Small intestinal T cells of celiac disease patients recognize a natural pepsin fragment of gliadin

A pepsin/trypsin digest of gluten was prepared as follows: 1 g of gluten (Fluka) was solubilized in 10 ml of 1 M acetic acid and boiled for 10 min. Pepsin (10 mg) (Sigma P-6887, purified, activity 3,200–4,500 units/mg) was added first, and the mixture was incubated for 4 h at 37°C. Subsequently, the pH was adjusted to 7.8 with NaOH, followed by the addition of 10 mg of trypsin (Sigma T-8253, type III, activity 10,000–13,000 benzoyl l-arginine ethyl ester units/mg). Then, the mixture was incubated for another 4 h at 25°C. Finally, 10 mg of trypsin inhibitor (Sigma) was used to stop further trypsin activity, followed by dialysis of the mixture against a large volume of water. Protein concentration was determined by using a bicinchoninic acid protein assay (Pierce). Synthetic peptides were made as described (8).

A small intestinal biopsy was taken from patient S., an adult Dutch CD patient on a gluten-free diet for several years. The patient gave informed consent for the study, which was approved by the hospital ethics committee. The patient was typed serologically to be HLA-DR3/4, DQ2/8, thus carrying both CD-associated DQ dimers. After treatment of the biopsy with 1 mM DTT (10 min) and 0.75 mM EDTA (4 h) in calcium/magnesium-free Hanks’ balanced salt solution (GIBCO) at 37°C under mild agitation, the biopsy was put into culture with autologous interleukin 4/granulocyte/macrophage-colony stimulating factor-cultured monocytes as a source of highly endocytic antigen-presenting cells (19). The antigen-presenting cells had been cultured in the presence of 250 μg/ml enzyme-treated gluten preparation for 48 h before the biopsy was taken. After 5 and 10 days of coculture, 20 units/ml r-interleukin 2 (Eurocetus, Amsterdam, The Netherlands) was added to the culture medium [RPMI medium 1640 (GIBCO)]. Subsequently, the cells were restimulated on an 8-day cycle with 20 units/ml r-interleukin 2 and 1 μg/ml phytohemagglutinin (Murex Diagnostics, Dartford, U.K.), antigen and autologous monocyte-enriched irradiated PBMCs as antigen-presenting cells. TCCs were established by limiting dilution (0.3 cell/well) in 150 μl of culture medium in 96-well, round-bottomed plates (Falcon) by using 10⁵ allogeneic PBMCs (3,000 rad), 1 μg/ml phytohemagglutinin, and 20 units/ml r-interleukin 2. This procedure resulted in the establishment of several HLA-DQ-restricted TCCs with a CD3+, CD4+, T cell antigen receptor α/β+ phenotype. Proliferation assays were performed in duplicate or triplicate in 150 μl of culture medium in 96-well, flat-bottomed plates (Falcon) by using 10⁴ T cells stimulated with 10⁵ PBMCs (3,000 rad) either in the absence or presence of antigen at several concentrations. After 48 h, cultures were pulsed with 0.5 μCi ³H-thymidine and harvested 18 h thereafter. The generation of gluten-specific TCCs from the Norwegian patient Å.W. has been described (6).

The enzymatic digest of gluten was purified in three successive rounds of HPLC as follows: first, ±3 mg of gluten was purified on an analytical HPLC system (Jasco, Easton, MD), using a gradient of 2% B/min (column Vydac 218TP1010, buffer B: acetonitrile, containing 0.1% trifluoric acid). Subsequently, bioactive fractions were separated further by micro-rp-HPLC (SMART, Pharmacia) using a gradient of 0.25% B/min [column C2/C18 sc 2.1/10 (Pharmacia), buffer B: acetonitrile, containing 0.1% trifluoric acid]. Finally, bioactive fractions were subfractionated by micro-rp-HPLC, using a gradient of 0.25% B/min (buffer B: acetonitrile, 0.1% heptafluorobutyric acid).

HPLC fractions were monitored for their peptide content by matrix-assisted laser desorption ionization time-of-flight mass spectrometry on a Lasermat (Finnigan-MAT, San Jose, CA) as described (8). Electrospray ionization mass spectrometry was performed on a hybrid quadrupole time-of-flight mass spectrometer, the Q-TOF (Micromass, Manchester, U.K.). The final bioactive fraction was introduced either by off-line nanoelectrospray using type A needles (Micromass) or by flow injection analysis using the on-line nanoflow electrospray interface (capillary tip 20-μm internal diameter × 90-μm outer diameter) with an approximate flow rate of 200 nl/min. In the latter case, 0.5- to 1.0-μl injections were done with a dedicated micro/nano HPLC autosampler, the FAMOS (LC Packings, Amsterdam, The Netherlands). MS/MS analyses were performed on the most abundant peptide peaks present in the bioactive fraction. Precursors (the triple and quadruple protonated molecules in case of the 35-mer) were selected with the quadrupole, and fragments were collected with high efficiency with the orthogonal time-of-flight mass spectrometer. The collision gas applied was argon (pressure 4 × 10⁻⁵ mbar), and the collision voltage was ≈30 V.

The program PeptideSearch was used for sequence elucidation. This program has been developed to identify sequences in databases using mass spectral information. This program is available on request from the authors of the program (20).

Gluten-specific T cell clones (TCCs) were isolated from a small intestine biopsy of the Dutch CD patient S. The reactivity of the TCCs was investigated by using a panel of HLA-typed peripheral blood mononuclear cells (PBMCs) in the presence of a gluten digest. On the basis of HLA-restriction, the TCCs could be divided into two categories: restricted either by HLA-DQ2 (represented by TCC S11 in Table 1) or by HLA-DQ8 (represented by TCC S2 in Table 1). In line with this, we observed that the gluten-specific proliferative response of these TCCs could be blocked by an HLA-DQ-specific mAb but not by an HLA-DR-specific mAb (Table 1).

To identify the gluten-derived epitopes that are recognized by the TCCs of patient S., a pepsin/trypsin digest of gluten was purified in consecutive rounds of HPLC. After each purification step, the peptide content of the fractions was monitored by time-of-flight mass spectrometry, and the fractions were screened for their bioactivity in a standard T cell proliferation assay. After three rounds of HPLC purification (Fig. 1A), one of the fractions (54) that strongly stimulated TCC S2 (DQ8-restricted) (Fig. 1B) was found to contain three dominant peptides with masses of 2,479, 2,679, and 3,897-Da (Fig. 1C). To obtain amino acid sequence information, these peptides were subjected individually to tandem mass spectral analyses using a hybrid quadrupole–time-of-flight mass spectrometer. Such an analysis yields a peptide fragmentation pattern that contains information on the nature and order of the amino acids in the peptide. As an example, part of the fragmentation pattern of the 3,897 Da fragment is shown in Fig. 2. The interpretation of this pattern yielded the partial amino acid sequence Q/K-V-S. This sequence is preceded by a peptide fragment of 857.5 Da, with unknown amino acid composition. On the basis of this information and the actual mass of the complete peptide (3,897 Da), a database search was carried out that identified gliadin (residues 198–232) as the source protein (gda09, SwissProt P18573). The source proteins for the other two peptides were identified in a similar fashion. The peptide of 2,679 Da was found to represent a glutenin sequence (residues 50–72), whereas the 2,479-Da peptide was derived from gliadin (residues 3–24) (Fig. 1C). Synthetic versions of the identified peptides showed that only the peptide corresponding to the 3,897-Da peak stimulated TCC S2 (data not shown). The stimulating capacity was found in a peptide corresponding to the first 25 aa of this gliadin fragment (residues 198–222). Next, we tested a set of overlapping peptides, which indicated that the epitope is located in the central region of the gliadin 198–222 peptide (Fig. 3A). Additional truncation analysis revealed that the minimal epitope comprises residues 206–216 (sequence: SGQGSFQPSQQ) (Fig. 3B). A sequence homology search with this peptide resulted in the identification of five highly similar gliadin sequences. Only one of these peptides (sequence: SSQGSFQPSQQN) was found to stimulate TCC S2 (not shown). Next, we tested a panel of HLA-DQ8-restricted, gluten-specific TCCs derived from the small intestinal mucosa of a Norwegian CD patient (CD282). Six of eight HLA-DQ8-restricted TCCs derived from this patient recognized the gliadin 198–222 peptide (three representative examples are shown in Table 2).