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Comparative Study
. 2006 Nov 15;177(10):7033-41.
doi: 10.4049/jimmunol.177.10.7033.

Genes within the Idd5 and Idd9/11 diabetes susceptibility loci affect the pathogenic activity of B cells in nonobese diabetic mice

Pablo A Silveira  1 , Harold D Chapman, Jessica Stolp, Ellis Johnson, S Lewis Cox, Kara Hunter, Linda S Wicker, David V Serreze
Affiliations
Comparative Study

Genes within the Idd5 and Idd9/11 diabetes susceptibility loci affect the pathogenic activity of B cells in nonobese diabetic mice

Pablo A Silveira et al. J Immunol. .

Abstract

Autoreactive T cells clearly mediate the pancreatic beta cell destruction causing type 1 diabetes (T1D). However, studies in NOD mice indicate that B cells also contribute to pathogenesis because their ablation by introduction of an Igmunull mutation elicits T1D resistance. T1D susceptibility is restored in NOD.Igmunull mice that are irradiated and reconstituted with syngeneic bone marrow plus NOD B cells, but not syngeneic bone marrow alone. Thus, we hypothesized some non-MHC T1D susceptibility (Idd) genes contribute to disease by allowing development of pathogenic B cells. Supporting this hypothesis was the finding that unlike those from NOD donors, engraftment with B cells from H2g7 MHC-matched, but T1D-resistant, nonobese-resistant (NOR) mice failed to restore full disease susceptibility in NOD.Igmunull recipients. T1D resistance in NOR mice is mainly encoded within the Idd13, Idd5.2, and Idd9/11 loci. B cells from NOD congenic stocks containing Idd9/11 or Idd5.1/5.2-resistance loci, respectively, derived from the NOR or C57BL/10 strains were characterized by suppressed diabetogenic activity. Immature autoreactive B cells in NOD mice have an impaired ability to be rendered anergic upon Ag engagement. Interestingly, both Idd5.1/5.2 and Idd9/11-resistance loci were found to normalize this B cell tolerogenic process, which may represent a mechanism contributing to the inhibition of T1D.

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Figures

Figure 1
Figure 1. Non-MHC Idd alleles from the T1D resistant NOR strain reduces the pathogenic capacity of B-cells
A. T1D incidence was compared in cohorts of female NOD.Igμnull mice that were lethally irradiated and reconstituted with SBM alone (closed squares, n=12) or admixed with purified splenic NOD (open squares, n=23) or NOR (closed circles, n=20) B-cells. (B) T1D incidence of female NOD.Igμnull mice reconstituted with SBM and purified splenic B-cells from either NOD.Idd13NOR (line D2Mit490-144NOR; open triangles, n=16) or NOD.Idd9/11NOR (closed diamonds, n = 16) mice and in (C) NOD.Idd5NOR (open triangles, n=12) or NOD.Idd5B10 (line R444; open diamonds, n=36) mice. For comparative purposes, disease development in NOD.Igμnull mice reconstituted with SBM and NOD (open squares, n=58) or NOR (closed circles, n=20) B-cells are also shown in graphs (B) and (C). All B-cell donor mice were 6 week old females. T1D development was assessed over 21 weeks post-reconstitution. As mice did not develop disease prior to 10 weeks after reconstitution, only weeks 10-21 are shown on graphs. Stars indicate a significant difference (p<0.05) in T1D development compared to NOD B-cell reconstituted controls using Kaplan-Meier Life-Table Analysis.
Figure 2
Figure 2. Sub-congenic analysis of the Idd5 locus reveals epistatic contributions to the diabetogenic activity of NOD B-cells
Schematic illustrations of Chr. 1 showing polymorphic microsatellite markers (on left) used to define congenic intervals in NOD.Idd5NOR (grey box) and various NOD.Idd5B10 (diagonally lined boxes) lines. Location of markers (in mega base pairs (Mb) from centromere) were determined using the ENSEMBL mouse genome database. On the right are shown boundaries of Idd5.1, 5.2 and 5.3 T1D susceptibility genes as defined by Wicker and colleagues. Numbers of female NOD.Igμnull mice reconstituted with SBM and various B-cells, and subsequent frequency of T1D over a 21 week follow up period are displayed above the Chr. maps. Stars are indicative of a significant difference (p<0.05) in T1D compared to the NOD B-cell reconstituted controls using Kaplan Meier Life-Table Analysis.
Figure 3
Figure 3. Idd5B10 and Idd9NOR congenic regions do not normalize subset distribution of splenic B-cells in NOD mice
Representative FACS profiles of CD21 and CD23 antibody staining on B220+ gated splenocytes from 6 week old NOD, C57BL/10, NOR, NOD.Idd5B10 and NOD.Idd9NOR female mice. Mean proportions and absolute cell numbers of total and gated B-cell subpopulations (including T1, T2/pre-MZ, FO and MZ) in spleens of four female mice of each strain are shown on Table II.
Figure 4
Figure 4. A gene(s) in the Idd9/11, but not the Idd5 region affects the proliferative responsiveness of NOD B-cells
B-cells were purified from pooled LN (cervical, axillary, mesenteric and inguinal) of three 6-8 week old NOD (black bars), NOD.Idd5B10 (white bars), NOD.Idd9/11NOR (grey bars) and C57BL/10 female mice (striped bars) in experiment (A) or four 6-8 week old female NOD (black bars), NOD.Idd9/11NOR (grey bars) and NOR (dotted bars) mice in a separate Experiment (B). Triplicate aliquots of 1×105 B-cells were stimulated with 1 μg/ml goat anti-mouse IgM F(ab’)2 fragments with or without 5μg/ml CD40 specific mAb. Proliferation over the final 24 hours of a 72 hour incubation period was measured by [3H] thymidine incorporation and is shown as mean Δ cpm (stimulated – non-stimulated) ± SEM for triplicate wells of each strain.
Figure 5
Figure 5. Neither the Idd5B10 or Idd9/11NOR regions can correct the deletion resistant phenotype of immature B-cells in NOD mice
T1 B-cells (B220+ CD21− CD23−) were purified from the pooled spleens of four 6-8-week-old NOD (open diamonds, solid line), NOD.Idd5B10 (closed triangles, dashed line), NOD.Idd9/11NOR (open squares, solid line) or B6 (closed circles, dashed line) female mice as described in Material and Methods. Triplicate aliquots of 1×105 T1 B-cells from each pool were cultured in with the indicated concentrations of anti-IgM F(ab’)2 fragments for 20 hours. Cells were subsequently harvested and analyzed by flow cytometry for viability characterized by negative staining for Annexin V and PI. Results are displayed as a percentage of viable T1 B-cells remaining in culture after 20 hours of IgM stimulation compared to non-stimulated controls ± SD. Two separate experiments are shown.
Figure 6
Figure 6. B-cell anergy induction is controlled by Idd5 and Idd9/11 region genes
B-cells were purified from pooled spleens of three 6-8 week old IgHEL single transgenic or IgHEL/sHEL double transgenic female mice. In each experiment, proliferation of purified B-cells from single (black bars) and double transgenic (grey bars) mice on the NOD genetic background were compared to single (white bars) and double transgenic mice (striped bars) on the (A) B6, (B) NOD.Idd5B10 or (C) NOD.Idd9/11NOR genetic background. B-cells were cultured with or without 1μg/ml anti-IgM F(ab’)2 fragments in combination with 5μg/ml anti-CD40 mAb. Proliferation over the final 24 hours of a 72 hour culture period was measured by [3H] thymidine incorporation and is presented as mean Δ cpm (stimulated – non-stimulated) ± SEM for triplicate wells of each strain. Each graph is representative of one of two experiments with similar results.
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