An insulin-IAPP hybrid peptide is an endogenous antigen for CD4 T cells in the non-obese diabetic mouse

Abstract

BDC-6.9, a diabetogenic CD4 T cell clone isolated from a non-obese diabetic (NOD) mouse, responds to pancreatic islet cells from NOD but not BALB/c mice. We recently reported that a hybrid insulin peptide (HIP), 6.9HIP, formed by linkage of an insulin C-peptide fragment and a fragment of islet amyloid polypeptide (IAPP), is the antigen for BDC-6.9. We report here that the core 12-mer peptide from 6.9HIP, centered on the hybrid peptide junction, is also highly antigenic for BDC-6.9. In agreement with the observation that BALB/c islet cells fail to stimulate the T cell clone, a single amino acid difference in the BALB/c IAPP sequence renders the BALB/c version of the HIP only weakly antigenic. Mutant peptide analysis indicates that each parent molecule-insulin C-peptide and IAPP-donates residues critical for antigenicity. Through mass spectrometric analysis, we determine the distribution of naturally occurring 6.9HIP across chromatographic fractions of proteins from pancreatic beta cells. This distribution closely matches the profile of the T cell response to the fractions, confirming that 6.9HIP is the endogenous islet antigen for the clone. Using a new MHC II tetramer reagent, 6.9HIP-tet, we show that T cells specific for the 6.9HIP peptide are prevalent in the pancreas of diabetic NOD mice. Further study of HIPs and HIP-reactive T cells could yield valuable insight into key factors driving progression to diabetes and thereby inform efforts to prevent or reverse this disease.

Keywords: Autoimmune type 1 diabetes; Hybrid insulin peptides (HIPs); Non-obese diabetic (NOD) mouse; Peptide fusion; Tetramer.

Figure 1. A core 12-mer peptide of 6.9HIP containing the NOD (but not BALB/c) IAPP sequence is highly antigenic for BDC-6.9

(A) T cell clones were cultured with APCs and 1×10⁴ dissociated islet cells from NOD, BALB/c, or NOD.IAPP−/− (IAPP−/−) mice; IFN-γ production was measured by ELISA as a readout of T cell activation. The average +/− S.D. of triplicate wells is reported. Results are representative of two independent experiments. (B-D) T cell clones were cultured with APCs and varying concentrations of the insulin 2 B chain peptide B:9-23, an unlinked combination of the insulin 2 C-peptide fragment C:1-26 and the NOD variant of IAPP2, 6.9HIP (EVEDPQVAQLELGGGPGAGDLQTLAL-NAARDPNRESLDFLLV), 6.9HIP:core (LQTLAL-NAARDP), or 6.9HIP:R→G (LQTLAL-NAAGDP). IFN-γ production was measured by ELISA. Results are representative of three independent experiments. (E) Splenocytes from BDC-6.9 TCR-Tg mice were labeled with CFSE and then cultured with varying concentrations of 6.9HIP:core or 6.9HIP:R→G. After four days, cells were analyzed by flow cytometry for CFSE dilution as an indicator of proliferation. The percentage of the original population that was induced to proliferate was calculated using the FlowJo (Tree Star) proliferation platform and is indicated in the figure. Results are representative of two independent experiments.

Figure 2. Amino acid residues in both the insulin and IAPP regions of 6.9HIP are critical for antigenicity

BDC-6.9 was cultured with APCs and varying concentrations of truncated or mutated 6.9HIP peptides to define a minimal epitope and residues critical for antigenicity. IFN-γ production was measured by ELISA. (A) Peptides truncated at the N-terminus. (B) Peptides truncated at the C-terminus. (C) Peptides with substitutions at critical residues. Results in (A-C) are from the same assay plate; accordingly, values for the LQTLAL-NAARDP peptide are identical in all three figures. Results are representative of three independent experiments. The peptide LQTLAL-NAARAP was only tested in two experiments.

Figure 3. The abundance of the insulin-IAPP hybrid in chromatographic fractions of beta cell proteins correlates with the magnitude of the BDC-9.3 response to the fractions

(A) Secretory granule proteins, isolated from pancreatic beta cell tumors of NOD.RIP-TAg mice, were fractionated by SEC. Fractions were then tested for antigenicity with BDC-9.3. In addition to measuring IFN-γ production, the absorbance at 280 nm was measured for each fraction as an indicator of total protein content. (B) Antigenic SEC fractions were pooled and further fractionated by reverse-phase HPLC; HPLC fractions were then tested for antigenicity with BDC-9.3. (C) Following digestion with the protease AspN, HPLC fractions were analyzed by mass spectrometry. Presence of the peptide DLQTLAL-NAAR in antigenic fractions was confirmed by MS/MS analysis. Samples were then run in MS mode, and the spectral intensity of the parent ion corresponding to the peptide DLQTLAL-NAAR was analyzed using MassProfiler Professional software (Agilent). Reported spectral intensities represent the mean of two duplicate MS runs of the same biological sample. The IFN-γ response to each fraction, as shown in (B), is also displayed here for ease of comparison. Results for (A-C) are representative of two independent experiments.

Figure 4. CD4 T cells specific for 6.9HIP are prevalent in the pancreas of diabetic NOD mice

(A) The control clones PD12-4.4 (insulin B:9-23-reactive), BDC-6.9, and BDC-9.3 were stained with either 6.9HIP-tet or the control insulin tetramer insp8G-tet. Gated on CD4⁺ cells. (B,C) Cells from the (B) pancreas (n=15) and (C) spleen (n=15) of diabetic NOD mice were stained with antibodies and tetramer (HEL-tet, insp8G-tet, or 6.9HIP-tet) and analyzed by flow cytometry. Representative flow plots and summary statistics are shown for each tissue. Data is pooled from eight independent experiments. (D,E) The (D) pancreas (n = 5) and (E) spleen (n = 5) of diabetic NOD.IAPP−/− mice were also analyzed. NOD.IAPP−/− data is pooled from three independent experiments. NOD data in (D) and (E) is the same as from (B) and (C), respectively. (F,G) Cells from the (F) pancreas (n = 7) and (G) spleen (n = 7) of non-diabetic 12-13-week old NOD mice were stained with antibodies and tetramer and analyzed by flow cytometry. Data is pooled from two independent experiments. (B-G) are gated on live CD4⁺CD45⁺CD8/CD11b/CD11c/CD19⁻ cells.