RESUMEN
To characterize the ligand binding properties of a naive T cell repertoire capable of responding to a foreign antigen, we analyzed T cell populations from T cell receptor (TCR) beta transgenic mice using a novel, single cell peptide/major histocompatibility complex (MHC) tetramer dissociation assay. The largely CD4+CD8(-/low) antigen-specific thymocyte repertoire exhibited a broad, bimodal distribution of tetramer binding half-lives (t(1/2)s), with a significant underrepresentation in the intermediate half-life range in which the majority of the peripheral repertoire lies. Thus, cells with the potential to bind foreign antigen with the lowest and highest stability are likely to be selectively removed from the repertoire prior to their establishment in the periphery. These studies provide direct evidence that thymic selection biases the naive peripheral T cell repertoire toward TCR-ligand interactions that fall within a moderate half-life "window."
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Reordenamiento Génico de Linfocito T/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Timo/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Linfocitos T CD4-Positivos/metabolismo , Células Clonales/inmunología , Células Clonales/metabolismo , Regiones Determinantes de Complementariedad/genética , Citometría de Flujo , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T/genética , Semivida , Cinética , Ratones , Ratones Transgénicos , Mutación/genética , Receptores de Antígenos de Linfocitos T/química , Termodinámica , Timo/citología , Timo/metabolismoRESUMEN
We identified circulating CD8+ T-cell populations specific for the tumor-associated antigens (TAAs) MART-1 (27-35) or tyrosinase (368-376) in six of eleven patients with metastatic melanoma using peptide/HLA-A*0201 tetramers. These TAA-specific populations were of two phenotypically distinct types: one, typical for memory/effector T cells; the other, a previously undescribed phenotype expressing both naive and effector cell markers. This latter type represented more than 2% of the total CD8+ T cells in one patient, permitting detailed phenotypic and functional analysis. Although these cells have many of the hallmarks of effector T cells, they were functionally unresponsive, unable to directly lyse melanoma target cells or produce cytokines in response to mitogens. In contrast, CD8+ T cells from the same patient were able to lyse EBV-pulsed target cells and showed robust allogeneic responses. Thus, the clonally expanded TAA-specific population seems to have been selectively rendered anergic in vivo. Peptide stimulation of the TAA-specific T-cell populations in other patients failed to induce substantial upregulation of CD69 expression, indicating that these cells may also have functional defects, leading to blunted activation responses. These data demonstrate that systemic TAA-specific T-cell responses can develop de novo in cancer patients, but that antigen-specific unresponsiveness may explain why such cells are unable to control tumor growth.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Melanoma/inmunología , Melanoma/patología , Linfocitos T/inmunología , Antígenos de Neoplasias/inmunología , Antineoplásicos/uso terapéutico , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Antígenos CD57/inmunología , Antígenos CD57/metabolismo , Linfocitos T CD8-positivos/inmunología , Femenino , Citometría de Flujo/métodos , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Humanos , Receptores de Hialuranos/inmunología , Receptores de Hialuranos/metabolismo , Antígenos Comunes de Leucocito/inmunología , Antígenos Comunes de Leucocito/metabolismo , Metástasis Linfática/inmunología , Metástasis Linfática/patología , Antígeno MART-1 , Melanoma/tratamiento farmacológico , Melanoma/secundario , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/inmunología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Antígeno gp100 del MelanomaRESUMEN
The basis for T cell antigen receptor (TCR) repertoire selection upon repeated antigenic challenge is unclear. We evaluated the avidity and dissociation kinetics of peptide/major histocompatibility complex (MHC) tetramer binding to antigen-specific T lymphocytes isolated following primary or secondary immunization. The data reveal a narrowing of the secondary repertoire relative to the primary repertoire, largely resulting from the loss of cells expressing TCRs with the fastest dissociation rates for peptide/MHC binding. In addition, T cells in the secondary response express TCRs of higher average affinity for peptide/MHC than cells in the primary response. These results provide a link between the kinetics and affinity of TCR-peptide/MHC interactions and TCR sequence selection during the course of an immune response.
Asunto(s)
Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Animales , Sitios de Unión/inmunología , Femenino , Inmunización Secundaria , Cinética , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Péptidos/inmunología , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/biosíntesis , Coloración y Etiquetado , Subgrupos de Linfocitos T/química , Subgrupos de Linfocitos T/metabolismoRESUMEN
Immunogenic peptides of human tumor Ag have been used to generate antigen-specific CTL. However, the vast majority of these peptide-specific CTL clones are of low avidity and are peptide, but not tumor, reactive. Peptide-MHC tetramers have been shown to bind specific TCRs with sufficient affinity to be useful reagents for flow cytometry. In this paper we demonstrate that peptide-MHC tetramers can also be used to selectively identify high avidity tumor-reactive CTL and enrich, from a heterogeneous population, the subpopulation of peptide-reactive T cells that can lyse tumor targets. The melanoma proteins, MART-1 and gp100, were used to induce potentially tumor-reactive T cells, and the intensity of T cell staining by TCR binding of specific peptide-MHC tetramers was assessed. A range of fluorescence intensity was detected, and the magnitude of tetramer binding was correlated with T cell avidity. The population of peptide-reactive T cells was phenotypically similar with regard to expression of TCR and adhesion molecules, suggesting that this differential avidity for tumor cells reflected differential affinity of the TCR for its peptide-MHC ligand. Sorting, cloning, and expansion of tetramerhigh CTL from a heterogeneous population of peptide-stimulated PBMCs enabled rapid selection of high avidity tumor-reactive CTL clones, which retained their functional and tetramerhigh phenotype on re-expansion. These results demonstrate that the avidity of a T cell for its tumor target is due to the specific affinity of the TCR for its peptide-MHC ligand, that this interaction can be described using peptide-MHC tetramers and used to isolate high avidity tumor-reactive CTL.
Asunto(s)
Pruebas Inmunológicas de Citotoxicidad , Antígeno HLA-A2/química , Melanoma/inmunología , Oligopéptidos/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Células Presentadoras de Antígenos/inmunología , Antígenos de Neoplasias/inmunología , Línea Celular , Separación Celular , Células Clonales , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Antígeno HLA-A2/inmunología , Humanos , Antígeno MART-1 , Glicoproteínas de Membrana/inmunología , Proteínas de Neoplasias/inmunología , Oligopéptidos/química , Coloración y Etiquetado , Subgrupos de Linfocitos T/química , Linfocitos T Citotóxicos/química , Células Tumorales Cultivadas , Antígeno gp100 del MelanomaAsunto(s)
Linfocitos T/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Comunicación Celular/inmunología , Variación Genética , Antígenos de Histocompatibilidad/metabolismo , Humanos , Cinética , Ligandos , Activación de Linfocitos , Ratones , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/metabolismoRESUMEN
A high-performance liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) method is presented that allows rapid and accurate determination of amino acid chiral purity in a peptide. Peptides are hydrolyzed in hydrochloric acid-d1/acetic acid-d4 and then converted to diastereomers by derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, Marfey's reagent). Mixtures of D- and L-amino acid diastereomeric pairs are resolved in one chromatographic separation using conventional reversed-phase high-performance liquid chromatography. Hydrolysis in a deuterated solvent is necessary because the original ratio of D-/L-amino acids present in a peptide changes during acid hydrolysis due to racemization. Peptide hydrolysis in deuterated acids circumvents this problem by labeling each amino acid that racemizes with one deuterium at the alpha-carbon. An increase in molecular mass of one atomic mass unit allows racemized amino acids to be distinguished from non-racemized amino acids by mass spectrometry. This procedure was used to determine the chiral purity of each amino acid in a purified, hexapeptide by-product (Arg-Lys-Lys-Asp-Val-Tyr) present in a kilogram batch of the synthetic pentapeptide, thymopentin (Arg-Lys-Asp-Val-Tyr).
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Péptidos/aislamiento & purificación , Alanina/análogos & derivados , Alanina/química , Secuencia de Aminoácidos , Aminoácidos/química , Deuterio , Dinitrobencenos/química , Hidrólisis , Indicadores y Reactivos , Datos de Secuencia Molecular , Péptidos/química , Conformación ProteicaRESUMEN
Chromosome band 11q23 is the site of recurring translocations with a variety of partner chromosomes in myeloid and lymphoid acute leukemias, infant leukemias, and treatment-induced secondary acute myelogenous leukemia. The translocation breakpoints cluster in a restricted region of the HRX gene resulting in fusion genes that encode an N-terminal portion of Hrx fused to various partner proteins. We have characterized the transcriptional transactivation properties of Enl, a protein that is fused to Hrx in t(11;19) leukemias. Enl is a nuclear protein that is capable of activating transcription from synthetic reporter genes in both lymphoid and myeloid cells, as well as in yeast. Deletion mutagenesis demonstrated that the minimal portion of Enl required for activation of transcription was localized to its C-terminal 90 amino acids. This region is highly conserved between Enl and the t(9;11) fusion partner Af-9 and is retained in all Hrx-Enl and Hrx-Af9 fusion proteins. Thus, the leukemogenic contribution and transcriptional activation potential of Enl colocalize to its highly conserved carboxy terminus, suggesting that Hrx-Enl chimeric proteins mediate alterations in the transcription program of t(11;19)-bearing cells.