B cell development during embryogenesis

Our previous results confirm the hypothesis that fetal liver (FL) and bone marrow (BM) lymphopoiesis follow distinct pathways and could indicate that HSC present in the P-Sp/AGM, FL and BM are distinct cell types that are independently generated. Alternatively, we could hypothesize that P-Sp/AGM derived HSC transit to the FL and then to the BM. This transition and the consequent diversity of signals received from the different stromal environment would modify and thereby condition the response of the hematopoietic precursors to growth factors.

To distinguish between the two possibilities, we will analyze the frequencies of TSLP and Flk2/Flt3 ligand responding HSC present in the P-Sp/AGM, FL and BM in comparison with the response to IL-7. This experiment will allow us to confirm the broad reactivity of embryonic precursors versus the more restricted pattern of response in their adult counterpart. We will determine the frequency of TSLP and Flk2/Flt3 ligand responding cells isolated from donor derived BM HSC from recipients transplanted with P-Sp/AGM HSC. This experiment will test the possibility of independent HSC generation versus changes in reactivity resulting when hematopoietic precursors of embryonic origin transited to the BM.

Adult IL-7-/- mice show undetectable levels of CD19+ cells in the BM. CD19 expression is controlled by the transcription factor Pax-5, responsible for the irreversible engagement of precursors in the B lineage pathway of differentiation. This observation suggests that IL-7 has a role at stages prior to lineage commitment. The decrease and eventual disappearance of committed B cell precursors, in the BM of IL-7-/- mice, contrasts with a constant number of T cell precursors in the thymus throughout life. We are presently analyzing different compartments of common lymphoid progenitors (CLP) in the IL-7-/- mouse, at different ages, and determine whether the absence of B lineage cells is correlated with the absence of CLP. Preliminary evidence indicates that the number of CLP present in the BM of IL-7-/- mice decreased with age in a curve that resembles that of B cell precursors in the BM. This results indicates that a precursor other than a CLP is likely to be responsible for the thymic input in these animals. We are presently characterizing different fractions of BM cells and using a combination of in vitro and in vivo approaches to identify the nature of the thymic migrants in the absence of IL-7. These experiments will further elucidate the role of interleukins in hematopoietic lineage commitment.

T cell development

T cell subsets (ab and gd) are generated in the thymus throughout life. The rules that govern the development of these two cell types remain ill-defined, although it is known that ab T cells can develop in the absence of IL-7 and require Notch 1 triggering, while ab T cells are absent in IL-7-/- mice due to impairment in the rearrangement process. We have determined that certain subsets of gd T cells produce IL-4 and express low levels of the Thy1 marker are oligoclonal and preferentially generated during foetal and neonatal life (Azuara et al J. Immunol 165:42, 2000 – Grigoriadou et al J. Immunol. 169:3736, 2002). To better define the rules that condition development of this gd T lymphocyte subset and to investigate the signals involved in ab versus gd commitment, we recently generated a stromal cell line (OP9) constitutively expressing delta-like-1 (a Notch 1 ligand). The OP-9-delta-1 cell line allows the development of ab and gd T lymphocytes from uncommitted precursors, in vitro. As an alternative approach, we also have generated OP9 cells that constitutively express delta 4, another Notch 1 ligand. This system (which we have already validated in the laboratory), will allow us to test the role of different signals and cytokines in T cell development. This study will be complemented by tests of T cell development in fetal thymic organ cultures and in vivo reconstitutions by grafts of embryonic thymic lobes from wild type and various mutant mice (interleukin and Notch 1 receptors) in adult recipients.

Web site created by Marie-Christine Vougny (04/2004)