CD99 plays an critical role in the diapedesis of monocytes, T cell differentiation, and the transport of MHC molecules. Engagement of CD99 by agonistic monoclonal antibodies has been reported to trigger multifactorial events including T cell activation as well as cell-cell adhesion during hematopoietic cell differentiation. In this study, to identify the functional domains participating in the cellular events, we mapped the epitopes of CD99, which are recognized by two agonistic CD99 monoclonal antibodies, DN16 and YG32. Using recombinant fusion proteins of GST with whole or parts of CD99, we found that both antibodies interact with CD99 molecules independently of sugar moieties. DN16 mAb detected a linear epitope located in the amino terminal region of CD99 while YG32 mAb bound another linear epitope in the center of the extracellular domain. To confirm that the identified epitopes of CD99 are actually recognized by the two mAbs, we showed the presence of physical interaction between the mAbs and the fusion proteins or synthetic peptides containing the corresponding epitopes using surface plasmon resonance analyses. The dissociation constants of DN16 and YG32 mAbs for the antigen were calculated as 1.27 X 10(-7) and 7.08 X 10(-9) M, respectively. These studies will help understand the functional domains and the subsequent signaling mechanism of CD99.
Amino Acid Sequence ; Antibodies, Monoclonal/*immunology ; Antigens, CD/*chemistry/*immunology ; Blotting, Western ; Cell Adhesion Molecules/*chemistry/*immunology ; *Epitope Mapping ; Epitopes/*chemistry/*immunology ; Glutathione Transferase ; Human ; Molecular Sequence Data ; Peptide Fragments/chemistry/immunology ; Recombinant Fusion Proteins/chemistry/immunology

PD-L1 is a member of the B7 protein family, most of whose members so far were identified as dimers in a solution and crystalline state, either complexed or uncomplexed with their ligand(s). The binding of PD-L1 with its receptor PD-1 (CD279) delivers an inhibitory signal regulating the T cell function. Simultaneously with the Garboczi group, we successfully solved another structure of human PD-L1 (hPD-L1). Our protein crystallized in the space group of C222(1) with two hPD-L1 molecules per asymmetric unit. After comparison of reported B7 structures, we have found some intrinsic factors involved in the interaction of these two molecules. Based on these results, we tend to believe this uncomplexed hPD-L1 structure demonstrated its potential dimeric state in solution, although it could just be an evolutionary relic, too weak to be detected under present technology, or still a functional unit deserved our attentions.
Antigens, CD ; chemistry ; immunology ; B7-H1 Antigen ; Crystallography, X-Ray ; Evolution, Molecular ; Humans ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Secondary ; T-Lymphocytes ; chemistry ; immunology

OBJECTIVETo study the regulation of Fc receptor expression by immune complexes (ICs) on neutrophils and U937 cells.

METHODSIgA ICs, IgG1 ICs, IgG2 ICs, IgG3 ICs, IgG4 ICs, and IgM ICs were incubated with neutrophils or U937 cells for 1 h. Then their surface Fc receptors were stained by anti-Fc gammaR I, anti-Fc gammaR II , anti-Fc gammaR III, and anti-Fc alphaR I monoclonal antibodies and analyzed by fluorescent activated cell sorting (FACS).

RESULTSIgG1 ICs and IgG3 ICs up-regulated Fc gammaR II and Fc gammaR III on U937 cells, Fc gammaR I and Fc alphaR I on neutrophils. Almost all ICs down-regulated Fc gammaR II on neutrophils.

CONCLUSIONSICs can regulate Fc receptor expression on neutrophils and U937 cells, among which IgG1 ICs and IgG3 ICs are most effective.

Antibodies, Anti-Idiotypic ; immunology ; Antibodies, Monoclonal ; pharmacology ; Antigen-Antibody Complex ; immunology ; metabolism ; Antigens, CD ; immunology ; Humans ; Immunoglobulin A ; classification ; immunology ; Immunoglobulin G ; chemistry ; classification ; immunology ; metabolism ; Neutrophils ; metabolism ; Receptors, Fc ; biosynthesis ; genetics ; Receptors, IgG ; immunology ; U937 Cells ; immunology

Although various functions of CD99 have been reported, such as apoptosis and homotypic aggregation of thymocyte and transendothelial migration of immune cells, biochemical/molecular natures of CD99 are still elusive. Using mouse CD99 gene, we show that CD99 forms homodimer through its extracellular domain. Expression of mouse CD99 is up-regulated on T cells after CD3-mediated activation, like the case for human CD99. The potential of CD99 to form homodimer was tested with a recently developed bimoleular fluorescence complementation analysis (BiFC). In BiFC analysis, the dimerization-induced fluorescence was strong near the perinuclear region and was faded at the cell membrane. However, surface expression of CD99 was still detected by flow cytometry, suggesting that CD99 either in monomer form or in association with other molecules exists on the cell surface. In BiFC analysis using CD99 mutants with its extracellular, transmembrane, or cytosolic domains changed to corresponding human CD4 domains, the mutant replaced with human CD4-extracellular domain did not produce fluorescence. Purified soluble CD99-Fc fusion proteins bound to CD99-Fc immobilized onto the gold sensor chip in surface plasmon resonance analysis, confirming that the extracellular domain was responsible for dimer formation. Intracytoplasmic staining for CD99 expression in the thymocytes and mature T cells showed that most of the cells, even the cells with low surface level of CD99, contained the molecule inside the cell. Our results suggest that majority of CD99 homodimers may exit in the cell and be exported to the cell surface, dissociating from each other, after a certain regulatory signal is delivered.
Animals ; Antigens, CD/chemistry/*isolation & purification ; Cell Adhesion Molecules/chemistry/*isolation & purification ; Flow Cytometry ; *Fluorescence ; Luminescent Measurements/*methods ; Mice ; Molecular Biology/*methods ; T-Lymphocytes/immunology

OBJECTIVETo compare the characteristics of immunomagnetic beads and hespan precipitation for isolation of mononuclear cells (MNCs) from umbilical cord blood and try to find a better isolation method for MNCs.

METHODSFifteen umbilical cord blood samples from healthy parturiens were collected between December 2007 and March 2008. MNCs were isolated using hespan precipitation and CD133 immunomagnetic beads, respectively. MNCs were identified using the surface marker CD34 by flow cytometry on the 30th of primary culture. Growth conditions and morphologic changes of primary cells were observed by an inverted microscope.

RESULTSThe number of MNCs from umbilical cord blood isolated by hespan precipitation (15.23 +/- 4.30 x 10(6)/mL) was significantly greater than that by CD133 immunomagnetic beads (0.066 +/- 0.027 x 10(6)/mL) (p<0.05). The MNCs isolated by hespan precipitation suspended at the culture medium and their growth was slow after passage. The growth of MNCs isolated by CD133 immunomagnetic beads was kept in a good condition. The CD34 positive rate of MNCs isolated by hespan precipitation and immunomagnetic beads was 10.1% and 0.5%, respectively.

CONCLUSIONSThe hespan precipitation is an effective method for MNCs isolation from human umbilical cord blood, but with a cell growth condition below the mark. The MNCs isolated by CD133 immunomagnetic beads are in a high purity quotient.

AC133 Antigen ; Antigens, CD ; immunology ; Antigens, CD34 ; analysis ; Cell Separation ; methods ; Chemical Precipitation ; Fetal Blood ; cytology ; Glycoproteins ; immunology ; Humans ; Hydroxyethyl Starch Derivatives ; chemistry ; Immunomagnetic Separation ; methods ; Infant, Newborn ; Leukocytes, Mononuclear ; cytology ; Peptides ; immunology

OBJECTIVETo investigate the expressions of costimulators on peripheral T and B lymphocytes in patients with idiopathic thrombocytopenic purpura (ITP).

METHODSThe expression of B7-CD(28) and CD(40) of peripheral lymphocytes was measured by flow cytometry in 21 ITP patients and 9 normal subjects. The expression of PAIgG was measured by ELISA method.

RESULTSThe expression of CD(4)(+)CD(28)(+) was lower in ITP patients than in normal controls, but the expression of CD(86)(+) and CD(86)(+)CD(19)(+) was higher in ITP patients than in normal controls, while the expression of CD(80)(+), CD(40)(+), CD(28)(+), CD(19)(+)CD(86)(+), CD(19)(+)CD(40)(+), CD(4)(+)CD(28)(+)/CD(4)(+), CD(19)(+)CD(80)(+)/CD(19)(+) and CD(19)(+)CD(40)(+)/CD(19)(+) in ITP patients was normal. The PAIgG level was higher in 16 patients with a mean of (184.62 +/- 38.00) ng/10(7) plt. A positive correlation was found between PAIgG and CD(19)(+)CD(86)(+)/CD(19)(+) expression.

CONCLUSIONThere is no deficiency in expression of CD(28) on CD(4)(+) T lymphocytes in ITP patients. The change of CD(86) expression on B lymphocytes is possibly involved in pathophysiology of ITP, which may provide a theoretical instruction for ITP patients immunological therapy.

Adolescent ; Adult ; Antigens, CD ; blood ; Blood Platelets ; immunology ; Female ; Humans ; Immunoglobulin G ; blood ; Lymphocyte Activation ; Lymphocytes ; chemistry ; Male ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; blood

After years of development, biosensors based on imaging ellipsometry and biosensors based on total internal reflection imaging ellipsometry have been successfully implemented in various engineering systems. Their experimental setups, detection principles, and biological and clinical applications are briefly reviewed.
Antibodies ; analysis ; immunology ; Antigens, CD ; analysis ; immunology ; Bacteria ; chemistry ; isolation & purification ; Biomarkers ; analysis ; Biosensing Techniques ; instrumentation ; methods ; Humans ; Ligands ; Microfluidic Analytical Techniques ; instrumentation ; methods ; Microfluidics ; instrumentation ; methods ; Molecular Imaging ; instrumentation ; methods ; Protein Array Analysis ; instrumentation ; methods ; Viruses ; chemistry ; isolation & purification

BACKGROUNDHepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. In order to investigate the molecular biologic mechanism of HCC's development, we studied the expressions of SE-1, CD105 and CD31 in tumor endothelial cells (TECs) of HCC and in the serum of rats.

METHODSWe analyzed the expressions of SE-1, CD31 and CD105 in rat HCC tumor tissues using immunohistochemistry (IHC). Twenty HCC bearing rats and eighteen normal rats were examined for the expressions of SE-1, CD31 and CD105 antigens in serum by enzyme-linked immunosorbent assay (ELISA).

RESULTSSE-1, CD31 and CD105 antigens were detected both in HCC tissue and in normal liver tissue with higher expressions of CD31 and CD105 in HCC while the SE-1 antigen expression was higher in normal liver. Similarly, serum CD31 and CD105 in rats with HCC were significantly increased compared with normal rats (t = 2.8628, P = 0.0086; t = 4.4922, P < 0.0001, respectively). In contrast, SE-1 antigen in HCC rat serum was significantly decreased compared with normal rats (t = 3.4983, P = 0.0011).

CONCLUSIONSE-1, CD31 and CD105 are closely related with liver tumor angiogenesis, which is similar to their performances in terms of their expressions in the serum.

Animals ; Antigens, CD ; blood ; Carcinoma, Hepatocellular ; blood supply ; chemistry ; Endothelial Cells ; chemistry ; immunology ; Enzyme-Linked Immunosorbent Assay ; Immunohistochemistry ; Liver Neoplasms, Experimental ; blood supply ; chemistry ; Male ; Neovascularization, Pathologic ; blood ; Platelet Endothelial Cell Adhesion Molecule-1 ; blood ; Rats ; Rats, Inbred BUF

OBJECTIVETo detect the expression of surface molecule CD96 on stem cell (LSC) in children with acute leukemia, and to explore its clinical significance.

METHODSBone marrow mononuclear cells were isolated in 69 children with newly diagnosed acute leukemia. CD34(+)CD38(-)CD123(+) LSCs were separated from these cells by flow cytometry (FCM) and then cultured, and CD96 expression on LSCs was detected by FCM. R-banding technique was used to analyze the karyotypes of the 69 children, and the data of their routine blood and immunological tests were collected.

RESULTSCD96 was mainly expressed in children with acute myelogenous leukemia, and expressed to a lesser extent in those with acute lymphoblastic leukemia (P<0.05). The median expression level of CD96 in Uyghur children was 23.4%, versus 21.2% in Han children (P>0.05). The majority of children with CD96-positive children presented poor-prognosis karyotypes. Compared with CD96-negative children, children with CD96-positive children had a significantly lower complete remission rate (P<0.05) and significantly higher infection and relapse rates after chemotherapy (P<0.05).

CONCLUSIONSChildren with acute leukemia who have CD96-positive LSCs have a poor prognosis. CD96 may be a new indicator of prognosis in children with acute leukemia.

Adolescent ; Antigens, CD ; analysis ; Child ; Child, Preschool ; Humans ; Infant ; Leukemia, Myeloid, Acute ; drug therapy ; genetics ; immunology ; pathology ; Neoplastic Stem Cells ; chemistry ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; genetics ; immunology ; pathology ; Prognosis

Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.
Amino Acid Sequence ; Antigens, CD ; chemistry ; genetics ; pharmacology ; Autoimmunity ; Biological Assay ; Cell Line ; Cytotoxicity, Immunologic ; genetics ; immunology ; Dose-Response Relationship, Immunologic ; Humans ; Immunoglobulins ; immunology ; metabolism ; Immunologic Factors ; chemistry ; genetics ; pharmacology ; Kinetics ; Leukocyte Immunoglobulin-like Receptor B1 ; Lymphocyte Activation ; genetics ; immunology ; Major Histocompatibility Complex ; genetics ; immunology ; Molecular Sequence Data ; Molecular Targeted Therapy ; Mutagenesis, Site-Directed ; Peptide Library ; Polyethylene Glycols ; Protein Binding ; genetics ; immunology ; Receptors, Immunologic ; chemistry ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism