In some T cell activation experiments, the freshly isolated PBMCs (5 105 cells) were seeded in a 24-well culture plate (1 ml/well) and stimulated by adding 2 106 Dynabeads goat anti-mouse-IgG (Invitrogen 110?33) precoated with anti-CD3 antibody (clone UCHT1, BD Biosciences, 2 g/107 beads) and anti-CD28 antibody (clone CD28.2, BD Biosciences, 2 g/107 beads) as described previously (31). Circulation cytometry and cell sorting In a typical experiment, 106 cells were incubated in 200 l of the antibody cocktail in FACS staining buffer (PBS containing 5% FBS and 0.1% sodium azide) for 1 h at 4C. in the intracellular domains. Human genes are preferentially expressed in matured B cells at numerous stages in response to antigen exposure, whereas is usually highly expressed in T cells (4). Even though natural ligands of FCRL proteins are not known, their potential for generating intracellular signaling and their stage-dependent expression on B cells suggest functions as receptors in tuning Cyclazodone of immune cell functions. Human genes are clustered on chromosome 1q21?23 near classical Fc receptors genes, but genes are not present at the corresponding gene locus in mice (2, 5). These human specific may be involved in the regulation of human specific immune responses. Using a mAb specific to FCRL3, this protein was recently reported to be expressed on NK cells in addition to B cells (6). The reactivity of this mAb Rabbit polyclonal to PDE3A to T cells has not been extensively analyzed although a low level of mRNA is usually detectable by RT-PCR in T cell fractions (7, 8). In this study, we investigated FCRL3 protein expression on the surface of human T cells in peripheral blood using a newly generated anti-FCRL3 mAb, and found its expression on regulatory T (Treg) cells. Naturally occurring CD4+ Treg (nTreg) cells, which characteristically express CD25 ( chain of high affinity IL-2R), are produced mainly in the thymus and engage in upkeep of self-tolerance (9-11). Forkhead box p3 (Foxp3) has been extensively studied as a transcription factor associated with the occurrence and function of CD4+CD25+ nTreg cells (12-14). Primarily in mouse studies, it has been shown that TGF- treatment induces Foxp3+ cells from na?ve T cells and that the Foxp3+ cells are functionally and phenotypically much like nTreg cells (15). Although an exclusive surface marker for nTreg cells has not yet been recognized, cytotoxic T-lymphocyte antigen 4 (CTLA-4) and glucocorticoid-induced TNF receptor family related protein (GITR) are suggested by their involvement in Treg cells suppressive activity (16, 17). Despite their potential importance in suppression mechanisms, conclusive identification of CD4+ nTreg cells by these surface markers is usually problematic because other T cells can express these markers upon activation. For example, CD25 is commonly used as an activation marker of any type of T cell because CD25 expression is usually transiently and strongly up-regulated on activated T cells (18). CTLA-4 and GITR are also known to be induced in na?ve T cells by activation Cyclazodone (19). An additional complication is that the marker distribution is usually considerably different between mice and humans. In mice, Foxp3 expression is usually purely correlated with suppressor activity, whereas human na?ve peripheral T cells without Treg cell activity readily express low levels of Foxp3 upon TCR stimulation (20, 21). About 6?12% of Cyclazodone mouse CD4+ T cells constitutively express CD25 and all are suppressive in the functional assay. However, the expression level of CD25 on human CD4+ peripheral T cells is rather variable and only 2?4% of human peripheral CD4+ cells with the highest level of CD25 (CD25hi) show Treg activity (22, 23). Recent searches for nTreg cell markers in human CD4+ cells revealed that down-regulation of CD127 (IL-7 receptor chain) is usually associated with suppressor function in human CD4+ cells (24, 25). The CD4+CD25+CD127low surface phenotype found in 5?8% of the human CD4+ T cell population is also utilized for identification of CD4+ nTreg cells in human peripheral blood (26). In this study, we found that 40% of human CD4+CD25+CD127low nTreg cells in peripheral blood express a human specific FCRL member, FCRL3, around the cell surface and that FCRL3 expression correlates with an hypoproliferative phenotype of cells in response to exogenous IL-2. Materials and Methods Production and characterization of anti-FCRL3 mAbs The cDNAs of human FCRL3 and other FCRLs were obtained by RT-PCR as explained previously (27, 28). The cDNAs were confirmed to encode the same proteins as the following research sequences: Genbank (http://www.ncbi.nlm.nih.gov/Genbank/) accession number, “type”:”entrez-nucleotide”,”attrs”:”text”:”AF459634″,”term_id”:”18140080″,”term_text”:”AF459634″AF459634 (FCRL1), “type”:”entrez-nucleotide”,”attrs”:”text”:”AF459633″,”term_id”:”18140078″,”term_text”:”AF459633″AF459633 (FCRL2), “type”:”entrez-nucleotide”,”attrs”:”text”:”AF459027″,”term_id”:”18092654″,”term_text”:”AF459027″AF459027 (FCRL3), “type”:”entrez-nucleotide”,”attrs”:”text”:”AF343659″,”term_id”:”13591703″,”term_text”:”AF343659″AF343659 (FCRL4), “type”:”entrez-nucleotide”,”attrs”:”text”:”AF343664″,”term_id”:”13591713″,”term_text”:”AF343664″AF343664 (FCRL5), and “type”:”entrez-nucleotide”,”attrs”:”text”:”AY513661″,”term_id”:”46241312″,”term_text”:”AY513661″AY513661 (FCRL6). Expression of full-length FCRLs in transfected 293T cells and Cyclazodone expression of their extracellular domains as human IgG Fc-fusion proteins were carried out as explained previously (27, 28). Production and characterization of anti-FCRL3 mAb were performed according to a series of our established protocols for obtaining mAbs to membrane proteins in their native conformation (27-30). The FCRL3-specific mAbs were screened in an ELISA using FCRL3-Fc protein as the coated.
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