Targeting Anti-Insulin B Cell Receptors Improves Receptor Editing in Type 1 Diabetes-Prone Mice

Abstract

Autoreactive B lymphocytes that commonly arise in the developing repertoire can be salvaged by receptor editing, a central tolerance mechanism that alters BCR specificity through continued L chain rearrangement. It is unknown whether autoantigens with weak cross-linking potential, such as insulin, elicit receptor editing, or whether this process is dysregulated in related autoimmunity. To resolve these issues, we developed an editing-competent model in which anti-insulin Vκ125 was targeted to the Igκ locus and paired with anti-insulin VH125Tg. Physiologic, circulating insulin increased RAG-2 expression and was associated with BCR replacement that eliminated autoantigen recognition in a proportion of developing anti-insulin B lymphocytes. The proportion of anti-insulin B cells that underwent receptor editing was reduced in the type 1 diabetes-prone NOD strain relative to a nonautoimmune strain. Resistance to editing was associated with increased surface IgM expression on immature (but not transitional or mature) anti-insulin B cells in the NOD strain. The actions of mAb123 on central tolerance were also investigated, because selective targeting of insulin-occupied BCR by mAb123 eliminates anti-insulin B lymphocytes and prevents type 1 diabetes. Autoantigen targeting by mAb123 increased RAG-2 expression and dramatically enhanced BCR replacement in newly developed B lymphocytes. Administering F(ab')2123 induced IgM downregulation and reduced the frequency of anti-insulin B lymphocytes within the polyclonal repertoire of VH125Tg/NOD mice, suggesting enhanced central tolerance by direct BCR interaction. These findings indicate that weak or faulty checkpoints for central tolerance can be overcome by autoantigen-specific immunomodulatory therapy.

Figure 1. A proportion of anti-insulin B cells lose insulin-binding specificity in the presence of endogenous insulin in the Igκ transgenic model, VH125Tg/Vκ125^SD

(A) Targeting vector schematic, showing WT (non-targeted), targeted Vκ125^SDNeo (Neo^R gene retained), or targeted Vκ125^SD (Neo^R gene removed) alleles. (B) ES cell clones (Left) or progeny mouse tail DNA (Right) were digested with SacI and the following alleles were identified by probe hybridization: WT (5.5 kb), Vκ125^SDNeo (6.3 kb), or Vκ125^SD (5.1 kb, Neo^R gene removed). (C) Splenocytes were freshly isolated from VH125Tg/Vκ125Tg (125Tg, Left), VH125Tg/Vκ125^SDNeo (Middle), and VH125Tg/Vκ125^SD (Right) C57BL/6 mice. Insulin-binding B cells were identified among B220⁺ IgM^a+ live lymphocytes using flow cytometry, confirming expression of anti-insulin Vκ125. Data are representative of progeny from 6 independent founder lines. (D) B cell subsets were identified among B220⁺ live lymphocytes as follows: bone marrow: immature (IgM⁺ CD23⁻) or mature recirculating (IgM⁺ CD23⁺); spleen: T1 (CD21^low CD23^low IgM^high), T2 (CD21^mid CD23^high IgM^high), FO (CD21^mid CD23^high IgM^mid), Pre-MZ (CD21^high CD23^high IgM^high), and MZ (CD21^high CD23^mid IgM^high), as shown by representative flow cytometry dot plots of VH125Tg/Vκ125^SD mice (Top). Flow cytometry was used to assess the average percentage ± SD of non-insulin-binding (edited) B cells within the indicated B cell subset in n = 3 VH125Tg/Vκ125^SD+/− (black) or n = 4 VH125Tg/Vκ125^SD+/+ (white) B6 mice (Bottom). ** p < 0.001, *** p < 0.001, two-tailed t-test.

Figure 2. Anti-insulin specificity correlates with increased RAG2-GFP expression in developing B lymphocytes

B cell subsets were identified in freshly isolated bone marrow and spleen as in Fig. 1D. (A) Representative flow cytometry plots of immature B cells depict Control (non-insulin-binding, Left), Edited (non-insulin-binding, Middle), or Insulin-Binding (Middle) B cells, based on reactivity with biotinylated insulin. Biotinylated mAb123 detects insulin-occupied BCR on immature VH125Tg/Vκ125^SD B cells using flow cytometry (Right). (B) Immature, T1, T2, or FO B cell subsets were identified in n ≥ 6 VH125Tg/Vκ125^SD/RAG2-GFP/B6 or control VH281Tg/Vκ125^SD/RAG2-GFP/B6 5–12 wk old mice, in which GFP expression is driven by the RAG-2 promoter. RAG2-GFP MFI is shown for Edited/Non-Insulin-Binding (black diamonds), Insulin-Binding (grey diamonds), or control (white squares) populations identified as in Panel A that were GFP⁺. Averages are indicated by bars. (C) The average percentage ± SD of Igλ⁺ cells in the indicated B cell subset was compared between n ≥ 7 VH125Tg/Vκ125^SD/B6 (black) and control VH281Tg/Vκ125^SD/B6 (white) mice. (D) Flow cytometry was used to measure surface IgM expression on the indicated B cell subsets in n ≥ 7 mice per group. The IgM MFI of VH125Tg/Vκ125^SD cells was divided by the IgM MFI of VH281Tg/Vκ125^SD B cells within the developmental subset indicated. A value < 1 indicates lower IgM expression in VH125Tg/Vκ125^SD B cells; the average ratio ± SD is shown. * p < 0.05, ** p < 0.001, *** p < 0.001, two-tailed t-test (B–C) or one sample, two-tailed t test (D).

Figure 3. Insulin autoantigen recognition is associated with replacement of Vκ125^SD in VH125Tg/Vκ125^SD B lymphocytes that lose the insulin-binding specificity

Flow cytometry sorting was used to purify B cell populations from freshly isolated spleens. Cells were sorted into RNA lysis buffer, RNA was purified, cDNA was generated, and PCR was used to amplify Igκ as in Methods. The number of clones (black circle, center) and percentage of isolates that expressed the targeted Vκ125^SD allele (Vκ4-74 Jκ5, white) or Novel Vκ/Jκ (grey) is indicated for each sorted population. (A) Total B cells (B220⁺ IgM^a+ live lymphocytes) were purified from n = 2 VH125Tg/Vκ125^SD/B6 (Left, GenBank Accession #KT250658- KT250681) or n = 2 VH281Tg/Vκ125^SD/B6 (Right, GenBank Accession # KT250637-KT250657) mice. (B) Non-insulin-binding (Left, GenBank Accession # KT250682-KT250703) or insulin-binding (Right, GenBank Accession # KT250704-KT250727) total B cells (gated as in Panel A) were purified from n = 3 VH125Tg/Vκ125^SD/B6 mice.

Figure 4. Anti-insulin B cells undergo receptor editing less efficiently in the type 1 diabetes-prone NOD strain

B cell subsets were identified using flow cytometry as in Fig. 1D in VH125Tg/Vκ125^SD bone marrow or splenocytes freshly isolated from 8 –12 week old B6 (black) or NOD (white) mice. (A–B) The percentage of non-insulin-binding (edited) B cells was assessed within the indicated subset using flow cytometry Representative dot plots (A) and the average ± SD (B) is shown for n = 6 B6 or n = 7 NOD VH125Tg/Vκ125^SD mice, n ≥ 2 experiments. (C) The percentage of Igλ⁺ B cells were further identified in total spleen as shown in representative plots (Top). The average total Igλ⁺ % ± SD in each subset is indicated for n ≥ 8 mice, n = 3 experiments (Bottom). (D) The IgM MFI of VH125Tg/Vκ125^SD/NOD B cells was divided by the IgM MFI of VH125Tg/Vκ125^SD/B6 B cells within the developmental subset indicated, n = 10 mice, n= 4 experiments. A value > 1 indicates higher IgM expression in NOD. * p < 0.05, ** p < 0.001, *** p < 0.001, two-tailed t-test (B–C), or one sample, two-tailed t test (D).

Figure 5. Igλ usage is reduced in NOD mice, even in the absence of H chain usage bias

B cell subsets were identified in freshly isolated bone marrow or spleen using flow cytometry as in Fig. 1D and the percentage of Igλ⁺ B cells was assessed in NOD or B6 mice, representative plots are shown in (A). The average Igλ % ± SD is shown for WT (B) or VH125Tg (C) B6 (black) or NOD (white) mice, n ≥ 6 8–12 week old mice per group, n ≥ 2 experiments. * p < 0.05, ** p < 0.001, *** p < 0.001, two-tailed t-test.

Figure 6. Faulty central tolerance in NOD mice is ameliorated by treatment with insulin autoantigen-targeted mAb123

6–13 week old VH125Tg/Vκ125^SD/NOD mice were injected i.p. once with 100 µg anti-insulin mAb123 (black, n = 5) or isotype control mAb (white, n = 4). Bone marrow and spleens were harvested 2–7 d later and B cell subsets were identified as in Fig. 1D, further gated as non-insulin-binding or insulin-binding B cells. (A) Representative flow cytometry plots for isotype control mAb (Left) or mAb123 (Right). (B) Individual mice are plotted, bars indicate the average. (C) The IgM MFI of insulin-binding (Insulin+) B cells was divided by the IgM MFI of non-insulin-binding (Insulin−) B cells in the same mouse. A ratio < 1 indicates reduced surface IgM expression in the insulin-binding population (relative to edited B cells). Bars indicate the average Insulin⁺ / Insulin⁻ IgM ratio ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, two tailed t test.

Figure 7. Targeting insulin-occupied BCR increases RAG2-GFP expression and the frequency of non-insulin-binding B cells in developing subsets

VH125Tg/Vκ125^SD/RAG2-GFP/B6 7–17 week old mice were injected i.p. with 100 µg anti-insulin mAb123 (black, n = 7) or isotype control mAb (white, n = 6) i.p. every 2 days for 1 week. Bone marrow and spleens were freshly isolated 1 d after the final injection and B cell subsets were identified as in Fig. 1D. (A) The frequency of non-insulin-binding B cells in individual mice is plotted, bars indicate the average. (B–D) The average percentage of RAG2-GFP⁺ cells (B), the average RAG2-GFP MFI of RAG2-GFP⁺ cells (C), or the IgM MFI (D) of non-insulin-binding B cells (Left) or insulin-binding (Right) in each subset is shown, error bars indicate SD. * p < 0.05, ** p < 0.01, *** p < 0.001, two tailed t test.

Figure 8. F(ab’)₂123 depletes developing anti-insulin B cells

VH125Tg/NOD mice were injected once i.p. with 75 µg F(ab’)₂123 or 100 µg full-length mAb123 (to achieve ~ molar equivalence). After 1–2 d, mice were sacrificed and bone marrow, spleen, and pancreatic draining lymph nodes were freshly harvested. (A) Representative flow cytometry plots depict the frequency of insulin-binding B cells within the T1 B cell subset in untreated, F(ab’)₂123-treated, or mAb123-treated mice. (B) The percentages of insulin-binding B cells in each subset (identified as in Fig. 1D) are plotted for n = 5 mice per group; untreated (white diamonds), F(ab’)₂123- treated (grey circles), or mAb123-treated (black triangles), n = 3 experiments. (C) Flow cytometry was used to compare the IgM MFI of insulin-binding (Insulin+) vs. non-insulin-binding (Insulin−) B cells in subsets identified in (A). The insulin⁺ / insulin⁻ ratio was calculated as in Fig. 6C. The average ratio ± SD is shown for untreated (white diamonds), F(ab’)₂123-treated (grey circles), or mAb123-treated (black triangles) mice. Mice were 6–10 wk of age.