Chronic GVH prevents anergy in bone marrow self-reactive B cells: a selective increase in post-endoplasmic reticulum processing and trafficking to the cell surface of autoreactive IgM receptors

Nili Feuerstein1, Debra Shivers1, Fangqi Chen2, Robert A. Eisenberg2 and Terri H. Finkel1,3

1 Division of Rheumatology, The Children’s Hospital of Philadelphia, andDepartments of 2 Medicine and 3 Pediatrics, Division of Rheumatology, University of Pennsylvania, Philadelphia, PA 19104, USA

Correspondence to: Nili Feuerstein, Division of Rheumatology, The Children’s Hospital of Philadelphia, 1107 ARC, Philadelphia, PA 19104, USA. E-mail: feuerstein{at}email.chop.edu
Transmitting editor: S. Izui


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
B cell autoreactivity is a component of chronic graft versus host (GVH) disease in humans and mice. Chronic GVH driven by I-A disparity results in loss of B cell tolerance in Ig/sHEL tolerant mice. In these mice, B cell anergy is characterized by down-modulation of sIgM mediated by intracellular retention in the endoplasmic reticulum (ER) and/or a block in post-ER processing of IgM receptors. Here, we report that GVH induces a selective increase in post-ER processing of the µ chain and trafficking to the cell surface of IgM receptors in B cells that bind HEL self-antigen. The increase in sIgM was detectable as early as 6 days post-GVH, before the appearance of circulating autoantibodies, and was particularly prominent in B cells that up-regulated surface I-A. A further increase in sIgM was found at later time points, along with circulating anti-HEL autoantibodies and a marked decrease in serum-free HEL, but no significant change in the amounts of HEL bound to B cells in vivo. These findings suggest that (i) abrogation of ER retention of IgM receptors in self-reactive B cells is an early event triggered by allogeneic T cells and (ii) at later stages of GVH disease the appearance of autoantibodies reduces the availability of free autoantigen, which may further escalate anergy escape of self-reactive B cells, and lead to exacerbation and perpetuation of autoimmunity.

Keywords: autoimmunity, B lymphocyte, BCR, graft versus host disease


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Manifestations of autoimmune diseases mediated by chronic graft versus host (GVH) disease are common among patients who have received allogeneic bone marrow transplantation. The protean manifestations of chronic GVH have been likened to those of progressive systemic sclerosis, systemic lupus erythematosus (SLE), lichen planus, Sjögren’s syndrome and others [reviewed in (1)]. Autoantibody formation has also been reported in experimental models of chronic GVH [reviewed in (2)] and clinical reports parallel these findings in humans (3,4). Specifically, anti-nuclear, anti-double-stranded DNA, anti-smooth muscle, anti-cytoskeletal and anti-nucleolar autoantibodies are found in patients with chronic GVH, and specific nucleolar phosphoproteins have been identified as GVH targets [reviewed in (1)]. In mice, chronic GVH reaction induced by the transfer of I-A-incompatible spleen cells results in an autoimmune syndrome that closely resembles SLE in the spectrum of autoantibodies and immunopathology (5,6). It is thought that in this GVH model, the allohelper T cells of the donor react against incompatible I-A structures of the host and generate abnormal help, which activates a subpopulation of B cells to become self-reactive (6,7). While evidence indicates that chronic GVH results in breakdown of B cell tolerance, the biochemical mechanisms that mediate this process are poorly understood.

The major limitation in elucidation of the biochemical basis for B cell autoreactivity results from the inability to detect the self-reactive B cells in the normal B cell repertoire and to track their fate during chronic GVH. In order to overcome these limitations, we have established an Ig transgenic model of chronic GVH (8). Transgenic mice carrying rearranged BCR genes have provided a window into the events associated with the development of B cell tolerance to self-antigens (912). Studies have demonstrated that B cells with rearranged BCR genes specific for hen egg lysozyme (HEL) that are exposed throughout development to soluble HEL antigen (Ig/sHEL double-transgenic mice) mature and populate the peripheral lymphoid organs (13). However, these B cells are functionally tolerant to HEL self-antigen and cannot differentiate into antibody-secreting cells in response to antigenic stimulation (13,14). Furthermore, antigen binding to Ig/sHEL B cells fails to induce proliferation (15), B7-2 expression (16) or resistance to Fas-mediated apoptosis (17). On the other hand, these cells can proliferate in response to lipopolysaccharide and retain responsiveness to stimulation via CD40 and IL-4 (15,18), indicating that the cells are tolerant only to stimulation via BCR. The anergic state in B cells from Ig/sHEL double-transgenic mice is characterized by a marked decrease in surface expression of IgM antigen receptors (14), and diminished tyrosine phosphorylation, mobilization of intracellular Ca2+ (15) and activation of distinct signaling pathways following ligation of IgD receptors (19).

We proposed that the ability to induce systemic autoimmunity in mice with a profoundly anergic and uniform population of B cells, by GVH, can create a unique model to examine cellular and molecular mechanisms associated with breakdown of B cell tolerance during chronic GVH. Strikingly, we found that induction of chronic GVH reaction resulted in breakdown of tolerance and systemic autoimmunity in anergic Ig/sHEL double-transgenic mice (8). The breakdown of B cell tolerance in these GVH-induced mice was evidenced by high and sustained levels of circulating anti-HEL autoantibodies, which were manifested as early as 2 weeks post-inoculation and remained elevated several months later. In addition, these mice develop glomerulonephritis with proteinuria (8). Furthermore, B cells from GVH-induced Ig/sHEL mice can proliferate in vitro in response to self-antigen. These data clearly demonstrate that chronic GVH resulted in abrogation of B cell tolerance in Ig/sHEL mice. This underscores the importance of this model in elucidating basic mechanisms of autoimmunity in chronic GVH. Thus, analysis of B cells in this murine model should provide important insights into the biochemical basis of loss of B cell tolerance during chronic GVH and possibly in autoimmune diseases such as SLE.

Anergy in Ig/sHEL self-reactive B cells is maintained by two distinct BCR mechanisms that are differentially associated with IgD and IgM antigen receptors: (i) desensitization of IgD receptors and (ii) down-modulation of surface IgM antigen receptors. In a previous report we showed that GVH abrogates IgD desensitization in self-reactive B cells (8). Thus, IgD receptor ligation in GVH-induced B cells results in rapid tyrosine phosphorylation of specific proteins that is not observed following BCR activation of tolerant B cells (8). In the current work we further examined the effect of GVH on the second BCR mechanism of anergy in Ig/sHEL B cells, i.e. modulation of IgM antigen receptors.

Anergy of self-reactive B cells, such as in Ig/sHEL and anti-double-stranded DNA transgenic mouse models, is acquired in the bone marrow and characterized by a marked decrease in surface expression of IgM antigen receptors (13,14,20,21). Previous studies in Ig/sHEL double-transgenic mice have elucidated the biochemical basis for the decreased surface expression of IgM antigen receptor (22). These studies showed that tolerant B cells expressed normal amounts of IgM mRNA, but demonstrated a selective decrease in transport to the cell surface due to a block in post-endoplasmic reticulum (ER) processing of IgM receptor complexes (22). In normal B cells, nascent unprocessed high mannose N-linked carbohydrate IgM chains are formed in the ER and then transported to the medial Golgi, where they are processed into mature IgM complexes that are resistant to cleavage by endoglycosidase H (Endo H) (23). However, in tolerant B cells, nascent IgM complexes are retained in the ER and fail to be processed into the mature Endo H-resistant form, suggesting a block in their transport from the ER to the medial Golgi (22).

The physiological significance of ER retention of IgM antigen receptors in self-reactive B cells during development in the bone marrow is unknown. Notably, ER retention of IgM receptors in anergic B cells resembles ER retention of pre-BCR found in pre-B cells (24). Developmental arrest is associated with antigenic stimulation of immature B cells in the bone marrow [reviewed in (25)] and characterizes anergic anti-double-stranded DNA B cells (20). Importantly, a dose-dependent correlation between the magnitude of modulation of IgM receptors by a panel of soluble antigens and the level of receptor editing in IgHEL B cells in bone marrow cultures suggests an intimate relationship between anergy and receptor editing in maintaining B cell tolerance (26). This conclusion is further supported by evidence of receptor editing in Ig/sHEL mice (26,27). Since modulation of IgM surface receptors plays an important role in anergy it was important to investigate whether the induction of systemic autoimmunity in chronic GVH in Ig/sHEL mice is associated with interference in the mechanisms that trigger intracellular retention of IgM receptors and anergy in self-reactive B cells.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
MD3 x ML5 transgenic mice expressing HEL-specific IgMa and IgDa and soluble HEL (Ig/sHEL double-transgenic mice) on a C57BL/6J (B6) background were originally provided by Dr C. Goodnow (Australian National University, Canberra, Australia), and were bred and typed in our mouse colony as previously described (28). B6 and co-isogenic B6.C-H2bm12KhEg (bm12) mice were obtained from Jackson Laboratories (Bar Harbor, ME) as pedigree-identified littermate pairs and maintained in our mouse colony.

Reagents and antibodies
Goat anti-mouse IgM, µ chain specific, was purchased from Jackson ImmunoResearch. Endo H deglycosylation kit (Boehringer Mannheim, Indianapolis, IN). Dodecyl-ß-maltoside (Anatrace, Maumee, HO). Polyclonal anti-Ig{alpha} was kindly provided by Dr Linda Matsuuchi (University of British Colombia, Vancouver, BC). Purified monoclonal anti-IgD antibody (H{delta}a/1) with specificity to the heavy chain of IgD (29) was kindly provided by Dr Fred Finkelman (University of Cincinnati Medical Center, Cincinnati, OH). Anti-HEL mAb 2D1 (mouse IgG1) (30).

Experimental chronic GVH protocol
Chronic GVH reaction was induced as previously described (31). Briefly, donor bm12 splenocytes (~1 x 108) were prepared by pressing spleens through a nylon mesh cell strainer into HBSS. The single-cell suspension was washed and injected i.p. into recipient Ig/sHEL mice at 2–4 months of age. The mice were sacrificed 4–5 weeks post-induction of GVH or as indicated in specific experiments.

Preparation of bone marrow cells
The mouse was sacrificed with CO2, and the femur and tibia were removed and placed in 5 ml RPMI containing 5% FBS. The bones were flushed with an 18-gauge needle into a Petri dish, resuspended with a 22-gauge needle and filtered through a 100-µm mesh filter. Red blood cells were lysed and the cells were stained with conjugated antibodies and analyzed by FACScan.

Immunofluorescence staining
Two-color flow cytometric staining of spleen lymphocytes or bone marrow cells was performed as previously described (32). The antibodies used for staining were purchased from PharMingen (San Diego, CA). In all experiments, 10,000 live events were acquired, and positive staining with specific antibodies was analyzed using FACScan flow cytometry and CellQuest software (Becton Dickinson, Mountain View, CA).

Detection of IgM and IgD complexes by immunoprecipitation and Western blot
Splenic lymphocytes (~15–25 x 106) were lysed at 4°C in 300 µl of buffer A containing 150 mM NaCl, 20 mM Tris, pH 8.0, 1 mM EDTA, 1% dodecyl-ß-maltoside, 1 mM PMSF and 1 mM leupeptin. Lysates containing equal amounts of B cells, as determined by FACS analysis, were used in the immunoprecipitation assay. The insoluble fractions were pelleted and the supernatants were further incubated with anti-IgM or anti-IgD antibodies (5 µg per 300 µl cell lysate) at 4°C for 3 h. Thereafter, Protein A–Sepharose was added and the samples were further incubated for 90 min. The immune complexes were pelleted, washed 3 times with buffer A, suspended in reducing SDS sample buffer and boiled for 5 min. The immunoprecipitates were analyzed by SDS–PAGE on a large 10% acrylamide gel. Proteins were transferred onto nitrocellulose membranes and blotted with goat anti-mouse IgM or goat anti-mouse IgD (3 µg/ml) as primary antibodies and horseradish peroxidase-conjugated anti-goat as a secondary antibody. The immunoreactive complexes were detected using the ECL system (33).

Determination of anti-HEL antibodies in the serum
Blood samples were obtained 6 weeks post-induction of GVH. Serum samples were stored at –20°C until further analysis. The levels of anti-HEL autoantibodies were determined by ELISA as previously described (8).

Determination of the amounts of HEL in the serum
Serum samples were obtained 6 weeks post-GVH and kept at –70°C until further analysis. Spleen cells (1 x 106) from IgHEL single-transgenic mice were prepared and incubated in FACS staining media (32) for 15 min. The cells were then re-suspended in FACS staining media plus serum (1:1 ratio) or with known amounts of HEL and incubated for 45 min on ice. The cells were washed and stained with fluorescent- conjugated antibodies to IgMa and HEL (2D1), and analyzed by FACScan. The levels of HEL bound to B cells [mean fluorescence intensity (MFI)] in serum were determined and compared to HEL standards.

End H treatment
IgM was immunoprecipitated from splenic lymphocytes as described above. The immune complexes were suspended in 10 µl of buffer A, and then 10 µl of reduced denaturation buffer was added and the complexes boiled for 5 min. Following centrifugation, the supernatant of each sample was divided into two tubes: control and Endo H treatment. Control tubes were treated with reaction buffer supplemented with protease inhibitors (1 mM PMSF and 1 mM leupeptin), and the experimental tubes were treated with an equal volume of reaction buffer and Endo H. The denaturation buffer, reaction buffer and Endo H were provided in the Endo H deglycosylation kit (Boehringer Mannheim)


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The surface expression of IgM antigen receptors is markedly increased in splenic self-reactive B cells during chronic GVH
Studies were initiated to investigate whether loss of tolerance in Ig/sHEL double-transgenic mice was associated with changes in the surface expression of IgM antigen receptors in splenic B lymphocytes. Induction of chronic GVH, by injection of I-A incompatible splenocytes from bm12 mice into Ig/sHEL double-transgenic mice, was carried out as described in Methods. Four weeks later, splenic B cells from the mice that received bm12 splenocytes (‘GVH’), from untreated Ig/sHEL double-transgenic (‘tolerant’) or from IgHEL single-transgenic mice (‘naive’) were stained for B220 and IgMa, IgMb or IgDa, and analyzed by flow cytometry.

As previously reported, tolerant B cells demonstrated a significant reduction in the expression of surface IgMa when compared to naive B cells (Fig. 1). Strikingly, in the GVH-induced mice, almost the entire population of B cells demonstrated a marked increase in the surface expression of IgMa, while only a small increase was seen in the expression of IgDa antigen receptor. In contrast to the changes observed in the expression of the transgenic IgMa, the surface expression of the endogenous gene, IgMb, did not increase at all in the GVH-induced mice. Furthermore, induction of GVH in non-transgenic mice did not result in an increase in surface IgM (Fig. 1B). Thus, the increase in surface IgM is not a general phenomenon in the entire B cell population in chronic GVH, but a specific effect on self-reactive B cells that down-modulates surface IgM as a function of induction of tolerance.



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Fig. 1. GVH induces a marked increase in surface expression of IgM antigen receptors in splenic self-reactive B cells. (A) Splenocytes were prepared from IgHEL single-transgenic mice (naive), Ig/sHEL double-transgenic mice (tolerant) or from GVH-induced Ig/sHEL mice (GVH) 4 weeks post-inoculation of donor bm12 spleen cells. Splenic lymphocytes were double stained with labeled antibodies to B220 plus IgMa, IgMb or IgDa. Flow cytometry analysis was gated on lymphocytes by scatter and on B220+ cells as shown. Histograms are representative of more than four experiments. (B) Splenocytes were prepared from B6 non-transgenic mice 4 weeks post-GVH induction. Splenic lymphocytes were double stained with labeled antibodies to B220 plus IgM. Flow cytometry analysis was gated on lymphocytes by scatter and on B220+ cells as shown.

 
These experiments demonstrate that loss of tolerance in chronic GVH is associated with abrogation of an important mechanism of B cell anergy, i.e. modulation of surface IgM.

GVH-induced increase in surface expression of IgM antigen receptors originates in immature self-reactive B cells in the bone marrow.
B cell tolerance in Ig/sHEL double-transgenic mice is acquired in the bone marrow, as evidenced by modulation of IgM antigen receptors (34,35). Therefore, further experiments addressed the question: does the increase in surface expression of IgM antigen receptors in self-reactive B cells during chronic GVH originate in the bone marrow or in the secondary lymphoid organs?

Analysis of IgM surface expression in the spleen and in the bone marrow of the same mice shows that during chronic GVH self-reactive B cells fail to modulate IgM antigen receptors in the bone marrow (Fig. 2). Notably, IgM surface expression is further decreased in the spleens of tolerant mice, but not in the spleens of GVH-induced mice (Fig. 2). This indicates that pathological processes in both the bone marrow and the spleen may participate in abrogation of intracellular retention of IgM antigen receptors in self-reactive B cells.



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Fig. 2. The increase in surface expression of IgM antigen receptors in chronic GVH originates in the bone marrow in immature self-reactive B cells. (A) Splenocytes and bone marrow cells were prepared from IgHEL single-transgenic mice (naive), Ig/sHEL double-transgenic mice (tolerant) or from GVH-induced Ig/sHEL mice (GVH). Cells were double stained with labeled antibodies to B220 and IgMa. Flow cytometry analysis demonstrates the expression of IgMa and B220 gated on lymphocytes by scatter. MFI of gated cells. Results are representative of four experiments. (B) Bone marrow cells were prepared from IgHEL single-transgenic mice (naive), Ig/sHEL double-transgenic mice (tolerant) or GVH-induced Ig/sHEL mice (GVH) 4 weeks post-inoculation of donor bm12 spleen cells. Cells were double stained with labeled antibodies to IgMa plus antibodies to CD23–FITC, IgDa–FITC and CD24–phycoerythrin. Results are representative of three experiments.

 
We further investigated whether the GVH-induced increase in surface expression of IgM antigen receptors in self-reactive B cell is initiated in mature or immature B cells in the bone marrow. Phenotypic analysis of bone marrow B cells demonstrates that the increase in IgM surface expression, in GVH mice, is seen in B cells that express phenotype markers of immature B cells: CD23, IgD and CD24high (Fig. 2B). The GVH-induced B cells undergo normal maturation in the spleen as evidenced by an increase in expression of CD23 and a decrease in expression of CD24 (8). These results indicate that abrogation of B cell tolerance in chronic GVH originates in the bone marrow by abrogation of intracellular retention in immature self-reactive B cells.

IgM antigen receptors in GVH-induced self-reactive B cells are functional in signaling for B cell activation
The presence of high levels of surface IgM antigen receptors in self-reactive B cells that are continuously exposed in vivo to HEL antigen raises the question: do these receptors become refractory to self-stimulation as do the IgD antigen receptors in tolerant B cells? We have previously shown that GVH restores immune reactivity to HEL antigen in Ig/sHEL B cells (8). This was demonstrated by an increase in tyrosine phosphorylation and proliferation of Ig/sHEL B cells from GVH-induced mice in response to HEL stimulation (8). The restoration of immune reactivity to HEL in the GVH-induced self-reactive B cells may be due in part to reversal of desensitization of IgD antigen receptors leading to activation of IgD receptors by HEL antigen (8). To address the role of IgM antigen receptors in loss of B cell tolerance to HEL self-antigen, we investigated whether specific ligation of surface IgM antigen receptors in the GVH-induced double-transgenic mice can also trigger B cell activation.

Increase in surface expression of the co-stimulatory molecule B7-2 is an important response to BCR activation that is repressed in anergic self-reactive B cells (16). Thus, we investigated whether IgM antigen receptor ligation can trigger an increase in B7-2 expression in self-reactive B cells from GVH-induced mice. To this end, tolerant or GVH-induced splenic B cells were stimulated in vitro with anti-µ-specific antibody and B cell expression of B7-2 was monitored by flow cytometry. Figure 3(A) shows data for three GVH-induced mice, all of which showed an increase in expression of surface IgM antigen receptor on splenic B cells. Upon stimulation with anti-IgM, a marked increase in surface expression of B7-2 was seen in ~80% of the B cells in each of the GVH-induced mice (Fig. 3B). This confirms that the surface IgM antigen receptors in the GVH-induced B cells are functional in inducing B cell activation.



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Fig. 3. Ligation of IgM antigen receptors in self-reactive B cells induces a marked increase in surface expression of B7-2. (A) Splenic lymphocytes from untreated Ig/sHEL mice (Tolerant) and from GVH-induced Ig/sHEL mice (GVH) were stained with anti-IgMa/B220. (B) Cells were incubated in the presence or absence of anti-µ-specific antibody (15 µg/ml) for 17 h. At the end of incubation, the cells were washed and double stained with B220 and B7-2 antibodies. Histograms demonstrate the expression of B7-2 gated on lymphocytes by scatter and on B220+ cells as shown at the top. Open histograms represent B cells stimulated with anti-µ antibody. Filled histograms represent non-stimulated B cells. The percentage of B cells that express B7-2 (R3) following stimulation with anti-µ-specific antibody is shown for each mouse.

 
A marked decrease in serum-free HEL, in the absence of a decrease in HEL bound to B cells, is found at late stages of GVH disease
We further investigated whether the increase in surface IgM in GVH-induced self-reactive B cells is due to a decrease in availability of the autoantigen. Analysis of serum 6 weeks post-induction of GVH revealed that the increase in serum anti-HEL autoantibodies (Fig. 4A) is correlated with a striking decrease (~98%) in free circulating HEL, which was evidenced in all the mice (Fig. 4B). Intriguingly, in spite of the marked decrease in serum-free HEL, the levels of HEL bound in vivo to spleen B cells did not change in the majority of the chronic GVH mice (Fig. 4C). The ~30% of the GVH mice which show reduced levels of HEL bound in vivo had even lower levels of serum-free HEL (below the level of detection). We further compared the levels of surface IgM in the group of GVH mice that showed either high or low levels of HEL bound in vivo (Fig. 4D). Notably, the GVH mice that demonstrated high levels of HEL bound to B cells (equal to tolerant mice) expressed ~5.9-fold increase in surface IgM (Fig. 4D). The GVH mice that demonstrated lower levels of HEL bound to B cells expressed a higher increase in surface IgM (~9.2-fold).



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Fig. 4. Serum HEL is markedly decreased in the absence of a significant change in HEL bound to B cells in vivo in chronic GVH. (A and B) Serum was derived from Ig/sHEL tolerant mice (n = 4) and from GVH-induced mice (n = 6), 6 weeks post-induction of GVH. (A) The amounts of anti-HEL antibodies in the serum were determined by ELISA. Results show means ± SEM of a representative experiment out of two experiments with similar results. (B) The amounts of free HEL in the serum were determined by the capacity of HEL to bind to B cells from IgHEL single-transgenic mice, as described in Methods. Results show means ± SEM of a representative experiment out of two experiments with similar results. (C) Spleen cells from untreated tolerant mice (n = 8) or from GVH-induced mice (n = 10) were stained with fluorescent antibodies to IgMa and to HEL (2D1 mAb) and analyzed by FACScan. The histogram shows the amount of HEL bound in vivo (MFI) to IgM+ cells in each mouse. Bars indicate the means. (D) Results show the mean ± SEM of HEL bound in vivo (MFI) and surface IgMa levels (MFI) of mice shown above (Fig. 4C): untreated tolerant mice, GVH mice that express high levels of HEL (GVH-H/H) and GVH mice that express low levels of HEL (GVH-L/H).

 
These results suggest that: (i) limiting levels of autoantigen contribute to the increase in surface IgM at later stages of GVH disease, at least in some of the mice, and (ii) BCR efficiently compete with the circulating soluble Ig(s) for binding of HEL autoantigen, resulting in high levels of autoantigen binding to BCR in the majority of the GVH-induced mice.

An increase in surface IgM on self-reactive B cells is an early event that precedes the appearance of autoantibodies and is associated with an increase in surface I-A
The marked decrease in serum-free HEL in the GVH mice could plausibly be due to sequestering of the autoantigen by circulating autoantibodies. Thus, a question of importance is whether the increase in surface IgM in the GVH-induced B cells precedes the appearance of autoantibodies in chronic GVH. To address this question we studied the kinetics of the change in surface IgM, serum autoantibodies and serum HEL at early days post-induction of GVH (Fig. 5A and B). We found that 3 days post-GVH there was no increase in surface IgM (data not shown). A small, but significant, increase in surface IgM could be detected 6 days post-GVH (eight out of eight mice tested demonstrated an increase in surface IgM at days 6–7 post-GVH). Conversely, at days 6 and 7 post-GVH there was no change in (i) circulating autoantibodies, (ii) serum-free HEL or (iii) HEL bound to B cells in vivo. Thus, the increase in surface IgM in GVH-induced self-reactive B cells is an early event that precedes autoantibody secretion and the subsequent decrease in autoantigen.



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Fig. 5. An increase in surface IgM precedes the increase in circulating autoantibodies and the decrease in autoantigen. (A) Experiment 1: Ig/sHEL double-transgenic control mice (n = 6) or mice inoculated with bm12 splenocytes (GVH) were sacrificed after 5 (n = 2), 6 (n = 6) or 7 (n = 2) days. In other experiments control Ig/sHEL mice (n = 4) and GVH mice (n = 6) were sacrificed after 6 weeks. For each mouse we determined the levels of serum HEL, serum anti-HEL antibodies and surface IgM in bone marrow cells. Results show mean ± SD. (B) Experiment 2: Ig/sHEL mice were sacrificed 6 days post-induction of GVH. For each mouse we determined the levels of serum HEL, anti-HEL autoantibodies, surface IgM in bone marrow cells and spleen B cells, and HEL bound in vivo to B cells. Results show mean ± SD of the percent change compared to control untreated Ig/sHEL mice (100%).

 
Parallel to the early increase in surface IgM 6–7 days post-GVH, there was also an increase in I-A surface expression in bone marrow (data not shown) and spleen self-reactive B cells (Fig. 6A). Importantly, elevated levels of surface I-A in a subset of self-reactive B cells, 6–7 days post-induction of GVH, was correlated with a prominent increase in surface IgM. Conversely, self-reactive B cells that did not up-regulate surface I-A show significant down-modulation of surface IgM (Fig. 6B). We have previously shown that self-reactive B cells that have lost tolerance in chronic GVH express elevated levels of surface I-A several weeks post-induction of GVH (8). We now demonstrate that the increase in surface I-A is an early event that occurs several days post-induction of GVH and that it appears to be correlated with failure to modulate surface IgM in self-reactive B cells.



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Fig. 6. An early increase in surface IgM is correlated with an increase in surface I-A on self-reactive B cells. (A) Spleen cells from control Ig/sHEL mice or GVH mice were derived 7 days post-GVH. Cells were stained with antibodies to I-Ab and IgMa, and analyzed by FACScan. (B) Histograms show the levels of surface IgM in I-A high-gated cells or I-A low-gated cells (as indicated in A above) in GVH mice (data are representative of five mice).

 
The increase in surface IgM antigen receptors in self-reactive B cells is dependent upon the number of donor splenocytes inoculated into the host
Induction of full-blown SLE-like syndrome is dependent on the number of spleen cells that are inoculated into the host (36). Thus, in further experiments, we examined the increase in IgM surface expression in Ig/sHEL mice that were inoculated with different number of splenocytes (60 or 120 x 106 cells). The surface expression of IgM antigen receptors in self-reactive splenic B cells was monitored at 2 and 6 weeks post-inoculation (Fig. 7). These studies demonstrate that the increase in surface expression of IgM in self-reactive B cells is apparent as early as 2 weeks post-inoculation of donor cells and is sustained after 6 weeks (Fig. 7). Importantly, the increase in surface IgM expression is ~3-fold higher in mice injected with 120 x 106 cells as compared to mice injected with 60 x 106 cells. Thus, the level of increase in surface IgM expression in self-reactive B cells is closely dependent upon the number of splenocytes that are inoculated into the host.



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Fig. 7. The increase in surface IgM antigen receptors in self-reactive B cells is dependent upon the number of donor splenocytes inoculated into the host. Ig/sHEL mice were injected with 60 or 120 x 106 spleen cells from bm12 mice and sacrificed after 2 or 6 weeks. Splenic lymphocytes were double stained with labeled antibodies to B220 and IgMa. Flow cytometry analysis demonstrates the expression of IgMa gated on lymphocytes by scatter and on B220+ cells as shown. Results are representative of two experiments with similar results.

 
GVH induces a selective up-regulation of a high-molecular weight µ chain in self-reactive B cells
Further studies explored the biochemical basis of the increase in expression of surface IgM in B cells that had lost tolerance by chronic GVH. Splenic lymphocytes from naive, tolerant and GVH mice were lysed, and IgM complexes were immunoprecipitated, analyzed by SDS–PAGE and further detected by Western blot using anti-IgM antibody. Figure 8 demonstrates that in naive B cells there are two major species of µ chain: a lower-mol.-wt chain at ~75 kDa (‘µ-1’) and a higher-mol.-wt chain at ~82 kDa (‘µ-2’). Densitometry scanning showed that the lower-mol.-wt species, ‘µ-1’, was found in similar abundance in naive and tolerant B cells, but the higher-mol.-wt species, ‘µ-2’, was completely absent in tolerant B cells. B cells from the GVH-induced Ig/sHEL mice showed no increase in abundance of the lower-mol.-wt species, ‘µ-1’, but showed a striking increase in the abundance of the higher-mol.-wt µ chain, ‘µ-2’. Further stripping of the nitrocellulose membrane and reblotting with anti-Ig{alpha} showed that similar amounts of Ig{alpha} were associated with IgM complexes in naive, tolerant or autoimmune B cells from GVH-induced mice (Fig. 8B). This corroborates previous studies that have demonstrated that, in tolerant B cells of Ig/sHEL mice, IgM complexes assemble normally into intact receptors containing Ig{alpha}/ß heterodimers (22).



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Fig. 8. Loss of B cell tolerance is associated with selective up-regulation of a high-mol.-wt species of µ chain. (A) IgM complexes were immunoprecipitated from lysates prepared from splenic lymphocytes from ‘naive’, ‘tolerant’ or ‘GVH’ mice (as defined in Fig. 1). The immunoprecipitates were resolved by SDS–PAGE and detected by Western blot analysis using anti-IgM antibody. (B) The nitrocellulose membrane shown in (A) was stripped and further blotted with anti-Ig{alpha}. (C) Lysates of splenic lymphocytes of ‘naive’, ‘tolerant’ and ‘GVH’ mice were divided in half, and were exposed to immunoprecipitation with either anti-IgD or with anti-IgM. The immune complexes were resolved by SDS–PAGE and detected by Western blot analysis using anti-IgM or anti-IgD antibodies. Arrows indicate two species of µ chain, ‘µ-1’ at ~75 kDa and ‘µ-2’at ~82 kDa, and two species of {delta} chains, ‘{delta}-1’ and ‘{delta}-2’. Results are representative of three experiments with similar results.

 
IgM and IgD complexes were then immunoprecipitated in parallel from splenic lymphocyte lysates of the same mice (Fig. 8C). Similar to µ chains, {delta} chains are also expressed as two species that differ in mol. wt: ‘{delta}-1’ and ‘{delta}-2’. However, while the GVH-induced B cells showed a selective increase in expression of the higher-mol.-wt species, ‘µ-2’, there was no increase in the abundance of the higher-mol.-wt species of IgD chain,‘{delta}-2’, in the GVH-induced B cells, as compared to tolerant B cells (Fig. 8C). These studies demonstrate that GVH is associated with a selective up-regulation of a higher-mol.-wt form of µ Ig heavy chain in self-reactive B cells.

The GVH-induced µ chain in self-reactive B cells is Endo H resistant, indicating post-ER processing
To further characterize the two species of µ chain, IgM was immunoprecipitated and then subjected to Endo H treatment. Endo H cleaves high mannose oligosaccharides borne by glycoproteins before their arrival to the medial Golgi, but fails to cleave carbohydrates following processing at this site. This allows use of the sensitivity to Endo H treatment to monitor polypeptide transport into the medial Golgi (23). IgM was immunoprecipitated from naive, tolerant or GVH-induced B cells and then incubated in the presence or absence of Endo H. The complexes were further resolved by SDS–PAGE and subsequently detected by Western blot using anti-IgM antibody (Fig. 9). These studies identified the 75-kDa species, ‘µ-1’, as the unprocessed µ chain located in the ER, which carries high mannose N-linked carbohydrates that are Endo H sensitive (as indicated by a shift to a triplet with apparent mol. wt of ~65/70 kDa following treatment with Endo H). Conversely, the 82-kDa species, ‘µ-2’, was identified as the mature µ chain bearing processed N-linked carbohydrates that were not cleavable by Endo H (Endo H resistant), and, therefore, were processed and matured in the Golgi. Previous studies using metabolic labeling followed by pulse–chase indicated that in tolerant B cells, nascent IgM complexes failed to be processed into the mature Endo H-resistant form, indicating that there is a block in their transport from the ER to the Golgi (22). Our work corroborates and extends these data with the novel observation that the block in processing of nascent IgM complexes into the mature Endo H-resistant form, which is found in tolerant B cells, is abrogated in B cells during chronic GVH, resulting in breakdown of tolerance.



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Fig. 9. The high-mol.-wt µ chain (µ-2) that is selectively up-regulated in self-reactive B cells is not cleavable by Endo H treatment. (A) IgM complexes were immunoprecipitated from splenic lymphocytes of ‘naive’, ’tolerant’ or ‘GVH’ mice. The immunoprecipitates were divided in half and further incubated in the presence or absence of Endo H as described in Methods. Thereafter, the IgM complexes were resolved by SDS–PAGE and detected by Western blot analysis using anti-IgM antibody. Arrows indicate the two species of µ chain in the absence or presence of Endo H respectively. (B) Densitometry: numbers represent densitometry of µ-1 and µ-2 in Endo H-treated naive, tolerant and GVH-induced mice. Results are representative of three experiments with similar results.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this work, we report that the breakdown of B cell tolerance, driven by inoculation of I-A-incompatible splenocytes into Ig/sHEL double-transgenic mice, is associated with abrogation of the hallmark mechanism of tolerance in these cells, i.e. the modulation of surface expression of IgM antigen receptors. This effect occurs several days post-induction of GVH, is correlated with an increase in surface I-A and is dependent on the number of allogeneic cells inoculated into the host. Biochemical analysis demonstrates that chronic GVH induces a strikingly selective increase in post-ER processing of IgM antigen receptors in self-reactive B cells, resulting in an increase in trafficking to the cell surface of IgM antigen receptors that are immunoreactive to self-antigen.

Failure to modulate surface IgM: early and late events in chronic GVH
Modulation of IgM antigen receptors is initially observed in the bone marrow in anergic self-reactive B cells (34,35). This is associated with ER retention of IgM antigen receptors, mediated by a block in the transport of IgM receptor complexes to the Golgi (22). The physiological significance of anergy in self-reactive B cells is underscored by the observation of B cell anergy in other Ig transgenic models. Thus, anergic self-reactive anti-doubled-stranded DNA B cells also modulate IgM antigen receptors in the bone marrow (20). Furthermore, similar to Ig/sHEL B cells, anergic anti-DNA B cells reside at the T–B interface of the splenic follicle and continue to exhibit low levels of IgM antigen receptors (20).

Previous evidence has shown that modulation of IgM in self-reactive B cells is a graded response to signaling via BCR. It is increased by signal enhancing mutations in Lyn (37) and in the SH2 domain of hemopoietic phosphatase 1 (38). Conversely, IgM modulation in self-reactive B cells is diminished by signal lowering mutations in CD45 (28). Thus, an issue of importance is the role of changes in autoantigen in the failure of self-reactive B cells to modulate surface IgM. Our findings demonstrate that a small, but significant, increase in surface IgM occurs several days post-inoculation of allogeneic T cells, before the appearance of autoantibodies and the subsequent decrease in serum-free autoantigen. Thus, the initial increase in surface IgM is driven by allogeneic T cells in the absence of a decrease in autoantigen. More importantly, this evidence suggests that reversal of modulation of surface IgM in self-reactive Ig/sHEL B cells is not a consequence of loss of tolerance but rather a primary early event in loss of tolerance in chronic GVH.

Our findings demonstrate that at later stages of the GVH disease there is a 98% decrease in free HEL in the serum. In spite of this decrease in free circulating HEL, the level of HEL bound in vivo to B cells in GVH mice is not significantly changed. This suggests that the BCR (surface Ig) are effectively competing with soluble Ig in binding HEL autoantigen in GVH mice. However, our results with individual mice indicate a negative correlation between the level of HEL bound to B cells in vivo and the magnitude of the increase in surface IgM. Thus, it is plausible that limitation in amounts of autoantigen may contribute to the increase in surface IgM in the late stages of GVH disease. This would in essence create a positive feedback mechanism to further exacerbate and perpetuate autoimmunity. Further studies will be required to establish the role of the decrease in autoantigen in driving B cell autoimmunity in the late stages of chronic GVH.

Taken collectively, these results suggest that two mechanisms affect the increase in surface IgM in self-reactive B cells in chronic GVH. The primary mechanism involves activation of B cells by allogeneic T cells, resulting in failure to modulate surface IgM and secretion of autoantibodies by some of the B cells. The secondary mechanism involves the appearance of circulating autoantibodies which consequently result in sequestering of autoantigen and limiting of free autoantigen in the serum. Thus, the two mechanisms, a decrease in autoantigen and persistent allo-T cell help, may act in synergy at later stages of chronic GVH to induce further increase in surface IgM and tolerance escape in self-reactive B cells.

The role of allogeneic T cells in abrogation of B cell anergy
Previous studies have established a crucial role for allogeneic T cells in inducing the GVH response [reviewed (1)] and loss of B cell tolerance in chronic GVH (2,6,31). Indeed, our studies show that the level of increase in IgM surface expression in self-reactive B cells, is closely dependent upon the number of donor spleen cells that are inoculated into the host. This corroborates previous studies establishing that sufficient numbers of allogeneic splenocytes are required to achieve a full-blown SLE like GVHD in mice (36). Furthermore, cognate T–B cell interaction was implicated in loss of B cell tolerance in chronic GVH (6). Strikingly, the early increase in surface IgM several days post-induction of GVH is correlated with an increase in surface I-A on bone marrow and spleen B cells. Thus, it is likely that cognate interaction with allogeneic T cells leads to B cell activation and up-regulation of surface I-A, which in turn enables further interaction with allo-T cells. Intriguingly, the early increase in surface IgM is prominent only in B cells that have elevated levels of I-A. Thus, allogeneic T cell-induced B cell activation is an early event in GVH that results in an increase in surface I-A in some self-reactive B cells, leading to abrogation of anergy and failure to down-modulate surface IgM. The increase in surface expression of immunoreactive IgM antigen receptors in those self-reactive B cells may facilitate antigen-specific B cell activation leading to autoantibody secretion and loss of tolerance.

Our present results demonstrate that failure to modulate surface IgM is initiated in immature bone marrow B cells. We further found that GVH abrogates another important mechanism of B cell tolerance, i.e. receptor editing (data not shown). This suggests that newly emerging B cells in the bone marrow escape central tolerance in GVH. Intriguingly, we found that chronic GVH induces a sustained increase in activation of T cells in the bone marrow (data not shown). Thus, it is possible that the continuous presence of activated T cells in the bone marrow in chronic GVH contributes to abrogation of tolerance in immature self-reactive B cells. Self-reactive B cells, which failed to be tolerized in the bone marrow, mature in the spleen and continue to express high levels of IgM receptors that are immunoreactive to self-antigen. It is, therefore, plausible that the marked increase in surface expression of immunoreactive IgM antigen receptors, in self-reactive B cells, facilitates antigen-specific B cell activation in the spleen. In collaboration with allogeneic T cell help in the spleen, this may promote autoimmune responses in chronic GVH.

Biochemical analyses in this work demonstrate that the increase in surface IgM, in GVH-induced self-reactive B cells, is associated with a strikingly selective increase in the abundance of IgM complexes that are processed in the Golgi (Endo H-resistant species). Conversely, there is no change in the abundance of IgM complexes in the ER (Endo H-sensitive species). These findings suggest that intracellular trafficking and/or post-ER processing of IgM antigen receptors can be tightly regulated by factors in the microenvironment of the bone marrow and the spleen in chronic GVH. While it has been well established that allogeneic T cells play a pivotal role in breakdown of B cell tolerance in chronic GVH, the mechanism of their effect is still unknown. The results of this work implicate a biochemical consequence of T–B cell interaction in chronic GVH. Thus, encounter with self-antigen under non-autoimmune conditions results in a block in post-ER processing of IgM antigen receptors and anergy. However, allogeneic T cell help during chronic GVH may abrogate this block leading to autoreactivity. Elucidation of the molecular mechanisms that regulate intracellular trafficking of IgM antigen receptors in B cells stimulated with self-antigen should shed light on the molecular processes that underlie tolerance and its abrogation in chronic GVH.


    Acknowledgements
 
The authors wish to thank Dr Chris Goodnow for providing the Ig/sHEL transgenic mice, Dr Fred Finkelman for the kind gift of purified monoclonal anti-IgD antibody, Dr Linda Matsuuchi for the kind gift of anti-Ig{alpha} antibody and Dr Steve Grupp for critical review of the manuscript. This work was supported by NIH grants RO1 A130575 (T. H. F.), RO1 AR34156 (R. A. E.) and by the Joseph Lee Hollander Chair (T. H. F.).


    Abbreviations
 
Endo H—endoglycosidase H

ER—endoplasmic reticulum

GVH—graft versus host

HEL—hen egg lysozyme

MFI—mean fluorescence intensity

SLE—systemic lupus erythematosus


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Sullivan, K. 1994. Graft versus host disease. In Forman, S., Blume, K. G. and Thomas, E. D., eds, Bone Marrow Transplantation, p. 339. Blackwell Scientific, Oxford.
  2. Via, C. S. and Shearer, G. M. 1988. T-cell interactions in autoimmunity: insights from a murine model of graft-versus-host disease. Immunol. Today 9:207.[CrossRef][ISI][Medline]
  3. Rouquette-Gally, A. M., Boyeldieu, D., Prost, A. C. and Gluckman, E. 1988. Autoimmunity after allogeneic bone marrow transplantation. A study of 53 long-term-surviving patients. Transplantation 46:238.[ISI][Medline]
  4. Lister, J., Messner, H., Keystone, E., Miller, R. and Fritzler, M. J. 1987. Autoantibody analysis of patients with graft versus host disease. J. Clin. Lab. Immunol. 24:19.[ISI][Medline]
  5. Morris, S. C., Cohen, P. L. and Eisenberg, R. A. 1990. Experimental induction of systemic lupus erythematosus by recognition of foreign Ia. Clin. Immunol. Immunopathol. 57:263.[ISI][Medline]
  6. Morris, S. C., Cheek, R. L., Cohen, P. L. and Eisenberg, R. A. 1990. Autoantibodies in chronic graft versus host result from cognate T–B interactions. J. Exp. Med. 171:503.[Abstract]
  7. Eisenberg, R. A. and Cohen, P. L. 1983. Class II major histocompatibility antigens and the etiology of systemic lupus erythematosus. Clin. Immunol. Immunopathol. 29:1.[ISI][Medline]
  8. Feuerstein, N., Chen, F., Madaio, M., Maldonado, M. and Eisenberg, R. A. 1999. Induction of autoimmunity in a transgenic model of B cell receptor peripheral tolerance: changes in coreceptors and B cell receptor-induced tyrosine-phosphoproteins. J. Immunol. 163:5287.[Abstract/Free Full Text]
  9. Erikson, J., Radic, M. Z., Camper, S. A., Hardy, R. R., Carmack, C. and Weigert, M. 1991. Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature 349:331.[CrossRef][ISI][Medline]
  10. Gay, D., Saunders, T., Camper, S. and Weigert, M. 1993. Receptor editing: an approach by autoreactive B cells to escape tolerance. J. Exp. Med. 177:999.[Abstract]
  11. Goodnow, C. C. 1992. Transgenic mice and analysis of B-cell tolerance. Annu. Rev. Immunol. 10:489.[CrossRef][ISI][Medline]
  12. Nemazee, D. A. and Burki, K. 1989. Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature 337:562.[CrossRef][ISI][Medline]
  13. Goodnow, C. C., Crosbie, J., Jorgensen, H., Brink, R. A. and Basten, A. 1989. Induction of self-tolerance in mature peripheral B lymphocytes [see Comments]. Nature 342:385.[CrossRef][ISI][Medline]
  14. Goodnow, C. C., Crosbie, J., Adelstein, S., Lavoie, T. B., Smith-Gill, S. J., Brink, R. A., Pritchard-Briscoe, H., Wotherspoon, J. S., Loblay, R. H., Raphael, K., et al. 1988. Altered immunoglobulin expression and functional silencing of self- reactive B lymphocytes in transgenic mice. Nature 334:676.[CrossRef][ISI][Medline]
  15. Cooke, M. P., Heath, A. W., Shokat, K. M., Zeng, Y., Finkelman, F. D., Linsley, P. S., Howard, M. and Goodnow, C. C. 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.[Abstract]
  16. Rathmell, J. C., Fournier, S., Weintraub, B. C., Allison, J. P. and Goodnow, C. C. 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.[Abstract/Free Full Text]
  17. Foote, L. C., Marshak-Rothstein, A. and Rothstein, T. L. 1998. Tolerant B lymphocytes acquire resistance to Fas-mediated apoptosis after treatment with interleukin 4 but not after treatment with specific antigen unless a surface immunoglobulin threshold is exceeded. J. Exp. Med. 187:847.[Abstract/Free Full Text]
  18. Eris, J. M., Basten, A., Brink, R., Doherty, K., Kehry, M. R. and Hodgkin, P. D. 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.[Abstract]
  19. Healy, J. I., Dolmetsch, R. E., Timmerman, L. A., Cyster, J. G., Thomas, M. L., Crabtree, G. R., Lewis, R. S. and Goodnow, C. C. 1997. Different nuclear signals are activated by the B cell receptor during positive versus negative signaling. Immunity 6:419.[ISI][Medline]
  20. Mandik-Nayak, L., Bui, A., Noorchashm, H., Eaton, A. and Erikson, J. 1997. Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T–B interface of the splenic follicle. J. Exp. Med. 186:1257.[Abstract/Free Full Text]
  21. Noorchashm, H., Bui, A., Li, H. L., Eaton, A., Mandik-Nayak, L., Sokol, C., Potts, K. M., Pure, E. and Erikson, J. 1999. Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process. Int. Immunol. 11:765.[Abstract/Free Full Text]
  22. Bell, S. E. and Goodnow, C. C. 1994. A selective defect in IgM antigen receptor synthesis and transport causes loss of cell surface IgM expression on tolerant B lymphocytes. EMBO J. 13:816.[Abstract]
  23. Dunphy, W. G., Brands, R. and Rothman, J. E. 1985. Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack. Cell 40:463.[ISI][Medline]
  24. Brouns, G. S., de Vries, E., Neefjes, J. J. and Borst, J. 1996. Assembled pre-B cell receptor complexes are retained in the endoplasmic reticulum by a mechanism that is not selective for the pseudo-light chain. J. Biol. Chem. 271:19272.[Abstract/Free Full Text]
  25. Nemazee, D. 2000. Receptor selection in B and T lymphocytes. Annu. Rev. Immunol. 18:19.[CrossRef][ISI][Medline]
  26. Tze, L. E., Baness, E. A., Hippen, K. L. and Behrens, T. W. 2000. Ig light chain receptor editing in anergic B cells. J. Immunol. 165:6796.[Abstract/Free Full Text]
  27. Morris, S. C., Moroldo, M., Giannini, E. H., Orekhova, T. and Finkelman, F. D. 2000. In vivo survival of autoreactive B cells: characterization of long-lived B cells. J. Immunol. 164:3035.[Abstract/Free Full Text]
  28. Cyster, J. G., Healy, J. I., Kishihara, K., Mak, T. W., Thomas, M. L. and Goodnow, C. C. 1996. Regulation of B-lymphocyte negative and positive selection by tyrosine phosphatase CD45. Nature 381:325.[CrossRef][ISI][Medline]
  29. Zitron, I. M. and Clevinger, B. L. 1980. Regulation of murine B cells through surface immunoglobulin. I. Monoclonal anti-delta antibody that induces allotype-specific proliferation. J. Exp. Med. 152:1135.[Abstract]
  30. Metzger, D. W., Ch’ng, L. K., Miller, A. and Sercarz, E. E. 1984. The expressed lysozyme-specific B cell repertoire. I. Heterogeneity in the monoclonal anti-hen egg white lysozyme specificity repertoire, and its difference from the in situ repertoire. Eur. J. Immunol. 14:87.[ISI][Medline]
  31. Morris, S. C., Cheek, R. L., Cohen, P. L. and Eisenberg, R. A. 1990. Allotype-specific immunoregulation of autoantibody production by host B cells in chronic graft-versus host disease. J. Immunol. 144:916.[Abstract/Free Full Text]
  32. Reap, E. A., Felix, N. J., Wolthusen, P. A., Kotzin, B. L., Cohen, P. L. and Eisenberg, R. A. 1995. bcl-2 transgenic Lpr mice show profound enhancement of lymphadenopathy. J. Immunol. 155:5455.[Abstract]
  33. Firestein, R. and Feuerstein, N. 1998. Association of activating transcription factor 2 (ATF2) with the ubiquitin-conjugating enzyme hUBC9. Implication of the ubiquitin/proteasome pathway in regulation of ATF2 in T cells. J. Biol. Chem. 273:5892.[Abstract/Free Full Text]
  34. Mason, D. Y., Jones, M. and Goodnow, C. C. 1992. Development and follicular localization of tolerant B lymphocytes in lysozyme/anti-lysozyme IgM/IgD transgenic mice. Int. Immunol. 4:163.[Abstract]
  35. Hartley, S. B., Cooke, M. P., Fulcher, D. A., Harris, A. W., Cory, S., Basten, A. and Goodnow, C. C. 1993. Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death. Cell 72:325.[ISI][Medline]
  36. Van Rappard-Van Der Veen, F. M., Radaszkiewicz, T., Terraneo, L. and Gleichmann, E. 1983. Attempts at standardization of lupus-like graft-vs-host disease: inadvertent repopulation by DBA/2 spleen cells of H-2-different nonirradiated F1 mice. J. Immunol. 130:2693.[Abstract/Free Full Text]
  37. Cornall, R. J., Cyster, J. G., Hibbs, M. L., Dunn, A. R., Otipoby, K. L., Clark, E. A. and Goodnow, C. C. 1998. Polygenic autoimmune traits: Lyn, CD22, and SHP-1 are limiting elements of a biochemical pathway regulating BCR signaling and selection. Immunity 8:497.[ISI][Medline]
  38. Cyster, J. G. and Goodnow, C. C. 1995. Protein tyrosine phosphatase 1C negatively regulates antigen receptor signaling in B lymphocytes and determines thresholds for negative selection. Immunity 2:13.[ISI][Medline]