|M.Sc Student||Kroner Amit|
|Subject||Redirecting regulatory T cells to common gut antigens|
for restoring immune tolerance in IBD
|Department||Department of Medicine||Supervisors||PROF. Nathan Karin|
|ASSOCIATE PROF. Gidi Gross|
|Full Thesis text|
inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis are
characterized by chronic inflammation in the intestinal tract which likely
results from disruption of immunological tolerance to commensal gut microbes. Using
CD4 T regulatory (Treg) cells for restoring immune tolerance holds great
promise in the treatment of IBD. However, this direction faces major obstacles:
1) Redirecting Tregs specifically to the inflamed gut tissue is hampered by the fact that no genuine antigen associated with IBD has been identified yet.
2) In the inflammatory milieu, Tregs are inherently unstable and may be converted to effector T cells and exacerbate disease.
3) Natural FOXP3 CD4 Tregs (nTregs) constitute only a small fraction of human CD4 T cells and must be extensively expanded ex-vivo prior to clinical use.
To overcome these challenges I examined several new gene-based strategies. For redirecting and activating Tregs in an antigen-specific manner that is restricted to the inflamed gut, I embarked on the development of a new type of Toll-like receptor-based chimeric antigen receptors (TLR-CARs). These were designed to redirect Tregs at common bacterial antigens which allegedly cross the breached gut epithelium in IBD. The antigens of choice are peptidoglycan (PGN), an abundant bacterial cell wall component, and flagellin, the structural component of bacterial flagella. For redirecting Tregs to those antigens I exploited the ectodomains of TLR-2 and TLR-5, natural receptors for PGN and flagellin, respectively. TLR-5 and TLR-2 ectodomains were fused to human TCR ζ chain. These new types of CAR are expected to confer on Tregs MHC-independent specificity to the affected gut tissue. A series of genetic constructs of TLR-CARs were assembled. Following mRNA and DNA transfections, membrane expression and function of the gene products could not be confirmed although they could be detected in cell lysates.
In an attempt to enhance Treg suppression capacity I have equipped human nTregs with constitutively active TLR4 (caTLR4) and CD40 (caCD40), two genetic adjuvants developed in our laboratory which had been shown to exert multiple costimulatory effects on different human T cell subpopulations. We hypothesized that the suppressive capacity of nTreg could be similarly augmented. While the electroporation of Tregs with caCD40 mRNA maintained high level of FOXP3 and upregulated CTLA4 and OX-40, caTLR4 downregulated FOXP3 and exerted no significant effect on OX-40 and CTLA-4.
For producing a sufficient number of functional Tregs for adoptive transfer, I examined the induction of T regulatory type 1 (Tr1) cells from human CD4 precursors through the expression of membrane-bound IL-10 (memIL-10). Constitutive expression of soluble IL-10 in human CD4 T cells was previously shown to induce and maintain a Tr1 phenotype and memIL-10 is expected to function in an autocrine manner and prevent systemic dissemination. Indeed, memIL-10 was highly expressed in mouse and human cell Lines and human CD4 cells and upregulated IL-10 receptor in human CD4 cells similarly to treatment with soluble IL-10.
Hopefully, this study will lay the ground for a new clinical protocol for the treatment of IBD.