RESUMEN

The structure of human CTLA-4 reveals that residues Met 99, Tyr 100 and Tyr 104 of the M99YPPPY104 motif are adjacent to a patch of charged surface residues on the A'GFCC' face of the protein. Mutation of these residues, which are conserved in the CTLA-4/CD28 family, significantly reduces binding to CD80 and/or CD86, implicating this patch as a ligand binding site.

Asunto(s)

Antígenos CD/metabolismo , Antígenos de Diferenciación/química , Antígenos de Diferenciación/metabolismo , Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Inmunoconjugados , Glicoproteínas de Membrana/metabolismo , Abatacept , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación/genética , Antígeno B7-2 , Sitios de Unión , Antígeno CTLA-4 , Secuencia Conservada , Dimerización , Humanos , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Soluciones , Sulfuros

RESUMEN

CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation. While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface. The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling. Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain. Using the yeast two-hybrid method, we demonstrate association of the mu2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28. The mu1 subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28. Sequences required for interaction of mu2 and CTLA-4 were localized to residues, 161TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165YVKM, involved in CTLA-4 signaling. Mu2 interacted preferentially with CTLA-4 when residue 165Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165Y was phosphorylated. In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 (165Y and 182Y) were phosphorylated by the T lymphocyte-associated tyrosine kinase, p56lck. Thus, phosphorylation of CTLA-4 residue 165Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail.

Asunto(s)

Complejo 1 de Proteína Adaptadora , Complejo 2 de Proteína Adaptadora , Complejo 3 de Proteína Adaptadora , Subunidades mu de Complejo de Proteína Adaptadora , Antígenos de Diferenciación/metabolismo , Inmunoconjugados , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Abatacept , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Antígenos CD , Antígenos de Diferenciación/química , Western Blotting , Compuestos de Boro/metabolismo , Antígeno CTLA-4 , Clatrina/química , Colorantes Fluorescentes/metabolismo , Genes Reporteros , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Fosforilación , Plásmidos/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , Linfocitos T/química , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo , Dominios Homologos src/genética

RESUMEN

T lymphocyte receptors CD28 and CTLA-4 bind costimulatory molecules CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells and regulate T cell activation. While distinct functional roles have been ascribed to each of these molecules, little is known about how they interact. To better characterize these interactions, we have used surface plasmon resonance to perform equilibrium and kinetic binding analyses of extracellular fragments of CD28/CTLA-4/CD80/CD86. We show that CTLA-4 and CD28 binding are both characterized by rapid kinetic on-rates and rapid dissociation rates. Native disulfide-linked homodimers of CD28 and CTLA-4 bound with two kinetically distinct binding sites, one of high avidity and slow dissociation and one of low avidity and more rapid dissociation. Monomeric CTLA-4 bound only with low affinity and rapid dissociation. Therefore, covalent dimerization of CTLA-4 is required for its high avidity binding. Oligomerization of CD80/CD86 is also required for high avidity CTLA-4 binding since CTLA-4 bound with low avidity to monomeric CD86. This contrasts with the ability of CD80/CD86 on antigen-presenting cells to bind CTLA4Ig with high avidity and predicts their organization as oligomers or clusters that permit multivalent binding. Thus, covalent receptor dimerization and ligand oligomerization are two key features of the CD28/CTLA-4/CD80/CD86 receptor system that control ligand binding and may regulate signal transduction by controlling the duration of receptor occupancy.

Asunto(s)

Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Inmunoconjugados , Glicoproteínas de Membrana/metabolismo , Abatacept , Secuencia de Aminoácidos , Antígeno B7-2 , Biopolímeros , Antígeno CTLA-4 , Espacio Extracelular/metabolismo , Cinética , Datos de Secuencia Molecular , Unión Proteica

RESUMEN

The B7-related molecules CD80 and CD86 are expressed on antigen-presenting cells, bind the homologous T cell receptors CD28 and CTLA-4, and trigger costimulatory signals important for optimal T cell activation. All four molecules are immunoglobulin superfamily members, each comprising an extracellular Ig variable-like (IgV) domain, with CD80 and CD86 containing an additional Ig constant-like (IgC) domain. Despite limited sequence identity, CD80 and CD86 share similar overall receptor binding properties and effector functions. We have identified, by site-directed mutagenesis of soluble forms of CD80 and CD86, residues in both the IgV and IgC domains that are important for CTLA4Ig and CD28Ig binding. Mutagenesis in the IgV domain of CD80 identified 11 amino acids that support receptor binding. Many of these residues are conserved in the B7 family, are hydrophobic, and approximately map to the GFCC'C" beta-sheet face of an IgV fold. Mutagenesis of corresponding residues in CD86 established that some, but not all, of these residues also played a role in CD86 receptor binding. In general, mutations had a similar effect on CTLA4Ig and CD28Ig binding, thereby indicating that both receptors bind to overlapping sites on CD80 and CD86. Further, mutagenesis of several conserved residues in the ABED beta-sheet face of the IgC domain of CD80 completely ablated receptor binding. Point mutagenesis had a more pronounced effect than complete truncation of the IgC domain. Thus, full CTLA4Ig and CD28Ig binding to B7 molecules is dependent upon residues in the GFC'C" face of the IgV domain and the ABED face of the IgC domain.

Asunto(s)

Antígenos de Diferenciación/metabolismo , Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Inmunoconjugados , Abatacept , Secuencia de Aminoácidos , Antígenos CD , Antígeno B7-1/genética , Secuencia de Bases , Antígeno CTLA-4 , Secuencia Conservada , Cartilla de ADN , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

We have examined the ability of CTLA4Ig to induce long-lasting Ag-specific tolerance to a T-dependent Ag. Treatment of mice with murine CTLA4Ig following immunization with SRBC induced immune unresponsiveness to SRBC that was only reversible upon repeated Ag exposure. This unresponsiveness was Ag specific, lasted > 90 days, was observed in thymectomized mice, and was maintained even during challenge with another strong T-dependent Ag. A third challenge with SRBC, however, led to an Ab response. Splenic T cells and B cells from unresponsive mice were functional upon transfer to irradiated hosts, indicating that they had been neither depleted nor rendered permanently "tolerant" by CTLA4Ig. The mechanism of immunosuppression by CTLA4Ig was investigated by measuring cytokine transcripts in spleens of immunized mice. CTLA4Ig treatment following primary immunization blocked the induction of IL-2 transcripts in splenic T cells and IL-4 transcripts in both T cells and non-B, non-T cells. Splenocytes from CTLA4Ig-treated, SRBC-unresponsive mice showed altered induction of IL-2 and IL-4 transcripts, but T cells nonetheless became primed for IL-4 mRNA synthesis despite the lack of a measurable Ab response. Anti-IL-4 mAb and human CTLA4Ig were synergistic in their ability to induce unresponsiveness, indicating that incomplete suppression of IL-4 production by CTLA4Ig limited its effectiveness.

Asunto(s)

Antígenos de Diferenciación/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Inmunoconjugados , Linfocitos T/efectos de los fármacos , Abatacept , Animales , Antígenos CD , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Antígeno CTLA-4 , Eritrocitos/inmunología , Femenino , Citometría de Flujo , Tolerancia Inmunológica/inmunología , Tolerancia Inmunológica/fisiología , Interleucina-2/antagonistas & inhibidores , Interleucina-4/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa/métodos , Ovinos , Linfocitos T/inmunología , Vacunación

RESUMEN

CD28 and CTLA-4 are homologous T cell receptors of the immunoglobulin (Ig) superfamily, which bind B7 molecules (CD80 and CD86) on antigen-presenting cells and transmit important costimulatory signals during T cell activation. Here we have investigated the subunit structure of CTLA-4 and the stoichiometry of its binding to B7 molecules. We demonstrate CTLA-4 is a homodimer interconnected by one disulfide bond in the extracellular domain at cysteine residue 120. Each monomeric polypeptide chain of CTLA-4 contains a high affinity binding site for B7 molecules; soluble CTLA-4 and CD86 form complexes containing equimolar amounts of monomeric CTLA-4 and CD86 (i.e. a 2:2 molecular complex). Thus, CTLA-4 and probably CD28 have a receptor structure consisting of preexisting covalent homodimers with two binding sites. Dimerization of CTLA-4 and CD28 is not required for B7 binding, nor is it sufficient to trigger signaling.

Asunto(s)

Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , Antígeno B7-1/metabolismo , Inmunoconjugados , Glicoproteínas de Membrana/metabolismo , Linfocitos T Citotóxicos/inmunología , Abatacept , Secuencia de Aminoácidos , Animales , Antígenos CD/aislamiento & purificación , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/aislamiento & purificación , Antígeno B7-1/aislamiento & purificación , Antígeno B7-2 , Sitios de Unión , Antígeno CTLA-4 , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Disulfuros , Citometría de Flujo , Humanos , Riñón , Cinética , Sustancias Macromoleculares , Glicoproteínas de Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Trombina , Transfección

RESUMEN

T cell surface receptors CD28 and CTLA-4 are homologous members of the immunoglobulin superfamily (IgSF), each comprising a single V-like extracellular domain. CD28 and CTLA-4 bind to the B7-1 and B7-2 counter-receptors on antigen presenting cells (APCs), thereby triggering a costimulatory pathway important for optimal T cell activation in vitro and in vivo. Soluble forms of CD28 and CTLA-4 in which the V-like extracellular domains were fused to Ig constant domains (CD28Ig and CTLA4Ig), have been used to study their interactions with B7-1 and B7-2, with CTLA4Ig binding B7-1 more strongly than CD28Ig (approximately 20-fold higher avidity). We have now, by site-specific and homologue mutagenesis, identified regions in CTLA4Ig important for strong binding to B7-1. A hexapeptide motif (MYPPPY) in the complementarity determining region 3 (CDR3)-like region is fully conserved in all CD28 and CTLA-4 family members. Alanine scanning mutagenesis through the motif in CTLA4Ig and at selected residues in CD28Ig reduced or abolished binding to B7-1. Chimeric molecules HS4, HS4-A, and HS4-B were constructed in which CDR3-like regions of CTLA-4, COOH-terminally extended to include nonconserved residues, were grafted onto CD28Ig. These homologue mutants showed stronger binding to B7-1 than did CD28Ig. Grafting of the CDR1-like region of CTLA-4, which is not conserved in CD28 and is predicted to be spatially adjacent to CDR3, into HS4 and HS4-A, resulted in chimeric molecules (HS7 and HS8) which bound B7-1 even better. Inclusion of the CDR2-like domain of CTLA-4 into HS7 and HS8 did not further increase binding. Thus, the MYPPPY motifs of CTLA4Ig and CD28Ig are important for their binding to B7-1, but the increased strength of this binding by CTLA4Ig is mediated by nonconserved residues in the CDR1- and CDR3-analogous regions.

Asunto(s)

Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/química , Antígenos CD28/biosíntesis , Antígenos CD28/química , Secuencia Conservada , Inmunoconjugados , Linfocitos T/inmunología , Abatacept , Secuencia de Aminoácidos , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos CD , Sitios de Unión , Células CHO , Antígeno CTLA-4 , Línea Celular , Chlorocebus aethiops , Cricetinae , Humanos , Activación de Linfocitos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores de Antígenos de Linfocitos T/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Homología de Secuencia de Aminoácido , Transfección

RESUMEN

Costimulation by the CD28 ligand B7/BB1 plays an important role during T cell proliferation primarily by augmenting synthesis of IL-2 and other cytokines. Resting CD4+ T cells express CD28 but not CTLA-4 on their surface. Costimulation of T cells with ICAM-1 or VCAM-1 induced CTLA-4 expression and up-regulated CD28 expression. CD28 and CTLA-4 were independently distributed on the surface of activated T lymphoblasts. When co-immobilized with anti-TCR mAb both anti-CD28 and anti-CTLA-4 mAb augmented T cell proliferation. Although anti-CD28-mediated augmentation of T cell proliferation was stronger than that seen with anti-CTLA-4 mAb, together these two mAb caused supraadditive augmentation of T cell proliferation. The augmentation of the effects of anti-CD28 mAb by anti-CTLA-4 mAb was greater at low occupancy of CD28 by anti-CD28 mAb. Costimulation of CD28+ CTLA-4+ T cells with anti-CTLA-4 caused three- to fivefold increase in IL-2 production, whereas similar treatment with anti-CD28 caused > 40-fold increase. The costimulatory effect of B7 on primed T cells was partially inhibited by Fab anti-CD28 mAb. Anti-CTLA-4 mAb alone did not inhibit B7-induced response but caused modest increase in the inhibitory effect of anti-CD28 Fab. On integrin-mediated costimulation, Ag-specific CD4+ T cell lines also up-regulated their CTLA-4 expression, and proliferation of these cells was augmented by anti-CTLA-4 mAb. Unlike that of CD28, ligation of CTLA-4 alone failed to mobilize intracellular [Ca2+]. However, coligation of CTLA-4 and TCR induced stronger [Ca2+] response in Ag-specific T cell lines than that seen with TCR alone. These results suggest that integrin-costimulated T cells express CTLA-4 and can be costimulated via CTLA-4. Optimal development of various immune functions may involve combined costimulation via both CD28 and CTLA-4.

Asunto(s)

Antígenos de Diferenciación/análisis , Moléculas de Adhesión Celular/farmacología , Inmunoconjugados , Activación de Linfocitos , Linfocitos T/inmunología , Abatacept , Antígenos CD , Antígenos de Diferenciación/fisiología , Antígeno B7-1/farmacología , Antígenos CD28/análisis , Antígenos CD28/fisiología , Antígeno CTLA-4 , Calcio/metabolismo , Citocinas/biosíntesis , Humanos , Integrinas/fisiología , Molécula 1 de Adhesión Intercelular , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Linfocitos T/química , Molécula 1 de Adhesión Celular Vascular

RESUMEN

Integrin ligands intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) can efficiently costimulate proliferation of resting T cells but not that of Ag-specific T cells. In contrast, CD28 ligand B7 and CD2 ligand leukocyte function-associated Ag (LFA-3) can support IL-2 synthesis and proliferation of Ag-specific T cells more efficiently than that of resting T cells. The molecular basis for this differential costimulation of T cells is poorly understood. In this study, using mAb and soluble IgC gamma 1 chimeras of these adhesion molecules, we demonstrate that coligation of the TCR and CD11a/CD18 (LFA-1/beta 2 integrin) or CD29/CD49d (very late activation Ag-4/beta 1 integrin) using anti-TCR mAb and either ICAM-1 or VCAM-1 induces activation-dependent death of DRw6-specific CD4+ T cells. Similar coligation of the TCR with CD2 or CD28 using either mAb or ligands LFA-3 or B7 not only lacked the ability to induce death but also failed to reverse or inhibit integrin-facilitated death of DRw6-specific T cells. Each of these ligands augmented anti-TCR mAb-induced transcription of IL-2 and IL-4 genes. Exogenous addition of IL-2 and IL-4 did not reverse the integrin-supported T cell death. The death-promoting costimulatory effects of ICAM-1 and VCAM-1 were observed with Ag-specific chronically stimulated T cells but not with either resting T cells or those activated in short-term cultures. Treatment of T cells with cyclosporin A or a protein tyrosine kinase inhibitor herbimycin A inhibited ICAM-1 or VCAM-1-promoted activation-induced T cell death. The Ag-specific T cells that survived death-promoting effects of ICAM-1 or VCAM-1 proliferated efficiently upon restimulation with these ligands. Exposure of DRw6-specific T cells to DRw6+ B7+ ICAM-1+ LFA-3+ VCAM-1+ APC but not DR3+ B7+ ICAM-1+ LFA-3+ VCAM-1+ APC induced death of these T cells. This effect was blocked by pretreatment of T cells with mAb directed at CD18 or CD29 but not with those against CD2 or CD28. Taken together, these results suggest that TCR-directed engagement of integrins by their ligands ICAM-1 or VCAM-1 induces activation-dependent death of some perhaps more differentiated Ag-specific T cells and this may be an important homeostatic mechanism by which functional expression of Ag-specific T cells is regulated during an ongoing immune response.

Asunto(s)

Linfocitos T CD4-Positivos/fisiología , Moléculas de Adhesión Celular/fisiología , Muerte Celular , Activación de Linfocitos , Células Presentadoras de Antígenos/fisiología , Secuencia de Bases , Benzoquinonas , Línea Celular , Células Cultivadas , Ciclosporina/farmacología , Humanos , Molécula 1 de Adhesión Intercelular , Interleucinas/fisiología , Lactamas Macrocíclicas , Datos de Secuencia Molecular , Quinonas/farmacología , Receptores de Antígenos de Linfocitos T/fisiología , Rifabutina/análogos & derivados , Molécula 1 de Adhesión Celular Vascular

RESUMEN

Staphylococcal enterotoxin superantigens (SAg) bind class II major histocompatibility complex (MHC) molecules on antigen-presenting cells (APC) and upon cell-to-cell contact stimulate proliferation of T cells expressing appropriate V beta gene products. In addition, SAg can also deliver negative signals to Ag-specific T cells resulting in a state of unresponsiveness or a loss of viability. The present study examines the functional consequences of a direct interaction of SAg with alloAg-specific class II MHC+ CD4+ T cell lines (TCL). Our results demonstrate that SAg induce programmed death (apoptosis) in a majority of Ag-specific CD4+ T cells accompanied by genomic DNA fragmentation. SAg binding to Ag-specific TCL resulted in a rapid mobilization of intracellular free calcium ([Ca2+]i) and transcription of a number of cytokine genes including interleukin-2(IL-2), IL-4, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granzyme B indicating the activation of primed T cells. Both SAg-induced cytokine gene expression as well as subsequent death were significantly inhibited by a tyrosine kinase inhibitor herbimycin A and also by cyclosporin A. SAg-induced death of primed T cells was also inhibited by monoclonal antibodies (mAb) directed at the CD11a/CD18 molecule but not those reactive with other T cell surface molecules such as CD2, CD7, CD28, CD29 or CD49d. None of these mAb, including anti-CD11a/CD18, had any effect on SAg-induced expression of IL-2 and IL-4 genes or SAg-induced [Ca2+]i response. Addition of cytokines such as IL-1 alpha, IL-2, IL-4, IL-6, GM-CSF, IFN-gamma, tumor necrosis factor (TNF-alpha, or TNF-beta), or neutralizing Ab to these cytokines had no effect on SAg-induced death of Ag-specific TCL. The T cells which survived the death-inducing effects of SAg showed down-regulation of the CD3/T cell receptor and up-regulation of CD2 and HLA-DR expression, and upon re-exposure to the same SAg upregulated expression of mRNA for IL-2 and IFN-gamma. Presentation of SAg by B7+ ICAM-1+ LFA-3+ DR+ professional APC was also able to induce the death of Ag-specific TCL. Together these results suggest that the activation with SAg causes programmed death of Ag-specific TCL cells via a mechanism that requires late participation of the CD11a/CD18 molecule.

Asunto(s)

Antígenos Bacterianos/inmunología , Antígenos CD/inmunología , Apoptosis , Linfocitos T CD4-Positivos/inmunología , Citocinas/genética , Enterotoxinas/inmunología , Activación de Linfocitos , Antígeno-1 Asociado a Función de Linfocito/inmunología , Staphylococcus aureus/inmunología , Secuencia de Bases , Antígenos CD18 , Calcio/metabolismo , Daño del ADN , Regulación de la Expresión Génica , Antígenos HLA-DR/inmunología , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Transcripción Genética

RESUMEN

Optimal stimulation of CD4+ T cells in an immune response requires not only signals transduced via the CD3/TCR complex but also costimulatory signals delivered as a consequence of interactions between T-cell surface-associated costimulatory R and their counter-R on APC. CD28 plays a crucial role as a dominant costimulatory R during the induction of CD4+ T-cell proliferation by interacting with counter-R B7 on APC to sustain IL-2 production. The absence of CD28-mediated costimulation has been postulated to result in T-cell anergy or unresponsiveness. The costimulatory effects of CD28 can be generated with its natural counter-R B7 or mAb directed at CD28. Using soluble C gamma 1 chimeras of B7, ICAM-1, and VCAM-1, we have recently shown that B7 costimulates TCR-dependent proliferation of Ag-primed CD4+ T cells more efficiently than that of resting nonactivated CD4+ T cells. In contrast, proliferation of resting CD4+ T cells can be efficiently costimulated by either ICAM-1 or VCAM-1 via interactions with their R CD11a/CD18 (LFA-1/beta 2 integrin) and CD29/CD49d (VLA-4/beta 1 integrin), respectively. TCR-directed preactivation of resting CD4+ T cells with ICAM-1 can induce increased responsiveness to B7 costimulation. In this study, we show that prior TCR-directed activation of resting CD4+ T cells with VCAM-1 induced increased responsiveness to B7 costimulation. VCAM-1 also synergized with B7 to bring about supraoptimal proliferation of CD4+ T cells. In addition, costimulation of resting T cells with VCAM-1 significantly increased not only surface expression of CD28 but also CD28-mediated mobilization of intracellular free [Ca2+]i. Similar activation of T cells with fibronectin also resulted in increased B7 responsiveness, suggesting the involvement of VLA-4 molecule. VCAM-1 costimulation induced hyperresponsiveness to B7 costimulation in both CD18+ (normal) and CD18- (leukocyte adhesion deficient) T cells. Thus, VCAM-1 may play an important costimulatory role during the activation of resting T cells and, by augmenting responsiveness to B7, facilitate optimal development of immunological memory in addition to various regulatory and effector functions.

Asunto(s)

Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Superficie/inmunología , Linfocitos T CD4-Positivos/inmunología , Moléculas de Adhesión Celular/fisiología , Receptores Inmunológicos/inmunología , Anticuerpos Monoclonales , Antígeno B7-1 , Antígenos CD28 , Calcio/sangre , Fibronectinas/fisiología , Humanos , Técnicas In Vitro , Transducción de Señal , Molécula 1 de Adhesión Celular Vascular

RESUMEN

Staphylococcal enterotoxins, also known as superantigens (SAg), bind class II MHC molecules on APC and upon direct cell-to-cell contact stimulate proliferation of T cells expressing appropriate V beta gene products. The T cell surface molecule CD28 binds its costimulatory counter-receptor, B7 expressed on APC, and augments IL-2 production and T cell growth. Although the role of B7 costimulation during Ag-specific responses of T cells is established, its involvement during the activation of T cells with SAg has not been examined. Using a soluble Ig C gamma 1 chimera of CTLA-4, a second receptor for B7 and a homologue of CD28, this study examines the role of B7 expressed on APC during the induction of proliferation of CD4+ T cells upon stimulation with SAg (SAg/staphylococcal enterotoxins). CTLA-4lg, which has a higher avidity for B7 than CD28, had no effect on the synthesis of IL-2 as well as proliferative responses of CD4+ T cells induced by SAg presented on allogeneic EBV-transformed B cells, and IFN-gamma-activated endothelial cells. In contrast, T cell proliferation induced by alloAg presentation by the same APC was significantly inhibited by CTLA-4lg. mAb directed at the CD11a/CD18 molecule inhibited both SAg-induced and alloAg-induced proliferation of T cells. AlloAg-primed CD4+ T cells, which expressed both class II MHC and intercellular adhesion molecule-1 but not B7, presented SAg to and induced proliferation of both resting and SAg-primed T cells. These responses were inhibited by mAb directed at CD11a/CD18 but not by CTLA-4 Rg. These results suggest that SAg-induced responses differ from those induced by alloAg in that they are not obligatorily dependent on the costimulation by B7. In contrast, adhesive interaction between CD11a/CD18 on T cells and its counter-receptor on SAg-presenting cells is necessary and probably sufficient to support SAg-induced proliferation of T cells.

Asunto(s)

Antígenos Bacterianos/inmunología , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos de Superficie/fisiología , Enterotoxinas/inmunología , Activación de Linfocitos , Staphylococcus aureus/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígeno B7-1 , Antígenos CD11 , Antígenos CD18 , Antígenos CD28 , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Humanos , Interferón gamma/farmacología , Ratones , Ratones Endogámicos BALB C