Production of a panel of recombinant gliadins for the characterisation of T cell reactivity in coeliac disease

Abstract

Background/aims: Coeliac disease is a chronic intestinal disorder most probably caused by an abnormal immune reaction to wheat gliadin. The identification of the HLA-DQ2 and HLA-DQ8 as the molecules responsible for the HLA association in coeliac disease strongly implicates a role for CD4 T cells in disease pathogenesis. Indeed, CD4 T cells specific for gliadin have been isolated from the small intestine of patients with coeliac disease. However, identification of T cell epitopes within gliadin has been hampered by the heterogeneous nature of the gliadin antigen. To aid the characterisation of gliadin T cell epitopes, multiple recombinant gliadins have been produced from a commercial Nordic wheat cultivar.

Methods: The alpha-gliadin and gamma-gliadin genes were amplified by polymerase chain reaction from cDNA and genomic DNA, cloned into a pET expression vector, and sequenced. Genes encoding mature gliadins were expressed in Escherichia coli and tested for recognition by T cells.

Results: In total, 16 alpha-gliadin genes with complete open reading frames were sequenced. These genes encoded 11 distinct gliadin proteins, only one of which was found in the Swiss-Prot database. Expression of these gliadin genes produced a panel of recombinant alpha-gliadin proteins of purity suitable for use as an antigen for T cell stimulation.

Conclusion: This study provides an insight into the complexity of the gliadin antigen present in a wheat strain and has defined a panel of pure gliadin antigens that should prove invaluable for the future mapping of epitopes recognised by intestinal T cells in coeliac disease.

Figure 1

Recognition of pepsin treated pW1621 recombinant gliadin by the T cell transfectant 60.6. Pepsin-trypsin treated crude gliadin from Sigma (PT gliadin) was used as positive control. T cell activation was measured as interleukin-2 release and is given as arbitrary europium (Eu) counts. T+APC, T cells plus antigen presenting cells.

Figure 2

Amino acid sequence alignment of the α-gliadin clones. The EMBL accession numbers of the DNA sequence and the clone names are indicated. A consensus amino acid sequence is given above the alignment. The N-terminal M and the C-terminal Y and R are non-gliadin sequences that are introduced as part of the expression vector. The sequences of the six N-terminal residues and the eight C-terminal residues are determined by the primers used for the PCR amplification.

Figure 3

SDS/PAGE of the recombinant gliadins, before and after purification. Lane 1, molecular mass markers; lane 2, bacterial lysate from batch 1; lane 3, purified recombinant gliadins from batch 1; lane 4, lysate from bacteria expressing a single gliadin (α-11, not present in batch 1); lane 5, purified α-11. The bacterial lysates were harvested 18 hours after induction with isopropyl β-D-thiogalactoside.

Figure 4

Recognition of batches of chymotrypsin digested and tissue transglutaminase (tTG) treated recombinant α-gliadins by the gut derived DQ2 restricted T cell clone (TCC) CD 412 R-5.32. Batches 1 and 2 each contain nine α-gliadin clones. Crude gliadin prepared from the wheat strain Kadett was used as a positive control. Similar data were obtained from the other T cell clones tested—that is, CD 380 E-27, CD 387 E-34, and CD 370 R2.3. T+APC, T cells plus antigen presenting cells.

Figure 5

Recognition of one of the recombinant gliadin subtypes (α-2) after chymotrypsin digestion and tissue transglutaminase (tTG) treatment of the T cell clone (TCC) CD 412 R-5.32. Similar data were obtained from the other T cell clones tested—that is, CD 380 E-27, CD 412 R-3, and CD 370 R2.3. T+APC, T cells plus antigen presenting cells.