Extramedullar B lymphopoiesis in liver schistosomal granulomas: presence of the early stages and inhibition of the full B cell differentiation.

Inflammatory granulomatous reactions in liver elicited by schistosomal infection have been shown to function as active extramedullar myelopoietic sites, producing potentially all the myeloid lineages. We have now addressed the question of the extramedullar B lymphopoiesis in these sites. We have shown the presence of early B cell precursors (pro-B cells) in the granulomas by immunophenotyping. Their total number in the liver was equivalent to the pro-B cells in the bone marrow of one femur. In agreement with their phenotype, the RT-PCR analysis showed that these cells expressed RAG-1 and lambda5 genes. However, the conversion of the pro-B to pre-B cells was not observed and no clonogenic B cell precursors could be detected in semi-solid cultures stimulated by IL-7. The granulomatous stroma was shown to produce IL-7 and express c-kit, and was able to sustain the full B lymphopoiesis in vitro. Conversely, the granuloma supernatant was shown to inhibit actively the development of B lymphocytes. We conclude that the granuloma environment elicits homing and proliferation of totipotent hematopoietic precursors, and that it is permissive for early commitment to the B cell lineage, but the full extramedullar production of B cell is abrogated by soluble factors produced inside the granulomas.     

Proliferation and differentiation of immature thymocytes induced by a thymic stromal cell clone

The monolayer of our established thymic stromal cell clone (MRL104.8a) exhibited the capacity to maintain immature double-negative thymocytes. Such capacity was also expressed by a factor produced by the MRL104.8a monolayer. This factor designated as thymic stroma-derived T cell growth factor (TSTGF) was found to be distinct from IL-2 or IL-4 but similar to IL-7 of the previously described cytokines from the functional and molecular aspects. The MRL104.8a monolayer also exerted its differentiation-promoting effect on double-negative thymocytes. Culture for one day of purified double-negative thymocytes on the monolayer resulted in the induction of an appreciable per cent of CD3-4-8+ cells. This differentiation could also be induced by a semipurified TSTGF sample but not by recombinant IL-7, suggesting that the MRL104.8a cells elaborate a factor(s) responsible for initiating the differentiation of double-negative cells in addition to the growth-promotion factor identical or closely related to IL-7. When the culture period of double-negative thymocytes was extended to 2 or 3 days, an appreciable number of double-positive (CD4+8+) and single-positive (CD4+8-) cells were generated on the MRL104.8a monolayer. Thus, these observations provide strong support for the proposition that a specialized thymic stromal component plays an essential role in the intrathymic T cell development in the context of T cell growth and differentiation.     

Effect of TH-lines and clones on the growth and differentiation of B cell clones in microculture

Antibody isotype expression by B cell clones was analyzed using in vitro microcultures containing low numbers of hapten-gelatin-enriched B cells and higher numbers of hemocyanin-specific helper T cell lines or clones. Twenty-eight to sixty-three percent of clones grown in microculture with haptenated hemocyanin and T cells from established lines expressed IgG and/or IgA isotypes in random mixtures, almost always accompanied by IgM. Helper T cells from hemocyanin-specific clones also supported the expression of non-IgM isotypes by the B cell clones, suggesting that a single specificity of T cell can provide sufficient growth and differentiation factors for the display of isotype switching. A positive correlation between the antibody output of clones and the expression of non-IgM isotypes indicated that the switching process may be associated with cell division. Although memory B cells that give clones expressing IgG and/or IgA in the absence of IgM are also enriched on haptenated gelatin, they are not stimulable under conditions of this microculture assay.     

Class switch recombination in B lymphopoiesis: a potential pathway for B cell autoimmunity

Pathogenic autoantibodies detected in autoimmune diseases are predominantly IgG isotypes, reflecting the generation and activation of an autoimmune memory B cell repertoire. It is not completely understood how such autoreactive cells are generated and escape central and/or peripheral tolerance mechanisms, and several models to explain this have been proposed. It is generally thought that B lymphocytes utilize IgM receptors for development and tolerance establishment, whereas IgG receptors are primarily used to promote memory formation and signal for memory-type responses. In here we review recent findings suggesting that spontaneously occurring class switch recombination in B lymphopoiesis confer B lymphocytes with a novel developmental pathway that is driven by non-IgM receptors. The physiological relevance of this developmental pathway in generating an autoimmune memory repertoire, as well as a Fas-dependent mechanism regulating it, is discussed.     

CD40 ligand-positive CD8+ T cell clones allow B cell growth and differentiation

A fraction of activated CD8⁺ T cells expresses CD40 ligand (CD40L), a molecule that plays a key role in T cell-dependent B cell stimulation. CD8⁺ T cell clones were examined for CD40L expression and for their capacity to allow the growth and differentiation of B cells, upon activation with immobilized anti-CD3. According to CD40L expression, CD8⁺ clones could be grouped into three subsets. CD8⁺ T cell clones expressing high levels of CD40L (≥80% CD40L⁺ cells) were equivalent to CD4⁺ T cell clones with regard to induction of tonsil B cell proliferation and immunoglobulin (Ig) production, provided the combination of interleukin (IL)-2 and IL-10 was added to cultures. CD8⁺ T cell clones, with intermediate levels of CD40L expression (10 to 30% CD40L⁺ cells), also stimulated B cell proliferation and Ig secretion with IL-2 and IL-10. B cell responses induced by these CD8⁺ T cell clones were neutralized by blocking monoclonal antibodies specific for either CD40L or CD40. By contrast, CD40L^?? T cell clones (?5 % CD40L⁺ cells), only induced marginal B cell responses even with IL-2 and IL-10. All three clone types were able to activate B cells as shown by up-regulation of CD25, CD80 and CD86 expression. A neutralizing anti-CD40L antibody indicated that T cell-dependent B cell activation was only partly dependent on CD40-CD40L interaction. These CD40L^?? clones had no inhibitory effects on B cell proliferation induced by CD40L-expressing CD8⁺ T cell clones. Taken together, these results indicate that CD8⁺ T cells can induce B cell growth and differentiation in a CD40L-CD40-dependent fashion.     

Human clones with natural killer function can activate B cells and secrete B cell differentiation factors

Large granular lymphocyte clones were prepared from normal human mononuclear cells. Seven clones were studied in detail. All had high cytotoxic activity against the natural killer (NK) cell target K562 and all were T11- and DR-positive but T4- and T8-negative: none released migration inhibitory factor, a characteristic of helper T cell clones. When these NK cell clones were co-cultured with autologous B cells in a 1:1 ratio, 4 of the 7 induced both IgM and IgG synthesis. Ig production could not be further enhanced by the addition of B cell differentiation factors. The cells stimulated included antigen-specific memory B cells, as the Ig induced contained specific antibody to an antigen, tetanus toxoid, to which the B cell donors had recently been immunized. Supernatants from the helper/NK clones contained B cell differentiation factors, and were able to induce IgG synthesis from the lymphoblastoid cell line CESS and from co-cultured T and B cells. However, NK clone supernatants, unlike the clones themselves, were not effective at inducing Ig synthesis from purified B cells. Instead it appears that NK clones first activate B cells and thus render them responsive to the factors that the clones secrete.     

Effect of different Ig transgenes on B cell differentiation in scid mice

In this study we show that the ability of bone marrow pre-B cells to differentiate into B cells in H/L chain transgenic scid mice correlates with the ability of the transgenes to inhibit initiation of endogenous kappa gene rearrangement. Initiation of rearrangement was scored by assaying for DNA double-strand breaks (DSB) at the recombination signal/coding borders of J kappa 1 and J kappa 2. In H/L chain transgenic scid mice that develop B cells, we found little or no DSB; whereas in H chain only transgenic scid mice, in which pre-B cells are unable to give rise to B cells, we found a normal level of DSB but no VJ kappa coding joints. As scid mice are deficient in the repair of DSB, we suggest that initiation of kappa gene rearrangement in H chain transgenic scid mice causes B lineage cells to die at the late pre-B stage. In one transgenic scid line (Y-Sp6), which falls to generate B cells despite containing a H and L chain transgene, we found evidence for loss of B lineage cells at two stages of development: the pro-B to pre-B transitional stage and the late pre-B stage.      

Immunophenotypes of malignant lymphoma centroblastic-centrocytic and malignant lymphoma centrocytic: an immunohistologic study indicating a derivation from different stages of B cell differentiation

Five cases of intermediate lymphocytic lymphoma (ILL), 13 cases of malignant lymphoma centrocytic (MLCC), and 27 cases of malignant lymphoma centroblastic centrocytic (MLCBCC) were studied morphologically and with the aid of a panel of monoclonal antibodies. The immunophenotypes of ILL and MLCC (IgM+/IgD+, MT1+, CALLA-) indicate a mantle zone or very early follicle center derivation. The immunophenotypes of MLCBCC (IgM+ or IgG/IgA+, MT1-, CALLA+) indicate a "true" follicle center derivation. The morphologic diversity of MLCBCC could not be related to specific immunophenotypes.     

Targeting the bone marrow with activin A-overexpressing embryonic multipotent stromal cells specifically modifies B lymphopoiesis

In vitro and in vivo studies implicate a series of cytokines in regulation of lymphohematopoiesis. However, direct indications for a local role of most of these cytokines within the bone marrow is lacking. In the present study, we aimed to test the contribution of a specific cytokine, activin A, a member of the transforming growth factor-beta (TGF-beta) family, to lymphohematopoiesis in mouse bone marrow. We show that mouse embryonic fibroblasts (MEFs) are indistinguishable from multipotent stromal cells (MSCs). Such MEFs overexpressing activin A, supported in vitro myelopoiesis in long-term bone marrow cultures as effectively as control MEFs. In contrast, activin A-overexpressing MEFs interfered with the in vitro generation of B lineage cells in such cultures. Thus, excessive expression in vitro of activin A, by supportive stromal cells, causes preferential maturation of myeloid rather than lymphoid cells. Moreover, the activin A-overexpressing MEFs caused an increased incidence in vivo of relatively immature B lineage cells; upon transplantation through the spleen route, MEFs engrafted the bone marrow specifically. Activin A-overexpressing MEFs accumulated in the bone marrow compartment and slowed down the progression of B cell precursors along the differentiation pathway, while sparing the myeloid population. The assay system described in this paper provides a means to assess the contribution of a wide range of molecules to hematopoiesis without perturbing the constitution of other organs.     

The earliest stages of B cell development require a chemokine stromal cell-derived factor/pre-B cell growth-stimulating factor

Environmental factors essential for the first stages of B lymphopoiesis remain elusive. Here, we report that immediately after commitment to B lineage, precursors become dependent on a chemokine SDF-1 and its receptor CXCR4 using mutant and radiation chimeric mice. In bone marrow, generation of the earliest identifiable B cell precursor populations requires CXCR4. In fetal liver, we identified Lin(-)CD19(-)c-kit(+)IL-7Ralpha(+)AA4.1(+), the earliest unipotent B cell precursor population, and found that its development was severely affected in SDF-1(-/-) embryos but not in IL-7(-/-) embryos. Lin(-) T cell progenitors appeared normal in SDF-1(-/-) embryos. Moreover, SDF-1 exhibited specific biologic activities on the earliest B cell precursors. SDF-1 provides the first example of a cytokine responsible for the earliest B lineage stages.     

Azathioprine inhibits vaccinia virus replication in both BSC-40 and RAG cell lines acting on different stages of virus cycle

In the present study we demonstrate that azathioprine (AZA) inhibits vaccinia virus (VV) replication in both BSC-40 and RAG cell lines, acting on different stages of virus cycle. In BSC-40 cells, early protein synthesis was not significantly affected, but late gene expression was severely impaired. In RAG cells all stages of gene expression were completed during synchronous infection in the presence of the drug. The onset of DNA replication was not affected in RAG cells, but a severe inhibition was observed in BSC-40 cells. Electron microscopic analysis of VV-infected RAG cells treated with AZA revealed brick-shaped particles presenting abnormal definition of the internal structure. Purified virions from AZA-treated RAG cells presented several modifications of the protein content, a lesser amount of DNA, and a lower PFU:particle ratio. Our results suggest that in VV-infected RAG cells AZA interfered with virus morphogenesis, whereas in BSC-40 cells the replicative cycle was inhibited at the DNA replication stage.     

Two models of murine B lymphopoiesis: a correlation

During B cell genesis in mouse bone marrow (BM), precursor B cells pass through a series of developmental stages that have been defined by changes in expression of various marker molecules. The use of dissimilar phenotypic criteria in different laboratories, however, has led to the formulation of disparate models of B lymphopoiesis not fully reconciled with one another. We have directly compared two such models, one based on expression of intracellular μ heavy chain of IgM (cμ) and terminal deoxynucleotidyl transferase (TdT), the other monitoring cell surface leukosialin (CD43), heat-stable antigen (HSA; CD24) and the ectopeptidase BP-1. Each model uses cell surface B220 glycoprotein (CD45RA) to denote the B cell lineage. We have examined the cellular composition of four sorted BM fractions by immunofluorescent labeling of CD43, HSA and BP-1, using immunofluorescence microscopy of cytocentrifuged fractions to quantitate precursor B cell populations expressing either cμ or TdT. The results reveal a range of B cell differentiation stages within individual sorted BM fractions, providing a cross-reference between these two analytical methods and contributing to a unified model of B cell development in mouse BM.     

Selection of B cell clones and memory B cells.

During a humoral immune response, a variety of histological, cellular, and molecular changes occur within the immune system. In this review, we would like to summarize and integrate these changes with a view toward gaining insight into the process of clonal selection. We describe here how antigen receptor number limits the sensitivity of a B cell to transduce a signal in response to antigen. We have also seen that this limitation can be compensated for by expressing a higher affinity receptor for antigen. These data suggest that the down regulation of antigen binding receptors in germinal center cells might be designed to exploit anticipated increases in affinity that result from somatic hypermutation in these tissues. This hypothesis is consistent with observed biological changes that occur during an immune response and has bearing on both the mechanism of affinity maturation and generation of immunologic memory.     

IL-7: a key regulator of B lymphopoiesis

Interleukin-7 plays important roles in the B cell developmental pathway including events leading to commitment, survival, proliferation, and maturation. Because of its central role in adult murine B lymphopoiesis, IL-7 is frequently used to generate B cell progenitors in vitro. We have shown that differentiation of IL-7-responsive cells in these cultures is influenced by CD45, pre-B cell receptor, and other downstream signaling molecules. A common, but often overlooked aspect of IL-7 containing cultures is the routine maturation of cells to the sIgM(+) stage. The production of B cells in IL-7 containing cultures is balanced by cell death, since such cells fail to survive for long without additional stimulation.