RESUMEN
Recent work has demonstrated that nonstimulatory endogenous peptides can enhance T cell recognition of antigen, but MHCI- and MHCII-restricted systems have generated very different results. MHCII-restricted TCRs need to interact with the nonstimulatory peptide-MHC (pMHC), showing peptide specificity for activation enhancers or coagonists. In contrast, the MHCI-restricted cells studied to date show no such peptide specificity for coagonists, suggesting that CD8 binding to noncognate MHCI is more important. Here we show how this dichotomy can be resolved by varying CD8 and TCR binding to agonist and coagonists coupled with computer simulations, and we identify two distinct mechanisms by which CD8 influences the peptide specificity of coagonism. Mechanism 1 identifies the requirement of CD8 binding to noncognate ligand and suggests a direct relationship between the magnitude of coagonism and CD8 affinity for coagonist pMHCI. Mechanism 2 describes how the affinity of CD8 for agonist pMHCI changes the requirement for specific coagonist peptides. MHCs that bind CD8 strongly were tolerant of all or most peptides as coagonists, but weaker CD8-binding MHCs required stronger TCR binding to coagonist, limiting the potential coagonist peptides. These findings in MHCI systems also explain peptide-specific coagonism in MHCII-restricted cells, as CD4-MHCII interaction is generally weaker than CD8-MHCI.
Asunto(s)
Epítopos/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/agonistas , Receptores de Antígenos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos T CD8-positivos/inmunología , Células CHO , Simulación por Computador , Cricetinae , Cricetulus , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Cinética , Activación de Linfocitos/inmunología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Ovalbúmina/inmunología , Péptidos/química , Péptidos/inmunología , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
T cells with specificity for antigens derived from Wilms Tumor gene (WT1), Proteinase3 (Pr3), and mucin1 (MUC1) have been demonstrated to lyse acute myeloid leukemia (AML) blasts and multiple-myeloma (MM) cells, and strategies to enhance or induce such tumor-specific T cells by vaccination are currently being explored in multiple clinical trials. To test safety and immunogenicity of a vaccine composed of WT1-, Pr3-, and MUC1-derived Class I-restricted peptides and the pan HLA-DR T helper cell epitope (PADRE) or MUC1-helper epitopes in combination with CpG7909 and MontanideISA51, four patients with AML and five with MM were repetitively vaccinated. No clinical responses were observed. Neither pre-existing nor naive WT1-/Pr3-/MUC1-specific CD8+ T cells expanded in vivo by vaccination. In contrast, a significant decline in vaccine-specific CD8+ T cells was observed. An increase in PADRE-specific CD4+ T helper cells was observed after vaccination but these appeared unable to produce IL2, and CD4+ T cells with a regulatory phenotype increased. Taken into considerations that multiple clinical trials with identical antigens but different adjuvants induced vaccine-specific T cell responses, our data caution that a vaccination with leukemia-associated antigens can be detrimental when combined with MontanideISA51 and CpG7909. Reflecting the time-consuming efforts of clinical trials and the fact that 1/3 of ongoing peptide vaccination trails use CpG and/or Montanide, our data need to be taken into consideration.
Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/uso terapéutico , Leucemia Mieloide Aguda/terapia , Manitol/análogos & derivados , Mieloma Múltiple/terapia , Ácidos Oléicos , Oligodesoxirribonucleótidos , Péptidos/uso terapéutico , Adolescente , Antígenos de Neoplasias/química , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/efectos adversos , Vacunas contra el Cáncer/inmunología , Femenino , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Masculino , Manitol/efectos adversos , Mucina-1/efectos adversos , Mucina-1/química , Mucina-1/inmunología , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Mieloblastina/efectos adversos , Mieloblastina/química , Mieloblastina/inmunología , Estadificación de Neoplasias , Neoplasia Residual/inmunología , Neoplasia Residual/patología , Neoplasia Residual/terapia , Ácidos Oléicos/efectos adversos , Oligodesoxirribonucleótidos/efectos adversos , Oligodesoxirribonucleótidos/inmunología , Péptidos/efectos adversos , Péptidos/inmunología , Proyectos Piloto , Resultado del Tratamiento , Proteínas WT1/efectos adversos , Proteínas WT1/química , Proteínas WT1/inmunologíaRESUMEN
The use of appropriate fluorescent proteins has allowed the use of FRET microscopy for investigation of intermolecular interactions in living cells. This method has the advantage of both being dynamic and of working at the subcellular level, so that the time and place where proteins interact can be visualized. We have used FRET microscopy to analyze the interactions between the T cell antigen receptor and the coreceptors CD4 and CD8. This chapter reviews data on how these coreceptors are recruited to the immunological synapse, and how they interact when the T cell is stimulated by different ligands.
Asunto(s)
Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Animales , Presentación de Antígeno , Humanos , Nanotubos , Linfocitos T/metabolismoRESUMEN
T cells are extremely sensitive in their ability to find minute amounts of antigenic peptide in the midst of many endogenous peptides presented on an antigen-presenting cell. The role of endogenous peptides in the recognition of foreign peptide and hence in T cell activation has remained controversial for CD8(+) T cell activation. We showed previously that in a CD8(+) T cell hybridoma, nonstimulatory endogenous peptides enhance T cell sensitivity to antigen by increasing the coreceptor function of CD8. However, others were not able to detect such enhancement in naive and activated CD8(+) T cells. Here, we show that endogenous peptides substantially enhance the ability of T cells to detect antigen, an effect measurable by up-regulation of activation or maturation markers and by increased effector function. This enhancement is most pronounced in thymocytes, moderate in naive T cells, and mild in effector T cells. The importance of endogenous peptides is inversely proportional to the agonist activity of the stimulatory peptide presented. Unlike for CD4(+) T cells, the T cell receptor of CD8(+) T cells does not distinguish between endogenous peptides for their ability to enhance antigen recognition.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos , Complejo Mayor de Histocompatibilidad , Linfocitos T/inmunología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Células Presentadoras de Antígenos/inmunología , Diferenciación Celular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/inmunologíaRESUMEN
We report a distinct microenvironment within the nuclear envelope (NE) in living cells revealed by a spectral shift of the fluorescent dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium 2Br). The dye readily translocated to the NE at physiological temperature where it exhibited enhanced fluorescence when excited at 620-650 nm in contrast to 480-520 nm excitation in the endocytic pathway and in the endoplasmic reticulum (ER). In vitro data indicated that the dye reveals an enrichment of negatively charged lipids, presumably due to local phospholipid synthesis. Dual-excitation imaging of FM4-64 in relation to lamina-associated polypeptide-1-green fluorescent protein during mitosis suggested that the disassembly of NE preserves microscale lipid complexes in the ER. Convolutions of NE in cancer or primary cells were readily visualized, and killing of tumor cells by T cells was marked by sudden loss of the long-wavelength excited fluorescence in the NE coincident with apoptosis. This report of FM4-64 as the first vital dye sensitive to the NE environment opens new ways for real-time visualization and functional studies of the NE.
Asunto(s)
Colorantes Fluorescentes/química , Membrana Nuclear/química , Compuestos de Piridinio/química , Compuestos de Amonio Cuaternario/química , Supervivencia Celular , Células Cultivadas , Detergentes , Colorantes Fluorescentes/farmacología , Concentración de Iones de Hidrógeno , Microscopía Fluorescente , Membrana Nuclear/efectos de los fármacos , Compuestos de Piridinio/farmacología , Compuestos de Amonio Cuaternario/farmacologíaRESUMEN
Growing evidence indicates that multiple myeloma (MM) and other malignancies are susceptible to CTL-based immune interventions. We studied whether transcription factors inherently involved in the terminal differentiation of mature B lymphocytes into malignant and nonmalignant plasma cells provide MM-associated CTL epitopes. HLA-A*0201 (A2.1) transgenic mice were used to identify A2.1-presented peptide Ag derived from the plasma cell-associated transcriptional regulators, positive regulatory domain I-binding factor 1 (PRDI-BF1) and X box-binding protein 1 (XBP-1). A2.1-restricted CTL specific for PRDI-BF1 and XBP-1 epitopes efficiently killed a variety of MM targets. PRDI-BF1- and XBP-1-reactive CTL were able to recognize primary MM cells from A2.1(+) patients. Consistent with the expression pattern of both transcription factors beyond malignant and nonmalignant plasma cells, PRDI-BF1- and XBP-1-specific CTL activity was not entirely limited to MM targets, but was also associated with lysis of certain other malignancies and, in defined instances, with low-to-intermediate level recognition of a few types of normal cells. Our results also indicate that the A2.1-restricted, PRDI-BF1- and XBP-1-specific human CD8(+) T cell repertoire is affected by partial self tolerance and may thus require the transfer of high-affinity TCR to break tolerance. We conclude that transcription factors governing terminal cellular differentiation may provide MM- and tumor-associated CTL epitopes.
Asunto(s)
Presentación de Antígeno , Citotoxicidad Inmunológica , Proteínas de Unión al ADN/inmunología , Mieloma Múltiple/inmunología , Proteínas Represoras/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Factores de Transcripción/inmunología , Animales , Presentación de Antígeno/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Muerte Celular/genética , Muerte Celular/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular Tumoral , Pruebas Inmunológicas de Citotoxicidad , Citotoxicidad Inmunológica/genética , Proteínas de Unión al ADN/metabolismo , Epítopos de Linfocito T/inmunología , Antígeno HLA-A2/biosíntesis , Antígeno HLA-A2/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Células 3T3 NIH , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Factores de Transcripción del Factor Regulador X , Proteínas Represoras/metabolismo , Autotolerancia/genética , Linfocitos T Citotóxicos/patología , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-BoxRESUMEN
The human tyrosinase (hTyr) (369-377) cytotoxic T lymphocyte (CTL) epitope is presented by malignant melanoma and various nontransformed cells in association with human leukocyte antigen (HLA)-A*0201 (A2.1) and used for vaccination-based immunotherapy of melanoma patients. Its mouse homologue, mTyr (369-377), is naturally processed and bound by A2.1 with equivalent efficacy and thus enabled us to explore the effect of self tolerance on Tyr-specific T cells in different lines of A2.1 transgenic (Tg) mice and man. We found that self Tyr-reactive CTL in Tg mice and, importantly, in man were affected by partial tolerance resulting in only residual T lymphocytes of higher avidity for self Tyr along with low-avidity T cells to be present in the periphery. Immunizing mice with the xenogeneic nonself Tyr peptide facilitated the generation of self Tyr-reactive CTL. As compared to Tyr-reactive CTL induced by high amounts of the self Tyr epitope, however, the nonself antigen (Ag) had no effect on improving the avidity of self Tyr-specific mouse and human T cells. Depleting mice of CD25(+) T cells with and without CTL-associated Ag 4 (CTLA-4) blockade demonstrated that tolerance of Tyr-specific CTL was not regulated by CD4(+)CD25(+) T regulatory cells (Treg) or CTLA-4. Our studies have important implications for the design of anti-Tyr-based immunotherapeutics.