Derivation and use of human thymic epithelial progenitor cell lines.

Dr. Clare Blackburn

Confirmed funding for a BBSRC Case studentship, sponsored by Stem Cell Sciences UK.

T-cells are central regulators of the adaptive immune system. They co-ordinate and effect the cellular and humoral immune responses which are required to resolve microbial and viral infections. The specialised organ, the thymus (1), is the principal site of T-cell development, therefore, athymic individuals are profoundly immunodeficient. The unique processes of T-cell differentation and T-cell repertoire selection are dependent on interactions between developing T cells and a number of highly specialised epithelial cell-types found only within the thymus (2). These epithelial cell-types are thought to provide the molecular niches required to support T-cells through the multiple stages of differentiation, selection and maturation that ensure emergence into the peripheral immune system of self-restricted, self-tolerant T-cells (3). T-cell development therefore requires interactions with multiple epithelial subpopulations.

The ability to generate T-cells from haematopoietic progenitor cells in vitro would be highly advantageous for many medical applications (4), particularly since it should be possible to customise in vitro-derived T-cell repertoires to be tolerant to defined MHC haplotypes. For instance, it would help reduce infection-related morbidity in treatments requiring transplantation of T-cell depleted bone marrow: in this case, patients could receive, at the same time as the transplant itself, in vitro-generated T-cells tolerant to both the transplant donor and recipient, thereby gaining an immediately functional adaptive immune system rather waiting in excess of 9 months for immune reconstitution to occur. However, the cellular complexity of the thymic epithelium has proved a stumbling block for attempts to generate large T-cell repertoires in vitro, and this is currently possible only in cultures based on limited amounts of ex vivo-thymic tissue (5).

Recent work in my laboratory has demonstrated that a population of cells in the thymic primordium, identified by two monoclonal antibodies (6), can differentiate into all thymic epithelial cell-types, attract lymphoid progenitors, support thymocyte differentiation into mature CD4+ and CD8+ T-cells, and confer thymus function on congenitally athymic nude mice. Neither other epithelial cell populations in the thymic primordium, which include cells expressing markers associated with mature cortical thymic epithelium, nor unfractionated dissociated-and-reaggregated thymus primordium cells, can fulfil all of these functions (7). These data indicate that our cell-population constitutes a thymic epithelial progenitor cell-type (TEPC; 6; 7) sufficient to generate a functional thymus in vivo. They provide the first functional evidence that all subpopulations of thymic epithelial cell may arise from a common multipotent progenitor, or stem, cell, and thus provide a model for understanding the mechanisms underlying thymus organogenesis. Further to this, they present the possibility of supporting T-cell differentiation in vitro and in vivo using a single TEPC line: such lines could be propagated in their undifferentiated state, but would differentiate into a functional thymus upon culture under appropriate conditions.

Aims:
The aims of this PhD project are to derive human TEPC lines and to demonstrate use of these lines to generate defined T-cell repertoires in vitro.

Further information is available from Dr Clare Blackburn (c.blackburn@ed.ac.uk), Centre for Genome Research/Institute of Cell, Animal and Population Biology, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH3 9JQ.

References for the project:

1) Miller, J.F.A.P., Immunological function of the thymus. Lancet 2, 748 - 749 (1961).

2) Anderson, G., Moore, N. C., Owen, J. J. T. & Jenkinson, E. J. (1996) Cellular interactions in thymocyte development. Annu. Rev. Immunol. 14, 73 - 99.

3) Boyd, R. L., et al. (1993) The thymic microenvironment. Immunol. Today 14, 445-459.

4) Markert, M. L., et al. (1999) Transplantation of thymus tissue in complete DiGeorge Syndrome. N. Engl. J. Med. 341, 1180-1189; Weissman, I.L., and Shizuru, J.A. (1999) Immune Reconstitution. N Engl J Med 341 1227-1229; Reisner, Y. & Martelli, M. F. (2000) Tolerance induction by 'megadose' transplants of CD34+ stem cells: a new option for leukemia patients without an HLA-matched donor. Curr. Opinion. Immunol. 12, 536-541; Slavin, S. (2000) New strategies for bone marrow transplantation. Curr. Opinion. Immunol. 12 542-551.

5) Poznansky, M. C. et al. (2000) Efficient generation of human T cells from a tissue-engineered thymic organoid. Nature Biotech. 18, 729-734.

6) Blackburn, C.C., et al. (1996) The nude gene acts cell-autonomously and is required for differentiation of thymic epithelial progenitors. Proc. Natl. Acad. Sci. USA,. 93 5742-5746.

7) Bennett, A.R., Gordon, J., Bennett, C.L., Boyd, R.L., and Blackburn, C.C. Identification and characterization of progenitor cells for the thymic epithelium (submitted to Nature Immunol.).

Recent publications:

Gordon, J., Bennett, A.R., Manley, N.R., and Blackburn, C.C. (2001) Gcm2 and Foxn1 mark parathyroid- and thymus-specific domains in the developing third pharyngeal pouch (Mech. Dev. in press).

Colledge, L., Sun, M-Y., Lin, W., Blackburn, C.C., and Reay, P.A. (2001) Characterization of the processing requirements of several recombinant antigens for presentation by MHC Class II (Immunol. in press)

Bennett, C.L., Misslitz, A., Colledge, L., Aebischer, T., and Blackburn, C.C. (2001) Silent infection of bone marrow-dendritic cells by Leishmania mexicana amastigotes. Eur. J. Immunol. 31 876-883.

Blackburn, C.C., Grogan, J., Augustine, C.L., Varigos, G., Miller, J.F.A.P., and Morahan, G. (2001) Effect of Localised Cytokine Dysregulation: Accelerated Rejection of IL-2-Expressing Skin Grafts. Immunol. Cell. Biol. 79 128-131.

Blackburn, C.C., Augustine, C.L., Li, R., Harvey, R.P., Malin, M.A., Boyd, R.L., Miller, J.F.A.P., and Morahan, G. (1996) The nude gene acts cell-autonomously and is required for differentiation of thymic epithelial progenitors. Proc. Natl. Acad. Sci. USA,. 93 5742-5746.

 

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