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The Journal of Immunology, 2002, 169: 6617-6624.
Copyright © 2002 by The American Association of Immunologists

The Natural Autoantibody Repertoire of Nonobese Diabetic Mice Is Highly Active1

James W. Thomas2, Peggy L. Kendall and Holly G. Mitchell

Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Analysis of spontaneous hybridomas generated from nonobese diabetic (NOD) mice indicates that the natural autoantibody repertoire of NOD mice is highly active compared with C57BL/6 and BALB/c mice. This property of increased B cell activity is present early in life (4 wk) and persists in older mice of both sexes. Even when selected for binding to a prototypic {beta} cell Ag, such as insulin, NOD mAb have characteristics of natural autoantibodies that include low avidity and broad specificity for multiple Ags. Analyses of the variable region of Ig H chain (VH) and variable region {kappa} L chain genes expressed by six insulin binding mAb show that V gene segments are often germline encoded and are identical with those used by autoantibodies, especially anti-dsDNA, from systemic autoimmune disease in MRL, NZB/W, and motheaten mice. VH genes used by four mAb are derived from the large J558 family and two mAb use VH7183 and VHQ52 genes. The third complementarity-determining region of Ig H chain of these mAb have limited N segment diversity, and some mAb contain DNA segments indicative of gene replacement. Genetic abnormalities in the regulation of self-reactive B cells may be a feature that is shared between NOD and conventional systemic autoimmune disorders. In NOD, the large pool of self-reactive B cells may fuel autoimmune {beta} cell destruction by facilitating T-B cell interactions, as evidenced by the identification of one mAb that has undergone Ag-driven somatic hypermutation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The nonobese diabetic (NOD)3 mouse is a useful model to examine the immunoregulatory abnormalities that lead to autoimmune {beta} cell destruction and type I or insulin-dependent diabetes mellitus (T1DM). As in human T1DM, the disorder in NOD is under polygenic control with a dominant contribution from genes in the MHC complex (1, 2). The principal mediators of {beta} cell destruction are believed to be T lymphocytes. Although a direct role for autoantibodies in disease has not been found, detection of Abs to {beta} cell Ags in the prodrome of T1DM suggests that loss of tolerance in the B cell compartment is one of the earliest indicators of the covert autoimmune process (3, 4, 5, 6). Autoantibodies to insulin have predictive value in both NOD and human T1DM (5, 7), and insulin-specific T cells can transfer disease (8). Studies using Ab reagents that block B cell development (9, 10) and genetic deletion of B cells in µMT mice provide evidence that B lymphocytes are necessary for the development of diabetes in NOD mice (11, 12). Elegant studies also show that I-Ag7 deficiency confined to the B cell compartment protects NOD mice from T1DM and suggest that B cells play a key role in mediating T cell responses as a consequence of Ag presentation (13).

Although the importance of B cells for the development of insulitis and diabetes in the NOD mouse model of type I diabetes is well-recognized, data on the expressed B cell repertoire in NOD are limited. Studies from the Holmberg laboratory used in situ hybridization to show that the neonatal bias for expression of D-proximal variable region of Ig H chain (VH) genes persists in adult NOD mice (14, 15). Pleau et al. (16) examined the VH and variable region {kappa} L chain (V{kappa}) structures of two IgG mAb selected for binding to insulin and peripherin in diabetic NOD mice and found nucleotide replacements suggesting Ag-driven somatic mutation. These observations indicate that both developmental abnormalities and autoantigen-driven selection may contribute to the functional B cell repertoire in NOD mice.

To better understand how developmental programs and Ag-driven selection contribute to the B cell repertoire in prediabetic NOD mice, we characterized the structure and function of a panel of autoantibodies generated by hybridoma formation. In the absence of mitogen or Ag stimulation, this technique favors the capture of spontaneously active B cells (17, 18). Using this approach, we find that the B cell repertoire of NOD mice is highly active when compared with that of nonautoimmune mice. This active repertoire is present from an early age (4 wk) and is not dependent on gender. Because insulin is a known autoantigen in T1DM, we examined a large number of NOD hybridomas in search of high-affinity autoantibodies. In contrast to expectation, we find a high proportion of insulin-binding mAb in NOD (including IgG mAb) have characteristics of a natural autoantibody repertoire. This observation is based on the low avidity of insulin-binding in competitive ELISA and on the ability of mAb captured for insulin-binding to interact with multiple other Ags including DNA and Ig. Further support for this conclusion is based on the nucleotide composition of V regions from these mAb. The V region genes expressed by these mAb are principally unmutated (germline) structures that are often identical with V genes expressed by Abs (especially anti-DNA) in systemic autoimmune disease such as murine lupus. Although most V genes are in germline configuration, data on one mAb show clear evidence for somatic hypermutation and indicate that the active B cell repertoire is available to interact with helper T cells. These findings suggest that the genetic background of NOD mice favors generation of autoreactive B cells and imply that defects in B cell selection may be present in NOD. The recognition that lupus-like syndromes may accompany therapies that protect NOD mice from developing diabetes (19) also indicates that the active B cell repertoire in NOD has a broad pathological potential. Thus, genetic abnormalities in the regulation of self-reactive B cells may be shared between NOD mice and strains of mice that have systemic autoimmune disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and production of hybridomas

NOD, BALB/c, and C57BL/6 mice were purchased from Taconic Farms (Germantown, NY) and used between the ages of 4 and 24 wk. To produce hybridomas, spleens were removed from mice following sacrifice and fused with the mouse myeloma line X63Ag8.653 in a ratio of 1:4 using polyethylene glycol (20). No immunization or mitogen activation was used. Following fusion, cells were plated at 2 x 106/ml in 96-well flat-bottom plates and selected in hypoxanthine-aminopterin-Tdr medium (Sigma-Aldrich, St. Louis, MO). After removal of selection media, growth-positive wells were screened for production of IgM or IgG Abs by a capture ELISA. To score for frequency of hybridoma production, the number of growth positive wells that produced Ab on two screens were counted and the results reported as number of hybridomas per 105 spleen cells used in the fusion. In some experiments, supernatants were also screened for insulin binding and positive wells were cloned at limiting dilution.

Immunoassays

ELISAs were used to identify Ab-producing hybridomas and to measure specific Ag Ab interactions. Briefly, to detect Ab production, microtiter plates coated with supernatants from growth positive wells were reacted with goat anti-mouse IgG or IgM Ab conjugated to alkaline phosphatase (Kirkegaard and Perry Laboratories, Rockville, MD). Wells producing Ig were cloned at limiting dilution in 96-well plates coated with 50 µl of FCS and then expanded for additional studies. For Ag-specific assays, plates were coated with the indicated Ag, including insulin (pork or human), calf thymus DNA, cardiolipin, dextran, thyroglobulin, tetanus toxoid, human gammaglobulin, or glucagon (Sigma-Aldrich). Human sera from patients with lupus or rheumatoid arthritis and immunized mouse sera were used as positive controls for multi-Ag panels. Hybridomas that stick to noncoated wells were excluded from study. For insulin binding assays, 96-well microtiter plates (Immulon II) were coated overnight at 4°C with 100 µl of human insulin (1 µg/ml) in PBS pH 8.0. Plates were washed extensively in PBS containing Tween (0.2%) and 1% BSA before use. Radiolabeled tracer studies indicate that each coated well contains ~30 ng of insulin. Hybridoma cultures producing Ig were screened for insulin binding using 2-fold serial dilutions to identify a nonsaturated binding (70% maximum binding), usually 1:10. For competitive inhibition with insulin, supernatants were assayed on dilutions from the linear portion of the binding curve and preincubated with insulin at 0.05–100 µg/ml for 2 h before transfer to insulin-coated plates (21). Insulin binding in the presence or absence of soluble inhibitor was measured using isotype-specific goat anti-mouse IgM or IgG conjugated to alkaline phosphatase. Anti-insulin clone 1F11 (mAb301) was used as a positive control for IgM anti-insulin (22) and mAb125 was used as a control for IgGs (23). All dilutions and washes were conducted at room temperature using 0.2% Tween 20 and 1% BSA. Results are reported as mean OD405 for triplicate determinations using paranitrophenyl phosphate (Sigma-Aldrich) as substrate and an automated ELISA reader. To simplify data presentation, results are presented as the OD of insulin binding in the absence or presence of soluble insulin (50 µg/ml) using the dilution of supernatant at 70% maximal binding.

RT-PCR and nucleotide sequences

Total cellular RNA was isolated from 107 hybridoma cells using the TRI Reagent protocol (Molecular Research Center, Cincinnati, OH). Ten micrograms of total cellular RNA was used as a substrate for cDNA synthesis using RNase H-reverse transcriptase (Life Technologies, Gaithersburg, MD). Oligo(dT) primers were used in first strand synthesis and one-tenth volume of the first strand material was then amplified directly using the previously described primers for Ig{kappa}, IgMµ, and IgG1,2,3 (3' end) and degenerate oligonucleotides for the 5' ends (22). Amplification was conducted using the PerkinElmer thermocycler (Cetus, Emeryville, CA) using 30 cycles of 1 min at 94°C, 2 min at 45°C, and 1 min at 72°C. PCR products were then separated on 1% agarose gels and purified with QIEAXII Ger Extraction kit (Qiagen, Chatsworth, CA). Cloned PCR products were sequenced using an Applied Biosystems DNA sequencer (Applied Biosystems, Foster City, CA). Using this approach to analyze duplicate PCR products, we estimate the error rate is 0.5/400 bp and is consistent with that expected from Taq polymerase (24). Nucleotide sequences were analyzed using BLASTN searches of European Molecular Biology Laboratory/GenBank databases (National Center for Biotechnology Information, Bethesda, MD) using sequences from V{kappa}, VH, and third complementarity-determining region of Ig H chain (CDRH3) regions. For purposes of comparison, amino acid residue assignments are based on the nomenclature of Kabat et al. (25). All sequences are available in GenBank database.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The B cell repertoire in NOD mice is highly active

These studies were initiated with the goal of identifying spontaneous insulin autoantibodies in NOD mice. In our initial experiments, we observed that the frequency of hybridoma production in naive NOD mice approached that of immunized mice. Because activated B cells are preferentially selected for hybridoma formation, we used this approach to examine the fusion frequency of spleens from NOD, C57BL/6, and BALB/c mice of different ages. The data are summarized in Fig. 1Go and show results as the frequency of Ab-producing wells (M + G) per 105 of spleen cells. The frequency of Ab-producing wells in NOD mice ranged from 4–20/105 spleen cells while the frequency in naive BALB/c and B6 mice ranged from 0.3–1.5/105 spleen cells. Thus, the frequency of B cell fusion events is 5- to 20-fold greater in unmanipulated NOD mice than in naive BALB/c or B6 mice. Increased B cell activity in NOD was observed from 4 wk (the youngest age tested) to 6 mo of age and was highest at 14 wk. The sex of NOD mice does not appear to be a major influence on B cell activation as indicated by the similar Ab capture frequency in male and female NOD mice. In diabetic NOD mice (blood sugar >300 mg/dl), the recovery of spleen cells was reduced 50%; however, the relative frequency of Ab capture was still greater than in BALB/c or B6 mice. mAb generated in 4- to 12-wk-old mice were chiefly IgM and <1% IgG. IgG or double producers (IgG and IgM) were observed in 5% of Ab-producing wells from older NOD mice (14–25 wk). These findings suggest that in addition to having a high basal level of B cell activity, the active B cell repertoire of NOD undergoes qualitative changes in isotype expression with age.



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  		FIGURE 1. Frequency of hybridoma generation is increased in naive NOD mice. The number of Ab-producing wells per 105 spleen cells is compared in NOD (N), diabetic NOD (Nd), C57BL/6 (B), and BALB/c (Bl) mice of the indicated age and sex. Spleens were obtained from nonimmunized mice and used directly in a standard fusion protocol without activation, as described in Materials and Methods.

 
Nature of insulin autoantibodies from NOD mice

To further characterize the active B cell repertoire in NOD mice, hybridomas captured from fusion at the peak of the response (14 wk) were screened for insulin binding in ELISA and cloned at limiting dilution. One of 12 insulin binding mAb was of the IgG2a isotype (EB6) and one (1B9) was a double secretor (IgM + IgG2a); the others were IgM Abs. All of these mAb-bound insulin in ELISA compared with noncoated wells (OD>1.0), and binding was removed by absorption on either insulin-conjugated Sepharose or by insulin-coated microtiter plates. However, at multiple dilutions binding was poorly inhibited by preincubation with an excess of soluble insulin (1–100 µg/ml). This was also true of the IgG mAbs. The results of several experiments are summarized in Fig. 2Go in which histograms show binding of individual mAb in the presence and absence of insulin (50 µg/ml). In contrast to mAb derived from NOD mice, the binding of anti-insulin IgM hybridoma (1F11) from an immune BALB/c mouse (22) is totally inhibited by insulin (IC50, 0.1 µM). For some mAb, the binding to insulin in ELISA is facilitated when soluble insulin is added as an inhibitor (e.g., mAb 7F5, Fig. 2Go). These findings are entirely representative of mAb generated in multiple fusions and indicate that the majority of active anti-insulin B cells captured by hybridoma production in NOD mice have low affinity for insulin in solution (IC50, >50 µM).



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  		FIGURE 2. Insulin-binding hybridomas captured from NOD mice have low avidity for soluble insulin. Supernatants from insulin binding hybridomas were used at 70% maximal binding and their binding measured by ELISA in the absence ({blacksquare}) or presence () of soluble insulin, 50 µg/ml. 1B9 and EB6 are IgG and other Abs are IgM. 1F11 is an IgM anti-insulin mAb from BALB/c that is >95% inhibitable with soluble insulin. Data are mean ± SD of binding OD405 on insulin-coated plates.

 
An example of preferential binding of insulin on a surface (i.e., ELISA plate) for a spontaneous IgG anti-insulin is shown in Fig. 3Go. Insulin binding at serial dilutions of mAb 1B9 (Fig. 3Go, {blacktriangleup}) were compared with binding of the same dilution after preincubated with 100 µg/ml of human insulin (Fig. 3Go, {triangleup}). The resulting binding profile in the presence or absence of excess insulin shows that insulin inhibition is <10% at all dilutions. In contrast, the binding of IgG mAb125 derived from insulin immunization is effectively inhibited at all dilutions by insulin in solution. These data indicated that although IgG mAb 1B9 has undergone isotype switch, its relative affinity is sufficiently low that it only binds insulin when the Ag is stabily displayed on a surface and not in solution. As indicated in Fig. 2Go, most spontaneous insulin-binding mAb from NOD have this characteristic avidity-dependent binding.



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  		FIGURE 3. Low avidity of a spontaneous IgG anti-insulin mAb captured from NOD. Serial dilutions of mAb125 from BALB/c (circles) and mAb1B9 from NOD (triangles) are compared for insulin binding in ELISA alone (filled symbols) or following preincubation with 100 µg/ml of human insulin (open symbols). More than 90% of insulin binding by IgG mAb125 is inhibited by excess insulin while excess insulin inhibits <10% of binding of 1B9. The staining dilution of mAb125 was 1/10 and that of 1B9 was 1/5 to equalize insulin binding.

 
Most anti-insulin mAb from NOD are part of the natural or polyreactive repertoire

The apparent low avidity of anti-insulin hybridomas captured from NOD spleen suggest that these mAb are part of the natural autoantibody repertoire. Because natural autoantibodies are characterized by low avidity and broad specificity, mAb from NOD were examined for reactivity on a panel of environmental and autoantigens. The results for four mAb are shown in Fig. 4Go. The findings indicate that all these mAb bind one or more Ags in addition to insulin, including thyroglobulin, Ig, and DNA. In all, 16 mAb identified by insulin binding in NOD were found to be widely cross-reactive with several Ags and are represented by the profiles of mAb 4A11 and F10B. The two mAb with selective (i.e., insulin preferential binding) represent a minority of the B cells captured by hybridoma formation in NOD. One mAb, 1B9, binds insulin preferentially when compared with other Ags but, as shown above, this binding is not inhibited by soluble insulin. Another IgG mAb, EB6, was identified by insulin binding but is observed to be more active as a rheumatoid factor (anti-Ig). Together the data show that most mAb captured from NOD spleen on the basis of insulin binding have characteristics of natural autoantibodies that include low avidity and multireactivity.



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  		FIGURE 4. NOD mAb selected for insulin binding are broadly cross reactive with multiple Ags. Histograms compare binding of the indicated mAb in ELISA to seven Ags in the following order: insulin, human Ig, calf thymus DNA, thyroglobulin, dextran, cardiolipin, and tetanus toxoid. 4A11 and F10B are IgM and represent a polyreactive pattern observed in 16 other mAb captured from NOD. 1B9 and EB6 are IgG and demonstrate more selective insulin binding but also react with other Ags. Binding is indicated as mean OD405 in triplicate.

 
NOD anti-insulin mAb share V{kappa} genes with Abs found in systemic autoimmune disease

To investigate the genetic origins of autoantibodies in NOD, we determined the nucleotide sequences of V{kappa} region genes expressed by six anti-insulin hybridomas. The V{kappa} and J{kappa} gene segments and third complementarity-determining region of Ig L chain region used by these hybridomas are summarized in Table IGo. Different V{kappa}2 genes are used by EB6 and F10B while the same V{kappa}1c gene is used by clonally unrelated 1B9 and 10E10. Neither V{kappa}2 nor V{kappa}8 genes observed in this study were found in our previous analyses of 20 anti-insulin mAb from BALB/c (22, 26, 27). The V{kappa}28 gene, also known as V{kappa} ser, has not been observed in other anti-insulin responses; however, this gene is closely related to the V{kappa}19 family that was found to dominate the secondary anti-insulin response (27). The V{kappa}1c gene used by 1B9 and 10E10 is almost identical with that expressed by anti-insulin mAb 301 derived from immunization of BALB/c with Beucella abortus Ag-insulin (Fig. 5Go). This same gene isalso expressed in germline (unmutated) configuration in two anti-DNA mAb from lupus prone mice, mAb 202s.38 and 111.68 (28). Further analysis shows the V{kappa} genes used by all NOD hybridomas were often identical with V{kappa} genes expressed by mAb (principally anti-DNA) from systemic autoimmune disease in MRL, NZB, and motheaten mice (Table IIGo).


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  		Table 1. VK genes used by NOD hybridomas

 


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  		FIGURE 5. V{kappa}1c genes are shared by anti-insulin and other autoantibodies. The nucleotide and predicted amino acid sequence from anti-insulin mAb 301 from BALB/c is compared with the nucleotide sequence from V{kappa} expressed by insulin binding mAb 1B9 and 10E10 from NOD mice. Also shown is the nucleotide sequence from anti-DNA mAb 111.68 (also identical with another anti-DNA 202s.38) that uses the same V{kappa}1c gene.

 

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  		Table 2. V{kappa} genes shared by autoantibodies

 
Anti-insulin VH genes expressed in NOD mice are identical with known germline genes

Analysis of the nucleotide sequences from the VH of six NOD hybridomas shows that four Abs were derived from different germline genes in the large J558 family, and one each was from the 7183 and Q52 families (Table IIIGo). Although a number of J558 genes and a few 7183 and Q52 genes are used by anti-insulin mAb from primary and secondary anti-insulin responses in BALB/c (22, 26, 27), none of the previously described anti-insulin VH genes were closely related to the family member expressed by these NOD mAb. Five of the six NOD VH genes were identical with known VH gene segments (Table IIIGo). Three of these VH, J558.B10, J558.B18, and J558.B20 were identified in LPS-activated B cells and are also found in transformed B cell lines such a 70Z/3 and CH14 (29). MAb 4A11 contained four nucleotide replacements that resulted in two amino acid substitutions in second complementarity-determining region of Ig H chanin (S54N and Y61N) when compared with a known chromosomal segment (RP23-197F8) and to anti-DNA mAb H11A3 from motheaten mice (30). The J{kappa}1 segment of 4A11 also contains a nucleotide replacement (L104V) in a potential hot spot for mutation (31). In contrast to the other mAb that are encoded directly by germline (unmutated) genes, mAb 4A11 has likely undergone Ag-driven selection and somatic mutation.


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  		Table 3. VH genes used by NOD hybridomas with insulin binding

 
Limited N segment diversity and unique recombination events characterize the CDRH3s of NOD anti-insulins

Generation of the third hypervariable region of H chain Ig reflects juxtapositioning of D and JH gene segments followed by the joining of a VH gene segment to the DJH complex. Extensive diversity within this region is created by the exonuclease removal of nucleotides as well as by the addition of palindromic and nontemplated addition on nucleotides by TdT. Adult repertoires are characterized by the presence of N segment addition in >75% of HC gene rearrangements (32, 33) while there is a bias against N segment addition in neonatal repertoires. The nucleotide and predicted amino acid from V-D-J gene segments that contribute to CDRH3 (residues 95–102) of NOD anti-insulins are shown in Fig. 6Go. The length of CDRH3 varied from 4–10 aa. Three mAb use JH4, two use JH1, and one uses JH2. Evidence for both 5' and 3' TdT activity (N segments) is seen only in mAb 1B9. For three mAb, a D gene donor segment was readily identified, two were DSP2 genes that used the third reading frame and one was a DQ52 gene that used the first reading frame. Two mAb, EB6 and EF10B, have no D segments. An interesting feature of mAb 10E10 and 4A11 is the potential for non-D gene nucleotides to contribute to CDRH3 diversity. A 12-nucleotide gene segment from plasmacytoma variant translocation-1 (pvt-1) can account for the nucleotides in CDRH3 of 4A11 (34) and the pvt-1 segment contains a possible recombination signal sequences flanking the donor segment. In addition, a donor sequence from framework three of an X24 anti-asialo GM1 mAb (35) is identical with the nucleotide sequence in CDRH3 of 10E10. The third framework region of this mAb contains an exact heptamer recombination signal sequence, consistent with a VH gene replacement event as originally described in B cell lymphomas (36). These observations suggest that gene replacement events may contribute to CDRH3 structures in NOD.



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  		FIGURE 6. Derivation of CDRH3 segments expressed by insulin-binding mAb from NOD. Nucleotide sequences from VH, D, JH and other gene segments that potentially contribute to CDRH3 in NOD mAb are shown. N and p segment additions are indicated in bold. mAb F10B shows direct VH-JH recombination without the use of a D segment. mAb 10E10 shows evidence for gene replacement as indicated by the presence of frame work 3 (FW3) nucleotides that are identical with an X24 anti-asialoGM1 mAb (35 ). mAb 4A11 contains sequences that are homologous to nucleotides from pvt-1, a translocation site in plasmacytomas.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The importance of B cells for the development of insulitis and diabetes in the NOD mouse model of type I diabetes is well-recognized in studies that use Ab reagents or genetic approaches to block B cell development (9, 10, 11, 12). Although evidence for a direct contribution of Abs to the disease process is limited, most studies indicate that the key role for B cells in NOD T1DM may reside in their ability to capture and present {beta} cell Ags to disease promoting T cells. Data addressing Ig V region expression in B cells from NOD mice are limited. Studies using RNA hybridization and amplification of genomic DNA show a disproportional retention of fetal-type VH7183 genes in adult NOD mice and suggest that regulation of the B cell repertoire may be abnormal in NOD (14, 15).

In this study, we use recognition of the {beta} cell Ag insulin as a tool to investigate the expressed B cell repertoire in NOD mice. Hybridoma generation from spleens of naive mice indicates that a larger component of the endogenous B cell pool is active in NOD compared with B6 and BALB/c mice. This property of increased B cell activity is present early in life (4 wk) and persists in older mice of both sexes. These differences are not easily attributable to qualitative differences in NOD B cells since markers of B cell developed and differentiation such as CD21, CD22, CD23, and B220 are not abnormal in NOD (our unpublished observations). However, we find NOD mice express the infrequent CD72c allele which is also used by some lupus prone strains and the functional consequences of CD72c in NOD is currently being examined.

A large majority of mAb captured from NOD spleen are IgM; however, the contribution of IgG Abs increases during the period when the autoimmune process of {beta} cell attack is expected to be intense (>14 wk). Even though selected for insulin binding in ELISA, these mAb have broad specificity and are of low avidity as indicated by their polyreactive nature and poor inhibition by soluble insulin. These mAb are functionally different from either monospecific insulin Abs that follow immunization or from spontaneous insulin Abs found in the prodrome of NOD and human T1DM. Rather, in NOD most spontaneously active B cells may not be Ag-driven but are expanded from a repertoire that produces natural type autoantibodies (37). Although we did not formally analyze the frequency of insulin binding in the small number of hybridomas from B6 and BALB/c mice, we do find anti-insulin activity as part of the natural mAb repertoire of these strains. Thus, insulin binding is not excluded from the natural repertoire of nonautoimmune strains and this is consistent with the detection of natural insulin autoantibodies in many studies of human and animal sera (38, 39). Based on our extensive search, we postulate that the production of high-affinity insulin autoantibody is a rare event in NOD spleen. Although high-affinity anti-insulin is detected in sera, B cells producing such autoantibodies may be short lived or reside in discrete anatomical locations and we are currently examining those possibilities. In prior studies on IgG anti-insulin mAb125 and 127, we introduced back mutations to germline structures and show that a single amino acid substitution can impart insulin binding to V regions with little or no insulin binding (23). Thus, a large pool of natural type insulin autoantibodies may provide a substrate to fuel the autoimmune process if appropriate T cell help is available. When examined in RIA, we find our panel of NOD mAb bind insulin poorly. However, the binding of some mAb, such as 4A11, is not negative. For example, 4A11 typically binds 200 cpm above background compared with 5000 cpm for mAb125. Because structural studies suggest that 4A11 has undergone somatic mutation, this mAb may have been captured during its evolution in the context of T1DM.

The proposition that natural Abs provide a starting repertoire for an insulin-specific response is supported by data on the genetic components of spontaneous NOD mAb that bind insulin. The V genes from NOD mAb are shared with monospecific insulin Abs from immunized mice and with autoantibodies detected in systemic autoimmune disease. V{kappa} genes with identical sequence are observed in other autoantibodies, principally anti-DNA, and the same V{kappa}1c gene is used by NOD mAb and by mAb from insulin immunization. Similarly, the V{kappa} Ox2 gene reported for a NOD IgG anti-insulin mAb (16) is the same V{kappa} gene used by mAb125 from BALB/c (26). Analysis of the V gene structures used by NOD mAb indicate they are derived directly from germline-encoded (i.e., unmutated) structures, and only one mAb shows evidence for Ag-driven selection. The VH genes used by insulin binding NOD mAb are not closely related to any of the previously reported VH genes expressed by anti-insulin mAb (22, 26, 27). These VH are often identical with genes used by anti-DNA mAb from lupus prone mouse strains and by B cell lymphomas. An important structural feature of NOD VH regions is that most mAb maintain CDRH3-length D segments without the use of N segment additions. Lack of N segments is associated with VH genes that originate in the fetal period and is consistent with reports on persistent fetal-type VH genes in NOD mice (15). Because the B1 subset has many characteristics of the fetal repertoire (32), we also examined our hybridomas for CD5 expression by both RT-PCR and FACS and do not find evidence that these mAb are derived from B1 B cells (our unpublished observations). In addition, the B1a population (CD5+) in the spleens of NOD mice is not significantly different from B6 mice (P. Kendall and J. W. Thomas, manuscript in preparation). Thus, increases in autoreactive B cells observed in NOD mice are not easily attributable to expansion of the B1 subset as occurs, for example, in motheaten mice (40).

Virtually all of the expressed VH and V{kappa} genes used by insulin-binding hybridomas from in NOD are also found in anti-DNA or other autoantibodies from mouse strains with systemic autoimmune disease such as MRL and NZB/W. Although databases are biased by the intense investigation of autoantibodies, the nearly exact nucleotide sequence homologies in many cases indicates that the same V genes are selected into the repertoire of NOD mice as in mice with different autoimmune processes. Interestingly, some treatments that protect NOD mice from developing diabetes are observed to induce lupus-like syndromes (19) and support a hypothesis that NOD and lupus prone strains may share genetic features with immunoregulatory abnormalities in the B cell repertoire. Among our mAb, 4A11 shows consistent evidence for Ag-driven somatic hypermutation. Because mAb 4A11 is the most insulin selective of our panel, the presence of mutations suggests it may have been evolving into a high-affinity Ab. One other mutated anti-insulin (Ig2b) from NOD has been reported and it also expresses a VH gene used by MRL mice (16). Thus, some components of the highly active natural autoantibody repertoire in NOD may be selected into T cell-dependent immune responses and fuel the autoimmune process. The recognition that high-affinity insulin Abs detected by RIA and not low avidity binding in ELISAs are more predictive of disease progression suggests that availability of Ag-specific T cell help is a critical feature of disease progression.

The abundant repertoire of autoantibodies in NOD mice raises the possibility that selection and regulation of the B cell repertoire is abnormal in NOD. Recent studies in mice that are deficient in key signaling components in the B cell receptor (BCR) pathway, such as lyn and CD45, show that signaling defects favor the selection of BCR that are autoreactive as a means to maintain B cell homeostasis (41, 42, 43). Because abnormalities in signal transduction are recognized in NOD T cells (44), the increased entry of autoreactive B cells into the repertoire of NOD may reflect a similar abnormality in BCR signaling in NOD. Our recent observation that mice harboring an Ig H chain transgene favor the development of insulin-binding B cells in NOD but not in B6 mice also suggests an altered threshold for selection of autoreactive B cells in NOD (45). The previously noted persistence of fetal VH genes in NOD may reflect an increased frequency of autoreactive V regions in the fetal repertoire that aids selection of B cells in the repertoire. A recent case report of a human T1DM patient with Bruton tyrosine kinase deficiency has been used to suggest that the role of B cells in NOD and human T1DM may be different (46). An alternative interpretation is that a signaling defect in Bruton tyrosine kinase deficiency may have favored selection of small numbers of autoreactive B cells in the human disease and our studies suggest that a qualitatively similar defect may be present in the B cell repertoire of NOD. Understanding the mechanisms responsible for these defects may aid in the identification of additional genetic components that contribute to susceptibility in T1DM.


   Acknowledgments
 
We thank Elaine Beeler for her help in preparing the manuscript and Andres Rojas for technical assistance.


   Footnotes
 
1 This work supported by Grants R01 DK43911 and R21 AI47763 from the National Institutes of Health. H.G.M. was supported by a fellowship from the Juvenile Diabetes Foundation. The Molecular Biology core lab used in these studies was supported by National Institutes of Health Grants P30-CA6848T and 5P60-DK20593 to the Vanderbilt Diabetes and Cancer Centers. Back

2 Address correspondence and reprint requests to Dr. James W. Thomas, Department of Medicine, Vanderbilt University, T-3219 Medical Center North, Nashville, TN 37232-2681. E-mail address: james.thomas{at}vanderbilt.edu Back

3 Abbreviations used in this paper: NOD, nonobese diabetic; BCR, B cell receptor; T1DM, type I or insulin-dependent diabetes mellitus; CDRH3, third complementarity-determining region of Ig H chain; V{kappa}, variable region {kappa} L chain; VH, variable region of Ig H chain; pvt-1, plasmacytoma variant translocation-1. Back

Received for publication May 28, 2002. Accepted for publication October 2, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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