B Cell Lymphogenesis in the Fetal Piglet

University Of Iowa, Iowa City IA

Investigators

Abstract

The heavy chain variable region (HVR) of swine antibodies is a surprisingly and comparatively simple system that is encoded by ~ 20 highly homologous VH genes, one single JH and apparently only 2 DH segments. Only 4 or 5 of these VH genes, clustered at the 3' end of the locus, are used in fetal and early neonatal life in non-mutated form. This simple system operates in a species in which it is believed that fetal repertoire development occurs in the absence of maternal regulatory factors and environmental antigens. Since combinatorial diversity in the HVR is restricted to 8-10 possibilities, i.e. <0.3% of that in mice and humans, swine may utilize other mechanisms to diversify their antibody repertoire. Considering that an extensive Vl repertoire has been reported for sheep, this project will determine whether extensive light chain diversity or gene conversion compensate for the restricted combinatorial diversity of the HVR in swine. Sequence analysis of VDJs from individual cells recovered by micromanipulation or single cell sorting, will test whether this species uses gene conversion. Light chain diversity will be addressed by cloning and characterizing the Vk and Vl loci and then characterizing the Vk and Vl repertoire used during fetal life. The 30-day fetal liver (114 day gestation) is the first site at which VDJ rearrangements can be found, of which an astonishing 95% are productive. This conflicts with the concept that combinatorial joining is random. Since the proportion of productive rearrangements progressively declines to "textbook" levels in older fetuses, this raises the question of whether early fetal liver B-cells comprise a distinct subset. Since pro-B-cell-like VDJ rearrangements also occur in the thymus, this project will test the hypothesis that B-cells developed in fetal liver, bone marrow and thymus, may each generate different B-cell subsets. Finally, the role of the ileal Peyers patches (IPP) of the fetal pig remains unresolved. Are they a site for: (a) primary B-cell development? (b) antigen-independent diversification of primary rearrangements? or (c) development of the mucosal immune system? It would be over-ambitious to think that this issue could be resolved in the proposed project period. However, this project is designed to gain greater insight into the role of the IPPs by analyzing the clones derived from individual follicles in the IPP. This project also addresses several testable hypotheses about B-cell lymphogenesis stemming from observations made during previous NSF-funded projects. These hypotheses challenge certain paradigms of immunoontogeny that are based on the mouse model. Addressing these hypotheses will not only provide a clearer picture of B-cell lymphogenesis in this non-traditional species but may also broaden the perspective of B-cell lymphogenesis in all species.