Human intestinal T lymphocytes : a comparative analysis of phenotype and function in normal and inflamed mucosa

Abstract: The epithelial lining of the gut must allow immediate contact with beneficial components as nutrients and normal microflora. At the same time it runs the constant risk of attack from pathogenic microbes and noxious agents. Only a singel layer of epithelial cells separates the body's largest lymphocyte population from foreign components in the gut lumen. I have addressed two fundamental questions: 1) How does the immune system in the gut specifically protect against infectious agents and other harmful substances and at the same time avoid overreaction against antigens in food and commensals? and 2) What causes the immune protection breakdown in inflammatory bowel disease? To this end, the phenotype, distribution and functions of T lymphocytes, key players in adaptive immunity, were compared to T lymphocytes in the chronically inflamed intestine of patients with ulcerative colitis (UC) and Crohn's disease (CD). Intestinal T lymphocytes are present both within the epithelium, intraepithelial lymphocytes (IEL), and in the underlying lamina propria (LP). Even though there are many T lymphocytes in normal intestine, there is a highly significant increase in inflamed intestine. They seem to be involved in the pathogenesis of both UC and CD. In UC, T lymphocytes populate the basal lymphoid aggregates, which occupy up to 45% of LP.Phenotype was analysed in situ by immunomorphometry and immunoelecton microscopy, and by immunoflow cytometry of isolated IEL and lamina propria lymphocytes (LPL). Cytokine production was monitored as the frequencies of cells expressing the proteins in situ and as mRNA expression in purified T lymphocytes using quantitative RT-PCR. Cytotoxicity was measured in functional assays using purified IEL and LPL as effector cells and as expression of cytotoxic effector molecules. The responsiveness to polyclonal T cell activators was assayed as proliferation and secretion of cytokines in vitro.Major findings were: (1) Interleukin-2 (IL-2), the Th1 cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), the down-regulatory cytokine transforming growth factor-β1 (TGF-β1) and the chemokine IL-8 are normally produced by IEL and LPL in both small and large intestine. Activation in vitro enhanced IFN-γ and TGF-β1 production and induced IL-10. These data indicate ongoing controlled cell mediated immune activity and readiness for both pro-inflammatory and immunosuppressive responses. (2) IEL and LPL of both small and large intestine exhibited a capacity to kill via the Fas/FasL pathway in a T cell antigen receptor (TCR)/CD3 independent manner. FasL expressing cells were present both intraepithelially and in LP and were most abundant in LP of colon, where they constitute 30% of the immune cells. These results suggest that local immune activity in the intestinal mucosa is controlled by activation induced cell death, AICD. Small intestinal mucosa harbours a small population of CD8+ αβ+T lymphocytes with cytolytic machinery that can be triggered through the TCR/CD3 complex. These T cells use the perforin/granzyme exocytosis pathway for killing. Such cells are not detected in colon. Most probably these cytotoxic T lymphocytes are "caught in the act" of eliminating virus infected epithelial cells. (3) Inflammation in colon leads to activation of γδ T cells. In UC, Vδ1+ γδ T cells, which normally reside within the epithelium, were numerous in LP. They constituted as much as 15% of the cells in the lymphoid aggregates and showed TCR-γδ internalisation and surface down-regulation, signs of receptor mediated activation. Thus, γδ T cells appear to be recruited from the epithelium in response to nominal antigens present in LP. (4) The cytokine profile of LP T cells was markedly distorted in UC patients and regulatory T cells were induced. IL-10 production was increased and expression levels correlated with disease activity while TGF-β1 levels were unchanged. CD4+ T cells were the major source of IL-10. Moreover, cells with the regulatory/suppressor phenotype CD4+CD28-TCR-αβ+ were frequent, particularly in the aggregates. IL-2 production was shut off and production of IFN-γ and TNF-α was decreased. However, we found no evidence for a corresponding induction of the Th2 cytokines. (5) There was a parallel accumulation of FasL expressing cells with retained capacity to kill via the Fas/FasL pathway and apoptosis resistant bcl-2 expressing lymphocytes in UC. Inflammation in ileum of CD patients led to an enhanced cytotoxicity. The frequency of perforin expressing LPL was increased and the capacity of LPL to execute TCR/CD3 mediated cytotoxicity via the perforin exocytosis pathway was elevated.In summary, it appears that intestinal T lymphocytes are constantly involved in cell mediated immune activities and that cytotoxic memory CD8+ αβ T cells accumulate in the small intestinal mucosa. IEL and LPL seem capable of down-regulatory actions in the context of proinflammatory cytokines and the local homeostasis of immune reactions to luminal components seems to be regulated by IL-2 dependent AICD. The cytokine profile in UC suggests a generalised activation of regulatory T cells along the intestine. Inflammation in colon, but not in ileum, point to the importance of colonic microflora for precipitating the proinflammatory actions of IL-10.