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


     
 

The Journal of Immunology, 2007, 179, 5564 -5570
Copyright © 2007 by The American Association of Immunologists, Inc.
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 Google Scholar
Google Scholar
Right arrow Articles by Choudhury, A.
Right arrow Articles by Eisenberg, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Choudhury, A.
Right arrow Articles by Eisenberg, R. A.

Mature B Cells Preferentially Lose Tolerance in the Chronic Graft-versus-Host Disease Model of Systemic Lupus Erythematosus1

Arpita Choudhury*, Philip L. Cohen*,{dagger} and Robert A. Eisenberg2,*

* Department of Medicine, Division of Rheumatology, University of Pennsylvania, Philadelphia, PA 19104 and {dagger} Veterans Affairs Medical Center, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chronic graft-vs-host (cGVH) disease is a well-characterized systemic lupus erythematosus (SLE) model. Induction of cGVH in anti-DNA H chain knockin (3H9KI) transgenic mice results in specific activation of anti-dsDNA B cells. In this study, we show that B cells from 3H9KI mice were activated by cGVH even when adoptively transferred into irradiated JHT–/– recipients that lack endogenous B cells. This process of activation was reflected by high autoantibody titers and changes in phenotypic markers. We have used this system to characterize the particular B cell subsets that were responsible for secreting autoantibodies during cGVH response. We isolated splenic B cell subsets based on their expression of specific cell surface markers and used them in our adoptive transfer studies. We found that mature B cells were the most vulnerable to the allostimulus and were the major source of autoantibodies compared with immature B cells. The greater susceptibility of mature B cells to become activated and thereby lose tolerance was unanticipated and has implications for maintenance of peripheral tolerance and for the development of autoimmunity. Furthermore, of the mature B cells, marginal zone B cells were particularly responsible for mounting the initial response to the cGVH stimulus. This observation underscores the critical role of marginal zone B cells in activation and production of autoantibodies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemic lupus erythematosus (SLE)3 is an autoimmune disease that affects multiple organ systems in association with a spectrum of autoantibodies that target normal proteins and nucleic acids. The understanding of SLE has been greatly facilitated by the chronic GVH (cGVH) model. Transfer of Ia-incompatible spleen cells from nonautoimmune B6.C-H2bm12/KhEg (bm12) mice into coisogenic C57BL/6 (B6) recipients results in cGVH reactions with features characteristic of SLE, including loss of B cell tolerance, high titers of autoantibodies and renal immunopathology (1).

To determine how allo-T cell help induces tolerance loss in anti-DNA B cells, we have combined the cGVH model with an H chain Ig transgene (tg), 3H9. The 3H9 is a "knockin" transgenic in which the normal chromosomal JH locus was replaced with a rearranged V(D)J tg, and this knockin (KI) H chain could combine with endogenous light chains to form both anti-dsDNA and nonautoreactive Abs (2, 3). Previous work from our laboratory showed that induction of cGVH in 3H9KI tg mice on a nonautoimmune B6 background resulted in specific activation of anti-dsDNA B cells (4). In a recent study using the cGVH model in anti-DNA H chain transgenic mice, 56R, Witsch et al. (5) have shown that B cells secreting pathogenic autoantibodies are probably located in the marginal zone (MZ) of the spleen. Although these studies helped in understanding some of the mechanisms by which allo-T cell help induces B cell autoreactivity, but they do not unambiguously establish the precise timing and the B cell subset that is most affected by such intervention.

We have, therefore, developed an adoptive transfer model to address these issues in our present study. We have utilized the expression of different cell surface markers in peripheral B cells to isolate particular subsets. Immature B cells that emigrate from the bone marrow circulate as transitional B cells that are HSAhighIgMhighB220lowIgD– and can be distinguished from mature splenic B cells by use of mAb AA4.1 that recognizes a protein of apparent m.w. 130–140, expressed exclusively on immature/transitional cells (6). The mature B cells can be broadly classified as follicular (FO) or MZ B cells that differ in their localization and cell surface markers. FO recirculating B cells are IgMintIgDhighCD21intCD23highCD1–CD9low, and resident MZ B cells located at the junction of white and red pulps are IgMhighIgDlowCD21+CD23–CD1highCD9high (7). Based on these variations in cell surface markers, we have isolated different B cell populations from the anti-DNA H chain transgenic mice, 3H9, and transferred them into irradiated B6.JHT–/– recipients, which lack endogenous B cells. We then induced cGVH in the recipients.

Our data indicate that adoptively transferred B cells are susceptible to allo-T cell help and produce autoantibodies. Most importantly, mature B cells were more susceptible to allo-T cell help than immature B cells. Furthermore, of the mature B cell subpopulation, MZ B cells responded more vigorously initially than did FO B cells.


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

The generation of C57BL/6.3H9 (3H9) and C57BL/6.3H9/56R (56R) mice has been described previously (3, 8). The C57BL/6.JHT mice, which lack JH segments and the intron enhancer in the IgH locus and therefore cannot generate B cells (9), were a gift from Dr. Sasha Tarakhovsky (Rockefeller University, New York City). C57BL/6J (B6) and coisogenic B6.C-H2bm12/KhEg (bm12) mice, which differ by only three amino acids in the beta-chain of I-A, were originally obtained from The Jackson Laboratory. All mice were bred and maintained in our colony at the University of Pennsylvania. Recipients and donors were sex and age matched within each experiment. All experimental procedures were conducted according to the guidelines of the Institutional Animal Care and Use Committee.

Adoptive cell transfers

JHT recipients were irradiated (3 Gy from a Cs-137 source) before adoptive cell-transfer experiments. All experiments were performed with 4–5 animals in each group. The experiments were repeated twice or thrice and had comparable results. Then 5–10 x 106 cells were transferred i.v. cGVH was induced on the following day by transferring i.p. 1 x 108 bm12 spleen cells. Blood samples were obtained from experimental mice before the induction of cGVH disease and at 2- to 4-wk intervals thereafter. Sera were stored at –20°C for later analysis.

Detection of autoantibodies and allotype specific antibodies

Autoantibodies were assessed by ELISA, as previously described (10). In brief, plates were coated with chicken erythrocyte-derived chromatin at 5 µg/ml; or with calf thymus-derived dsDNA at 3 µg/ml. Serum samples, diluted 1/250 in buffer, were added in duplicate and incubated overnight at 4°C. For the anti-dsDNA ELISA, plates were first coated with poly-L-lysine (1 µg/ml) (Sigma-Aldrich) before incubating with the autoantigen. Biotinylated goat anti-mouse IgG (pFc' specific; Jackson ImmunoResearch Laboratories) was added as secondary Ab. For reference, standard serum from a diseased MRL/lpr mouse with high titer autoantibodies was tested at serial 2-fold dilutions from 1/250 to 1/128,000. The plates were washed and incubated for 1 h at room temperature with avidin-alkaline phosphatase (Zymed Laboratories). The plates were washed again, and 1 mg/ml para-nitrophenyl phosphate substrate (Sigma-Aldrich) in 0.01 M diethanolamine (pH 9.8) was added. For allotype-specific ELISAs, the assays were similar to those described above, except that they were developed with rabbit anti-mouse preabsorbed allotype reagents (anti-IgG2aa or anti-IgG2ab (also known as IgG2c); Accurate Chemical and Scientific) and detected with avidin-alkaline phosphatase-conjugated anti-rabbit IgG Ab (Jackson ImmunoResearch Laboratories). For "a" allotype standard, serum from an old MRL/lpr was used, and for "b" allotype standard, serum from an old B6/lpr (Ighb) was used. Both the reference sera were used at serial 2-fold dilutions starting from 1/250 to 1/128,000. For estimation of total IgM, plates were coated with goat anti-mouse IgM (Jackson ImmunoResearch Laboratories) at 4 µg/ml. Biotinylated Bet-2 F(ab')2 was used as the second Ab, and mouse IgM (clone CBPC 112) was used as standard in these assays. The plates were read at various time points with an automated ELISA reader (Molecular Devices).

Immunofluorescence staining

The following conjugated Abs and reagents were purchased from BD Pharmingen: allophycocyanin anti-CD19 (1D3), FITC anti-CD21 (7G6), FITC anti-B7.1 (16–10A1), FITC anti-class II (AF6–120.1), PE anti-B7.2 (GL1), PE anti-CD23 (B3B4), and PE anti-Fas (Jo2). Anti-Fc{gamma}R (2.4G2), used for blocking, was grown in our laboratory. Cell surface staining was routinely performed with age- and sex-matched controls, as previously described (4, 10). Cells were fixed in PBS containing 1% paraformaldehyde and analyzed on a BD Biosciences FACScan. Relative fluorescence intensity was plotted on a logarithmic scale using Flow-Jo software.

Cell separation using magnetic beads

Magnetic beads, anti-CD43, and anti-biotin were purchased from Miltenyi Biotec, and the AutoMACS was used for cell separation as described previously (10). Peripheral B cells were isolated by using anti-CD43 magnetic beads and the depletion cycle. Mature and immature B cells were further separated by staining with biotinylated mAb AA4.1 (obtained from Dr. Dave Allman, University of Pennsylvania) and anti-biotin magnetic beads. Mature B cells were AA4.1– and immature B cells were AA4.1+. For separation of FO and MZ B cells, the CD43– B cells were labeled with biotinylated anti-CD23, and AA4.1 Ab and anti-biotin beads were used for separation. The CD23–AA4.1– fraction consisted of MZ B cells, and the CD23+AA4.1+ fraction contained immature and FO B cells. The purity of cell separation was checked by flow cytometry.

Statistical analysis

Statistical analyses were performed using Student’s t test. A value of p < 0.05 was considered to be significant. The bars indicate SEs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
We have previously established that induction of cGVH in 3H9KI tg mice resulted in specific activation of anti-dsDNA B cells (4). In the present studies, we have isolated B cell subsets to determine when, in ontogeny, B cell tolerance can be lost in the cGVH model.

Induction of cGVH in B cell knockout recipients

In our initial studies, we have used sIgM knockout mice (µMT) as recipients in our adoptive transfer experiments. Unlike JHT mice, which completely lack the J locus, µMT mice lack only the IgM transmembrane exons, so their B cell development is blocked at the pro-B cell stage (11). Adoptive transfer of 3H9 spleen cells into lightly irradiated (3 Gy) µMT recipients, followed by transfer of bm12 cells, resulted in production of IgG2aa autoantibodies in the recipients, indicating activation of the donor 3H9 cells (data not shown). However, the control µMT recipients given only bm12 cells also made low levels of IgG2aa Ig, although no autoantibodies (Fig. 1A). This suggested "leakiness" in µMT recipients, presumably due to the strong stimulus of cGVH, as has been reported in other experimental systems (12). We, therefore, used JHT mice as recipients in additional experiments, because these animals are incapable of producing Ig under any circumstances. The JHT recipients were also lightly irradiated (3 Gy) before being subjected to adoptive transfers. The experimental group of JHT recipients received 5–10 x 106 spleen cells from 3H9 tg mice followed by 1 x 108 bm12 cells. One control group of JHT recipients received only 3H9 spleen cells, whereas the other control group had only bm12 spleen cells. Each group consisted of 4–5 mice. Fig. 1B shows that the JHT recipients secreted Ig exclusively of the donor cell IgG2aa allotype. Importantly, unlike µMT mice, JHT recipients with only bm12 cells did not produce any IgG of "a" allotype but only IgG2ab IgG (presumably from the bm12 B cells), clearly illustrating the key point that JHT recipients do not show any leakiness (Fig. 1C). The experimental JHT recipients secreted total Ig of both allotypes, reflecting the presence of both 3H9 cells and bm12 cells; however, only the 3H9 tg-derived cells produced anti-dsDNA Abs (IgG2aa allotype (Fig. 1D) but not IgG2ab (Fig. 2E)). This is consistent with our previous observations that the cGVH response could be initiated only in the B cells recognized by alloreactive T cells (13, 14). These data establish that the process of loss of tolerance and induction of cGVH, as previously seen in 3H9 tg mice, could be reproduced in JHT recipients following adoptive cell transfer.


Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 1. Loss of tolerance by peripheral B cells from 3H9KI tg mice after adoptive transfer. 5–10 x 106 splenic cells from 3H9KI tg mice were transferred into lightly irradiated (3 Gy) JHT and µMT recipients followed by challenge with 1 x 108 bm12 spleen cells. A, Total IgG2aa allotype Ig in µMT recipients (n = 4) with adoptively transferred 3H9 spleen cells and bm12 cells (•) and in control µMT recipients with only bm12 cells ({triangleup}). B, Total IgG2aa allotype Ig in JHT recipients (n = 5) that received both 3H9 and bm12 spleen cells (•), and controls that received only 3H9 spleen cells ({circ}) or only bm12 spleen cells ({triangleup}). C, IgG2ab allotype Ig in the same JHT recipients as in B. D, anti-dsDNA Abs of allotype IgG2aa from 3H9 tg B cells in JHT recipients upon induction of cGVH. E, anti-dsDNA Abs of IgG2ab allotype in JHT recipients. The results are representative of two experiments.

 

Figure 2
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 2. Phenotypic changes of B cells after induction of cGVH. Spleen cells from JHT mice were analyzed by flow cytometry a week after transfer of B cells from 3H9 tg mice and induction of cGVH. Histograms represent B cells gated by scatter on the lymphocyte region and CD19 and IgMa positivity. A, Expression of activation markers MHC II, B7.2, and Fas, and developmental markers, CD23 and CD21, in JHT recipients after cGVH (heavy line) and JHT recipients with only donor 3H9 B cells (thin line) and isotype control (filled histograms). The y-axis represents the percentage of B cells gated on CD19 and IgMa positivity. B, MFI of B cells from experimental group ({blacksquare}) and the control recipients ({square}). Four to five mice per group were analyzed. The p values of MHC II (p = 0.010), B7.2 (p = 0.004), Fas (p = 0.002), CD23 (p = 0.003), and CD21 (p = 0.005) give the statistical significance between control JHT recipients and those with cGVH.

 
We have also previously found that the B cells in 3H9 tg mice show up-regulation of cell surface markers, such as MHC class II, Fas, and B7.2, upon induction of cGVH. This suggested a general activation of B cells by allostimulation (4). We asked whether such phenotypic changes also occurred when 3H9 tg B cells were transferred to JHT recipients and were provided with an allostimulus. The JHT recipients were sacrificed 1 wk after adoptive cell transfer and induction of cGVH, and their splenic B cells of 3H9 origin were analyzed. As seen in Fig. 2A, following induction of cGVH, adoptively transferred 3H9 B cells were activated as reflected by up-regulation of surface MHC II, B7.2, and Fas, whereas CD21 was down-regulated. This difference in CD21 expression is consistent with our previous findings in cGVH model (4). Similar changes in CD21 levels have been reported in other murine models (15) and in SLE patients (16). The increase in CD23 levels has also been observed in other models of cGVH such as in B6.56R (5). Fig. 2B illustrates the differences in mean fluorescence intensity (MFI) of various B cell activation markers between experimental recipients and control recipients (those with only 3H9 spleen cells). These data clearly indicate that the 3H9 donor cells are activated after cGVH in JHT recipients, and the cell surface marker profiles are consistent with our previous observations of activation of B cells in 3H9 mice following induction of cGVH (4).

Mature B cells are more susceptible to activation and loss of tolerance

After ascertaining that adoptively transferred spleen cells from 3H9 tg mice could be activated and produced autoantibodies in JHT recipients, we dissected the particular B cell subsets that were affected by the allointervention. We separated mature B cells from immature B cells in the adult spleen based on the expression of AA4.1 on the latter. Surprisingly, JHT recipients of mature B cells produced quantitatively more autoantibodies than those with immature B cells (Fig. 3). It is possible that the significantly lower response in immature B cells could be attributed to their more rapid turnover rate (17).


Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 3. Adoptively transferred mature 3H9 tg+ B cells initially lose tolerance under cGVH. Peripheral B cells were isolated from 3H9 tg mice by using CD43 magnetic beads. Immature B cells were sorted out by using mAb AA4.1. 5 x 106 immature ({circ}) or mature B cells (•) were transferred into lightly irradiated (3 Gy) JHT recipients. A positive control group received equal numbers of unfractionated B cells ({triangleup}). All the groups were challenged with 1 x 108 bm12 spleen cells. A, Anti-dsDNA Abs (p = 0.043 at 2 wk and p = 0.001 at 4 wk); anti-chromatin Abs (p = 0.1 at 2 wk, p = 0.3 at 4 wk) were measured by ELISA (B). The p values refer to the difference in autoantibody titers produced by mature and immature B cells at 2 and 4 wk. Each group consisted of 4–5 mice. The data are representative of two independent experiments. Error bars, SE of mean of the samples.

 
MZ B cells initially respond to allostimulation

Once it became apparent that the cGVH stimulus preferentially activated mature B cells, we further assessed the proclivity to lose tolerance in different populations of mature B cells. Based on their phenotype, location, and functional characteristics, mature B cells have been subdivided as recirculating FO B cells (CD21intCD23highCD1–), and resident MZ B cells (CD21+CD23–CD1high). Because MZ B cells have been found in some systems to play a role in the spontaneous development of autoantibodies (5, 18), we hypothesized that they might show a special susceptibility to allo-T help in the cGVH. Our data show that the JHT recipients of MZ B cells developed high titers of both anti-dsDNA Abs (Fig. 4A) and anti-chromatin Abs (Fig. 4B). The autoantibody titers in JHT recipients of FO B cells progressed more slowly and peaked at around 4 wk, when the autoantibodies in the MZ group were already declining (Fig. 4). As seen in Fig. 4C, total IgM titers also followed a similar pattern. These data illustrate that the initial autoantibody production in cGVH probably comes mainly from autoreactive B cells residing in the MZ. This interpretation of our data would apparently seem inconsistent with our flow cytometry data in Fig. 2 where it is shown that CD21 expression is down-regulated upon induction of cGVH, and this is similar to our previous findings in cGVH models (4). Also studies from Weigert’s group showed a decrease in MZ B cells following induction of cGVH in 56R strain of mice (5). However, we hypothesize that MZ B cells in situ are initially activated by cGVH and change their phenotype and/or their anatomical location. This would explain the down-regulation of CD21 expression as seen upon induction of cGVH in intact animals and why adoptively transferred MZ B cells mount the initial response to cGVH compared with FO B cells. At this point, we do not have data to show that adoptively transferred MZ B cells also populate the MZ area. Further elucidation of the involvement of MZ B cells in the SLE autoantibody response will be the subject of future studies.


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 4. Adoptively transferred MZ B cells from 3H9KI tg mice are the first to lose tolerance and produce autoantibodies upon induction of cGVH. MZ B cells were sorted on the basis of expression of cell surface marker CD23 using magnetic beads. Both fractions CD23+ FO B cells ({circ}) and CD23– MZ B cells (•) were transferred into different lightly irradiated (3 Gy) JHT recipients (n = 5). The FO fraction also had immature (AA4.1+) cells. Autoantibody titers were measured after induction of cGVH. Anti-dsDNA Abs (p = 0.03) (A), anti-chromatin Abs (p = 0.028) (B), and total IgM (p = 0.032) (C) after induction of cGVH. The results are representative of three experiments. Error bars, SEM of the samples. The p values refer to the difference in autoantibody titers produced by JHT recipients with MZ B cells and those with FO B cells at 2 wk.

 
In subsequent experiments, we examined B cells from another anti-DNA knockin transgenic mouse, 56R. 56R is similar to 3H9, except for an additional arginine in the CDR2 of the 3H9 H chain, which enhances the antibodies’ affinity for dsDNA (8). JHT recipients of MZ B cells from 56R mice showed high titers of anti-dsDNA Abs (Fig. 5A) and anti-chromatin Abs (Fig. 5B), compared with other B cell fractions. These data reinforce our findings that MZ B cells are more likely activated in the initial phase of cGVH reactions.


Figure 5
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 5. MZ B cells isolated from wild-type B6 or 56R mice show similar trends of initially losing tolerance under cGVH. MZ B cells and FO B cells were sorted by expression of CD23 on mature splenic B cells by using magnetic beads. CD23– fraction was considered as MZ B cells, whereas FO B cells were CD23+. The FO fraction also had immature (AA4.1+) cells. A total of 10 x 106 of MZ B cells (•) and FO B cells ({circ}) were adoptively transferred into lightly irradiated (3 Gy) JHT recipients (n = 5), and cGVH was induced by transferring 1 x 108 bm12 cells. Anti-dsDNA Abs (p = 0.001) (A) and anti-chromatin Abs (p = 0.003) (B) with adoptively transferred 56R B cells following induction of cGVH. Anti-dsDNA (p = 0.007) (C) and anti-chromatin Abs (p = 0.006) (D) secreted by B6 B cells. Error bars, SEM samples. The p values refer to the difference in autoantibody titers secreted by JHT recipients with MZ B cells and those with FO B cells.

 
It was possible that the Ig transgenic KIs skewed our model toward autoreactive MZ compartment, especially in light of recent reports that describe accumulation of autoreactive B cells in the MZ area of 56R mice (5, 19). To rule out such a possibility, we also isolated MZ and FO B cells from wild-type B6 mice. As seen in Fig. 5, C and D, transferred MZ B cells from wild-type mice preferentially secrete autoantibodies early under the influence of an allostimulus. Although the FO B cells were also activated and produce autoantibodies, their response peaked later, around 2 wk. These data confirm that MZ B cells are important in the initial response to activation and loss of tolerance in the cGVH model. Because we have used the expression of CD23 to isolate FO and MZ B cells, our FO B cells also include immature (AA4.1+) B cells. It is therefore, possible to argue that the quantitative difference in the response of FO B cells could be due to the presence of immature B cells. However, the proportion of immature B cells in the total CD23+ population is very small, (5–8% of the total spleen cells are AA4.1+ immature B cells), therefore, it is unlikely that the presence of such a small population of immature B cells would affect the outcome of the greater proportion of FO B cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The autoimmune syndrome of cGVH results from cognate recognition of host B cells by alloreactive donor T cells. The subsequent loss of B cell tolerance leads to production of typical SLE autoantibodies. The induced nature of this model permitted us to investigate the stage of ontogeny at which autoreactive B cells can be influenced by abnormal T cell help. Normally, autoreactivity is controlled at various checkpoints through mechanisms of deletion, anergy, and receptor editing (20, 21). By isolating B cells through the expression of characteristic surface markers, we could determine when in development they could lose self-tolerance. Somewhat surprisingly, we found that mature B cells were most amenable to loss of tolerance, particularly the MZ subset.

Our previous studies with 3H9KI mice showed that allo-T help triggered specific activation of anti-dsDNA B cells. In our present studies, we wanted to utilize this cGVH system in an adoptive transfer model to determine which particular B cell populations are activated by the allostimulus. A successful adoptive transfer model is greatly dependent on the choice of the host recipient. Our initial studies were conducted using sIgM knockout mice (µMT) as recipients. These mice lack only the IgM transmembrane exons, so their B cell development is blocked at the pro-B cell stage (11). Our data (Fig. 1) showed that adoptively transferred 3H9 spleen cells were activated by allostimulus and could secrete autoantibodies. However, the strong allostimulus also elicited endogenous Ig (but not autoantibody) production from the host recipient as seen in Fig. 1A. This implied leakiness in our recipient and suggested that allostimulus could circumvent the block in B cell development as seen in other studies (12). This finding led us to use JHT knockout mice in subsequent experiments. Unlike µMT mice where it is possible to have Ig expression by isotype class switching of H chain genes downstream of Cµ, the JH defect is irreparable and these JHT mice do not produce Ig under any circumstances (9). As seen from our data, the allostimulus activated only the 3H9 donor cells and did not generate Ig production from the host (Fig. 1, B and C). Furthermore, the autoantibodies were produced exclusively by the transferred donor 3H9 B cells as seen in Fig. 1, D and E.

Our previous studies in 3H9KI mice have also shown that the B cell activation that characterized cGVH was a complex, multistep process. The phenotypic changes most likely corresponded to polyclonal activation affecting all B cells, while the autoantibody response resulted from specific activation of anti-dsDNA B cells (4). In the adoptive transfer system, 3H9 B cells not only retained their ability to be activated by allo-T help and secrete anti-dsDNA Abs, but also up-regulated their cell surface markers, especially MHC II and B7.2, as seen in the cGVH in the intact recipient (3H9KI). However, these findings contrast with what is seen in other adoptive transfer studies in which B cells only undergo homeostatic proliferation in lymphopenic hosts in response to an antigenic or mitogenic stimulus, and the B cells retain their native phenotype and do not differentiate into effector cells (22).

In our previous studies with 3H9 and 56R mice, we showed that the cGVH favored the production of autoab that resulted from receptor editing and extensive somatic mutation (4). One interpretation of these findings was that the allostimulus would induce the re-expression of the rag genes. Our current results, whereby mature B cells readily lose tolerance in this model, are consistent with this idea. The peripheral re-expression of rag has been a subject of some controversy (23); nevertheless, it is possible that the strong stimulus of the cGVH could result in changes in gene regulation that would not normally be observed. Alternatively, the mature B cell population that loses tolerance in the cGVH could represent a population that has already been edited, perhaps with a resultant failure of allelic exclusion (5, 24). It has also been shown that transitional immature B cells up-regulate rag genes in response to BCR cross-linking and are thus capable of undergoing secondary Ig gene rearrangement (25). These observations might have predicted that under allostimulus, immature B cells would preferentially become autoreactive, but this was contrary to our present data. In future work, it will be important to study the extent of receptor editing and secondary rearrangements that occur in the transferred populations of anti-dsDNA B cells upon stimulation with allo-T cells in the secondary JHT host.

Our studies further suggest that MZ B cells played an important role in the pathogenesis of the disease. We found that transferred MZ B cells responded more strongly to allostimulation than did FO B cells. Although, the FO B cell population also contained immature B cells (AA1.4+), but it is only a small fraction and we have shown that immature B cells are less responsive to cGVH stimulation. Therefore, any contribution from immature B cells would not dilute the response of FO B cells significantly (data not shown). MZ B cells occupy a unique niche in the splenic architecture and have been under scrutiny because of their possible role in autoimmunity. It has been shown that MZ area is enriched in autoreactive B cells in certain transgenic models and autoimmune-prone mice. CD1high B cells have been reported to produce large amounts of IgM anti-dsDNA Abs in lupus-prone (New Zealand Black x New Zealand White)F1 mice (26), which have an increased number of MZ B cells (27). It has also been suggested that defective removal of apoptotic cells causes activation of autoreactive anti-Sm MZ B cells in anti-Sm tg mice (28). Using the K/BxN mouse model of rheumatoid arthritis, Mandik-Nayak et al. (18) have shown that autoreactive MZ B cells are spontaneously activated. These studies collectively implicate a role for MZ B cells in autoimmunity.

Studies with VH3H9 Ig transgenic mice have shown that anti-dsDNA B cells are excluded from the follicles (4, 29). We hypothesize that these anti-dsDNA B cells probably accumulate in the MZ area. Recent work from Weigert’s laboratory using the 56R model also indicated that dual receptor expressing autoreactive B cells home to the MZ area, and these B cells may contribute to the autoimmune response generated in cGVH (5). Autoreactive B cells tend to concentrate in MZ region, and MZ B cells can undergo class-switching and somatic hypermutation in response to certain Ags. In fact, for some Ags, MZ B cells may be the most important source of somatically mutated post germinal center B cells (30). It has also been shown that MZ B cells, not FO B cells, are most potent activators of CD4 T cells (31). In another recent study, it was suggested that autoreactive B cells are retained for a prolonged period in the MZ area because they over-express BAFF, and BAFF-tg mice have an expanded MZ B cell pool and develop lupus-like disease (32). Based on these studies and our own data, we therefore propose two possible reasons why the onset of the disease is initiated from the MZ region. First, it is possible that most anti-dsDNA-specific B cells that are barred from entering the follicles "home" to the MZ area where they may persist for a long time. Second, the MZ B cells that arise from precursor transitional cells probably have an intrinsic potential to become autoreactive when they encounter suitable Ag and T cell-derived signals.

Our previous studies in 3H9 and 56R mice showed that upon induction of cGVH, CD23 expression level was significantly reduced in activated B cells, especially in {lambda}+ dsDNA B cell population, and the MZ B cell population was reduced (4, 5). We postulate that the MZ cells in situ were preferentially affected by the cGVH, and changed their phenotype and/or their anatomical location. Our adoptive transfer studies also indicate that MZ B cells indeed have autoreactive potential and are preferably activated by alloreactive T cells.

In our study, the production of autoantibodies by adoptively transferred FO B cells followed a slower time course than did MZ cells in the cGVH. Previous reports have shown that adoptive transfer of highly purified FO B cells in lymphopenic hosts, such as RAG–/– mice, led to their differentiation into MZ B cells (33). It is possible that the autoantibodies in our model may be the product of newly differentiated MZ B cells that arise from the FO B cells. Alternatively, the FO B cells could be activated by a mechanism that differs from that operating on MZ cells. We cannot rule out a role for the contaminating immature B cells in our FO population, but we doubt that they were important, because their numbers were relatively few, and our data had already indicated that they were even less susceptible to autoreactivity in this model.

In conclusion, we have established that cGVH reactions could be successfully initiated in adoptive transfer studies. We have also shown that mature B cells from 3H9 mice are more predisposed to loss of tolerance than immature B cells. Furthermore, we show that MZ cells are most readily activated by allo-T help and differentiate to become autoantibody-producing B cells. Because loss of tolerance and autoantibody production may commence from MZ B cells, it is possible that they could represent a useful target for therapeutic interventions.


   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 National Institutes of Health Grants R01-AR34156, R01-AI063626, and U19-AI-46358; the Lupus Research Institute, Alliance for Lupus Research, U.S. Department of Veteran Affairs, and the Lupus Foundation of South Jersey. A.C. was supported by a postdoctoral fellowship from the Arthritis Foundation. Back

2 Address correspondence and reprint requests to Dr. Robert A. Eisenberg, Division of Rheumatology, University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA 19104. E-mail address: raemd{at}mail.med.upenn.edu Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; GVH, graft-vs-host; cGVH, chronic GVH; bm12, B6.C-H2bm12/KhEg mice; B6, C57BL/6; KI, knockin; tg, transgene; MZ, marginal zone; FO, follicular; MFI, mean fluorescence intensity. Back

Received for publication March 22, 2007. Accepted for publication August 7, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Morris, S. C., P. L. Cohen, R. A. Eisenberg. 1990. Experimental induction of systemic lupus erythematosus by recognition of foreign Ia. Clin. Immunol. Immunopathol. 57: 263-273. [Medline]
  2. Li, H., Y. Jiang, E. L. Prak, M. Radic, M. Weigert. 2001. Editors and editing of anti-DNA receptors. Immunity 15: 947-957. [Medline]
  3. Chen, C., Z. Nagy, E. L. Prak, M. Weigert. 1995. Immunoglobulin heavy chain gene replacement: a mechanism of receptor editing. Immunity 3: 747-755. [Medline]
  4. Sekiguchi, D. R., S. M. Jainandunsing, M. L. Fields, M. A. Maldonado, M. P. Madaio, J. Erikson, M. Weigert, R. A. Eisenberg. 2002. Chronic graft-versus-host in Ig knockin transgenic mice abrogates B cell tolerance in anti-dsDNA B cells. J. Immunol. 168: 4142-4153. [Abstract/Free Full Text]
  5. Witsch, E. J., H. Cao, H. Fukuyama, M. Weigert. 2006. Light chain editing generates polyreactive Abs in chronic graft-versus-host reaction. J. Exp. Med. 203: 1761-1772. [Abstract/Free Full Text]
  6. Allman, D., R. C. Lindsley, W. DeMuth, K. Rudd, S. A. Shinton, R. R. Hardy. 2001. Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation. J. Immunol. 167: 6834-6840. [Abstract/Free Full Text]
  7. Martin, F., J. F. Kearney. 2002. Marginal-zone B cells. Nat. Rev. Immunol. 2: 323-335. [Medline]
  8. Erikson, J., M. Z. Radic, S. A. Camper, R. R. Hardy, C. Carmack, M. Weigert. 1991. Expression of anti-DNA immunoglobulin transgenes in nonautoimmune mice. Nature 349: 331-334. [Medline]
  9. Gu, H., Y. R. Zou, K. Rajewsky. 1993. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73: 1155-1164. [Medline]
  10. Choudhury, A., M. A. Maldonado, P. L. Cohen, R. A. Eisenberg. 2005. The role of host CD4 T cells in the pathogenesis of the chronic graft-versus-host model of systemic lupus erythematosus. J. Immunol. 174: 7600-7609. [Abstract/Free Full Text]
  11. Kitamura, D., J. Roes, R. Kuhn, K. Rajewsky. 1991. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin µ-chain gene. Nature 350: 423-426. [Medline]
  12. Melamed, D., E. Miri, N. Leider, D. Nemazee. 2000. Unexpected autoAb production in membrane Ig-µ-deficient/lpr mice. J. Immunol. 165: 4353-4358. [Abstract/Free Full Text]
  13. Morris, S. C., R. L. Cheek, P. L. Cohen, R. A. Eisenberg. 1990. Allotype-specific immunoregulation of autoAb production by host B cells in chronic graft-versus host disease. J. Immunol. 144: 916-922. [Abstract]
  14. Morris, S. C., R. L. Cheek, P. L. Cohen, R. A. Eisenberg. 1990. Autoantibodies in chronic graft versus host result from cognate T-B interactions. J. Exp. Med. 171: 503-517. [Abstract/Free Full Text]
  15. Takahashi, K., Y. Kozono, T. J. Waldschmidt, D. Berthiaume, R. J. Quigg, A. Baron, V. M. Holers. 1997. Mouse complement receptors type 1 (CR1; CD35) and type 2 (CR2; CD21): expression on normal B cell subpopulations and decreased levels during the development of autoimmunity in MRL/lpr mice. J. Immunol. 159: 1557-1569. [Abstract]
  16. Wilson, J. G., W. D. Ratnoff, P. H. Schur, D. T. Fearon. 1986. Decreased expression of the C3b/C4b receptor (CR1) and the C3d receptor (CR2) on B lymphocytes and of CR1 on neutrophils of patients with systemic lupus erythematosus. Arthritis Rheum. 29: 739-747. [Medline]
  17. Sandel, P. C., J. G. Monroe. 1999. Negative selection of immature B cells by receptor editing or deletion is determined by site of Ag encounter. Immunity 10: 289-299. [Medline]
  18. Mandik-Nayak, L., J. Racz, B. P. Sleckman, P. M. Allen. 2006. Autoreactive marginal zone B cells are spontaneously activated but lymph node B cells, require T cell help. J. Exp. Med. 203: 1985-1998. [Abstract/Free Full Text]
  19. Li, Y., H. Li, M. Weigert. 2002. Autoreactive B cells in the marginal zone that express dual receptors. J. Exp. Med. 195: 181-188. [Abstract/Free Full Text]
  20. Goodnow, C. C., R. Brink, E. Adams. 1991. Breakdown of self-tolerance in anergic B lymphocytes. Nature 352: 532-536. [Medline]
  21. Nemazee, D.. 2000. Receptor editing in B cells. Adv. Immunol. 74: 89-126. [Medline]
  22. Cabatingan, M. S., M. R. Schmidt, R. Sen, R. T. Woodland. 2002. Naive B lymphocytes undergo homeostatic proliferation in response to B cell deficit. J. Immunol. 169: 6795-6805. [Abstract/Free Full Text]
  23. Jankovic, M., R. Casellas, N. Yannoutsos, H. Wardemann, M. C. Nussenzweig. 2004. RAGs and regulation of autoantibodies. Annu. Rev. Immunol. 22: 485-501. [Medline]
  24. Casellas, R., Q. Zhang, N. Y. Zheng, M. D. Mathias, K. Smith, P. C. Wilson. 2007. Ig{kappa} allelic inclusion is a consequence of receptor editing. J. Exp. Med. 204: 153-160. [Abstract/Free Full Text]
  25. King, L. B., J. G. Monroe. 2000. Immunobiology of the immature B cell: plasticity in the B cell Ag receptor-induced response fine tunes negative selection. Immunol. Rev. 176: 86-104. [Medline]
  26. 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]
  27. Zeng, D., M. K. Lee, J. Tung, A. Brendolan, S. Strober. 2000. Cutting edge: a role for CD1 in the pathogenesis of lupus in NZB/NZW mice. J. Immunol. 164: 5000-5004. [Abstract/Free Full Text]
  28. Qian, Y., H. Wang, S. H. Clarke. 2004. Impaired clearance of apoptotic cells induces the activation of autoreactive anti-Sm marginal zone and B-1 B cells. J. Immunol. 172: 625-635. [Abstract/Free Full Text]
  29. Mandik-Nayak, L., S. J. Seo, C. Sokol, K. M. Potts, A. Bui, J. Erikson. 1999. MRL-lpr/lpr mice exhibit a defect in maintaining developmental arrest and follicular exclusion of anti-dsDNA B cells. J. Exp. Med. 189: 1799-1814. [Abstract/Free Full Text]
  30. Pillai, S., A. Cariappa, S. T. Moran. 2005. Marginal zone B cells. Annu. Rev. Immunol. 23: 161-196. [Medline]
  31. Attanavanich, K., J. F. Kearney. 2004. Marginal zone, but not follicular B cells, are potent activators of naive CD4 T cells. J. Immunol. 172: 803-811. [Abstract/Free Full Text]
  32. Enzler, T., G. Bonizzi, G. J. Silverman, D. C. Otero, G. F. Widhopf, A. Anzelon-Mills, R. C. Rickert, M. Karin. 2006. Alternative and classical NF-{kappa}B signaling retain autoreactive B cells in the splenic marginal zone and result in lupus-like disease. Immunity 25: 403-415. [Medline]
  33. Srivastava, B., W. J. Quinn, III, K. Hazard, J. Erikson, D. Allman. 2005. Characterization of marginal zone B cell precursors. J. Exp. Med. 202: 1225-1234. [Abstract/Free Full Text]




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 Google Scholar
Google Scholar
Right arrow Articles by Choudhury, A.
Right arrow Articles by Eisenberg, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Choudhury, A.
Right arrow Articles by Eisenberg, R. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS