Proteins specifically hyperexpressed in a coeliac disease patient with aberrant T cells

Abstract

An aberrant T cell population is the basis for diagnosis of refractory coeliac disease and determines the risk of enteropathy-associated T cell lymphoma. This disease is serious with a poor survival. Pathogenetic mechanisms sustaining aberrant T cell proliferation remain unknown. Recently, alemtuzumab has been proposed as a promising new approach to treat these patients. Only few single cases have been tested at present; nevertheless, in all the cases a clinical improvement was observed. However, whether intraepithelial lymphocytes have been targeted effectively by alemtuzumab is still debated. This study reports, using two-dimensional difference gel electrophoresis (2D DIGE), hyperexpressed proteins associated specifically with aberrant T cells found in a patient with coeliac disease by comparison of the protein expression of this sample with that of patients with coeliac disease and polyclonal T cells or with control subjects. The data demonstrated a significantly higher expression of IgM, apolipoprotein C-III and Charcot-Leyden crystal proteins in a duodenal biopsy specimen of the patient with clonal T cells compared with that of other patients. These preliminary results allow hypothesizing different clinical effects of alemtuzumab in patients with coeliac disease and aberrant T cell proliferation, because as well as the probable effect on T cells, alemtuzumab could exert its effect by acting on inflammatory associated CD52(+) IgM(+) B cells and eosinophil cells, known to produce IgM and Charcot-Leyden crystal proteins, that we demonstrated to be altered in this patient. The results also emphasize the possible association of apolipoprotein with aberrant T cell proliferation.

Fig. 1

Comparison of the fluorescence-labelled T cell receptor–polymerase chain reaction (TCR–PCR) products by GeneScan analysis. The x-axes represent molecular size (base pairs) and the y-axes fluorescence intensity. (a) Duodenal tissue; (b) peripheral blood mononuclear cells of patient 1. In the duodenal sample PCR results show two overexpanded TCR-γ clones, while in peripheral blood mononuclear cells a single TCR-γ clone was found. Base pair size, NCBI GeneBank accession number and the corresponding V, D, J germline segments are reported next to the corresponding TCR-γ clonal peaks.

Fig. 2

Two-dimensional (2D) difference gel electrophoresis between refractory type II CD (RCD-II) and controls. (i) 2D pattern from gut biopsy of (a) an RCD-II patient; (b) coeliac disease (CD) patients and (c) patients with no DQ2/8 variants. Proteins were separated on the basis of pI (x-axis) and molecular mass (y-axis). Highlighted in rectangles are those areas where consistent alterations of protein expression were identified. (ii) Enlarged portions and (iii) 3D view of regions highlighted in (i). Each protein was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and shown as abbreviations on the gels. (iv) Graphical representation of the intergel distribution of apo C3, IgM and Charcot–Leyden crystal protein (CLC) expression levels in RCD-II patient and in CD and normal patients determined with bva version 6·5 software.

Fig. 3

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identifying apo C-3, IgM and Charcot–Leyden crystal protein (CLC) proteins. Matching peptides are listed in Table 3.