Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta , Linfocitos T , Humanos , Citometría de Flujo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Receptores de Antígenos de Linfocitos T/genética , Células ClonalesRESUMEN
In jawed vertebrates, adaptive immunity depends on the process of V(D)J recombination creating vast numbers of T and B lymphocytes that each expresses unique Ag receptors of uniform specificity. The asynchronous initiation of V-to-(D)J rearrangement between alleles and the resulting protein from one allele signaling feedback inhibition of V recombination on the other allele ensures homogeneous receptor specificity of individual cells. Upon productive Vß-to-DßJß rearrangements in noncycling double-negative thymocytes, TCRß protein signals induction of the cyclin D3 protein to accelerate cell cycle entry, thereby driving proliferative expansion of developing αß T cells. Through undetermined mechanisms, the inactivation of cyclin D3 in mice causes an increased frequency of αß T cells that express TCRß proteins from both alleles, producing lymphocytes of heterogeneous specificities. To determine how cyclin D3 enforces monogenic TCRß expression, we used our mouse lines with enhanced rearrangement of specific Vß segments due to replacement of their poor-quality recombination signal sequence (RSS) DNA elements with a better RSS. We show that cyclin D3 inactivation in these mice elevates the frequencies of αß T cells that display proteins from RSS-augmented Vß segments on both alleles. By assaying mature αß T cells, we find that cyclin D3 deficiency increases the levels of Vß rearrangements that occur within developing thymocytes. Our data demonstrate that a component of the cell cycle machinery mediates TCRß protein-signaled feedback inhibition in thymocytes to achieve monogenic TCRß expression and resulting uniform specificity of individual αß T cells.
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Receptores de Antígenos de Linfocitos T alfa-beta , Timocitos , Animales , Ratones , Alelos , Ciclina D3/genética , Retroalimentación , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Linfocitos , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
The ß chain rearrangement in T cells is a two-step process where first Dß and Jß bind, and only then Vß is joined to the complex. We here show that the frequency of human and mouse Vß Jß combinations deviates from the one expected based on each gene usage frequency. This bias is observed mainly in functional (F) rearrangements, but also slightly in non-functional (NF) rearrangements. Preferred Vß Jß combinations in F clones are shared between donors and samples, suggesting a common structural mechanism for these biases in addition to any host-specific antigen-induced peripheral selection. The sharing holds even in clones with J ß 1 that share the same Dß 1 gene. Vß Jß usage is correlated with the Molecular Weight and Isoelectric Point in F clones. The pairing is also observed in the Double Positive cells in mice thymocytes, suggesting that the selection leading to such a pairing occurs before thymic selection. These results suggest an additional structural checkpoint in the beta chain development prior to thymic selection during the T cell receptor expression. Understanding this structural selection is important for the distinction between normal and aberrant T cell development, and crucial for the design of engineered TCRs.
Asunto(s)
Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico , Animales , Sesgo , Humanos , Ratones , Linfocitos TRESUMEN
The monoallelic expression (allelic exclusion) of diverse lymphocyte Ag receptor genes enables specific immune responses. Allelic exclusion is achieved by asynchronous initiation of V(D)J recombination between alleles and protein encoded by successful rearrangement on the first allele signaling permanent inhibition of V rearrangement on the other allele. The ATM kinase that guides DNA repair and transiently suppresses V(D)J recombination also helps impose allelic exclusion through undetermined mechanisms. At the TCRß locus, one Vß gene segment (V31) rearranges only by inversion, whereas all other Vß segments rearrange by deletion except for rare cases in which they rearrange through inversion following V31 rearrangement. The poor-quality recombination signal sequences (RSSs) of V31 and V2 help establish TCRß gene repertoire and allelic exclusion by stochastically limiting initiation of Vß rearrangements before TCRß protein-signaled permanent silencing of Vß recombination. We show in this study in mice that ATM functions with these RSSs and the weak V1 RSS to shape TCRß gene repertoire by restricting their Vß segments from initiating recombination and hindering aberrant nonfunctional Vß recombination products, especially during inversional V31 rearrangements. We find that ATM collaborates with the V1 and V2 RSSs to help enforce allelic exclusion by facilitating competition between alleles for initiation and functional completion of rearrangements of these Vß segments. Our data demonstrate that the fundamental genetic DNA elements that underlie inefficient Vß recombination cooperate with ATM-mediated rapid DNA damage responses to help establish diversity and allelic exclusion of TCRß genes.
Asunto(s)
Señales de Clasificación de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta , Alelos , Animales , Daño del ADN , Reparación del ADN/genética , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/genética , Ratones , Señales de Clasificación de Proteína/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Recombinación V(D)J/genéticaRESUMEN
T cells play an important role in adaptive immunity. An enormous clonal diversity of T cells with a different specificity, encoded by the T cell receptor (TCR), protect the body against infection. Most TCRß chains are generated from a V, D, and J segment during recombination in the thymus. Although complete absence of the D segment is not easily detectable from sequencing data, we find convincing evidence for a substantial proportion of TCRß rearrangements lacking a D segment. Additionally, sequences without a D segment are more likely to be abundant within individuals and/or shared between individuals. Our analysis indicates that such sequences are preferentially generated during fetal development and persist within the elderly. Summarizing, TCRß rearrangements without a D segment are not uncommon, and tend to allow for TCRß chains with a high abundance in the naive repertoire.
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Inmunidad Adaptativa , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunología , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , Glicina/deficiencia , HumanosRESUMEN
Recent advances in T cell repertoire (TCR) sequencing allow for the characterization of repertoire properties, as well as the frequency and sharing of specific TCR. However, there is no efficient measure for the local density of a given TCR. TCRs are often described either through their Complementary Determining region 3 (CDR3) sequences, or theirV/J usage, or their clone size. We here show that the local repertoire density can be estimated using a combined representation of these components through distance conserving autoencoders and Kernel Density Estimates (KDE). We present ELATE-an Encoder-based LocAl Tcr dEnsity and show that the resulting density of a sample can be used as a novel measure to study repertoire properties. The cross-density between two samples can be used as a similarity matrix to fully characterize samples from the same host. Finally, the same projection in combination with machine learning algorithms can be used to predict TCR-peptide binding through the local density of known TCRs binding a specific target.
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Receptores de Antígenos de Linfocitos T/clasificación , Receptores de Antígenos de Linfocitos T/genética , Programas Informáticos , Algoritmos , Secuencia de Aminoácidos , Regiones Determinantes de Complementariedad/clasificación , Regiones Determinantes de Complementariedad/genética , Biología Computacional , Bases de Datos Genéticas , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Humanos , Región Variable de Inmunoglobulina/genética , Aprendizaje Automático , Receptores de Antígenos de Linfocitos T alfa-beta/clasificación , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
T cell development is predicated on the successful rearrangement of the TCR gene loci, which encode for Ag-specific receptors. Recombination-activating gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T cell development. In this study, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCR ß-chain in mediating ß-selection during human T cell development. In stark contrast to mice, human RAG2-KO T lineage progenitors progressed to the CD4+CD8+ double-positive (DP) stage in the absence of TCRß rearrangements. Nonetheless, RAG2-KO DPs retrovirally transduced to express a rearranged TCR ß-chain showed increased survival and proliferation as compared with control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRß- and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide important insights as to the distinct requirement for the TCR ß-chain during human T cell development.
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Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Diferenciación Celular/genética , Células Madre Embrionarias Humanas/citología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Técnicas de Inactivación de Genes , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/genética , Hematopoyesis/genética , Humanos , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Transducción GenéticaRESUMEN
Allogeneic immune responses underlie the graft-versus-leukaemia effect of stem cell transplantation, but disease relapse occurs in many patients. Minor histocompatibility antigen (mHAg) peptides mediate alloreactive T cell responses and induce graft-versus-leukaemia responses when expressed on patient haematopoietic tissue. We vaccinated nine HA-1-negative donors against HA-1 with a 'prime-boost' protocol of either two or three DNA 'priming' vaccinations prior to 'boost' with modified vaccinia Ankara (MVA). HA-1-specific CD8+ T cell responses were observed in seven donors with magnitude up to 1·5% of total CD8+ T cell repertoire. HA-1-specific responses peaked two weeks post-MVA challenge and were measurable in most donors after 12 months. HA-1-specific T cells demonstrated strong cytotoxic activity and lysed target cells with endogenous HA-1 protein expression. The pattern of T cell receptor (TCR) usage by HA-1-specific T cells revealed strong conservation of T cell receptor beta variable 7-9 (TRBV7-9) usage between donors. These findings describe one of the strongest primary peptide-specific CD8+ T cell responses yet recorded to a DNA-MVA prime-boost regimen and this may reflect the strong immunogenicity of mHAg peptides. Prime-boost vaccination in donors or patients may prove of substantial benefit in boosting graft-versus-leukaemia responses.
Asunto(s)
Antígenos de Neoplasias/inmunología , Efecto Injerto vs Leucemia/inmunología , Antígenos de Histocompatibilidad Menor/inmunología , Oligopéptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunación , Vacunas de ADN/uso terapéutico , Virus Vaccinia/inmunología , Vacunas Virales/uso terapéutico , Adulto , Anciano , Aloinjertos , Citotoxicidad Inmunológica , Epítopos/inmunología , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Antígeno HLA-A2/inmunología , Trasplante de Células Madre Hematopoyéticas , Humanos , Inmunogenicidad Vacunal , Memoria Inmunológica , Masculino , Persona de Mediana Edad , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Vacunas Atenuadas , Vacunas de ADN/inmunología , Vacunas Virales/inmunologíaRESUMEN
The bottlenose dolphin (Tursiops truncatus) belongs to the Cetartiodactyla and, similarly to other cetaceans, represents the most successful mammalian colonization of the aquatic environment. Here we report a genomic, evolutionary, and expression study of T. truncatus T cell receptor beta (TRB) genes. Although the organization of the dolphin TRB locus is similar to that of the other artiodactyl species, with three in tandem D-J-C clusters located at its 3' end, its uniqueness is given by the reduction of the total length due essentially to the absence of duplications and to the deletions that have drastically reduced the number of the germline TRBV genes. We have analyzed the relevant mature transcripts from two subjects. The simultaneous availability of rearranged T cell receptor α (TRA) and TRB cDNA from the peripheral blood of one of the two specimens, and the human/dolphin amino acids multi-sequence alignments, allowed us to calculate the most likely interactions at the protein interface between the alpha/beta heterodimer in complex with major histocompatibility class I (MH1) protein. Interacting amino acids located in the complementarity-determining region according to IMGT numbering (CDR-IMGT) of the dolphin variable V-alpha and beta domains were identified. According to comparative modelization, the atom pair contact sites analysis between the human MH1 grove (G) domains and the T cell receptor (TR) V domains confirms conservation of the structure of the dolphin TR/pMH.
Asunto(s)
Delfín Mular/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de Proteína/métodos , Animales , Mapeo Cromosómico , Femenino , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Masculino , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Alineación de Secuencia , Microglobulina beta-2/metabolismoRESUMEN
Restoration of T cell repertoire diversity after allogeneic bone marrow transplantation (allo-BMT) is crucial for immune recovery. T cell diversity is produced by rearrangements of germline gene segments (V (D) and J) of the T cell receptor (TCR) α and ß chains, and selection induced by binding of TCRs to MHC-peptide complexes. Multiple measures were proposed for this diversity. We here focus on the V-gene usage and the CDR3 sequences of the beta chain. We compared multiple T cell repertoires to follow T cell repertoire changes post-allo-BMT in HLA-matched related donor and recipient pairs. Our analyses of the differences between donor and recipient complementarity determining region 3 (CDR3) beta composition and V-gene profile show that the CDR3 sequence composition does not change during restoration, implying its dependence on the HLA typing. In contrast, V-gene usage followed a time-dependent pattern, initially following the donor profile and then shifting back to the recipients' profile. The final long-term repertoire was more similar to that of the recipient's original one than the donor's; some recipients converged within months, while others took multiple years. Based on the results of our analyses, we propose that donor-recipient V-gene distribution differences may serve as clinical biomarkers for monitoring immune recovery.
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Trasplante de Médula Ósea , Regiones Determinantes de Complementariedad/genética , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/genética , Linfocitos T/inmunología , Adulto , Femenino , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Prueba de Histocompatibilidad , Humanos , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Donantes de Tejidos , Trasplante HomólogoRESUMEN
In hematopoietic cell transplantation (HCT), permissive HLA-DPB1 mismatches between patients and their unrelated donors are associated with improved outcomes compared with nonpermissive mismatches, but the underlying mechanism is incompletely understood. Here, we used mass spectrometry, T-cell receptor-ß (TCRß) deep sequencing, and cellular in vitro models of alloreactivity to interrogate the HLA-DP immunopeptidome and its role in alloreactive T-cell responses. We find that permissive HLA-DPB1 mismatches display significantly higher peptide repertoire overlaps compared with their nonpermissive counterparts, resulting in lower frequency and diversity of alloreactive TCRß clonotypes in healthy individuals and transplanted patients. Permissiveness can be reversed by the absence of the peptide editor HLA-DM or the presence of its antagonist, HLA-DO, through significant broadening of the peptide repertoire. Our data establish the degree of immunopeptidome divergence between donor and recipient as the mechanistic basis for the clinically relevant permissive HLA-DPB1 mismatches in HCT and show that permissiveness is dependent on HLA-DM-mediated peptide editing. Its key role for harnessing T-cell alloreactivity to HLA-DP highlights HLA-DM as a potential novel target for cellular and immunotherapy of leukemia.
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Epítopos/inmunología , Antígenos HLA-D/inmunología , Cadenas beta de HLA-DP/inmunología , Histocompatibilidad/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Aloinjertos , Antígenos de Diferenciación de Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Endosomas/metabolismo , Epítopos/metabolismo , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Células HeLa , Trasplante de Células Madre Hematopoyéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Histocompatibilidad/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Espectrometría de Masas , Chaperonas Moleculares , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Donante no EmparentadoRESUMEN
BACKGROUND: We investigated T cell clonality (TCC) and T cell fraction (TCF) in human papilloma virus associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) progressors [cases] vs. non-progressors [controls]. METHODS: This nested case-control study included patients undergoing intent-to-cure surgery ± adjuvant therapy from 6/1/2007-10/3/2016. Patients experiencing local/regional/distant disease (progressors), and a consecutive sample of non-progressors were matched (2 controls: 1 case) on tumor subsite, T-stage and number of metastatic lymph nodes. We performed imunosequencing of the CDR3 regions of human TCRß chains. RESULTS: 34 progressors and 65 non-progressors were included. There was no statistically significant difference in baseline TCF (range: 0.039-1.084) and TCC (range: 0.007-0.240) (p > 0.05). Female sex was associated with higher TCF (p = 0.03), while extranodal extension (ENE) was associated with lower TCF (p = 0.01). There was a positive correlation between tumor size and clonality (R = 0.34, p < 0.01). The strongest predictor of progression-free survival (PFS) was TCF (HR 0.80, 95%CI 0.66-0.96, p = 0.02). The strongest predictors of cancer specific survival (CSS) were TCF (HR0.69, 95%CI 0.47-1.00, p < 0.05) and Adult Comorbidity Evaluation-27 (ACE-27) score (p < 0.05). Similarly, the strongest predictors of overall survival (OS) were TCF (HR 0.62, 95%CI 0.43-0.91, p = 0.01) and ACE-27 score (p = 0.03). On multivariable modeling, TCF ≥ 0.4 was independently associated with PFS (HR 0.34, 95%CI 0.14-0.85, p = 0.02) while an ACE-27 score of ≥ 2 independently predicted CSS (HR 3.85, 95%CI 1.07-13.85, p = 0.04) and OS (HR 3.51, 95%CI 1.10-11.20, p = 0.03). CONCLUSIONS: In patients with HPV(+)OPSCC, TCF was higher in female patients and those without ENE, suggesting differential immune responses. Lower TCF was significantly and independently associated with disease progression. Better ACE-27 scores appear to predict improved oncologic control.
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Alphapapillomavirus/inmunología , Neoplasias Orofaríngeas/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Linfocitos T/citología , Microambiente Tumoral/inmunología , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/inmunología , Papillomavirus Humano 16/inmunología , Humanos , Estimación de Kaplan-Meier , Ganglios Linfáticos/patología , Metástasis Linfática , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Orofaríngeas/mortalidad , Neoplasias Orofaríngeas/patología , Neoplasias Orofaríngeas/virología , Supervivencia sin Progresión , Factores Sexuales , Carcinoma de Células Escamosas de Cabeza y Cuello/mortalidad , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Carga TumoralRESUMEN
Deep sequencing of T-cell receptor (TCR) genes is powerful at profiling immune repertoire. To prepare a TCR sequencing library, multiplex polymerase chain reaction (mPCR) is widely applied and is highly efficient. That is, most mPCR products contain the region critical for antigen recognition, which also indicates regular V(D)J recombination. Multiplex PCR, however, may suffer from primer bias. A promising alternative is 5'-RACE, which avoids primer bias by applying only one primer pair. In 5'-RACE data, however, non-regular V(D)J recombination (e.g., TCR sequences without a V gene segment) has been observed and the frequency varies (30-80%) between studies. This suggests that the cause of or how to reduce non-regular TCR sequences is not yet well known by the science community. Although it is possible to speculate the cause by comparing the 5'-RACE protocols, careful experimental confirmation is needed and such a systematic study is still not available. Here, we examined the 5'-RACE protocol of a commercial kit and demonstrated how a modification increased the fraction of regular TCR-ß sequences to >85%. We also found a strong linear correlation between the fraction of short DNA fragments and the percentage of non-regular TCR-ß sequences, indicating that the presence of short DNA fragments in the library was the main cause of non-regular TCR-ß sequences. Therefore, thorough removal of short DNA fragments from a 5'-RACE library is the key to high data efficiency. We highly recommend conducting a fragment length analysis before sequencing, and the fraction of short DNA fragments can be used to estimate the percentage of non-regular TCR sequences. As deep sequencing of TCR genes is still relatively expensive, good quality control should be valuable.
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Secuencia de Aminoácidos/genética , ADN/genética , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Secuencia de Bases , Fragmentación del ADN , ADN Complementario/genética , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/inmunología , Humanos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunologíaRESUMEN
BACKGROUND: Human T-lymphotropic virus-1 (HTLV-1)+ Hodgkin lymphoma (HL) is difficult to differentiate from adult T-cell leukemia/lymphoma (ATLL) with HL-like histology (HL-like ATLL). METHODS: Cytological and immunohistological features, HTLV-1 proviral DNA integration, and rearrangements of the T-cell receptor (TCR) Cß1 gene were examined in 11 HTLV-1+ patients with HL-like disease. RESULTS: Six patients were classified as HTLV-1+ HL and five as HL-like ATLL in accordance with genetic findings of HTLV-1 proviral DNA integration and rearrangements of the TCR Cß1 gene. Small ordinary looking lymphocytes with round nuclei were detected in the background of six patients with HTLV-1+ HL, which were immunohistochemically negative for CD25 and CC chemokine receptor (CCR)4 and had a low MIB1 labeling index (mean: 28.3%). In the HL-like ATLL specimens, small- and medium-sized atypical lymphocytes with indented and irregular-shaped nuclei were found, and were diffusely positive for CD25 and CCR4, with high MIB1 labeling (mean: 76%). Both groups had scattered CD30+ and CD15+ Hodgkin and Reed Sternberg (RS) giant cells, with or without CD20 expression and Epstein-Barr virus infection. The 50% overall survival period was significantly longer for the HTLV-1+ HL group (180 months) than for the HL-like ATLL group (7.8 months; P = .004). CONCLUSIONS: HTLV-1+ HL showed typical small lymphoid cells with a low MIB1 labeling index in a background of Hodgkin and RS cells, with some scattered CD25+ and CCR4+ lymphocytes. In HTLV-1 endemic areas, distinguishing HTLV-1+ HL from HL-like ATLL is important because of their differing treatment strategies and prognoses.
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Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Enfermedad de Hodgkin , Virus Linfotrópico T Tipo 1 Humano , Leucemia-Linfoma de Células T del Adulto , Adulto , Anciano , Antígenos CD20/metabolismo , Tamaño de la Célula , ADN Viral , Infecciones por Virus de Epstein-Barr , Femenino , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/mortalidad , Enfermedad de Hodgkin/patología , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/mortalidad , Leucemia-Linfoma de Células T del Adulto/patología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Masculino , Persona de Mediana Edad , Células de Reed-Sternberg , Integración Viral/genéticaRESUMEN
T-cell receptor gene beta (TCRß) gene rearrangement represents a complex, tightly regulated molecular mechanism involving excision, deletion and recombination of DNA during T-cell development. RUNX1, a well-known transcription factor for T-cell differentiation, has recently been described to act in addition as a recombinase cofactor for TCRδ gene rearrangements. In this work we employed a RUNX1 knock-out mouse model and demonstrate by deep TCRß sequencing, immunostaining and chromatin immunoprecipitation that RUNX1 binds to the initiation site of TCRß rearrangement and its homozygous inactivation induces severe structural changes of the rearranged TCRß gene, whereas heterozygous inactivation has almost no impact. To compare the mouse model results to the situation in Acute Lymphoblastic Leukemia (ALL) we analyzed TCRß gene rearrangements in T-ALL samples harboring heterozygous Runx1 mutations. Comparable to the Runx1+/- mouse model, heterozygous Runx1 mutations in T-ALL patients displayed no detectable impact on TCRß rearrangements. Furthermore, we reanalyzed published sequence data from recurrent deletion borders of ALL patients carrying an ETV6-RUNX1 translocation. RUNX1 motifs were significantly overrepresented at the deletion ends arguing for a role of RUNX1 in the deletion mechanism. Collectively, our data imply a role of RUNX1 as recombinase cofactor for both physiological and aberrant deletions.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Eliminación de Gen , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas c-ets/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Proteínas Represoras/genética , Animales , Linfocitos B , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Recuento de Linfocitos , Ratones Noqueados , Linfocitos T , Timo/patología , Proteína ETS de Variante de Translocación 6RESUMEN
Polymerase chain reaction (PCR) analysis of rearranged T-cell receptor (TCR) genes is a valuable diagnostic tool for differential diagnosis of T-cell large granular lymphocytic (T-LGL) leukemia and reactive lymphocytosis. Age-related narrowing of T-cells repertoire and expansion of immune or autoimmune clones may lead to false-positive results. The objective of this study was to evaluate the specificity and positive predictive value of PCR-based clonality assessment for a differential diagnostics of T-LGL leukemia. Rearrangements of TCRG and TCRB genes using the BIOMED-2 protocol were assessed in healthy individuals including the elderly (n = 62) and patients with rheumatic diseases (n = 14), transitory reactive CD8+ lymphocytosis (n = 17), and T-LGL leukemia (n = 42). Monoclonal TCRG/TCRB rearrangements in blood were identified in 11.3%/4.8% (7/3 of 62) of healthy individuals; 21.4%/14.3% (3/2 of 14) of patients with rheumatic diseases, and 17.6%/11.8% (3/2 of 17) of patients with reactive lymphocytosis. Immunomagnetic selection of lymphocytes in healthy individuals (31 of 33) revealed that clonal T-cells belong to CD8+ and CD57+ population. No clonal Vß-Jß TCRB rearrangements were found in the control group, only Dß-Jß TCRB and TCRG. Given the high detectability (96.7%) of Vß-Jß TCRB monoclonal rearrangements in patients with αß-T-LGL leukemia, this marker had the greatest specificity and positive predictive value (100%; 99.2%). The presence of clonal CD8+CD57+ cells in blood is common for healthy individuals and patients with reactive conditions and may not associate with any malignancy. Different specificity of TCRG/ Dß-Jß TRB/ Vß-Jß TCRB PCR reactions should be taken into account for T-cell clonality data interpretation.
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Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Neoplasias Hematológicas , Reacción en Cadena de la Polimerasa , Adolescente , Adulto , Anciano , Linfocitos T CD8-positivos/patología , Diagnóstico Diferencial , Femenino , Estudios de Seguimiento , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patología , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Estudios RetrospectivosRESUMEN
RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development, from early CD7-CD5- to CD4+CD8+. The impaired differentiation was accompanied by an increase in CD7-CD56+CD33+ natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells, whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements, normalized T cell development, and corrected the NK cell-like phenotype. In conclusion, we succeeded in generating an iPSC-based RAG2-SCID model, which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks.
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Proteínas de Unión al ADN/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/patología , Linfocitos T/inmunología , Animales , Antígenos CD/metabolismo , Diferenciación Celular , Linaje de la Célula , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Hematopoyesis , Humanos , Células Asesinas Naturales/inmunología , Ratones SCIDRESUMEN
The domestic ferret, Mustela putorius furo, is an important mammalian animal model to study human respiratory infection. However, insufficient genomic annotation hampers detailed studies of ferret T cell responses. In this study, we analyzed the published T cell receptor beta (TRB) locus and performed high-throughput sequencing (HTS) of peripheral blood of four healthy adult ferrets to identify expressed V, D, J, and C genes. The HTS data is used as a guide to manually curate the expressed V, D, J, and C genes. The ferret locus appears to be most similar to that of the dog. Like other mammalian TRB loci, the ferret TRB locus contains a library of variable genes located upstream of two D-J-C gene clusters, followed by a (in the ferret non-functional) V gene with an inverted transcriptional orientation. All TRB genes (expressed or not) reported here have been approved by the IMGT/WHO-IUIS nomenclature committee.
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Regulación de la Expresión Génica , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Animales , Hurones , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
PURPOSE: NCT01725165 was a phase II prospective trial in which patients with non-small cell lung cancer were randomized to local consolidative therapy (LCT) versus maintenance therapy or observation (MT/O). METHODS AND MATERIALS: Peripheral blood from patients enrolled on NCT01725165 were labeled as (1) baseline, (2) early follow-up (FU) if obtained in the first or second FU evaluation (6-18 weeks), and (3) late FU if obtained in the third to sixth FU evaluations (22-50 weeks). All patients who underwent LCT and were included in this analysis received radiation. Among 49 randomized patients, 21 patients underwent T cell CDR3 variable region sequencing using immunoSEQ, 31 patients underwent circulating tumor DNA (ctDNA) analysis using next-generation sequencing with a 1021 cancer gene panel, and cytokine concentration was assayed in 19 patients using enzyme-linked immunosorbent assay. All analyses were exploratory and not corrected for multiple testing. RESULTS: No associations were identified between baseline T cell repertoire and ctDNA metrics with patient outcomes. Among baseline cytokines, interleukin 1α was the only cytokine associated with both overall survival (hazard ratio, 0.02; 95% confidence interval, 0.1-0.5; P = .0006) and progression-free survival (hazard ratio, 0.5; 95% confidence interval, 0.2-0.9; P = .03). At early FU, LCT was associated with decreased ctDNA burden, including lower number of detected mutations (median, 2 [interquartile range {IQR}, 1-6] vs 6 [IQR, 4-18]) and decreased average variable allele frequency (VAF; median, 0.006 [IQR, 0.003-0.010] vs 0.011 [IQR, 0.007-0.014]) compared with MT/O. Among 6 patients with serial ctDNA analysis, a rise in ctDNA detected mutation burden preceded clinical progression by 6.7 months. At early FU, LCT was associated with changes in T cell clonality that suggested oligoclonal expansion specifically increased T cell clonality (median, 0.15 [IQR, 0.12-0.24] vs 0.10 [IQR, 0.05-0.13]) and frequency of top 10 clones (median, 0.14 [IQR, 0.06-0.18] vs 0.21[IQR, 0.19-0.28]). CONCLUSION: LCT was associated with decreased ctDNA burden and oligoclonal expansion at early FU timepoints. Baseline interleukin 1α was associated with improved patient outcomes.
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Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , ADN Tumoral Circulante/sangre , Citocinas/sangre , Neoplasias Pulmonares/radioterapia , Alelos , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , ADN Tumoral Circulante/genética , Dermatoglifia del ADN , Ensayo de Inmunoadsorción Enzimática , Estudios de Seguimiento , Perfilación de la Expresión Génica/métodos , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Mutación , Supervivencia sin Progresión , Estudios Prospectivos , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/efectos de la radiación , Factores de Tiempo , Investigación Biomédica Traslacional , Espera VigilanteRESUMEN
Signal strength controls the outcome of αß T cell selection in the thymus, resulting in death if the affinity of the rearranged TCR is below the threshold for positive selection, or if the affinity of the TCR is above the threshold for negative selection. Here we show that deletion of the GTPase RRAS2 results in exacerbated negative selection and above-normal expression of positive selection markers. Furthermore, Rras2-/- mice are resistant to autoimmunity both in a model of inflammatory bowel disease (IBD) and in a model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). We show that MOG-specific T cells in Rras2-/- mice have reduced affinity for MOG/I-Ab tetramers, suggesting that enhanced negative selection leads to selection of TCRs with lower affinity for the self-MOG peptide. An analysis of the TCR repertoire shows alterations that mostly affect the TCRα variable (TRAV) locus with specific VJ combinations and CDR3α sequences that are absent in Rras2-/- mice, suggesting their involvement in autoimmunity.