Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes - PubMed (original) (raw)

. 2003 Jul 8;100(14):8384-8.

doi: 10.1073/pnas.0932778100. Epub 2003 Jun 18.

Anne M Evans, Bingye Han, Toshiyuki Takaki, Yuliya Vinnitskaya, Jennifer A Caldwell, David V Serreze, Jeffrey Shabanowitz, Donald F Hunt, Stanley G Nathenson, Pere Santamaria, Teresa P DiLorenzo

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Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes

Scott M Lieberman et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Type 1 diabetes is an autoimmune disease in which autoreactive T cells attack and destroy the insulin-producing pancreatic beta cells. CD8+ T cells are essential for this beta cell destruction, yet their specific antigenic targets are largely unknown. Here, we reveal that the autoantigen targeted by a prevalent population of pathogenic CD8+ T cells in nonobese diabetic mice is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Through tetramer technology, IGRP-reactive T cells are readily detected in islets and peripheral blood directly ex vivo. The human IGRP gene maps to a diabetes susceptibility locus, suggesting that IGRP also may be an antigen for pathogenic T cells in human type 1 diabetes and, thus, a new, potential target for diagnostic and therapeutic approaches.

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Figures

Fig. 1.

Fig. 1.

Identification of IGRP206–214 as the β cell peptide recognized by the pathogenic T cell clone 8.3. (A–C) Epitope reconstitution activity of first-dimension (A), second-dimension (B), and third-dimension (C) HPLC fractions of H-2Kd-eluted NIT-1 peptides. (D) Determination of candidate peptides by correlation of ion abundance curves (solid lines; plotted on the left and bottom axes) with epitope reconstitution activity of third-dimension HPLC fractions (broken line, ⋄; plotted on the right and top axes). Peptide m/z values are 548.845 (red diamonds), 513.285 (▴), 509.78 (+), 555.805 (-), and 555.835 (▪). (E) Collision-activated dissociation mass spectrum of candidate peptide (M + 2H)+2 ion with monoisotopic m/z of 548.845. X represents I or L. Ions observed in the spectrum are underlined; the b ions originate from the N terminus of the peptide, and the y ions originate from the C terminus. (F) Response of 8.3 CTL toward RMA-S/Kdtarget cells pulsed with varying concentrations of VYLKTNVFL (▴), IYQKAFDLI (▪), NRP-V7 (⋄), or NRP-A7 (+). (G) Response of 8.3 CTL toward COS-7 cells cotransfected (solid lines) with varying concentrations of an IGRP expression construct (▴), or empty vector (▪), together with 10 ng/ml H-2Kd expression construct, or toward separate cultures of COS-7 cells transfected with the H-2Kdconstruct alone and pulsed with varying concentrations of IGRP206–214 peptide (broken line, ⋄). T cell response was measured as IFN-γ release by ELISA and is presented as absorbance at 405 nm (A405).

Fig. 2.

Fig. 2.

Multiple early insulitic T cell clones recognize IGRP206–214.58α-β-transfectants expressing the indicated TCRs were cultured with RMA-S/Kd cells pulsed with 1.0 (solid bars), 0.1 (open bars), or 0.01 μM (striped bars) of the indicated peptides, and IL-2 release was measured by ELISA. The partial TCRα and TCRβ chain sequences for 8.3 and for the early insulitic T cell clones AI12.B1.3 and AI15.F5 have been reported (3, 10, 13). The AI4, AI12.B1.1, AI12.B1.2, and AI15.A10 lines, all of which express non-Vα17 TCRα chains, did not respond to any of the peptides tested, although they were capable of signaling through the transfected TCR as evidenced by their release of IL-2 in response to plate-bound anti-CD3ε (data not shown). IGRP, IGRP206–214; INS, INS B15–23; FW, framework residues; CDR, complementarity-determining region.

Fig. 3.

Fig. 3.

IGRP206–214 does not demonstrate poor peptide binding to H-2Kd. RMA-S/Kd cells were pulsed with 1.0 (solid bars), 0.1 (open bars), or 0.01 μM (striped bars) of the indicated peptides, stained with an anti-H-2Kd antibody, and analyzed by flow cytometry. IGRP, IGRP206–214; INS, INS B15–23; MFI, mean fluorescence intensity.

Fig. 4.

Fig. 4.

IGRP206–214-reactive T cells are readily detected in islets and peripheral blood of NOD mice directly ex vivo. Cells from islets or peripheral blood of 9- or 20-week-old nondiabetic NOD mice were stained with anti-CD8 antibody and the indicated peptide/H-2Kdtetramers. (Left) Representative tetramer staining patterns of samples from 20-week-old mice gated on the CD8+ population. Numbers indicate percentage of tetramer-positive cells within the CD8+population. (Right) The percentage of IGRP (solid bars) or NRP-V7 (open bars) tetramer-positive cells within the CD8+ population for five individual mice per age group. TUM/H-2Kd tetramers stained 0% of cells in each group. IGRP, IGRP206–214.

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