Jerome G. Weidner

Characterization of T-cell Receptor Repertoire in Inflamed Tissues of Patients with Crohn's Disease Through Deep Sequencing

Background:Intestinal tissues of patients with Crohns disease (CD) contain expanded populations of T cells which are believed to mediate inflammation. We performed a detailed characterization of these T-cell repertoires. Methods:We obtained biopsies from the neoterminal ileum of 12 patients undergoing evaluation for postoperative recurrent CD and 4 individuals with normal terminal ileum and no history of inflammatory bowel disease (controls). Samples of diseased terminal ileum were obtained from 5 patients undergoing surgery for stricturing or penetrating CD. Total RNA was extracted from tissues and peripheral blood mononuclear cells, and cDNAs were generated. We used next-generation sequencing to characterize T-cell receptor (TCR)-&agr; and TCR-&bgr; cDNAs in ileal mucosal tissue and matched peripheral blood mononuclear cells of 17 patients with CD to identify oligoclonal expansions of T-cell populations associated with CD. Results:TCR diversity in mucosal tissue was significantly lower than that of matched peripheral blood mononuclear cells, indicating expansion of certain T-cell populations in inflamed intestinal tissue. A single TCR-&bgr; clonotype, CASSWTNGEQYF (TRBV10-1-TRBJ2-7), was enriched at a frequency of 7.0% to 28.9% in the neoterminal ileum of 4 of 12 patients with recurrent CD. The abundance of this clonotype significantly correlated with the severity of disease recurrence, based on Rutgeerts score (P = 0.015). Conclusions:Specific populations of T cells are expanded in the inflamed intestinal mucosa of patients with CD; their abundance correlates with severity of disease recurrence. Studies of these T cells could provide information about mechanisms of CD pathogenesis. Deep TCR sequencing is a powerful tool that rapidly provides in-depth, real-time assessment of the T-cell repertoire.

EDTA is superior to DTT treatment for overcoming the prozone effect in HLA antibody testing

A limitation of solid‐phase human leukocyte antigen (HLA) antibody assays is the falsely low/negative result of samples with high‐titer antibodies, a phenomenon known as the prozone effect. Here we compared the efficacy of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect. A total of 21 serum samples were treated with either EDTA or DTT before HLA single antigen bead assay. The efficacy of prozone effect reversal, compared with untreated samples, was examined on fourfold, serially diluted samples, from neat to 1:256, using PBS as diluent. EDTA reversed the prozone effect in all tested samples, with an efficiency of greater than 84%, estimated by the ratio of undiluted sample mean fluorescence intensity (MFI) to peak MFI, for any given dilution. In contrast, the efficiency of DTT treatment was as low as 47%. These results show superior prozone effect reversal with EDTA treatment, compared with DTT.