In Vivo Survival of Autoreactive B Cells: Characterization of Long-Lived B Cells1 Suzanne C. Morris,2*† Marta Moroldo,‡ Edward H. Giannini,‡ Tatyana Orekhova,*‡ and Fred D. Finkelman*† To determine the effects of chronic Ag stimulation on B cell survival and phenotype, we compared survival and surface markers of hen egg lysozyme (HEL)-specific B cells in Ig transgenic (Tgn) mice, which lack HEL, and in HEL-Ig transgenic mice, which express soluble HEL. Serum HEL levels were maximized in HEL-Ig Tgn mice by feeding them zinc, which activates the metallothionein promoter that regulates HEL expression. B cell age was characterized by expression of heat-stable Ag, and B220 and B cell survival was studied by evaluating changes in B cell number when lymphopoiesis was suppressed with anti-IL-7 mAb and by identifying newly generated B cells through 5-bromo-2ⴕ-deoxyuridine incorporation. Our observations show that the mean B cell life span is considerably reduced in HEL-Ig Tgn compared with Ig Tgn mice, but also demonstrate that some HEL-Ig Tgn B cells survive to maturity. Some of these surviving B cells have undergone receptor editing (substitution of an endogenous Ig light chain for the transgenic Ig light chain), so that their ability to bind HEL is decreased or absent. Surviving HEL-Ig Tgn B cells that retain HEL specificity express decreased mIgD and little or no mIgM. mIgD expression progressively decreases with increasing HEL-Ig Tgn B cell age. These observations suggest that self Ag-specific B cells can survive in the presence of soluble self Ag by down-regulating mIg expression, which should limit B cell signaling by Ag that might otherwise cause deletion of these cells. The Journal of Immunology, 2000, 164: 3035–3046.
olerization of autoreactive B cells that results from an interaction between autoantigen and B cell mIg in the absence of T cell help is an important constraint on the development of Ab-mediated autoimmune disease (1–3). Two forms of Ag-induced B cell tolerance have been described: 1) exposure of newly developed bone marrow B cells to multivalent, cell membrane-bound Ag, in the absence of T cell help leads to B cell apoptosis before the cells can migrate to secondary lymphoid organs (3–7); and 2) exposure to soluble Ag, in the absence of T cell help, leads to a more subtle type of B cell tolerance that is termed anergy (1, 8). Anergic B cells have been characterized most thoroughly in a transgenic (Tgn)3 mouse system in which mice, whose B cells nearly all express mIgM and mIgD that binds hen egg lysozyme (HEL) with high affinity (Ig Tgn mice), were bred to mice that secrete soluble HEL (HEL Tgn mice) (8, 9). HEL-specific B cells and T cells in their offspring, which express both transgenes (HEL-Ig Tgn mice), are exposed throughout their life span to soluble HEL and exhibit tolerance to this Ag (9, 10).
*Division of Immunology, University of Cincinnati College of Medicine, Cincinnati, OH 45267; †Cincinnati Veterans Administration Medical Center, Cincinnati, OH 45220; and ‡William S. Rowe Division of Rheumatology, Department of Pediatrics, Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45267 Received for publication April 9, 1999. Accepted for publication January 11, 2000. 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 a Biomedical Science Award from the National Arthritis Foundation, National Institutes of Health Grant P60-AR-44059-02, the Cincinnati Veterans Administration Medical Center, and the Children’s Hospital Research Foundation. 2 Address correspondence and reprint requests to Dr. Suzanne C. Morris, Department of Veterans Affairs, Research Service 151, 3200 Vine Street, Cincinnati, OH 45220. E-mail address: [email protected]
3 Abbreviations used in this paper: Tgn, transgenic; BrdU, 5-bromo-2⬘-deoxyuridine; HEL, hen egg lysozyme; HSA, heat-stable Ag; m, cell membrane; S-PE, streptavidinR-PE; S-PharRed, streptavidin-PharRed.
Copyright © 2000 by The American Association of Immunologists
Splenic B cells in HEL-Ig Tgn mice have been reported to have very low levels of mIgM but near normal levels of mIgD (8, 9, 11) and to proliferate well in response to stimulation with CD40 ligand or bacterial LPS but poorly in response to mIg cross-linking (12– 14). Because signals that result from mIg cross-linking allow B cells to survive the Fas ligand stimulation that occurs during cognate interactions with activated T cells (15), failure of anergic B cells to respond fully to mIg cross-linking can cause them to die rather than clonally expand during cognate B-T interactions (16 –18). Most recent studies have also concluded that B cell anergy in the HEL-Ig system is accompanied by a dramatic decrease in life span that results in a considerable decrease in splenic B cell number (19 –21), although the serum HEL concentration (20, 21), competition with B cells that are not autoreactive (22–24), and the presence of CD4⫹ T cells (24) have all been reported to influence the survival of the autoreactive B cells. Few of these studies, however, have examined the possibility that some autoreactive B cells in HEL-Ig Tgn mice survive for a considerable period of time, and with the exception of a report that some B cells in HEL-Ig Tgn mice lack autoreactivity because they express an endogenous, rather than the transgenic, Ig heavy chain gene (8), none has attempted to characterize long-lived HEL-Ig Tgn B cells. Because previous studies have not ruled out the possibility that some autoreactive B cells survive for a long time in HEL-Ig Tgn mice, and long term survival of autoreactive B cells would have important implications for the development of autoimmune disorders, we have restudied the issue of B cell survival in HEL-Ig Tgn mice and have looked for evidence of long-lived autoreactive B cells in a system in which the serum HEL concentration is maintained at a high level. The results of these experiments, which used three different techniques to evaluate B cell survival, reveal that while most B cells in HEL-Ig Tgn mice have a decreased life span, some survive 0022-1767/00/$02.00
3036 for a relatively long time. The survival of some of these long-lived B cells appears to be associated with receptor editing (25–27), which decreases or eliminates their affinity for HEL. A larger population of long-lived HEL-Ig Tgn splenic B cells, however, appears to retain mIg that has the ability to bind HEL, but has downregulated its expression of both mIgM and mIgD. Thus, mIg expression down-regulation may be a mechanism that allows autoreactive B cell survival and creates a reservoir of long-lived autoreactive B cells that, if activated, might induce humoral autoimmune disease.
Materials and Methods Animals C57BL/6 female mice, obtained from the Small Animals Division of the National Cancer Institute, National Institutes of Health (Bethesda, MD), and CB20 mice, obtained from Dr. Michael Potter (National Cancer Institute, National Institutes of Health) were bred in the Cincinnati Veterans Administration Medical Center animal facility (Cincinnati, OH). C57BL/6 female mice were bred to C57BL/6 male mice that were hemizygous for both the MD4 anti-HEL Ig H and L transgene and the ML5 soluble HEL transgene (a gift from Chris Goodnow, Australian National University, Canberra, Australia) to generate mice that carry only the MD4 transgene (Ig Tgn mice) and mice that carry both the MD4 and ML5 transgenes (HEL-Ig Tgn mice). Mice were used at 8 – 43 wk of age. Groups of mice subjected to different treatments were age and sex matched in individual experiments.
Typing of transgenic mice Mice that expressed the HEL transgene and/or the anti-HEL transgene were identified by PCR (28). DNA was isolated with QIAamp tissue kits for DNA isolation (Qiagen, Santa Clarita, CA). PCR reactions were performed as previously described (5). Goodnow (unpublished observation). Briefly, the following five oligonucleotides were used in PCR reactions: IgHF1, 5⬘-GCGACTCCATCACCAGCGAT-3⬘; IgHF2, 5⬘-CTGGAGCCCT AGCCAAGGAT-3⬘; IgHR1, 5⬘-ACCACAGACCAGCAGGCAGA-3⬘; HEL3F, 5⬘-GAGCGTGAACTGCGCGAAGA-3⬘; and HEL4R, 5⬘-TCGG TACCCTTGCAGCGGTT-3⬘. HEL-Ig Tgn mice have three bands, corresponding to the 264-bp endogenous Ig band, the 430-bp Ig transgene band, and the 160-bp lysozyme transgenic band. Ig Tgn mice have two bands, the 264-bp band and the 430-bp band. Oligonucleotide primers were produced by the BIC synthesis center at the Uniformed Services University of the Health Science (Bethesda, MD).
Experimental conditions All mice were maintained on drinking water that contained 25 mM ZnCl2 for at least 3 days before the initiation of other treatments and for the duration of each experiment to maximize serum HEL levels in HEL-Ig Tgn mice (21). In experiments in which newly generated B cells were identified by 5-bromo-2⬘-deoxyuridine (BrdU) incorporation (7, 19), 0.8 mg/ml of BrdU (Sigma, St. Louis, MO) was also added to drinking water for a defined period of time. BrdU-containing water was shielded from light and changed every third day.
Abs and immunological reagents The following hybridomas and plasmacytomas were obtained and grown as ascites in Pristane-primed athymic nude, BALB/c, or CB20 mice, and mAbs were purified from ascites by (NH4)2SO4 precipitation and DE-52 (Whatman, Clifton, NJ) cation exchange column chromatography, unless otherwise stated: RA3-6B2 (rat IgG2a anti-mouse CD45R/B220) (29), DS-1 (mouse IgG1 of the b allotype specific for mouse IgM of the a allotype) (30), H␦a/1 (mouse IgG2b of the b allotype specific for mouse IgD of the a allotype) (31), AF3.33 (mouse IgG2a of the a allotype specific for mouse IgD of the b allotype) (32), 5E4 and 2D1 (mouse IgG1 anti-HEL mAbs; gift from Dennis Metzger, Toledo, OH) (33), 24G2 (rat IgG2b anti-mouse Fc␥RII/III) (34), GK1.5 (rat IgG2b anti-mouse CD4) (35), M25 (mouse IgG2b anti-human IL-7 that cross-reacts with mouse IL-7) (36), and MOPC-352 (a gift from Dr. Michael Potter, National Cancer Institute, National Institutes of Health), a mouse IgG2b that does not bind to mouse proteins and that was used as a control for M25. Some of these Abs were labeled with FITC (37) (Calbiochem-Behring, La Jolla, CA), biotin-Nhydroxysuccinimide (38) (Calbiochem-Behring), or Cy5 reactive dye (Research Organics, Cleveland, OH), as suggested by the manufacturer. Biotin- or FITC-labeled M1/69 (anti-HSA) (39), PE-labeled 1D3 (anti-CD19)
SURVIVAL OF AUTOREACTIVE B CELLS (40), and PerCP-labeled RA3– 6B2 were purchased from PharMingen (San Diego, CA). FITC-anti-BrdU was purchased from Becton Dickinson (San Jose, CA). HEL (lysozyme from chicken egg white) was purchased from Sigma (St. Louis, MO).
Immunofluorescence staining Spleen or bone marrow cells were depleted of erythrocytes, filtered through nylon gauze, and suspended at 20 ⫻ 106 cells/ml in HBSS with 10% newborn bovine serum and 0.2% NaN3 (HNA). One hundred microliters of cell suspension was stained for 30 min on ice with 1 g each of appropriately labeled Abs. Cells were washed twice with HNA, then, if appropriate, were exposed to either streptavidin-R-PE (S-PE; purchased from Life Technologies (Gaithersburg, MD) or Becton Dickinson Immunocytometry Systems) or to streptavidin-PharRed (S-PharRed; purchased from PharMingen) for 30 min on ice. All staining was performed in the presence of 1 g of unlabeled anti-Fc␥RII/III (24G2). After washing once with HNA, all samples, except those that required staining for BrdU incorporation, were washed once with HBSS/0.2% sodium azide, then fixed in PBS/2% paraformaldehyde. Staining for BrdU was modified from the procedure described by Allman (39). Samples that required staining for BrdU were washed in PBS and resuspended in 0.5 ml of ice-cold 0.15 M NaCl, after which 1.2 ml of ice-cold 95% ethanol was slowly added while gently vortex mixing the cells. Cells were incubated on ice for 30 min, then washed with PBS. One milliliter of PBS/1% paraformaldehyde/0.01% Tween 20 was then added, and cells were incubated for 30 min at room temperature, followed by overnight storage at 4°C. The following day cells were pelleted by centrifugation (1500 rpm for 15 min), then incubated for 10 min at room temperature with 1 ml of 0.15 M saline that contained 4.2 mM MgCl2 and 50 Kunitz units/ml of DNase I (Sigma). Samples were then washed with PBS, stained with FITC-anti-BrdU (30 min, room temperature), washed with PBS, and analyzed by flow cytometry. All samples were analyzed with either a FACScan, FACSCalibur Analyzer equipped with a red diode laser, or with a FACS Vantage equipped with a red diode laser (Becton Dickinson, Mountain View, CA). Data analysis was performed with LYSIS II or CellQuest software (Becton Dickinson). Light scatter gates were set to exclude most nonlymphoid cells and cells that had died before fixation. Cells that had been stained with a single fluorochromelabeled Ab were used to determine compensation for overlap between emission spectra. The percentages of specifically stained cells and the mean fluorescence intensities of specifically stained cells were determined.
Cell counts Nucleated cells were counted with a Coulter counter (Coulter, Miami, FL) that was set to exclude dead cells. Absolute numbers of cells that had a defined phenotype were determined by multiplying the percentage of cells that expressed that phenotype by the total spleen cell number.
Results B cell mIg is nearly saturated with HEL in ZnCl2-treated HELIg Tgn mice Preliminary studies demonstrated considerable variability in serum HEL levels and B cell numbers in HEL-Ig Tgn mice (data not shown). Because B cell survival in HEL-Ig Tgn mice may depend on the serum HEL concentration (20, 21), and HEL gene expression in these mice is regulated by the metallothionein promoter, ZnCl2 was added to the drinking water of all of our mice at least 3 days before initiation of each experiment and was continued for the duration of each experiment to maintain the serum HEL concentration at a consistently high level. In contrast to results obtained in another study in which HEL-Ig Tgn mice were not treated with zinc (18), surface mIg on splenic B cells from ZnCl2-treated HEL-Ig Tgn mice was nearly saturated with HEL (Fig. 1). B cells from HEL-Ig Tgn mice were found to bind considerably less HEL than B cells from Ig Tgn mice; thus, mIg expression is considerably down-regulated on HEL-specific B cells in HEL-Ig Tgn mice. Splenic B cells that have a mature phenotype are selectively depleted in HEL-Ig Tgn mice Most, but not all, previous studies of HEL-Ig Tgn mice have suggested that the B cell life span is considerably decreased in these
The Journal of Immunology
FIGURE 1. B cell mIg is decreased and nearly saturated with HEL in HEL-Ig Tgn mice. Spleen cells from individual Ig Tgn and HEL-Ig Tgn mice were counted and incubated in HNA that did not contain HEL (filled bars) or in HNA that contained 100 ng/ml of HEL (hatched bars) for 1 h at 4°C. Cells were washed, then stained with PerCP-labeled anti-B220, biotin-labeled anti-HSA, and FITC-labeled anti-HEL mAbs, followed by PE-labeled anti-CD19 and S-PharRed, and were analyzed by flow cytometry to determine HEL fluorescence of B220⫹CD19⫹HSA⫹ spleen cells. Upper panels show histograms of HEL fluorescence of B220⫹ CD19⫹HSA⫹ spleen cells from a representative HEL-Ig Tgn mouse and an Ig Tgn mice. Scales on the ordinates differ for the two panels. The lower panel shows mean HEL fluorescence intensity for B cells from the single Ig Tgn mouse analyzed and for B cells from four HEL-Ig Tgn mice. Means and SEs are shown.
mice (19 –21). Because most immature B cells express considerable HSA, and expression of this surface marker decreases as B cells mature (39, 41), we compared HSA expression on splenic B cells from Ig Tgn and HEL-Ig Tgn mice (Fig. 2). Although numbers of HSAbright B cells were similar in HEL-Ig Tgn and Ig Tgn mice, numbers of HSAint and HSAdull B cells were considerably decreased in HEL-Ig Tgn mice. This suggests that B cell production is at least normal in HEL-Ig Tgn mice, but that the mean survival of B cells in these mice is decreased. The presence of some HSAint and HSAdull splenic B cells in HEL-Ig Tgn mice, however, suggests that some B cells in these mice have a long life span. Characterization of splenic B cell life span by in vivo BrdU labeling To confirm our conclusions about B cell life span that were based on B cell HSA expression, Ig Tgn and HEL-Ig Tgn mice were fed BrdU in their drinking water for 14 days before sacrifice, and BrdU labeling of HSAbright, HSAint, and HSAdull splenic B cells was determined by flow cytometry (Fig. 3). As expected, most HSAbright B cells in both Ig Tgn and HEL-Ig Tgn mice had incorporated BrdU into DNA, indicating cell division during the past 14 days, while only a minority of HSAdull B cells in either strain were BrdU⫹. Because BrdU labels dividing cells, and pro-B cells divide before differentiating into B cells, these results confirm that strong HSA expression for the most part identifies immature B cells, while low HSA expression for the most part identifies a relatively mature B cell population. In addition, the increased percentage of BrdU⫹ B cells and the decreased absolute number of BrdU⫺ B cells in every HEL-Ig Tgn HSA-defined B cell population, compared with the corresponding populations in Ig Tgn mice, are consistent with decreased B cell life span in HEL-Ig Tgn mice. To confirm this observation, we pulsed mice with BrdU-containing drinking water for 3 days, then provided them with water
FIGURE 2. Survival of most, but not all, B cells is decreased in HEL-Ig Tgn mice. Spleen cells from individual Ig Tgn and HEL-Ig Tgn mice were counted and stained with FITC-labeled anti-HSA and biotin-labeled antiB220 mAbs, followed by S-PE, and were analyzed by flow cytometry to determine the percentages of B220⫹ cells that exhibit bright, intermediate, or dull staining for HSA. The numbers of HSA bright, intermediate, and dull spleen cells were determined by multiplying the percentage of each cell type in spleen by the total number of spleen cells. The upper panel shows HSA histograms of B220⫹ spleen cells from representative Ig Tgn and HEL-Ig Tgn mice. Boundaries of bright, intermediate, and dull populations are indicated. The bottom panel shows mean numbers and SEs of HSA bright, intermediate, and dull spleen cells in 25 Ig Tgn and 29 HEL-Ig Tgn mice.
that lacked BrdU for the next 11 days, after which mice were sacrificed. Most BrdU⫹ B cells from both Ig Tgn and HEL-Ig Tgn mice were in the HSAint population at the time of sacrifice; however, there were 5 times as many HSAint BrdU⫹ splenic B cells in Ig Tgn as in HEL-Ig Tgn mice (Fig. 4). Because B cell production is at least as great in HEL-Ig Tgn as in Ig Tgn mice (Figs. 2 and 3), this suggests that only one-fifth as many HEL-Ig Tgn B cells as Ig Tgn B cells survive for 11–14 days. Because a substantial, albeit considerably reduced, number of BrdU⫺ HSAint and HSAdull B cells is still found in HEL-Ig Tgn mice in the experiment shown in Fig. 3, and a substantial, albeit considerably reduced, number of BrdU⫹ HSAint and HSAdull B cells is still found in HEL-Ig Tgn mice in the experiment shown in Fig. 4, some HEL-Ig Tgn B cells must survive for a relatively long time (at least 11–14 days). Characterization of splenic B cell life span by in vivo anti-IL-7 mAb treatment To compare splenic B cell life span in Ig Tgn and HEL-Ig Tgn mice by a third, independent method, we evaluated the effects of treating Ig Tgn and HEL-Ig Tgn mice with anti-IL-7 mAb (3 mg i.p., three times per week, a dose that has previously been shown to strongly inhibit B lymphopoiesis in the bone marrow and T
FIGURE 3. HEL-Ig Tgn mice have a higher percentage of recently generated splenic B cells than Ig Tgn mice. Three Ig Tgn and five HEL-Ig Tgn mice were administered BrdU-containing drinking water for 14 days before sacrifice. Spleen cells from individual mice were counted; stained with biotin-labeled anti-HSA mAb and Cy5-labeled anti-B220 mAb, followed by S-PE; then fixed, permeabilized, and stained with FITC-anti-BrdU mAb. Contour plots demonstrate gates drawn to identify HSA⫹B220⫹ BrdU⫹or⫺ cells that were then determined to be either HSA bright, intermediate, or dull by the indicated markers on fluorescence histograms. The percentages of BrdU⫺ and BrdU⫹ HSA bright, intermediate, and dull B220⫹ spleen cells were determined by flow cytometry and multiplied by total spleen cell numbers to determine the number of spleen cells of each type. Means and SEs are shown.
lymphopoiesis in the thymus (36, 42, 43)) on numbers of HSAbright, HSAint, and HSAdull splenic B cells (Fig. 5). To distinguish B cells generated before initiation of anti-IL-7 mAb treatment from B cells generated after anti-IL-7 mAb treatment had begun, mice were fed drinking water that contained BrdU, starting on the first day of anti-IL-7 mAb treatment. Anti-IL-7 mAb treatment decreased the number of recently generated (BrdU⫹) HSAbright splenic B cells in both strains, but had a considerably greater effect in HEL-Ig Tgn than in Ig Tgn mice (89.1 vs 56.1%, respectively). Anti-IL-7 mAb treatment also decreased HSAint BrdU⫹ splenic B cell number in HEL-Ig Tgn, but not in Ig Tgn mice. It is likely that these effects resulted from the suppression of B lymphopoiesis, rather than from reduced B cell survival, because anti-IL-7 had no effect on the number of splenic B cells generated before the initiation of treatment with this mAb (BrdU⫺ B cells). Anti-IL-7 mAb treatment also had little effect on the small number of BrdU⫹ HSAdull B cells in Ig Tgn or HEL-Ig Tgn mice, suggesting that most BrdU⫹ HSAdull B cells were mature, proliferating cells rather than newly generated cells. Treatment of Ig Tgn or HEL-Ig Tgn mice with an isotype-matched control mAb, instead of anti-IL-7, did not decrease the number of HSAbright or HSAint spleen cells (data not shown). Taken together, our experimental data provide evidence by three independent techniques that the average B cell life span is shorter in HEL-Ig Tgn mice than in Ig Tgn mice when HEL is maintained at a high level.
SURVIVAL OF AUTOREACTIVE B CELLS
FIGURE 4. Characterization of B cell survival in Ig Tgn and HEL-Ig Tgn mice by pulsing mice with BrdU. Ig Tgn and HEL-Ig Tgn mice (four per group) were administered BrdU-containing drinking water on days 0 –2 followed by regular drinking water on days 3–13 and were sacrificed on day 14. Spleen cells from all mice were individually counted and stained with biotin-anti-HSA mAb and Cy5-anti-B220 mAb, followed by S-PE, then fixed, permeabilized, and stained with FITC-anti-BrdU mAb. Cells were analyzed by flow cytometry as described in Fig. 3 to determine the percentages of BrdU⫹ and BrdU⫺B220⫹ cells that demonstrated bright, intermediate, or dull HSA staining; the number of cells of each phenotype was determined by multiplying percentages by total spleen cell number. Means and SEs are shown. (Note the log scale on the abscissa.) Numbers of HSAdullBrdU⫹, HSAintBrdU⫹, and HSAbrightBrdU⫹ spleen cells were 25, 20, and 65% as great, respectively, in HEL-Ig Tgn as in Ig Tgn mice.
Surface Ig expression and HEL binding by B cells from Ig Tgn and HEL-Ig Tgn mice Although our studies indicate that average splenic B cell life span is decreased in HEL-Ig Tgn mice, they also demonstrate the existence of long-lived B cells in these mice. This long-lived population might represent B cells that were insensitive to the relatively high concentrations of HEL in zinc-treated mice because they expressed endogenous, rather than transgenic, Ig heavy chains (8) or because they expressed an endogenous Ig light chain as a result of receptor editing (25–27). Alternatively, B cells that retained their HEL specificity might have survived by modifying their phenotype in a way that decreased sensitivity to Ag signaling, or survival may have been selective for a subset of B cells that is relatively insensitive to deletion. Finally, some HEL-specific B cells may have survived by chance regardless of and without altering their properties. To investigate these possibilities, experiments were performed to characterize mature splenic B cells in zinc-treated HEL-Ig Tgn mice. The possibility that the long-lived B cells in these mice were unresponsive to HEL because they expressed endogenous, rather than transgenic, Ig heavy chains was eliminated because splenic B cells that expressed IgM or IgD of the endogenous b allotype were not detected in the great majority (⬎95%) of HEL-Ig Tgn mice in our colony (data not shown); the small number of mice that exhibited substantial numbers of b allotype B cells was excluded from further study. To compare the extent of receptor editing in splenic B cells from Ig Tgn and HEL-Ig Tgn mice, spleen cells
The Journal of Immunology
FIGURE 5. Effect of anti-IL-7 mAb on B cell number in Ig Tgn and HEL-Ig Tgn mice. Ig Tgn and HEL-Ig Tgn mice were administered BrdU in their drinking water for 2 wk. Mice of each type (four per group) were treated for the same 2-wk period with anti-IL-7 mAb or received no mAb. Spleen cells from all mice were individually counted and stained with biotin-anti-HSA mAb and Cy5-anti-B220 mAb, followed by S-PE, then fixed, permeabilized, and stained with FITC-anti-BrdU mAb. Cells were analyzed by flow cytometry as described in Fig. 3 to determine the percentages of BrdU⫹ and BrdU⫺ B220⫹ cells that demonstrated bright, intermediate or dull HSA staining. The number of cells of each phenotype was determined by multiplying percentages by total spleen cell number. Means and SEs are shown.
from these mice were stained for B220, HSA, transgenic mIg (IgM plus IgD), and HEL (after in vitro incubation with HEL on ice) and were evaluated for mIg and HEL expression by B cells that had a mature (B220brightHSAdull) or an immature (B220dullHSAbright) phenotype (Fig. 6). Although immature B cells from HEL-Ig Tgn mice (the R2 population in Fig. 6) expressed less mIg than the same cell population from Ig Tgn mice, nearly all immature B cells from both strains expressed proportionate quantities of mIg and HEL binding. This is evidence against considerable receptor editing, which would decrease the HEL binding capacity without decreasing mIg expression. In contrast, B cells that showed evidence of receptor editing (decreased binding of HEL relative to mIg expression) were easily detected in the mature splenic B cell population in HEL-Tgn, but not Ig Tgn, mice. The percentage of mature splenic B cells that showed evidence of receptor editing varied considerably among different HEL-Ig Tgn mice (note error bars in Fig. 6, lower bar graph). Many HEL-Ig B cells that had undergone receptor editing still bound some HEL, suggesting that receptor editing had reduced, but not eliminated, the affinity for HEL, although the HEL binding capacity of receptor-edited B cells also varied considerably among different HEL-Ig Tgn mice. Most interestingly, the mature splenic B220⫹ cell population in zinctreated HEL-Ig Tgn mice included a substantial number of cells that expressed little or no mIg and failed to bind detectable HEL; this mIg⫺ population was smaller in immature splenic B cells from HEL-Ig Tgn mice and was difficult to detect in immature or mature B cells from Ig Tgn mice. We further characterized the B220⫹mIg⫺ spleen cells in HEL-Ig Tgn mice by studying 1) whether these cells were truly B cells, and 2) whether the number of these cells increased when mean B cell age was increased by treating mice with anti-IL-7 mAb to suppress B lymphopoiesis. To investigate the first issue, we examined HEL and mIg expression on spleen cells that bore B220 and a second B cell marker, CD19. As was seen when only B220 was used to identify spleen cells as B cells, B220⫹CD19⫹ spleen cells from HEL-Ig Tgn mice included cells that lacked detectable mIg (Fig. 7, third contour plot in second row and right bar
FIGURE 6. Receptor editing does not account for the survival of most mature HEL-Ig Tgn splenic B cells. Spleen cells from individual Ig Tgn and HEL-Ig Tgn mice (four per group) were counted and incubated for 1 h with 100 ng/ml of HEL at 4°C and then stained with PerCP-anti-B220 mAb, FITC-anti-HSA mAb, biotin-anti-HEL-mAb, and a mixture of Cy5labeled anti-IgMa and anti-IgDa mAbs. Upper contour plots show B220 and HSA staining of representative mice; middle contour plots show mIg(Da⫹Ma) and HEL staining of mature (B220bright, HSAint⫹dull) spleen cells (R1) from these same mice; lower contour plots show mIg(Da⫹Ma) and HEL staining of immature (B220dull, HSAbright) spleen cells (R2) from these same mice. Splenic B cells that stain less brightly for HEL than would be expected from their staining for mIg(Da⫹Ma) are considered to have undergone receptor editing. Bar graphs indicate percentages or numbers of mature (B220brightHSAint⫹dull; R1) and immature (B220dullHSAbright; R2) mIg⫹ splenic B cells that lack evidence of receptor editing (Ig⫹HEL⫹), mIg⫹ splenic B cells that show evidence of receptor editing, and mIg⫺ splenic B cells in these mice. Percentages were determined by flow cytometry and were multiplied by total spleen cell numbers to determine the number of spleen cells of each type. Means and SEs are shown.
graph). Thus, the mIg⫺B220⫹ spleen cells that are present in HEL-Ig Tgn mice truly appear to be B cells. We investigated the second issue by treating a group of HEL-Ig Tgn mice for 2 wk with a dose of anti-IL-7 mAb that strongly suppresses bone marrow B
SURVIVAL OF AUTOREACTIVE B CELLS
FIGURE 7. Anti-IL-7 treatment is associated with loss of mIg by splenic HEL-Ig Tgn B cells. HEL-Ig Tgn mice (four per group) were left untreated or were injected i.p with 3 mg of either anti-IL-7 mAb (to suppress new B cell production) or a control isotype-matched mAb three times per week for 2 wk or were injected i.v. with 1 mg of anti-CD4 mAb once a week (to block T cell help). Mice were sacrificed on day 14, and their spleen cells were counted and incubated at 4°C for 1 h with either 100 ng/ml of HEL or buffer only and then stained with PerCP-labeled anti-B220, Cy5-labeled anti-IgMa and/or anti-IgDa, and FITC-labeled anti-HSA or FITC-labeled anti-HEL, followed by PE-labeled anti-CD19. Spleen cells from an Ig Tgn mouse and a CB20 mouse (Ig b allotype) were similarly treated. Cells were then analyzed by flow cytometry. Upper contour plots show spleen cells from a representative mouse, gated for scatter and for the CD19⫹B220⫹ (B cell) population. The second row of contour plots shows HEL and mIg fluorescence for the CD19⫹B220⫹ population. Gates define the receptor-edited (R-E), non-receptor-edited mIg⫹HEL⫹ (mIg⫹), and mIg⫺ splenic B cell populations. The third row of contour plots show B220 and HSA fluorescence for CD19⫹B220⫹ spleen cells. Numbers of mature, immature, mIg⫹, mIg⫺, and receptor-edited spleen cells were determined by multiplying the appropriate percentages by the total spleen cell number. Bar graphs show means and SEs. By the criteria shown, a single Ig Tgn mouse that was simultaneously studied had 20.6 ⫻ 106 mIg⫹ cells, 0.12 ⫻ 106 mIg⫺ cells, and 0.11 ⫻ 106 receptor-edited cells in its spleen.
lymphopoiesis (8, 36, 42). Anti-IL-7 mAb treatment selectively decreased the number of immature splenic B cells (Fig. 7, second vs third contour plot in third row and bar graph on left), decreased the number of mIg⫹ B cells, and increased the number of mIg⫺ B cells (Fig. 7, third vs fourth contour plot in second row and bar graph on right). These effects most likely resulted from inhibition of B lymphopoiesis rather than from a nonspecific effect of injection of a large quantity of mouse IgG or from inhibition of IL-7 effects on mature T cells, which may have decreased T cell help. Injection of an isotype-matched control mAb at the same dose as anti-IL-7 had no significant effect on any of these populations (Fig. 7, third vs fifth contour plots in second row; second vs fourth
contour plots in third row and bar graphs), and injection of an anti-CD4 mAb at a dose that effectively suppresses T cell help (35, 42) selectively decreased the number of mature B cells and failed to increase the percentage of splenic B cells that were mIg⫺ (Fig. 7, second vs fifth contour plots in third row, third vs sixth contour plot in second row, and bar graphs). Thus, the effects of anti-IL-7 mAb on HEL-Ig Tgn splenic B cells are specific and are not related to inhibition of T cell help. Membrane IgM and IgD expression by HEL-Ig Tgn B cells The presence of a B220bright HSAdull mIgdull-negative spleen cell population in HEL-Ig Tgn mice raised the question of which Ig
The Journal of Immunology
3041 mice, HSAint splenic B cells from HEL-Ig Tgn mice expressed only one-half as much mIgD, on the average, as HSAint Ig Tgn splenic B cells, and HSAdull HEL-Ig Tgn B cells averaged only one-third as much mIgD as the corresponding Ig Tgn B cell population. Consistent observations were made in studies of bone marrow B cells from Ig Tgn and HEL-Ig Tgn mice (Fig. 9). Three bone marrow B-lineage populations were defined in Ig Tgn and HEL-Ig Tgn mice by staining for HSA and B220: a mature, B220brightHSAdull population that most likely represents recirculating B cells (19, 39, 42, 44, 45), an immature B220dullHSAbright B cell population, and a population of pre- and pro-B cells that is B220⫹/⫺HSAbright (Fig. 9, A and B). The Ig Tgn B220brightHSAdull B cells express considerable mIgM and mIgD, most B220dullHSAbright B cells express considerable mIgM and relatively low amounts of mIgD, and most B220⫹/⫺HSAbright B cells lack mIg (Fig. 9, A–C). In contrast, mIgM expression was almost undetectable on all bone marrow B cells from HEL-Ig Tgn mice, and mIgD expression, while normal on the immature B220dullHSAbright subset of HEL-Ig Tgn bone marrow cells, was reduced by a factor of 3– 4 on the small number of mature B220brightHSAdull bone marrow B cells in these mice (Fig. 9C). Anti-IL-7 mAb treatment reduces mIgD expression to barely detectable levels on HEL-Ig Tgn splenic B cells
FIGURE 8. Splenic B cells from HEL-Ig Tgn mice express low levels of mIgM and lose mIgD as they age. Spleen cells from Ig Tgn and HEL-Ig Tgn mice (four per group) were stained with biotin-anti-B220 mAb, FITCanti-HSA mAb, and either Cy5-anti-IgM or Cy5-anti-IgD mAbs, followed by S-PE. mIgM and mIgD expression on HSAbright, HSAint, and HSAdull B220⫹ cells was determined by flow cytometry. Contour plots in upper panels illustrate staining for B220 and HSA by spleen cells from representative mice; histograms in the second row of panels illustrate mIgM staining of HSAbright, HSAint, and HSAdull splenic B cells from these mice; histograms in the third row of panels illustrate mIgD staining of splenic B cells from these mice; and the dot plot in the lowest panel indicates means and SEs for mIgM and mIgD staining of the HSA/B220-defined splenic B cell populations.
isotype(s) accounted for the decrease in mIg expression among mature HEL-Ig splenic B cells. This question was addressed by evaluating mIgM and mIgD expression on splenic B cells from Ig Tgn and HEL-Ig Tgn mice following staining for B220, HSA, and IgM or IgD. Consistent with previous reports (8, 9, 11), all splenic B cells from HEL-Ig Tgn mice regardless of the level of apparent maturity expressed little or no mIgM (Fig. 8). However, while immature (HSAbright) splenic B cells from HEL-Ig Tgn mice expressed at least as much mIgD as the same population from Ig Tgn
To specifically examine the effect of anti-IL-7 mAb treatment on mIgM and mIgD expression by HEL-Ig Tgn splenic B cells, an experiment was performed in which spleen cells from untreated or anti-IL-7 mAb-treated Ig Tgn and HEL-Ig Tgn mice were stained for B220 and CD19 as well as for HSA, HEL, and mIgMa, mIgDa, or mIgMa plus mIgDa. Untreated CB20 mice (IgH b allotype) were similarly stained as a negative control. Only spleen cells that stained positively for both CD19 and B220 were considered to be B cells and were evaluated further. Representative contour plots and histograms from this experiment are shown in Fig. 10; results are summarized in a scatter graph and bar graphs shown in Fig. 11, A and B. Although simultaneous staining with five fluorochromes required the use of a system that detected individual fluorochromes with reduced sensitivity; sufficient sensitivity was retained to fulfill the goal of the experiment. All populations of HEL-Ig Tgn B cells expressed barely detectable mIgM (Fig. 11B). Immature (HSAbright) B cells from untreated Ig Tgn and HEL-Ig Tgn mice expressed similar amounts of mIgD, while mIgD expression was reduced on HEL-Ig Tgn mature (HSAint-dull) B cells compared with mature Ig Tgn B cells (Fig. 11B). Most importantly, 2 wk of treatment with anti-IL-7 mAb was accompanied by a substantial loss of mIgD and HEL binding capacity by HEL-Ig Tgn splenic B cells (Fig. 10, second and fourth histograms in second row and Fig. 11, A and B), so that most splenic B cells from these mice stained no more brightly for mIgMa, mIgDa, or HEL than B cells from nontransgenic, Ig b allotype, CB20 mice (Fig. 10, third row of histograms). In contrast, mIgM, mIgD, and HEL staining were clearly positive on mature, B220⫹CD19⫹ spleen cells from Ig Tgn mice (Fig. 10, fourth row of histograms, and Fig. 11, A and B). To confirm these observations, an additional experiment was performed that examined the effects of anti-IL-7 mAb, anti-CD4 mAb, or a control mAb on mIgM and mIgD expression and HEL binding capacity of HEL-Ig Tgn splenic B cells. B cells were identified by their expression of both B220 and CD19; B cells that had an immature or mature phenotype were identified by their expression of B220 and HSA (Fig. 7). Of the mAbs tested, only the anti-IL-7 mAb reduced HEL binding by HEL-Ig Tgn splenic B cells (Fig. 11C). This reduction in HEL binding capacity was accompanied by a selective reduction in mIgD expression (Fig. 11D).
SURVIVAL OF AUTOREACTIVE B CELLS
FIGURE 9. mIg expression by bone marrow B cells in Ig Tgn and HEL-Ig Tgn mice. Bone marrow cells from Ig Tgn and HEL-Ig Tgn mice (four per group) were stained with biotin-anti-HSA, FITC-anti-B220, and either Cy5-anti-IgMa or Cy5-anti-IgDa mAbs, followed by S-PE. mIgM and mIgD expression on B220brightHSAdull, B220dullHSAbright, and B220⫹/⫺HSAbright cells was determined by flow cytometry. A, Contour plots illustrate the light scatter and B220 and HSA staining of the bone marrow cells from a representative Ig Tgn mouse and show the gates used; histograms illustrate mIgM and mIgD staining of the B220brightHSAdull, B220dullHSAbright, and B220⫹/⫺HSAbright cells from the same mouse (marker indicates mIg⫹ cells). B, Bar graphs indicate the percentages of Ig Tgn and HEL-Ig Tgn bone marrow cells that are B220brightHSAdull, B220dullHSAbright, or B220⫹/⫺HSAbright and the percentages of bone marrow cells that have these phenotypes and also express mIgM or mIgD. Means and SEs are shown. C, The means and SEs for mIgM and mIgD staining of the HSA/B220defined bone marrow B cell populations are shown (open symbols represent Ig Tgn mice and closed symbols represent HEL-Ig Tgn mice).
Taken together, these experiments demonstrate that in the absence of receptor editing, long term survival of Ag-specific B cells in the presence of a relatively high concentration of Ag is accompanied by down-regulation of mIgD as well as mIgM and/or by the selection of B cells that express little mIg of either isotype.
Discussion We have used three different methods to confirm previous observations that exposure of autoreactive B cells to near-saturating quantities of soluble Ag causes most of these B cells to die while
The Journal of Immunology
FIGURE 10. Effect of anti-IL-7 mAb treatment on B cell mIg expression in Ig Tgn and HEL-Ig Tgn mice. Ig Tgn and HEL-Ig Tgn mice (three or four per group) were left untreated or were injected i.p. with 3 mg of anti-IL-7 mAb three times per week for 2 wk to suppress new B cell production. Spleen cells from individual Ig Tgn and HEL-Ig Tgn mice as well as from a CB20 mouse (Ig b allotype) were counted and incubated for 1 h with 100 ng/ml of HEL at 4°C and stained with PerCP-anti-B220, biotin-anti-HSA, FITC-anti-HEL, and Cy5-labeled anti-IgMa and/or anti-IgDa mAb, followed by PE-labeled anti-CD19 and S-PharRed, then analyzed by flow cytometry. Upper panels show contour plots from representative animals of the type labeled. Gates used for analysis are shown. Histograms in the second row show the intensity of fluorescence staining of B220⫹CD19⫹ spleen cells from an untreated mouse (thin line) or an anti-IL-7 mAbtreated mouse (bold line) for IgMa, IgDa, IgMa and IgDa, or HEL. In histograms in the third and fourth rows spleen cells from a CB20 mouse (Ig b allotype) are used as a negative control for staining with the anti-Ig a allotype reagents and for staining with HEL. The scale on the ordinate differs among panels.
still immature (19, 46 – 49). 1) The decreased number of splenic B cells in HEL-Ig Tgn mice, as opposed to Ig Tgn mice, was shown to result from a selective deficiency in B cells that express surface markers characteristic of mature B cells (relatively high quantities of B220 and relatively low quantities of HSA) (Figs. 2 and 6). 2) Labeling of dividing B cells, including newly generated B cells, with BrdU demonstrated that similar numbers of splenic B cells were labeled in Ig Tgn and HEL-Ig Tgn mice when the mice were treated with BrdU for 14 days (Fig. 3), but fewer labeled splenic B cells were present in HEL-Ig Tgn than in Ig Tgn mice 11 days after a 3-day labeling period (Fig. 4). Furthermore, B cells that had survived for ⬎14 days were particularly deficient in HEL-Ig Tgn mice regardless of their apparent maturity, as characterized by surface expression of B220 and HSA (Fig. 3). 3) Treatment with anti-IL-7 mAb, which suppresses B lymphopoiesis, decreased splenic B cell numbers more in HEL-Ig Tgn than in Ig Tgn mice (Fig. 5), while treatment with an isotype-matched control mAb did not decrease splenic B cell numbers (data not shown). The effect of anti-IL-7 mAb on splenic B cell number most likely resulted from inhibition of B lymphopoiesis rather than from nonspecific effects of injecting mice with a large quantity of mouse IgG2b or blocking IL-7 stimulation of T cells or other cells that could provide help to B cells, because anti-IL-7 mAb treatment had no effect on the survival of B cells that had been produced before the initiation of anti-IL-7 treatment (i.e., the BrdU⫺ B cells in Fig. 5), and the effects of anti-IL-7 mAb treatment were not reproduced by injecting mice with an isotype-matched control Ab or an anti-CD4 mAb (Fig. 7).
In view of previous debate about the survival of autoreactive B cells in HEL-Ig Tgn mice (8, 13, 19) and the potential problems with each of the methods that have been used to evaluate survival, we believe that investigation of B cell life span by these three methods was necessary to securely demonstrate decreased survival of most HEL-Ig Tgn B cells. 1) Although B220 and HSA expression can be used to characterize B cells as mature or immature (19, 39, 42), this characterization is not foolproof. Previous studies have shown, for example, that mature marginal zone B cells can be HSAbright (50), and we found considerable numbers of HSAbright B cells in Ig Tgn mice that failed to label after mice were treated for 14 days with BrdU (Fig. 3). 2) B cells that label with BrdU may be proliferating mature B cells rather than newly generated B cells. 3) Suppression of B lymphopoiesis by anti-IL-7 mAb may be incomplete, and decreased B cell production in the bone marrow appears to be partially compensated for by an increased likelihood that newly produced bone marrow B cells will migrate to the spleen (Fig. 5 and data not shown). Furthermore, neutralization of IL-7 might affect B cells indirectly by inhibiting the activating effects of IL-7 on T cells (51–54). Despite the potential problems with each of these three methods, our observation that all the methods lead to similar conclusions about B cell survival reinforces the likelihood that these conclusions are correct. In addition to confirming that the binding of soluble Ag to Agspecific B cells decreases the average life span of these cells, our observations demonstrate that this decrease in B cell survival results not only from decreased survival of newly generated B cells, but also from decreased survival of more mature B cells. Ig Tgn
SURVIVAL OF AUTOREACTIVE B CELLS
FIGURE 11. Effect of anti-IL-7 mAb treatment on B cell mIg expression in Ig Tgn and HEL-Ig Tgn mice. A, Means and SEs of the number of mature and immature spleen cells were determined for untreated and anti-IL-7treated Ig Tgn and HEL-Ig Tgn mice in the same experiment that is illustrated in Fig. 10 (left panel). Numbers were determined by multiplying the percentage of each cell type in the spleen by the total number of spleen cells. Means and SEs of HEL mean fluorescence intensities for the same populations of splenic B cells are shown in the right panel. B, Means and SEs for mIgM and mIgD staining of CD19⫹HSA/B220-defined splenic B cells from untreated and anti-IL-7-treated Ig Tgn and HEL-Ig Tgn mice are shown for the same experiment that is illustrated in Fig. 10 (filled symbols represent anti-IL-7 mAb-treated mice). C, Means and SEs of HEL mean fluorescence intensity for mature and immature splenic B cells from untreated, anti-IL-7 mAbtreated, control mAb-treated, or anti-CD4 mAb-treated HEL-Ig Tgn mice and a single CB20 and Ig Tgn mouse were determined in the same experiment that is illustrated in Fig. 7. Following incubation with HEL, spleen cells were stained with PerCP-labeled antiB220, Cy5-labeled anti-HEL, and FITC-labeled anti-HSA, followed by PE-labeled antiCD19. D, Means and SEs are shown for mIgM and mIgD staining of CD19⫹HSA/B220-defined splenic B cell populations from the same mice described in C and illustrated in Fig. 7.
and HEL-Ig Tgn mice that have been fed BrdU-containing water for 14 days have similar numbers of BrdU⫹ HSAint B cells (Figs. 3 and 5). However, most of the HEL-Ig Tgn BrdU⫹HSAint splenic B cells are newly produced cells rather than proliferating mature cells, because many fewer BrdU⫹ HSAint B cells are present in anti-IL-7 mAb-treated HEL-Ig Tgn mice than in untreated HEL-Ig Tgn mice (Fig. 5). Thus, at least equal numbers of B cells enter the
predominantly mature HSAint population in untreated HEL-Ig Tgn mice as in untreated Ig Tgn mice. In contrast, the numbers of BrdU⫺, HSAint splenic B cells were strikingly lower in HEL-Ig Tgn than in Ig Tgn mice in the same experiments. If approximately equal numbers of cells enter the HSAint population in HEL-Ig Tgn mice as in Ig Tgn mice, but the size of this population is considerably smaller in HEL-Ig Tgn than in Ig Tgn mice, the survival
The Journal of Immunology time of cells that enter this population must be shorter in HEL-Ig Tgn than in Ig Tgn mice. These observations contradict previous suggestions that B cells experience a single period of susceptibility to Ag-induced deletion (47), but support studies performed in normal mice demonstrating that mIgD cross-linking shortens the life span of most mature B cells (42). The most novel observation of our studies concerns the minority of B cells that survive to maturity in HEL-Ig Tgn mice. The presence of a mature B cell population in these mice is demonstrated by the existence of HSAint and HSAdull splenic B cells in these mice that fail to label when the mice are fed BrdU for 2 wk and persist when B lymphopoiesis is suppressed for 2 wk with antiIL-7 mAb. Few of these B cells have lost the ability to bind HEL by expressing endogenous Ig heavy chains, because B cells that express Ig heavy chains of the endogenous b allotype are undetectable in most HEL-Ig mice in our colony, even when we selectively examine HSAint-dull splenic B cells (data not shown). In contrast, a considerable, but variable, percentage of HEL-Ig splenic B cells has undergone receptor editing. Many of these receptor-edited B cells still bind HEL, but bind it with lower affinity than do B cells that express the transgenic Ig light chain, as demonstrated by a reduction in the ratio of HEL binding to mIg expression (Fig. 6). Most HSAint-dull splenic B cells in HEL-Ig Tgn mice, however, appear to remain HEL specific, but have not undergone receptor editing; rather, their reduced expression of mIgD as well as mIgM may promote survival in the presence of HEL by limiting their ability to interact with this Ag. Recently produced bone marrow or splenic B cells in HEL-Ig Tgn mice express normal quantities of mIgD, but appear to progressively lose mIgD as they age. This loss of mIgD becomes particularly apparent when the average age of B cells in HEL-Ig Tgn mice is increased by treating the mice for 2 wk with anti-IL-7 mAb; most splenic B cells in these mice expressed barely detectable mIgM or mIgD and bound little HEL (Figs. 10 and 11, A and B). It is not certain whether the low mIgD expression by mature HEL-Ig Tgn B cells results from selective survival of B cells that express low levels of mIgD or is induced by prolonged mIg-mediated signaling. It is likely, however, that prolonged mIg-mediated signaling, rather than selection, causes at least some of the decrease in mIgD expression that follows treatment with anti-IL-7 mAb, because antiIL-7 mAb treatment of HEL-Ig Tgn mice appears to cause an increase in the absolute number of splenic Ig⫺ B cells (Fig. 7). B cells that have escaped Ag-induced deletion by decreasing mIg expression might provide a reservoir of autoreactive cells that could promote protective immunity by allowing Ab responses to be generated to a pathogen-associated Ag that cross-reacts with an autoantigen. Alternatively, activation of these B cells by autoimmune T cells or by pathogen-induced inflammation might lead to production of disease-causing autoantibodies. Identification of the stimuli that can activate these mIg-deficient autoreactive B cells to increase mIg expression, proliferate, and/or differentiate into Absecreting cells and determination of whether autoreactive B cells can survive as mIglow or mIg⫺ cells in normal, nontransgenic animals should facilitate determination of whether these cells may be important in protective immunity or autoimmune disorders.
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