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Early Requirement for B Cells for Development of Spontaneous Autoimmune Thyroiditis in NOD.H-2h4 Mice¹

Helen Braley-Mullen^2,*,, and Shiguang Yu^*

Departments of ^* Internal Medicine and Medical Microbiology and Immunology, University of Missouri, and Veteran’s Administration Research Service, Columbia, MO 65212

	Abstract

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B cells are known to play an important role in the pathogenesis of several autoimmune diseases. NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) and anti-mouse thyroglobulin (MTg) autoantibodies, the levels of which correlate closely with the severity of thyroid lesions. NOD.H-2h4 mice genetically deficient in B cells (NOD.Kµ^null) or rendered B cell-deficient by treatment from birth with anti-IgM develop minimal SAT. B cells were required some time in the first 4–6 wk after birth, because NOD.Kµ^null or NOD.H-2h4 mice did not develop SAT when they were reconstituted with B cells as adults. The requirement for B cells was apparently not solely to produce anti-MTg autoantibodies, because passive transfer of anti-MTg Ab did not enable B cell-deficient mice to develop SAT, and mice given B cells as adults produced autoantibodies but did not develop SAT. B cell-deficient mice developed SAT if their T cells developed from bone marrow precursors in the presence of B cells. Because B cells are required early in life and their function cannot be replaced by anti-MTg autoantibodies, B cells may be required for the activation or selection of autoreactive T cells. These autoreactive T cells are apparently unable to respond to Ag if B cells are absent in the first 4–6 wk after birth.

	Introduction

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Autoimmune thyroiditis is a chronic organ-specific autoimmune disease characterized by infiltration of the thyroid gland by mononuclear cells and destruction of thyroid follicles by infiltrating inflammatory cells (1). NOD.H-2h4 mice, which express the thyroiditis susceptibility I-A^k allele on the NOD background (2), develop spontaneous autoimmune thyroiditis (SAT)³ after administration of NaI in their drinking water (3, 4, 5). Previous studies have demonstrated that both CD4⁺ and CD8⁺ T cells are required for the development of SAT in NOD.H-2h4 mice (3, 5). All mice that develop SAT produce detectable anti-MTg autoantibodies, and levels of both IgG1 and IgG2b anti-MTg autoantibodies generally correlate with the severity of thyroid lesions (3). However, it is not known whether the anti-MTg autoantibodies and/or B cells that produce anti-MTg autoantibodies are required for development of SAT.

B cells are important for the development of several spontaneous autoimmune diseases including diabetes in NOD mice (6, 7, 8), SLE in MRL/Mp-lpr/lpr mice (9, 10) and arthritis in K/BxN mice (11). In addition, some experimentally induced autoimmune diseases, including experimental autoimmune thyroiditis, collagen-induced arthritis and experimental allergic encephalomyelitis (EAE) either do not develop or develop suboptimally in B cell-deficient mice (Refs. 12, 13, 14, 15, 16 and H.B.-M., unpublished results). The purpose of these studies was to determine whether B cells and/or autoantibodies produced by B cells were required for the development of SAT in NOD.H-2h4 mice. The results indicate that development of SAT is severely compromised in B cell-deficient mice. B cells are apparently required some time during the first 4–6 wk after birth, and their function cannot be replaced by anti-MTg autoantibodies.

	Materials and Methods

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Mice

NOD.H-2h4 mice, derived by crossing NOD mice with B10.A(4R) mice, with repetitive backcrosses to NOD using progeny expressing the MHC haplotype of B10.A(4R), were provided by Dr. Linda Wicker (Merck, Rahway, NJ), and subsequently bred and maintained under specific pathogen-free conditions in the animal facilities at the University of Missouri (3). B lymphocyte-deficient NOD.Igµ^null mice (6) were provided by Dr. David Serreze (The Jackson Laboratory, Bar Harbor, ME). NOD.Igµ^null males were crossed with NOD.H-2h4 females and the resulting F₁ mice were bred to produce F₂ mice. The F₂ progeny were selected for homozygosity at H-2K^k and lack of expression of H-2K^d and for expression of the B cell markers B220 and IgM (Igµ⁺) or lack of expression of B220 and IgM (Igµ^null) by flow cytometric analysis of PBLs. Homozygous Igµ⁺ mice were then selected by PCR analysis of tail DNA using published PCR primer sequences (6). The resultant homozygous Igµ⁺ or Igµ^null H-2K mice, hereafter designated NOD.Kµ⁺ and NOD.Kµ^null, were bred and maintained under specific pathogen-free conditions, and the offspring were used for the studies reported here. Mice were age- and sex-matched for each individual experiment; both male and female mice were used for these experiments. All mice received 0.05% NaI in their drinking water beginning at 7–8 wk of age (3), or in the bone marrow reconstitution experiments, at 12 wk of age.

B cell depletion with anti-IgM

For some experiments, B cells were depleted from newborn mice using rabbit anti-mouse IgM (Southern Biotechnology Associates, Birmingham, AL or Jackson ImmunoResearch, West Grove, PA) as described previously (17). Mice received anti-IgM s.c. within the first 24 h after birth and three times weekly thereafter (50 µg/injection), either throughout the experiment (16 wk) or for 1–4 wk after birth as specified in the Tables. When mice were 3 wk old, anti-IgM was administered i.p. instead of s.c. This regimen of anti-IgM treatment, if initiated within 24 h of birth, results in essentially complete depletion of peripheral B cells (IgM⁺ and B220⁺ cells) as determined by flow cytometry, and has no apparent effects on T cells. This reagent is not effective for depletion of B cells when treatment is begun later (Ref. 17 and our unpublished observations). In the experiments shown here, control mice were injected with saline. Previous studies (Ref. 17 and our unpublished observations) have shown that mice given a control rabbit Ig have normal numbers of B lymphocytes, and development of SAT and immune responses to exogenous Ags are indistinguishable from those of saline-injected mice.

Irradiation, bone marrow, and B cell reconstitution

Male NOD.Kµ⁺ or µ^null mice were lethally irradiated (1000 rad) at 5 wk of age and reconstituted with 9 x 10⁶ T cell-depleted syngeneic bone marrow cells from syngeneic NOD.Kµ⁺ or µ^null donors. Some mice also received 2 x 10⁷ B cells (CD4- and CD8-depleted splenocytes) from adult (3- to 4-mo-old) NOD.Kµ⁺ mice. Analysis of PBLs 9 days after irradiation and bone marrow reconstitution indicated that the irradiation adequately depleted mature CD4⁺ T cells and B cells in the recipient mice (irradiated bone-marrow reconstituted mice had <2% CD4⁺ T cells and <3% B220⁺ cells in peripheral blood, similar to background values using isotype control Abs). Six weeks after irradiation and bone marrow reconstitution, CD4⁺ T cells and B220⁺ cells substantially increased in the peripheral blood, but were still reduced compared with unirradiated controls (see Table V). Mice that received NOD.Kµ^null bone marrow plus B cells had variable, but clearly detectable, levels of circulating B220⁺ cells at this time; B220⁺ cells were only slightly lower than the B220⁺ cells in mice reconstituted with µ⁺ bone marrow (Table V). One week later (7 wk after irradiation and bone marrow reconstitution), all mice were given NaI water, and were assessed for SAT 12 wk later. The time the mice were on NaI water was increased to 12 wk because preliminary experiments indicated that development of SAT was suboptimal at 8 wk in NOD.Kµ⁺ recipients of µ⁺ bone marrow, presumably due to immunosuppression from the irradiation.

Eight to 9 wk (or 12 wk in Table V) after mice began receiving NaI in their water, thyroids were collected, fixed in formalin, sectioned, and stained with hematoxylin and eosin as previously described (3, 18). As shown previously (3), thyroid lesions reach maximal severity 7–9 wk after NOD.H-2h4 mice are given NaI in their drinking water beginning at 2 mo of age; the lesions are chronic and remain relatively unchanged in severity for at least the next 20 wk (our unpublished observations). The NOD.Kµ⁺ mice described above developed SAT with similar severity and kinetics to the NOD.H-2h4 mice. Thyroids were scored for the extent of follicle destruction (SAT severity) using a scale of 1⁺ to 4⁺ as previously described (3). Briefly, 1⁺ thyroiditis is defined as an infiltrate of at least 125 cells in one or several foci, 2⁺ represents 10–20 foci of cellular infiltration, each the size of several follicles and involving up to 1/4 of the gland, 3⁺ indicates that 1/4 to 1/2 of the gland is destroyed by infiltrating inflammatory cells, and 4⁺ indicates that greater than 1/2 of the gland is destroyed. Qualitatively, the thyroid inflammatory cell infiltrate was typical of that seen in conventional lymphocytic experimental autoimmune thyroiditis, consisting primarily of lymphocytes and other mononuclear cells with occasional polymorphonuclear and plasma cells (3, 18). Most thyroids also had some proliferation and enlargement of thyroid follicular cells. Follicular cell enlargement was evident even in many thyroids with insufficient inflammatory cell infiltration to receive a severity score of 1⁺, and was probably a consequence of the increased dietary iodine. All slides were coded before being scored by two individuals, one of whom had no knowledge of the experimental protocol.

Autoantibody determination

MTg-specific autoantibodies were assessed by ELISA using serum from individual mice as previously described (3, 19). Alkaline phosphatase-conjugated secondary Abs specific for total mouse IgG or for mouse IgG1 and IgG2B were used at previously determined optimal dilutions (1:6000 or 1:8000), which gave an OD <0.05 with 1:50 diluted normal mouse serum on MTg-coated plates or with a 1:50 dilution of each test serum on plates coated with an irrelevant protein (OVA). Plates coated either with highly purified MTg or with less purified MTg preparations always gave identical results in this assay, suggesting that the IgG1 and IgG2b autoantibodies are directed against thyroglobulin, and not against another thyroid protein (3).

Flow cytometry

Spleen cells or PBL from experimental mice were analyzed for cell surface expression of B220, IgM, CD4, and CD8 by flow cytometry (FACSvantage; Becton Dickinson, San Jose, CA) as previously described (17). FITC-conjugated Abs specific for mouse B220, CD4, and CD8 were obtained from Caltag (South San Francisco, CA) and FITC-conjugated anti-mouse IgM was obtained from Southern Biotechnology Associates. MHC class I expression by PBL to identify NOD.K mice that were homozygous for H-2K^k and that lacked the H-2K^d allele expressed by NOD mice was determined using the mAbs 16-1-11N (anti-K^k; HB-16; American Type Culture Collection, Manassas, VA) and SF-1.1.1 (anti-K^d; HB-159; American Type Culture Collection) followed by a FITC-conjugated goat anti-mouse IgG (Boehringer Mannheim, Indianapolis, IN).

Statistical analysis

A two-tailed Student’s t test was used to determine the significance of differences in SAT severity between different groups. The p values are given in the footnotes in the tables.

	Results

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B cell-depleted mice develop minimal SAT

To determine whether B cells were required for development of SAT, NOD.H-2h4 mice were given anti-IgM beginning at birth and continuing through the entire 16 wk of the experiment as described in Materials and Methods. As shown in Table I, lines 1 and 2, 6 of 11 anti-IgM treated mice did not develop SAT, and the other 5 mice had mild (1) thyroiditis. Most of the thyroids of B cell-deficient mice with 1⁺ severity scores had only one to two foci of inflammatory cells, the minimal criteria for receiving a 1⁺ score (see Materials and Methods). In contrast, all 10 age-matched saline-injected control mice developed SAT, with 9 of 10 mice having a severity score of 2⁺-4⁺. All control mice had anti-MTg IgG1 and IgG2b autoantibodies, while anti-MTg autoantibodies were not significantly above background in sera of anti-IgM treated mice. Similar results were observed for mice genetically lacking B cells due to deletion of the Igµ gene (Table I, lines 3 and 4). Both anti-IgM treated NOD.H-2h4 mice and NOD.Kµ^null mice had essentially undetectable B220⁺ and IgM⁺ cells both in peripheral blood (data not shown) and spleen (Fig. 1 and footnote, Table I), with increased percentages of CD4⁺ (Fig. 1) and CD8⁺ T cells (data not shown) compared with controls. These results indicate that B cells are required for optimal development of SAT in NOD.H-2h4 and NOD.K mice, although mild thyroid lesions do develop in some mice in the absence of B cells and autoantibodies.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Flow cytometry analysis of spleen cells from a B cell-intact NOD.H-2h4 mouse (A and D), from a NOD.H-2h4 mouse treated from birth with anti-IgM (B and E), and from a NOD.H-2h4 mouse treated for the first 3 wk after birth with anti-IgM (C and F). Spleens were obtained from mice at 16 wk of age; the mice received 0.05% NaI in their drinking water beginning at 8 wk of age; at 6 wk of age, mice treated with anti-IgM for 3 wk (C and F) had normal numbers of B220+ cells in peripheral blood (Table II). Staining of cells is described in Materials and Methods. Results are representative of all experiments of this type.

To begin to define the function of B cells in SAT, it was of interest to determine whether B cell reconstitution at 6–8 wk of age (before mice begin NaI water, and several weeks before thyroid lesions develop in B cell-intact mice; Ref. 3) would enable NOD.H-2h4 mice to develop SAT. Adult NOD.Kµ^null mice could not be reconstituted with B cells from NOD.Kµ⁺ mice (Table III, lines 6 and 7, and data not shown), presumably because the transferred B cells were rejected by CD8⁺ T cells (20) or inhibited by CD4⁺ T cells (21) of the B cell-deficient mice. Therefore, the approach we used to reconstitute B cell-depleted mice with B cells was to stop the anti-IgM treatment at various times (1–3 wk) after birth and allow peripheral B cells (B220 and IgM⁺ cells) to gradually repopulate the mice. Although there was some variation in the rate of B cell reconstitution with different lots of anti-IgM, B cell percentages in peripheral blood usually returned to near normal levels (as determined by flow cytometry) 2–3 wk after stopping the anti-IgM treatment. In the first experiment (Table II, lines 1–3), mice were treated with anti-IgM for the first 3 wk after birth. When mice were 6 wk old (2 wk before starting NaI water and 4–5 wk before detectable thyroid lesions would develop in B cell-intact mice; Ref. 3), percentages of B220⁺ (Table II, line 3), IgM⁺, and CD4⁺ cells (not shown) in peripheral blood were similar to those of controls (line 1). However, mice treated with anti-IgM for only 3 wk after birth (line 3) had an incidence and severity of SAT comparable to mice in which B cell depletion was maintained throughout the experiment (line 2). In a second experiment, mice that received anti-IgM for only 1 wk (Table II, line 5) had normal percentages of peripheral B cells when tested at 6 wk of age, whereas mice given anti-IgM for 3 wk still had slightly lower percentages of peripheral B220⁺ cells at 6 wk (line 6). Mice given anti-IgM for only 1 wk were as resistant to SAT as mice treated with anti-IgM for 3 wk (line 6) or, in other experiments, through the entire 16 wk of the experiment. These results suggest that B cells are required sometime during the first 4–6 wk after birth for optimal development of SAT in adults, and the presence of normal numbers of B cells at 6 wk of age is not sufficient to promote development of SAT. Another possible interpretation of these results is that SAT development could simply be delayed in mice given anti-IgM for only 1–3 wk after birth. To address this possibility, half of the controls and half of the mice treated with anti-IgM for 3 wk in the second experiment in Table II were maintained on NaI water an additional 8 wk. As shown in Table II, lines 7 and 8, SAT severity scores in both groups assessed at 24 wk were not significantly different from those of each respective group at 16 wk (lines 4 and 6). This suggests the early absence of B cells does not simply delay development of SAT.

Mice in which B cell depletion was maintained through 16 wk did not produce detectable anti-MTg autoantibodies (Table I and Table II, line 2). However, when anti-IgM treatment was stopped after 1–3 wk, most mice produced detectable anti-MTg autoantibodies, even though many of them did not develop thyroid lesions. Although the results shown in the Tables represent the mean ELISA results of the group, the mice having SAT severity scores of 1⁺ did not contribute all the autoantibody to the group mean. In all these experiments, anti-IgM treated mice with 1⁺ SAT severity scores had autoantibody responses that were indistinguishable from those of mice in the same group that had scores of 0 (data not shown). Anti-MTg autoantibodies were always much lower in the 1–3 wk B cell-depleted mice compared with controls, possibly because their B cells did not develop for several weeks after birth and, therefore, became activated later. When such mice were maintained on NaI water through 24 wk (Table II, line 8), their anti-MTg IgG2b responses were comparable to those of controls (Table II, line 7), and IgG1 responses were higher than at 16 wk. Because these mice developed minimal or no SAT, the decreased autoantibody levels probably do not explain their relative resistance to SAT. In other experiments, mice depleted of B cells for 4 wk after birth were given serum containing anti-MTg autoantibodies from NOD.H-2h4 mice with SAT. Ab injections were begun at 11 wk (3 wk after the mice began NaI water, when autoantibodies first become detectable in controls; Ref. 3) and were continued weekly through 16 wk (Table III, lines 1–3). Although IgG1 autoantibodies in mice receiving passive anti-MTg were lower than in controls, anti-MTg IgG2b levels were comparable to those of controls. However, mice given passive anti-MTg did not develop SAT, suggesting that a lack of circulating anti-MTg autoantibody may not be the critical factor responsible for the relative resistance of B cell-depleted mice to SAT. In another experiment, B cell-deficient NOD.Kµ^null mice were given B cells from NOD.Kµ⁺ mice at 8 and 10 wk of age, and weekly injections of serum from MTg-immunized CBA/J mice containing high amounts of anti-MTg (Table III, lines 4–7). As noted above, B cell reconstitution in adult NOD.Kµ^null mice was not effective (20, 21), and the mice did not develop SAT (line 6). Mice receiving passive anti-MTg autoantibodies had higher levels of anti-MTg than controls, but they did not develop SAT (line 7). These results suggest the sole requirement for B cells is probably not to produce anti-MTg autoantibodies, although we cannot exclude the possibility that the transferred Ab differs in some important way, or does not gain access to the thyroid in the same way, as anti-MTg autoantibodies that develop spontaneously. In the experiment shown in Table IV, adult NOD.Kµ^null mice could be stably repopulated with B cells from NOD.Kµ⁺ mice when they were irradiated (500 rad) before B cell transfer (line 3 vs line 4). Although many of these mice had fewer splenic B cells than control µ⁺ mice, the B cells were functional because the B cell-repopulated mice spontaneously produced as much anti-MTg IgG1 and IgG2b autoantibody as control µ⁺ mice (Table IV, line 4 vs lines 1 and 2). However, even though anti-MTg autoantibodies developed spontaneously and reached levels comparable to those of NOD.Kµ⁺ mice, B cell-repopulated irradiated NOD.Kµ^null mice did not develop SAT. In contrast, 9 of 10 irradiated and nonirradiated NOD.Kµ⁺ mice developed SAT (i.e., 500 rad irradiation did not inhibit development of SAT in µ⁺ mice). As shown above, unirradiated NOD.Kµ^null mice could not be reconstituted with B cells, and they did not develop SAT or produce anti-MTg autoantibodies (Table IV, line 3). These results indicate that mice given B cells as adults can spontaneously produce normal levels of circulating anti-MTg autoantibodies, but this does not enable them to develop SAT.

The results presented above indicate that B cells are required during the first 4–6 wk of life for optimal development of SAT, and this is probably not due solely to a requirement for circulating anti-MTg autoantibodies. If the inability of B cell-deficient mice to develop SAT is due to a lack of B cells during a critical time in the first 4–6 wk after birth, NOD.Kµ^null mice should develop SAT if they are given B cells within 1–2 wk after birth. However, we were unable to achieve stable reconstitution of peripheral B cells when NOD.Kµ^null mice were given adult B cells at 1 or 2 wk of age (not shown). This is presumably due to the inhibitory effects of NOD.Kµ^null T cells on transferred B cells mentioned above (20, 21). However, Ighµ^null mice can be stably reconstituted with B cells when their T cells mature from immature bone marrow precursors in the presence of B cells (20). This provided an alternative means to determine whether B cell-deficient NOD.Kµ^null mice could develop SAT if B cells were available when their T cells were maturing. To address this question, lethally irradiated mice were repopulated with bone marrow from either NOD.Kµ⁺ or µ^null mice as a source of precursor T cells. Some mice also received B cells from adult NOD.Kµ⁺ mice (Table V). Analysis of peripheral blood cells 9 days after irradiation and bone marrow reconstitution indicated that mature T and B lymphocytes resident in recipients were adequately destroyed by the irradiation (see Materials and Methods). Six weeks after bone marrow reconstitution, peripheral blood CD4⁺ T cells were present at >50% of control values in most mice. Mice that received µ⁺ bone marrow (lines 2 and 5) or µ^null bone marrow plus B cells from µ⁺ mice (lines 4 and 6) had variable, but clearly detectable, B220⁺ cells (Table V). Mice that received NOD.Kµ^null bone marrow plus B cells (lines 4 and 6) developed SAT comparable in severity to the mice reconstituted with bone marrow from NOD.K µ⁺ donors (lines 2 and 5), indicating that T cells from B cell-deficient mice can induce SAT if B cells are available when they are maturing from bone marrow precursors. Similar results were obtained using either µ⁺ or µ^null mice as bone marrow recipients. Mice reconstituted with NOD.Kµ^null bone marrow plus B cells produced IgG1 and IgG2b anti-MTg autoantibodies, and autoantibody levels were comparable to those of mice reconstituted with µ⁺ bone marrow. In contrast, unirradiated NOD.Kµ^null mice (line 7) and three of four irradiated mice reconstituted with µ^null bone marrow (but no B cells; line 3) developed minimal or no SAT. With the exception of one animal, mice in the latter group had virtually no detectable splenic B cells, indicating that the majority of the mature lymphocytes were derived from the bone marrow inoculum. The one animal in line 3 that developed SAT was the only animal in this group that produced clearly detectable anti-MTg autoantibody. Apparently some B cells in this animal survived the irradiation, and although splenic B cell numbers were low compared with the mice receiving the same bone marrow cells with NOD.Kµ⁺ B cells (lines 4 and 6), there were apparently sufficient B cells for development of SAT and production of autoantibody. There were insufficient NOD.Kµ^null mice available to transfer µ^null bone marrow to µ^null recipients.

	Discussion

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These results demonstrate that B cells are required for optimal development of SAT in NOD.H-2h4 mice. Because anti-MTg IgG1 and IgG2b autoantibody levels correlate with SAT severity in this model (3), it is probably not surprising that depletion of B cells largely prevented development of thyroid lesions. One explanation for the requirement for B cells for optimal development of SAT is that anti-MTg or other autoantibodies produced by B cells could play a role in destruction of the thyroid gland. Autoantibodies have direct pathogenic effects in several autoimmune disease models (11, 22, 23), and there can also be synergism between T cells and B cells or autoantibodies (11, 14, 16, 23). However, autoantibody production is probably not the only function of B cells in SAT because B cells were required before detectable autoantibodies were produced (Table II), and passive transfer of anti-MTg or reconstitution of adult mice with B cells that spontaneously produced anti-MTg autoantibodies did not enable B cell-deficient mice to develop SAT (Tables III and IV). Although passively transferred autoantibodies may not be comparable to naturally produced autoantibodies with respect to concentration, time of development, access to the thyroid, or epitope specificity, irradiated adult NOD.Kµ^null reconstituted with B cells spontaneously produced as much anti-MTg as controls and did not develop SAT (Table IV). Although our results do not exclude a secondary role for autoantibodies, they do suggest B cells have another important function(s) in SAT.

The fact that B cells had to be present during the first few-wk after birth or when T cells were developing from bone marrow precursors (Tables II-V) places some constraints on the potential mechanisms by which B cells could function in SAT. Previous studies have shown that CD4⁺ T cells from B cell-deficient mice can be activated by some foreign proteins or peptides (16, 17, 20, 24, 25, 26), but not others (17, 20, 24, 27, 28, 29, 30). The nature of the Ag, the activation state of the T cells, and the T cell function (e.g. proliferation, help to B cells) being assessed can all influence whether T cell activation occurs in B cell-deficient mice (16, 17, 20, 24, 26, 31). Dendritic cells are usually the most efficient APC for the initial sensitization of naive T cells, and B cells may function to amplify or sustain T cell responses initiated by other APC (9, 23, 32, 33, 35). In some previous studies, T cells from B cell-deficient mice could be activated when adult mice were reconstituted with B cells at the time of T cell priming (27, 28, 29, 30, 31), suggesting that B cells were necessary for initial T cell activation.

A role for B cells in activation of autoreactive T cells has been demonstrated in some autoimmune disease models. For example, autoreactive T cells were not activated spontaneously in SLE-prone B cell-deficient mice (9, 10), and T cells from B cell-deficient NOD mice could not be sensitized to the glutamate decarboxylase (GAD) autoantigen (20, 34). Because B cell-deficient mice also did not develop autoimmune disease, these studies suggested B cells might function as APC for the activation or diversification of autoreactive T cells (20, 23, 34, 35). However, in a model of EAE induced by myelin oligodendrocyte glycoprotein (MOG), B cell-deficient mice did not develop EAE, but their T cells were apparently activated because they produced similar amounts of Th1 cytokines upon restimulation by the major encephalitogenic epitope (MOG 35–55) as did T cells from similarly immunized B cell-intact mice, which did develop EAE (16). Another study showed that islet-reactive T cells were apparently activated and could migrate to the pancreas in B cell-deficient NOD mice, but the inflammatory response was mild, and diabetes did not develop (8). These two studies suggested autoreactive T cells could be activated in the absence of B cells, but the T cells were not pathogenic. Falcone et al. (34) showed that T cells from adult B cell-deficient NOD mice spontaneously proliferated to GAD when B cells were added in vitro, suggesting that initial sensitization to GAD did not require B cells, but B cells were required as APC for amplification of the response. However, NOD B cells were required for initial sensitization to GAD after immunization with GAD in adjuvant (20). None of these studies have determined when B cells were required for development of autoimmune disease. One study showed that activated T cells could transfer diabetes to B cell-deficient mice (36), suggesting B cells were not required as APC for reactivation of T cells in the target organ or for mediating damage to the pancreas. Consistent with the results reported here (Table V), T cells from B cell-deficient mice could induce diabetes if they developed from bone marrow precursors in the presence of B cells (20).

Our attempts to determine whether peripheral MTg-reactive T cells were spontaneously activated in B cell-deficient mice were inconclusive, possibly because MTg is a large protein with multiple potential T cell epitopes, only some of which are pathogenic and/or may require B cells for activation (37). If B cells function primarily as APC for activation of pathogenic autoreactive T cells in SAT, it is surprising the T cells were apparently not activated when B cells became available in mice 6–8 wk of age (Tables II and IV). In the experiment shown in Table IV, B cells given to adult mice spontaneously produced anti-MTg autoantibodies comparable to NOD.Kµ⁺ mice, suggesting that failure to spontaneously produce sufficient anti-MTg autoantibody does not explain their inability to develop SAT. Although we cannot completely exclude the possibility that B cell-deficient mice might have eventually developed SAT in our experiments, a period of 10–11 wk after B cell reconstitution until assessment of SAT (Table II) should have been sufficient for T and B cells to migrate to the thyroid (2, 3, 4, 5). In addition, one group of mice treated with anti-IgM for 3 wk after birth was maintained on NaI water for 16 wk instead of the usual 8 wk. These mice had autoantibody responses only slightly lower than B cell-intact mice, but they developed minimal or no SAT (Table II, line 8). Although T cells from B cell-deficient NOD.K mice could not be activated to induce SAT when B cells were made available in adults, their T cells did induce SAT when they matured from bone marrow precursors in the presence of B cells (Table V). These results indicate that T cells from B cell-deficient NOD.K mice can be effectively activated to become effector cells for SAT if B cells are present during their maturation.

The fact that mice reconstituted with B cells as adults do not develop SAT, while those given B cells during the maturation of T cells from bone marrow precursors do develop SAT, suggests the initial activation of autoreactive T cells may be an early event. If T cell activation does not occur before mice are about 6 wk of age, the T cells apparently are unable to respond even if B cells become available in adults. It is also possible, although probably less likely, that the T cell epitope that initiates SAT is only present in neonatal mice. Although tolerance to self Ags expressed in the thymus develops during ontogeny, T cells specific for self Ags not expressed in the thymus or expressed at levels that escape negative selection are present in the periphery at birth (38, 39, 40, 41, 42, 43, 44). These nontolerant, potentially autoreactive T cells can become activated in certain strains of mice, resulting in spontaneous autoimmune disease (44, 45); activation of such cells can be facilitated by B cells (9, 10, 23, 35, 46). Garza et al. (43, 44) showed that T cells able to respond to a physiologically expressed self Ag ZP3, expressed by the ovary are present in neonatal mice. Tolerance to ZP3 develops in the first week of life in female mice that express the Ag, but not in males that do not express ZP3 (44). Our results would be compatible with such a mechanism if B cells either produce or are required for presentation of the epitope that initiates SAT in NOD.H-2h4 mice. B cells that become available in adults may be unable to activate autoreactive T cells because the T cells able to respond to the epitope that initiates T cell activation in this model were eliminated or inactivated in the absence of B cells in neonates. Experiments to test this hypothesis are in progress.

Because thyroids of mice with SAT have many infiltrating B cells found in clusters with CD4⁺ T cells, (54), B cells could also be required after T cells have migrated to the thyroid. Although B cells clearly have an early role in SAT (Tables II-IV), B cells could also produce autoantibodies or present Ag to T cells to amplify or sustain the inflammatory response in the target organ. Ags presented to T cells in the target organ could be proteins released during initial damage to the thyroid or proteins that become iodinated when mice receive NaI in their water. These Ags may differ from the Ag that initiates autoreactive T cell activation, because B cells were initially required well before cells migrated to the thyroid, and several weeks before acceleration of SAT by NaI water (Tables II and IV). Studies are in progress to determine whether B cells are required in the target organ after autoreactive T cells have been activated. Although it seems unlikely, the possibility that B cells that become available 6–8 wk after birth differ from B cells of neonatal mice in their ability to migrate to the thyroid cannot be ruled out. Some B cell-deficient mice repopulated with B cells have mild thyroid lesions (0⁺-1⁺). A few B220⁺ cells are present in these thyroids, whereas no B220⁺ cells were detected in the 0 ± 1⁺ lesions of B cell-deficient mice not repopulated with B cells (S.Y., unpublished observations). These results may suggest B cells given to adult mice are not inherently unable to migrate to the thyroid, although this is difficult to prove, as the numbers of infiltrating cells (T or B) in mice with such mild lesions are very low.

To our knowledge, this is the first study to demonstrate an early role for B cells for development of a spontaneous autoimmune disease. A requirement for B cells or B cell Ig early after birth for activation of particular subsets of T cells has been demonstrated previously (17, 47, 48, 49, 50). B cells can also present Ig determinants to T cells (51), and natural IgM has been shown to play a role in maturation of the immune response (52). We have previously shown that normal mouse Ig could substitute for B cells in T cell activation (17). Normal mouse Ig given to anti-IgM treated or NOD.Kµ^null mice using the regimen that was successful in our earlier study (17) had no effect on the ability of B cell-deficient mice to develop SAT (data not shown). This suggests that B cells probably do not simply provide Ig that could promote T cell activation or selection of the T cell repertoire by various mechanisms (47, 48, 49, 50, 51, 52), unless the Ig component is an autoantibody (53) and/or is unique to NOD.H-2h4 mice. To address some of these questions, we are generating NOD.Kµ^null mice that express transgenic surface Ig⁺ B cells that do not secrete Ig (10) to provide a model in which B cells will be present at birth, but autoantibody will not be produced.

	Acknowledgments

 
The authors thank Patti Mierzwa for skilled technical assistance and Louise Barnett for performing the flow cytometry analyses. We also thank Dr. Linda Wicker for providing the breeding stock of NOD.H-2h4 mice, Dr. David Serreze for providing the breeding stock of NOD.Igh^null mice, and Eric Greidinger, Mark Estes, and Gordon Sharp for critical reading of the manuscript.

	Footnotes

 
¹ This work was supported by a Merit Review Grant from the Veteran’s Administration. S.Y. was partially supported by the Orscheln Foundation.

² Address correspondence and reprint requests to Dr. Helen Mullen, Division of Immunology, Department of Medicine, M450 Medical Sciences, University of Missouri School of Medicine, Columbia, MO 65212.

³ Abbreviations used in this paper: SAT, spontaneous autoimmune thyroiditis; MTg, mouse thyroglobulin; EAE, experimental allergic encephalomyelitis; GAD, glutamate decarboxylase; MOG, myelin oligodendrocyte glycoprotein.

Received for publication June 30, 2000. Accepted for publication September 18, 2000.

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