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* The Jackson Laboratory, Bar Harbor, ME 04609; and
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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cell Ags to autoreactive CD4 T cells. This results from their ability to use cell surface Ig to specifically capture cell Ags. Hence, we postulated a diabetogenic role for defects in the tolerance mechanisms normally blocking the maturation and/or activation of B cells expressing autoreactive Ig receptors. We compared B cell tolerance mechanisms in NOD mice with nonautoimmune strains by using the IgHEL and Ig3-83 transgenic systems, in which the majority of B cells recognize one defined Ag. NOD- and nonautoimmune-prone mice did not differ in ability to delete or receptor edit B cells recognizing membrane-bound self Ags. However, in contrast to the nonautoimmune-prone background, B cells recognizing soluble self Ags in NOD mice did not undergo partial deletion and were also not efficiently anergized. The defective induction of B cell tolerance to soluble autoantigens is most likely responsible for the generation of diabetogenic APC in NOD mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells leading to type 1 diabetes (T1D)4 (1). However, studies using the nonobese diabetic (NOD) mouse and Bio-breeding rat models have demonstrated that T1D is primarily mediated by T cells (2, 3). Thus, it was surprising to find that NOD mice rendered deficient in B cells, either through introduction of an Ig H chain knockout (Igµnull) (4) or treatment with anti-IgM Abs (5), were markedly T1D resistant. These results demonstrated that B cells are important contributors to the diabetogenic process.
One mechanism whereby B cells contribute to T1D in NOD mice is through production of maternally transmitted autoantibodies (6). However, there are several reasons that autoantibody production is unlikely to be the sole pathogenic role of B cells. First, disease resistance was not abrogated in NOD.Igµnull mice chronically infused with Ig from overtly diabetic NOD donors (7). Second, NOD.Igµnull mice born to B cell intact NOD.Igµnull/+ mothers remained T1D resistant (4). Third, due to their ability to use an Ig-mediated capture mechanism, B cells serve as the subset of APC that most efficiently processes and presents certain pancreatic cell Ags in a MHC class II-restricted fashion to diabetogenic CD4 T cells in NOD mice (7, 8, 9). The importance of this Ig-mediated capture mechanism was demonstrated by analyses of NOD mice in which the combined presence of a transgenic (tg) Ig construct and the Igµnull mutation restricted the B cell repertoire to recognition of the pathologically irrelevant hen egg lysozyme (HEL) Ag (10). T cell responses to pancreatic cell autoantigens and T1D development were abrogated in such NOD.IgHEL.Igµnull mice. Conversely, T1D was accelerated in NOD mice carrying an IgM/D H chain transgene that increased the frequency of B cells specific for the candidate autoantigen, insulin (11). An important implication of these findings is that in addition to defects allowing the generation of autoreactive T cells, another fundamental component of T1D may involve the aberrant development of B cells expressing Ig molecules capable of capturing cell Ags.
Significant insights into B cell tolerance induction mechanisms have come from analyses of nonautoimmune-prone mouse strains expressing Ig transgenes specific for HEL or MHC class I H-2Kk,b Ags (designated IgHEL and Ig3-83, respectively). According to these analyses, the majority of immature B cells within bone marrow (BM)-expressing IgM molecules that recognize a multivalent membrane-bound form of self Ag undergo apoptosis-mediated deletion (12, 13, 14). In some cases, B cells recognizing a membrane-bound self Ag in the BM undergo receptor editing, a process by which clones can revise IgM specificity by initiating rearrangements of alternative L chain genes (15). The IgHEL system has also demonstrated that differentiating B cells expressing IgM molecules that recognize a weakly cross-linking soluble self Ag are rendered functionally anergic (16, 17). Anergic B cells can emigrate from the BM, but are relatively short-lived because they cannot properly home to lymphoid follicular areas producing factors necessary for their survival (18, 19, 20, 21). Self-reactive B cells may not necessarily encounter their cognate Ag within the BM. To avoid autoimmunity to peripherally expressed self Ags, recent B cell emigrants from the BM reach the spleen in a still immature state termed the transitional (T)1 stage, at which they remain highly susceptible to apoptosis upon Ig cross-linking with self Ag (22, 23). These combined tolerogenic mechanisms guard against B cell autoimmunity. In this study, we tested whether the capacity of autoreactive B cells to serve as a preferential subset of pathogenic APC in NOD mice may be due to their failure to undergo deletion, anergy, or receptor editing following what should normally be a tolerogenic encounter with self Ags.
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Materials and Methods |
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C57BL/6 (B6) mice carrying transgenes encoding HEL-reactive Ig molecules (IgHEL, MD4), or HEL protein in either a soluble (sHEL, ML5) or membrane-bound (mHEL, KLK3) form (described in Refs. 13 and 16) were kindly supplied by C. Goodnow (John Curtin School of Medical Research, Canberra, Australian Capital Territory, Australia). Derivation of a N9 NOD.IgHEL congenic stock has also been reported (10). The sHEL and mHEL transgenes were also congenically transferred to the NOD/Lt genetic background (designated NOD.sHEL and NOD.mHEL, respectively). In the course of developing these congenic stocks, backcross segregants carrying the sHEL or mHEL transgene were identified through PCR analyses of DNA from PBL using the primer set: 5'-GAGCGTGAACTGCGCGAAGA-3' and 5'-TCGGTACCCTTGCAGCGGTT-3'. The N11NOD.sHEL and NOD.mHEL congenic stocks are fixed to homozygosity for a panel of microsatellite markers (4, 24) delineating all known NOD-derived genetic loci influencing T1D development (Idd loci).
The Ig3-83µ
transgene construct was also congenically transferred from the previously described B10.D2.Ig3-83 stock (25) onto the NOD/Lt background (designated NOD.Ig3-83). Ig3-83µ transgene carriers at each backcross generation were detected by PCR using the primers 5'-CAGCTTCCTGCTAATCAGTGCC-3' and 5'-TGGTCCCCCCTCCGAACGTG-3.The N11 NOD.Ig3-83 congenic stock is fixed to homozygosity for microsatellite markers delineating all known Idd loci of NOD origin. Some studies used a previously described (26) N21 backcross stock of T1D-resistant NOD mice congenic for the H2nb1 MHC haplotype (Kb, Anb1, Ek, Db).
Female mice were used for all experiments. Mice were housed at The Jackson Laboratory (Bar Harbor, ME) under specific pathogen-free conditions with free access to food (National Institutes of Health 31A/6% fat diet; Ralston Purina, Richmond, IN) and acidified water. Experimental procedures involving mice were all approved by The Jackson Laboratory’s Animal Care and Use Committee.
Flow cytometric analyses
BM or splenic leukocyte suspensions from the indicated mice were assessed for proportions of B cell subsets by multicolor flow cytometry using the FACSCalibur instrument with CellQuest 3.3 software (BD Biosciences, San Jose, CA). Total B cells were enumerated with a FITC-conjugated goat polyclonal antiserum specific for mouse Ig (Southern Biotechnology Associates, Birmingham, AL) combined with a CD45R/B220-specific mAb (RA36B2) conjugated to either PE, PerCP, or APC fluorochromes (BD Biosciences). The proportion of B cells expressing the IgHEL or Ig3-83 transgenes was determined using the FITC-conjugated or biotinylated mAbs DS-1 and AMS9.1, specific for IgMa and IgDa, respectively. B cells expressing endogenous Ig chains were detected using the PE-conjugated AF6-78 and 217-170 Abs specific respectively for IgMb and IgDb molecules. Those expressing Ig
or 
L chains were determined with the biotinylated or PE-labeled R26-46 Ab or the FITC-conjugated R5-240 Ab, respectively (BD Biosciences). A biotinylated HEL construct was made using the EZ-link sulfo-normal human serum biotinylation kit (Pierce, Rockford, IL) and used for the detection of HEL-specific B cells. Various splenic B cell populations were distinguished using the PE-conjugated 7G6 Ab specific for CD21/CD35, the FITC-conjugated B3B4 Ab specific for CD23, and the biotinylated M1-69 Ab specific for CD24 (BD Biosciences). The clonotype-specific Ab 54.1 was used to detect B cells expressing Ig3-83 molecules. Biotinylated reagents were detected by means of streptavidin linked to FITC, PE, or APC fluorochromes. Propidium iodide was used to determine and gate out dead cells. Total numbers of particular B cell populations in the BM or spleen were calculated by multiplying the percentage of each population determined by FACS by the total cell count.
Determination of soluble HEL levels in serum
Splenocytes (1 x 106) from NOD.IgHEL or NOD (negative control) mice were incubated for 30 min at 4°C with 10 µl of the indicated serum samples or HEL (Sigma-Aldrich, St. Louis, MO) at concentrations ranging from 50 µg/ml to 5 pg/ml. Cells were washed and incubated with a biotinylated HEL-specific polyclonal Ab (Polysciences, Warrington, PA) and a PE-conjugated B220-specific mAb (RA36B2). A streptavidin-FITC conjugate was used to detect the biotinylated anti-HEL Abs. Increasing concentrations of the HEL standards could be detected as increases of mean fluorescence intensity (MFI) in the FITC channel on B220+ B cells and were graphed as a standard curve. The concentration of HEL within the serum samples was calculated by plotting the MFI they yielded against the standard curve.
In vitro proliferative responses of stimulated B cells
B cells were purified from the spleens of the indicated mice using a previously described (7) magnetic bead system (Miltenyi Biotec, Auburn, CA). Subsequent FACS analysis indicated B cell purity was >95%. Triplicate aliquots of 5 x 105 B cells were seeded into flat-bottom 96-well culture plates (Corning Glass, Corning, NY) in a final volume of 200 µl of complete RPMI 1640 medium (27) containing either 10 µg/ml LPS or 5 µg/ml CD40-specific mAb (HM40-3; BD Biosciences) in combination with 20 µg/ml AffiniPure goat anti-mouse IgG + IgM (H + L) (Jackson ImmunoResearch Laboratories, West Grove, PA) or the IgDa-specific mAb Ha-1 (kindly supplied by F. Finkelman, Cincinnati Veterans Affairs Medical Center, Cincinnati, OH). Control wells contained no stimulatory agents. The cultures were pulsed with 1 µCi/well [3H]thymidine for the final 24 h of a 72-h incubation period at 37°C. The cultures were then harvested, and [3H]thymidine incorporation was determined using an LKB Betaplate 1205 system (LKB Instruments, Gaithersburg, MD). B cell proliferative responses are presented as mean cpm ± SEM.
ELISA quantitation of Abs
Triplicate wells with 5 x 105 purified B cells from the indicated strains were cultured in complete RPMI 1640 with or without anti-IgDa and anti-CD40, as described above. After 96 h of incubation, supernatants from triplicate wells for each strain were pooled. The concentration of tg IgMa in the supernatants was determined by ELISA. Briefly, 96-well EIA/RIA high binding polystyrene plates (Corning Glass) were coated at 4°C overnight with a polyclonal goat anti-mouse IgM (Southern Biotechnology Associates) in 0.1 M carbonate buffer (pH 6.9) and blocked the next day with 10% BSA in PBS at room temperature for 1 h. The experimental supernatants were diluted 2-fold and added to the anti-IgM-coated wells for 1 h at 37°C. After washing with PBS containing 0.2% Tween 20 and 1% BSA, a biotinylated IgMa-specific Ab (DS-1) was added as a secondary detection reagent to each well, and the plate was incubated for 1 h at 37°C. Following another wash, streptavidin-conjugated alkaline phosphatase (Southern Biotechnology Associates) was added to each well, and the plate was incubated for another hour at 37°C. Each well was then developed by the addition of para-nitrophenyl phosphate substrate (Sigma-Aldrich) for 15 min and assessed for OD at 405 nm using a SPECTRAmax 250 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). The concentration of IgMa in each of the supernatants was calculated from a standard curve generated with an isotype/allotype control Ab (TEPC 183; Sigma-Aldrich). IgMa Ab concentrations in sera were determined in the same fashion. To induce the production of anti-HEL Abs, (CBA x B6.sHEL)F1 and NOD.sHEL mice as well as non-tg littermates were immunized i.p. with 50 µg/ml HEL in CFA. HEL-specific Abs in sera obtained 21 days later were measured by ELISA, as described above, except: 1) plates were coated with 1 µg/ml HEL, and 2) serum anti-HEL Abs were detected with an Ig L chain-specific Ab conjugated to alkaline phosphatase. No HEL-specific mAbs were commercially available to serve as a control, and therefore serum levels are reported as the OD405.
Tolerance induction assays of immature and T1 B cell populations
Immature B cell tolerance assays were performed using BM from NOD.IgHEL and B6.IgHEL female mice. BM cell suspensions were analyzed by flow cytometry to calculate the proportions of immature B cells (IgM+ IgD−). BM suspensions containing 2.0 x 105 immature B cells were incubated in complete RPMI 1640 medium with the indicated concentrations of soluble HEL for 24 h at 37°C. The remaining viable (propidium iodide-negative) immature B cells in each sample were then determined by flow cytometry. For the T1 B cell tolerance assays, total B cells were initially purified from the spleens of NOD or B6 mice using the magnetic bead system described above. Purified B cells were subsequently stained with an Ab mixture containing RA36B2 PerCP (anti-B220), M1-69 PE (anti-CD24), and 7G6 FITC (anti-CD21; BD Biosciences). A FACSVantage flow cytometer (BD Biosciences) was used to sort and collect T1 B cells, defined by a B220+ CD21low CD24high phenotype. Aliquots of 2 x 105 T1 B cells were seeded in complete RPMI 1640 into 24-well plates previously coated with 0, 10, or 50 µg/ml AffiniPure goat anti-mouse IgG + IgM (H + L). Cells were incubated for either 6 or 24 h, and then harvested and stained with an Ab mixture containing CD24 PE, CD21 FITC, propidium iodide, and annexin V biotin in the presence of annexin-friendly FACS buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, 2% dialyzed FBS, 0.02% NaN3). Streptavidin APC was used as a secondary reagent for the biotinylated annexin reagent. Samples were analyzed for T1 B cell apoptosis and viability using the aforementioned flow cytometric procedures.
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Results |
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We initially compared the ability of B cells from NOD mice and a nonautoimmune-prone strain to undergo deletion following exposure to a membrane-bound self Ag. Hemizygous NOD.mHEL and B6.mHEL mice were respectively crossed with homozygous NOD.IgHEL and B6.IgHEL mice (previously described in Refs. 10 and 16) to yield IgHEL/mHEL double-tg and control IgHEL single-tg progeny. All (Ig+) and tg (IgMa+) B cells were measured in the spleens of 6-wk-old IgHEL/mHEL vs IgHEL mice of both genetic backgrounds (Fig. 1A). Consistent with previous reports (10, 16), allelic exclusion resulted in greater than 98% of the splenic B cells in both NOD.IgHEL and B6.IgHEL mice expressing tg IgM/IgD molecules, while 94% retained the capacity to bind HEL biotin (Fig. 1B). In contrast, very few IgMa+ B cells were detected in the spleens of IgHEL/mHEL double-tg B6 or NOD mice (5.9 and 1.1% compared with IgHEL single-tg controls, respectively; Fig. 1A), and essentially none of these were capable of binding HEL (
0.2% of B220+ B cells in all mice; Fig. 1B). Coexpression of IgHEL and mHEL transgenes on either background was not associated with an increase in B cells expressing endogenous b-allotypic IgM molecules. Thus, NOD mice did not differ from nonautoimmune-prone B6 mice in their ability to eliminate B cells recognizing a multivalent membrane-bound protein.
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L chains (data not shown), whose presence would indicate receptor editing. Therefore, the non-HEL-binding IgMa+ B cells detected in the spleens of IgHEL/mHEL double-tg mice may have arisen either as a result of exclusive receptor editing by endogenous Ig L chains or due to the enrichment of cells in which the tg L chain failed to elicit allelic exclusion. Supporting the latter explanation are the findings of Fang et al. (28), who detected very low overall levels of receptor editing in B cells from B6 IgHEL/mHEL double-tg mice.
Although most B cells expressing the Ig3-83 transgene on the B10.D2 genetic background are also deleted upon encountering their cognate membrane-bound H-2Kk,b Ag (14), in contrast to the IgHEL/mHEL system, they demonstrate a greater capacity to use receptor editing as an alternate tolerance mechanism (15). We used the Ig3-83 tg system to compare the ability of NOD and B6 B cells to use receptor editing as an alternative tolerance mechanism when encountering membrane-bound self Ags. NOD.Ig3-83 mice were crossed with H-2Kb-expressing NOD.H2nb1 mice to produce progeny for studying B cell tolerance. Nonautoimmune-prone controls consisted of (B10.D2.Ig3-83 x B10(H-2Kb))F1 progeny. NOD.Ig3-83 and B10.D2.Ig3-83 mice were used as nontolerance-inducing controls. As expected, a high percentage of tg B cells was found in the spleens of NOD.Ig3-83 and B10.D2.Ig3-83 mice (Fig. 2A). Although lower than the total number of B cells detected using the IgMa-specific Ab (DS-1; Fig. 2B), >88% of the B cells from mice of both backgrounds stained with the Ig3-83 clonotype-specific Ab 54.1, indicative of fairly stringent allelic exclusion mediated by the tg H and L chains. Compared with what was observed when using the IgHEL/mHEL double-tg system, a greater number of IgMa+ B cells was detected in the spleens of (NOD.Ig3-83x NOD.H2nb1) and (B10.D2.Ig3-83 x B10) F1 mice (Fig. 2A). Nevertheless, very few of the remaining splenic IgMa+ B cells (
4%) from either background stained with the Ig3-83 clonotype-specific 54.1 Ab. This suggested they had changed their autoreactive Ig specificity by pairing tg H chains with endogenous L chains. Although <0.3% of IgMa+ B cells in the spleens of NOD.Ig3-83 and B10.D2.Ig3-83 expressed an endogenous L chain, 60% of the remaining 54.1− IgMa+ B cells in the spleens of (NOD.Ig3-83 x NOD.H2nb1) and (B10.D2.Ig3-83 x B10) F1 mice did so (Fig. 2B), implying a high proportion of these cells had undergone receptor editing. However, there were no significant differences in the numbers of IgMa+ 54.1− B cells remaining in the spleens of (NOD.Ig3-83 x NOD.H2nb1) and (B10.D2.Ig3-83 x B10) F1 mice (Fig. 2A), nor in their levels of endogenous Ig chain usage (Fig. 2B). Hence, we conclude that the induction of the signaling pathways mediating deletional tolerance or receptor editing in B cells that engage strong Ig cross-linking membrane-bound autoantigens remains intact in NOD mice.
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Given that they are presented through the MHC class II pathway, cell autoantigens recognized by diabetogenic CD4 T cells in NOD mice are likely to be soluble in nature. Ig-mediated capture allows B cells to be the subset of APC that most efficiently presents soluble cell autoantigens to diabetogenic CD4 T cells in NOD mice (10). Therefore, even though B cell tolerance to membrane-bound Ags appears to be intact in NOD mice, this might not be the case for soluble self Ags. To test this possibility, hemizygous NOD.sHEL and B6.sHEL mice were respectively crossed with homozygous NOD.IgHEL and B6.IgHEL mice to produce IgHEL/sHEL double-tg and control IgHEL single-tg progeny. Compared with B6.IgHEL controls, total numbers of splenic IgMa+ B cells were decreased by 30% in B6.IgHEL/sHEL mice (Fig. 3A). In contrast, there was no reduction in the total numbers of splenic IgMa+ B cells in NOD.IgHEL/sHEL mice compared with controls (Fig. 3A). This suggested NOD mice are indeed defective in a mechanism leading to the partial deletion of B cells recognizing soluble self Ags. However, it was important to ensure that the different pattern of autoreactive B cell development observed in B6.IgHEL/sHEL and NOD.IgHEL/sHEL mice was not due to lowered levels of soluble HEL in the latter stock. As shown in Fig. 3B, this was not the case because serum HEL concentrations were actually marginally, but statistically higher in NOD.sHEL than B6.sHEL mice. This difference was less pronounced in the NOD vs the B6 IgHEL/sHEL double-tg mice. The lower serum HEL levels observed in both strains of IgHEL/sHEL double-tg vs sHEL single-tg mice were probably due to the presence of residual nondeleted HEL-binding B cells in the former set of mice. Therefore, the reduction in IgHEL B cell deletion observed in NOD vs B6 IgHEL/sHEL double-tg mice could not be explained in terms of different levels of Ag expression. Collectively, these results indicate an intrinsic defect in the ability of NOD B cells to be deleted upon recognizing soluble self Ag.
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Previous reports have demonstrated the majority of IgHEL B cells in B6.IgHEL/sHEL double-tg mice are rendered functionally anergic rather than undergoing deletion (16, 17). Thus, it remained possible that while NOD B cells recognizing a soluble self Ag during their development do not undergo a normal level of deletion, they could still be functionally anergized. This possibility was initially examined by comparing surface Ig expression on splenic B cells that had escaped deletion in the NOD and B6 IgHEL/sHEL double-tg stocks as a surrogate marker of anergy. Splenic B cells from both NOD and B6 IgHEL/sHEL double-tg mice displayed an 11- to 14-fold reduction in surface IgMa compared with those from IgHEL single-tg controls (Fig. 4A). Although surface IgDa was up-regulated 1.7-fold in the B6.IgHEL/sHEL splenic B cells compared with levels in B6.IgHEL controls, this was not observed in the NOD background stocks (p < 0.05, Student’s t test; Fig. 4A). The relevance of this modest difference in surface IgD expression is not known.
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We next evaluated whether anergy was reversible in peripheral B cells from NOD or B6 IgHEL/sHEL double-tg mice. Splenic B cells were purified from the double-tg stocks and IgHEL single-tg controls and stimulated in vitro through Ag receptor-specific (anti-IgM/G + anti-CD40 or anti-IgDa + anti-CD40) or nonspecific pathways (LPS). Anti-IgM/G, anti-IgDa, or anti-CD40 stimulation on their own only produced minimal responses in all tested B cell populations (data not shown). Conversely, costimulation through the B cell receptor (BCR) and CD40 induced an optimum response by purified B cells from both NOD and B6 IgHEL single-tg mice (Fig. 5A). Consistent with the preservation of anergy, the proliferative responses of B6.IgHEL/sHEL B cells to anti-IgM/G + anti-CD40 or anti-IgDa + anti-CD40 costimulation were markedly reduced compared with B6.IgHEL control B cells (49 and 21%, respectively). The greater degree of proliferation in anti-IgM/G + anti-CD40-stimulated B6.IgHEL/sHEL B cells compared with anti-IgDa + anti-CD40 costimulation was probably due to activation of the small proportion of nonanergic B cells expressing endogenous Ig chains in these mice. In contrast, the reduction in proliferation of NOD.IgHEL/sHEL B cells stimulated with anti-IgM/G + anti-CD40 or anti-IgDa + anti-CD40 was much less marked (86 and 70% of the NOD.IgHEL control response, respectively). A similar trend was observed when IgMa Ab was measured. Thus, compared with the IgHEL single-tg controls, Ab production following BCR and CD40 costimulation was less suppressed in NOD than B6 IgHEL/sHEL double-tg mice (2.6- and 4.5-fold lower, respectively; Fig. 5B). The difference in LPS-induced proliferation between B cells from NOD and B6 IgHEL/sHEL double-tg mice was not as pronounced as that seen when using Ag receptor stimulation. Nevertheless, in comparison with IgHEL single-tg controls, the ability to proliferate in response to LPS was suppressed to a greater extent in B cells from B6 than NOD IgHEL/sHEL double-tg mice (Fig. 5A).
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cell autoantigen during their development. Such B cells could then serve as APCs that efficiently amplify diabetogenic CD4 T cell responses. NOD B cell tolerance defects occur at two developmental stages
B cells recognizing self Ag can be tolerized either when at an immature state in the BM or at the T1 stage in the spleen. Hence, we assessed at which of these development points autoreactive B cells from NOD mice fail to become tolerant. For this purpose, an in vitro assay was performed in which BM suspensions from NOD.IgHEL and B6.IgHEL mice (normalized for IgMa+ IgDa− immature B cell numbers) were exposed to different concentrations of soluble HEL ranging from 0.01 to 5 ng/ml. The extent to which immature B cells were deleted after 24 h of exposure to HEL was determined by FACS and compared with cells not encountering Ag. Under these in vitro conditions, the immature B cells from B6.IgHEL mice were eliminated at lower concentrations of HEL than those from NOD.IgHEL mice (Fig. 6). It should be noted that in the predisease stage, the circulating concentration of insulin, which is the antigenic target of an important population of diabetogenic B cells in NOD mice (11), is in the range of 1–2 ng/ml. Thus, the 0.5–2 ng/ml Ag dose range that differentially induced the deletion of immature BM-derived B cells from NOD.IgHEL and B6.IgHEL mice is likely to be physiologically relevant.
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Discussion |
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cells. Thus, it is not surprising that there have been numerous reports describing disruptions in both central and peripheral T cell tolerance induction mechanisms in NOD mice, which ultimately lead to the production of both CD4 and CD8 autoreactive effectors (34, 35, 36, 37, 38, 39). By using Ig tg models, we have demonstrated in the current study that NOD mice are also defective in their ability to both delete and sustain anergy in B cells that recognize soluble self Ags. These defects are likely to underlie the development of B cells expressing Ig molecules specific for pancreatic cell autoantigens. The ability of such B cells to take up autoantigens by an Ig-mediated capture mechanism allows them to be the APC subpopulation that most efficiently expands diabetogenic CD4 T cell responses in NOD mice (10) and that also contributes to disease through the secretion of maternally transmitted autoantibodies (6). Defects leading to impaired deletion of immature B cells in NOD mice were manifest at both the immature developmental stage in BM, as well as the later T1 stage in the spleen. The survival or deletion of B cells at various developmental stages is largely regulated by the relative ratio between pro- and antiapoptotic molecules, such as those of the Bcl-2 or TNFR families (reviewed in Refs. 40 and 41). Therefore, it is possible that the impaired deletion of B cells in the NOD strain may be due to an imbalance in the expression of these proteins. It is also interesting to note that exposure to soluble self Ag in the ng/ml range primarily caused immature B cells of NOD and B6 origin to undergo differential levels of deletion in vitro (Figs. 6 and 7), while the acquisition of functional anergy appeared to be a more predominant mode of tolerance induction in vivo (Figs. 3 and 4). This may be due to the in vivo environment providing survival factors that are perhaps not present or as accessible when the cells are dispersed in the in vitro cultures, thus allowing B cells to be anergized, but not eliminated at high levels through deletional processes. Alternatively, the impaired ability of developing B cells in NOD mice to be deleted and/or anergized may be caused by a decrease in the strength of signaling through the BCR upon encountering weakly cross-linking soluble self Ags. This possibility is consistent with the enlarged total numbers of MZ B cells and slightly decreased follicular B cells in the spleens of NOD mice (Table I), a profile that has been observed in various mice with targeted mutations or transgenes that attenuate BCR signaling, e.g., Btk and CD21 knockout mice (32, 42).
Mechanistic studies to determine whether BM-derived immature and splenic T1 B cells from NOD mice and control strains are characterized either by differing balances of various pro- and antiapoptotic molecules and/or a disparity in BCR signaling after exposure to putatively tolerogenic Ags will be the subject of future studies. It is also of interest to note that abnormal balances in apoptotic regulatory molecules and impaired intrinsic signaling defects have also been reported to be a key factor in the development of autoreactive diabetogenic T cells in NOD mice (43, 44, 45). Hence, it is certainly possible that the impaired ability of both autoreactive T and B cells in NOD mice to be deleted or anergized when encountering soluble self Ags during their development is under common or largely overlapping genetic control, with both defects being necessary for T1D development.
It is tempting to speculate that autoreactive B cells also contribute to T1D in humans, and that they arise due to the same kind of tolerance induction defects characterizing NOD mice. There has been a report of a single human T1D patient who also has X-linked agammaglobulinemia, a disease characterized by markedly decreased numbers of functional B cells (46). However, without appropriate epidemiological studies, it is unknown whether the patient examined was characterized by a normal or exceptional pathogenic basis of T1D development. Indeed, we have previously reported that while most B cell-deficient NOD.Igµnull mice are T1D resistant, there are occasional exceptions (10). Moreover, in NOD mice, the preferential role of B cells in expanding diabetogenic T cell responses appears to be due to developmental defects in other APC subtypes (reviewed in Ref. 3). Similarly, defects in APC subtypes other than B lymphocytes also characterize many, but not all, humans with T1D (reviewed in Ref. 3). Perhaps the single T1D patient with X-linked agammaglobulinemia represented one of the minority cases in which other APC populations differentiated normally, and hence bypassed a pathogenic need for B cells. Hence, the possibility is still open that B cells may contribute to T1D in most humans in the same way as in NOD mice. Because of this, gaining an increased understanding of the B cell tolerance induction defects in NOD mice may ultimately aid in developing protocols that might block progression to overt T1D in otherwise susceptible individuals.
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Acknowledgments |
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Footnotes |
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2 Current address: Centenary Institute of Cancer Medicine and Cell Biology, Building 93, Royal Prince Alfred Hospital, Missenden Road, Camperdown NSW 2050, Australia.
3 Address correspondence and reprint requests to Dr. David V. Serreze, The Jackson Laboratory, Bar Harbor, ME 04609. E-mail address: dvs{at}jax.org
4 Abbreviations used in this paper: T1D, type 1 diabetes; BCR, B cell receptor; BM, bone marrow; HEL, hen egg lysozyme; m, membrane bound; MFI, mean fluorescence intensity; MZ, marginal zone; NOD, nonobese diabetic; s, soluble; T, transitional; tg, transgenic.
Received for publication September 15, 2003. Accepted for publication February 5, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells of nonobese diabetic mice. J. Immunol. 163:743.This article has been cited by other articles:
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  A. K. Panigrahi, N. G. Goodman, R. A. Eisenberg, M. R. Rickels, A. Naji, and E. T. Luning Prak RS rearrangement frequency as a marker of receptor editing in lupus and type 1 diabetes J. Exp. Med., December 22, 2008; 205(13): 2985 - 2994. [Abstract] [Full Text] [PDF]
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 A. M. Marleau, K. L. Summers, and B. Singh Differential Contributions of APC Subsets to T Cell Activation in Nonobese Diabetic Mice J. Immunol., April 15, 2008; 180(8): 5235 - 5249. [Abstract] [Full Text] [PDF]
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V. Saxena, J. K. Ondr, A. F. Magnusen, D. H. Munn, and J. D. Katz The Countervailing Actions of Myeloid and Plasmacytoid Dendritic Cells Control Autoimmune Diabetes in the Nonobese Diabetic Mouse J. Immunol., October 15, 2007; 179(8): 5041 - 5053. [Abstract] [Full Text] [PDF]
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H. Bour-Jordan, B. L. Salomon, H. L. Thompson, R. Santos, A. K. Abbas, and J. A. Bluestone Constitutive Expression of B7-1 on B Cells Uncovers Autoimmunity toward the B Cell Compartment in the Nonobese Diabetic Mouse J. Immunol., July 15, 2007; 179(2): 1004 - 1012. [Abstract] [Full Text] [PDF]
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D. V. Serreze, M. A. Osborne, Y.-G. Chen, H. D. Chapman, T. Pearson, M. A. Brehm, and D. L. Greiner Partial versus Full Allogeneic Hemopoietic Chimerization Is a Preferential Means to Inhibit Type 1 Diabetes as the Latter Induces Generalized Immunosuppression J. Immunol., November 15, 2006; 177(10): 6675 - 6684. [Abstract] [Full Text] [PDF]
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P. A. Silveira, H. D. Chapman, J. Stolp, E. Johnson, S. L. Cox, K. Hunter, L. S. Wicker, and D. V. Serreze Genes within the Idd5 and Idd9/11 Diabetes Susceptibility Loci Affect the Pathogenic Activity of B Cells in Nonobese Diabetic Mice J. Immunol., November 15, 2006; 177(10): 7033 - 7041. [Abstract] [Full Text] [PDF]
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W. J. Quinn III, N. Noorchashm, J. E. Crowley, A. J. Reed, H. Noorchashm, A. Naji, and M. P. Cancro Cutting edge: impaired transitional B cell production and selection in the nonobese diabetic mouse. J. Immunol., June 15, 2006; 176(12): 7159 - 7164. [Abstract] [Full Text] [PDF]
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S. Yu, P. K. Maiti, M. Dyson, R. Jain, and H. Braley-Mullen B cell-deficient NOD.H-2h4 mice have CD4+CD25+ T regulatory cells that inhibit the development of spontaneous autoimmune thyroiditis J. Exp. Med., February 21, 2006; 203(2): 349 - 358. [Abstract] [Full Text] [PDF]
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P. K. A. Mongini, J. K. Inman, H. Han, S. L. Kalled, R. J. Fattah, and S. McCormick Innate Immunity and Human B Cell Clonal Expansion: Effects on the Recirculating B2 Subpopulation J. Immunol., November 1, 2005; 175(9): 6143 - 6154. [Abstract] [Full Text] [PDF]
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E. J. Woodward and J. W. Thomas Multiple Germline {kappa} Light Chains Generate Anti-Insulin B Cells in Nonobese Diabetic Mice J. Immunol., July 15, 2005; 175(2): 1073 - 1079. [Abstract] [Full Text] [PDF]
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J. Rolf, V. Motta, N. Duarte, M. Lundholm, E. Berntman, M.-L. Bergman, L. Sorokin, S. L. Cardell, and D. Holmberg The Enlarged Population of Marginal Zone/CD1dhigh B Lymphocytes in Nonobese Diabetic Mice Maps to Diabetes Susceptibility Region Idd11 J. Immunol., April 15, 2005; 174(8): 4821 - 4827. [Abstract] [Full Text] [PDF]
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C. H. Koonce and E. K. Bikoff Dissecting MHC Class II Export, B Cell Maturation, and DM Stability Defects in Invariant Chain Mutant Mice J. Immunol., September 1, 2004; 173(5): 3271 - 3280. [Abstract] [Full Text] [PDF]
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), NOD.IgHEL/mHEL (
), B6.IgHEL (
), and B6.IgHEL/mHEL (
) female mice. In both backgrounds, * signifies expression of membrane-bound Ag results in a significant reduction in tg B cells (p < 0.05, Student’s t test). B, Representative FACS plots of live splenocytes with a capacity to bind HEL, as determined by the protocol described in Materials and Methods. Dot plots are shown for NOD and B6 IgHEL single-tg and IgHEL/mHEL or IgHEL/sHEL double-tg mice. Percentages of gated cells in the upper left (non-HEL-binding B cells) and upper right (HEL-binding B cells) quadrants are displayed.
