Jens Brimnes

Defects in CD8+ Regulatory T Cells in the Lamina Propria of Patients with Inflammatory Bowel Disease

Mucosal tolerance is believed to be partly mediated by regulatory T cells. Intestinal epithelial cells (IECs) may play an important role in the generation of such regulatory cells, because they are able to process and present Ag to T cells. Furthermore, we have previously demonstrated that IECs are able to generate regulatory CD8+ T cells in vitro. In the present study, we have analyzed lamina propria (LP) lymphocytes for the presence of such regulatory CD8+ T cells in normal individuals as well as in patients with inflammatory bowel disease (IBD). The results of the present study show that LP CD8+ T cells derived from normal controls possess regulatory activity, whereas both unfractionated LP lymphocytes and purified LP CD4+ T cells do not. The LP CD8+ T cells suppress Ig production by pokeweed mitogen-stimulated PBMCs by 31–80%, in a cell contact-dependent manner. No significant difference in suppression between CD28+ and CD28−CD8+ LP T cells was observed. In contrast to CD8+ T cells from normal LP, CD8+ T cells isolated from LP of IBD patients, did not suppress Ig production by pokeweed mitogen-stimulated PBMC (five of six ulcerative colitis specimens; six of six Crohn’s disease specimens). Furthermore, we demonstrate that the frequency of TCR Vβ5.1-positive CD8+ T cells, which we previously have demonstrated to be regulatory and to be expanded by IECs in vitro, is decreased in IBD LP compared with normal LP. In conclusion, this study demonstrates that CD8+ T cells with regulatory activity are present in the LP of normal healthy individuals, but not in patients with IBD, suggesting that these cells might play an active role in mucosal tolerance.

Induction of mucosal tolerance in Peyer‘s patch—deficient, ligated small bowel loops

To explore the requirement for M cells and the Peyers patch (PP) in induction of oral tolerance and address the potential in vivo role of intestinal epithelial cells as nonprofessional APCs, we have attempted to induce tolerance in mice with ligated small bowel loops without M cells and Peyers patches. A 2-centimeter section of vascularized small bowel was spliced away from the gut without disruption of the mesenteric attachments. We introduced OVA directly into the lumen of the loop prior to footpad immunization. By excising segments of bowel that contain PPs in some mice and segments without patches in others, we could study the necessity of the M cell and the underlying patch versus epithelial cells in induction of mucosal tolerance. We show that OVA-specific T cell proliferation and serum antibody responses are reduced in mice that have previously been given OVA both in PP-containing loops and in loops without patches. Furthermore, both high- and low-dose tolerance could be induced in the absence of PPs. Low-dose tolerance is associated with bystander suppression and requires IL-10, which indicates active suppression and the induction of regulatory cells. These data suggest that there is a critical role for components of the mucosal immune system other than PPs in antigen sampling and induction of oral tolerance.

Activation of a Unique Population of CD8+ T Cells by Intestinal Epithelial Cells

Abstract: Intestinal epithelial cells may play a role in the regulation of immune responses toward luminal antigens. We show that a subset of CD8+ T cells undergoes oligoclonal expansion in the intestinal mucosa, probably through interaction with a unique complex expressed on epithelial cells, formed by a CEA subfamily member (gp180) and CD1d. This subset, which is regulatory in vitro, may play a role in the control of intestinal immune responses toward luminal antigens. A lack of expansion of these CD8+ regulatory T cells, probably related to the defective expression of the gp180/CD1d complex, is observed in inflammatory bowel disease.

Enhanced Oral Tolerance in Transgenic Mice with Hepatocyte Secretion of IL-10

Several cytokines derived from Th3 and Tr1 cells, including IL-10, are believed to regulate oral tolerance, but direct evidence is lacking. We have explored the potential role of IL-10 by generating transgenic (TG) mice with sustained hepatocyte-specific expression of rat IL-10. TG mice expressed rat IL-10 downstream of a transthyretin promoter, which led to serum levels that were increased 10- to 100-fold compared with normal animals. Animals were orally administered 1 mg of whole OVA for 5 consecutive days, with control animals receiving PBS. There were six animal groups: Either OVA or PBS were fed orally to rat IL-10 TG mice, non-TG wild-type mice without IL-10 administration, and non-TG wild-type mice administered rat IL-10 systemically. On day 8, all mice were immunized with two injections of OVA, and then analyzed on day 18. T cell proliferation responses were reduced by 65.8 ± 14.3% after feeding of OVA in rIL-10 TG animals, compared with 39.4 ± 15.6% in the non-TG mice (p = 0.02). Anti-OVA titers were expressed as fold increase over naive non-TG mice. After feeding, titers decreased by ∼33% (from 3- to 2-fold) in TG animals and, to a lesser extent, in non-TG animals. IFN-γ secretion by cultured popliteal lymphocytes decreased in TG animals by 83% after feeding and by 69% in non-TG animals. IL-4 secretion increased 4-fold in TG-fed mice, but did not significantly change in non-TG OVA-fed animals. In contrast to hepatic TG expression of rIL-10, systemic administration of rIL-10 had only a modest effect on tolerance. IL-10, when transgenically expressed in the liver enhances mucosal tolerance to an oral Ag.

IL-10 is necessary for the induction of low dose tolerance

Oral Tolerance (OT) is defined as the systemic unresponsiveness to an antigen administered by the oral route. Feeding high doses of antigen is believed to lead to deletion or anergy of Ag-specific T-cells, whereas low doses of antigen leads to the generation of regulatory cells producing cytokines such as IL-4, IL-IO and TGF-beta. In the present study we have investigated the role of IL-IO in the induction of high and low dose tolerance in intact mice as well as in murine small bowel loops with or without Peyers Patches (PP). Tolerance was induced by admtinstration of OVA-peptide 323-339 (OVA-pep) by gastric intubation or into the loops for 5 days, followed by immuni,ation with OVA-peptide in Complete Freunds Adjuvant and subsequent restimulation of spleen and lymph node cells in vitro. Low dose tolerance could not be induced in the absence of IL-10, as feeding a low dose of OVA-pxp (0.5 rag) to 1L-10 KO mice, did not result in a sigmflcant reduction of T-cell proliferation or IFN-gamma production compared to nonfed mice. In contrast, feeding the same low dose to wild type C57B1/6 mice resulted in both reduced T-cell proliferation and lFN-gamma production. We then investigated whether IL-10 was necessary for the induction of either low or high dose tolerance in small bowel loops with and without PP. Administration of a low dose of OVA-pep (0.01 mg) into the loops of IL-10 KO mice did not result in the induction of oral tolerance, regardless of whether the loops contained PP or not. However, giving a high dose of OVA-pep (0.5 mg)~to the loops of IL-10 KO mice did induce oral tolerance characterized by reduced T-cell proliferation as well as reduced production of IFN-gamma and IL-2, compared to nonfed mice Again, no differences were observed between loops with and without PP, indicating that high dose tolerance is independent of [L-IO both in the presence and absence of PP. Taken together our results show that IL-10 is necessary for the generation of low dose tolerance, both in the presence and absence of PP, whereas high dose tolerance can be induced independently of IL-IO.