In-vitro analyses of mechanisms of B-cell development.

B-cell lymphopoiesis in vivo is very complex due to the influences of cooperating cells, cytokines and other receptor-ligand interactions which appear to occur developmentally at different 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 products of rearranged Ig-loci in the developmentally different cellular stages will be discussed.

Roles of IgH and L chains and of surrogate H and L chains in the development of cells of the B lymphocyte lineage.

Proteins expressed from productively rearranged H and L chain gene loci have been implied in the regulation of Ig gene rearrangements during B lymphopoiesis. However, recent findings suggest that early B cell development can occur without expression of surrogate L chain, without deposition of microH chains into membranes, without productive H chain gene rearrangements, and even without any rearrangements of Ig gene loci. In bone marrow, 2-5% of all B220-, sIgM-, c-kit+ cells are pro B cells that undergo differentiation from B220- via B220+, c-kit+, CD43+, clonable long-term proliferating pre B-I cells to B220+, c-kit-, CD43-, IL-2 receptor+ pre B-II cells and immature B cells, only to die by apoptosis in situ within less than 4 days. A membrane-bound complex of surrogate H chain (gp130/gp35-65) and surrogate L chain expressed on pro B and pre B-I cells has apparently no influence on this early development. Pre B-I cells carrying DHJH-rearrangements in reading frame (rf) II are counter-selected, probably because they can express an Ig-like complex of truncated DHJHC mu-protein and surrogate L chain, while pre B-I cells DHJH-rearranged in rf I or III are not suppressed. Immature sIg+ B cells, also from bcl-2 transgenic mice, can continue to rearrange L chain gene loci. Thus, mere membrane deposition of Ig, even with concomitant expression of bcl-2, terminates neither expression of RAG-1 and 2, nor secondary L chain gene rearrangements, nor does it allow the development of mature B cells. Membrane-bound expression of an Ig-like complex of microH chains and surrogate L chains appears to be needed to generate the 50-70 million pre B-II cells in bone marrow. However, the membrane-bound expression of Ig is mandatory for negative and positive selection of immature B cells. Autoantigens delete or anergize self-reactive B cells. We speculate that all mature, resting, primary antigen-reactive B cells in the periphery have been selected from immature sIg+ B cells by unknown antigens and have, thereby, changed their lifestyle from rapid death by apoptosis to longevity.

Influence of surrogate L chain on DHJH-reading frame 2 suppression in mouse precursor B cells.

DHJH rearrangements start in progenitor and precursor B cells and occur in three reading frames (rf). A strong bias for rf I has been noticed in murine and chicken antibodies, while the representation of rf II has been found suppressed both in peripheral as well as in precursor B cells. H chain gene loci DHJH rearranged in rf II are potentially capable of expressing a truncated DHJHC mu protein on the cell surface. Mice incapable of expressing this protein on the surface have previously been shown to have all reading frames represented in near equal frequency, suggesting that membrane-bound DHJHC mu protein is involved in the suppression of rf II. In this paper we show that suppression of rf II is not yet established in c-kit+ CD43+ IL-7/stromal cell-reactive pre-B I cells of fetal liver at day 15 of gestation, but becomes established when such precursor cell populations are expanded in vitro on stromal cells in the presence of IL-7. H chain gene loci using the DQ52 segment for rearrangements (which contains a stop codon in rf II, thus being unable to make DHJHC mu protein) do not show rf II suppression under these conditions. The same type of fetal liver-derived pre B-I cells from lambda 5 deficient mice also do not show rf II suppression after in vitro expansion. Bone marrow-derived pre B-I cells from normal mice assayed ex vivo and expanded in vivo show rf II suppression, while the corresponding pre-B I cells from lambda 5T mice do not. Collectively these experiments suggest that surrogate L chain is involved in rf II suppression. This may happen by inhibition of proliferation of pre-B cells expressing a complex of DHJHC mu protein and surrogate L chain.

Rearrangement and expression of kappa light chain genes can occur without mu heavy chain expression during differentiation of pre-B cells.

The kinetics of kappa light (kappa L) chain gene rearrangement and expression on mRNA and protein level has been studied with four stromal cell/IL-7 reactive, long-term in vitro proliferating pre-B cell lines and clones, two from fetal liver of normal mice and two from fetal liver of E microH-bcl-2 transgenic (bcl-2-tg) mice. These pre-B cell lines and clones are DJH-rearranged on both H chain alleles. Two of the clones harbor H chain rearrangements which do not allow the expression of VHDJH rearranged H chain genes as microH chain proteins. Upon removal of IL-7 from the pre-B cell cultures all four cell lines rearrange VH-DJH and VL-JL gene segments, loose the surface expression of c-kit, CD43, and surrogate light chain, as well as the capacity to be clonable on stromal cells in the presence of IL-7. Pre-B cells from normal mice die by apoptosis during differentiation, while those from bcl-2-tg mice do not. All four lines and clones express comparable levels of mRNA for microH and kappa L chains with the same time kinetics during 3 days of differentiation. However, only two of the four pre-B cell lines and clones express microH chain protein, whereas all four pre-B cell lines and clones express kappa L chain protein at comparable levels between 2 x 10(5) and 1.4 x 10(6) kappa L chain molecules per cell. These results suggest that microH chain expression is not mandatory for rearrangement and normal expression of kappa L chain genes when pre-B cells differentiate to B cells.

Immature surface Ig+ B cells can continue to rearrange kappa and lambda L chain gene loci.

Pro and pre B cells possess the long-term capacity to proliferate in vitro on stromal cells and interleukin 7 (IL-7) and can differentiate to surface immunoglobulin (sIg+) cells upon removal of IL-7 from the cultures. A key event in this differentiation is the extensive cell loss due to apoptosis. Because the proto-oncogene bcl-2 can promote cell survival, we established pre-B cell lines from E mu-bcl-2 transgenic mice. These pre-B cells have the same properties as those derived from non-bcl-2 transgenic mice except that they do not die by apoptosis. This allowed us to study the fate of newly formed B cells in vitro for a longer period of time. Here we show that early during the differentiation of pre-B cells, upregulation of RAG-1 and RAG-2 expression go hand in hand with rearrangements of the Ig gene loci. Moreover, the newly formed sIg+ B cells continue to express RAG-1 and RAG-2 and continue to rearrange L chain gene loci, even in the absence of proliferation, in an orderly fashion, so that kappa L+ sIg+ cells can become lambda L+ sIg+ or sIg- cells, whereas lambda L+ sIg+ cells can become sIg-, but not kappa L+ sIg+ cells. Thus, deposition of a complete Ig molecule on the surface of a B cell does not automatically stop the Ig-rearrangement machinery.

Lymphoproliferative disorders in an IL-7 transgenic mouse line.

A high incidence of severe lymphoproliferative disease was observed in a newly generated strain of mice carrying murine IL-7 as a transgene under the control of the E alpha (MHC class II) promoter. An analysis of the cells from lesions in these mice shows the selective expansion of cells at an early stage of B cell development and, more interestingly, expansion of cells phenotypically identical to the recently reported bipotent (B/macrophage) stem cell populations described in midgestation embryonic liver. Such cells can be propagated (and remain dependent upon) bone marrow feeder cell lines obtained from IL-7 transgenic mice. A molecular analysis of fresh and cultured cells reveals that the lesions are oligoclonal, or in rare cases monoclonal, and include clones of cells with unrearranged Ig heavy chain loci. These data suggest that IL-7 acts at multiple stages of B cell development. Furthermore cell lines derived from IL-7 transgenic mice may provide a novel source of rare factor-dependent bipotent stem cells.

B cell development in mice with a defective lambda 5 gene.

The surrogate light chain encoded by the two pre-B cell-specific genes VpreB and lambda 5 plays a critical role in B cell development of the mouse. It has been shown that targeted disruption of the lambda 5 gene results in a depletion of B220+ CD43- IgM-pre-B cells in bone marrow, and in a delayed appearance both of CD5+ as well as CD5- surface immunoglobulin (sIg)+ B cells in the periphery. In this report we show that DHJH-rearranged B220- and B220+, CD43+, c-kit+, sIgM- pro- and pre-B-I cells with long-term capacity to proliferate in vitro on stromal cells in the presence of interleukin-7 are present in normal numbers in the bone marrow of lambda 5 T/lambda 5 T mice at various ages. They express normal levels of VpreB mRNA but, in contrast to normal pre-B-I cells, do not express surrogate light chain on their surface. Pre-B-I cells from fetal liver and bone marrow of lambda 5 T/lambda 5 T mice differentiate with normal kinetics and in normal numbers to sIg+, mitogen-reactive B cells. These results suggest that the delayed generation of sIg+ B cells in the peripheral, mature compartments of CD5+ and CD5- cells could be accounted for by the daily production of approximately 5 x 10(5) sIg+ B cells from the pre-B-I cell pool in the absence of a normal pool of pre-B-II cells.

Changes in frequencies of clonable pre B cells during life in different lymphoid organs of mice.

Progenitor and precursor B lymphocytes with the capacity of long-term proliferation on stromal cells in the presence of interleukin-7 (IL-7) can be cloned ex vivo from fetal liver, neonatal blood, and spleen, and from adult bone marrow (BM) in frequencies that are similar in different strains of mice and that change with age. A wave of clonable cells appears before birth and disappears after birth in liver. Up to 2 weeks after birth, high frequencies of clonable cells are present in spleen but become undetectable at 6 to 8 weeks of age. In BM, high frequencies (1 in 50) of clonable cells are present early after birth, and then decrease continuously to 10- to 20-fold lower levels at 6 to 8 months of age. The earliest clonable cells have at least part of their IgH genes in germline configuration. Clones of pro/pre B cells apparently continue to rearrange DH to JH segments on both chromosomes. Rearrangements without insertion of N-sequences at the DHJH joints are found in fetal liver, while DHJH joints in pre B cells of spleen and BM throughout life have N-regions inserted. At least half of all primary pre B-cell clones develop mitogen-reactive B cells after differentiation to sIg+ B cells. Clonable pro and pre B cells are enriched in B220- c-kit(low) as well as in B220+ c-kit+ and B220+ CD43+ cell populations of BM. The frequencies of clonable cells in the B220- c-kit(low) BM cell population decrease 10- to 20-fold during 8 months of life, while those in the B220+ c-kit+ population remain constant, although their absolute numbers drop 5- to 10-fold during that time. All long-term proliferating clones express the surrogate L chain VpreB/lambda 5 as well as c-kit and CD43 on all cells. The number of total clonable pro and pre B cells is at best 10% of the number of cells required to produce the estimated daily output of 5 x 10(7) B-lineage cells in a mouse. This suggests that the production of a relatively constant number of B cells during adulthood may be effected by precursors, which are not clonable on stromal cells and IL-7 with long-term proliferative capacity. On the other hand, BM transplantation experiments indicate that a mouse retains B220- progenitors throughout life, from which pre B and B cells can be generated in old mice in frequencies characteristic of young mice.

The surrogate light chain in B-cell development.

The proteins encoded by the VpreB and lambda 5 genes associate with each other to form a light (L) chain-like structure, the surrogate L chain. It can form Ig-like complexes with three partners-the classical heavy (H) chain, the DHJHC mu-protein, or the newly discovered p55 chain; these are expressed on the surface of pre-B cells at different stages of development. Here, Fritz Melchers and colleagues review the structures of the VpreB and lambda 5 genes in mouse and their relatives in humans, describe their pattern of expression, and speculate on their possible evolution and functions.