Peter D. Burrows

Fc receptor homologs: newest members of a remarkably diverse Fc receptor gene family.

Summary: Newfound relatives of the classical Fc receptors (FcR) have been provisionally named the Fc receptor homologs (FcRH). The recent identification of eight human and six mouse FcRH genes substantially increases the size and functional potential of the FcR family. The extended family of FcR and FcRH genes spans ∼15 Mb of the human chromosome 1q21–23 region, whereas in mice this family is split between chromosomes 1 and 3. The FcRH genes encode molecules with variable combinations of five subtypes of immunoglobulin (Ig) domains. The presence of a conserved sequence motif in one Ig domain subtype implies Ig Fc binding capability for many FcRH family members that are preferentially expressed by B lineage cells. In addition, most FcRH family members have consensus tyrosine‐based activating and inhibitory motifs in their cytoplasmic domains, while the others lack features typical of transmembrane receptors. The FcRH family members, like the classical FcRs, come in multiple isoforms and allelic variations. The unique individual and polymorphic properties of the FcR/FcRH members indicate a remarkably diverse Fc receptor gene family with immunoregulatory function.

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.

Transcription factor Pax5 (BSAP) transactivates the RAG-mediated V H -to-DJ H rearrangement of immunoglobulin genes

Immunoglobulin rearrangement from variable heavy chain (VH) to diversity (D)–joining heavy chain (JH), which occurs exclusively in B lineage cells, is impaired in mice deficient for the B lineage–specific transcription factor Pax5. Conversely, ectopic Pax5 expression in thymocytes promotes the rearrangement of DH-proximal VH7183 genes. In exploring the mechanism for Pax5 regulation of VH-to-DJH recombination, we have identified multiple Pax5 binding sites in the coding regions of human and mouse VH gene segments. Pax5 bound to those sites in vitro and occupied VH genes in early human and mouse B lineage cells. Moreover, Pax5 interacted with the recombination-activating gene 1 (RAG1)–RAG2 complex to enhance RAG-mediated VH recombination signal sequence cleavage and recombination of a VH gene substrate. These findings indicate a direct activating function for Pax5 in RAG-mediated immunoglobulin VH-to-DJH recombination.

The transient expression of pre-B cell receptors governs B cell development.

Only a subpopulation of relatively large pre-B cells express pre-B cell receptors (preBCR) that can be seen with very sensitive immunofluorescence methods. Inefficient assembly of the multicomponent preBCR coupled with their ligand-induced endocytosis may account for the remarkably low in vivo levels of preBCR expression. Signaling initiated via the preBCR promotes cellular proliferation and RAG-1 and RAG-2 downregulation to interrupt the immunoglobulin V(D)J gene rearrangement process. Silencing of the surrogate light chain genes, VpreB and lambda5, then terminates preBCR expression to permit cell cycle exit, recombinase gene upregulation, and VJ(L) rearrangement by small pre-B cells destined to become B cells.

New nomenclature for Fc receptor-like molecules

To the editor: Newly identified Fc receptor–like genes are referred to in various publications as Fc receptor homologs (FcRH)1, immunoglobulin superfamily receptor translocation-associated genes (IRTA)2, immunoglobulin-Fc-gp42– related genes (IFGP)3, Src homology 2 domain– containing phosphatase anchor proteins (SPAP) or B cell crosslinked by anti–immunoglobulin M–activating sequences (BXMAS). Eight human and six mouse Fc receptor–like genes have been identified. Correspondence organized by the International Committee on Standardized Genetic Nomenclature for Mice, the Mouse Genomic Nomenclature Committee and the Human Genome Organisation Gene Nomenclature Committee has emphasized the need for a unified nomenclature to classify these genes and has proposed the term ‘Fc receptor–like’ (‘FCRL’ or ‘Fcrl’). The chromosomal position and genomic organization of ‘FCRL’ family is conserved with that of the ‘classical’ Fc receptor (‘FCR’) gene family. FCRL1–FCRL5 are tandemly located in the 1q21–23 region near FCGR1, whereas FCRL6 is located closer to FCER1A. FCRL1–FCRL6 encode type I transmembrane glycoproteins containing three to nine extracellular immunoglobulin domains and cytoplasmic immunoreceptor tyrosine-based activation–like motifs and/or immunoreceptor tyrosine-based inhibition motifs (Fig. 1, top). FCRL1 contains a charged residue in its transmembrane region, but the transmembrane portions of FCRL2–FCRL6 are hydrophobic and uncharged. FCRL1–FCRL5 are ‘preferentially’ expressed by B cells, whereas FCRL6 is expressed mainly by T cells and natural killer cells. Two additional human ‘FCRL’ genes, originally called FcRL (also known as FREB or FcRX) and FcRL2 (also known as FREB2 or FcRY)4, have unusual features that justify their designation as a separate subfamily. These genes are located in the low-affinity ‘FCR’ locus on chromosome 1q23 and contain two or three immunoglobulin domains (Fig. 1, top). However, FcRL lacks exons encoding a split signal peptide, a genomic organization characteristic of other ‘FCR’ and ‘FCRL’ genes. Both FcRL and FcRL2 are expressed by B cells as well as nonlymphoid cells and encode immunoglobulinlike molecules that lack transmembrane regions and tyrosine-based signaling motifs, but distinctly have C-terminal mucin-like regions. The nomenclature we suggest for these genes is FCRLA (for FcRL) and FCRLB (for FcRL2). The mouse ‘Fcr’ locus is divided between chromosomes 1 and 3. The genes originally called FcRH1 (also known as Ifgp1), FcRH2 (also known as Ifgp2 and Msr2) and FcRH3 are positioned near Fcgr1 on chromosome 3 and encode proteins containing two to five immunoglobulin domains with or without transmembrane regions5 (Fig. 1, bottom). FcRH1 and FcRH3 are expressed by B cells and encode molecules containing cytoplasmic tyrosine-based signaling motifs. Uniquely, FcRH2 does not cluster with FcRH1 and FcRH3, lacks a human ortholog or lymphoid expression and encodes a molecule containing a C-terminal type B scavenger receptor cysteine-rich domain without a 1 L R C F

The molecular basis and biological significance of VH replacement

Summary:  First observed in mouse pre‐B‐cell lines and then in knock‐in mice carrying self‐reactive IgH transgenes, VH replacement has now been shown to contribute to the primary B‐cell repertoire in humans. Through recombination‐activating gene (RAG)‐mediated recombination between a cryptic recombination signal sequence (RSS) present in almost all VH genes and the flanking 23 base pair RSS of an upstream VH gene, VH replacement renews the entire VH‐coding region, while leaving behind a short stretch of nucleotides as a VH replacement footprint. In addition to extending the CDR3 region, the VH replacement footprints preferentially contribute charged amino acids. VH replacement rearrangement in immature B cells may either eliminate a self‐reactive B‐cell receptor or contribute to the generation of self‐reactive antibodies. VH replacement may also rescue non‐productive or dysfunctional VHDJH rearrangement in pro‐B and pre‐B cells. Conversely, VH replacement of a productive immunoglobulin H gene may generate non‐productive VH replacement to disrupt or temporarily reverse the B‐cell differentiation process. VH replacement can thus play a complex role in the generation of the primary B‐cell repertoire.

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.

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.

Molecular Mechanism of Serial VH Gene Replacement

Abstract: The molecular mechanism of serial VH replacement was analyzed using a human B cell line, EU12, that undergoes continuous spontaneous differentiation from pro‐B to pre‐B and then to B cell stage. In earlier studies, we found that this cell line undergoes intraclonal V(D)J diversification. Analysis of the IgH gene sequences in EU12 cells predicted the occurrence of serial VH replacement involving the cryptic recombination signal sequences (cRSS) embedded within framework 3 regions and concurrent extension of the CDR3 region. Detection of double‐stranded DNA breaks at the cRSS site and different VH replacement excision circles confirm the ongoing nature of this diversification process in the EU12 cells. In vitro binding and cleavage assays using recombinant RAG‐1 and RAG‐2 proteins further validated the cRSS participation in this RAG‐mediated recombination process. Serial VH replacements may represent an additional mechanism for diversification of the primary B cell repertoire.

FCRLA is a resident endoplasmic reticulum protein that associates with intracellular Igs, IgM, IgG and IgA

Fc receptor-like A (FCRLA) is an unusual member of the extended Fc receptor family. FCRLA has homology to receptors for the Fc portion of Ig (FCR) and to other FCRL proteins. However, unlike these other family representatives, which are typically transmembrane receptors with extracellular ligand-binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is an intracellular protein. We show by confocal microscopy and biochemical assays that FCRLA is a soluble resident endoplasmic reticulum (ER) protein, but it does not possess the amino acid sequence KDEL as an ER retention motif in its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal partial Ig-like domain. We have identified ER-localized Ig as the FCRLA ligand. FCRLA is unique among the large family of Fc receptors, in that it is capable of associating with multiple Ig isotypes, IgM, IgG and IgA. Among hemopoietic cells, FCRLA expression is restricted to the B lineage and is most abundant in germinal center B lymphocytes. The studies reported here demonstrate that FCRLA is more broadly expressed among human B lineage cells than originally reported; it is found at significant levels in resting blood B cells and at varying levels in all B-cell subsets in tonsil.