P-190 Use of a Novel Activation Assay to Detect Increased Flagellin-Reactive CD4+ T cells in Peripheral Blood from Crohnʼs Disease Patients

Anti-commensal immune responses are abnormally active in patients with IBD. About half of people with Crohn's disease (CD) produce antibodies to bacterial flagellins. However, previous attempts to demonstrate T cell responses against these flagellins have been largely unsuccessful. We used a newly described dual activation-marker flow cytometric assay to quantify flagellin-reactive CD4+ T cells in patients with CD compared to controls. Consenting patients with CD were recruited from the McGill University Health Center IBC clinic. Clinical data collected included age, sex, disease phenotype, and current treatment. Controls were healthy volunteers without a history of IBD. Whole blood from venipuncture was stimulated with 1 µg/mL of LPS-free flagellin (A4-Fla2 or E. coli H18 FliC) for 48 hour and then stained for flow cytometry. The CD3+CD4+ population within the live cell gate was analyzed for expression of CD25 and CD134 (OX40) and the percentage of double positive cells was recorded. Cells were stimulated with tetanus toxoid or phytohemagglutinin as positive controls. Kruskal-Wallis and Mann-Whitney tests were used to compare % CD25+CD134+ cells between groups. The assay has low background, with unstimulated samples showing very few double positive cells (median 0.07%, IQR 0.118%). Tetanus toxoid stimulation led to double positive cells in about half of tested subjects (median 0.425%, IQR 0.87%). LPS stimulation at up to 10 µg/mL did not produce double positive cells, suggesting that the assay reflects TCR-dependent stimulation rather than nonspecific toll-like receptor effects. This is supported by our observation that responses to E. coli FliC (a strong TLR5 agonist) were undistinguishable from those against a transposon mutant of FliC, 2H3, which lacks TLR5 stimulatory activity. Most healthy controls had no discernible increase in double-positive CD4+ cells after stimulation with Fla2 (median 0.08%, IQR 0.32%) or FliC (median 0.11%, IQR 0.431%). In contrast, CD patients had significantly higher percentages of double positive cells after stimulation with Fla2 (median 0.24, IQR .505; P = 0.002) and FliC (median 0.30, IQR 0.53; P = 0.004). Elevated double positive cells were seen in patients with isolated luminal disease (Fla2: P = 0.010; FliC: P = 0.012) as well as those with complicated stricturing/perforating disease, in whom anti-flagellin antibodies are reported to be more prevalent (Fla2: P = 0.046; FliC: P = 0.016). Differences between these 2 groups were not significant. The type of current treatment (5-ASA, immunomodulator, or anti-TNF) also did not significantly affect the percentage of double-positive cells after flagellin stimulation. We used a highly sensitive and specific assay for CD4+ T cell activation to demonstrate for the first time a statistically significant increase in anti-flagellin T cell responses in patients with CD. Responses were similarly directed against E. coli FliC and the A4-Fla2 flagellins, unlike antibody responses, which more strongly target the Fla2-like Clostridiales flagellins. Since helper T cells are important both in optimal production of antibodies and cytotoxic T cell responses, we hypothesize that flagellin-reactive CD4+ cells play a pathogenic role in CD. Future studies will be needed to examine the phenotype of these double-positive cells and their prevalence in intestinal tissue and draining lymph nodes.

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