Analysis of FOXP3 Isoform Expression by Regulatory T Cells in Human Inflammatory Bowel Disease

Abstract

For nearly fifty years, azathioprine has been a reliable therapy for IBD. However, the mechanism by which this purine antimetabolite controls intestinal inflammation remains obscure. FOXP3+ regulatory T cells (Tregs) are critical for limiting intestinal inflammation in mice and humans, so we evaluated the effect of azathioprine on intramucosal FOXP3+ Treg frequency. A blinded, cross-sectional, retrospective analysis of archived colon biopsies from IBD patients was performed. Using immunohistochemical (IHC) quantification of FOXP3+ and CD4+ T cells, we found that a significantly higher percent of CD4+ T cells were FOXP3+ in the mucosa of patients taking azathioprine at the time of biopsy. This increase was primarily due to a decrease in the relative number of CD4+, but not FOXP3+, T cells in the mucosa of azathioprine recipients. To determine the effect of azathioprine on FOXP3+ versus FOXP3T cells In Vitro, we cultured T cells from healthy donor blood in the presence or absence of 6-thioguanine nucleotide (6-TGN)—the active metabolite of azathioprine. We found that the proliferation of both naive and effector/memory CD4+ T cells was inhibited by similar concentrations of 6-TGN. Likewise, proliferation of both FOXP3+ and FOXP3T cells was inhibited by similar concentrations of 6-TGN, in the presence or absence of IL-2. However, de novo activation-induced expression of FOXP3 in sorted CD25T cells was much less sensitive to inhibition by 6-TGN. Thus, 6-TGN was able to dissociate FOXP3 induction fromT cell proliferation.We hypothesize that azathioprine selectively enriches FOXP3+ T cells in the inflamed bowel by inhibiting T cell proliferation without inhibiting induced expression of FOXP3. Such local enrichment of induced Tregs (“iTregs”) may be a major mechanism by which azathioprine controls intestinal inflammation in IBD.