Regulation of peripheral B cell maturation

Although it is clear that the final phases of B cell maturation occur after newly formed B cells exit the bone marrow, the mechanisms underpinning the maturation, selection, and long-term survival of immature peripheral B cells remain poorly understood. Here, we review recent advances in our understanding of how B cell receptor (BCR)-mediated signaling events integrate with additional environmental cues to promote the selection and differentiation of immature B cells into functionally distinct subpopulations of mature B cells. We pay particular attention to the role of the Baff cytokine family and the Notch receptor-ligand family and their unique roles in promoting B cell survival and differentiation into follicular and marginal zone B cells.     

B cell selection and affinity maturation during an antibody response in the mouse with limited B cell diversity

The quasi-monoclonal mouse has limited B cell diversity, whose major (approximately 80%) B cell Ag receptors are comprised of the knockin V(H) 17.2.25 (V(H)T)-encoded H chain and the lambda1 or lambda2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although V(H)T/lambda1 and V(H)T/lambda2 IgM were equally produced, V(H)T/lambda2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 of V(H)T (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated V(H)T-encoded gamma-chains could form lambda1-bearing IgG in Chinese hamster ovary cells, apparent absence of V(H)T/lambda1 anti-pNP IgG may not be due to the incompatibility between the gamma-chains and the lambda1-chain, but may be explained by the fact that V(H)T/lambda1 B cells showed 50- to 100-fold lower affinity for pNP than V(H)T/lambda2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an affinity maturation process.     

Peripheral B cell maturation. I. Immature peripheral B cells in adults are heat-stable antigenhi and exhibit unique signaling characteristics.

Whether recently generated peripheral B cells in adults are functionally equivalent to immature B cells in the neonatal spleen is unknown. We have identified a splenic B cell subpopulation in adults whose phenotypic and in vitro characteristics closely resemble those of neonatal B cells. These cells are defined by the cell surface phenotype heat-stable Aghi (HSAhi), and make up 10 to 15% of the adult splenic B cell pool. HSAhi B cells in adults bear the immature phenotype B220lo sIgMhi, and are 50% sIgD+. Furthermore, after sublethal irradiation, the initial wave of newly generated splenic B cells in self-reconstituting adults express a similar phenotype. In keeping with previous data on neonatal B cells, HSAhi cells from either normal or self-reconstituting mice are refractory to stimulation with anti-IgM antibodies, yet proliferate upon LPS stimulation, and generate primary antibody responses if given appropriate T cell help. In contrast to neonatal cells, HSAhi adult B cells are refractory to stimulation with PMA plus ionomycin. Together, these data suggest that peripheral HSAhi B cells in adults correspond to recently generated B cells, whose signaling characteristics are distinct from previously described B cell subsets.     

Direct involvement of surface IA/E molecules during B cell maturation using an antigen-specific B cell clone

TP67.14 is a subclone of a resulting B cell hybridoma established by somatic hybridization between splenic B cells of A/J mice immunized with TNP-LPS and 2.52 M, a HAT medium-sensitive mutant of a B cell line; it expresses IgM, B220, IAk, and IEk on the cell membrane and also possesses a receptor molecule for TNP on its surface derived from TNP-reactive B cells of A/J mice used for cell fusion. As shown previously, TP67.14 could be induced to generate a significant amount of anti-TNP antibodies when treated with TNP-conjugated protein such as TNP-BSA and TNP-keyhole limpet hemocyanin without T cell help as well as LPS. Our study was undertaken to investigate direct involvement of surface MHC class II molecules on B cells during B cell maturation by analysis with this Ag-specific B cell clone. The data demonstrate that mAb against IAk and IEk molecules, but not IAd and H-2k, markedly inhibited the differentiative effects of LPS on TP67.14. In contrast, both antibodies specifically augmented the secretion of anti-TNP antibodies by TP67.14 treated with TNP-BSA, although these antibodies alone failed to induce the generation of anti-TNP antibodies. Interestingly, TP67.14 significantly differentiated into anti-TNP antibody secreting cells when incubated with TNP-conjugated monoclonal anti-IAk or anti-IEk antibodies alone; this differentiative effect was much greater than that of TNP-conjugated anti-IAd mAb or purified mouse IgG under the same conditions. Our result suggests that surface IA/E molecules on B cells may be directly involved in a transductional signal for B cell maturation mediated by the cross-linkage of receptor molecules on B cells with Ag.     

Split tolerance in peripheral B cell subsets in mice expressing a low level of Igkappa-reactive ligand

Peripheral B cell tolerance differs from central tolerance in anatomic location, in the stage of B cell development, and in the diversity of Ag-responsive cells. B cells in secondary lymphoid organs are heterogeneous, including numerous subtypes such as B-1, marginal zone, transitional, and follicular B cells, which likely respond differently from one another to ligand encounter. We showed recently that central B cell tolerance mediated by receptor editing was induced in mice carrying high levels of a ubiquitously expressed kappa-macroself Ag, a synthetic superantigen reactive to Igkappa. In this study, we characterize a new transgenic line that has a distinctly lower expression pattern from those described previously; the B cell tolerance phenotype of these mice is characterized by the presence of significant numbers of immature kappa+ B cells in the spleen, the loss of mature follicular and marginal zone B cells, the persistence of kappa+ B-1 cells in the peritoneal cavity, and significant levels of serum IgM,kappa. These findings suggest distinct signaling thresholds for tolerance among peripheral B cell subsets reactive with an identical ligand.     

Reduced responsiveness of immature B cells to the B cell mitogen, lipoprotein.

When pre-B cells are isolated from bone marrow and incubated in vitro for several days, they spontaneously differentiate into LPS-reactive B cells. These newly produced B cells do not respond to another B cell mitogen, lipoprotein (Lp). The lack of Lp reactivity of newly produced B cells represents a qualitative difference between these cells and B cells in peripheral tissues that respond equally well to both LPS and Lp. Thus, responsiveness to different mitogens provides unique markers to identify B cells at early stages of development.     

Extrathymic T cell maturation. Phenotypic analysis of T cell subsets in nude mice as a function of age.

T cell maturation in an extrathymic environment has been studied using as a model the congenitally athymic nude mouse. Phenotypic analyses as a function of age were conducted on lymphocytes obtained from the spleens and lymph nodes of nude mice through use of mAb recognizing T cell surface markers and multiparameter flow cytometry. The data show that nude mice accumulate increasing numbers of lymphocytes bearing Thy-1, CD3, CD4, and CD8 with age characterized by a progression from heterogeneous dim to more homogeneous bright expression. In contrast, the expression of heat-stable Ag (HSA), a marker of immature thymocytes, decreases with age. By analogy to intrathymic maturation, spleens and lymph nodes in nude mice contain T cells defined as immature, transitional, and mature based on the expression of these markers. Although the proportion of CD4+ and CD8+ T cells associated with bright CD3 expression increases with age, at no age are significant numbers of CD4+8+ cells observed, in contrast to intrathymic T cell maturation. In addition to the frequently observed inversion in the ratio of CD4 to CD8, the CD8 T cell subpopulation in older nude mice contains mainly mature cells (CD8+, CD3+, HSA-) whereas only 50% of CD4+ T cells express the mature (CD4+, CD3+, HSA-) phenotype. At any age, the spectrum of phenotypes observed indicates that lymph nodes contain more mature T cells than spleen, suggesting a role for environmental Ag in driving extrathymic maturation, a process occurring most efficiently among CD8+ T cells. Because extrathymic maturation mirrors some but not all aspects of the intrathymic pathway, we propose that the nude mouse may be a useful model for further dissecting those interactions crucial to establishing the T cell repertoire in euthymic individuals as well as elucidating the contribution of extrathymically derived T cells to the peripheral immune system.     

Distinct responses of splenic dendritic cell subsets to infection with Listeria monocytogenes: maturation phenotype, level of infection, and T cell priming capacity ex vivo

To determine the relative contributions of DC subsets in the development of protective immunity to Listeria monocytogenes we examined the relationship between maturation, bacterial burden, and T cell priming capacity of four well characterized subsets of splenic DC following infection with Lm. CD8α⁺, CD4⁺, and CD8α⁻CD4⁻ DC and the B220⁺ plasmacytoid DC (pDC) were compared for abundance and costimulatory molecule expression at 24, 48, and 72 h post i.v. infection. We further determined the bacterial burden associated with each DC subset and their relative capacities to prime CD8⁺ T cells at 24 hpi. The CD8α⁺ DC displayed the highest level of maturation, association with live bacteria, and T cell activation potential. Second, the CD4⁺ DC were also mature, yet were associated with fewer bacteria, and stimulated T cell proliferation, but not IFN-γ production. The CD8α⁻CD4⁻ DC showed a modest maturation response and were associated with a high number of bacteria, but failed to induce T cell proliferation ex vivo. pDC displayed a strong maturation response, but were not associated with detectable bacteria and also failed to stimulate T cell activation. Finally, we measured the cytokine responses in these subsets and determined that IL-12 was produced predominantly by the CD8⁺ DC, correlating with the ability of this subset DC to induce IFN-γ production in T cells. We conclude that Listeria-specific CD8⁺ T cell activation in the spleen is most effectively achieved by infection-induced maturation of the CD8α⁺ DC subset.     

The asynchrony of gamma- and beta-chain synthesis during human erythroid cell maturation. III. gamma- and beta-mRNA in immature and mature erythroid clones

The synthesis of the β-crystallin polypeptides has been studied in different regions of the embryonic chicken lens. Seven β-crystallin polypeptides ranging in molecular weight from approximately 19,000 (19K) to 35,000 (35K) daltons were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each polypeptide was synthesized in a rabbit reticulocyte cell-free system supplemented with RNA from the embryonic lens fiber cells suggesting that each is encoded by a separate mRNA. Analysis of the cell-free translation products of the RNAs from 6-, 15-, and 19-day-old embryonic chicken lens fibers demonstrated that all seven polypeptides are translated at each of the stages and that the proportion of β-crystallin mRNAs increases as the chicken embryo matures. Fingerprints of methionine-containing tryptic peptides indicated that the three predominant β-crystallin polypeptides synthesized in the reticulocyte lysate (20K, 26K, and 35K) have related but distinct primary structures. Surprisingly, both the 35K β-crystallin polypeptide and its mRNA were selectively absent from the cells in the central region of the epithelium. Synthesis of this polypeptide from extracted RNAs was detected in the elongating cells of the equatorial region of the epithelium and from the fiber cells. In contrast to the 35K polypeptide, the six lower-molecular-weight β-crystallin polypeptides were synthesized in a reticulocyte lysate directed by RNAs extracted from all three regions of the lens. These data indicate that lens cell elongation and fiber cell differentiation in the embryonic chicken are accompanied by the appearance of the mRNA for the 35K polypeptide.     

Distinct effector cytokine profiles of memory and naive human B cell subsets and implication in multiple sclerosis

Although recent animal studies have fuelled growing interest in Ab-independent functions of B cells, relatively little is known about how human B cells and their subsets may contribute to the regulation of immune responses in either health or disease. In this study, we first confirm that effector cytokine production by normal human B cells is context dependent and demonstrate that this involves the reciprocal regulation of proinflammatory and anti-inflammatory cytokines. We further report that this cytokine network is dysregulated in patients with the autoimmune disease multiple sclerosis, whose B cells exhibit a decreased average production of the down-regulatory cytokine IL-10. Treatment with the approved chemotherapeutic agent mitoxantrone reciprocally modulated B cell proinflammatory and anti-inflammatory cytokines, establishing that the B cell cytokine network can be targeted in vivo. Prospective studies of human B cells reconstituting following in vivo depletion suggested that different B cell subsets produced distinct effector cytokines. We confirmed in normal human B cell subsets that IL-10 is produced almost exclusively by naive B cells while the proinflammatory cytokines lymphotoxin and TNF-alpha are largely produced by memory B cells. These results point to an in vivo switch in the cytokine "program" of human B cells transitioning from the naive pool to the memory pool. We propose a model that ascribes distinct and proactive roles to memory and naive human B cell subsets in the regulation of memory immune responses and in autoimmunity. Our findings are of particular relevance at a time when B cell directed therapies are being applied to clinical trials of several autoimmune diseases.     

Differentiation of an immature T cell line: a model of thymic positive selection.

Thymocyte differentiation is dependent upon recognition of major histocompatibility complex (MHC) molecules on thymic stroma, a process called positive selection. Here we describe an immature CD4+8+ T cell line derived from a TCR transgenic mouse that differentiates into CD4+8- cells in response to antigen and nonthymic antigen-presenting cells. When injected intrathymically, these cells differentiate in the absence of antigen. The ability of immature T cells to recognize MHC molecules in the absence of foreign antigen in the thymus can thus be attributed to a unique property of thymic antigen-presenting cells. These studies also demonstrate the phenotypic and functional changes associated with TCR-mediated T cell maturation and establish an in vitro model system of positive selection.     

Role of I-A molecules in early stages of B cell maturation.

The role of the Ia molecule in the early phase of B cell development remains controversial. In contrast to previous studies, we have detected minute amounts of Ia (I-A) molecule on early B lineage (B220+IgM-) cells from normal bone marrow, using ELISA. The presence of the I-A molecule even on pro-B cells was deduced from experiments in which a monoclonal anti-I-A antibody completely blocked the generation of pre-B cells from B progenitor (B220-) cells in stromal cell-dependent B cell culture. Inasmuch as this antibody did not inhibit the maturation of pre-B cells to IgM+ B cells in culture, the I-A molecule on early B lineage cells probably plays a role in their maturation. We also examined the role of the I-A molecule in early B cell development, using transgenic mice harboring the antisense DNA to I-A beta-chain gene. The amount of I-A molecule on splenic B cells from the young transgenic mice decreased in the presence of abundant amounts of the antisense RNA. B cell development was perturbed in spleen from the transgenic mice. Stromal cell-dependent B cell cultures from these mice clearly showed that the maturation of B lineage cells was delayed at a very early stage of development (B220- to B220+). We propose that the I-A molecule on early B lineage cells may play an essential role in their maturation.     

Incomplete activation of CD4 T cells by antigen-presenting transitional immature B cells: implications for peripheral B and T cell responsiveness

B cells leave the bone marrow as transitional B cells. Transitional B cells represent a target of negative selection and peripheral tolerance, both of which are abrogated in vitro by mediators of T cell help. In vitro, transitional and mature B cells differ in their responses to B cell receptor ligation. Whereas mature B cells up-regulate the T cell costimulatory molecule CD86 (B7.2) and are activated, transitional B cells do not and undergo apoptosis. The ability of transitional B cells to process and present Ag to CD4 T cells and to elicit protective signals in the absence of CD86 up-regulation was investigated. We report that transitional B cells can process and present Ag as peptide:MHC class II complexes. However, their ability to activate T cells and elicit help signals from CD4-expressing Th cells was compromised compared with mature B cells, unless exogenous T cell costimulation was provided. A stringent requirement for CD28 costimulation was not evident in interactions between transitional B cells and preactivated CD4-expressing T cells, indicating that T cells involved in vivo in an ongoing immune response might rescue Ag-specific transitional B cells from negative selection. These data suggest that during an immune response, immature B cells may be able to sustain the responses of preactivated CD4(+) T cells, while being unable to initiate activation of naive T cells. Furthermore, the ability of preactivated, but not naive T cells to provide survival signals to B cell receptor-engaged transitional immature B cells argues that these B cells may be directed toward activation rather than negative selection when encountering Ag in the context of a pre-existing immune response.     

Interleukin 4 induces the maturation of idiotype-specific regulatory B cell populations.

CD5+ B cells have been shown to be disproportionately associated with autoimmune diseases and transformation. In many cases, their apparent ability to bypass self-tolerance is manifested by the production of autoantibodies. These observations, plus the hypothesis that CD5+ B cells represent a distinct B cell lineage, encourage studies into the conditions and factors that influence their development. In the present study, we employed a well-established assay for murine CD5+ B cell function, i.e., their ability to augment the responses of IgHb-linked idiotypic determinants on anti-(4-hydroxy-3-nitrophenyl) acetyl (NP) antibody (Nbb) idiotype-bearing CD5- B cells to a T-independent antigen, together with multiple methods of cell surface phenotyping, to evaluate the potential for interleukin (IL) 4 to effect maturation of CD5+ B cells, CD5+, IgM+, Thy-1-, and NPb idiotype-specific cells panned on antibody-coated petri dishes or sorted by flow cytometry from spleen were capable of augmenting NPb idiotypic responses of NP-KLH-primed responder cells to NP-Ficoll. Splenic B cell populations depleted of CD5+ B cells failed to affect idiotype expression even after 2 days in culture, a time when a small percentage of CD5+ B cells appeared to be regenerated. However, idiotype-specific regulatory activity could be restored in CD5- splenic B cell populations by culture for 2 days with recombinant IL-4. Cells responsible for idiotype-specific regulatory activity after culture with IL-4 were in fact CD5+, IgM+, and Thy-1.2- B cells, demonstrating that IL-4 can drive the functional, if not the phenotypic, maturation of splenic B cells associated with the CD5+ B cell lineage. The results illustrate one possible mechanism by which T cells could control the maturation of cells belonging to the CD5+ B cell lineage.     

Expression profiling of a transformed thymocyte cell line undergoing maturation in vitro identifies multiple genes involved in positive selection

Biochemical and genetic studies of thymocyte maturation would be facilitated by the development of cultured cell lines that reflect stages of positive selection. We have derived a CD4(+)CD8(+)TCR(+) T-lymphoid cell line (M20) from a murine thymic tumor induced by a retrovirus carrying the v-myc oncogene (M-MuLV(myc)). M20 subclones undergo several aspects of positive selection in response to co-culture with a thymic stromal cell line (St3), including down-regulation of CD4 and CD8, and up-regulation of CD5 and TCR. M20 possesses a functional TCR complex, and ligation of this complex produces changes similar to co-culture with St3 stroma. Expression profiling of M20 cells in this system identified 23 genes previously shown to be important in thymocyte maturation, as well as several novel candidate genes. This system provides a new model to elucidate the molecular mechanisms of thymocyte maturation and TCR-mediated cell signaling in double-positive thymocytes.     

Single-cell transcriptome analysis reveals three sequential phases of gene expression during zebrafish sensory hair cell regeneration

1. Download : Download high-res image (244KB)
2. Download : Download full-size image

     

B lymphocyte differentiation induced by lipopolysaccharide. III. Suppression of B cell maturation by anti-mouse immunoglobulin antibodies.

Lipopolysaccharide-(LPS) induced differentiation of mouse B lymphocytes to cells synthesizing large amounts of cytoplasmic IgM and IgG2 could be suppressed by antibodies to mu-chains. Maximal inhibition of LPS-induced differentiation was associated with increased cellular proliferation as measured by incorporation of 3H-thymidine, whereas treatment with anti-mu alone over a wide dosage range did not stimulate cellular proliferation. Spleen cells from newborn mice were suppressed by concentrations of anti-mu several hundred-fold lower than required for adult spleen cells; the adult pattern of susceptibility to suppression was acquired by 1 week of age. No significant differences in susceptibility to anti-mu were found in comparisons of adult spleen, lymph node, bone marrow, and Peyer's patch lymphocytes.     

Differential modulation of human epidermal Langerhans cell maturation by ultraviolet B radiation.

UVB irradiation of the skin causes immunosuppression and Ag-specific tolerance in which Langerhans cells (LC) are involved. We tested the effect of UVB on LC that had migrated out of cultured epidermal sheets derived from the skin that was irradiated ex vivo (200, 400, 800, or 1600 J/m2). Two separate subpopulations of LC were distinguished: large-sized LC with high HLA-DR expression, and HLA-DR-low, small LC. UVB stimulated the maturation of the former LC subset as demonstrated by enhanced up-regulation of CD80, CD86, CD54, CD40, and CD83 and reduced CD1a expression in comparison with unirradiated controls. In contrast, the latter LC exhibited little or no up-regulation of these molecules except for high CD1a expression and high binding of annexin V, indicating that they were apoptotic, although their CD95 expression was relatively low. Stimulation of enriched LC with CD40 ligand-transfected cells and IFN-gamma revealed that the release of IL-1beta, IL-6, IL-8, and TNF-alpha was enhanced by UVB. In comparison with HLA-DR-low LC, HLA-DR-high LC were the principal IL-8 producers as demonstrated by intracellular cytokine staining, and they retained more accessory function. There was no detectable secretion of IL-12 p70, and IL-18 production was neither affected by any stimulus nor by UVB. These results suggest a dual action of UVB on LC when irradiated in situ: 1) immunosuppression by preventing maturation and inducing apoptotic cell death in part of LC, and 2) immunopotentiation by enhancing the up-regulation of costimulatory molecules and the production of proinflammatory cytokines in another part.