZBc1L, as compared with B6.NZBc1S, mice appears to be driven by interactions with these functionally altered self-reactive B cells, and it is probable that the enhanced histone-specific proliferative response of B6.NZBc1L T cells is due to the same process.

Despite the presence of activated self-reactive congenic B cells in B6.NZBc1L + B6.Thy1aIgHa mixed chimeric mice, activation of B6 T cells is not elevated above that seen in B6 + B6.Thy1aIgHa mixed chimeric mice. This observation suggests that the ability of B cells to drive T cell activation is dependent on the presence of an intrinsic T cell defect. Thus, in B6.NZBc1L mice, interactions between at least two independent susceptibility loci lead to a positive feedback loop in which functionally altered T cells activate functionally altered B cells and vice versa, resulting in amplification of the autoimmune phenotype. These data provide an important experimental confirmation of the role of self-reinforcing B and T cell interactions in the pathogenesis of lupus, as originally proposed by Shlomchik et al. (28).

The lack of autoantibodies of B6 origin in B6.NZBc1L + B6.Thy1aIgHa chimeric mice suggests that activation of histone-reactive T cells in these mice is insufficient to break tolerance to chromatin. This finding contrasts with previous work indicating that anergic chromatin/dsDNA-reactive B cells can become activated to enter germinal centers and differentiate into autoantibody producing cells, if provided with a source of cognate T cell help (29, 30, 31). It is possible that T cell help in B6.NZBc1L + B6.Thy1aIgHa chimeric mice differs qualitatively or quantitatively from that seen in these studies. As outlined above, B6.NZBc1L and B6.NZBc1S mice mount a predominant Th1 response to histones, producing IFN-{gamma} but not IL-4. It has been shown that Th1 cells producing high levels of IFN-{gamma} can support anti-chromatin Ab production and germinal center formation, whereas T cells with lower levels of IFN-{gamma} provide only limited help for entry into germinal centers and do not support Ab production (32). Our findings for normal B6 B cells in B6.NZBc1L + B6.Thy1aIgHa chimeric mice are similar to those observed for T cells with low IFN-{gamma} levels, suggesting that the production of IFN-{gamma} may be limiting in these mice. This limited T cell help may be sufficient to provide support only for the functionally abnormal congenic B cells. Alternatively, regulatory T (Treg) cells could inhibit production of autoantibodies by normally tolerant B6 B cells in B6.NZBc1L + B6.Thy1aIgHa chimeric mice. There are increasing data, from a variety of experimental systems, where generation of Treg cells has been impaired, implicating Treg cells in the regulation of lupus autoantibody production (26, 33). In studies demonstrating a T cell-dependent loss of B cell tolerance to chromatin/dsDNA by normal B cells, tolerant B cells were experimentally manipulated to present nonself-peptides to T cells, thus evading normal T cell tolerance mechanisms (30, 31). In one of these experimental systems, addition of Treg cells prevented differentiation of chromatin/dsDNA B cells into Ab-forming cells as well as entry into germinal centers (31). In B6.NZBc1L + B6.Thy1aIgHa chimeric mice, B6 B cells may be more susceptible than congenic B cells to the effects of this Treg-mediated inhibition.

The preferential ability of congenic B cells to be recruited into autoreactive germinal centers and differentiate into autoantibody-forming cells in B6.NZBc1L + B6.Thy1aIgHa chimeric mice suggests that these B cells have an intrinsic defect that disturbs tolerance. B cell tolerance to chromatin and dsDNA is maintained by multiple central and peripheral tolerance mechanisms, including clonal deletion, receptor editing, clonal anergy, follicular exclusion, down-regulation of the IgR, and germinal center tolerance mechanisms (34, 35, 36, 37). Our results do not support a clonal deletion and/or receptor editing defect in B6.NZBc1L mice. Up-regulation of CD69 in B6.NZBc1L and B6.NZBc1S B cells is dependent upon IgR engagement and interaction with T cells. In B6.NZBc1L + B6.Thy1aIgHa chimeric mice, increased proportions of both B6.NZBc1L and B6.Thy1aIgHa B cells express elevated levels of CD69, suggesting that similar proportions of B6 and congenic B cells have engaged self-Ags and received signals from autoreactive T cells. If clonal deletion and/or receptor editing was defective in congenic B cells, then the proportion of self-reactive congenic B cells, and thus CD69+ B cells, should have been increased compared with B6 B cells.

In normal mice dsDNA- and chromatin-reactive B cells that have evaded central deletion or editing are rendered anergic and/or down-regulate their IgRs (34, 35, 36). Although these cells can be recruited into germinal centers, in unmanipulated nonautoimmune mice they fail to differentiate into autoantibody-producing cells, undergo class-switching, or develop into memory cells (35). In vitro anergic B cells demonstrate impaired responses to IgR engagement (38, 39). For example, anergic B cells do not up-regulate costimulatory molecules following IgR cross-linking. It has been proposed this reduced expression of costimulatory molecules results in reduced T cell costimulation leading to decreased cytokine production and ineffective T cell help (40). Our results demonstrating differential expression of costimulatory molecules, but not CD69, on congenic, as compared with B6, B cells in B6.NZBc1L + B6.Thy1aIgHa chimeric mice, suggest that self-reactive congenic B cells may be "less" anergic than their B6 counterparts. As a consequence of reduced anergy, congenic B cells may more effectively activate self-reactive T cells leading to enhanced cytokine production and support for entry into germinal centers, Ab production, and class-switching.

The T cell- and IgR-dependent enhanced expression of costimulatory molecules in B6.NZBc1L mice was most marked in the CD21low B cell subset. Although transitional, CD5+, and germinal center B cells, as well as, plasmablasts, are found in the CD21low B cell subset, analysis of this subset in B6.NZBc1L mice has revealed that the majority of these cells are transitional and germinal center B cells (Y.-H. Cheung and J. E. Wither, unpublished observations). The increased proportion of CD21low B cells that we have previously identified in B6.NZBc1L mice is due to an increased number of germinal center B cells accumulating in this compartment and these cells have high levels of costimulatory molecules (data not shown). Therefore, it is probable that the increased proportion of congenic CD21low B cells with high levels of costimulatory molecules in B6.NZBc1L + B6.Thy1aIgHa chimeric mice reflects increased recruitment of these cells into germinal centers. Although we also noted a trend to increased expression of B7.1 on AA4.1+ IgMb, as compared with IgMa, immature B cells in these mice (data not shown).

Several candidate genes have been proposed for the ~82–106 cM interval found in B6.NZBc1S mice. These include Ifi202, Fcgr2b, and the SLAM/CD2 gene cluster (14, 15, 16, 17). Ifi202, a transcriptional regulator, has been proposed to promote the development of autoimmunity in NZB chromosome 1 congenic mice through impaired B cell apoptosis (14). As outlined above, our data are not consistent with a central B cell deletion defect. Furthermore, although we can readily demonstrate reduced apoptosis of NZB transitional B cells following IgM cross-linking, this process was normal in B6.NZBc1L mice (Ref. 50 and data not shown) Thus, if Ifi202 promotes autoimmunity in NZB mice, it is probable that it does so through some other mechanism.

A promoter polymorphism of the Fcgr2b gene, which leads to decreased expression of Fc{gamma}RIIB on germinal center B cells, has also been proposed to promote autoantibody production in NZB mice (15, 16). However, recent work indicates that the Fc{gamma}RIIB receptor has little impact on germinal center selection mechanisms and instead appears to play an important role in providing a negative feedback signal to germinal center B cells limiting progression of class-switched B cells to plasma cells (41). Therefore, it is unlikely that the B cell tolerance defect in B6.NZBc1L and B6.NZBc1S mice is due solely the Fcgr2b polymorphism in this interval.

Extensive polymorphisms in the SLAM/CD2 gene cluster were identified in B6.Slel congenic mice, with an introgressed NZW chromosome 1 interval, and proposed as candidate genes for the NZM mouse strain (17). NZB mice have the same SLAM/CD2 haplotype and likely share the same T cell signaling abnormality as this strain. It should be noted that B6.Sle1 mice have a similar T cell activation phenotype to B6.NZBc1S mice, with a B cell-independent increase in T cell activation and evidence of spontaneous priming of histone-reactive T cells (21, 42). However, recent work suggests that in B6.Sle1 mice this phenotype is derived from the Sle1a and Sle1c loci, and not the SLAM/CD2 cluster-containing Sle1b locus (43). Thus, B6.NZBc1S mice may share more than one susceptibility allele with B6.Sle1 mice. If this is the case, then the most likely additional susceptibility allele is Sle1a because the polymorphism in Cr2 that has been proposed as a strong candidate gene for Sle1c is not found in NZB mice (44). Aberrant activation of B cells with increased IL-6 secretion, STAT3/SOCS activation, and ras/MAPK activation has also been described for B6.Sle1ab mice (45). The impact of these signaling abnormalities on B cell tolerance is currently unclear, but our data suggest that it is likely that similar abnormalities are present in B6.NZBc1S mice. Ongoing experiments in the laboratory are seeking to determine which aspects of the B cell tolerance defect that we have described for B6.NZBc1L B cells can be localized to the ~82–106 interval.

Although no candidate genes have been proposed for the ~35–82 interval, this interval contains a number of potential candidates including bcl-2, ship, and several tyrosine-phosphatase genes and regulators of G-protein signaling. Subcongenic mice are currently being generated to more precisely locate the region containing the susceptibility gene within this interval before further investigation of these genes.

The data outlined in this study clearly demonstrate that intrinsic B and T cell defects must act in concert to produce the autoimmune phenotype in B6.NZBc1L and B6.NZBc1S mice. They further indicate that epistatic genetic interactions can occur when functionally abnormal B and T cells interact with each other producing a positive feedback loop. Notably, humans with lupus share many of the features of B6.NZBc1L mice, including activation of histone-reactive B cells, increased B cell costimulatory molecule expression, and increased recruitment of B cells into germinal centers (46, 47, 48, 49). Therefore, it is tempting to speculate that similar functional abnormalities will be required for the development of human SLE.


   Acknowledgments
 
We thank Phillipe Poussier for critical reading of the manuscript and Andrew Paterson for assistance with the statistical analysis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and the Arthritis Society of Canada. J.E.W. is the recipient of an Arthritis Society/CIHR Investigator Award, and Y.-H.C. is the recipient of an Arthritis Centre of Excellence studentship. Back

2 Address correspondence and reprint requests to Dr. Joan E. Wither, 1E429C, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada. E-mail address: jwither{at}uhnres.utoronto.ca Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; NZB, New Zealand Black; NZW, New Zealand White; HEL, hen egg white lysozyme; Tg, transgenic; NMS, normal mouse serum; PNA, peanut agglutinin; MFI, mean fluorescent intensity; Treg, T regulatory; NZM, New Zealand mixed. Back

Received for publication July 21, 2005. Accepted for publication October 11, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Burlingame, R. W., M. L. Boey, G. Starkebaum, R. L. Rubin. 1994. The central role of chromatin in autoimmune responses to histones and DNA in systemic lupus erythematosus. J. Clin. Invest. 91: 1687-1696.
  2. Hahn, B. N.. 1998. Antibodies to DNA. N. Engl. J. Med. 338: 1359-1368. [Free Full Text]
  3. Wakeland, E. K., K. Liu, R. R. Graham, T. W. Behrens. 2001. Delineating the genetic basis of systemic lupus erythematosus. Immunity 15: 397-408. [Medline]
  4. Theofilopoulos, A. N., F. J. Dixon. 1985. Murine models of systemic lupus erythematosus. Adv. Immunol. 37: 269-390. [Medline]
  5. Kono, D. H., R. W. Burlingame, D. G. Owens, A. Kuramochi, R. S. Balderas, D. Balomenos, A. N. Theofilopoulos. 1994. Lupus susceptibility loci in New Zealand mice. Proc. Natl. Acad. Sci. USA 91: 10168-10172. [Abstract/Free Full Text]
  6. Drake, C. G., S. K. Babcock, E. Palmer, B. L. Kotzin. 1994. Genetic analysis of the NZB contribution to lupus-like autoimmune disease in (NZB x NZW)F1 mice. Proc. Natl. Acad. Sci. USA 91: 4062-4066. [Abstract/Free Full Text]
  7. Drake, C. G., S. J. Rozzo, H. F. Hirschfeld, N. P. Smarnworawong, E. Palmer, B. L. Kotzin. 1995. Analysis of the New Zealand Black contribution to lupus-like renal disease: multiple genes that operate in a threshold manner. J. Immunol. 154: 2441-2447. [Abstract]
  8. Vyse, T. J., C. G. Drake, S. J. Rozzo, E. Roper, S. Izui, B. L. Kotzin. 1996. Genetic linkage of IgG autoantibody production in relation to lupus nephritis in New Zealand hybrid mice. J. Clin. Invest. 98: 1762-1772. [Medline]
  9. Rozzo, S. J., T. J. Vyse, C. G. Drake, B. L. Kotzin. 1996. Effect of genetic background on the contribution of New Zealand Black loci to autoimmune lupus nephritis. Proc. Natl. Acad. Sci. USA 93: 15164-15168. [Abstract/Free Full Text]
  10. Vyse, T., S. J. Rozzo, C. G. Drake, S. Izui, B. L. Kotzin. 1997. Control of multiple autoantibodies linked with a lupus nephritis susceptibility locus in New Zealand Black mice. J. Immunol. 158: 5566-5574. [Abstract]
  11. Vyse, T. J., S. J. Rozzo, C. G. Drake, V. B. Appel, M. Lemeur, S. Izui, E. Palmer, B. L. Kotzin. 1998. Contributions of Eaz and Ebz MHC genes to lupus susceptibility in New Zealand mice. J. Immunol. 160: 2757-2766. [Abstract/Free Full Text]
  12. Kono, D. H., A. N. Theofilopoulos. 2000. Genetics of systemic autoimmunity in mouse models of lupus. Int. Rev. Immunol. 19: 367-387. [Medline]
  13. Wither, J. E., A. D. Paterson, B. Vukusic. 2000. Genetic dissection of B cell traits in New Zealand black mice: the expanded population of B cells expressing up-regulated costimulatory molecules shows linkage to Nba2. Eur. J. Immunol. 30: 356-365. [Medline]
  14. Rozzo, S. J., J. D. Allard, D. Choubey, T. J. Vyse, S. Izui, G. Peltz, B. L. Kotzin. 2001. Evidence for an interferon-inducible gene, Ifi202, in the susceptibility to systemic lupus. Immunity 15: 435-443. [Medline]
  15. Jiang, Y., S. Hirose, R. Sanokawa-Akakura, M. Abe, X. Mi, N. Li, Y. Miura, J. Shirai, D. Zhang, Y. Hamano, T. Shirai. 1999. Genetically determined aberrant down-regulation of Fc{gamma}RIIB1 in germinal center B cells associated with hyper-IgG and IgG autoantibodies in murine systemic lupus erythematosus. Int. Immunol. 11: 1685-1691. [Abstract/Free Full Text]
  16. Yan, X., K. Nakamura, M. Abe, N. Li, X. S. Wen, Y. Jiang, D. Zhang, H. Tsurui, S. Matsuoka, Y. Hamano, et al 2002. Transcriptional regulation of Fcgr2b gene by polymorphic promoter region and its contribution to humoral immune responses. J. Immunol. 169: 4340-4346. [Abstract/Free Full Text]
  17. Wandstrat, A. E., C. Nguyen, N. Limaye, A. Y. Chan, S. Subramanian, X. H. Tian, Y. S. Yim, A. Pertsemlidis, H. R. Garner, Jr, L. Morel, E. K. Wakeland. 2004. Association of extensive polymorphisms in the SLAM/CD2 gene cluster with murine lupus. Immunity 21: 769-780. [Medline]
  18. Wither, J. E., G. Lajoie, S. Heinrichs, Y. C. Cai, N. Chang, A. Ciofani, Y. H. Cheung, R. MacLeod. 2003. Functional dissection of lupus susceptibility loci on the New Zealand black mouse chromosome 1: evidence for independent genetic loci affecting T and B cell activation. J. Immunol. 171: 1697-1706. [Abstract/Free Full Text]
  19. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, et al 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334: 676-682. [Medline]
  20. Yager, T. D., C. T. McMurray, K. E. van Holde. 1989. Salt-induced release of DNA from nucleosome core particles. Biochemistry 28: 2271-2281. [Medline]
  21. Sobel, E. S., S. Minoru, Y. Chen, E. K. Wakeland, L. Morel. 2002. The major murine systemic lupus erythematosus susceptibility locus Sle1 results in abnormal functions of both B and T cells. J. Immunol. 169: 2694-2700. [Abstract/Free Full Text]
  22. Sobel, E. S., L. Morel, R. Baert, C. Mohan, J. Schiffenbauer, E. K. Wakeland. 2002. Genetic dissection of systemic lupus erythematosus pathogenesis: evidence for functional expression of Sle3/5 by non-T cells. J. Immunol. 169: 4025-4032. [Abstract/Free Full Text]
  23. Kaliyapermual, A., C. Mohan, W. Wu, S. K. Datta. 1996. Nucleosomal peptide epitopes for nephritis-inducing T helper cells of murine lupus. J. Exp. Med. 183: 2459-2469. [Abstract/Free Full Text]
  24. Kaliyaperumal, A., M. A. Michaels, S. K. Datta. 2002. Naturally processed chromatin peptides reveal a major autoepitope that primes pathogenic T and B cells of lupus. J. Immunol. 168: 2530-2537. [Abstract/Free Full Text]
  25. Wither, J. E., V. Roy, L. A. Brennan. 2000. Activated B cells express increased levels of costimulatory molecules in young autoimmune NZB and (NZB x NZW)F1 mice. Clin. Immunol. 94: 51-63. [Medline]
  26. Wen, L., S. J. Roberts, J. L. Vinery, F. S. Wong, C. Mallick, R. C. Findly, Q. Peng, J. E. Craft, M. J. Owen, A. C. Hayday. 1994. Immunoglobulin synthesis and generalized autoimmunity in mice congenitally deficient in {alpha}{beta}+ T cells. Nature 369: 654-658. [Medline]
  27. Wen, L., W. Pao, F. S. Wong, Q. Peng, J. Craft, B. Zhang, G. Kelsoe, L. Dianda, M. J. Owen, A. C. Hayday. 1996. Germinal center formation, immunoglobulin class switching, and autoantibody production driven by "non-{alpha}/{beta}" T cells. J. Exp. Med. 183: 2271-2282. [Abstract/Free Full Text]
  28. Shlomchik, M. J., J. E. Craft, M. J. Mamula. 2001. From T to B and back again: positive feedback in systemic autoimmune disease. Nat. Rev. Immunol. 1: 147-153. [Medline]
  29. Putterman, C., B. Diamond. 1998. Immunization with a peptide surrogate for double-stranded DNA (dsDNA) induces autoantibody production and renal immunoglobulin deposition. J. Exp. Med. 188: 29-38. [Abstract/Free Full Text]
  30. Khalil, M., K. Inaba, R. Steinman, J. Ravetch, B. Diamond. 2001. T cell studies in a peptide-induce model of systemic lupus erythematosus. J. Immunol. 166: 1667-1674. [Abstract/Free Full Text]
  31. Seo, S. J., M. L. Fields, J. L. Buckler, A. J. Reed, L. Mandik-Nayak, S. A. Nish, R. J. Noelle, L. A. Turka, F. D. Finkelman, A. J. Caton, J. Erikson. 2002. The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells. Immunity 16: 535-546. [Medline]
  32. Fields, M. L., S. A. Nish, B. D. Hondowicz, M. H. Metzgar, G. N. Wharton, A. J. Caton, J. Erikson. 2005. The influence of effector T cells and fas ligand on lupus-associated B cells. J. Immunol. 174: 104-111.
  33. Shih, F. F., L. Mandik-Nayak, B. T. Wipke, P. M. Allen. 2004. Massive thymic deletion results in systemic autoimmunity through elimination of CD4+CD25+ T regulatory cells. J. Exp. Med. 199: 323-335. [Abstract/Free Full Text]
  34. Fields, M. L., J. Erikson. 2003. The regulation of lupus-associated autoantibodies: immunoglobulin transgenic models. Curr. Opin. Immunol. 15: 709-717. [Medline]
  35. Heltemes-Harris, L., L. Xiaohe, T. Manser. 2004. Progressive surface B cell antigen receptor down-regulation accompanies efficient development of antinuclear antigen B cells in mature, follicular phenotype. J. Immunol. 172: 823-833. [Abstract/Free Full Text]
  36. Paul, E., J. Lutz, J. Erikson, M. C. Carroll. 2004. Germinal center checkpoints in B cell tolerance in 3H9 transgenic mice. Int. Immunol. 16: 377-384. [Abstract/Free Full Text]
  37. Rice, J. S., J. Newman, C. Wang, D. J. Michael, B. Diamond. 2005. Receptor editing in peripheral B cell tolerance. Proc. Natl. Acad. Sci. USA 102: 1608-1613. [Abstract/Free Full Text]
  38. Cooke, M. P., A. W. Heath, K. M. Shokat, Y. Zeng, F. D. Finkelman, P. S. Linsley, M. Howard, C. C. Goodnow. 1994. Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells. J. Exp. Med. 179: 425-438. [Abstract/Free Full Text]
  39. Eris, J. M., A. Basten, R. Brink, K. Doherty, M. R. Kehry, P. D. Hodgkin. 1994. Anergic self-reactive B cells present self antigen and respond normally to CD40-dependent T cell signals but are defective in antigen-receptor-mediated functions. Proc. Natl. Acad. Sci. USA 91: 4392-4396. [Abstract/Free Full Text]
  40. Rathmell, J. C., S. Fournier, B. C. Weintraub, J. P. Allison, C. C. Goodnow. 1998. Repression of B7.2 on self-reactive B cells is essential to prevent proliferation and allow fas-mediated deletion by CD4+ T cells. J. Exp. Med. 188: 651-659. [Abstract/Free Full Text]
  41. Fukuyama, H., F. Nimmerjahn, J. V. Ravetch. 2005. The inhibitory Fc{gamma} receptor modulates autoimmunity by limiting the accumulation of immunoglobulin G+ anti-DNA plasma cells. Nat. Immunol. 6: 99-106. [Medline]
  42. Mohan, C., E. Alas, L. Morel, P. Yang, E. K. Wakeland. 1998. Genetic dissection of SLE pathogenesis-Sle1 on murine chromosome 1 leads to selective loss of tolerance to H2A/H2B/DNA subnucleosomes. J. Clin. Invest. 101: 1362-1372. [Medline]
  43. Chen, Y., C. Cuda, L. Morel. 2005. Genetic determination of T cell help in loss of tolerance to nuclear antigens. J. Immunol. 174: 7692-7702. [Abstract/Free Full Text]
  44. Boackle, S., V. M. Holers, X. Chen, G. Szakonyi, D. R. Karp, E. K. Wakeland, L. Morel. 2001. Cr2, a candidate gene in the murine Sle1c lupus susceptibility locus, encodes a dysfunctional protein. Immunity 15: 775-785. [Medline]
  45. Liu, K., C. Liang, Z. Liang, K. Tus, E. K. Wakeland. 2005. Sleab mediates the aberrant activation of STAT3 and Ras-ERK signaling pathways in B lymphocytes. J. Immunol. 174: 1630-1637. [Abstract/Free Full Text]
  46. Datta, S. K.. 2003. Major peptide autoepitopes for nucleosome-centered T and B cell interaction in human and murine lupus. Ann. NY Acad. Sci. 987: 79-90. [Medline]
  47. Folzenlogen, D., M. F. Hofer, D. Y. Leung, J. H. Freed, M. K. Newell. 1997. Analysis of CD80 and CD86 expression on peripheral blood B lymphocytes reveals increased expression of CD86 in lupus patients. Clin. Immunol. Immunopathol. 83: 199-204. [Medline]
  48. Bijl, M., G. Horst, P. C. Limburg, C. G. Kallenberg. 2001. Expression of costimulatory molecules on peripheral blood lymphocytes of patients with systemic lupus erythematosus. Ann. Rheum. Dis. 60: 523-526. [Abstract/Free Full Text]
  49. Grammer, A. C., R. Slota, R. Fischer, H. Gur, H. Girschick, C. Yarboro, C. G. Illei, P. E. Lipsky. 2003. Abnormal germinal center reactions in systemic lupus erythematosus demonstrated by blockade of CD154-CD40 interactions. J. Clin. Invest. 112: 1506-1520. [Medline]
  50. Roy, V., N.-H. Chang, Y. Cai, G. Bonventi, J. Wither. 2005. Aberrant IgM signaling promotes survival of transitional T1 B cells and prevents tolerance induction in lupus-prone New Zealand Black mice. J. Immunol. 175: 7363-7371. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
N.-H. Chang, T. McKenzie, G. Bonventi, C. Landolt-Marticorena, P. R. Fortin, D. Gladman, M. Urowitz, and J. E. Wither
Expanded Population of Activated Antigen-Engaged Cells within the Naive B Cell Compartment of Patients with Systemic Lupus Erythematosus
J. Immunol., January 15, 2008; 180(2): 1276 - 1284.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheung, Y.-H.
Right arrow Articles by Wither, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheung, Y.-H.
Right arrow Articles by Wither, J. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS