Development of the immune system from stem cells

Clonable, transgenetically mutable, transplantable precursor B cells

PreB-I cells from wildtype and a wide variety of mutant mice, which are DH-Jh-rearranged at both alleles of their Ig-heavy(H) chain loci, can proliferate 'in vitro' on stromal cells in the presence of interleukin (IL)-7 for very long periods of time [16]. Some of the cell lines have been carried for over 300 divisions without losing their status of differentiation as preB-I cells Since the efficiency of plating of these preB-I cells is near 100%, it is very easy to derive large numbers of individually DH/DH-rearranged, i.e. genetically marked preB cell clones, from which the differentiated progeny of cells can unambiguously identified as products of the original clone. Since these clones can also be retrovirally transfected with high efficiencies (e.g. with a gene encoding a fluorescent protein) they are suitable recipients of any transgenes with potential activities in B cell development and responsiveness. Neither the thymus nor the bone marrow of the recipient mice are populated from the wildtype-preB cell clones, nor are mature T cells or myeloid cells detectable. Hence wildtype-preB cells do not home back to their original sites in the bone morrow, and they appear committed to the development of one wave of mature, peripheral, long-lived B cells [16]. Transplanted into SCID or RAG-deficient hosts they mainly populate B1 compartments, which can respond to T cell-independent antigens. When helper T cells are co-transplanted conventional B cell compartments are also populated. The hosts become responsive also to T cell-dependent antigens in germinal centers and develop hypermutated, Ig-class-switched antibody responses.

Clonable precursors of myeloid and T lymphoid cell lineages

PAX-5-deficient mice are blocked in B cell development after the preB-I cell stage but develop all other hematopoietic cell lineages normally [18, 19]. PAX-5-deficient preB-I cells, like their wildtype counterparts, are clonable and proliferate for several hundred divisions on stromal cells in the presence of IL-7 [20]. 'In vitro' they differentiate to NK-cells and to different types of myeloid cells, and the direction of differentiation is induced by selective environmental influences of cytokines and cell-cell contacts to which they are induced after the removal of IL-7 [19, 21]. 'In vivo', after transplantation into sublethally irradiated hosts, erythrocytes, all lineages of myeloid cells, NK-cells and T cells develop with varying efficiencies [22, 23]. Thymocytes and peripheral T cells are generated at normal rates in normal numbers at normal sites. Importantly, the PAX-5-deficient preB cells home back to the bone marrow, from where they can be re-isolated and again grown on stromal cells in the presence of IL-7 - and this 'ex vivo/ in vitro' propagation can be repeated for several passages. In summary, with single, genetically DHJH/DHJH-marked and transgenically (e.g. GFP-markable) preBI cell clones from wild-type and from PAX5-/-mice, we are now able to follow homing, self renewal and differentiation into practically all hematopoietic and lymphopoietic cell lineages, in this laboratory specifically to B cells [14]. It should also allow to follow the cooperation of wildtype-derived B cells with PAX5-/--derived T cells and antigen presenting cells in transplanted hosts, and to test immune responses to a variety of experimental and naturally occurring antigens in histocompatible, wildtype, mutant or transgenically altered, reconstituted immune systems of the mouse.