Calcitonin gene-related peptide is a potential autoantigen for CD4 T cells in type 1 diabetes

Abstract

The calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide with critical roles in the development of peripheral sensitization and pain. One of the CGRP family peptides, islet amyloid polypeptide (IAPP), is an important autoantigen in type 1 diabetes. Due to the high structural and chemical similarity between CGRP and IAPP, we expected that the CGRP peptide could be recognized by IAPP-specific CD4 T cells. However, there was no cross-reactivity between the CGRP peptide and the diabetogenic IAPP-reactive T cells. A set of CGRP-specific CD4 T cells was isolated from non-obese diabetic (NOD) mice. The T-cell receptor (TCR) variable regions of both α and β chains were highly skewed towards TRAV13 and TRBV13, respectively. The clonal expansion of T cells suggested that the presence of activated T cells responded to CGRP stimulation. None of the CGRP-specific CD4 T cells were able to be activated by the IAPP peptide. This established that CGRP-reactive CD4 T cells are a unique type of autoantigen-specific T cells in NOD mice. Using IA^g7-CGRP tetramers, we found that CGRP-specific T cells were present in the pancreas of both prediabetic and diabetic NOD mice. The percentages of CGRP-reactive T cells in the pancreas of NOD mice were correlated to the diabetic progression. We showed that the human CGRP peptide presented by IA^g7 elicited strong CGRP-specific T-cell responses. These findings suggested that CGRP is a potential autoantigen for CD4 T cells in NOD mice and probably in humans. The CGRP-specific CD4 T cells could be a unique marker for type 1 diabetes. Given the ubiquity of CGRP in nervous systems, it could potentially play an important role in diabetic neuropathy.

Keywords: CD4 T cells; CGRP; MHC; thiol regulation; type 1 diabetes (T1D).

Figure 1

Amino acid sequence alignment of the CGRP family proteins in humans (h) and mice (m) using PRALINE.

Figure 2

The cross-reaction between the CGRP peptide and IAPP reactive T cell, BDC5.2.9. (A) M12C3G7 incubated with SG20 or KS20 peptides stimulated BDC5.2.9 at different concentrations, 0, 0.4, 4 and 40μg/mL. 24 hours later, supernatants were collected for HT-2 assay. Results are the means ± SEM of triplicate wells. (B) ICAM+B7⁺ SF9 insect cells expressing IA^g7-WT KS20, IA^g7-KS20△1-5, or IA^g7-SG20 were used as APCs to stimulate BDC5.2.9 T cells. Results are the means ± SEM of triplicate wells. (C) IA^g7-SG20 tetramers were used to stain BDC5.2.9. IA^g7-KS20 was used as a positive control and IA^g7-KS20△1-5, as a negative control. *P< 0.05, **P< 0.01, ***P< 0.001.

Figure 3

The essential amino acids of KS20 peptide for the activation of T cell BDC5.2.9 compared with the SG20 peptide. (A) Amino acid sequence alignment of mouse CGRP and IAPP segments studied here. (B) mAb LD96.24 bound weakly to IA^g7-SG20 molecules. The binding between mAb LD96.24 and IAg7-SG20 molecules was measured by ELISA. Results are the means ± SEM of triplicate wells. (C) Viruses encoding WT KS20, WT SG20, KS20 mutants, and SG20 mutants were used to infect mouse ICAM⁺B7⁺ SF9 insect cells. KS20△1-5 and non-infected wells were used as controls. 3 days later, these insect cells were used as APCs to stimulate BDC5.2.9 T cells. Results are the means ± SEM of triplicate wells. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

IA^g7-SG20 tet⁺ cells were present in the pancreas of prediabetic and diabetic NOD mice. Single-cell suspensions were prepared from the whole pancreas of each prediabetic (n=5; 12 weeks) or diabetic NOD mice (n=5; 12-23 weeks). Female BALB/c mice (n=5; 12-14 weeks) were used as the negative control. The cells were stained with IA^g7-SG20 tetramers, anti-B220, anti-F4/80, anti-CD44, anti-CD4, and anti-CD8. For tetramer analysis, cells were pregated on live, B220−, F4/80−, CD8−, CD4⁺, and CD44^high cells. (A) Representative scatter diagram of IA^g7-SG20 tet⁺CD44⁺ cells in different groups. (B) The percentages of IAg7-SG20 tet+CD44+ in CD4+ T cells of each group. Each symbol represented an individual mouse. Results are the means ± SEM. *P < 0.05, **P < 0.01. ***P < 0.001.

Figure 5

CGRP-specific T cell hybridomas did not cross-react with the KS20 peptide. 20 CGRP-specific T cell hybridomas were generated as described in materials and methods. (A) IA ^g7 -SG20S17E tetramers and IA ^g7 -KS20V17E tetramers were used to stain these CGRP reactive T cell hybridomas and unstained T cells as the negative control. The table showed the tetramer binding with the CGRP reactive T cell hybridomas. “-” represents no binding; “+” represents binding significantly. (B) ICAM ⁺ B7 ⁺ SF9 insect cells expressing IA ^g7 -WT SG20 or IA ^g7 -SG20S17E were used as APCs to stimulate three representative CGRP-reactive T cell hybridomas 22, 37, and 155. Results are the means ± SEM of triplicate wells. (C) M12C3 ^G7 cells incubated with different concentrations of SG20S17E peptide stimulated the CGRP reactive T cell hybridomas. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

TCR V gene usages of CGRP specific T cell hybridomas. (A) Pie charts showed the frequency of TRAV (left) and TRBV (right) gene segment usage of 13 CGRP specific T cell hybridomas. (B) The paired CDR1, CDR2, and CDR3 amino acid sequences of TRAV and TRBV of CGRP specific T cell hybridomas.

Figure 7

Human AG20 peptide cross-reacted with CGRP reactive T cell hybridomas. (A) M12C3^g7 cells incubated with different concentrations of human AG20S17E peptide stimulated mouse CGRP reactive T cell hybridomas 22, 37, and 155. (B) The three representative CGRP reactive T cell hybridomas were stimulated with M12C3-DQ8 incubated with 1 μg/mL hAG20P17E or mSG20P17E and M12C3 cells were used as the negative control. Results are the means ± SEM of triplicate wells. *P P < 0.05, **P < 0.01, ***P < 0.001.