seminars in IMMUNOLOGY, Vol 7, 1995: pp 155-167 In-vitro analyses of mechanisms of B-cell development Antonius Rolink, Paolo Ghia, ulf Grawundq Dirk Haasner; Hajime Karasuyama, Christian Kalbereq Thomas Winkler and Ftitx Melchers B-cell lymphopoiesis in vivo is vq complex due to the influences of cooperating cells, qtokines and other receptor-ligand interactions which appear to occur develojnnentally at daffment cellular stages. Therefore in-vitro models will help to unravel this complex situation. Here, we review our and others’ work on in-vitro models of B-cell development. The role of stromal cells, cytokines, surrogate light chain and wducts of rearranged Ig-loci in the o!evela,bmentally different cellular stages will be discussed. Key words: pre-B cells / suomal cells / interleukins / Ig-rearrangements / surrogate light chain THE DIFFERENTIATION of precursor cells along the pathway of B-lymphocyte development from the most immature, B-lineage-committed progenitor to the mature, antigen-sensitive, surface immunoglobulin (sIg) expressing cell can be followed: (1) by changes in the expression of intracellular and surface-bound molecular markers; (2) by the status of Ig gene rearrangements in the Ig heavy (H) and light (L) chain gene loci, and their expression’ as RNA and protein products; (3) by the capacities of cells to populate severely immunodeficient (SCID, RAGlT, RAG2T) mice and (4) by growth and differentiation properties of cells, isolated ex vivo, in tissue culture. In-vivo analyses in bone marrow of normal and of immunodeficient, mutant mice have defined the major cellular stages of at least two possible pathways of pre-B-cell development’,* (Figure 1). Growth of the different cellular stages in tissue culture and the differentiation to mature stages in vitro under the influence of cooperating cells, cytokines and ligand- specific antibodies with normal and mutant precursor B cells have allowed us to study some of the molecular mechanisms which influence the differentiation of From the Base1 Institute for Immunology, Grenzachtrasse 487 CH4005 Basel, Swikerland 01995 Academic Ptess Ltd 10445323/95/030155 + 13$8.00/O B-lineage cells. In particular, they have clarified the role of IgH and L chains, and of surrogate H and L chains on the one side, and the role of cell-cell contacts and cytokines on the other, in this process. This paper reviews the in-vitro experimental approaches which we have used to study the mecha- nistic aspects of B-lymphocyte development. The Ig heavy (H) chain gene locus on chromosome 12 of the mouse consists of probably > 100 VH gene segments, 15 DH gene segments, and four JH gene segments. ‘** Based on sequence homology, the 15 Dn segments are grouped into three different families. The Dn family comprises of two segments (Dnls.i and Dn1s.2), the D,, family includes 12 members (Dsrs.i-DSPsrs.1t plus Dsz~.~), while the DQ5* segment ‘constitutes’ a separate family. During differentiation of progenitors (pro B cells) to precursors of the Elineage pathway (pre B I cells; for nomenclature see ref 5), DH segments are first rearranged to Jn segments. This is followed by rearrangements of V, to Dr.&-segments. Vn-segments are chosen from more than 100 segments grouped in 12 families. The VH gene segments’ repertoire expressed in developing fetal and neonatal B cells, and in B cells during regeneration after bone marrow transplantation is biased towards Vr+-segments located in the mouse VH cluster near the 3’ end (represented by the VH’7183 and VHQ52 families). The adult repertoire appears to be randomized, so that in the first approximation, individual VKgene family mem- bers are expressed proportionally to the size of the corresponding VKgene family.6S7 One sIg+ B cell expresses only one productively VHDKrearranged IgH gene locus, while the com- plete, productive rearrangement of the other allele appears to be disfavored. The molecular mechanisms underlying the allelic exclusion are presently unknown. The mouse Ig XL chain gene locus consists of 50 to 300 V, segments, five Jx segments (one pseudo gene- segment J,3) and one C, exon**’ while the Igk chain locus comprises three functional C, exons, each with one Jh segment. JhlGl and J&& use the same V,, segment, while Jh2C~2 and the nonfunctional J&l4 155