ABSTRACT

This dissertation is based on studies performed in the facilities of the V-laboratory, Department of Medicine V, Aarhus University Hospital.

Crohn's disease (CD) is a chronic granulomatous inflammation in the gut, a complex polygenic disease characterized by an inappropriate immune response to gut bacteria in a genetically susceptible host. Daily clinical management of CD resorts to a large number of therapeutic regimens, and today's intensive research into cellular immunotherapies has been spurred by the development of treatment-resistance and refractory CD.

The principal aim of this dissertation was to evaluate the possible therapeutic potential of ex vivo generated gut-derived T cells in the treatment of CD, including the adoptive transfer of autologous regulatory T cells by a two-step approach: To isolate and characterize candidates for disease-associated and/or regulatory T cell populations in our T cell culture system and to determine whether cultured gut-derived T cells show selective homing to the gut mucosa after autologous infusion.

The project is based on a T cell culture principle, in which whole biopsies of colonic mucosa are placed into culture medium containing high amounts of IL-2 and IL-4. Using this approach we determined the V β repertoire in mucosal T cell cultures by reverse transcriptase polymerase reaction (RT-PCR) and the V β receptor prevalence by flow cytometry. Early T lymphocyte cultures from CD-patients and healthy controls showed a polyclonal V β gene expression that narrowed during culture, which in CD cultures (grown for more than 12 weeks) lead to a significant overexpression of the V β 5.1 and V β 8 gene segments.

These T cell clones may represent driver clones implicated in the development of CD and suggest a strategy of specific targeting of these TCRV β subpopulations by therapeutic intervention.

Further we demonstrated that gut-derived CD4 + CD25 + GITR + phenotype T cells from both CD-patients and healthy individuals show suppressive properties comparable to that of freshly isolated CD4 + CD25 + . Regulatory T cells from peripheral blood can be propagated ex vivo. This observation does not support an intrinsic regulatory T cell defect in CD.

We investigated whether homing of cultured T cells in the gut mucosa can be accomplished upon intravenous infusion. We made the important observation that cultured gut-derived T cells can be infused without side effects. Despite a strong adhesion to MAdCAM-1 (Mucosal Addressin Cellular Adhesion Molecule 1) in vitro, however, in vivo the T cells showed no preferential distribution to the intestinal compartment in healthy individuals, but migrated to the liver, bone marrow and spleen. Studies on the selective expansion of regulatory T cells and lymphocyte trafficking are warranted before considering a therapeutical use.