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The diversity in antibody repertoire relies on different B cell populations working efficiently to fulfil distinct specific functions. We recently described an innate-like CD19(+)CD45R(-/lo) (19(+)45R(lo)) cell population in postnatal unstimulated adult mice, a heterogeneous population containing cells expressing immunoglobulin M (IgM) and others behaving as differentiated mature B lymphocytes (intracytoplasmic IgG1, AID(+), Blimp-1(+)RAG2(-)). In the present study, we characterized the Ig repertoire expressed by splenic 19(+)45R(lo) cells, assuming that they would bear a restricted repertoire biased for germline rearrangements and low mutation rates similar to other innate-like cells. Sequences from 19(+)45R(lo) cells displayed a variety of V, D and J regions, and the analysis of the CDR-H3 region revealed an intermediate overall CDR-H3 length and moderate hydrophobicity. Both IgM and switched sequences of PD15 19(+)45R(lo) cells had shorter CDR-H3 region and fewer non-template N nucleotides than adult sequences, as expected for profiles that correspond to an immature phenotype. Regarding the mutation rate in the VH regions, IgG1 sequences already carried a high rate of replacement mutations at PD15, which increased further in the sequences obtained from adult mice. Moreover, statistical models suggest that a proportion of the switched sequences in adult 19(+)45R(lo) cells had experienced antigen selection, unlike other innate-like B cell compartments., (© 2014 S. Karger AG, Basel.)

Teleost fish are among the most ancient vertebrates possessing an adaptive immune system with B and T lymphocytes that produce memory responses to pathogens. Most bony fish, however, have only 2 types of B lymphocytes, in contrast to the 4 types available to mammals. To better understand the evolution of adaptive immunity, we generated transgenic zebrafish in which the major immunoglobulin M (IgM(+)) B-cell subset expresses green fluorescence protein (GFP) (IgM1:eGFP). We discovered that the earliest IgM(+) B cells appear between the dorsal aorta and posterior cardinal vein and also in the kidney around 20 days postfertilization. We also examined B-cell ontogeny in adult IgM1:eGFP;rag2:DsRed animals, where we defined pro-B, pre-B, and immature/mature B cells in the adult kidney. Sites of B-cell development that shift between the embryo and adult have previously been described in birds and mammals. Our results suggest that this developmental shift occurs in all jawed vertebrates. Finally, we used IgM1:eGFP and cd45DsRed;blimp1:eGFP zebrafish to characterize plasma B cells and investigate B-cell function. The IgM1:eGFP reporter fish are the first nonmammalian B-cell reporter animals to be described. They will be important for further investigation of immune cell evolution and development and host-pathogen interactions in zebrafish.

Artiodactyls possess GALT that appears in fetal life and is located at the extreme end of the ileum. These IPP contain mostly B cells and involute early in postnatal life. Rabbits have a similarly located lymphoid organ, called the sacculus rotundus. Studies in sheep and rabbits have led to the concept that the lower hindgut GALT represents primary lymphoid tissue for B cells and is necessary for normal B cell development, analogous to the bursa of Fabricius. This review traces the history of the observations and theories that have led to the existing concept concerning the role of lower GALT. We then review recent data from piglets with resected IPP that challenges the concept that the IPP is primary B cell lymphoid tissue and that artiodactyls and rabbits are members of the GALT group in the same context as gallinaceous birds. Eliminating the IPP as the primary lymphoid tissue for B cells leads to the hypothesis that the IPP acts as first-responder mucosal lymphoid tissue.

Antigen-independent B-cell development occurs in several stages that depend on the expression of Ig heavy and light chain. We identified a line of mice that lacked mature B cells in the spleen. This mouse line carried approximately 11 copies of a transgene of the murine heavy chain constant region locus, and B-lineage cells expressed excessive amounts of the intracellular μ heavy chain. B-cell development failed in the bone marrow at the pro/pre B-cell transition, and examination of other lines with various copy numbers of the same transgene suggested that deficiencies in B-cell development increased with increased transgene copy number. Expression of a transgenic (Tg) light chain along with the Tg μ heavy chain led to minimal rescue of B-cell development in the bone marrow and B cells in the spleen. There are several potential mechanisms for the death of pro/pre B cells as a consequence of excess heavy chain expression.

B lymphocytes are characterized by a unique and highly specialized developmental pathway that is responsible for their vast phenotypic and function diversity. B cell development is strictly regulated to ensure sufficient specific humoral immunity while at the same time avoiding any errors that would compromise B cell functionality. The generation and maintenance of mature B cells from the constant flux of bone marrow progenitors is a complex process that is generally poorly understood, although great progress has been made in recent years. B cells have for long been considered mainly as antibody-producing cells and therefore believed to play an important role in the pathophysiology of allergic diseases, primarily through their ability to produce IgE antibodies. However, recent findings have revealed new aspects of their role in immune responses that place them again under the spotlight as important immune regulators, independent of antibody production. This review focuses on the developmental processes responsible for the numerous phenotypes and functions of the B-lymphocyte pool and the different aspects of effector B cell functionality in the context of allergy.

Germinal centres are receiving renewed attention following recent intravital multi-photon imaging studies. These data have shed new light on longstanding questions about the spatial organisation of germinal centres, B-cell migration, selection and differentiation. Mathematical models have proven invaluable in the analysis of intravital motility data, and have predicted novel B-cell selection mechanisms that are now supported by experimental findings. We argue that mathematical modelling adds a different vantage point to experimental data and provides a quantitative and systematic analysis of hypotheses and theories in immunology. Furthermore, the well-characterised nature of the germinal centre provides an excellent proving ground for mathematical modelling.

Cellular and humoral aspects of the immune response develop sequentially in the fetus. During the ontogeny, the pluripotent stem cells emerge and differentiate into all hematopoietic lineages. Basic questions including the identification of the first lympho-hematopoietic sites, the origin of T and B lymphocytes, the development of different subpopulations of alphabeta T, gammadelta T and B lymphocytes as well as development of innate immunity and the acquisition of full immunological capacities are discussed here for swine and compared with other species. The description of related topics such as fertilization, morphogenesis, maternal-fetal-neonatal physiology and early neonatal development are also discussed.

Trophic and instructive models have been invoked to explain how various cytokines influence the survival, proliferation and differentiation of lymphoid progenitors. There is evidence that IL-7 can act in either a trophic or an instructive manner during B and T cell development although its roles in B cell development in the fetus versus the adult mouse and in the mouse versus the human may vary. Here we outline the stages of B cell development and the conventional model of the role of IL-7 in the transitions from pro-B cells to pre-B cells and then to immature B cells. We also discuss the implications of recent data that have led to a new model for this process.

The nerve growth factor (NGF) receptor TrkA is widely expressed in non-neural tissues suggesting pleiotropic functions outside the nervous system. Based on pharmacological and immuno-depletion experiments, it has been hypothesized that NGF plays an important role in the normal development and function of the immune system. However, attempts to unravel these functions by conventional gene targeting in mice have been hampered by the early postnatal lethality caused by null mutations. We have developed a novel 'reverse conditional' gene targeting strategy by which TrkA function is restored specifically in the nervous system. Mice lacking TrkA in non-neuronal tissues are viable and appear grossly normal. All major immune system cell populations are present in normal numbers and distributions. However, mutant mice have elevated serum levels of certain immunoglobulin classes and accumulate B1 cells with aging. These data, confirmed in a classical reconstitution model using embryonic fetal liver from TrkA-null mice, demonstrate that endogenous NGF modulates B cell development through TrkA in vivo. Furthermore, they demonstrate that many of the dramatic effects previously reported by pharmacological or immuno-depletion approaches do not reflect physiological developmental roles of TrkA in the immune system.

Current models of adult hematopoiesis propose that pluripotent hematopoietic stem cells (PHSC) differentiate into common myeloid- and lymphoid-committed precursors and that this establishes an early separation between the myeloid and lymphoid lineages. However, the description of rare, and previously unidentified, B/macrophage progenitors in postnatal bone marrow is not consistent with this model. Hypotheses to explain and reconcile their existence are discussed with particular emphasis on the possibility that fetal B/macrophage progenitors, whose development is well accepted, may persist into adult life. The existence of a normally occurring adult B/macrophage progenitor has implications for understanding the origin of tumors with B cell and myeloid characteristics.

B cells are present in human fetal intestine from approximately 14 weeks of gestation. Here we show that this population includes mature, dividing B cells. These are large cells with dendritic processes, resembling human thymic B cells. In addition, we observed IgM+, light chain-, and CD20- cells and local expression of V pre-B, demonstrating that the human fetal intestine is a site of B cell development. Ig V(H)DJ(H) gene sequencing can confirm clonal identity of B cells. Identification of the same IgV(H)4-34 sequence in serial sections in two fetuses confirmed local accumulation of related cells in each case. IgV(H)4-34 was also amplified from an additional two samples, and the D and J repertoire compared with a unique database of unselected V(H)4-34 genes from postnatal gut. Distinguishing characteristics of Ig lambda genes in postnatal gut were also studied in the fetus. According to these parameters, fetal and postnatal B cells are unrelated.

B cell development is a highly regulated process whereby functional peripheral subsets are produced from hematopoietic stem cells, in the fetal liver before birth and in the bone marrow afterward. Here we review progress in understanding some aspects of this process in the mouse bone marrow, focusing on delineation of the earliest stages of commitment, on pre-B cell receptor selection, and B cell tolerance during the immature-to-mature B cell transition. Then we note some of the distinctions in hematopoiesis and pre-B selection between fetal liver and adult bone marrow, drawing a connection from fetal development to B-1/CD5(+) B cells. Finally, focusing on CD5(+) cells, we consider the forces that influence the generation and maintenance of this distinctive peripheral B cell population, enriched for natural autoreactive specificities that are encoded by particular germline V(H)-V(L) combinations.

Immune reconstitution during bone marrow transplantation has been proposed to produce a fetal-type immune system. This characteristic may contribute to the relative immunodeficiency that occurs in the early post-transplant period. This review reappraises recent studies of immunoglobulin heavy chain genes produced by the recovering immune system. Comparison of these genes to those that are generated by fetal and adult B cells, demonstrates that there is no evidence to support the conclusion that adult lymphocytes in the graft reverse to a fetal stage of differentiation. In terms of lymphocyte diversity, the inadequacy of the recovering immune system is more likely to be explained by a combination of other factors - such as the delayed occurrence of somatic hypermutation and class switching, and clonal dominance.

During neonatal life, Ig diversity is limited in many respects. The absence of terminal deoxynucleotidyl transferase (TdT) expression with the consequent lack of nontemplated addition during the neonatal period, coupled with the predominant usage of a single D(H) reading frame (RF), leads to severe limitations of diversity in the CDR3 region of Ig heavy (H) chains. The neonatal Ig H chain repertoire is also characterized by restricted V(H) usage, with predominant expression of certain V(H) segments, such as V(H)81x, that are rarely evident during adult life. In this report, we examine the effect of enforced TdT expression on the neonatal repertoire of V(H)81xDJ(H) rearrangements. We find that TdT synthesis abrogates D(H) RF bias during the fetal/neonatal period through a Ig-receptor-independent mechanism. These findings suggest that D(H) RF bias during neonatal life is determined largely by homology-directed joining. We also find that TdT synthesis alters the selection of productively rearranged V(H)81xDJ(H) alleles in the neonatal spleen through a Ig-receptor-dependent mechanism. Analysis of predicted CDR3 amino acid sequences indicates that positive selection of V(H)81x-encoded H chains is correlated with the presence of a consensus sequence immediately adjacent to the V(H) segment. These data support the hypothesis that the CDR3 region is critical in determining the ability of V(H)81x-encoded H chains to form functional receptors that support positive selection of B lymphocytes. Together, our results demonstrate that TdT can indirectly influence the Ig repertoire by influencing both receptor-dependent and receptor-independent selection processes.

Human B lymphocytes share one major distinctive feature with B cells of other higher animals, namely the ability to generate and secrete immunoglobulins. These highly specialized proteins are capable of tremendous diversity, and thereby account for much of our immune protection against invading organisms. Despite the great potential diversity possible in the specificities of immunoglobulin molecules, however, the binding of antibody to antigen initiates a limited spectrum of biologically important effector functions, such as complement activation and/or adherence of the immune complex to receptors on leukocytes. A variety of mechanisms have been elucidated that account for this, not all of which are shared by the different types of animals capable of making these proteins. The purpose of this chapter is to review the genetic, developmental, and physiologic mechanisms critical for human B cell expression of immunoglobulin.

Development of B cells in chickens proceeds via a series of discrete developmental stages that includes the maturation of committed B cell progenitors in the specialized microenvironment of the bursa of Fabricius. The bursa has been shown to be required for the amplification of the B cell pool and selects for cells with productive immunoglobulin rearrangement events. Other events regulating chicken B cell development such as lymphocyte trafficking and apoptosis are just beginning to be elucidated. Within the bursa, the variable regions of immunoglobulin genes of B cell progenitors are diversified by a process of intrachromosomal gene conversion, where blocks of sequence information are transferred from pseudo-V regions to the recombined variable regions of the immunoglobulin genes. Recently gene conversion has been determined to play a role in the diversification of the immune repertoire in other species. In this review we focus on the current understanding and recent advances of B cell development in the chicken.

The development and establishment of the B Cell Repertoire is the net result of both genetic and environmental forces. The primary event at the genetic level is Ig gene rearrangement resulting in numerous possible combination of genes which can be further modified by somatic events such as N segment addition and somatic mutation. Environmental forces in the form of self and exogenous Ags also shape the repertoire by positively or negatively selecting B cells according to the specificity of their Ig receptors. These are dynamic processes beginning with the earliest expression of immunoglobulins in fetal life and continuing throughout life. In this review we discuss the genetic and selective mechanisms responsible for differences in the early immune system compared to that of the adult.

The mouse B-cell repertoire early in ontogeny contains B cells with receptor immunoglobulins exhibiting high connectivity, multi/self-reactivity, and generally low affinity. This is due structurally to extensive restriction in the germline components used to generate the B-cell receptor. The selective pressure acting on the nascent repetoire has both negative and positive components as seen in our in vivo models. VH81X-bearing B cells from the VH81X transgenic mice (and probably from normal mice) are subject to self-selective pressure with two components: a positive one favoring a certain (self-reactive) specificity in the CD23-IgM+ population and a negative one preventing the entry of B cells with this specificity into the CD23+IgM+ compartment.

C-myb encodes a transcriptional activator that is essential for the development of the hematopoietic system but appears to lack major roles in non-hematopoietic cells. The identification of two conserved myb-related genes, designated A-myb and B-myb, has raised the possibility that these genes are functional equivalents of c-myb in non-hematopoietic cells. Here, we report the isolation and preliminary characterization of the mouse A-myb gene. Mouse A-myb maps to the proximal region of chromosome 1 and encodes a transcriptional activator with properties similar to those of the c-myb and v-myb proteins. During embryo-genesis A-myb is predominantly expressed in several regions of the developing central nervous system (CNS) and the urogenital ridge. Expression in the CNS is confined to the neural tube, the hindbrain, the neural retina and the olfactory epithelium, and coincides with the presence of proliferating immature neuronal precursor cells. In the adult mouse, A-myb is expressed during the early stages of sperm cell differentiation and in B lymphocytes located in germinal centers of the spleen. Taken together, these results suggest a role for A-myb in the proliferation and/or differentiation of neurogenic, spermatogenic and B-lymphoid cells.

To study the ontogeny of lymphocytes in the lizard, Chalcides ocellatus antisera were raised in rabbits against thymocytes collected from adult lizards (AATS) or from embryos of stages 40 and 41 (AETS), and against serum immunoglobulins (AGGS). AATS recognized in adult lizards a surface membrane antigenic system (TA) specific to thymocytes and thymus-derived (T-) cells in spleen, and a distinct antigenic entity (Tt) found exclusively on intrathymic lymphocytes. After exhaustive absorption with adult thymocytes, AETS defined a further antigen (TE). On the other hand, AGGS combined with the immunoglobulin (Ig) determinants present in the cytoplasm and surface membrane of lizard presumptive B lymphocytes. Immunofluorescence assays during the development of the T and B lineage showed that TA+ and Tt+ thymocytes increased from 35% at stage 37 to 96% at stage 41, and were exported precociously to the embryonic splenic environment. Embryonic thymocytes were shown to totally lack surface Ig, whereas 40-50% of splenocytes carried cytoplasmic and/or surface membrane Ig determinants. The data revealed further the presence of a subset of splenocytes bearing the specific markers of both T and B cells.