RESUMEN

Thymic stromal lymphopoietin (TSLP) is a mucosal tissue-associated cytokine that has been widely studied in the context of T helper type 2 (Th2)-driven inflammatory disorders. Although TSLP is also produced upon viral infection in vitro, the role of TSLP in antiviral immunity is unknown. In this study we report a novel role for TSLP in promoting viral clearance and virus-specific CD8+ T-cell responses during influenza A infection. Comparing the immune responses of wild-type and TSLP receptor (TSLPR)-deficient mice, we show that TSLP was required for the expansion and activation of virus-specific effector CD8+ T cells in the lung, but not the lymph node. The mechanism involved TSLPR signaling on newly recruited CD11b+ inflammatory dendritic cells (DCs) that acted to enhance interleukin-15 production and expression of the costimulatory molecule CD70. Taken together, these data highlight the pleiotropic activities of TSLP and provide evidence for its beneficial role in antiviral immunity.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Citocinas/metabolismo , Células Dendríticas/inmunología , Inflamación/inmunología , Inflamación/metabolismo , Virus de la Influenza A/inmunología , Animales , Presentación de Antígeno , Antígenos/inmunología , Antígenos/metabolismo , Supervivencia Celular , Pulmón/inmunología , Pulmón/metabolismo , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Ratones , Modelos Inmunológicos , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Linfopoyetina del Estroma Tímico

RESUMEN

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

Asunto(s)

Antígenos CD8/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Microdominios de Membrana/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Secuencia de Aminoácidos , Animales , Complejo CD3/metabolismo , Células CHO , Calcio/metabolismo , Cricetinae , Datos de Secuencia Molecular , Fosforilación

RESUMEN

We have determined high-resolution crystal structures of the complexes of HLA-A2 molecules with two modified immunodominant peptides from the melanoma tumor-associated protein Melan-A/Melanoma Ag recognized by T cells-1. The two peptides, a decamer and nonamer with overlapping sequences (ELAGIGILTV and ALGIGILTV), are modified in the second residue to increase their affinity for HLA-A2. The modified decamer is more immunogenic than the natural peptide and a candidate for peptide-based melanoma immunotherapy. The crystal structures at 1.8 and 2.15 A resolution define the differences in binding modes of the modified peptides, including different clusters of water molecules that appear to stabilize the peptide-HLA interaction. The structures suggest both how the wild-type peptides would bind and how three categories of cytotoxic T lymphocytes with differing fine specificity might recognize the two peptides.

Asunto(s)

Antígenos de Neoplasias/química , Antígeno HLA-A2/química , Proteínas de Neoplasias/química , Secuencia de Aminoácidos , Antígenos de Neoplasias/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Antígeno HLA-A2/metabolismo , Humanos , Antígeno MART-1 , Sustancias Macromoleculares , Melanoma/inmunología , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Docilidad , Unión Proteica , Conformación Proteica , Linfocitos T Citotóxicos/inmunología , Agua

RESUMEN

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56lck. They are also inhibited by methyl-beta-cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR.

Asunto(s)

Antígenos CD8/metabolismo , Activación de Linfocitos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Microdominios de Membrana/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , beta-Ciclodextrinas , Animales , Antígenos de Protozoos/inmunología , Complejo CD3/metabolismo , Calcio/metabolismo , Células Cultivadas , Reactivos de Enlaces Cruzados , Ciclodextrinas/farmacología , Detergentes , Dimerización , Activación Enzimática , Citometría de Flujo , Humanos , Activación de Linfocitos/efectos de los fármacos , Sustancias Macromoleculares , Microdominios de Membrana/enzimología , Ácido Palmítico/metabolismo , Plasmodium berghei/inmunología , Transporte de Proteínas , Transducción de Señal , Solubilidad , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/metabolismo

Asunto(s)

Activación de Linfocitos/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Fosfatidiletanolaminas/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Animales , Glicerofosfolípidos/farmacología , Humanos , Microdominios de Membrana/fisiología , Ratones , Transducción de Señal , Linfocitos T Citotóxicos/fisiología , Células Tumorales Cultivadas

RESUMEN

Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide-major histocompatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen.These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.

Asunto(s)

Antígenos de Histocompatibilidad Clase I/inmunología , Activación de Linfocitos , Proteínas de la Nucleocápside , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos/genética , Antígenos/inmunología , Citocinas/biosíntesis , Semivida , Antígenos de Histocompatibilidad Clase I/genética , Hibridomas , Cinética , Mutagénesis Sitio-Dirigida , Nucleocápside/genética , Nucleocápside/inmunología , Péptidos/genética , Receptores de Antígenos de Linfocitos T/genética

RESUMEN

To combine the advantage of both the tumor targeting capacity of high affinity monoclonal antibodies (mAbs) and the potent killing properties of cytotoxic T lymphocytes (CTL), we investigated the activity of conjugates made by coupling single Fab' fragments, from mAbs specific for tumor cell surface antigens, to monomeric HLA-A2 complexes containing the immunodominant influenza-matrix peptide 58-66. In solution, the monovalent 95 kDa Fab-HLA-A2/Flu conjugates did not activate influenza-specific CTL. However, when targeted to tumor cells expressing the relevant tumor-associated antigen, the conjugates induced CTL activation and efficient tumor cell lysis, as a result of MHC/peptide surface oligomerization. The highly specific and sensitive in vitro cytotoxicity results presented suggest that injection of Fab-MHC/peptide conjugates could represent a new form of immunotherapy, bridging antibody and T lymphocyte attack on cancer cells.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Antígeno HLA-A2/inmunología , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/inmunología , Anticuerpos Monoclonales/farmacología , Calcio/metabolismo , Técnicas de Cocultivo , Citotoxicidad Inmunológica/efectos de los fármacos , Citometría de Flujo/métodos , Antígeno HLA-A2/genética , Humanos , Inmunoconjugados/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Concentración 50 Inhibidora , Neoplasias/inmunología , Neoplasias/patología , Proteínas Recombinantes/inmunología , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/inmunología

RESUMEN

To demonstrate that antibody-guided targeting of antigenic MHC class I-peptide tetramer on tumor cells can render them susceptible to lysis by relevant cytotoxic T lymphocytes (CTL), biotinylated HLA-A*0201/Flu matrix peptide complexes were tetramerized on streptavidin molecules previously coupled to Fab' fragments from monoclonal antibodies (mAb) specific for cell surface markers such as carcinoembryonic antigen (CEA), ErbB-2 or CD20. Flow cytometry analysis showed that coating of the HLA-A2-peptide complexes on the four HLA-A2-negative human cancer lines tested (including a CEA-positive colon carcinoma, an ErbB-2(+) breast carcinoma and two CD20(+) B lymphomas) was entirely dependent upon the specificity of the conjugated antibody fragments. More importantly, HLA-A2-restricted Flu matrix peptide-specific CTL were then found to lyse specifically and efficiently the MHC-coated target cells. These results open the way to the development of new immunotherapy strategies based on antibody targeting of MHC class I-peptide complexes.

Asunto(s)

Anticuerpos Antineoplásicos/inmunología , Citotoxicidad Inmunológica , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoconjugados , Inmunoterapia , Linfocitos T/inmunología , Antígenos de Neoplasias/inmunología , Humanos , Células Tumorales Cultivadas

RESUMEN

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

Asunto(s)

Antígenos CD8/metabolismo , Ácido Palmítico/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Ácido 4-Aminobenzoico/inmunología , Secuencia de Aminoácidos , Animales , Complejo CD3/metabolismo , Antígenos CD8/inmunología , Antígenos CD8/fisiología , Detergentes , Activación Enzimática/inmunología , Antígenos H-2/fisiología , Hibridomas , Activación de Linfocitos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fosforilación , Plasmodium berghei/inmunología , Estructura Terciaria de Proteína , Proteínas Protozoarias/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Linfocitos T/metabolismo

RESUMEN

The methodology for generating a homology model of the T1 TCR-PbCS-K(d) class I major histocompatibility complex (MHC) class I complex is presented. The resulting model provides a qualitative explanation of the effect of over 50 different mutations in the region of the complementarity determining region (CDR) loops of the T cell receptor (TCR), the peptide and the MHC's alpha(1)/alpha(2) helices. The peptide is modified by an azido benzoic acid photoreactive group, which is part of the epitope recognized by the TCR. The construction of the model makes use of closely related homologs (the A6 TCR-Tax-HLA A2 complex, the 2C TCR, the 14.3.d TCR Vbeta chain, the 1934.4 TCR Valpha chain, and the H-2 K(b)-ovalbumine peptide), ab initio sampling of CDR loops conformations and experimental data to select from the set of possibilities. The model shows a complex arrangement of the CDR3alpha, CDR1beta, CDR2beta and CDR3beta loops that leads to the highly specific recognition of the photoreactive group. The protocol can be applied systematically to a series of related sequences, permitting the analysis at the structural level of the large TCR repertoire specific for a given peptide-MHC complex.

Asunto(s)

Simulación por Computador , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Modelos Moleculares , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Algoritmos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Sitios de Unión , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Enlace de Hidrógeno , Datos de Secuencia Molecular , Mutación/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Reproducibilidad de los Resultados , Alineación de Secuencia , Programas Informáticos , Electricidad Estática , Especificidad por Sustrato , Termodinámica

RESUMEN

To elucidate the structural basis of T cell recognition of hapten-modified antigenic peptides, we studied the interaction of the T1 T cell antigen receptor (TCR) with its ligand, the H-2Kd-bound Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid (ABA) on P. berghei circumsporozoite Lys259. The photoaffinity-labeled TCR residue(s) were mapped as Tyr48 and/or Tyr50 of complementary determining region 2beta (CDR2beta). Other TCR-ligand contacts were identified by mutational analysis. Molecular modeling, based on crystallographic coordinates of closely related TCR and major histocompatibility complex I molecules, indicated that ABA binds strongly and specifically in a cavity between CDR3alpha and CDR2beta. We conclude that TCR expressing selective Vbeta and CDR3alpha sequences form a binding domain between CDR3alpha and CDR2beta that can accommodate nonpeptidic moieties conjugated at the C-terminal portion of peptides binding to major histocompatibility complex (MHC) encoded proteins.

Asunto(s)

Antígenos H-2/química , Haptenos/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Línea Celular , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Mapeo Peptídico , Péptidos/química , Etiquetas de Fotoafinidad , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T Citotóxicos/inmunología

RESUMEN

This study describes a form of partial agonism for a CD8+ CTL clone, S15, in which perforin-dependent killing and IFN-gamma production were lost but Fas (APO1 or CD95)-dependent cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted and specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). The presence of a photoactivatable group in the epitope permitted assessment of TCR-ligand binding by TCR photoaffinity labeling. Selective activation of Fas-dependent killing was observed for a peptide-derivative variant containing a modified photoreactive group. A similar functional response was obtained after binding of the wild-type peptide derivative upon blocking of CD8 participation in TCR-ligand binding. The epitope modification or blocking of CD8 resulted in an > or = 8-fold decrease in TCR-ligand binding. In both cases, phosphorylation of zeta-chain and ZAP-70, as well as calcium mobilization were reduced close to background levels, indicating that activation of Fas-dependent cytotoxicity required weaker TCR signaling than activation of perforin-dependent killing or IFN-gamma production. Consistent with this, we observed that depletion of the protein tyrosine kinase p56(lck) by preincubation of S15 CTL with herbimycin A severely impaired perforin- but not Fas-dependent cytotoxicity. Together with the observation that S15 CTL constitutively express Fas ligand, these results indicate that TCR signaling too weak to elicit perforin-dependent cytotoxicity or cytokine production can induce Fas-dependent cytotoxicity, possibly by translocation of preformed Fas ligand to the cell surface.

Asunto(s)

Antígenos CD8/fisiología , Señalización del Calcio , Interferón gamma/biosíntesis , Activación de Linfocitos , Glicoproteínas de Membrana/farmacología , Fragmentos de Péptidos/inmunología , Proteínas Protozoarias/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Receptor fas/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Azidas , Benzoquinonas , Antígenos CD8/efectos de los fármacos , Células Clonales , Citotoxicidad Inmunológica , Inhibidores Enzimáticos/farmacología , Epítopos/química , Epítopos/efectos de los fármacos , Epítopos/inmunología , Proteína Ligando Fas , Antígenos H-2/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Interferón gamma/genética , Lactamas Macrocíclicas , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/fisiología , Sarcoma de Mastocitos/patología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Fragmentos de Péptidos/química , Perforina , Fosforilación/efectos de los fármacos , Etiquetas de Fotoafinidad , Plasmodium berghei/inmunología , Proteínas Citotóxicas Formadoras de Poros , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteínas Protozoarias/química , Quinonas/farmacología , Receptores de Antígenos de Linfocitos T/metabolismo , Rifabutina/análogos & derivados , Salicilatos , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/metabolismo , Proteína Tirosina Quinasa ZAP-70

RESUMEN

Using H-2Kd-restricted CTL clones, which are specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS(252-260) (SYIPSAEKI) and permit assessment of TCR-ligand interactions by TCR photoaffinity labeling, we have previously identified several peptide derivative variants for which TCR-ligand binding and the efficiency of Ag recognition deviated by fivefold or more. Here we report that the functional CTL response (cytotoxicity and IFN-gamma production) correlated with the rate of TCR-ligand complex dissociation, but not the avidity of TCR-ligand binding. While peptide antagonists exhibited very rapid TCR-ligand complex dissociation, slightly slower dissociation was observed for strong agonists. Conversely and surprisingly, weak agonists typically displayed slower dissociation than the wild-type agonists. Acceleration of TCR-ligand complex dissociation by blocking CD8 participation in TCR-ligand binding increased the efficiency of Ag recognition in cases where dissociation was slow. In addition, permanent TCR engagement by TCR-ligand photocross-linking completely abolished sustained intracellular calcium mobilization, which is required for T cell activation. These results indicate that the functional CTL response depends on the frequency of serial TCR engagement, which, in turn, is determined by the rate of TCR-ligand complex dissociation.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Epítopos de Linfocito T/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Linfocitos T CD8-positivos/metabolismo , Células Clonales , Pruebas Inmunológicas de Citotoxicidad , Fragmentos Fab de Inmunoglobulinas/farmacología , Ligandos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Péptidos/agonistas , Péptidos/inmunología , Péptidos/metabolismo , Fosforilación , Transducción de Señal/inmunología

RESUMEN

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.

Asunto(s)

Antígenos H-2/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Protozoarias/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos T/metabolismo , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Apicomplexa , Células Clonales/inmunología , Simulación por Computador , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mapeo Peptídico , Fotoquímica , Plasmodium berghei , Receptores de Antígenos de Linfocitos T alfa-beta/química

RESUMEN

We tested for antigen recognition and T cell receptor (TCR)-ligand binding 12 peptide derivative variants on seven H-2Kd-restricted cytotoxic T lymphocytes (CTL) clones specific for a bifunctional photoreactive derivative of the Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI). The derivative contained iodo-4-azidosalicylic acid in place of PbCS S-252 and 4-azidobenzoic acid on PbCS K-259. Selective photoactivation of the N-terminal photoreactive group allowed crosslinking to Kd molecules and photoactivation of the orthogonal group to TCR. TCR photoaffinity labeling with covalent Kd-peptide derivative complexes allowed direct assessment of TCR-ligand binding on living CTL. In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold. The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity. There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response. An unexpected and novel finding was that CD8-dependent clones clearly incline more to TCR antagonism than CD8-independent ones. As there was no correlation between CD8 dependence and the avidity of TCR-ligand binding, the possibility is suggested that CD8 plays a critical role in aberrant CTL function.

Asunto(s)

Antígenos H-2/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Marcadores de Afinidad , Secuencia de Aminoácidos , Linfocitos T CD8-positivos , Línea Celular , Células Clonales , Glicoproteínas de Membrana/inmunología , Datos de Secuencia Molecular , Péptidos/química , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/inmunología , Receptor fas/inmunología

RESUMEN

Using H-2Kd-restricted photoprobe-specific cytotoxic T lymphocyte (CTL) clones, which permit assessment of T cell receptor (TCR)-ligand interactions by TCR photoaffinity labeling, we observed that the efficiency of antigen recognition by CTL was critically dependent on the half-life of TCR-ligand complexes. We show here that antigen recognition by CTL is essentially determined by the frequency of serial TCR engagement, except for very rapid dissociations, which resulted in aberrant TCR signaling and antagonism. Thus agonists that were efficiently recognized exhibited rapid TCR-ligand complex dissociation, and hence a high frequency of serial TCR engagement, whereas the opposite was true for weak agonists. Surprisingly, these differences were largely accounted for by the coreceptor CD8. While it was known that CD8 substantially decreases TCR-ligand complex dissociation, we observed in this study that this effect varied considerably among ligand variants, indicating that epitope modifications can alter the CD8 contribution to TCR-ligand binding, and hence the efficiency of antigen recognition by CTL.

Asunto(s)

Antígenos CD8/fisiología , Linfocitos T Citotóxicos/fisiología , Marcadores de Afinidad , Animales , Reacciones Antígeno-Anticuerpo , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Citotoxicidad Inmunológica , Cinética , Ligandos , Ratones , Fotoquímica , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Células Tumorales Cultivadas

RESUMEN

To study the role of CD8 beta in T cell function, we derived a CD8 alpha/beta-(CD8-/-) T cell hybridoma of the H-2Kd-restricted N9 cytotoxic T lymphocyte clone specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260. This hybridoma was transfected either with CD8 alpha alone or together with CD8 beta. All three hybridomas released interleukin 2 upon incubation with L cells expressing Kd-peptide derivative complexes, though CD8 alpha/beta cells did so more efficiently than CD8 alpha/alpha and especially CD8-/- cells. More strikingly, only CD8 alpha/beta cells were able to recognize a weak agonist peptide derivative variant. This recognition was abolished by Fab' fragments of the anti-Kd alpha 3 monoclonal antibody SF1-1.1.1 or substitution of Kd D-227 with K, both conditions known to impair CD8 coreceptor function. T cell receptor (TCR) photoaffinity labeling indicated that TCR-ligand binding on CD8 alpha/beta cells was approximately 5- and 20-fold more avid than on CD8 alpha/a and CD8-/- cells, respectively. SF1-1.1.1 Fab' or Kd mutation D227K reduced the TCR photoaffinity labeling on CD8 alpha/beta cells to approximately the same low levels observed on CD8-/- cells. These results indicate that CD8 alpha/beta is a more efficient coreceptor than CD8alpha/alpha, because it more avidly strengthens TCR-ligand binding.

Asunto(s)

Antígenos CD8/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular , Citometría de Flujo , Antígenos H-2/inmunología , Hibridomas/inmunología , Ratones , Fragmentos de Péptidos/inmunología , Plasmodium berghei/inmunología , Proteínas Protozoarias/inmunología , Linfocitos T Citotóxicos/inmunología , Timoma , Neoplasias del Timo

RESUMEN

T cell activation is triggered by the specific recognition of cognate peptides presented by MHC molecules. Altered peptide ligands are analogs of cognate peptides which have a high affinity for MHC molecules. Some of them induce complete T cell responses, i.e. they act as agonists, whereas others behave as partial agonists or even as antagonists. Here, we analyzed both early (intracellular Ca2+ mobilization), and late (interleukin-2 production) signal transduction events induced by a cognate peptide or a corresponding altered peptide ligand using T cell hybridomas expressing or not the CD8 alpha and beta chains. With a video imaging system, we showed that the intracellular Ca2+ response to an altered peptide ligand induces the appearance of a characteristic sustained intracellular Ca2+ concentration gradient which can be detected shortly after T cell interaction with antigen-presenting cells. We also provide evidence that the same altered peptide ligand can be seen either as an agonist or a partial agonist, depending on the presence of CD8beta in the CD8 co-receptor dimers expressed at the T cell surface.

Asunto(s)

Presentación de Antígeno/efectos de los fármacos , Antígenos CD8/farmacología , Péptidos/agonistas , Péptidos/inmunología , Péptidos/farmacología , Animales , Calcio/metabolismo , Línea Celular , Interleucina-2/biosíntesis , Células L , Ligandos , Ratones , Transducción de Señal/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo

RESUMEN

We used stepwise photochemical cross-linking for specifically assembling soluble and covalent complexes made of a T-cell antigen receptor (TCR) and a class I molecule of the major histocompatibility complex (MHC) bound to an antigenic peptide. For that purpose, we have produced in myeloma cells a single-chain Fv construct of a TCR specific for a photoreactive H-2Kd-peptide complex. Photochemical cross-linking of this TCR single-chain Fv with a soluble form of the photoreactive H-2Kd-peptide ligand resulted in the formation of a ternary covalent complex. We have characterized the soluble ternary complex and showed that it reacted with antibodies specific for epitopes located either on the native TCR or on the Kd molecules. By preventing the fast dissociation kinetics observed with most T cell receptors, this approach provides a means of preparing soluble TCR-peptide-MHC complexes on large-scale levels.

Asunto(s)

Antígenos H-2/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Línea Celular , Reactivos de Enlaces Cruzados , Cartilla de ADN , Antígenos H-2/aislamiento & purificación , Antígenos H-2/metabolismo , Hibridomas , Cinética , Complejo Mayor de Histocompatibilidad , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T/aislamiento & purificación , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/aislamiento & purificación , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Células Tumorales Cultivadas

RESUMEN

Melanoma-associated genes (MAGEs) encode tumor-specific antigens that can be recognized by CD8+ cytotoxic T lymphocytes. To investigate the interaction of the HLA-A1-restricted MAGE-1 peptide 161-169 (EADPT-GHSY) with HLA class I molecules, photoreactive derivatives were prepared by single amino acid substitution with N beta-[iodo-4-azidosalicyloyl]-L-2,3-diaminopropionic acid. These derivatives were tested for their ability to bind to, and to photoaffinity-label, HLA-A1 on C1R.A1 cells. Only the derivatives containing the photoreactive amino acid in position 1 or 7 fulfilled both criteria. Testing the former derivative on 14 lymphoid cell lines expressing over 44 different HLA class I molecules indicated that it efficiently photoaffinity-labeled not only HLA-A1, but possibility also HLA-A29 and HLA-B44. MAGE peptide binding by HLA-A29 and HLA-B44 was confirmed by photoaffinity labeling with photoreactive MAGE-3 peptide derivatives on C1R.A29 and C1R.B44 cells, respectively. The different photoaffinity labeling systems were used to access the ability of the homologous peptides derived from MAGE-1, -2, -3, -4a, -4b, -6, and -12 to bind to HLA-A1, HLA-A29, and HLA-B44. All but the MAGE-2 and MAGE-12 nonapeptides efficiently inhibited photoaffinity labeling of HLA-A1, which is in agreement with the known HLA-A1 peptide-binding motif (acidic residue in P3 and C-terminal tyrosine). In contrast, photoaffinity labeling of HLA-A29 was efficiently inhibited by these as well as by the MAGE-3 and MAGE-6 nonapeptides. Finally, the HLA-B44 photoaffinity labeling, unlike the HLA-A1 and HLA-A29 labeling, was inhibited more efficiently by the corresponding MAGE decapeptides, which is consistent with the reported HLA-B44 peptide-binding motif (glutamic acid in P2, and C-terminal tyrosine or phenylalanine). The overlapping binding of homologous MAGE peptides by HLA-A1, A29, and B44 is based on different binding principles and may have implications for immunotherapy of MAGE-positive tumors.

Asunto(s)

Antígenos de Neoplasias/metabolismo , Antígenos HLA-A/metabolismo , Antígeno HLA-A1/metabolismo , Antígenos HLA-B/metabolismo , Melanoma/genética , Marcadores de Afinidad , Secuencia de Aminoácidos , Línea Celular , Antígeno HLA-B44 , Humanos , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido