Priming and effector dependence on insulin B:9-23 peptide in NOD islet autoimmunity

Abstract

NOD mice with knockout of both native insulin genes and a mutated proinsulin transgene, alanine at position B16 in preproinsulin (B16:A-dKO mice), do not develop diabetes. Transplantation of NOD islets, but not bone marrow, expressing native insulin sequences (tyrosine at position B16) into B16:A-dKO mice rapidly restored development of insulin autoantibodies (IAAs) and insulitis, despite the recipients' pancreatic islets lacking native insulin sequences. Splenocytes from B16:A-dKO mice that received native insulin-positive islets induced diabetes when transferred into wild-type NOD/SCID or B16:A-dKO NOD/SCID mice. Splenocytes from mice immunized with native insulin B chain amino acids 9-23 (insulin B:9-23) peptide in CFA induced rapid diabetes upon transfer only in recipients expressing the native insulin B:9-23 sequence in their pancreata. Additionally, CD4(+) T cells from B16:A-dKO mice immunized with native insulin B:9-23 peptide promoted IAAs in NOD/SCID mice. These results indicate that the provision of native insulin B:9-23 sequences is sufficient to prime anti-insulin autoimmunity and that subsequent transfer of diabetes following peptide immunization requires native insulin B:9-23 expression in islets. Our findings demonstrate dependence on B16 alanine versus tyrosine of insulin B:9-23 for both the initial priming and the effector phase of NOD anti-islet autoimmunity.

Figure 1. Bone marrow transplant with native B16:Y insulin genes is not sufficient to restore insulin autoimmunity.

Four-week-old irradiated B16:A-dKO (A) or wild-type B16:Y mice (B) were transplanted (Tx) with bone marrow derived from 4-week-old wild-type NOD mice. Native insulin-positive B16:Y bone marrow cells did not induce IAAs in B16:A-dKO mice but did induce IAAs in B16:Y mice. Each line represents an individual recipient. The y axis represents the micro-IAA assay (mIAA) index in log scale.

Figure 2. Development of IAAs after B16:Y NOD/SCID islet transplant.

(A) B16:A-dKO mice, when transplanted with native B16:Y insulin NOD/SCID islets, developed IAAs. (B) Mice receiving B16:A-dKO islets did not express insulin antibodies after transplant, with the exception of 1 mouse. Each line represents an individual mouse. The y axis represents the micro-IAA assay index in log scale.

Figure 3. Native B16:Y islet transplants induce graft insulitis and severe transient insulitis in endogenous pancreatic islets.

(A–D) B16:Y NOD/SCID (A and C) and B16:A-dKO (B and D) islets were transplanted under the kidney capsule of B16:A-dKO mice. (A and B) H&E stain. (C and D) Insulin stain. Two weeks after transplant, the B16:Y NOD/SCID islet graft showed very little insulin staining and severe lymphocytic infiltration, whereas the B16:A-dKO islet graft was intact. (E and F) Endogenous pancreatic islets of B16:Y islet recipients (E), but not B16:A-dKO islet recipients (F), showed marked lymphocytic infiltration 18 weeks after islet transplant. Original magnification, ×100 (A, B, E, and F); ×200 (C and D). (G) More than 10 pancreatic islets from each B16:A-dKO mouse receiving B16:Y NOD/SCID or B16:A-dKO islet transplant were evaluated for lymphocytic infiltration less than 20 weeks or more than 30 weeks after transplant (n = 5–10). Pancreatic islets from an age-matched unmanipulated B16:A-dKO mouse were also evaluated. The y axis represents the mean ± SD of the insulitis score.

Figure 4. Summary of experiments with transfer of splenocytes into NOD/SCID recipients for mice immunized with islets (A) or insulin B:9–23 peptides (B).

DM, diabetes mellitus.

Figure 5. Rapid induction of diabetes with splenocytes from mice transplanted with B16:Y islets transferred into NOD/SCID mice with B16:Y.

Splenocytes from B16:A-dKO mice that received B16:Y NOD/SCID islets, B16:A-dKO islets, or no transplant (unmanipulated) were transferred into wild-type B16:Y NOD/SCID mice (A) or B16:A-dKO NOD/SCID mice (B). P < 0.01, B16:Y islets versus B16:A-dKO islets in B16:Y NOD/SCID recipients.

Figure 6. Development of IAAs but not insulitis by immunization with insulin B:9–23 peptide.

(A–D) Serum from B16:A-dKO mice immunized with native B16:Y insulin B:9–23 peptide (A), B16:A-dKO mice immunized with altered B16:A insulin B:9–23 peptide (B), wild-type B16:Y NOD mice immunized with native B16:Y insulin B:9–23 peptide (C), and wild-type B16:Y NOD/SCID mice that received splenocytes from insulin B:9–23 peptide–immunized B16:A-dKO mice (D) was incubated with I¹²⁵-insulin in the presence of tetanus toxin peptide (TT), native B16:Y insulin B:9–23 peptide, B16:A insulin B:9–23 peptide, or human insulin. Each line represents an individual mouse. (E) More than 10 pancreatic islets from each B16:A-dKO mouse immunized with B16:Y insulin B:9–23 peptide (n = 10), B16:A insulin B:9–23 peptide (n = 4), or PBS (n = 10) in CFA were evaluated for lymphocytic infiltration. The y axis represents the mean ± SD of the insulitis score.

Figure 7. Only wild-type B16:Y NOD/SCID mice receiving splenocytes of B16:A-dKO mice immunized with native B16:Y insulin B:9–23 peptide rapidly produce IAAs.

Splenocytes from B16:A-dKO mice immunized with native insulin B:9–23 peptide (A and B) or mutated B16:A insulin B:9–23 peptide (C and D) were transferred to wild-type B16:Y NOD/SCID (A and C) or B16:A-dKO NOD/SCID mice (B and D). IAAs were measured weekly after splenocyte transfer. Each line represents an individual mouse.

Figure 8. Development of diabetes after splenocyte transfer from insulin B:9–23 peptide–immunized B16:A-dKO mice.

Splenocytes from mice immunized with native B16:Y insulin B:9–23 peptide, but not with mutated B16:A insulin B:9–23 peptide, rapidly transferred diabetes to wild-type B16:Y NOD/SCID mice. B16:A-dKO NOD/SCID mice did not develop diabetes following transfer of splenocytes from mice immunized with either native B16:Y insulin B:9–23 or mutated B16:A insulin B:9–23 peptides.

Figure 9. Development of IAAs in wild-type B16:Y NOD/SCID mice after splenocyte transfer.

(A) Splenic CD4⁺ T cells from B16:A-dKO mice immunized with native B16:Y insulin B:9–23 peptide were transferred to wild-type B16:Y NOD/SCID mice along with non-CD4⁺ splenocytes from unmanipulated double insulin-knockout mice. (B) Splenic CD4⁺ T cells from unmanipulated mice were transferred to wild-type B16:Y NOD/SCID mice along with a non-CD4⁺ splenocyte population from mice immunized with B16:Y insulin B:9–23 peptide. Each line represents an individual mouse.

Figure 10. Provision of the native B16:Y insulin B:9–23 sequence by transgenesis induces IAAs and insulitis.

(A) B16:A-dKO and B16:Y-dKO mice were measured for the development of IAAs every 2–3 weeks between 4 and 30 weeks of age. Each symbol represents the peak level of mIAA index for individual mice. B16:Y-dKO mice developed IAAs (P < 0.01 versus B16:A-dKO). (B) Insulitis scoring of B16:A-dKO mice, B16:Y-dKO mice, and wild-type NOD mice between 10 and 22 weeks of age. B16:Y-dKO mice developed insulitis significantly more severe than did B16:A-dKO mice (P < 0.01) and as severely as did wild-type NOD mice. (C and D) Pancreatic histology (H&E; original magnification, ×100) of B16:A-dKO (C) and B16:Y-dKO (D) mice.