Michael Ratcliffe

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 μ chain that lacks antigen-binding capacity, in association with the Igα and Igβ components of the complex, is sufficient to drive the early stages of B cell development. Moreover, the μ chain can be replaced in its entirety by a chimeric receptor in which the intracellular domain of Igα is fused to the extracellular and transmembrane domain of an irrelevant protein such as CD8. This suggests that the μ chain in early B cell development simply functions as a chaperone to bring the Igα/β complex to the B cell surface and that surface Igα/β expression in the absence of receptor ligation is sufficient to provide signals for B cell maturation.

Antigen-dependent B cell development.
While a truncated μ 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 related receptors of defined specificity into developing B cell precursors, using fusions of the diversity region of lamprey VLR receptors with the chicken truncated μ. While expression of such VLR:Tμ receptors with specificity for the self-protein hen egg lysozyme demonstrates negative selection of self-reactive B cells, expression of a VLR:Tμ receptor with specificity for phycoerythrin demonstrates positive selection of specific B cells when phycoerythrin is introduced into the bursa.

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. The observation 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, 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. We are currently interested in the evolution of distinct mechanisms for the generation of antibody diversity following immunoglobulin gene rearrangement.

Recent Publications

1. 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).
2. 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).
3. 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).
4. Pike, K.A. and Ratcliffe, M.J.H. Cell surface immunoglobulin receptors in B cell development. Semin. Immunol. 2002. 14: 351-358.
5. Pike, K.A., Baig, E. and Ratcliffe, M.J.H. Distinct roles for Igα and Igβ in chicken B cell development. Immunol. Rev. 2004. 197: 10-15.
6. Pike, K.A., Iacampo, S., Friedmann, J.E. and Ratcliffe, M.J.H. The cytoplasmic domain of Igα is necessary and sufficient to support efficient early B cell development. J. Immunol. 2004. 172: 2210-2218.
7. Pike, K.A. and Ratcliffe, M.J.H. Dual requirement for the Igα immunoreceptor tyrosine-based activation motif (ITAM) and a conserved non-Igα ITAM tyrosine in supporting Igαβ-mediated B cell development. J. Immunol. 2005. 174: 2012-2020.
8. Ratcliffe, M.J.H. Antibodies, immunoglobulin genes and the bursa of Fabricius in chicken B cell development. Dev. Comp. Immunol. 2006. 30: 101-118.
9. Kothlow, S., Schenk-Weibhauser, K., Ratcliffe, M.J.H. and Kaspers, B. The RCAS retroviral gene transfer technology: an efficient system for functional in vivo studies with soluble proteins in the chicken. Mol. Immunol.2010. 47: 1619-1628.
10. Ratcliffe, M.J.H. and Härtle, S. B cells, the bursa of Fabricius and the generation of antibody repertoires. In Avian Immunology 2nd Edition, Schat, K.A., Kaspers, B. and Kaiser, P. (Eds.) Elsevier Ltd. 2013. pp65-89.

Appointments

Vice-Provost and Dean of Arts at Trinity College