Immature B cells from human and mouse bone marrow can change their surface light chain expression

The capacity of bone marrow-derived surface immunoglobulin-positive (sIg⁺) human and mouse immature B cells, generated either in vitro or in vivo, to change their light (L) chain expression, has been assayed by the number of cells which change in vitro from one type of L chain to the other type, or to no sIg at all. Immature sIg⁺ B cells were generated in vitro from sIg^? precursor cells from human or mouse bone marrow. The immature sIg⁺ cells expressed RAG-1. Human sIg⁺ cells expressed xfr; and λ L chains in ratios between 1:1 and 3:1, whereas in mouse cells, this ratio ranged from 10:1 to 20:1. Upon reculture of the human and mouse xfr;⁺sIg⁺ cells, about half of them remained xfr;⁺, a quarter became λ⁺, and another quarter became sIg^?. Between 1 and 3% expressed both xfr; and λ chains. Of the human λ⁺ cells, about two-thirds remained λ⁺, only 1 to 2% became xfr;⁺, while the other third became sIg^?. Again, between 1 and 3% expressed both xfr; and λ L chains. These results indicate that expression of sIgM in the B cell membrane does not terminate L chain gene rearrangement, and that some order exists in xfr; versus λ gene rearrangements. Hence, human and mouse xfr;⁺ immature B cells can become λ⁺, but very few of the λ⁺ cells can become xfr;⁺, and both can become sIg^?. Further, human CD10⁺/sIg⁺ xfr;⁺ and λ⁺ cells and mouse B220^low/sIg^low xfr;⁺ cells enriched from bone marrow, i.e. immature B cells differentiated in vivo, changed their Ig phenotype upon in vitro culture, but in lower frequencies. By contrast, human and mouse mature B cells did not change their L chain or Ig phenotype. Hence, at least a part of the sIg⁺ immature B cells in bone marrow retain the capacity to change their L chain and Ig phenotype, and this capacity is lost when they become mature, peripheral B cells.     

A B220+ CD117+ CD19- hematopoietic progenitor with potent lymphoid and myeloid developmental potential

In this report, we identify in the bone marrow (BM) of normal mice a subpopulation of B220+ CD117+ CD19- NK1.1- cells with potent lymphoid and myeloid developmental potential. These cells represent 0.1-0.2% of nucleated BM cells. By limiting dilution analysis in the presence of the appropriate combination of stromal cells and cytokines, 1 in 5-10 sorted cells formed B cells, 1 in 10-15 formed T cells and 1 in 5-10 generated macrophages. When cultured on a mixture of OP9 stroma and OP9 stromal cells expressing the Notch ligand Delta-like-1, single cells generated both T and B cells. Following intravenous infusion, freshly sorted cells transiently reconstituted both the T and B cell progenitor compartments, generating cohorts of mature T and B lymphocytes. The relationship between B220+ CD117+ CD19- NK1.1- cells of wild-type mice and other multi-lineage BM progenitors is discussed.     

The anti-apoptotic factor Bcl-2 can functionally substitute for the B cell survival but not for the marginal zone B cell differentiation activity of BAFF

The TNF family ligand B cell-activating factor (BAFF, BLyS, TALL-1) is an essential factor for B cell development. BAFF binds to three receptors, BAFF-R, transmembrane activator and CAML interactor (TACI), and B cell maturation antigen (BCMA), but only BAFF-R is required for successful survival and maturation of splenic B cells. To test whether the effect of BAFF is due to the up-regulation of anti-apoptotic factors, TACI-Ig-transgenic mice, in which BAFF function is inhibited, were crossed with transgenic mice expressing FLICE-inhibitory protein (FLIP) or Bcl-2 in the B cell compartment. FLIP expression did not rescue B cells, while enforced Bcl-2 expression restored peripheral B cells and the ability to mount T-dependent antibody responses. However, many B cells retained immaturity markers and failed to express normal amounts of CD21. Marginal zone B cells were not restored and the T-independent IgG3, but not IgM, response was impaired in the TACI-IgxBcl-2 mice. These results suggest that BAFF is required not only to inhibit apoptosis of maturating B cells, but also to promote differentiation events, in particular those leading to the generation of marginal zone B cells.     

Characterization of the interleukin 5-reactive splenic B cell population

The characteristics of the interleukin (IL) 5-reactive splenic B cell population of C57BL/6 nu/nu mice, with respect to IL 5/IL2 reactivity, cell surface phenotype, VH gene family usage, autoreactivity and the structure of the IL5 receptor (IL5R), were analyzed. It was found that 2%-4% of splenic B cells express relatively high levels of IL 5R as determined by the binding of the anti-IL 5R monoclonal antibody R52.120. Over 90% of the splenic B cells that mature to IgM secretion upon activation with IL5 are comprised in this small subpopulation of B cells. Moreover, the vast majority of splenic B cells that mature to IgM-secreting cells when activated by IL2 also reside in this IL5R+B cell population. The cell surface phenotype of the IL5R+ splenic B cells is IgM+, B220+, Ly-1- and IL2R p55-. Upon activation with IL5 this cell surface phenotype changes, in that a vast majority of the B cells then express the p55 chain of the IL2R, whereas the level of IL5R decreases. VH gene family usage in the IL5-activated splenic B cells was analyzed by in situ hybridization. VH gene family usage was found to be random and not different from the VH genes expressed in LPS-activated B cells. Hybridoma collections from IL5-activated splenic B cells and LPS-activated B cells were screened and compared for the production of autoantibodies and antibodies directed against the haptens (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) and 2,4,6-trinitrophenyl (TNP). In both collections high, but not significantly different frequencies of autoantibody-(32% IL5, 31.4% LPS) and of anti-hapten antibody (27.8% IL5, 18.6% LPS)-producing hybridomas were found. The structure of the IL5R on IL5-activated B cells was analyzed by 125I-labeled IL5 binding and cross-linking. About 100 high-affinity (10(-11) M) and 1000 low-affinity (10(-9) M) IL5-binding sites are present on IL5-activated splenic B cells, and both high- and low-affinity IL5R are similar to those expressed on the IL5-dependent B13 cell line. Cross-linking of 125I-labeled IL5 to the receptors on IL5-activated B cells revealed one major IL5-binding protein of 45-50 kDa molecular mass and another minor binding protein of 130-140 kDa. The same IL5-binding proteins are present on the IL5-dependent B13 cell line.(ABSTRACT TRUNCATED AT 400 WORDS)     

Extensive in vivo self-renewal, long-term reconstitution capacity, and hematopoietic multipotency of Pax5-deficient precursor B-cell clones

Self-renewal, pluripotency, and long-term reconstitution are defining characteristics of single hematopoietic stem cells. Pax5(-/-) precursor B cells apparently possess similar characteristics. Here, using serial transplantations, with in vitro recloning and growth of the bone marrow-homed donor cells occurring after all transplantations, we analyzed the extent of self-renewal and hematopoietic multipotency of Pax5(-/-) precursor B-cell clones. Moreover, telomere length and telomerase activity in these clones was analyzed at various time points. Thus far, 5 successive transplantations have been performed. Clones transplanted for the fifth time, which have proliferated for more than 150 cell divisions in vitro, still repopulate the bone marrow with precursor B cells and reconstitute these recipients with lymphoid and myeloid cells. During this extensive proliferation, Pax5(-/-) precursor B cells shorten their telomeres at 70 to 90 base pairs per division. Their telomerase activity remains at 3% of that of HEK293 cancer cells during all serial in vivo transplantations/in vitro expansions. Together, these data show that Pax5(-/-) precursor B-cell clones possess extensive in vivo self-renewal capacity, long-term reconstitution capacity, and hematopoietic multipotency, with their telomeres shortening at the normal rate.     

The development of autoimmune features in aging mice is closely associated with alterations of the peripheral CD4+ T‐cell compartment

Some signs of potential autoimmunity, such as the appearance of antinuclear antibodies (ANAs) become prevalent with age. In most cases, elderly people with ANAs remain healthy. Here, we investigated whether the same holds true for inbred strains of mice. Indeed, we show that most mice of the C57BL/6 (B6) strain spontaneously produced IgG ANA at 8–12 months of age, showed IgM deposition in kidneys and lymphocyte infiltrates in submandibular salivary glands. Despite all of this, the mice remained healthy. ANA production is likely CD4⁺ T‐cell dependent, since old (40–50 weeks of age) B6 mice deficient for MHC class II do not produce IgG ANAs. BM chimeras showed that ANA production was not determined by age‐related changes in radiosensitive, hematopoietic progenitor cells, and that the CD4⁺ T cells that promote ANA production were radioresistant. Thymectomy of B6 mice at 5 weeks of age led to premature alterations in T‐cell homeostasis and ANA production, by 15 weeks of age, similar to that in old mice. Our findings suggest that a disturbed T‐cell homeostasis may drive the onset of some autoimmune features.     

Three chemokines with potential functions in T lymphocyte-independent and -dependent B lymphocyte stimulation.

Three clustered mouse chemokine genes, ABCD-1, -2 and -3, are all expressed highly in dendritic cells and, at various levels, in activated B cells. T cell-independently activated B cells express ABCD-1 and -2, but not -3. T cell-dependently activated B cells express all three. ABCD-1 attracts activated CD8+ cytotoxic T cells and CD4+ helper T cells of type 1 and 2. ABCD-2 preferentially attracts type 2 helper T cells, while ABCD-3 does not attract T cells at all. Both ABCD-1 and ABCD-2 bind to the same receptor (CCR4). In addition, ABCD-1 binds to a second, unknown, receptor on a separate T cell population. The three chemokines might guide T cell-independent as well as -dependent responses with two types of CD4+ T cells.     

Mutation of the BAFF furin cleavage site impairs B-cell homeostasis and antibody responses

B-cell-activating factor of the TNF family (BAFF)/BLyS contributes to B-cell homeostasis and function in the periphery. BAFF is expressed as a membrane-bound protein or released by proteolytic cleavage, but the functional importance of this processing event is poorly understood. Mice expressing BAFF with a mutated furin consensus cleavage site, i.e. furin-mutant BAFF (fmBAFF), were not different from BAFF-deficient mice with regard to their B-cell populations and responses to immunization. It is however noteworthy that an alternative processing event releases some soluble BAFF in fmBAFF mice. Mild overexpression (～ 5-fold) of fmBAFF alone generated intermediate levels of B cells without improving humoral responses to immunization. Processed BAFF was however important for B-cell homeostasis, as peripheral B-cell populations and antibody responses were readily restored by administration of soluble BAFF trimers in BAFF-deficient mice. However, the rescue of CD23 expression in B cells of BAFF-deficient mice required both soluble BAFF trimers and fmBAFF, or a polymeric form of soluble BAFF (BAFF 60-mer). These results point to a predominant role of processed BAFF for B-cell homeostasis and function, and indicate possible accessory roles for membrane-bound BAFF.