PMC

Insulin B:9–23 mimotopes define two types of insulin-reactive CD4⁺ T cells. (A) Various natural and mimotope variants of the insulin B:9–23 peptide are shown. The amino acid mutations introduced into the mimotope peptides are highlighted in yellow. The red amino acids are predicted to fill the four anchor positions in the IA^g7 groove, when the peptides are bound in a particular register. (B) As described in , seven NOD insulin B:9–23 reactive T-cell hybridomas were tested for their responses to various concentrations of the three insulin B:9–23 peptide mimotopes listed in A. The NOD chromogranin A-specific T-cell hybridoma, BDC-2.5, was used as a negative control. The peptides were presented by paraformaldehyde-fixed, M12.C3 lymphoma B cells transfected with IA^g7. The averaged results from three experiments are shown as the amount of IL-2 produced in 24 h ± SEM vs. the peptide concentration.

Fluorescent tetramers produced with register 3 trapped peptides detect both types of T-cell hybridomas. The six constructs shown in were altered to replace the baculovirus gp64 transmembrane region and cytoplasmic tail with a peptide substrate for the BirA biotinylation enzyme. Soluble biotinylated proteins were prepared and incorporated into PE-streptavidin tetramers as described in . (A) Examples of insulin-IA^g7 tetramer staining of a type I (AS91, Left) and type II (PCR1–10, Right) T-cell hybridoma are shown using the p8G (green) and p8E (blue) tetramer trapped in register 3 by a p6 to IA^g7 disulfide. Staining with a negative control tetramer (CLIP-IA^b) is also shown (gray). (B) The seven insulin reactive T-cell hybridomas in were stained with all six of the insulin peptide-IA^g7 PE-streptavidin tetramers. The net mean fluorescent intensity (MFI) of staining was calculated as the MFI for the insulin-IA^g7 tetramer minus the MFI for the negative control tetramer (CLIP-IA^b). The bars show the average (± SEM) net MFI from three to five independent experiments expressed as the percentage of the net MFI obtained with a high-affinity APC-labeled 57–597 TCR Cβ Mab.

IA^g7-insulin tetramers detect insulin-reactive CD4⁺ T cells in the pancreases of prediabetic NOD mice. Two mixtures of fluorescent IA^g7 tetramers were prepared: (i) an equimolar mixture of two PE-streptavidin insulin tetramers, one containing p8G with no disulfide and the second containing p8E with a p6 to IA^g7 disulfide and an AF647-streptavidin tetramer containing a mimotope (SRLGLWSRMD) for NOD chromogranin A (CHGA) reactive CD4⁺ T cells; and (ii) an equimolar mixture of a PE-streptavidin tetramer containing the IA^g7 presented dominant epitope (MKRHGLDNYRGY) from hen egg lysozyme (HEL) and the AF647 version of the same HEL tetramer. Pooled pancreatic islet cells were isolated from 8-wk-old prediabetic NOD mice and stained with the two tetramer mixtures. The cells were stained as well with 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) Zebra contour plots showing 2D simultaneous staining with the insulin and CHGA tetramer (Left) or with the two HEL tetramers (Right). The percentage of cell in each quadrant is shown in B. One-dimensional histogram overlay of the PE-labeled insulin (red) and HEL(blue) tetramers (Left) or AF647-labeled CHGA (red) and HEL (blue) tetramers (Right). The percentage of CD4⁺ and CD44^high T cells staining with the insulin or CHGA tetramers was calculated from the difference between the two histograms.

Register trapping of the insulin peptides confirms their binding in register 3. (A) Schematic representations of six versions of the insulin peptide expressed in baculovirus linked to a cell surface expressed version of IA^g7 via a linker to the N terminus of the β-chain. The amino acid mutations introduced into the peptide or IA^g7 are highlighted in yellow. The red amino acids are predicted to fill the four anchor positions in the IA^g7 groove. See text for further description. (B) SF9 insect cells bearing mouse ICAM and B7 were infected with virus encoding each of the six constructs. Three days after infection these cells were used as APCs for the seven type I and type II insulin-specific T-cell hybridomas in . IL-2 production was assayed at 24 h. Stimulation of the hybridomas with an immobilized TCR Cβ-specifc Mab was used as a positive control. Each bar is the average of the results of three independent experiments ± SEM.