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Cutting Edge |
Departments of Medicine, Microbiology and Immunology, Vanderbilt University, Nashville, TN 37232
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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cells in the pancreas. In the nonobese diabetic (NOD) model of TIDM,
insulitis and diabetes are dependent on the presence of B lymphocytes;
however, the requirement for specificity within the B cell repertoire
is not known. To determine the role of Ag-specific B cells in TIDM,
VH genes with different potential for insulin
binding were introduced into NOD as H chain transgenes. VH125 H chain
combines with endogenous L chains to produce a repertoire in
which 1–3% of mature B cells are insulin specific, and these mice
develop accelerated diabetes. In contrast, NOD mice harboring a similar
transgene, VH281, with limited insulin binding develop
insulitis but are protected from TIDM. The data indicate that
Ag-specific components in the B cell repertoire may alter the course of
TIDM. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells in the pancreatic islets. Many aspects of
the human disease are shared with the nonobese diabetic (NOD) mouse
model of the disorder, including a role for class II MHC polymorphisms
(1, 2), T lymphocytes in the pancreatic infiltrates
(3), and shared T cell regulatory abnormalities (4, 5). These observations and the ability of T cells to transfer
the disease in NOD focus most studies on T lymphocytes. However, loss
of tolerance in the B cell compartment, as manifested by the detection
of autoantibodies to cell Ags, is one of the earliest
indicators of TIDM. Among these, insulin autoantibodies have predictive
value in both NOD and human TIDM (3, 6, 7), and
insulin-specific T cells can transfer disease in NOD
(8). Direct evidence for the importance of B lymphocytes in TIDM comes from NOD mice rendered deficient in B lymphocytes by homozygous disruption of membrane-Ig µ (µMT) (9, 10). Likewise, passive transfer of anti-IgM Ab that blocks B cell development is also effective at preventing insulitis and eliminating diabetes in NOD (11, 12). In B cell knockout mice, direct reconstitution with mature B cells was not possible because of the strong CTL response against donor B lymphocytes by µMT-NOD mice. When B cells were introduced using bone marrow chimeras, TIDM susceptibility was restored in the µMT animals while transfer of sera failed to show a role for circulating Ab in the disease (13). Although these studies clearly demonstrate the importance of B cells in the development of TIDM in NOD, there are no data on how specificity of the B cell receptor for Ag contributes to the process. The Ag receptors on B lymphocytes can capture low abundance Ag for presentation to T cells (14), and in vitro studies suggest that the Ag presenting function of B cells (15, 16) or specific Abs (17) is uniquely able to induce responses to islet Ags such as glutamic acid decarboxylase.
To examine the role of Ag-specific B lymphocytes in insulin-dependent diabetes mellitus (IDDM), we introduced VH genes related to anti-insulin mab125 into the germline of NOD mice as IgMa transgenes (Tg). In previous studies using site-directed mutagenesis, VH125 is shown to contain two amino acid replacements in CDRH2 that are necessary for insulin binding (18). VH281 is identical except for an unmutated CDRH2 and it has no measurable insulin binding when expressed as a soluble Ab (18). Thus, NOD mice harboring the VH125 Tg (VH125Tg) have a B cell repertoire with increased potential to bind insulin, while mice harboring the VH281 Tg (VH281Tg) have a repertoire with a decreased insulin-binding potential. Accordingly, we used these mice to examine the impact of the B cell repertoire on the development of insulitis and diabetes in NOD mice. The data show that skewing the B cell repertoire toward an islet Ag (i.e., insulin) promotes the development of diabetes, whereas a repertoire with impaired recognition of insulin limits the progression of the disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice expressing IgMa H chain (HC) Tgs VH125 and VH281 were produced in C57BL/6 as previously described (19) and were introduced into NOD mice (Taconic Farms, Germantown, NY) by multiple backcrosses. After the sixth and eight backcrosses (N7 and N9, respectively), animals were genotyped using 40 oligonucleotide primer pairs (Research Genetics, Huntsville, AL) for 13 known diabetes susceptibility loci (9). The animals were found to be homozygous for all NOD alleles. To eliminate expression of B cells that escape allelic exclusion, NOD.MuMT mice were obtained from Dr. D. Serreze (The Jackson Laboratory, Bar Harbor, ME) and crossed with the HC-transgenic lines to produce F2 generations that carry HC Tgs in the presence (µMT-/+) or absence (µMT-/-) of endogenous B cells. The deficiency of endogenous B cells was confirmed by the lack of circulating IgMb in ELISA and by lack of IgMb B cells in FACS. Female mice were monitored for the development of diabetes for a period of 40 wk. Mice were considered to be diabetic if two consecutive blood sugars were >200 mg/dl. All animals were housed in facilities at Vanderbilt under specific pathogen-free conditions.
Immunoassays
Spleen cells or PBLs were analyzed using FACScan (BD Biosciences, Mountain View, CA) and mAb to B220 (6B2), IgMa (RS-3.1), and IgMb (AF6) (BD PharMingen, San Diego, CA). Insulin-binding B cells were identified using biotin-insulin (5 ng) and FITC-streptavidin (Sigma-Aldrich, St. Louis, MO) in FACScan (19). To exclude nonspecific interactions, insulin-specific B cells were identified by inhibition with unmodified human insulin (50 ng). Serum allotypes and specific Abs were measured in ELISA as described elsewhere (19).
Histology
Pancreata from transgenic NOD or nontransgenic littermates ages 6–40 wk were analyzed for the presence of islet infiltrates after Formalin fixation and H&E staining. Islets (25–50) were examined on nonsequential sections from each pancreas. Islets that had either peri-insulitis or insulitis were scored as positive for infiltration and the percentage of islets with infiltration was determined for each mouse.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IgMa HC Tgs were introduced into NOD by
multiple backcrosses and their expression was examined by flow
cytometry. Representative histograms for NOD and VH125Tg NOD mice are
shown in Fig. 1
. B cells from
nontransgenic NOD mice do not express IgMa
(Fig. 1A) and are all IgMb+
(Fig. 1B). In contrast, >90% of B cells
(B220+) in VH125Tg NOD mice express the
IgMa Tg (Fig. 1C) and only a small
number of endogenous IgMb B cells are present
(Fig. 1D). As in B6 mice, Tg expression was stable for over
40 wk in NOD spleen (94 ± 4% IgMa,
n = 11). Similar results were obtained in VH281Tg NOD
mice, although the degree of allelic exclusion is less (82 ± 5%
IgMa, n = 10).
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To determine how B cell repertoires with different potentials for
insulin binding effect the development of diabetes in NOD mice, cohorts
of HC-transgenic female mice and their nontransgenic littermates were
monitored for hyperglycemia (Fig. 2). The
data show that mice harboring VH125Tg (n = 22) develop
diabetes at an accelerated rate compared with the other lines.
Twenty-one of 22 mice (95%) in this cohort developed diabetes by 40
wk. In contrast, mice that express VH281Tg (n = 19) are
protected from developing diabetes and only 3 of 19 mice (16%) in this
cohort developed diabetes. The differences are not attributable to
circulating insulin Abs produced by the Tgs which are negligible
(OD < 0.05) in both lines. Additional studies from different
founders and at later backcrosses have identical results and support
the observation that a skewed B cell repertoire can alter the outcome
of diabetes in NOD mice.
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Although VH281Tg NOD are protected from developing diabetes, by 40
wk all animals had extensive islet infiltrates. Since mice that carry
Ig Tgs routinely have populations of endogenous B cells that escape
allelic exclusion, this additional source of B cells may contribute to
the development of disease in transgenic NOD. Therefore, studies on
islet infiltration were conducted using mice that expressed HC Tg in
the presence (µMT-/+) or absence
(µMT-/-) of a fixed mutation in Ig-µ to
eliminate endogenous B cells (Fig. 3).
All NOD mice harboring HC Tgs showed evidence of islet infiltration in
the late prediabetic period (14 wk) and at this time point the extent
of disease (percentage of islets with infiltration) is only slightly
less in VH281Tg compared with VH125Tg NOD mice. When pancreata were
examined at an earlier time point (6 wk), differences in the incidence
of infiltration are more apparent, VH125Tg (75–85%) vs VH281Tg
(40–50%). The incidence of infiltration in each Tg line was not
effected by the presence of the µMT mutation. However, the extent of
involvement (percentage of islets infiltrated) was slightly greater in
both VH125 and VH281Tg NOD mice when endogenous B cells are present
(µMT+/-). Thus, the additional diversity
provided by endogenous B cells may enhance islet infiltration in
HC-transgenic NOD mice; however, the repertoire of
VH281Tg.µMT-/- is sufficient to generate
insulitis in the absence of endogenous B cells.
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and indicate that mice whose HC repertoire is composed only of VH125
develop diabetes at a rate comparable to the original cohort. In the
presence of endogenous B cells
(VH125Tg.µMT-/+), the onset of diabetes occurs
slightly earlier (12 wk) but the overall incidence compared with
VH125Tg.µMT-/- NOD mice is similar by 17 wk.
This outcome is consistent with the insulitis data and suggests that
residual B cells escaping allelic exclusion have only a modest effect
on diabetes development in VH125Tg.
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To characterize the functional potential of the B cell repertoire
of nontransgenic and HC-transgenic NOD mice, spleens were examined for
insulin binding (Fig. 5). In nontransgenic NOD
mice, some insulin-binding B cells are observed (Fig. 5A);
however, binding is dim (mean fluorescence intensity, <200) and
principally in B220-low or -negative regions. This binding is not Ag
specific as indicated by the inability of insulin to inhibit binding
(Fig. 5B). Low levels of noninhibitable insulin binding are
also seen in spleens from VH281Tg mice (Fig. 5, C and
D). The insulin-binding profile of B cells was different in
VH125Tg mice (Fig. 5, E and F). In these mice, a
distinct population of B220+ cells is shown to
bind insulin (mean fluorescence intensity, >200; Fig, 5E),
and inhibition with soluble insulin (Fig. 5F) indicates
binding is specific. This population accounts for 3.1 ± 1.3% of
B220+ cells in VH125Tg NOD mice
(n = 9). Insulin-specific B cells were uniformly
IgMa+ and similar results were obtained in mice
with the µMT mutation (data not shown). Insulin-specific cells were
very rare in VH281Tg NOD (1 of 9 at 0.5%) and in nontransgenic NOD (1
of 21 at 0.6%). Since NOD mice are known to produce insulin Abs
(7), we interpret these findings to indicate that the
frequency of insulin-specific B cell is increased at least 10–20 times
in VH125Tg compared with nontransgenic NOD mice. This finding in NOD
mice differs from our observations in B6 mice harboring VH125Tg where
insulin-specific B cells were not observed (19).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cell
destruction (7, 20). Thus, the selection of
insulin-specific B cells into the repertoire of VH125Tg NOD and the
differentiation of B cells to produce high-affinity IgG
anti-insulin Ab in human pre-IDDM are similar processes that denote
a critical threshold for cell destruction has been reached. Since
our fixed HC Tgs do not produce measurable insulin Abs, the role of
anti-insulin B cells may be as APCs to expand insulin-specific T
cells which are known to contribute to TIDM (8).
In contrast, a B cell repertoire that is impaired for insulin
binding by a HC Tg that differs in two amino acids protects VH281Tg NOD
mice from diabetes. These mice, however, still develop islet
infiltration (both peri-insulitis and insulitis). Since Ig Tgs do not
completely exclude the expression of endogenous B cells
(IgMb) that may include other specificities, we
intercrossed our HC-transgenic NOD with B cell-deficient NOD.µMT to
examine the role of endogenous B cells. The data show that additional
diversity provided by residual endogenous B cells contributes modestly
to insulitis in VH281Tg NOD; however, insulitis develops when VH281 is
the only HC available. Likewise in VH125Tg mice, endogenous B cells may
have a small effect on insulitis and diabetes but the overall outcome
is determined principally by the function of the Tg. Recent studies
examining anti-hen egg lysozyme Tgs in NOD also show the
protection from diabetes in the presence of insulitis (D.
Serreze, unpublished data). This observation is consistent with
the data in VH281Tg NOD and indicate that a B cell repertoire with
impaired recognition of islet Ags has limited the progression of
diabetes. Ag-specific B cells may not be required for early islet
infiltration but their presence may be associated with enhanced
cell destruction. The current findings suggest that diverting even a
small portion of the B cell repertoire may be beneficial in preserving
cell function. Since insulin is only one of several autoantigens in
TIDM, the approach may be readily extended to other islet Ags in the
future.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James W. Thomas, T-3219 MCN Vanderbilt University, Nashville, TN 37232-2681. E-mail address: James. Thomas{at}mcmail.vanderbilt.edu
3 Abbreviations used in this paper: TIDM, type I diabetes mellitus; IDMM, insulin-dependent diabetes mellitus; HC, H chain; VH125, variable region HC gene from mAb125; VH281, unmutated HC progenitor of mAb125; NOD, nonobese diabetic; Tg, transgene; µMT, homozygous disruption of membrane IgM.
Received for publication August 6, 2001. Accepted for publication September 20, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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24JQ T cells in type 1 diabetes. Nature 391:177.[Medline]
cells of nonobese diabetic mice. J. Immunol. 163:743.This article has been cited by other articles:
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M. R. Gilbert, D. G. Carnathan, P. C. Cogswell, L. Lin, A. S. Baldwin Jr, and B. J. Vilen Dendritic Cells from Lupus-Prone Mice Are Defective in Repressing Immunoglobulin Secretion J. Immunol., April 15, 2007; 178(8): 4803 - 4810. [Abstract] [Full Text] [PDF]
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J. W. Thomas, P. L. Kendall, and H. G. Mitchell The Natural Autoantibody Repertoire of Nonobese Diabetic Mice Is Highly Active J. Immunol., December 1, 2002; 169(11): 6617 - 6624. [Abstract] [Full Text] [PDF]
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J. C. Jaume, S. L. Parry, A.-M. Madec, G. Sonderstrup, and S. Baekkeskov Suppressive Effect of Glutamic Acid Decarboxylase 65-Specific Autoimmune B Lymphocytes on Processing of T Cell Determinants Located Within the Antibody Epitope J. Immunol., July 15, 2002; 169(2): 665 - 672. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, n = 22), VH281HC Tg (
,
n = 19), and nontransgenic littermates (
,
n = 40). VH125 contains mutations in CDRH2 that
favor insulin binding while VH281 is unmutated. Mice were considered
diabetic if two consecutive blood sugars were >200 mg/dl.
, 10–50%; and 
,
1–10%. VH125 and VH281 indicate the HC Tgs and µMT-/+
or µMT-/- indicate the presence or absence of
endogenous B cells after introduction of the NOD.µMT mutant
alleles.
) or absence (
) of endogenous B cells. VH125Tg NOD mice were
intercrossed and then backcrossed with NOD.µMT to produce cohorts of
female mice without endogenous B cells (µMT-/-,
n = 10) or without endogenous B cells
(µMT-/+, n = 15).