Cellular maturation defects in Bruton's tyrosine kinase-deficient immature B cells are amplified by premature B cell receptor expression and reduced by receptor editing

In the mouse, Bruton's tyrosine kinase (Btk) is essential for efficient developmental progression of CD43(+)CD2(-) large cycling into CD43(-)CD2(+) small resting pre-B cells in the bone marrow and of IgM(high) transitional type 2 B cells into IgM(low) mature B cells in the spleen. In this study, we show that the impaired induction of cell surface changes in Btk-deficient pre-B cells was still noticeable in kappa(+) immature B cells, but was largely corrected in lambda(+) immature B cells. As lambda gene rearrangements are programmed to follow kappa rearrangements and lambda expression is associated with receptor editing, we hypothesized that the transit time through the pre-B cell compartment or receptor editing may affect the extent of the cellular maturation defects in Btk-deficient B cells. To address this issue, we used 3-83 mu delta transgenic mice, which prematurely express a complete B cell receptor and therefore manifest accelerated B cell development. In Btk-deficient 3-83 mu delta mice, the IgM(+) B cells in the bone marrow exhibited a very immature phenotype (pre-BCR(+)CD43(+)CD2(-)) and were arrested at the transitional type 1 B cell stage upon arrival in the spleen. However, these cellular maturation defects were largely restored when Btk-deficient 3-83 mu delta B cells were on a centrally deleting background and therefore targeted for receptor editing. Providing an extended time window for developing B cells by enforced expression of the antiapoptotic gene Bcl-2 did not alter the Btk dependence of their cellular maturation. We conclude that premature B cell receptor expression amplifies the cellular maturation defects in Btk-deficient B cells, while extensive receptor editing reduces these defects.     

Expression, distribution and specificity of Fc receptors for IgM on murine B cells

Subpopulations of normal adult murine splenic B cells and a panel of murine B cell tumors were examined for their ability to bind murine IgM specifically. By using two-color flow cytometric analyses, we have demonstrated that 90 to 95% of surface (s)IgD+ B cells express surface membrane receptors for IgM (Fc mu R). The binding of pentameric murine IgM to splenocyte Fc mu R was IgM-specific since it was totally inhibited by other polymeric IgM proteins, but not by Ig of other H chain classes or by mAb specific for the murine IgG or IgE FcR. Binding of IgM to splenic cells was saturable. Fc mu R were co-expressed with the Fc gamma R as well as the Fc epsilon R on the majority of splenic B cells. Minor populations of splenic mononuclear cells expressed only an Fc mu R, Fc gamma R or Fc epsilon R. In a survey of B tumor cell lines representing different stages of B cell development, we observed that the Fc mu R was expressed on pre-B cell lines and that Fc mu R detection was maximal on immature B cell lines that expressed sIgM and low amounts of sIgD and Ia. Fc mu R were not detected on cell lines that had switched from sIgM to the expression of another sIg, or on plasmacytomas and hybridomas. The studies with normal splenocytes establish that the majority of sIgD+ B lymphocytes in adult BALB/c mice express surface membrane receptors that specifically bind IgM. The studies with B lineage tumor cells suggest that the expression of Fc mu R on B cells is developmentally regulated and that the pattern of expression exhibited by Fc mu R during B cell ontogeny differs from the patterns that have been previously found for IgG and IgE FcR. These observations raise the possibility that Fc mu R might have a functional significance in some aspect of B cell maturation and activation. By using a family of IgM H chain constant region domain deletional mutants, we have further demonstrated that, like the T cell Fc mu R, the B cell Fc mu R also requires a C mu 3 domain for binding to occur, raising the possibility that the T and B cell Fc mu R in mice may be structurally related molecules.     

Cutting edge: signaling and cell surface expression of a mu H chain in the absence of lambda 5: a paradigm revisited

Pre-B cell receptor (pre-BCR) signals are essential for pro-B cells to mature efficiently into pre-B cells. The pre-BCR is an Ig-like transmembrane complex that is assembled from two mu H chains (mu HC) and two surrogate L chains consisting of the non-covalently associated polypeptides VpreB and lambda5. In lambda5(-/-) mice, pro-B cell maturation is impaired, but not completely blocked, implying that a mu HC induces differentiation signals in the absence of lambda5. Using a mouse model, in which transgenic mu HC expression can be controlled by tetracycline, we show that in the absence of lambda5, the transgenic mu HC promotes in vivo differentiation of pro-B cells, induces IL-7-dependent cell growth, and is expressed on the surface of pre-B cells. Our findings not only show that an incomplete pre-BCR can initiate signals, but also challenge the paradigm that an IgHC must associate with an IgLC or a SLC to gain transport and signaling competency.     

Association of Ig L chain-like protein lambda 5 with a 16-kilodalton protein in mouse pre-B cell lines is not dependent on the presence of Ig H chain protein

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.     

B lymphocyte development in rabbit: progenitor B cells and waning of B lymphopoiesis

In mammals that use gut-associated lymphoid tissues for expansion and somatic diversification of the B cell repertoire, B lymphopoiesis occurs early in ontogeny and does not appear to continue throughout life. In these species, including sheep, rabbit, and cattle, little is known about the pathway of B cell development and the time at which B lymphopoiesis wanes. We examined rabbit bone marrow by immunofluorescence with anti-CD79a and anti-mu and identified both proB and preB cells. The proB cells represent the vast majority of B-lineage cells in the bone marrow at birth and by incorporation of 5-bromo-2'-deoxyuridine, they appear to be a dynamic population. PreB cells reach maximum levels in the bone marrow at 3 wk of age, and B cells begin to accumulate at 7 wk of age. We cloned two VpreB and one lambda5 gene and demonstrated that they are expressed within B-lineage cells in bone marrow. VpreB and lambda5 coimmunoprecipitated with the mu-chain in lysates of 293T cells transfected with VpreB, lambda5, and mu, indicating that VpreB, lambda5, and mu-chains associate in a preB cell receptor-like complex. By 16 wk of age, essentially no proB or preB cells are found in bone marrow and by PCR amplification, B cell recombination excision circles were reduced 200-fold. By 18 mo of age, B cell recombination excision circles were reduced 500- to 1000-fold. We suggest that B cell development in the rabbit occurs primarily through the classical, or ordered, pathway and show that B lymphopoiesis is reduced over 99% by 16 wk of age.     

Identification of IL-7-dependent bone marrow-derived Thy-1-B220- lymphoid cell clones that rearrange and express both Ig and T cell receptor genes.

Bone marrow stromal cell lines and lymphoid cell lines were co-established from the Whitlock-Witte type of long term liquid cultures of MRL/1 and C57BL/10 (B10) (Thy-1.1) bone marrow cells. The present study investigates the immunologic nature of parental and cloned lymphoid cell lines. Both strains of parental lines and their clones did not grow alone but proliferated on the monolayers of co-established parental stromal cell lines from a syngeneic or alternative strain. When various lymphokines or cytokines were tested for their capacity to support the growth of these lymphoid cell clones, only IL-7 could substitute for the growth-promoting function of stromal cells. These IL-7-dependent clones expressed neither Thy-1 nor B220 Ag. However, all of them from two strains were found to rearrange synchronously H chain of Ig as well as gamma chain of TCR genes. Some of the clones transcribed a mature size of IgH mRNA. Co-expression of mRNA for lambda 5 but not for IgL chain (kappa, lambda) genes resulted in the generation of cell surface mu chain in these clones. Other clones expressed a smaller size of IgH mRNA without exhibiting surface mu chain. Irrespective of the differences in IgH rearrangements and its mRNA expression, a mature size TCR gamma mRNA was detected in all of the clones. Thus, these results demonstrate the existence of untransformed (IL-7-dependent) immature lymphoid cells rearranging both Ig and TCR genes. Their unique features concerning cell surface markers (B220- mu+), specific growth factor requirement, and various modes of Ig/TCR gene rearrangements are discussed in the context of early lymphoid development.     

Rearrangements and deletions of immunoglobulin heavy chain genes in the double-producing B cell lymphoma I.29.

The B cell lymphoma I.29 consists of a mixture of cells expressing membrane-bound immunoglobulin M (IgM) (lambda) and IgA (lambda) of identical idiotypes. Whereas most of the cells express either IgM or IgA alone, 1 to 5% of the cells in this tumor express IgM and IgA simultaneously within the cytoplasm and on the cell membrane (R. Sitia et al., J. Immunol. 127:1388-1394, 1981; R. Sitia, unpublished data). When IgM+ cells are purified from the lymphoma and passaged in mice or cultured, a portion of the cells convert to IgA+. These properties suggest that some cells of the I.29 lymphoma may undergo immunoglobulin heavy chain switching, although it is also possible that the mixed population was derived by a prior switching event in a clone of cells. We performed Southern blotting experiments on genomic DNAs isolated from populations of I.29 cells containing variable proportions of IgM+ and IgA+ cells and on a number of cell lines derived from the lymphoma. The results were consistent with the deletion model for heavy chain switching, as the IgM+ cells contained rearranged mu genes and alpha genes in the germ line configuration on both the expressed and nonexpressed heavy chain chromosomes, whereas the IgA+ cells had deleted both mu genes and contained one rearranged and one germ line alpha gene. In addition, segments of DNA located within the intervening sequence 5' to the mu gene, near the site of switch recombination, were deleted from both the expressed and the nonexpressed chromosomes. Although mu genes were deleted from both chromosomes in the IgA+ cells, the sites of DNA recombination differed on the two chromosomes. On the expressed chromosome, Smu sequences were recombined with S alpha sequences, whereas on the nonexpressed chromosome, Smu sequences were recombined with S gamma 3 sequences.     

Surface mu heavy chain expressed on pre-B lymphomas transduces Ca2+ signals but fails to cause growth arrest of pre-B lymphomas.

Little is known about the role of signals transduced by cell surface IgM (sIgM) expressed during early B cell development. A subclone (1.6) of the late pre-B cell lymphoma 70Z/3.12 was used to study signal transduction by surface mu heavy (H) chain before and after transition to the early immature B cell stage, and the functional consequences thereof. Although kappa L chain expression can be induced on 1.6 cells by LPS or cytokines, immunoprecipitations indicated that the non-induced 1.6 cells expressed mu H chain with an alternative protein(s) which may be a surrogate light chain(s). Consistent with this, anti-mu but not anti-kappa or anti-lambda antibodies caused transient Ca2+ mobilization in noninduced 1.6 cells. The Ca2+ signal was derived from both intracellular stores and Ca2+ influx in either noninduced cells or in cells that had been preinduced to express kappa L chain. Thus, the ability of mu H chain to mobilize Ca2+ as a second messenger does not depend upon the expression of mature L chains. The immature B lymphomas, WEHI-231 and CH1, express mature forms of IgM and undergo growth arrest when stimulated by anti-mu antibody. In contrast, signals generated by mu H chain on either noninduced or preinduced 1.6 cells or in the sIgM+ pre-B cell transfectant 300-19 mu lambda 36/8 did not cause growth arrest. These results suggest that mu H chain expressed on pre-B cells is capable of mobilizing Ca2+, but that this signal alone is insufficient to induce growth arrest in the pre-B cell.     

B cell abnormalities induced by a mu Ig transgene extend to L chain isotype usage

We have analyzed the phenotype of B cell populations from mice transgenic for a rearranged Ig mu H chain gene. We find a decrease in the number of B cells in the spleens of these mice. Transgenic B cells have decreased surface levels of both IgM and IgD. The circulating IgM in these mice is 3- to 10-fold enriched in lambda L chains, compared with that in non-transgenic mice. Analysis of IgM-producing hybridomas, from transgenic mice that express the transgene at high levels, demonstrates that this higher lambda frequency is observed in transgene-nonexpressing as well as transgene-expressing hybridomas. A partial loss of L chain isotype exclusion is also noted in these hybridomas, and a significant proportion of primary B cells expressing both kappa and lambda L chains on their surface can be demonstrated. These findings suggest an ability of the transgenic Ig H chain to affect events in B cell ontogeny beyond the H chain locus. Our results support a quantitative model of exclusion for both the H chain alleles and the L chain isotypes.     

Murine pro-B cells require IL-7 and its receptor complex to up-regulate IL-7R alpha, terminal deoxynucleotidyltransferase, and c mu expression

Phenotypic analysis of bone marrow cells from IL-7 knockout (KO) mice revealed that B cell development is blocked precisely at the transition between pro-B cells and pre-B cells. In contrast, the generation of pre-pro-B cells and pro-B cells appeared to be normal, as judged by total cell numbers, proliferative indexes, D-JH and V-DJH gene rearrangements, and mRNA for recombinase-activating gene-1 (RAG-1), RAG-2, TdT, Ig mu, lambda 5, and VpreB. However, upon closer inspection, several abnormalities in pro-B cell development were identified that could be corrected by injection of rIL-7 in vivo. These included the absence of the subset of late pro-B cells that initiates cmu expression for pre-B cell Ag receptor (BCR) formation, and the failure of pro-B cells to up-regulate TdT and the IL-7R alpha (but not the common gamma-chain) chain. Similar defects were present in common gamma-chain and Jak3 KO mice, but not in lambda 5 or (excluding cytoplasmic Ig mu heavy chain (c mu)) RAG-1 KO mice, all of which also arrest at the late pro-B cell stage. Consequently, up-regulation of TdT and IL-7R alpha expression requires signaling through the high affinity IL-7R, but does not require cmu expression or a functional pre-BCR. Taken together, these results suggest that IL-7 and its receptor complex are essential for 1) up-regulating the expression of TdT and IL-7R alpha, 2) initiating the production of cmu and 3) promoting the formation of a functional pre-BCR in/on pro-B cells. These key events, in turn, appear to be prerequisite both for differentiation of pro-B cells to pre-B cells and for proliferation of these cell subsets upon continued stimulation with IL-7.     

B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.     

Analysis of marginal zone B cell development in the mouse with limited B cell diversity: role of the antigen receptor signals in the recruitment of B cells to the marginal zone

The quasimonoclonal (QM) mouse provides an intelligible model to analyze the B cell selection as the competition between two major 4-hydroxy-3-nitrophenylacetyl-specific B cell populations whose BCR are comprised of the knockin V(H)17.2.25 (V(H)T)-encoded H chain and the lambda1 or lambda2 L chain. In this study, we show the QM system is useful to examine how BCR signals guide a subset of B cells to the marginal zone (MZ). Compared with the control C57BL/6 mice, the QM mice had approximately 2.7-fold increased number of B cells exhibiting the MZ B cell phenotype and a larger MZ area in the spleen. Interestingly, V(H)T/lambda2 B cells significantly predominated over V(H)T/lambda1 B cells in MZ-(V(H)T/lambda1:V(H)T/lambda2 approximately 3:7) and transitional 2-B cell subsets, while these two populations were comparable in immature, transitional 1, and mature counterparts. Thus, the biased use of lambda2 in the MZ B cells may be the result of selection in the periphery. The enlargement of MZ B cell compartment and the preferred recruitment of the V(H)T/lambda2 B cells were further augmented by doubling the V(H)T gene, but dampened by the dysfunction of Bruton's tyrosine kinase, suggesting a positive role of BCR signaling in this selection. Comparison of Ag specificity between V(H)T/lambda1 and V(H)T/lambda2 IgM mAbs revealed a polyreactive nature of the V(H)T/lambda2 BCR, including the reactivity with ssDNA. Taken together, it is suggested that polyreactivity (including self-reactivity) of BCR is crucial in driving B cells to differentiate into the MZ phenotype.     

Analysis of VpreB expression during B lineage differentiation in lambda5-deficient mice

The VpreB/lambda5 surrogate L chain complex is an essential component of the pre-B cell receptor, the expression of which serves as an important checkpoint in B cell development. Surrogate L chains also may serve as components of murine pro-B cell receptors whose function is unknown. We have produced two new mAbs, R3 and R5, that recognize a different VpreB epitope than the one recognized by the previously described VP245 anti-mouse VpreB Ab. These Abs were used to confirm the expression of surrogate L chains on wild-type pro-B and pre-B cell lines. Although undetectable on the cell surface, VpreB was found to be normally expressed within B lineage cells of lambda5-deficient mice. Nevertheless, VpreB expression was extinguished at the B cell stage of differentiation in these mice. The normal pattern of VpreB expression in lambda5-deficient mice excludes an essential role for pro-B and pre-B cell receptors in VpreB regulation.     

B cells expressing a natural polyreactive autoantibody have a distinct phenotype and are overrepresented in immunoglobulin heavy chain transgenic mice

Despite stringent regulation of disease-associated autoantibodies, a substantial proportion of circulating Abs in sera of healthy individuals exhibit self-reactivity. These Abs are referred to as naturally occurring or natural autoantibodies (NAAs). To understand the origin and function of NAAs, we have generated a new site-directed transgenic mouse model in which a prerearranged VDJ gene coding for the H chain of a typical polyreactive NAA, ppc1-5, is inserted into the IgH locus. This H chain, when combined with its original L chain, the lambda1 L chain, yields a NAA that characteristically binds a variety of self and non-self Ags including ssDNA, actin, ubiquitin, and nitrophenyl phosphocholine. Despite their autoreactivity, B cells expressing ppc1-5H/lambda1 NAA are not negatively selected, but rather are overrepresented in the transgenic mice. The shift toward lambda1 expression mainly occurs during the transition of immature to mature B cells in the spleen, suggesting a BCR selection process. The ppc1-5H/lambda1 B cells exhibit a phenotype that is different from those of the known mature B cell populations, and they are located predominantly in the lymphoid follicles of the spleen and the lymph nodes. These B cells are functionally active, producing high levels of Abs in vivo and responding well to BCR stimulation in vitro. The findings indicate that the ppc1-5/lambda1 natural autoantibodies originate from a distinct B cell subset that may be positively selected by virtue of its poly/autoreactivity.     

Relationship between the rearrangement of immunoglobulin genes, the appearance of a B lymphocyte antigen, and immunoglobulin synthesis in murine pre-B cell lines

Eighteen Abelson virus-transformed immature B cell lines were established and immunoglobulin biosynthesis, expression of a B lymphocyte antigen detected by a monoclonal antibody, and rearrangement of immunoglobulin genes in these cell lines were studied. Only one cell line (A1) synthesized micro-chains but no light chains, and the other cell lines synthesized no detectable immunoglobulins. None of the cell lines established had detectable membrane-associated IgM. Fifteen cell lines expressed a B lymphocyte antigen on their cell surfaces. In three cell lines, however, the majority (greater than 99%) of cells did not express this antigen. Heavy chain genes were rearranged on both chromosomes in all the cell lines, although one heavy chain gene was deleted in three cell lines. In 12 of 18 cell lines, one or both kappa-chain genes were rearranged. In six cell lines, however, both kappa-chain genes remained in embryonic form; lambda-chain genes were in embryonic form in all the cell lines. These results suggested the hierarchy of Ig gene rearrangements, beginning with mu and proceeding to kappa and then to lambda. JH rearrangement was also shown to precede the appearance of a B lymphocyte antigen. In three cell lines (A1-A3), which were considered subclones derived from a single common precursor, it was suggested that one rearranged JH gene was functional, and the other was nonfunctional, indicating that allelic exclusion already operated in pre-B cells.