RESUMO
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.
Assuntos
Antígenos de Diferenciação/imunologia , Antígenos de Superfície/metabolismo , Linfócitos B/imunologia , Imunoconjugados , Ativação Linfocitária , Receptores Imunológicos , Linfócitos T/imunologia , Abatacepte , Sequência de Aminoácidos , Animais , Antígenos CD , Antígenos de Diferenciação/genética , Antígeno B7-1 , Sequência de Bases , Antígeno CTLA-4 , Linhagem Celular , Técnicas In Vitro , Ligantes , Cooperação Linfocítica , Dados de Sequência Molecular , Oligonucleotídeos/química , Testes de Precipitina , Proteínas Recombinantes de FusãoRESUMO
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.
Assuntos
Ligação Genética , Hipergamaglobulinemia/genética , Imunoglobulina M/sangue , Cromossomo X , Adolescente , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Antígenos CD40 , Ligante de CD40 , Feminino , Heterozigoto , Humanos , Masculino , Glicoproteínas de Membrana/fisiologia , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Antígenos CD4/fisiologia , Antígenos CD8/fisiologia , Antígenos Comuns de Leucócito/fisiologia , Ativação Linfocitária/fisiologia , Transdução de Sinais/fisiologia , Subpopulações de Linfócitos T/imunologia , Animais , Ativação Enzimática , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Proteínas de Membrana/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-fyn , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Fosfolipases Tipo C/fisiologia , Proteína-Tirosina Quinase ZAP-70RESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Antígenos CD40/imunologia , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células Cultivadas , Clonagem Molecular , Endotélio/citologia , Expressão Gênica , Humanos , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Monócitos/imunologia , NF-kappa B/imunologiaRESUMO
A subclone that had lost its IgG1 heavy chain was derived from a hybridoma cell line G17-2 that produces an anti-CD4 monoclonal antibody. This subclone was found to secrete a kappa light chain dimer (LCD) that could bind to the CD4 antigen expressed on a subset of human T lymphocytes. The light chain dimer bound to the same or similar epitope as the parental antibody since it blocked the binding of the parenteral anti-CD4 MAb but not the binding of another anti-CD4 MAb G19-2 that recognizes a different epitope. A rabbit anti-idiotype prepared against G17-2 crossreacted with the LCD and could block the antigen binding of both G17-2 and the LCD. Scatchard analysis performed with 35S-methionine or 3H-leucine labelled LCD showed an association constant Ka of 2.2 x 10(7) M-1, whereas the G17-2 parental antibody showed an association constant Ka of 2.5 x 10(9) M-1. These data indicate that the antigen specificity of the G17-2 parental MAb is conferred to a large extent by its light chain. The LCD was expressed on the surface of the LCD-producing hybridoma cells. Southern blot analysis with C kappa and J kappa probes demonstrated a single kappa transcription units which does not have any unusual DNA rearrangements and is distinct from the NS-1 kappa genes. To our knowledge, this LCD is unique in its ability to bind to a large (55,000 mol. wt) glycoprotein antigen.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Epitopos/análise , Cadeias kappa de Imunoglobulina/imunologia , Animais , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Signal transduction occurs through multiple receptors expressed on mature, resting T cells. In addition to the CD3-T cell receptor complex, the CD2, CD4, CD5, CD7, CD8 and CD28 receptors mobilize cytoplasmic calcium within minutes of binding with monoclonal antibodies and additional crosslinking occurs on the cell surface. As an approach to study the interactions between these receptors and their transduced signals, monoclonal antibodies to each of these receptors were covalently coupled as heteroconjugates and investigated for activity in cytoplasmic calcium mobilization using indo-1 and flow cytometry. Of a total of 35 conjugates studied, there were seven heteroconjugates that showed an increase in activity and these consisted of either certain conjugates of anti-CD3 or certain conjugates of anti-CD5. The CD3-CD2, CD3-CD4, CD3-CD6 and CD3-CD8 heteroconjugates each gained two to three orders of magnitude in titer in calcium mobilization compared to unconjugated CD3 or the CD3-CD3 conjugate. The increase in activity was not accompanied by an increase in binding titer, indicating that signal transduction occurred at lower levels of receptor occupancy. The increased activity was dependent in each case on the relevant second receptor, since unconjugated CD2, CD4, CD6 or CD8 MAb could block the activity of the corresponding heteroconjugate. Neither CD3-CD5, CD3-CD28 or CD3-CD3 conjugates gained activity, whereas CD3-CD7 heteroconjugates gained slightly in activity. The heteroconjugates with CD5 that acquired ability to mobilize calcium at low concns (less than 5 micrograms/ml) were CD5-CD4, CD5-CD8 and CD5-CD6. Their activity could be inhibited by either CD5 MAb or the second MAb of the heteroconjugate. The increased activity of CD3 or CD5 heteroconjugates was observed in the absence of extracellular calcium. Size exclusion chromatography of heteroconjugates demonstrated that 1:1 ratios were optimal, but larger conjugates were also active. These results suggest that certain receptors are capable for molecular interactions on the cell surface to form complexes with enhanced activity in signal transduction leading to calcium mobilization.
Assuntos
Proteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Animais , Anticorpos Monoclonais/imunologia , Cálcio/metabolismo , Membrana Celular/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
The binding of normal cat IgG, heat-aggregated cat IgG and specific immune complexes (IC) containing cat IgG to a silica matrix containing covalently bound Staphylococcus aureus protein A was evaluated. The amounts of serum relative to protein A-silica, the flow rates and the perfusion times were representative of those existing when protein A-silica columns are used for therapeutic extracorporeal immunoadsorption of IgG and IC from humans and animals. When cat IgG was present in a large excess, approximately one molecule was bound to the matrix per molecule of solid-phase protein A with a KA of 1.5 X 10(6) 1/mol. Aggregated and immune complexed IgG bound to the matrix with relatively higher affinity. IC prepared in vitro between the purified envelope glycoprotein of the feline leukemia virus (FeLV gp70) and affinity-purified cat antibodies bound to the matrix even though normal IgG was present in greater than 10,000-fold excess. Once bound, IC were not eluted from columns upon further perfusion with normal serum. However, bound IgG was eluted from columns by further perfusion of normal serum or IC. IC were at least five-fold more efficient than normal IgG in exerting this effect. The results suggest that protein A-silica columns can be used for preferential removal of IC from plasma in a clinical or experimental setting.
Assuntos
Complexo Antígeno-Anticorpo/análise , Imunoglobulina G/isolamento & purificação , Dióxido de Silício/farmacologia , Proteína Estafilocócica A/farmacologia , Adsorção , Animais , Sítios de Ligação , Gatos , Concentração de Íons de Hidrogênio , Imunoglobulina G/metabolismo , Vírus da Leucemia Felina/imunologia , Proteína Estafilocócica A/metabolismoRESUMO
The inhibitory effect of anti-HLA class I monoclonal antibodies on lymphocyte proliferation has been well documented. However, recent data suggest that anti-HLA class I monoclonal antibodies can enhance lymphocyte proliferation via both anti-CD3-induced (1,2) and anti-CD2-induced (3) activation pathways. Here we demonstrate that both inhibition and activation can be regulated by the degree of aggregation of HLA class I antigens. Crosslinking of monoclonal antibodies specific for HLA-A, HLA-B, or monomorphic determinants (using anti-IgG2 and/or anti-Ig kappa "second step" monoclonal antibodies) increased the capacity of anti-HLA class I monoclonal antibodies to inhibit phytohemagglutinin-induced proliferation. However, the cytosolic free calcium concentration was increased in CD4+ cells, CD8+ cells, B cells, and CD16+ cells when anti-HLA class I monoclonal antibodies were crosslinked, suggesting that an activation signal was generated by aggregation of the corresponding antigens. Indeed, inositol 1,4,5-trisphosphate could be detected in peripheral blood lymphocytes following crosslinking of anti-HLA class I monoclonal antibodies. Class I aggregation also induced proliferation of peripheral blood mononuclear cells in the presence of submitogenic doses of phorbol 12-myristate 13-acetate. Strong conditions of crosslinking (monomorphic monoclonal antibody plus both anti-IgG2 and anti-Ig kappa) induced CD25 expression and responsiveness to recombinant interleukin 2. Our results suggest that aggregation of HLA class I antigens primed cells to become activated in the presence of progression signals including phorbol 12-myristate 13-acetate, recombinant interleukin 2, or anti-CD5 plus anti-CD28 monoclonal antibodies.
Assuntos
Antígenos HLA , Ativação Linfocitária , Transdução de Sinais , Anticorpos Monoclonais , Antígenos de Diferenciação , Antígenos CD5 , Cálcio/metabolismo , Reagentes de Ligações Cruzadas , Humanos , Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/classificação , Linfócitos/imunologia , Linfócitos/metabolismo , Receptores de Interleucina-2 , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
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.
Assuntos
Antígenos CD/metabolismo , Cálcio/metabolismo , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Benzoquinonas , Antígenos CD4/metabolismo , Células Cultivadas , Células Clonais , Ativação Enzimática , Humanos , Lactamas Macrocíclicas , Antígenos Comuns de Leucócito , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinonas/farmacologia , Rifabutina/análogos & derivados , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
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).
Assuntos
Antígenos CD , Leucócitos/imunologia , Suínos/imunologia , AnimaisRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Fosfolipases Tipo C/metabolismo , Cálcio/metabolismo , Ativação Enzimática , Humanos , Técnicas In Vitro , Ativação Linfocitária , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina , Agregação de Receptores , Transdução de Sinais , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
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.
Assuntos
Anticorpos Monoclonais/farmacologia , Linfócitos B/metabolismo , Ciclosporina/farmacologia , Glicoproteínas de Membrana/metabolismo , Linfócitos T/metabolismo , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Sequência de Bases , Ligante de CD40 , Proteínas de Transporte , Células Cultivadas , Citometria de Fluxo , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/imunologia , Camundongos , Dados de Sequência Molecular , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Molecule CD28 (Tp44) is expressed on the surface of majority of human T cells and has been implicated to play an active role in the regulation of T cell growth. The present study examines the effect of antibody binding to the CD28 molecule during T cell activation. Anti-CD28 but not isotype-matched anti-CD5 mAb consistently augmented anti-CD3-induced and IL-2-induced T cell proliferation and subsequent release of soluble CD25 molecule. When added together, mAb anti-CD28 and anti-CD5 acted synergistically to cause 2- to 7-fold enhancement of T cell activation induced by anti-CD3 mAb or IL-2 with no effect on the development of non-MHC-restricted IL-2-activated killer T cells. In contrast, alloantigen-induced T cell proliferation, soluble CD25 release, and the subsequent development of CTL were all inhibited by anti-CD28 mAb. Moreover, alloantigen-induced proliferative response of both CD4+ and CD8+ T cells was inhibited by anti-CD28 without affecting the cytolytic effect of CTL. Because valency of anti-CD28 binding has been implicated as an important factor in signal transduction, this was explored in the allogeneic MLR by using Fab and F(ab')2 fragments of anti-CD28 mAb and anti-mouse kappa mAb. The inhibitory effect of anti-CD28 mAb in the MLR was reversed by cross-linking of anti-CD28 mAb with anti-mouse kappa mAb. In addition, cross-linking of the CD28 molecule on alloactivated T lymphoblasts but not that on resting T cells with anti-CD28 and anti-mouse kappa induced their proliferation in the absence of the priming alloantigen. These results indicate that stimulatory or inhibitory signals delivered through the CD28 molecule are determined by the degree of cross-linking of this molecule. In addition, these results also suggest that Ag-induced CD3-TCR-mediated T cell responses are more dependent on signals delivered through the CD28 molecule than those induced with anti-CD3, and thus these results have implications for potential use of anti-CD28 in sustained propagation of Ag-specific T cells.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação de Linfócitos T/fisiologia , Ativação Linfocitária , Linfócitos T/imunologia , Anticorpos Anti-Idiotípicos/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Humanos , Cadeias kappa de Imunoglobulina/imunologia , Isoantígenos/imunologia , Teste de Cultura Mista de LinfócitosRESUMO
Antibodies binding to a large subset of T-cell differentiation antigens, including CD2, CD4, CD5, CD6, CD7, CD8, Tp44, and CDw18, cause an increase in the cytoplasmic calcium concentration [( Ca2+]i) after the antigens are crosslinked on the cell surface. Similar crosslinking-induced signals were seen for a subset of mouse thymocyte differentiation antigens. The various antigens on human T cells differed in the extent of crosslinking required for generating the calcium signal, as evidenced by comparisons with monoclonal versus polyclonal second-step antibody. The [Ca2+]i increase that occurs after crosslinking represents mobilization of cytoplasmic calcium since the initial component of the signal is resistant to depletion of extracellular calcium by chelation with EGTA. The [Ca2+]i increase is completely inhibited by pretreatment of cells with pertussis toxin, indicating that a substrate for pertussis toxin regulates the signal transduction. Crosslinking of antigens other than the CD3/T-cell receptor complex did not result in T-cell proliferation. Crosslinking of CD2 and Tp44, but not other antigens, resulted in expression of functional interleukin 2 receptors. Comparisons of three different anti-CD3 antibodies showed that a second calcium signal was generated by crosslinking, even when the anti-CD3 antibodies were used at optimal concentrations.
Assuntos
Antígenos de Superfície/imunologia , Cálcio/metabolismo , Linfócitos T/imunologia , Animais , Anticorpos , Anticorpos Monoclonais , Antígenos de Diferenciação de Linfócitos B , Células Cultivadas , Ácido Egtázico/farmacologia , Camundongos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
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.
Assuntos
Anticorpos Antineoplásicos/uso terapêutico , Engenharia Genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Imunoterapia/métodos , Interleucina-2/uso terapêutico , Neoplasias/terapia , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Células CHO , Células COS , Cricetinae , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Interleucina-2/genética , Interleucina-2/imunologia , Neoplasias/genética , Neoplasias/imunologia , Proteínas Recombinantes de Fusão/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologiaRESUMO
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)
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos T/fisiologia , Antígenos de Diferenciação/fisiologia , Antígenos de Histocompatibilidade/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores de Interleucina-2/fisiologia , Transdução de Sinais/imunologia , Anticorpos Monoclonais , Complexo CD3 , Reagentes de Ligações Cruzadas , Humanos , Antígenos Comuns de Leucócito , Ativação Linfocitária/imunologia , Peso Molecular , Fosforilação , Proteínas Tirosina Quinases/metabolismoRESUMO
The present study was undertaken to determine whether human PBL can be specifically focused to lyse cells infected with HIV-1 by mAb heteroconjugates that can bridge target and effector cells. A mAb directed against the central portion of HIV-1 glycoprotein gp110 was chemically cross-linked to a mAb directed against the CD3/TCR complex or to a mAb directed against the CD16 Fc gamma-R expressed on large granular lymphocytes (LGL). HIV-1-infected cells, but not uninfected cells, were found to be lysed to a greater extent by PBL in the presence of the gp110 X CD3 or the gp110 X CD16 antibody heteroconjugate than in the presence of the single antibodies or a mixture of the mAb comprising the heteroconjugates. Pretreatment of PBL with anti-CD3 or IL-2 augments their ability to lyse HIV-1-infected cells in the presence of the heteroconjugates. Lysis by anti-CD3-activated PBL in the presence of the gp110 X CD3 heteroconjugate was found to be mediated by CD8+-enriched T cells, whereas lysis by IL-2-treated PBL in the presence of the gp110 X CD16 heteroconjugate is mediated by PBL enriched for CD16+ cells, which are primarily LGL. Furthermore, PBL from asymptomatic, HIV-1-infected seropositive donors were found to be functional in lysing HIV-1-infected cells in the presence of the antibody heteroconjugates. Such antibody heteroconjugates, which can target T cells or LGL to lyse HIV-1-infected cells, may be of prophylactic or therapeutic value in HIV-1-infected individuals.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos HIV , HIV/imunologia , Linfócitos/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Complexo CD3 , Citotoxicidade Imunológica/efeitos dos fármacos , Anticorpos Anti-HIV , Humanos , Interleucina-2/farmacologia , Ativação Linfocitária , Receptores Fc/imunologia , Receptores de IgG , Proteínas dos Retroviridae/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas do Envelope Viral/imunologiaRESUMO
The relationship between the increase of intracellular free Ca2+ concentration ([Ca2+]i) in resting T cells after stimulation with monoclonal antibody (mAb) to CD2 (E rosette receptor) and the subsequent proliferation response was investigated. Although the combination of 9.6 plus 9-1 mAb to CD2 was both mitogenic and induced an increase in [Ca2+]i, cross-linking of an individual CD2 mAb on the cell surface induced an increase in [Ca2+]i without directly stimulating T cell proliferation. The [Ca2+]i response from cross-linking an individual mAb was not epitope dependent, since 21 of 21 mAb to CD2 were effective when cross-linked with a polyclonal goat anti-mouse Ig second step. The kinetics of calcium mobilization was highly dependent upon the procedure for cross-linking, since the binding of biotin-conjugated 9.6 mAb followed by avidin gave a large and rapid response, whereas cross-linking of 9.6 with an anti-kappa mAb, 187.1, caused a minimal response, and the cross-linking of 9.6 followed by a polyclonal goat anti-mouse Ig gave an intermediate response. In addition, ligation of CD2 by rosetting with sheep red blood cells alone was sufficient to cause increased [Ca2+]i. In functional studies only the procedures associated with minimal CD2 cross-linking induced proliferation of resting T cells in combination with interleukin (IL)2. The proliferation also required IL 1 or accessory cells. Cross-linking 9.6 on the cell surface also enhanced proliferation in the presence of phorbol myristate acetate or a CD28 mAb, 9.3, under conditions that were accessory cell independent. In contrast to the proliferation following stimulation with 9.6 plus 9-1, the combination of 9.6 plus 9-1 F(ab')2 fragments lost mitogenic activity. The 9.6 plus 9-1 F(ab')2 combination was similar to 9.6 cross-linking in that either could induce responsiveness to recombinant IL2 or CD28 mAb 9.3. The combination of 9.6 plus 9-1 F(ab')2 fragments was still able to increase [Ca2+]i in T cells with slow kinetics. Together these results suggest that the binding of mAb to CD2 under conditions that cause a slow rather than a rapid increase in [Ca2+]i is associated with T cell activation. Furthermore, they suggest that in studies of T cell activation, sheep erythrocyte rosette formation should not be used to isolate T cells since rosetting may effect [Ca2+]i.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Cálcio/fisiologia , Ativação Linfocitária , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos/fisiologia , Linfócitos T/fisiologia , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Antígenos CD2 , Antígenos CD28 , Epitopos , Interleucina-1/farmacologia , Interleucina-2/farmacologia , Medições Luminescentes , Substâncias Macromoleculares , Receptores Fc/fisiologia , Formação de RosetaRESUMO
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.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Moléculas de Adesão Celular , Citoplasma/imunologia , Citoplasma/metabolismo , Imunização/métodos , Lectinas , Proteínas Tirosina Quinases/análise , Receptores de Antígenos/imunologia , Transporte Biológico/imunologia , Linfoma de Burkitt/patologia , Humanos , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Pironas/farmacologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Células Tumorais Cultivadas , Vanadatos/farmacologiaRESUMO
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.