Olle Korsgren’s projects – Induction of immunological tolerance
Regulatory T cells for treatment of transplantation induced immune reactions
In collaboration with Björn Carlsson, Clinicla Immunology, and Gunnar Tufveson, Dept. of Surgical Sciences
The objective of this project is to develop methods to isolate and/or expand regulatory T cells ex vivo, for potential use in restoring or inducing transplantation tolerance. The logistics of isolating, expanding, and administering the cells are as important as the biological characteristics of the resulting regulatory T cells.
Biologically viewed, the ideal candidate for adoptive immunotherapy will be a highly stable, long-lived, potently suppressive, non-immunogenic, and preferably Ag-specific Treg population of regulatory T cells. Practically, it must be possible to isolate or induce large numbers of these cells by in vitro manipulation, expand them without loss of function, and administer them in the periphery with the knowledge that they will migrate to the anatomic location required for optimal function. The developed protocol should also be feasible in clinical use.
The activities of T-regulatory cells (Tregs) are now recognized as fundamental to the development and maintenance of immune tolerance. Several different types of Tregs exist in humans, including specialized subsets of CD4+, CD8+, double negative CD3+ CD4−CD8−, γδ T cells, and natural killer T (NKT) cells. Research into how Tregs can be manipulated therapeutically in humans is most advanced for two main types of CD4+ Tregs: forkhead box protein 3 (FOXP3)+ Tregs and interleukin-10 (IL-10)-producing type 1 Tregs (Tr1 cells).
Tr1 cells arise in the periphery when naive CD4+ T cells are activated by tolerogenic antigen-presenting cells (APCs) in the presence of IL-10. Much effort has gone into developing protocols to generate IL-10-producing cells in vitro for cellular therapy. Using a system involving artificial APCs, it has been demonstrated that repetitive stimulation of human naive CD4+ T cells in the presence of exogenous IL-10 resulted in a relatively small increase in IL-10-producing Tr1 cells, whereas co-addition of interferon-α (IFN-α) resulted in a larger and more stable population of Tr1 cells. Addition of TGF-β to this system resulted in generalized suppression of cytokine production and did not enhance Tr1 cell development.
Repetitive stimulation of human naive cord blood CD4+ T cells or peripheral blood CD4+ T cells with allogeneic immature myeloid DCs induces the differentiation of human Tr1 cells in vitro. Similarly, tolerogenic myeloid DCs can be made by exposure to immunomodulatory cytokines, such as IL-10, either alone or in combination with IFN-α, granulocyte colony-stimulating factor (G-CSF), or hepatocyte growth factor. Treatment of DCs with immunosuppressive agents such as vitamin D3 either alone or in combination with dexamethasone also results in tolerogenic DCs, which produce high amounts of IL-10 and induce Tr1 cells.
FOXP3+ Tregs may either emerge from the thymus as fully functional suppressor T cells or differentiate in the periphery from naive precursors. Of particular interest is the fact that human T effector (Teff) cells transiently express FOXP3 following TCR-mediated activation but do not acquire suppressive activity. Thus, functional assays are still required for characterization of human FOXP3+ cells as Tregs. Several groups have defined a variety of culture conditions in which human FOXP3+ Tregs can expand 100–13 000-fold and their findings suggest that the key requirements are strong TCR-mediated stimulation and high doses of exogenous IL-2.
In the murine system, specific subsets of APCs provide optimal signals for Foxp3+ Treg expansion, and limited data suggest that this may also be true for human FOXP3+ Tregs. It is also possible to selectively expand FOXP3+ Tregs in vitro using rapamycin. Ag-specific Foxp3+ Tregs are up to 100 times more potent than polyclonal populations at preventing disease, inhibiting established disease, and preventing immune destruction of transplanted islets.
FOXP3+ vs Tr1 cells
Owing to the lack of cell surface makers for Tr1 cells and the consequent difficulty in isolating and tracking these cells, the vast majority of basic research directed toward the development of cellular therapy has been conducted with FOXP3+ Tregs. Tr1 cells may nevertheless have some advantages, through their capacity to secrete IL-10 and thus efficiently mediate infectious tolerance. Indeed, in humans, it has long been known that spontaneous transplantation tolerance can be attributed to Tr1 cells.
So far no clinical trial has been presented using adoptive transfer of regulatory T cells. Results have been presented in mice where adoptive transfer of regulatory T cells has been shown to inhibit both autoimmune diseases and transplantation induced immune reactions. The optimization of the protocol to culture human regulatory T cells is still ongoing and several alternative methods are being exploited with an aim to initiate clinical studies in 1-2 years.