Development of the immune system from stem cells

Differentiation of clonable and transplantable precursors of B-, and of myeloid, NK- and T-lymphoid lineages from embryonic stem cells of the mouse

A major limitation of pluripotent hematopoietic stem cells and of clonable, transplantable precursor of B-, and of myeloid-, NK- and T-lymphoid lineages in analyses of the function of genes expressed during hematopoietic development and in immune responses of mature cells of the immune system is our apparent inability to stably transfect and homologously recombine mutated forms of genes at their proper, or at chosen sites of the mouse genome. This is in contrast to mouse embryonic stem cells, in which targeted integration of a gene on both alleles is an established method, which is used for the generation of homologously recombined, targeted mutant mouse strains. Therefore, we intend to develop efficient experimental protocols to generate clonable, transplantable pre B cell lines from wildtype and from PAX5-/- embryonic stem cells by' in vitro' differentiation. On the one side we plan to introduce regulatable forms of transgenes, which might favor autonomous development as well as easy detection of pHSC and lymphoid/myeloid progenitors [14]. On the other side recent publications have indicated that a favorable environments can be generated for hematopoietic development by certain stromal cell lines expressing ligands for the selection and preferred differentiation of hematopoietic cell development [24, 25]. If successful these tissue culture procedures should allow for a rapid screening of a much larger number of candidate genes [26] with potential functions of pHSC, of progenitors and of mature cells of the innate and adaptive immune system, as well as for a functional analysis of genes in positive and negative responses of the immune system.

Retroviral tagging of genes active in the development of hematopoietic cell lineages and immune respnoses

Using retroviral vectors as single insertions into the mouse genome to detect endogenous promotors or polyadenylation sites of actively transcribed genes we plan to identify those genes which are active in DHJH-rearranged preB-I cells from wildtype and from PAX5-/- mice. Puromycin resistance transferred by the vectors is used to select stabily transfected preB cell lines. We follow the expression of tagged, active genes by the green fluorescent protein-encoding reporter gene also transferred by the vector as wildtype preB-I cells are induced to differentiate to B-lineage lymphocytes in vitro and in vivo. We will do the same with PAX5-/- preB-I cells as they are induced to differentiate to natural killer cells and to myeloid cell lineages in vitro and in vivo, and to T-lymphoid lineage cells in vivo. We will also attempt to identify genes, which are not active in preB-I cells but are only turned on when they differentiate along these pathways of differentiation. Special attention will be given to dominant-negative insertional mutations, which might become detectable as a loss-of-function in these hematopoietic differentiation lineages. This mutational analysis is intended to complement the efforts to define the molecular programs of hematopoietic development currently undertaken by subtractive hybridizations of cDNA libraries of different cellular stages of this differentiation, by chip analyses of such cDNA libraries, and by mutational analyses with embryonic stem cells, or of Zebra fish, developing into the hematopoietic cell lineages. So far two retroviral vectors have been constructed which infect mouse preB-I cell lines particularly well. Approximately one in 2x10e4 to 1x 10e5 puromycin-resistant preB cell express GFP. The GFP-expressing cells have been cloned and were shown to have the vector integrated into specific sites of the mouse genome. Several integration sites have already been characterized. The project is being funded at the Biozentrum of the University of Basel, Department of Cell Biology, to F.M. by the Swiss National Founds and is a collaboration with Drs Ulf Grawunder and Ton Rolink at the Department of Molecular Immunology, Pharmazentrum of the University of Basel, Switzerland.

Biochemical and functional characterization of the mouse preB cell receptor

PreB cell receptors (preBcR's) are complexes of mu-heavy chains and surrogate light (SL) chain, the latter being composed of the VpreB-and lambda 5-proteins [13, 27]. Single lambda 5-deficient, double VpreB1-and VpreB2-deficient, and triple VpreB1-, VpreB2-and lambda 5-deficient mice have been generated, and all of them appear deficient in signal transduction from the preBcR, so that preBII do not expand by proliferation. However allelic exclusion of H chains expressed from the second H chain allele is not affected [28]. Recent mutational analysis of the non Ig-like, aminoterminal portion of the lambda 5 gene have shown that this part of the SL-chain is responsible for a preB cell-autonomous, constitutive signalling of the preBcR [29]. Future mutational analyses will attempt to define the role(s) of the non Ig-like, carboxyterminal portion of the VpreB-genes in preBcR signalling, and will analyze the biochemical changes of preBcR leading to the apparently pre B cell autonomous, pre BcR- ligand- independent function of this receptor.