HLA-DQ2 and -DQ8 signatures of gluten T cell epitopes in celiac disease

Abstract

Celiac disease is associated with HLA-DQ2 and, to a lesser extent, HLA-DQ8. Type 1 diabetes is associated with the same DQ molecules in the opposite order and with possible involvement of trans-encoded DQ heterodimers. T cells that are reactive with gluten peptides deamidated by transglutaminase 2 and invariably restricted by DQ2 or DQ8 can be isolated from celiac lesions. We used intestinal T cells from celiac patients to map DQ2 and DQ8 epitopes within 2 representative gluten proteins, alpha-gliadin AJ133612 and gamma-gliadin M36999. For alpha-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of 2 separate regions. For gamma-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of the same region. Some gamma-gliadin peptides were recognized by T cells in the context of DQ2 or DQ8 when bound in exactly the same registers, but with different requirements for deamidation; deamidation at peptide position 4 (P4) was important for DQ2-restricted T cells, whereas deamidation at P1 and/or P9 was important for DQ8-restricted T cells. Peptides combining the DQ2 and DQ8 signatures could be presented by DQ2, DQ8, and trans-encoded DQ heterodimers. Our findings shed light on the basis for the HLA associations in celiac disease and type 1 diabetes.

Figure 1. Testing of T cell lines (TCL) isolated from biopsy specimens of a DQ2⁺ DQ8⁺ patient (CD465) stimulated in parallel with (A) chymotrypsin-treated gluten, (B) chymotrypsin-treated recombinant AJ133612 α-gliadin, and (C) overlapping AJ133612 α-gliadin peptides against the whole panel of TG2-treated AJ133612 α-gliadin peptides (1409–1457).

Peptides were tested at 10 μM, and CD114 (DQA1*0501/DQB1*0201) B lymphoblastoid cells were used as APCs. TG2-treated gluten and TG2-treated recombinant AJ133612 α-gliadin were used as positive controls. Responses are given as the stimulation index. α-GPs, α-gliadin peptides; r–α-gli, recombinant α-gliadin.

Figure 2. T cell lines derived from biopsy specimens of 3 different patients stimulated with chymotrypsin-treated recombinant AJ133612 α-gliadin were tested against the panel of TG2-treated AJ133612 α-gliadin peptides as in Figure 1.

Patient CD506 is DQ2⁺DQ8^– (A), patient CD559 is DQ2⁺DQ8⁺ (B), and patient CD489 is DQ2^–DQ8⁺ (C). TG2-treated gluten and TG2-treated recombinant AJ133612 α-gliadin were used as positive controls. Black bars (CD114 cells as APCs) indicate DQ2-restricted T cell responses, and white bars (9092 cells as APCs) indicate DQ8-restricted responses.

Figure 3. Testing for T cell recognition of variants of the²²⁸ SGQGSFQPSQQ NPQ²⁴¹ peptide of AJ133612 α-gliadin.

The peptide harbors the DQ8–α-I epitope. A peptide with the native sequence and peptides with Q→E substitutions in P1 (position 230), P9 (position 238), and P1/P9 were tested for their ability to stimulate 2 DQ8-restricted T cell clones, TCC489.2.1.4 (A) and TCC360-HTLR8 (B), which were derived from 2 different DQ2^–DQ8⁺ patients. Responses are shown as cpm × 10³. TG2-treated, native sequence treated with TG2, used as a control.

Figure 4. Testing of T cell lines isolated from biopsy specimens of a DQ2⁺ DQ8⁺ patient (CD548) stimulated in parallel with chymotrypsin-treated gluten (A ) and overlapping M36999 γ-gliadin peptides (B ) against the panel of TG2-treated M36999 peptides (1369–1527).

Peptides were tested at 10 μM. TG2-treated gluten was used as a positive control. Black (CD114 cells as APCs) and white bars (9092 cells as APCs) indicate DQ2-restricted and DQ8-restricted T cell responses, respectively.

Figure 5. T cell recognition of variants of the⁶¹ QFPQTQQPQQPFPQ PQQTFP⁸⁰ peptide of M36999 γ-gliadin.

The peptide harbors the DQ2–γ-VII and DQ8–γ-I epitopes. A peptide with the native sequence and shorter peptides (aa 63–76) with Q→E substitutions in P1 (position 66), P4 (position 69), and P1/P4 were tested for their ability to stimulate 2 DQ8-restricted T cell clones (A and B) and 1 DQ2-restricted T cell clone (C). TCC544.1.1.2 (TCR Vβ17) and TCC544.1.3.2 (TCR Vβ5.1) originate from a DQ2^–DQ8⁺ CD patient. TCC387.19 originates from a DQ2⁺DQ8^– patient. Responses are shown as cpm × 103.

Figure 6. T cell recognition of variants of the⁶⁶ FPQQPQQPYPQQPQQ⁸⁰ peptide of AJ416339 γ-gliadin.

The peptide harbors the DQ2–γ-III and DQ8–γ-I epitopes. A peptide with the native sequence and peptides with Q→E substitutions in positions P1 (position 68), P4 (position 71), P9 (position 76), and P1/P9 were tested for their ability to stimulate 2 DQ8-restricted T cell clones (A and B) and 2 DQ2-restricted T cell clones (C and D). TCC430.1.145 (TCR Vβ2) and TCC430.1.134 (TCR Vβ2) originate from a DQ2⁺DQ8^– patient. Responses are given in cpm × 103.

Figure 7. T cell recognition of peptides presented bycis- andtrans- encoded DQ heterodimers.

(A) Recognition of variants of the ⁶³PQTQQPQQPFPQPQ⁷⁶ peptide (described in Figure 5) by the T cell clone TCC387.19. (B) Recognition of variants of the ⁶⁶FPQQPQQPYPQQPQQ⁸⁰ peptide (described in Figure 6) by the T cell clone TCC544.1.1.2.