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1.
Vet Immunol Immunopathol ; 80(1-2): 5-23, 2001 Jul 20.
Article in English | MEDLINE | ID: mdl-11445215

ABSTRACT

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with "w". As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).


Subject(s)
Antigens, CD , Leukocytes/immunology , Swine/immunology , Animals
2.
J Immunol ; 165(2): 1004-12, 2000 Jul 15.
Article in English | MEDLINE | ID: mdl-10878377

ABSTRACT

The transcription factor NF-kappa B regulates many genes involved in proinflammatory and immune responses. The transport of NF-kappa B into the nucleus is essential for its biologic activity. We describe a novel, potent, and selective NF-kappa B inhibitor composed of a cell-permeable peptide carrying two nuclear localization sequences (NLS). This peptide blocks NF-kappa B nuclear localization, resulting in inhibition of cell surface protein expression, cytokine production, and T cell proliferation. The peptide is efficacious in vivo in a mouse septic shock model as well as a mouse model of inflammatory bowel disease, demonstrating that NF-kappa B nuclear import plays a role in these acute inflammatory disease models.


Subject(s)
NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Nuclear Localization Signals , Peptides/pharmacology , Shock, Septic/metabolism , Amino Acid Sequence , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cell Line , Cytokines/antagonists & inhibitors , Cytokines/biosynthesis , Disease Models, Animal , Humans , Immunoglobulin kappa-Chains/biosynthesis , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/pharmacology , Inflammation/immunology , Inflammation/metabolism , Inflammation/prevention & control , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Nuclear Localization Signals/drug effects , Peptides/administration & dosage , Peptides/chemical synthesis , Receptors, Antigen, B-Cell/biosynthesis , Shock, Septic/immunology , Shock, Septic/pathology , Shock, Septic/prevention & control , T-Lymphocytes/drug effects , T-Lymphocytes/immunology
3.
J Hematother Stem Cell Res ; 8(4): 393-9, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10634177

ABSTRACT

Local production of cytokines by genetically engineered tumor cells decreases their tumorigenicity and elicits protective immune responses against the parental tumor cells. An alternative approach to elicit a therapeutic immune response is to use fusion proteins that can target tumor cells and simultaneously activate effector cells. Fusion proteins between human IL-2, murine or human GM-CSF, and sFv of antihuman carcinoma antibody L6 have been constructed, expressed in both COS and Chinese hamster ovary (CHO) cells, and purified by affinity chromatography. The biologic activity of L6 sFV-hIL-2, L6 sFv-mGM-CSF, and L6 sFv-hGM-CSF was tested on human T cell blasts, factor-dependent FDCP-1, and TF-1 cells, respectively. The ability of soluble L6 sFv-hIL-2, L6 sFv-mGM-CSF, and L6 sFv-hGM-CSF to stimulate the proliferation of the indicator cells was found to be comparable to that of recombinant hIL-2, mGM-CSF, or hGM-CSF. Tumor cells coated with L6 sFV-mGM-CSF or L6 sFv-hGM-CSF were also tested in this way and were found to be potent stimulators, indicating that the cytokines were functionally active when bound to the tumor cell surface. This work demonstrates the feasibility of targeting sFv-cytokine fusion proteins for the activation of effector cells as an alternative to cytokine gene therapy.


Subject(s)
Antibodies, Neoplasm/therapeutic use , Genetic Engineering , Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use , Immunotherapy/methods , Interleukin-2/therapeutic use , Neoplasms/therapy , Recombinant Fusion Proteins/therapeutic use , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neoplasm/genetics , Antibodies, Neoplasm/immunology , CHO Cells , COS Cells , Cricetinae , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Humans , Interleukin-2/genetics , Interleukin-2/immunology , Neoplasms/genetics , Neoplasms/immunology , Recombinant Fusion Proteins/genetics , T-Lymphocytes/drug effects , T-Lymphocytes/pathology
4.
Crit Rev Immunol ; 17(5-6): 427-35, 1997.
Article in English | MEDLINE | ID: mdl-9419430

ABSTRACT

A single-chain Fv (sFv) was expressed from the variable regions of the CD40-specific mAb G28-5. The molecule bound CD40 with a high affinity (2.2 nM) and was a monomer in solution. Surprisingly, G28-5 sFv was a potent CD40 agonist that rapidly crosslinked CD40 on the cell surface but did not crosslink CD40-Ig in solution. G28-5 sFv was a more potent agonist than G28-5 IgG and was able to stimulate CD40 responses by B cells and monocytes. G28-5 IgG partially blocked, whereas G28-5 sFv augmented CD40 responses during stimulation with natural ligand (gp39-CD8 fusion protein). These results indicate that the functional activity of ligands built from the binding site of G28-5 is highly dependent upon the size and physical properties of the molecule both in solution and on the cell surfaces.


Subject(s)
Antibodies, Monoclonal/immunology , B-Lymphocytes/immunology , CD40 Antigens/immunology , Immunoglobulin Fragments/immunology , Immunoglobulin Variable Region/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/genetics , Cells, Cultured , Cloning, Molecular , Endothelium/cytology , Gene Expression , Humans , Immunoglobulin Fragments/biosynthesis , Immunoglobulin Fragments/genetics , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Monocytes/immunology , NF-kappa B/immunology
5.
J Immunol ; 157(10): 4390-8, 1996 Nov 15.
Article in English | MEDLINE | ID: mdl-8906814

ABSTRACT

CD22 is a key accessory molecule for Ag receptor signaling in B cells that becomes tyrosine phosphorylated in the signaling process. CD22 associates with sIg and strongly amplifies sIg-induced signals. During B cell development, CD22 is initially expressed intracellularly, with surface expression appearing with IgD expression. We used confocal laser-scanning microscopy and flow cytometry to analyze CD22 translocation responses to signaling events. Cross-linking surface IgM (sIgM) induced rapid movement of CD22 to the cell surface in both Ramos and Daudi B cells, with a 50 to 100% increase in surface expression observed within 5 min of stimulation. The increase in CD22 surface expression was specific in that CD19 expression was not affected. Both cell surface and intracellular CD22 were directed toward the site of sIgM stimulation. Treatment with the phosphotyrosine phosphatase inhibitor bis(maltolato)oxovanadium(IV) also increased CD22 surface expression. The tyrosine kinase inhibitor tyrphostin A10 inhibited CD22 movement at concentrations that inhibited tyrosine phosphorylation of CD22 and other cellular proteins. In contrast to the B cell lines, mature peripheral blood B cells contained very little intracellular CD22 and showed no significant increase in surface expression following sIgM stimulation. The rapid directed movement of intracellular CD22 provides a new mechanism to dynamically regulate Ag receptor signaling, as well as CD22-mediated adhesion.


Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, B-Lymphocyte/metabolism , Antigens, Surface/immunology , Antigens, Surface/metabolism , Cell Adhesion Molecules , Cytoplasm/immunology , Cytoplasm/metabolism , Immunization/methods , Lectins , Protein-Tyrosine Kinases/analysis , Receptors, Antigen/immunology , Biological Transport/immunology , Burkitt Lymphoma/pathology , Humans , Protein Tyrosine Phosphatases/antagonists & inhibitors , Pyrones/pharmacology , Sialic Acid Binding Ig-like Lectin 2 , Tumor Cells, Cultured , Vanadates/pharmacology
6.
Tissue Antigens ; 48(4 Pt 1): 242-54, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8946677

ABSTRACT

Interaction of the CD80 (B7-1) and CD86 (B7-2) molecules on antigen presenting cells with the receptors CD28 and CTLA-4 on T cells generates signals important in the regulation of immune responses. Because this receptor system involves multiple receptor-ligand interactions, determining the function for individual receptors has been difficult. One approach is the use of antibodies and their derivatives with singular specificity as substitute ligands to explore the activities of these molecules. We have constructed recombinant mono- and bi-specific sFv molecules specific for the CD28 receptor that are capable of binding and generating costimulatory signals to activate T cells. We demonstrate that these soluble molecules are capable of higher levels of costimulation than soluble CD80Ig at equivalent concentrations. We also constructed artificial adhesion receptors on the cell surface using two different CD28-specific sFvIgs fused to the CD80 cytoplasmic and transmembrane domains. In this report, we compared costimulation by a soluble bispecific (alpha CD28-alpha L6) single chain sFvIg fusion protein to that generated by L6 antigen positive (L6+) H3347 tumor cells transduced with cell surface expressed forms of alpha CD28 sFv's. We show that the bispecific protein can target potent CD28 costimulatory activity to L6+ tumor cells in vitro. We also show that transfection of the cell surface forms of the two different CD28 sFvIgs into H3347 tumor cells allows them to generate significant costimulatory signals to activated T cells. Finally, we demonstrate that tumor cell presentation of either the soluble bispecific or transduced cell surface sFv generate similar costimulatory effects resulting in T cell activation.


Subject(s)
Antigens, Surface/immunology , CD28 Antigens/immunology , Immunoglobulin Fragments/immunology , Neoplasm Proteins/immunology , T-Lymphocytes/immunology , Amino Acid Sequence , Animals , Antigens, Surface/genetics , B7-1 Antigen/immunology , CHO Cells , COS Cells , Cloning, Molecular , Cricetinae , Cross-Linking Reagents/pharmacology , DNA , Gene Expression , Humans , Immunoglobulin Fragments/genetics , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Mitogens , Molecular Sequence Data , Neoplasm Proteins/genetics , Phytohemagglutinins/pharmacology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Tumor Cells, Cultured
8.
Tissue Antigens ; 46(3 ( Pt 1)): 145-54, 1995 Sep.
Article in English | MEDLINE | ID: mdl-8525473

ABSTRACT

Ligation of major histocompatibility complex (MHC) class II antigens expressed on antigen-activated human CD4+ T-lymphocytes induces early signal transduction events including the activation of tyrosine kinases, the tyrosine phosphorylation of phospholipase-C gamma 1 and the mobilization of intracellular calcium. Similar responses have been observed in B-cells following stimulation of MHC class II molecules, including the increased production of intracellular cAMP. In this report, we demonstrate that the ZAP-70 tyrosine kinase is a responsive signaling element following cross-linking of HLA-DR in class II+ T-cells, and that the homologous tyrosine kinase p72syk is stimulated in B-cells following ligation of class II antigens. Antibody mediated co-ligation of the T-cell antigen receptor (TCR/CD3) with class II molecules resulted in augmented tyrosine phosphorylation of ZAP-70. Comparable to antibody induced receptor ligation, bacterial superantigen (SEA and SEB) treatment of HLA-DR+ T-cells stimulated ZAP-70 tyrosine phosphorylation, consistent with class II transmembrane signaling by ligation of HLA-DR and V beta in cis. Modulation of the TCR/CD3 led to abrogation of class II induced ZAP-70 tyrosine phosphorylation, but did not result in sequestering of ZAP-70 from the cellular cytoplasm. Hyperphosphorylated ZAP-70 was associated with TCR/CD3 zeta-chain following cross-linking of HLA-DR, suggesting a mechanism for the TCR/CD3-dependence of class II induced signals in alloantigen-activated human T-cells. In both tonsillar B-lymphocytes and B-cell leukemia lines, p72syk was rapidly phosphorylated on tyrosine residues following HLA-DR cross-linking. Tyrosine phosphorylation of p72syk induced through ligation of either the B-cell antigen receptor or class II molecules was potently inhibited by herbimycin A. MHC class II ligation on B-lymphocytes resulted in cell death, which was both qualitatively distinct from Fas-induced apoptosis and partially protected by herbimycin A pretreatment. Thus, ligation of MHC class II molecules expressed on human lymphocytes stimulates the ZAP-70/p72syk family of tyrosine kinases, leading functionally to a tyrosine kinase-dependent pathway of receptor-induced cell death.


Subject(s)
Enzyme Precursors/analysis , Protein-Tyrosine Kinases/analysis , Signal Transduction/immunology , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/immunology , Enzyme Activation/immunology , Enzyme Precursors/chemistry , Enzyme Precursors/immunology , HLA-DR Antigens/chemistry , HLA-DR Antigens/immunology , Humans , Intracellular Signaling Peptides and Proteins , Lymphocyte Activation , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/immunology , Receptors, Antigen, T-Cell/physiology , Syk Kinase , ZAP-70 Protein-Tyrosine Kinase
9.
Circ Shock ; 44(2): 67-72, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7743602

ABSTRACT

CD40 functional responses can be triggered by binding of mAb G28-5. Here we show that G28-5 induces partial CD40 responses and functions as a partial antagonist of natural CD40 ligand, gp39, by preventing gp39 binding. Fab fragments of G28-5 retain inhibitory activity but lose crosslinking-dependent stimulatory activity. The synergistic interaction of CD40 signals with PMA or CD20 show differential requirements for CD40 crosslinking and different sensitivity to cyclosporine A, suggesting that CD40 receptor may use different effector mechanisms for synergy with calcium-dependent CD20 signals or with calcium-independent signals from PMA. Activation of NF-kappa B occurred in RAJI cells by G28-5 or by gp39 treatment, and was CD40 crosslinking-dependent. These results suggest that activation of NF-kappa B is involved in some CD40 receptor signals and may be related to CD40 effects on stimulation or inhibition of apotosis.


Subject(s)
Antibodies, Monoclonal/pharmacology , B-Lymphocytes/metabolism , Cyclosporine/pharmacology , Membrane Glycoproteins/metabolism , T-Lymphocytes/metabolism , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Base Sequence , CD40 Ligand , Carrier Proteins , Cells, Cultured , Flow Cytometry , Membrane Glycoproteins/drug effects , Membrane Glycoproteins/immunology , Mice , Molecular Sequence Data , NF-kappa B/drug effects , NF-kappa B/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/immunology
10.
J Clin Invest ; 94(2): 616-22, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7518839

ABSTRACT

The molecular origin of X-linked hyper IgM syndrome has recently been identified as a defect in the ligand of CD40, gp39, a protein expressed on the surface of activated T cells. The availability of detailed pedigrees for three families with affected males allowed assessment of the random or nonrandom nature of the inactivation of the defective X chromosome as well as a determination of the origin of the mutation. X chromosome inactivation was studied because of the relevance to the ability to detect carriers of HIGM1 and the potential for phenotypic effect in the carriers. Using immunostaining, PCR, and DNA sequencing, we found that the defective gene for gp39 is not selectively inactivated. Even in the presence of extremely skewed inactivation, normal levels of serum Ig were found. In carriers in which the defective gene is predominantly expressed, staining alone revealed the carrier status reliably while cloning and sequencing of the cDNA was necessary when the normal gene was predominantly expressed. Unlike some other X-linked defects where extreme Lyonization may lead to disease, a small population of cells expressing the wild-type gp39 is sufficient to maintain normal humoral immunity and prevent the clinical symptoms of X-HIM.


Subject(s)
Genetic Linkage , Hypergammaglobulinemia/genetics , Immunoglobulin M/blood , X Chromosome , Adolescent , Antigens, CD/physiology , Antigens, Differentiation, B-Lymphocyte/physiology , Base Sequence , CD4-Positive T-Lymphocytes/immunology , CD40 Antigens , CD40 Ligand , Female , Heterozygote , Humans , Male , Membrane Glycoproteins/physiology , Molecular Sequence Data , Pedigree , Polymerase Chain Reaction
11.
Proc Natl Acad Sci U S A ; 91(3): 1099-103, 1994 Feb 01.
Article in English | MEDLINE | ID: mdl-7508119

ABSTRACT

Common variable immunodeficiency (CVI) is characterized by hypogammaglobulinemia and recurrent bacterial infections due to failure of CVI B cells to differentiate in vivo into immunoglobulin-secreting plasma cells. We hypothesized that T-cell dysfunction resulting in abnormal contact-mediated B-cell activation may play a prominent role in the failure of CVI B cells to produce specific antibody. We have previously shown that B-cell proliferation and IgE production after stimulation with anti-CD40 and interleukin (IL) 4 were normal in 22 CVI patients evaluated, indicating that CVI B cells respond to signals delivered via CD40. Here we report that CD40 ligand (gp39) mRNA expression by activated lymphocytes from CVI patients (n = 31) as a group was significantly depressed (P < 0.0001) compared with normal controls (n = 32). gp39 mRNA expression by activated lymphocytes from 13 CVI patients fell below the normal control range. T-cell surface expression of functional gp39 protein was correspondingly low in those patients with gp39 mRNA levels below normal control range and normal in patients with gp39 mRNA levels within normal control range. In CVI patients as a group, gp39 mRNA levels correlated with IL-2 mRNA levels (P < 0.002, r = 0.6) and production (P < 0.001, r = 0.7) but not with gene expression or production of other lymphokines evaluated, suggesting an as-yet-undetermined association between gp39 and IL-2 gene regulation. Of the 13 patients whose activated T cells exhibited gp39 mRNA expression below the normal control range, 2 had normal T-cell-derived lymphokine production, whereas the remaining 11 exhibited broader T-cell dysfunction, resulting in IL-2 deficiency, and in some patients deficient production of other lymphokines as well, reflecting a heterogeneity in the underlying mechanisms leading to depressed gp39 expression in these patients. The observation that both gene and surface expression of gp39 by activated T cells is depressed in a subgroup of CVI patients suggests that inefficient signaling via CD40 may be responsible, in part, for failure of B-cell differentiation in these patients.


Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, B-Lymphocyte/metabolism , Common Variable Immunodeficiency/immunology , Membrane Glycoproteins/metabolism , Adolescent , Adult , Aged , B-Lymphocytes/immunology , CD40 Antigens , CD40 Ligand , Child , Common Variable Immunodeficiency/genetics , Common Variable Immunodeficiency/metabolism , Female , Gene Expression , Humans , Interleukin-2/biosynthesis , Interleukin-2/genetics , Lymphocyte Activation , Lymphokines/biosynthesis , Male , Membrane Glycoproteins/genetics , Middle Aged , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
12.
Proc Natl Acad Sci U S A ; 90(15): 7099-103, 1993 Aug 01.
Article in English | MEDLINE | ID: mdl-7688472

ABSTRACT

One of the beta 2-integrins found on hematopoietic cells is lymphocyte function-associated antigen 1 (LFA-1), a lymphocyte/myeloid cell-specific receptor that binds to members of the intercellular adhesion molecule (ICAM) family on antigen-presenting cells. Stimulation of LFA-1 with antibodies or purified ICAMs induces augmentation of T-cell antigen receptor (TCR)-directed T-cell responsiveness. In the present study, LFA-1 was shown to be linked to the tyrosine kinase signaling pathway that stimulates tyrosine phosphorylation and activation of phospholipase C-gamma 1 (PLC-gamma 1). Integrin beta-chain (CD18) crosslinking independently induced downstream mobilization of intracellular Ca2+ and potently costimulated TCR-induced Ca2+ flux with an increase in both amplitude and kinetics. beta 2-Integrin signaling through this pathway was completely inhibited by herbimycin A and was prevented by TCR modulation. Coligation of the TCR via antibody and LFA-1 with a counter-receptor in the form of a soluble ICAM-1/Rg fusion protein resulted in prolonged tyrosine phosphorylation of PLC-gamma 1. Monoclonal antibodies to both the alpha chain (CD11a) and the beta chain (CD18) of LFA-1 induced Ca2+ mobilization to different levels, suggesting epitope specificity for activation potential. In addition to PLC-gamma 1, tyrosine phosphorylation of an 80-kDa protein substrate was augmented following CD18 crosslinking but was not TCR-dependent. The beta 2-integrin LFA-1 on T cells is therefore directly linked to a tyrosine kinase pathway that stimulates signaling by phosphatidylinositol-specific PLC-gamma 1.


Subject(s)
CD4-Positive T-Lymphocytes/physiology , Lymphocyte Function-Associated Antigen-1/metabolism , Receptors, Antigen, T-Cell/metabolism , Type C Phospholipases/metabolism , Calcium/metabolism , Enzyme Activation , Humans , In Vitro Techniques , Lymphocyte Activation , Phosphoproteins/metabolism , Phosphorylation , Phosphotyrosine , Receptor Aggregation , Signal Transduction , Tyrosine/analogs & derivatives , Tyrosine/metabolism
13.
Curr Opin Immunol ; 5(3): 334-40, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8347296

ABSTRACT

CD4, CD8 and CD45 regulate the coupling of the T-cell receptor complex (CD3-TCR) to tyrosine kinase activation and phosphorylation of key substrates such as phospholipase C gamma 1. CD4 and CD8 contribute to activation signals through their cytoplasmic association with p56lck. Expression of the zeta-chain is required for functional synergy of the T-cell receptor with CD4 in the activation of phospholipase C gamma 1, which probably reflects an interaction between p56lck and zeta-associated kinase ZAP-70. CD45 expression is required for CD3-TCR signaling. CD45 may positively regulate signaling by dephosphorylating the carboxyl-terminal tyrosine of p56lck and p59fyn, and negatively regulate signaling by dephosphorylation of other TCR-associated substrates directly. One ligand for CD45 receptor has been identified as the B cell CD22 molecule. The positive and negative effects of CD45 are sensitive to the composition of CD45 in receptor complexes, and may be regulated by specific associations of CD45 isoforms with other receptors such as CD3-TCR, CD2 and CD4.


Subject(s)
CD4 Antigens/physiology , CD8 Antigens/physiology , Leukocyte Common Antigens/physiology , Lymphocyte Activation/physiology , Signal Transduction/physiology , T-Lymphocyte Subsets/immunology , Animals , Enzyme Activation , Lymphocyte Specific Protein Tyrosine Kinase p56(lck) , Membrane Proteins/physiology , Phosphorylation , Protein Processing, Post-Translational , Protein-Tyrosine Kinases/physiology , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-fyn , Receptor-CD3 Complex, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/physiology , Type C Phospholipases/physiology , ZAP-70 Protein-Tyrosine Kinase
14.
Cell ; 72(2): 291-300, 1993 Jan 29.
Article in English | MEDLINE | ID: mdl-7678782

ABSTRACT

The prominent role of the CD40 receptor in B cell responses led us to investigate the role of the gp39-CD40 interaction in a group of primary immunodeficient patients with defective antibody production. Here we report that patients with hyper-IgM syndrome (HIM) have a defective gp39-CD40 interaction. B cells from HIM patients express functional CD40, but their T cells do not bind CD40-Ig. These patients expressed normal levels of gp39 mRNA, but these mRNAs encode defective gp39 proteins owing to mutations in the extracellular domain of gp39. Soluble recombinant forms of gp39 containing these mutations were unable to bind CD40 and drive normal B cell proliferation. The gene encoding gp39 was mapped to Xq26, the X chromosome region where the gene responsible for HIM had previously been mapped. These data suggest that a defect in gp39 is the basis of X-linked HIM.


Subject(s)
Antigens, Differentiation, T-Lymphocyte/genetics , Hypergammaglobulinemia/genetics , Immunoglobulin M/blood , Immunologic Deficiency Syndromes/genetics , Lymphocyte Activation , Membrane Glycoproteins/genetics , T-Lymphocytes/immunology , X Chromosome , Adolescent , Adult , Amino Acid Sequence , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, Differentiation, B-Lymphocyte/genetics , Antigens, Differentiation, B-Lymphocyte/metabolism , B-Lymphocytes/immunology , Base Sequence , Blotting, Northern , CD40 Antigens , CD40 Ligand , Chromosome Mapping , DNA/genetics , DNA/isolation & purification , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin Switch Region , Male , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Mutation , Oligodeoxyribonucleotides , Protein Structure, Secondary , RNA, Messenger/genetics , RNA, Messenger/metabolism
15.
EMBO J ; 11(12): 4313-21, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1385114

ABSTRACT

Signals delivered to B cells via CD40 can synergize with those provided by other B cell surface receptors to induce B cell proliferation and antibody class switching as well as modulate cytokine production and cell adhesion. Recently, it has been shown that the ligand for CD40 is a cell surface protein of approximately 39 kDa expressed by activated T cells, gp39. Here we report on the isolation and characterization of a cDNA clone encoding human gp39, a type II membrane protein with homology to TNF, and the construction and characterization of a soluble recombinant form of gp39. COS cell transfectants expressing gp39 synergized with either anti-CD20 mAb or PMA to drive strong B cell proliferation and alone were able to drive B cells to proliferate weakly. In all cases the B cell proliferation induced by gp39-expressing COS cells was reduced to background levels by the addition of soluble CD40. Unlike gp39-expressing COS cells, recombinant soluble gp39 was not mitogenic alone and required co-stimulation to drive B cell proliferation. These results suggest that B cells require a second signal besides gp39-CD40 to drive proliferation and that soluble gp39 alone in a non-membrane bound form is able to provide co-stimulatory signals to B cells.


Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, B-Lymphocyte/metabolism , Membrane Proteins/metabolism , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/genetics , Amino Acid Sequence , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Base Sequence , CD40 Antigens , CD40 Ligand , Cell Division , Cell Line , Cells, Cultured , DNA , Humans , Membrane Proteins/genetics , Molecular Sequence Data , Multigene Family , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , T-Lymphocytes/metabolism
16.
Hum Immunol ; 32(2): 85-94, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1835971

ABSTRACT

Recently, it was shown that HLA class II molecules on B cells and activated human T cells can transmit signals involving tyrosine phosphorylation of specific proteins, activation of the inositol phospholipid pathway, and release of cytosolic free Ca2+(Ca2+)i. The regulation of class II induced signals is poorly understood, however, and it remained unknown whether these pathways were coupled or activated independently. Here we show that a specific inhibitor of protein tyrosine kinases (PTK), herbimycin, abrogated DR-induced elevation of (Ca2+)i in activated human T cells. Genistein, belonging to another family of PTK inhibitors, had weaker but significant inhibitory effects on DR-induced (Ca2+)i responses. CD45 crosslinking with DR almost completely abrogated DR-induced (Ca2+)i responses and profoundly changed the PTK profiles. In contrast, CD4 crosslinking with DR enhanced the (Ca2+)i responses, but the inhibitory effect of CD45 dominated over the enhancing effect of CD4. These data indicate that PTK activation is obligatory for DR-induced (Ca2+)i responses, suggesting a linkage between these pathways in class II signal transduction. This conclusion is consistent with our observation that in activated human T cells, class II signals are up regulated by CD4, which is associated with p56lck, and down regulated by CD45, which is a tyrosine phosphatase.


Subject(s)
Antigens, CD/metabolism , Calcium/metabolism , HLA-DR Antigens/metabolism , Histocompatibility Antigens/metabolism , Protein-Tyrosine Kinases/metabolism , Signal Transduction , T-Lymphocytes/metabolism , Benzoquinones , CD4 Antigens/metabolism , Cells, Cultured , Clone Cells , Enzyme Activation , Humans , Lactams, Macrocyclic , Leukocyte Common Antigens , Protein-Tyrosine Kinases/antagonists & inhibitors , Quinones/pharmacology , Rifabutin/analogs & derivatives , T-Lymphocytes/drug effects , T-Lymphocytes/immunology
17.
J Exp Med ; 174(3): 561-9, 1991 Sep 01.
Article in English | MEDLINE | ID: mdl-1714933

ABSTRACT

Functional interactions between T and B lymphocytes are necessary for optimal activation of an immune response. Recently, the T lymphocyte receptor CD28 was shown to bind the B7 counter-receptor on activated B lymphocytes, and subsequently to costimulate interleukin 2 production and T cell proliferation. CTLA-4 is a predicted membrane receptor from cytotoxic T cells that is homologous to CD28 and whose gene maps to the same chromosomal band as the gene for CD28. It is not known, however, if CD28 and CTLA-4 also share functional properties. To investigate functional properties of CTLA-4, we have produced a soluble genetic fusion between the extracellular domain of CTLA-4 and an immunoglobulin C gamma chain. Here, we show that the fusion protein encoded by this construct, CTLA4Ig, bound specifically to B7-transfected Chinese hamster ovary cells and to lymphoblastoid cells. CTLA4Ig also immunoprecipitated B7 from cell surface 125I-labeled extracts of these cells. The avidity of 125I-labeled B7Ig fusion protein for immobilized CTLA4Ig was estimated (Kd approximately 12 nM). Finally, we show that CTLA4Ig was a potent inhibitor of in vitro immune responses dependent upon cellular interactions between T and B lymphocytes. These findings provide direct evidence that, like its structural homologue CD28, CTLA-4 is able to bind the B7 counter-receptor on activated B cells. Lymphocyte interactions involving the B7 counter-receptor are functionally important for alloantigen responses in vitro.


Subject(s)
Antigens, Differentiation/immunology , Antigens, Surface/metabolism , B-Lymphocytes/immunology , Immunoconjugates , Lymphocyte Activation , Receptors, Immunologic , T-Lymphocytes/immunology , Abatacept , Amino Acid Sequence , Animals , Antigens, CD , Antigens, Differentiation/genetics , B7-1 Antigen , Base Sequence , CTLA-4 Antigen , Cell Line , In Vitro Techniques , Ligands , Lymphocyte Cooperation , Molecular Sequence Data , Oligonucleotides/chemistry , Precipitin Tests , Recombinant Fusion Proteins
19.
Blood ; 75(7): 1531-9, 1990 Apr 01.
Article in English | MEDLINE | ID: mdl-2156582

ABSTRACT

The CD28 homodimer is thought to function as a signal transducing receptor during activation of T cells. Evidence is presented that the degree of aggregation of CD28 on the cell surface regulates two distinct CD28-associated signals. Binding of bivalent CD28 monoclonal antibody (MoAb) 9.3 upregulates lymphokine production by messenger RNA (mRNA) stabilization, without direct initiation of lymphokine mRNA transcription. This signal was not dependent on inositol phospholipid production or activation of a protein tyrosine kinase (PTK). In contrast, further crosslinking of CD28 on the cell surface rapidly induced formation of large amounts of inositol trisphosphate (InsP3) and increased cytoplasmic calcium concentration [( Ca2+]i), but did not stimulate PTK. CD28 crosslinking directly activated a subset of resting T cells, since CD25 (interleukin [IL]-2 receptor alpha chain) mRNA was rapidly induced in purified T cells, and proliferation, even without addition of exogenous IL-2, was sometimes observed. CD25 expression was detected on the cell surface of approximately 20% of CD4+ T cells. The degree of CD28 aggregation required for activation was investigated by preparing soluble 9.3 x 9.3 conjugates ranging in size from approximately 300 Kd to greater than 1,000 Kd, and comparing their function in T-cell proliferation assays with phorbol-12-myristate-13-acetate (PMA), anti-CD3, or IL-2. There was a correlation between conjugate size and proliferation with IL-2, whereas costimulation with PMA or CD3 was optimized at a lower degree of CD28 aggregation. The inositol phospholipid (InsP) generation and increase in [Ca2+]i after CD28 receptor aggregation appeared to proceed through a pathway different from the CD3/T-cell receptor (TCR) pathway since it was enhanced by pretreatment with PMA, while the InsP and [Ca2+]i signal from crosslinking CD3 was suppressed by PMA. Furthermore, the proliferation response to CD28 aggregation was resistant to inhibition by CD3 modulation. Thus, CD28 aggregation appears to trigger a phospholipase C activation pathway that differs from the CD3/TCR-linked pathway.


Subject(s)
Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/immunology , Calcium/metabolism , Lymphocyte Activation , Signal Transduction , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal , CD28 Antigens , Cell Line , Fluorescent Dyes , Gene Expression , Humans , Indoles , Kinetics , Lymphokines/genetics , Mice , Mice, Inbred BALB C/immunology , RNA, Messenger/genetics , Rats/immunology
20.
Tissue Antigens ; 35(3): 128-35, 1990 Mar.
Article in English | MEDLINE | ID: mdl-2142837

ABSTRACT

The cytoplasmic domain of the CD45 leukocyte cell surface antigen has recently been shown to possess protein tyrosine phosphatase (PTPase) activity. The existence of a cell membrane-bound PTPase may represent a mechanism by which an activation signal, initiated by ligand binding to a surface receptor, is down-regulated following delivery of the signal. Both the interleukin-2 (IL2) growth factor receptor and the CD3/Ti T-cell antigen receptor contain a subunit which is phosphorylated on tyrosine by an activated protein kinase (PTK) during T-cell activation. We compared the effect of CD45 ligation on signal transduction mediated by the binding of IL2 or anti-CD3 to these two receptors. Immunoblotting with anti-phosphotyrosine antiserum was used to investigate the effect of CD45 ligation on anti-CD3- or IL2-induced protein tyrosine phosphorylation. When CD3 and CD45 were triggered together, changes in the pattern of tyrosine phosphorylation of specific substrates was observed in comparison to the stimulus triggered through CD3 alone. In contrast, CD45 ligation did not alter the pattern of tyrosine-phosphorylated proteins in "resting" T-cell blasts responding to IL2, except for a mobility shift of a 55 kDa protein and increased phosphorylation of a 112 kDa substrate. The proliferative response of T cells to both anti-CD3 or IL2 was inhibited by ligating CD45. The CD45 molecule down-regulated CD3-induced T-cell activation when the CD45 and CD3 molecules were ligated simultaneously with immobilized antibodies. In contrast, immobilized CD45 mAb alone inhibited IL2-induced proliferation, and the inhibition was not potentiated by simultaneously using a CD25 mAb which was non-competitive for IL2-binding.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Antigens, CD/physiology , Antigens, Differentiation, T-Lymphocyte/physiology , Antigens, Differentiation/physiology , Histocompatibility Antigens/physiology , Receptors, Antigen, T-Cell/physiology , Receptors, Interleukin-2/physiology , Signal Transduction/immunology , Antibodies, Monoclonal , CD3 Complex , Cross-Linking Reagents , Humans , Leukocyte Common Antigens , Lymphocyte Activation/immunology , Molecular Weight , Phosphorylation , Protein-Tyrosine Kinases/metabolism
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