Nikolai A. Chikaev

Identification of CD16-2, a novel mouse receptor homologous to CD16/FcγRIII

Abstract. It is believed that mouse FcγRIII arose by an evolutionarily recent recombination, which brought together the extracellular domains from FcγRII with the transmembrane/cytoplasmic region from the ancestor FcγRIII. Here, we report identification of a mouse gene encoding a transmembrane receptor that may be regarded as the true ortholog of nonrodent CD16/FcγRIII. Designated CD16-2, the novel protein is highly similar to human FcγRIIIA in the signal peptide (60% identical residues), and in the extracellular domains (65%). Although the similarity between the two proteins is less conspicuous in the transmembrane/cytoplasmic region (54%), it is higher than between human FcγRIIIA and mouse FcγRIII (44%). However, the conserved transmembrane motif LFAVDTGL shared by rodent and human FcγRIII and FcεRI has two replacements in CD16-2. The CD16-2 gene is tightly linked to the FcγRIII and FcγRII genes and consists of five exons. Northern blot analysis revealed that CD16-2 is expressed in peripheral blood leukocytes, as well as in spleen, thymus, colon and intestine. RT-PCR showed prominent expression in macrophage cell line J774. Based on sequence comparisons, it is suggested that the modern repertoire of the mammalian low affinity Fc receptors has resulted from repetitive duplications and/or recombinations of three ancestral genes.

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.

Expression patterns of the human and mouse IFGP family genes

The IFGP gene family has recently been found in human and mouse cells and is structurally related to the leukocytic Fc receptor genes. Expression of six human and four mouse IFGP genes was studied. Apart from mouse IFGP2, the genes of the family are expressed predominantly in hematopoietic cells. Expression of human IFGP1-IFGP5 and mouse IFGP3 is restricted to B cells. Mouse IFGP1 is expressed in B cells and, possibly, in nonlymphoid cells. IFGP6 is specifically expressed in CD8+ T cells and natural killers. Alternative splicing was demonstrated for the first time for the human IFGP1 and IFGP6 mRNAs. The alternative transcripts code for an IGFP1 isoform devoid of the transmembrane domain and an IFGP6 isoform with a changed cytoplasmic tail. It was assumed that the receptors of the family play a role in controlling differentiation and/or function of effector lymphocytes of the three main types: B cells, CD8+ T cells, and natural killers.

FCRL6 receptor: Expression and associated proteins

FCRL6 receptor is a more recently identified representative of the FCRL family. We generated a panel of mouse mAbs to baculovirus-derived recombinant FCRL6 protein. The clone 7B2 was found to specifically recognize a 63kDa protein expressed preferentially on the surface of CD8 T and CD56 NK cells in human peripheral blood and spleen. The clone 7B2 reacts with FCRL6 in Western blotting, FACS, and immunohistochemistry. In the T cell lineage, FCRL6 functions in antigen-experienced cells. Mitogenic stimulation of PB leukocytes in vitro resulted in an abrogation of the FCRL6 gene expression. We found a significant decrease in the FCRL6 gene expression in peripheral T cells of patients with certain autoimmune and blood diseases, and its upregulation at the late stages of HIV infection. Study of the FCRL6 association with signaling molecules showed its ability to recruit SHP-1, SHP-2, SHIP-1, and SHIP-2 phosphatases, and also adaptor protein Grb2 through phosphorylated cytoplasmic tyrosines. The current results demonstrate inhibitory potential of FCRL6 and suggest its possible involvement in modulation of CTL effector functions in various immune disorders.

Differential expression of FCRLA in naïve and activated mouse B cells

FCRLA is an intracellular B cell protein that belongs to the FcR-like family. Using newly generated FCRLA-specific antibodies, we studied the constitutive expression pattern of mouse FCRLA and monitored changes during an immune response and following in vitro B cell activation. All B cell subpopulations examined expressed FCRLA. However, the level of FCRLA expression is determined by the stage of B cell differentiation. Low expression of FCRLA is characteristic of naïve follicular and marginal zone B cells. High expression was detected in a small fraction of activated B cells scattered along migratory pathways in the lymphoid tissues. FCRLA-bright cells could be subdivided into two subpopulations, with high and low/undetectable level of intracellular immunoglobulins, which phenotypically resemble either plasma or memory B cells. High expression of FCRLA in subset(s) of terminally differentiated B-cells suggests that, being an ER protein, FCRLA may participate in the regulation of immunoglobulin assembly and secretion.

A direct antigen-binding assay for detection of antibodies against native epitopes using alkaline phosphatase-tagged proteins

We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.

Characterization of human FCRLA isoforms

FCRLA is an ER-resident B-cell specific protein. The exact function of this protein remains unclear although human FCRLA has been recently shown to interact with IgM, IgG and IgA. The retention of FCRLA in ER is mediated by the N-terminal domain. The major human FCRLA isoform is encoded by five exons, of which one encodes a short signal peptide (SSP) and the others code four protein domains. Here we show that human tissues also produce transcripts which contain an additional exon and encode proteins with signal peptide that is six residues longer (LSP). Transfection experiments demonstrated that the extension of the signal peptide had no visible effect on the topology and molecular mass of the processed four-domain FCRLA isoform. However, the length of the signal peptide was found to affect processing of two-domain FCRLA isoforms composed of the third and fourth domains (FCRLAd2). The signal peptide was not cleaved in the SSP-FCRLAd2 and this isoform was found to accumulate in the ER. In contrast, the LSP-containing FCRLAd2 isoform was processed, O-glycosylated and secreted. The secreted FCRLAd2 isoform did not interact with IgG- or IgM-immunosorbents.

Expression of human B-Cell specific receptor FCRL1 in healthy individuals and in patients with autoimmune diseases

The expression levels of the FCRL1 gene, which encodes a human B-cell surface receptor, were compared in healthy individuals and patients with autoimmune diseases. The expression levels were evaluated using DNA dot hybridization on membranes with spotted cDNA samples derived from blood-cell sub-populations of patients with autoimmune diseases. Quantification of the hybridization signals showed that FCRL1 expression in peripheral blood B-lymphocytes of patients with multiple sclerosis, lupus anticoagulants, Takayasu’s arteritis, and von Willebrand disease was significantly higher than in healthy individuals. Monoclonal and polyclonal FCRL1-specific antibodies that enable FCRL1 detection in Western blotting, immunohistochemistry, and flow-cytometry assays were generated. It was found that FCRL1 is expressed on the surfaces of mature CD19+ B-cells. In the tonsils, FCRL1-positive cells were located in the crypt area, i.e., in the mantle zone of secondary lymphoid follicles and among the cells of lymphoid epithelium. FCRL1-positive cells were also found in B-cell follicles of the spleen.

Diversity of Immunoglobulin Light Chain Genes in Non-Teleost Ray-Finned Fish Uncovers IgL Subdivision into Five Ancient Isotypes

The aim of this study was to fill important gaps in the evolutionary history of immunoglobulins by examining the structure and diversity of IgL genes in non-teleost ray-finned fish. First, based on the bioinformatic analysis of recent transcriptomic and genomic resources, we experimentally characterized the IgL genes in the chondrostean fish, Acipenser ruthenus (sterlet). We show that this species has three loci encoding IgL kappa-like chains with a translocon-type gene organization and a single VJC cluster, encoding homogeneous lambda-like light chain. In addition, sterlet possesses sigma-like VL and J-CL genes, which are transcribed separately and both encode protein products with cleavable leader peptides. The Acipenseriformes IgL dataset was extended by the sequences mined in the databases of species belonging to other non-teleost lineages of ray-finned fish: Holostei and Polypteriformes. Inclusion of these new data into phylogenetic analysis showed a clear subdivision of IgL chains into five groups. The isotype described previously as the teleostean IgL lambda turned out to be a kappa and lambda chain paralog that emerged before the radiation of ray-finned fish. We designate this isotype as lambda-2. The phylogeny also showed that sigma-2 IgL chains initially regarded as specific for cartilaginous fish are present in holosteans, polypterids, and even in turtles. We conclude that there were five ancient IgL isotypes, which evolved differentially in various lineages of jawed vertebrates.

Modular lentiviral vector system for chimeric antigen receptor design optimization

The article reports the development of a collection of lentiviral vector constructs enabling time-efficient production and testing of different variants of chimeric antigen receptors (CAR). These artificial surface proteins make it possible to redirect the activity of immune cytotoxic T-cells towards cancer cells. Chimeric antigen receptors usually encompass four functional modules, namely, antigen recognition, flexible linker, transmembrane, and signal modules. The use of modules with different properties allows modulating the affinity and specificity of CAR interaction with target antigens, as well as intensity and quality of activation signaling, which determines the cytotoxic properties of CAR T-cells, as well as their proliferation rate and time of persistence in the organism. The proposed vector system make it possible to easily test various combinations of CAR modules while its being open to distribution allows the direct comparison of the results obtained by different scientific groups.