Emerging roles for the FCRL family members in lymphocyte biology and disease.

Members of the extended Fc receptor-like (FCRL) family in humans and mice are preferentially expressed by B cells and possess tyrosine-based immunoregulatory function. Although the majority of these proteins repress B cell receptor-mediated activation, there is an emerging evidence for their bifunctionality and capacity to counter-regulate adaptive and innate signaling pathways. In light of these findings, the recent discovery of ligands for several of these molecules has begun to reveal exciting potential for them in normal lymphocyte biology and is launching a new phase of FCRL investigation. Importantly, these fundamental developments are also setting the stage for defining their altered roles in the pathogenesis of a growing number of immune-mediated diseases. Here we review recent advances in the FCRL field and highlight the significance of these intriguing receptors in normal and perturbed immunobiology.

Immunoglobulin recombinase gene activity is modulated reciprocally by interleukin 7 and CD19 in B cell progenitors.

Bone marrow stromal cells promote B cell development involving recombinase gene-directed rearrangement of the immunoglobulin genes. We observed that the stromal cell-derived cytokine interleukin 7 (IL-7) enhances the expression of CD19 molecules on progenitor B-lineage cells in human bone marrow samples and downregulates the expression of terminal deoxynucleotidyl transferase (TdT) and the recombinase-activating genes RAG-1 and RAG-2. Initiation of the TdT downregulation on the first day of treatment, CD19 upregulation during the second day, and RAG-1 and RAG-2 downmodulation during the third day implied a cascade of IL-7 effects. While CD19 ligation by divalent antibodies had no direct effect on TdT or RAG gene expression, CD19 cross-linkage complete blocked the IL-7 downregulation of RAG expression without affecting the earlier TdT response. These results suggest that signals generated through CD19 and the IL-7 receptor could modulate immunoglobulin gene rearrangement and repertoire diversification during the early stages of B cell differentiation.

Helper T cell subsets for immunoglobulin A responses: oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosa associated tissues.

Antigen-specific B cell responses to mucosally delivered proteins are dependent upon CD4-positive T helper (Th) cells, and the frequency of Th1 and Th2 cell responses after oral immunization may determine the level and isotype of mucosal antibody responses. We have used a protein-based vaccine, tetanus toxoid (TT), together with the mucosal adjuvant cholera toxin (CT), for oral immunization of mice to study the nature of antigen-specific Th cell subsets induced in Peyer's patches (PP) of the gastrointestinal (GI) tract and in the spleen (SP) during peak antibody responses. Mice orally immunized with TT and CT responded with antigen-specific secretory immunoglobulin A (S-IgA) antibodies in the GI tract, and with both IgG and IgA antibody responses in serum. PP and SP CD4+ T cells from mice orally immunized with TT plus CT were cultured with antigen-coated latex microspheres for induction of proliferative responses and for enumeration of cytokine producing CD4+ T cells. Interestingly, both PP and SP CD4+ T cell cultures showed increased numbers of IL-4- and IL-5 (Th2-type)-producing, spot-forming cells (SFCs) after 21 d of immunization, while essentially no interferon-gamma (IFN-gamma) or IL-2 (Th1-type) SFCs were noted. Cytokine-specific Northern blots and RT-PCR also revealed that significant IL-4 and IL-5 mRNA levels, but not IFN-gamma or IL-2 mRNA, were present in CD4+ T cells isolated from antigen-stimulated cultures. However, systemic immunization with TT and CT induced antigen-specific IgG and IgM but not IgA antibodies in serum. Further, both IL-2 and IFN-gamma-producing Th1-type cells as well as IL-4- and IL-5-secreting Th2-type cells were generated in SP. Our results show that oral immunization with TT and the mucosal adjuvant CT selectively induced antigen-specific Th2-type responses which may represent the major helper cell phenotype involved in mucosal IgA responses in the GI tract.

B-cell development in man.

The development of B-lineage cells requires a series of complex interactions with hemopoietic stromal cell elements during the generative phase, and with antigen and T lymphocytes during the subsequent proliferative/differentiative phases in lymphoid tissues. Recent advances have been made in defining developmental changes in structure and assembly of the antigen receptors and in identifying protein kinases involved in signal transduction via these receptors. The mechanism of T-cell help has also come into much clearer focus through elucidation of the interaction between CD40 on B cells and the CD40 ligand on activated T cells. Finally, progress has been made with the recent identification of defects in a cytoplasmic protein tyrosine kinase and in the CD40 ligand as causes of two B-cell immunodeficiencies in man.

B cell development and differentiation.

The initial phases of B cell development depend on interactions between the cell surface molecules and secreted products of stromal cells with their receptor-ligand partners on lymphoid progenitors. Recent research in this area has greatly advanced our understanding of B cell development and differentiation. Antigen receptors on pre-B and B cells play key roles in the progression of this differentiation process, as revealed by targeted and inherited gene mutations that disrupt B cell development and by the transgenic repair of these mutations in mice.

The neuronal EGF-related genes NELL1 and NELL2 are expressed in hemopoietic cells and developmentally regulated in the B lineage.

NELL1 and NELL2 (neural epidermal growth factor-like 1 and 2) are recently described members of the epidermal growth factor gene family that have previously been shown to be expressed almost exclusively in brain tissue. Here we demonstrate regulated expression of NELL1 and NELL2 in human hematopoietic cells. Mature NELL1 mRNA is not detected in any normal hemopoietic cell type, although the gene is transcribed during a narrow window of pre-B cell development, and cell lines at the same developmental stage express the NELL1 mRNA. The related NELL2 gene is expressed by all nucleated peripheral blood cells examined (B, T, monocyte, and natural killer cells), but not in any of the bone marrow B lineage cells at earlier stages of development. However, leukemic cell lines corresponding to the same early differentiation stages express abundant NELL2 mRNA. These results suggest normal and possible oncogenic roles for the NELL proteins in hemopoietic cells.

The human PD-1 gene: complete cDNA, genomic organization, and developmentally regulated expression in B cell progenitors.

We report the complete cDNA sequence and the genomic structure of the human PD-1 homologue. An analysis of the expression pattern of the human PD-1 gene (hPD-1) and the murine PD-1 gene (mPD-1) in developing bone marrow B-lineage cells was also undertaken. The full length hPD-1 cDNA is 2106 nucleotides long and encodes a predicted protein of 288 amino acid residues. The hPD-1 and mPD-1 genes share 70% homology at the nucleotide level and 60% homology at the amino acid level. Four potential sites for N-linked glycosylation are conserved, as are a stretch of amino acids between two cysteine residues resembling a V-set immunoglobulin domain, and another region containing a motif similar to an immunoreceptor tyrosine-based inhibitory motif. Isolation of the genomic locus of the hPD-1 gene reveals that the gene is composed of five exons located on human chromosome 2 at band q37. The 5' flanking region lacks TATA and CAAT cis-acting elements, but includes a number of potential transcription factor binding sites and a dominant transcription start site. The mPD-1 gene was preferentially expressed in pro-B cells from murine adult bone marrow. Although hPD-1 was not preferentially expressed in pro-B cells from human fetal bone marrow, treatment of isolated pro-B cells with interleukin-7 resulted in a dramatic increase in expression. These data suggest that PD-1 may play a role in B-cell differentiation during the pro-B cell stage.

Normal B-lineage cells: their differentiation and identification.

This article reviews the normal differentiation of B-lineage cells. The scheme of normal differentiation of the B lineage provides a context in which to analyze patients with immunodeficiencies and B-lymphoproliferative disorders.

A rapid and quantitative method for the evaluation of V gene usage, specificities and the clonal size of B cell repertoires.

The quantitative simultaneous description of both variable region gene usage and antigen specificity of immunoglobulin repertoires is a major goal in immunology. Current quantitative assays are labor intensive and depend on extensive gene expression cloning prior to screening for antigen specificity. Here we describe an alternative method based on high efficiency single B cell cultures coupled with RT-PCR that can be used for rapid characterization of immunoglobulin gene segment usage, clonal size and antigen specificity. This simplified approach should facilitate the study of antibody repertoires expressed by defined B cell subpopulations, the analysis of immune responses to self and nonself-antigens, the development and screening of synthetic antibodies and the accelerated study and screening of neutralizing antibodies to pathogenic threats.

Stage-specific transcription of germline IgH C gamma and C alpha regions during human B cell differentiation.

Previous studies have suggested that transcription of germline heavy chain constant region (CH) genes in murine B cells may determine the potential of their different CH regions to undergo isotype switch recombination. We have examined the transcriptional activity across the immunoglobulin heavy chain (IgH) locus in human B lineage cells. Transcription of germline C gamma and C alpha was observed in every surface IgM+ or surface IgM+/IgD+ B cell stage cell line and malignancy. In contrast, such transcription could not be detected in pre-B cells and only low levels of C alpha but not C gamma transcription were evident in IgM-secreting plasmablast cells. Transcriptional activity of germline IgH C epsilon was singularly absent at all stages of B cell development. Our results suggest that germline transcription of the C gamma and C alpha regions may be a constitutive feature of the human B cell differentiation program. Because this transcriptional activity is limited primarily to the B cell stage and occurs prior to the actual isotype switch, the induction of C gamma and C alpha transcription may represent preparation of the downstream IgH chromatin for potential switch recombination.

Expression of immunoglobulin heavy chain at a high level in the absence of a proposed immunoglobulin enhancer element in cis.

The major intron between the J and C gene segments of the immunoglobulin heavy (H) chain locus contains an enhancer-like sequence, and it has been proposed that this enhancer is necessary to achieve high levels of H chain expression. We have isolated a subclone of the lymphoid pre-B cell line 18-81 that lacks this enhancer but nevertheless produces mu chain at the level characteristic of pre-B cells. Another subclone with a larger deletion does not produce mu chain, but upon fusion with a myeloma that does not produce any immunoglobulin chain, mu chain is expressed by the homolog from the pre-B subclone. The hybridoma lacks the proposed enhancer element in cis; nevertheless it produces as much mu chain as other plasma cell hybridomas. Therefore, this enhancer element is not obligatory for a high level of H chain production.

Regulated expression of cell surface antigens during B cell development.

The progression of lymphocytes along the B cell developmental pathway is marked by the regulated appearance and disappearance of a variety of cell surface markers including immunoglobulin. In this review, several of these antigens are discussed in the context of their possible functions during normal B cell differentiation.

Activation of self-reactive B cells and autoimmune diseases.

The development of antigen-specific cells of the immune system, the T and B lymphocytes, creates a dilemma. On the one hand, survival of the organism depends upon the generation of a nearly limitless repertoire of potential antigen-binding specificities so that cells able to respond to pathogens are present prior to contact. However, by devising genetic strategies to maximize receptor diversity, the generation of T and B cells with autoreactive receptors is inevitable. B cells have an even greater opportunity than T cells to become autoreactive, since they may randomly alter the amino acid sequence and hence the specificity of their receptors during an immune response. Observing the system, one might wonder not why autoimmune diseases occasionally develop, but rather why they are not more frequent or even unavoidable. In this review, we examine the generation of B cells and their repertoire of antigen receptors, describe mechanisms that have evolved to prevent self-reactive B cells from causing autoimmune diseases, and discuss scenarios that may lead to a breakdown of self tolerance.