Conserved T cell receptor alpha-chain induces insulin autoantibodies

Abstract

A fundamental question is what are the molecular determinants that lead to spontaneous preferential targeting of specific autoantigens in autoimmune diseases, such as the insulin B:9-23 peptide sequence in type 1 diabetes. Anti-insulin B:9-23 T cell clones isolated from prediabetic NOD islets have a conserved Valpha-segment/Jalpha-segment, but no conservation of the alpha-chain N region and no conservation of the Vbeta-chain. Here, we show that the conserved T cell receptor alpha-chain generates insulin autoantibodies when transgenically or retrogenically introduced into mice without its corresponding Vbeta. We suggest that a major part of the mystery as to why islet autoimmunity develops relates to recognition of a primary insulin peptide by a conserved alpha chain T cell receptor.

Fig. 1.

Serum anti-insulin autoantibody levels. (A–D) Mice with the 12-4.1 α-chain transgene. (E–H) Mice lacking the 12-4.1 α-chain. (A) BDC 12-4.1 TCR α+β−, Cα−/−, n = 37. (B) BDC 12-4.1 TCR α+β+, Cα−/−, n = 19. (C) BDC 12–4.1 TCR α+β−, Cα+/−, n = 16. (D) BDC 12-4.1 TCR α+β+, Cα+/−, n = 16. (E) BDC 12-4.1 TCR α−β−, Cα−/−, n = 17. (F) BDC 12-4.1 TCR α−β+, Cα−/−, n = 10. (G) BDC 12-4.1 TCR α−β−, Cα+/−, n = 10. (H) BDC 12-4.1 TCR α−β+, Cα+/−, n = 14. Lines connect results for individual mice. Upper-right number is the IAA positive mouse number out of the total mouse number. mIAA, micro insulin autoantibodies.

Fig. 2.

Flow cytometry of peripheral blood mononuclear cells with FITC-conjugated Vβ 2 and APC-conjugated CD4 antibodies (Right) and FITC-conjugated Vβ 8.1–8.2 and APC-conjugated CD4 (Left). One of three different representative flow cytometry analyses was shown. Upper-right quadrant number shown represents percentage Vβ (2 or 8.1–8.2) of CD3-gated cells.

Fig. 3.

Dose–response to B:9–23 peptide, expressed as IFN-γ spots per million by ELISPOT assay of splenocytes. We tested three mice with only BDC 12-4.1 TCR α (Cα−/−), two mice with only BDC 12-4.1 TCR β (Cα +/−), and four wild-type NOD mice. Graphs represent typical results from one assay showing the number of IFN-γ-producing cells in response to a different dose of B:9–23 peptide and tetanus toxin (TT) peptide. All mice were immunized with B:9–23(100 μg per mouse) in complete Freund's adjuvant (CFA) by s.c. route 2 weeks before the assay. Splenocytes were stimulated by different concentrations of B:9–23 peptide (Left) or TT peptide (Right). Each line represents an individual mouse.

Fig. 4.

Histology of transgenic mice showing islets stained for glucagon. We evaluated 15 mice (315 total islets, age range: 5–30 weeks of age) with only the 12-4.1 α-chain (Cα−/−); eight mice (89 total islets, age range: 8–29 weeks of age) with the 12-4.1 α-chain (Cα+/−); five mice (103 total islets, age range: 16–29 weeks of age) without both 12-4.1 α- and β-chain (Cα+/−); five mice (69 total islets, age range: 8–23 weeks of age) with both 12-4.1 α- and β-chain (Cα−/−); six mice (74 total islets, age range: 7–25 weeks of age) with both α- and β-chain (Cα+/−); seven mice (139 total islets, age range: 9–29 weeks of age) with 12-4.1 β-chain (Cα+/−). (A) BDC 12-4.1 TCR α+β−, Cα−/−, evaluated at 23 weeks. (B) BDC 12-4.1 TCR α+β−, Cα+/−, evaluated at 20 weeks. (C) BDC 12-4.1 TCR α−β−, Cα+/−, evaluated at 27 weeks. (D) BDC 12-4.1 TCR α+β+, Cα−/−, evaluated at 23 weeks. (E) BDC 12-4.1 TCR α+β+, Cα+/−, evaluated at 13 weeks. (F) BDC 12-4.1 TCR α-β+, Cα+/−, evaluated at 29 weeks.

Fig. 5.

Adoptive transfer of diabetes. Diabetic splenocytes (1.5 × 10⁷ per recipient) from wild-type NOD mice were transferred into NOD scid mice (8–10 weeks of age) alone (filled circles, n = 6) or together with a double number of splenocytes from the mice with only BDC 12-4.1 TCR α-chain (Cα−/−) (30–52 weeks of age) (filled squares, n = 8), or young NOD mice (8–10 weeks of age) (filled triangles, n = 10).

Fig. 6.

Serum anti-insulin autoantibody levels of 12-4.4 α-chain retrogenic NOD mice. Three of six NOD scid mice retrogenic for the 12-4.4 α-chain in Cα−/− bone marrow cells developed insulin autoantibodies. Lines connect results for individual mice. mIAA, micro insulin autoantibodies (data for two mIAA negative mice overlap).