The function of the |iv|/LC complex and its ligand

The function of the pvj/LC complex has been investigated by creating mice that do not express the molecule or that express a transgenic rearranged p allele. The effects of these changes on B cell development were then studied.

Targeted disruption of the pm exon produced a pmT mouse, which has a complete block of B cell development at the transition from the pre-BI to pre-BII cell stage About 6% of splenic B cells in the heterozygous mice produce p heavy chain from both the mutant and the wild type alleles while only 0.3% of B cells in normal mice express both heavy chain alleles This indicates that surface expression of pm heavy chain mediates heavy chain allelic exclusion. A rearranged pm human transgene causes allelic exclusion.

demonstrated by the fact that no murine |i heavy chains are produced by cells that express

the human protein. A rearranged ps transgene does not have this effect The

PH/LC complex therefore appears to mediate immunoglobulin heavy chain allelic exclusion.

The RAG-2T mouse (produced by targeted disruption of the RAG-2 gene) cannot rearrange immunoglobulin genes and has a complete block in B cell development at the pro-B cell stage Introduction of a p transgene into the RAG-2T mouse allows expression of the pv)/LC complex enabling the mouse to generate CD25+ and CD43- CD45R+ large, cycling pre-BII cells This, together with the developmental block in the pmT mouse at the pre-BI stage, indicates that the pvj/LC complex is required for pre- BI cells to become pre-BII cells. It then permits amplification of large pre-BII cells and generation of small resting pre-BII cells.

Targeted disruption of the X5 gene also prevents expression o f the pvj/LC complex, but there are significant differences in B cell development between pmT and X5T mice. The ^5T mice have a partial block in B cell development at the pre-BI cell stage, with the numbers of small pre-B cells being reduced by a factor of at least 40 In the first few weeks after birth they have a 10- to 30-fold reduction in numbers of splenic B cells compared to littermate controls. By the age of 4 months the levels are only 3- to 5-fold lower than controls. The B cells are allelically excluded and respond to antigen.

Furthermore, pro-B cells and pre-BI cells from the X5T mouse proliferate and

rate as normal cells The leakiness of the X5T mouse raises questions about the importance of the pn/LC complex and the signal it delivers to the cell.

Immunoglobulin k light chain genes rearrange slowly from the pro-B cell stage of normal,

pmT, JrT and X5T mice, irrespective of whether the heavy chain gene is rearranged or

expressed As a result, a small number of progenitors in the bone marrow of

normal mice achieve a productive light chain rearrangement before a productive heavy chain gene rearrangement. On productive rearrangement of a heavy chain gene they pass directly from the pro-B to the B cell stage. This pathway of B cell generation is proposed to give rise to about 5% of B cells and would account for the slow generation of B cells in

the X5T mouse Allelic exclusion of X5T B cells is presumably mediated by IgM when

one heavy chain allele is productively rearranged. This pathway is disabled in the pmT mouse, as no heavy chain expression is possible. The phenotype of the X5T mouse is thus compatible with a role for the pvj/LC complex in allelic exclusion, as well as in the production and proliferation of pre-BII cells. The pvj/LC complex is not required however for light chain gene rearrangement and expression in normal B cell development

B cell precursors from mice with targeted disruption of p, RAG-2 and X5 genes appear to develop more normally in vitro than in vivo. In the stromal cell/ IL-7 culture system, lines that cannot make productive heavy chain rearrangements are still able to rearrange the k

gene and differentiate to the small pre-BII cell stage on withdrawal of IL-7 insofar as they stop proliferating, acquire CD25 and k germline transcripts, and lose c-kit, X5 and CD43

vitro development of ^5T pro-B and pre-BI cells is also puzzling. Other differences have been noted between in vivo and in vitro B cell development. For example, pre-BII cells that develop from normal pro-B cell clones in vitro do not express the pvj/LC complex on their cell surface and fail to proliferate just prior to light chain gene rearrangement The in vitro culture system may lack elements responsible for positive or negative selection, or may only support a slow rate of development of even normal cells, making differences between normal and 15T or RAG-2T cells unnoticeable.

An alternative explanation for the in vitro growth ofX5T pro-B cells and in vivo X5T leakiness is that a complex of p with VpreB or the newly described VpreB-like molecule 8HS-20 may perform the function of the pyLC complex in X5T B cell precursors

8HS-20 is a B lineage-specific gene expressed in murine bone marrow and weakly in spleen. Its product has amino acid homology to VpreBl and it is expressed in the same precursor B cell lines and at the same time as VpreB and X5. Its size is between 13.5 and

16 kD and it is associated with p heavy chain in pre-B cell lines. Its function is unknown. Some human pro-B and pre-B cell lines contain a 15 or 16 kD protein that is

immunoprecipitated with the v|/LC and is cross-reactive with antibodies specific for

VpreB, which may represent the human equivalent of 8HS-20 Another candidate for

replacement of the function ofX5 in the À5T mouse is surrogate k chain. The 15 kD JCk

chain, expressed in normal and leukaemic human B cell precursors, derives from transcription of the germline k gene and the protein has been shown to associate

Expression of the pyLC complex by pre-B cell lines is reported to trigger light chain gene rearrangement, but light chain gene rearrangement in the bone marrow does not require

pvj/LC complex expression 5i>’62,i83,]84 more likely that the p\|/LC complex promotes survival of B cell precursors that will go on to rearrange light chain genes. The pvj;LC complex may also transduce a signal that accelerates the rate of light chain gene

rearrangement. Ligation of the pyLC complex in vitro does not induce CD25 or k light

chain expression by normal cells, nor cause a decrease in c-kit or v|/LC expression, indicating that signalling through the |liv)/LC complex does not trigger differentiation of pre-B cells

It has been postulated that the pyLC complex may be involved in negative selection of strongly self-reactive p heavy chains. In this case the signal received through pvj/LC would initiate cell death or anergy. Incubation of human and mouse pre-B cell lines with anti-ij/LC or anti-p antibodies results in down-regulation of the p\)/LC complex, but appears not to affect growth or survival of the cells. Treatment of neonatal mice for 1 to 2 weeks with antibody raised against the vj/LC causes no discernible effect on B cell

development, therefore ligation of the pvj/LC complex probably does not generate a negative signal

Another suggestion is that the pv|/LC complex on pre-B cells and IgM on immature B cells may be positively selected for weak self-reactivity and polyreactivity This would provide an explanation for some puzzling observations, such as the preferential usage of certain Vh segments in pre-B and B cells, the existence of so many germline Vh segments

with self-reactivity and the high prevalence of weak autoantibodies at all ages from the fetus to the adult. The advantage of low affinity self-reactive antibody specificities, may be their polyreactive quality for binding to invading pathogens. A strongly self-destructive immune response would be prevented by the absence of high affinity T cell help and by the process o f affinity maturation which would steer the immune response away from self-reactivity and towards a high affinity for pathogen.

The ligand for the pij/LC complex is unknown. The stromal cells of the bone marrow are required during early stages of B cell development in the mouse and may express the ligand of the pvj/LC complex However, once pre-B cells express p heavy chain they no longer require stromal cell contact for survival This argues against a stromal cell- bound ligand for the pv|/LC complex, or suggests that the signal is not required for cell survival. Furthermore, transfected A-MuLV lines undergo k gene rearrangement on

expression o f the pvj/LC complex in the absence of stromal cells The ligand may be

expressed by B cell precursors or in culture medium. The ligand could equally be a soluble factor or extracellular matrix.