RESUMEN
Immune deficiency disorders are a heterogeneous group of diseases of variable genetic aetiology. While the hallmark of immunodeficiency is susceptibility to infection, it is increasingly clear that autoimmunity is prevalent, suggestive of a more general immune dysregulation in some cases. With the increasing use of genetic technologies, the underlying causes of immune dysregulation are beginning to emerge. Here we provide a review of the heterozygous mutations found in the immune checkpoint protein CTLA-4, identified in cases of common variable immunodeficiency disorders (CVID) with accompanying autoimmunity. Study of these mutations provides insights into the biology of CTLA-4 as well as suggesting approaches for rational treatment of these patients.
Asunto(s)
Antígeno CTLA-4/genética , Inmunodeficiencia Variable Común/inmunología , Inmunoterapia/tendencias , Mutación/genética , Linfocitos T/inmunología , Animales , Autoinmunidad/genética , Antígeno CTLA-4/inmunología , Inmunodeficiencia Variable Común/genética , Humanos , Activación de Linfocitos , Transducción de SeñalRESUMEN
T cell activation is a key event in the adaptive immune system and vital in the generation of protective cellular and humoral immunity. Activation is required to generate CD4 effector T cell responses and provide help for B cell and cytotoxic T cell responses. While defective T responses to foreign antigen result in infectious pathology, over-reactive T cell responses against self-antigens result in autoimmunity and, in a transplantation setting, tissue rejection. Understanding how T cell activation is normally regulated is critical to therapeutic intervention and the CD28/CTLA-4 (CD152) pathway represents the initial activation checkpoint in molecular terms. In particular, while the CTLA-4 pathway is well established as an essential regulator of self-reactivity, its mechanism of action is still uncertain. Such mechanistic issues are important given its central position in T cell activation and the increasing number of therapeutic modalities aimed at manipulating the CD28/CTLA-4 pathway. Here, we provide an updated view of CTLA-4 biology, reviewing the established features of the system and highlighting its interplay with CD28. We then discuss how recent progress in our understanding of this pathway affects our interpretations following intervention.
Asunto(s)
Antígenos CD28/inmunología , Antígeno CTLA-4/inmunología , Formación de Anticuerpos , Autoanticuerpos/inmunología , Humanos , Ligandos , Activación de Linfocitos , Linfocitos T/inmunologíaRESUMEN
Educational immune tolerance to self-antigens is induced primarily in the thymus where tissue-restricted antigens (TRAs) are presented to T lymphocytes by cells of the thymic stroma - a process known as central tolerance. The expression of these TRAs is controlled in part by a transcription factor encoded by the autoimmune regulatory (Aire) gene. Patients with a mutation of this gene develop a condition known as autoimmune-polyendocrinopathy-candidiasis-ectodermal-dystrophy (APECED), characterized by autoimmune destruction of endocrine organs, fungal infection and dental abnormalities. There is now evidence for TRA expression and for mechanisms of functional tolerance outside the thymus. This has led to a number of studies examining Aire expression and function at these extra-thymic sites. These investigations have been conducted across different animal models using different techniques and have often shown discrepant results. Here we review the studies of extra thymic Aire and discuss the evidence for its expression and function in both human and murine systems.
Asunto(s)
Estructuras Animales/metabolismo , Tejido Linfoide/metabolismo , Factores de Transcripción/fisiología , Estructuras Animales/inmunología , Animales , Humanos , Tejido Linfoide/inmunología , Ratones , Factores de Transcripción/química , Proteína AIRERESUMEN
The exon 2 nucleotide sequences of HLA-DQwl-associated and DQw3-associated HLA-DR"Br" alleles were determined from genomic DNA amplified by the Taq polymerase chain reaction technique. Both alleles reveal identical exon 2 nucleotide sequences. Comparison with other DR alleles suggests that DR"Br" may have originated from DR1 by gene conversion with DR4-Dw10, DR5, or DRw6-Dw18 third hypervariable region sequences.
Asunto(s)
Exones , Conversión Génica , Antígenos HLA-DR/genética , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN , ADN Polimerasa Dirigida por ADN , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Felty's syndrome (FS) is a rare complication of rheumatoid arthritis (RA) previously shown to be strongly associated with HLA-DR4 and less significantly with HLA-DQw7. To map more precisely the HLA locus responsible for susceptibility to FS, we have examined HLA-DR4 and DQ beta-chain polymorphisms in FS patients and controls using restriction fragment length polymorphism analysis and polymerase chain reaction amplification in conjunction with oligonucleotide probing. The increased frequency of DR4 in FS (93% vs. 32% controls) was due almost entirely to enrichment for the Dw4 subtype (88% vs. 20% controls) with a secondary increase of the Dw14 subtype. Dw10 and Dw13 subtypes of DR4 were absent from the patient group. Increase in DQw7 frequency among DR4 FS patients could be accounted for by linkage disequilibrium between Dw4 and DQw7 alleles. Whereas susceptibility to RA is strongly associated with a conserved HLA-DR beta epitope associated with several DRB1 alleles, it is primarily the Dw4 allele which is associated with progression to Felty's syndrome. The finding that amino acid sequence variation at the DR4B1 locus rather than DQB1 is associated with development of FS will have important implications for the development of novel immunotherapies which are major histocompatibility complex allele-dependent.
Asunto(s)
Síndrome de Felty/genética , Antígenos HLA-DQ/genética , Antígeno HLA-DR4/genética , Aminoácidos/genética , Secuencia de Bases , Southern Blotting , Mapeo Cromosómico , Síndrome de Felty/inmunología , Haplotipos , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
HLA DQ alpha and DQ beta cDNA probes were used to study TaqI generated restriction fragment length polymorphisms (RFLPs) in DR4-positive patients with Felty's syndrome (FS), seropositive rheumatoid arthritis (RA), and in HLA-DR4 positive controls. The results of this analysis revealed two DQ beta RFLP patterns (DQ beta 3a and DQ beta 3b) associated with DR4, of which DQ beta 3b was found at significantly higher frequency in patients with FS (73%) or with RA (52%) than in DR4 controls (29%). Hind III generated RFLPs provide evidence that DQ beta 3b is in strong linkage disequilibrium with the gene encoding the serologically recognized epitope TA10. Results obtained using a DQ alpha chain probe revealed polymorphic differences between DQ alpha chain genes associated with different DR types, thereby providing a possible explanation for the lack of association between RA and other DR haplotypes in linkage disequilibrium with TA10. We conclude that both DQ alpha and DQ beta genes may be important in determining HLA-linked susceptibility to severe forms of RA.
Asunto(s)
Artritis Reumatoide/genética , Desoxirribonucleasas de Localización Especificada Tipo II , Síndrome de Felty/genética , Antígenos HLA-D/genética , Antígenos HLA-DQ/genética , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , Artritis Reumatoide/complicaciones , Artritis Reumatoide/inmunología , ADN/genética , Enzimas de Restricción del ADN , ADN Recombinante , Epítopos/genética , Síndrome de Felty/etiología , Síndrome de Felty/inmunología , Ligamiento Genético , Marcadores Genéticos , HumanosRESUMEN
Southern blot analysis with DR beta and DQ beta cDNA probes was used to compare genomic DNA from Felty's syndrome patients with HLA-DR-matched normal controls. We describe two restriction fragment length polymorphisms putatively associated with Felty's syndrome.
Asunto(s)
Síndrome de Felty/genética , Marcadores Genéticos , Antígenos HLA-D/genética , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , ADN/análisis , ADN/genética , Síndrome de Felty/inmunología , HumanosRESUMEN
The activation of T cells is now thought to require at least two distinct signals. One signal is delivered through the interaction of the antigen-specific T cell receptor with major histocompatibility complex (MHC) molecules and peptide, while the other is received from interactions with less precisely defined accessory or costimulatory molecules. In the absence of this second costimulatory signal, some T cells subsequently become unresponsive to antigenic stimulation. One of the major candidates for providing such a second signal to T cells is the molecule B7 interacting with the T cell glycoprotein CD28. In the present study we have investigated whether B7 is expressed on human T cell lines and clones, since these cells have the capacity to present antigen to each other by expressing MHC class II molecules. Our results demonstrate that B7 can be detected on T cell clones and on repeatedly activated but not freshly isolated peripheral blood T cells. The expression of B7 is dependent on the state of activation of the cells, being maximally expressed shortly after restimulation and becoming undetectable as the cells quiesce. Together, these results suggest that B7 expression may be of importance to T cells, perhaps in the avoidance of anergy.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos de Superficie/análisis , Activación de Linfocitos , Linfocitos T/inmunología , Antígeno B7-1 , Antígenos CD28 , Línea Celular , HumanosRESUMEN
The invariant chain is a membrane protein associated with the major histocompatibility complex class II antigens both intra- and extracellularly. The extracellular portion of the human invariant chain (Ii) was expressed in Escherichia coli as a fusion protein with a polyhistidine tail and purified by metal affinity chromatography. The recombinant Ii was used as a ligand to probe binding to the cell surface of Chinese hamster ovary cells stably transfected with human class II alpha and beta genes of the DR4 isotype. We show that recombinant Ii inhibits peptide loading on class II polypeptides and also the converse; the presence of peptide in the antigen groove prevents binding of fluorescein-conjugated Ii. Moreover, blocking of Ii binding by peptide did not require a transition of the class II dimers to an SDS-stable state. A monoclonal antibody, L243, known to bind to (or close to) the peptide pocket of the class II molecule likewise blocked Ii-fluorescein binding. Further, we investigated whether or not the Ii, a variety of bacterial superantigens or the CD4 molecule, have overlapping binding sites on the class II heterodimer. Of the class II ligands tested, reduced binding was detected for the Staphylococcus superantigen type SEB on cells precincubated with soluble Ii while the binding of the other ligands was either unchanged or marginally changed. These data clarify by a direct biochemical approach the binding characteristics of Ii in comparison with other class II ligands.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B , Antígenos de Histocompatibilidad Clase II/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Sitios de Unión , Células CHO , Línea Celular , Cricetinae , Proteína gp120 de Envoltorio del VIH/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Superantígenos/metabolismoRESUMEN
The requirement for co-stimulation in T-cell activation has become firmly established, whilst the precise identity of the molecules involved remains uncertain. Some of the major co-stimulatory molecules include ICAM-1, LFA-3 and B7. We have investigated the abilities of both LFA-3 and B7 to co-stimulate T-cell proliferation under a number of conditions using transfected Chinese hamster ovary cells. Using anti-CD3 antibodies we observed that B7 but not LFA-3 transfectants were capable of co-stimulating proliferation in purified peripheral blood T cells. In addition, both LFA-3 and B7 could induce proliferation in response to phytohaemagglutinin (PHA) and we obtained additive effects using both B7 and LFA-3 together. Using the superantigen staphylococcal enterotoxin B (SEB), we observed that presentation to purified T cells required the presence of class II-positive transfectants and that sensitivity to antigen was increased approximately 100-fold by the co-transfection of either B7 or LFA-3. However, when co-stimulatory molecules were provided by cells separate from those engaging the T-cell receptor (TcR), only B7 was capable of enhancing proliferation. Kinetic studies which investigated the time dependence for co-stimulation revealed that T cells responding to anti-CD3 antibodies required the B7 co-stimulation within the first few hours, for proliferation to be effective. Our data differentiate between the co-stimulatory abilities of B7 and LFA-3 and support the concept of a pivotal role for B7 in T-cell proliferation.
Asunto(s)
Antígeno B7-1/inmunología , Antígenos CD28/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos CD2 , Complejo CD3/inmunología , Células Cultivadas , Humanos , Antígeno-1 Asociado a Función de Linfocito/inmunología , Fitohemaglutininas/inmunología , Receptores Inmunológicos/inmunología , Factores de Tiempo , TransfecciónRESUMEN
The CD28 cytoplasmic tail contains several potential phosphorylation sites for the serine/threonine kinase protein kinase C (PKC) and/or proline-directed serine/threonine kinases, such as extracellular signal-regulated kinases. We demonstrate that ligation of CD28 by B7.1 results in strong serine/threonine phosphorylation of CD28. It is unlikely that ligation-stimulated phosphorylation of CD28 is mediated via activation of PKC, since it was not prevented by pre-treatment of Jurkat cells with inhibitors of PKC, and it was not mimicked by treatment with PKC activators such as PMA. Nevertheless, despite for lack of detectable effects of PMA treatment on CD28 phosphorylation, PMA did partially inhibit the association of CD28 with the putative signalling molecule phosphatidylinositol 3-kinase (PI 3-kinase) and the subsequent accumulation of PtdIns(3,4,5)P3. PI 3-kinase exhibits dual specificity as both a lipid kinase and a protein serine kinase, and site-specific mutagenesis of the Tyr173 residue in the CD28 cytoplasmic tail, which abolishes CD28 coupling to PI 3-kinase [Pages, Ragueneau, Rottapel, Truneh, Nunes, Imbert and Olive (1994) Nature (London) 369, 327-329], also prevents ligation-stimulated phosphorylation of CD28. However, the two PI 3-kinase inhibitors wortmannin and LY294002 had no effect on phosphorylation of CD28 after ligation by B7.1. This study therefore demonstrates that (1) a CD28-activated serine/threonine kinase distinct from both PKC and PI 3-kinase mediates ligation-stimulated CD28 phosphorylation, and (2) the PMA-stimulated down-regulation of the coupling of CD28 to PI 3-kinase is not due to PMA-stimulated phosphorylation of CD28.
Asunto(s)
Antígenos CD28/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Proteína Quinasa C/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/fisiología , Antígeno B7-1/metabolismo , Antígeno B7-1/fisiología , Antígenos CD28/genética , Antígenos CD28/fisiología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Células Jurkat , Ligandos , Mutagénesis Sitio-Dirigida , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Fosfotirosina/genética , Proteína Quinasa C/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
CD4+ T-lymphocytes require two signals to become activated--antigen receptor (TcR) occupancy and an antigen-presenting cell (APC)-derived costimulus. The latter may be provided by B7.1 (CD80) or B7.2 (CD86) on APC interacting with CD28 on T-cells. We have studied the expression of these costimulatory molecules in rheumatoid and osteoarthritic synovial membrane. Very few B7.1-positive cells were seen in synovial tissue from either established or early rheumatoid disease, or in rheumatoid arthritis (RA) or osteoarthritis (OA) synovia at arthroplasty. In contrast, B7.2 was readily detected in rheumatoid synovia, predominantly in the lining layer, in a pattern of expression that corresponded to the presence of CD68-positive macrophages. Only occasional B7.2-positive cells were seen in OA synovia. The presence of B7.2 but the relative lack of expression of B7.1 may be partly responsible for the observations of 'frustrated' T-cell activation or T-cell hyporesponsiveness in the rheumatoid synovium.
Asunto(s)
Antígenos CD/metabolismo , Artritis Reumatoide/inmunología , Antígeno B7-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Membrana Sinovial/inmunología , Antígeno B7-2 , HumanosRESUMEN
T lymphocyte activation requires at least two signals, one via the antigen-specific T cell receptor and a second via the surface molecule CD28 which provides signals critical to interleukin-2 (IL-2) production and T cell proliferation. We have previously shown (Ward S. G., Westwick, J., Hall N. and Sansom D. M. Eur. J. Immunol. 1993. 23: 2572) that CD28 stimulates phosphoinositide (PI) 3-kinase activity, indicating that D-3 phosphoinositides may act as mediators of CD28-induced T cell costimulation. Here, we report that immunoprecipitation of CD28 molecules from Jurkat cells stimulated with the CD28-ligand B7, results in a ligand-dependent association of CD28 with PI 3-kinase. This association correlates with the appearance of PI 3-kinase enzymatic activity in CD28 immunoprecipitates and the formation of D-3 phosphoinositides. Consistent with the hypothesis that D-3 phosphoinositides are important mediators of CD28 signaling, treatment of T cells with the PI 3-kinase inhibitor wortmannin, inhibited both T cell proliferation and production of IL-2, but not the response of T cells to exogenous IL-2. Hence, abrogation of PI 3-kinase activity by wortmannin, appears sufficient to disrupt the costimulatory pathway utilized by CD28, indicating a central role for this enzyme in the CD28 signaling pathway.
Asunto(s)
Androstadienos/farmacología , Antígenos CD28/fisiología , Activación de Linfocitos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Linfocitos T/fisiología , Calcio/metabolismo , Células Cultivadas , Humanos , Interleucina-2/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas , Fosfatos de Fosfatidilinositol/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , WortmaninaRESUMEN
Ag recognition is an essential component for an effective T cell response. However, T cell activation is also subject to additional regulation by accessory molecules. CD28 provides essential costimulatory signals that allow T cells to proliferate, whereas molecules such as CTLA-4 and CD95 (Fas) appear to be negative regulators. Currently, which outcome predominates under conditions of antigenic challenge is poorly understood. In particular it has been suggested that one consequence of antigenic activation of T cells is the up-regulation of both CD95 and CD95 ligand, thereby exposing activated T cells to apoptotic death. We have investigated this possibility in normal human peripheral blood T cells triggered by the superantigen SEB either in the presence of endogenous APCs or transfectants expressing DR4 and CD80. In either case, we find that such activation does not expose the majority of T cells to anti-CD95-induced apoptosis as detected by annexin V externalization and DNA fragmentation. Furthermore, by phenotypically identifying, by flow cytometry, those cells that received both antigenic and costimulatory signals from those cells that did not, we observed that CD95-induced apoptosis was not seen in activated T cells receiving Ag and costimulatory signals via CD28. However, while not all T cells were stimulated by superantigen, CD95 expression was found to be homogeneously up-regulated, suggesting a mechanism whereby bystander cells might be made susceptible to CD95-induced death. We conclude that antigenic activation of T cells via the TCR and CD28 engagement provides protection from CD95-induced apoptosis.
Asunto(s)
Apoptosis/inmunología , Antígenos CD28/farmacología , Enterotoxinas/inmunología , Activación de Linfocitos , Superantígenos/farmacología , Receptor fas/fisiología , Animales , Antígeno B7-1/fisiología , Antígenos CD28/biosíntesis , Células CHO , Células Cultivadas , Cricetinae , Enterotoxinas/farmacología , Antígeno HLA-DR4/fisiología , Humanos , Inmunidad Innata , Staphylococcus aureus/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
The co-stimulatory role of B7/CD28 interactions is important in promoting T cell activation. Very little is known about the intracellular events that follow CD28 engagement although recent evidence has implicated coupling of CD28 to a protein tyrosine kinase signal transduction pathway. In this study we have investigated the putative role of D-3 phosphoinositides as mediators of CD28 receptor signaling, since phosphoinositide (PI) 3-kinase, the enzyme responsible for D-3 phosphoinositide formation, is a known substrate for protein tyrosine kinases associated with certain T cell surface receptors such as CD4 and interleukin-2 receptor. The lipid products of PI 3-kinase activity have been suggested to play a role in mitogenic signaling and growth regulation in other cells. Chinese hamster ovary cells (CHO) previously transfected with B7 cDNA, induced time-dependent elevation above basal levels of phosphatidylinositol(3,4)-bisphosphate (PtdIns(3,4)P2) and PtdIns(3,4,5)P3, while parental CHO cells that did not express B7 had no effect on these lipids. Moreover, the elevation of these same lipids by CD3 ligation was potentiated in an additive manner by CHO-B7+ but not by CHO-B7- cells. CHO-B7+ and CHO-B7- cells did not activate phospholipase C as evidenced by their inability to modulate basal or CD3-induced changes in the levels of phosphatidic acid or D-4 and D-5 phosphoinositides. These data imply that PI 3-kinase but not phospholipase C, may be an important signal transduction molecule with respect to CD28-mediated co-stimulation and T cell activation following ligation by B7.
Asunto(s)
Antígenos CD28/metabolismo , Fosfatidilinositoles/biosíntesis , Receptores Inmunológicos/metabolismo , Linfocitos T/inmunología , Animales , Complejo CD3/metabolismo , Células CHO/inmunología , Células CHO/metabolismo , Línea Celular , Membrana Celular/inmunología , Membrana Celular/metabolismo , Cricetinae , Humanos , Activación de Linfocitos , Ácidos Fosfatidicos/metabolismo , Fosfatos de Fosfatidilinositol/biosíntesis , Linfocitos T/metabolismoRESUMEN
The intracellular signaling pathways activated upon ligation of the co-stimulatory receptor CD28 remain relatively ill-defined, although CD28 ligation does result in the strong association with, and activation of, phosphatidylinositol (PI) 3-kinase. The downstream effector targets of the CD28-activated PI 3-kinase-dependent signaling pathway remain poorly defined, but recent evidence from other systems has shown that Akt/protein kinase B (PKB) is a major target of PI 3-kinase and have indicated that a major function of PKB is the regulation of cell survival events. Given the strong coupling of CD28 to PI 3-kinase and the known protective effects of both CD28 and PI 3-kinase against apoptosis in different cell models, we investigated the effects of CD28 on PKB activation. We demonstrate that ligation of CD28 by either anti-CD28 monoclonal antibodies or the natural ligand B7.1, results in the marked activation of PKB in both the leukemic T cell line Jurkat and freshly isolated human peripheral blood-derived normal T lymphocytes. Our data suggest therefore, that PKB may be an important intracellular signal involved in CD28 signal transduction and demonstrate CD28 coupling to downstream elements of a signaling cascade known to promote cell survival.
Asunto(s)
Antígeno B7-1/inmunología , Antígenos CD28/inmunología , Activación de Linfocitos/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal/fisiología , Linfocitos T/inmunología , Androstadienos/farmacología , Anticuerpos Monoclonales/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Leucemia-Linfoma de Células T del Adulto/patología , Proteínas de Neoplasias/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales Cultivadas , WortmaninaRESUMEN
The CD7 40-kDa glycoprotein is present on a major subset of human T cells and in the presence of phorbol esters mediates an accessory pathway of T cell activation. Hitherto, the intracellular events elicited by CD7 have been ill-defined. This report demonstrates that cross-linking of CD7 results in the formation of phosphatidic acid in the absence of phosphatidylinositol-4,5-bisphosphate metabolism and also the formation of D-3 phosphoinositides lipids which have been postulated to act as intracellular regulatory molecules. The magnitude of D-3 phosphoinositide formation was similar to that induced by CD3. Both the CD7- and CD3-induced elevation of phosphatidylinositol 3,4,5-trisphosphate approximately 5-10 fold less than that elicited by ligation of the costimulatory molecule CD28 by its counter receptor CD80. The formation of D-3 phosphoinositides following ligation of CD7 coincided with the co-association of CD7 with phosphoinositide 3-kinase, the enzyme which mediates the formation of D-3 phosphoinositide lipids. In contrast, ligation of another reported T cell accessory molecule CD5, failed to elicit formation of D-3 phosphoinositides, implying that phosphoinositide 3-kinase is not coupled to all T cell molecules with accessory functions. Since D-3 phosphoinositides have been suggested to play a pivotal role in T cell costimulatory signals induced by CD28, the results presented in this study suggest that CD7 may also influence T cell activation via this pathway.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/fisiología , Fosfatos de Fosfatidilinositol/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Linfocitos T/metabolismo , Androstadienos/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD/análisis , Antígenos CD7 , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos CD28/fisiología , Antígenos CD5 , Células CHO , Cricetinae , Humanos , Activación de Linfocitos , Ácidos Fosfatidicos/análisis , Fosfatidilinositol 3-Quinasas , Fosfatidilinositoles/análisis , WortmaninaRESUMEN
CD28 and CTLA-4 are related receptors that differentially regulate T cell activation. Despite the fact that they bind the same ligands, CD28 is a classical costimulator enhancing proliferation whereas CTLA-4 appears to perform negative regulatory functions. In this study, we have utilized the natural ligand for CD28 and CTLA-4 (CD80) to determine under what circumstances positive and negative effects are operative. We show here that the stimulation of purified human T cells with phorbol ester and ionomycin is inhibited in the presence of Chinese hamster ovary (CHO) cells expressing CD80. This inhibition is reversed by blocking with both anti-CD80 or Fab fragments of anti-CTLA-4 but also requires CD28 engagement. Furthermore, we show that the inhibitory function of CD80 requires elevated intracellular calcium since inhibition was observed only in the presence of ionomycin. In the absence of intracellular calcium elevation, CTLA-4 was not expressed at the cell surface, and CD80 acted positively as a costimulator of T cells, via CD28. These results demonstrate that the natural ligand CD80 can either costimulate or inhibit T cell responses depending on the conditions of T cell stimulation.
Asunto(s)
Antígenos de Diferenciación/inmunología , Antígeno B7-1/inmunología , Antígenos CD28/inmunología , Inmunoconjugados , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Abatacept , Animales , Antígenos CD , Células CHO , Antígeno CTLA-4 , Cricetinae , Humanos , Ligandos , Transducción de Señal/inmunologíaRESUMEN
Following antigen engagement of the T-cell receptor (TCR), T-cell survival is largely dictated by the provision of additional signals, such as those from costimulatory receptors and cytokine receptors. Whilst CD28-mediated signalling is increasingly associated with survival, ligation of alternative T-cell antigens, such as Fas (CD95), can trigger apoptosis. The T-cell response following antigen engagement may therefore be influenced by the relative expression levels of these coreceptors as well as by the availability of their ligands (CD80/86 and Fas-L). In this study we demonstrate functional interplay between the death receptor Fas and the costimulatory receptor CD28 in human T cells. In Jurkat T cells, we show that Fas signalling leads to rapid and selective CD28 down-regulation, and that this is associated with a specific decrease in mRNA for CD28, indicating that mechanisms exist which target CD28 at a transcriptional level. Moreover, cells that down-regulate CD28 also undergo apoptosis. Studies on activated human peripheral blood T cells demonstrate that cells expressing high levels of CD28 are resistant to Fas-mediated apoptosis whereas cells expressing low levels are more susceptible, implicating CD28 in the provision of anti-apoptotic signals. Consistent with this hypothesis, direct ligation of CD28 using B7 transfectants concomitant with anti-Fas challenge protects from apoptosis. Since antigen-presenting cells may express Fas-L under certain circumstances, the maintenance of T-cell CD28 expression may be crucial for the prevention of Fas-mediated apoptosis during the course of antigen engagement.
Asunto(s)
Apoptosis/inmunología , Antígenos CD28/inmunología , Regulación hacia Abajo/inmunología , Linfocitos T/inmunología , Receptor fas/inmunología , Apoptosis/efectos de los fármacos , Antígenos CD28/genética , Técnicas de Cultivo de Célula , Regulación hacia Abajo/efectos de los fármacos , Etopósido/farmacología , Humanos , Proteína Quinasa C/fisiología , ARN Mensajero/genética , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Although the role of CD28 in T cell costimulation is firmly established, the mechanisms by which it exerts its costimulatory actions are less clear. In many circumstances it is difficult to distinguish the effects of CD28 from subsequent actions of cytokines, such as IL-2, on T cell proliferation. Here, we report a model of CD28 costimulation using PMA plus the natural ligand CD80 that resulted in very limited stimulation of IL-2, as evidenced by both cytokine production and IL-2 promoter stimulation. Promoter assays revealed CD28-dependent effects on both NF-kappaB and AP-1, but not on NF-AT or the intact IL-2 promoter. In addition, T cell proliferation was completely resistant to the actions of the immunosuppressant cyclosporin A (CsA). Moreover T cell proliferation was unaffected by the addition of blocking Abs to both IL-2 and the IL-2 receptor, demonstrating that this form of costimulation by CD28 was independent of IL-2. We also investigated the effects of stimulating T cell blasts with CD80 alone and found that there was a limited requirement for IL-2 in this system. We conclude that CD28 costimulation can cause substantial T cell proliferation in the absence of IL-2, which is driven by a soluble factor independent of NF-AT transactivation.