Fritz Melchers

A critical role of λ5 protein in B cell development.

The lambda 5 gene is a homolog of immunoglobulin J lambda-C lambda genes, expressed specifically in immature B-lineage cells. Lambda 5-encoded molecules form membrane complexes with mu or D mu proteins in association with an additional protein specifically expressed in immature B cells that is encoded by the Vpre-B gene. We have generated mice in which the lambda 5 gene is inactivated by targeted gene disruption in embryonic stem cells. In these mice, B cell development in the bone marrow is blocked at the pre-B cell stage. However, the blockade is leaky, allowing B cells to populate the peripheral immune system at a low rate. These cells are allelically excluded and able to respond to antigen.

The SCID but Not the RAG-2 Gene Product Is Required for Sμ–Sε Heavy Chain Class Switching

We have investigated the capacity of precursor B cells from normal (BDF1) and V(D)J recombinase-deficient (RAG-27) or defective (SCID) mice to be induced by a CD40-specific monoclonal antibody and IL-4 to epsilon H chain gene transcription and to S mu-S epsilon switch recombination. In differentiating precursor B cells from all three strains of mice, the development of similar numbers of CD19+, CD23+, CD40+, and MHC class II+ expressing B lineage cells and similar levels of epsilon H chain gene transcription were induced. Efficient S mu-S epsilon switching occurred in normal and RAG-2-deficient, but not in SCID, precursor B cells. Thus, the transcription of the epsilon H chain is independent of the RAG-2 and the SCID gene product, while the S mu-S epsilon switch recombination requires the SCID gene-encoded DNA-dependent protein kinase, but not the RAG-2 protein.

Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors

The mechanisms controlling the commitment of haematopoietic progenitors to the B-lymphoid lineage are poorly understood. The observations that mice deficient in E2A and EBF lack B-lineage cells have implicated these two transcription factors in the commitment process. Moreover, the expression of genes encoding components of the rearrangement machinery (RAG1, RAG2, TdT) or pre-B-cell receptor (λ5, VpreB, Igα, Igβ) has been considered to indicate B-lineage commitment. All these genes including E2A and EBF are expressed in pro-B cells lacking the transcription factor Pax5 (refs 5,6,7). Here we show that cloned Pax5-deficient pro-B cells transferred into RAG2-deficient mice provide long-term reconstitution of the thymus and give rise to mature T cells expressing α/β-T-cell receptors. The bone marrow of these mice contains a population of cells of Pax5-/- origin with the same phenotype as the donor pro-B cells. When transferred into secondary recipients, these pro-B cells again home to the bone marrow and reconstitute the thymus. Hence, B-lineage commitment is determined neither by immunoglobulin DJ rearrangement nor by the expression of E2A, EBF, λ5, VpreB, Igα and Igβ. Instead, our data implicate Pax5 in the control of B-lineage commitment.

Continued RAG expression in late stages of B cell development and no apparent re-induction after immunization.

Models of B-cell development in the immune system suggest that only those immature B cells in the bone marrow that undergo receptor editing express V (D)J -recombination-activating genes (RAGs). Here we investigate the regulation of RAG expression in transgenic mice carrying a bacterial artificial chromosome that encodes a green fluorescent protein reporter instead of RAG2 (ref. 4). We find that the reporter is expressed in all immature B cells in the bone marrow and spleen. Endogenous RAG messenger RNA is expressed in immature B cells in bone marrow and spleen and decreases by two orders of magnitude as they acquire higher levels of surface immunoglobulin M (IgM). Once RAG expression is stopped it is not re-induced during immune responses. Our findings may help to reconcile a series of apparently contradictory observations, and suggest a new model for the mechanisms that regulate allelic exclusion, receptor editing and tolerance.

Down-regulation of RAG1 and RAG2 gene expression in preB cells after functional immunoglobulin heavy chain rearrangement.

Two waves of immunoglobulin gene rearrangements, first of the heavy, then of the light chain chain gene loci form functional immunoglobulin genes during B cell development. In mouse bone marrow the differential surface expression of B220 (CD45R), c-kit, CD25, and surrogate light chain as well as the cell cycle status allows FACS separation of the cells in which these two waves of rearrangements occur. The gene products of two recombination activating genes, RAG1 and RAG2 are crucial for this rearrangement process. Here, we show that the expression of the RAG genes is twice up- and down-regulated, at the transcriptional level for RAG1 and RAG2, and at the postranscriptional level for RAG2 protein. Expression levels are high in D-->JH and VH-->DJH rearranging proB and preB-I cells, low in preB cells expressing the preB cell receptor on the cell surface, and high again in VL-->JL rearranging small preB-II cells. In immature B cells expressing on the cell surface RAG1 and RAG2 mRNA is down-regulated, whereas RAG2 protein levels are maintained. Down-regulation of RAG1 and RAG2 gene expression after productive rearrangement at one heavy chain allele might be part of the mechanisms that prevent further rearrangements at the other allele.

Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo

Cell lines and clones were established from PB76‐positive mouse fetal liver at day 13 and 14 of gestation, which proliferated with division times of a day in serum‐substituted cultures under the stimulatory influence of adherent stromal cells and the cytokine IL‐7 for periods longer than half a year. These lines expressed varying levels of the B lymphocyte lineage related markers PB76, B220, BP‐1, VpreB and lambda 5, but no surface Ig or MHC class II molecules. All clones expressed PB76, VpreB and lambda 5 in a high percentage of cells, while B220 and/or BP‐1 expression was low or undetectable in some. A cell line, and several clones established from it, all had kappa and lambda light chain genes in germ‐line configuration. Either one or both of their H‐chain‐gene containing chromosomes carried a DH to JH. These pre B cell lines and clones could be induced to VH to DH and VL to JL rearrangements. This resulted in the development of varying percentages of sIg‐positive surface, MHC class II negative, LPS‐reactive B cells within 2–3 days, in the absence of contacts with stromal cells and/or IL‐7. When injected into SCID mice, the cultured pre B cells populated the spleen of these mice to 5% with surface Ig‐, MHC class II‐positive LPS‐reactive cells for greater than 25 weeks. The long‐term in vitro proliferative capacity of these DH‐JH rearranged pre B cell clones makes them major candidates for committed stem cells of the B lineage.

Molecular and cellular origins of B lymphocyte diversity

The diverse repertoire of antibodies or immunoglobulins (Igs) in the immune system is created by B lymphocytes. The specificity of an lg for an antigen is made up by three complementarity-determining regions (CD%) in the variable (V) regions of the heavy(H) and light (L) chains. Each B lymphocyte displays a single combination of H and L chains with a unique set of CDRs, out of millions of possible combinations in the total repertoire of lg molecules. At the molecular level, the diversity of antigen-binding V regions of lg molecules is generated by successive rearrangements and potentially by replacement events at both alleles of the H and L loci, as well as by N and P regions in H but not L chains, which are introduced in the joining reaction. At every step in this generation of diversity, it appears that cells of the B lymphocyte lineage check whether they have rearranged nonproductively or productively by depositing the product of the productively rearranged gene in the surface membrane. The importance of this process for pre-B cells is indicated by the fact that products of certain genes expressed specifically in

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.

Clonal growth and maturation to immunoglobulin secretion in vitro of every growth-inducible B lymphocyte

The frequency of normal murine B lymphocytes initiating growth in diluted suspension cultures in the presence of a B cell mitogen, such as lipopolysaccharide, can be increased approximately 10(4) fold by the addition of 2 X 10(6) normal thymus cells per ml. This increase in the frequency of growing cells by thymus cells can also be observed with X63-AG8 myeloma tumor cells secreting IgG1. Thus thymus cells may not contribute growth-stimulating factors, but may supply growth-supporting factors. Culture medium and plastic dishes can be conditioned by preincubation with thymus cells for a day after which the thymus cells may be omitted from further culture for maximal B cell growth. Irradiation of thymus cell abolishes their growth-enhancing properties. Thymus cells can be syngeneic and allogeneic with the growing B cells. The frequency of growing LPS-reactive, normal B cells in spleen of 6-8 week old C3H/Tif mice was determined by limiting dilution analysis to be one of three splenic B cells. With this limiting dilution analysis, it was also shown that the cloning efficiency of XB3-AG8 myeloma tumor cells in suspension culture in the presence of thymus cells is practically 100%. Analysis of the growth kinetics of single clones of LPS-reactive, normal B cells shown that these B cells divide every 18 hr. Within the first 126 hr of growth, every B cell in the clone divides, and every dividing B cell in this clone secretes sufficient immonoglobulin to form a hemolytic plaque. The conditions of in vitro suspension cultures of murine B lymphocytes are therefore perfect to the extent that every B cell capable of growth will grow as a single clone.

Characterization of immature B cells by a novel monoclonal antibody, by turnover and by mitogen reactivity

The transit of immature to mature sIgM+ B cells, the life span, maturation kinetics and response to polyclonal activators have been analyzed with the help of a new mAb (493), that distinguishes immature, 493+ from mature, 493− B cells in a variety of mouse strains tested. Analysis of the turnover of immature 493+ B cells by bromodeoxyuridine (BrdU) labeling kinetics indicate that only 10 – 20 % of the cells reach the spleen as immature 493+ cells. The life span of 493+ B cells in bone marrow and spleen is around 4 days. BrdU chase experiments show that most of the immature cells in spleen enter the pool of mature, 493− B cells where they gain a longer life span of 15 – 20 weeks. Immature and mature B cells respond equally well to LPS stimulation; anti‐CD40, however, stimulates mature B cells better than immature B cells. IgM cross‐linking of mature B cells results in proliferation, while it induces apoptosis in immature B cells. This apoptosis of immature cells can be inhibited by co‐stimulation with anti‐CD40 or by overexpression of bcl‐2. We speculate that Ig receptor ligand‐mediated apoptosis (negative selection) plays a major role in the transit of immature B cells from bone marrow to spleen, but only a minor role in the transit from immature B cells to mature B cells in the spleen.