DR. MICHAEL RATCLIFFE

The molecular regulation of B lymphocyte development and the molecular mechanisms underlying antibody diversification be somatic gene conversion.

Chair, Department of Immunology, University of Toronto and Senior scientist, Molecular and Cellular Biology, Sunnybrook Research Institute 2075 Bayview Avenue Toronto, Ontario M4N 3MS Tel: (416) 978-6382 Lab: (416) 480 6103 FAX: (416) 978-1938 Email: michael.ratcliffe@utoronto.ca http://sunnybrook.ca/team/member.asp?t=12&m=140&page=172

The antibody response to immunizing antigen results from the selective activation of antigen-specific B lymphocytes from within a pre-immune B cell repertoire. The major interest within the laboratory is to determine how this pre-immune B cell repertoire is generated and how its generation is controlled. We use the chick embryo as a model for the early stages of B lymphocyte development where the bursa of Fabricius provides a discrete site of B cell lymphopoesis and is accessible to experimental manipulation.

Early B cell development.
The formation of an intact surface immunoglobulin receptor complex is required for the early stages of normal B lymphocyte progression. We have demonstrated, using retroviral gene transfer in vivo, that expression of a truncated m chain that lacks antigen-binding capacity, in association with the Iga and Igb components of the complex, is sufficient to drive the early stages of B cell development. Moreover, the m chain can be replaced in its entity by a chimeric receptor in which the intracellular domain of Iga is fused to the extracellular and transmembrane domain of an irrelevant protein such as CD8. This suggests that the m chain in early B cell development simply functions as a chaperone to bring the Iga/b complex to the B cell surface and that surface Iga/b expression in the absence of receptor ligation is sufficient to provide signals for B cell maturation.

Antigen-dependent B cell development.
While a truncated m chain is sufficient to support early B cell development, expression of this receptor is not sufficient to support the normal redistribution of developing B cells in bursal follicles or their emigration to the periphery. The bursa is a gut associated lymphoid organ and gut derived antigen is taken up into bursal follicles. Redistribution and/or emigration of bursal cells may therefore require surface Ig receptor ligation as distinct from receptor expression. We are exploring this possibility by using retroviral gene transfer to introduce surface Ig receptors of defined specificity into developing B cell precursors.

Antibody diversification by gene conversion.
Antibody diversity in the chicken is generated by gene conversion events involving the replacement of Ig variable region sequences with sequences from upstream families of variable region pseudogenes. We have recently demonstrated that Activation Induced Cytidine Deaminidase, an enzyme required for somatic hypermutation in mouse and human B cells is expressed in chicken B cells that are undergoing somatic gene conversion. This observation, together with our demonstration that the border of gene conversion events is frequently associated with hotspots of somatic hypermutation suggests a linkage between the mechanisms of hypermutation and gene conversion.

RELEVENT PUBLICATIONS

1. Ratcliffe, M.J.H. Generation of immunoglobulin heavy chain diversity subsequent to cell surface immunoglobulin expression in the avian bursa of Fabricius. J. Exp. Med. 170: 1165-1173 (1989).
2. Benatar, T., Tkalec, L., and Ratcliffe, M.J.H. Stochastic rearrangement of immunoglobulin variable region genes in chicken B cell development. Proc. Natl. Acad. Sci. USA 89: 7615-7619 (1992).
3. Marmor, M.D., Benatar, T. and Ratcliffe, M.J.H. Retroviral transformation in vitro of chicken T cells expressing either ab or gd T cell receptors by reticuloendotheliosis virus strain T. J. Exp. Med. 177: 647-656 (1993).
4. Paramithiotis, E., Jacobsen, K.A., and Ratcliffe, M.J.H. Loss of surface immunoglobulin expression precedes B cell death by apoptosis in the bursa of Fabricius. J. Exp. Med. 181: 105-113 (1995).
5. Sayegh, C.E., Demaries, S.L., Iacampo, S. and Ratcliffe, M.J.H. Development of B cells expressing surface immunoglobulin molecules that lack V(D)J-encoded determinants in the avian bursa of Fabricius. Proc. Natl. Acad. Sci. USA 96: 10806-10811 (1999).
6. Sayegh, C.E., Drury, G. and Ratcliffe, M.J.H. Efficient antibody diversification by gene conversion in vivo in the absence of selection for V(D)J-encoded determinants. EMBO J. 18: 6310-6328 (1999).
7. Sayegh, C.E., Demaries, S.L., Pike, K.A., Friedmann, J.E. and Ratcliffe, M.J.H. The chicken B-cell receptor complex and its role in avian B-cell development. Immunol. Rev. 175: 187-200 (2000).