RESUMEN
The production of T cell receptor αß(+) (TCRαß(+) ) T lymphocytes in the thymus is a tightly regulated process that can be monitored by the regulated expression of several surface molecules, including CD4, CD8, cKit, CD25 and the TCR itself, after TCR genes have been assembled from discrete V, D (for TCR-ß) and J gene segments by a site-directed genetic recombination. Thymocyte differentiation is the result of a delicate balance between cell death and survival: developing thymocytes die unless they receive a positive signal to proceed to the next stage. This equilibrium is altered in response to various physiological or physical stresses such as ionizing radiation, which induces a massive p53-dependent apoptosis of CD4(+) CD8(+) double-positive (DP) thymocytes. Interestingly, these cells are actively rearranging their TCR-α chain genes. To unravel an eventual link between V(D)J recombination activity and thymocyte radio-sensitivity, we analysed the dynamics of thymocyte apoptosis and regeneration following exposure of wild-type and p53-deficient mice to different doses of γ-radiation. p53-dependent radio-sensitivity was already found to be high in immature CD4(-) CD8(-) (double-negative, DN) cKit(+) CD25(+) thymocytes, where TCR-ß gene rearrangement is initiated. However, TCR-αß(-) CD8(+) immature single-positive thymocytes, an actively cycling intermediate population between the DN and DP stages, are the most radio-sensitive cells in the thymus, even though their apoptosis is only partially p53-dependent. Within the DP population, TCR-αß(+) thymocytes that completed TCR-α gene recombination are more radio-resistant than their TCR-αß(-) progenitors. Finally, we found no correlation between p53 activation and thymocyte sensitivity to radiation-induced apoptosis.
Asunto(s)
Apoptosis/genética , Apoptosis/efectos de la radiación , Timocitos/metabolismo , Timocitos/efectos de la radiación , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis/inmunología , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Regulación de la Expresión Génica/efectos de la radiación , Inmunofenotipificación , Ratones , Ratones Noqueados , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/efectos de la radiación , Timocitos/inmunología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
T cell receptor genes are assembled in developing T lymphocytes from discrete V, D, and J genes by a site-specific somatic rearrangement mechanism. A flanking recombination signal, composed of a conserved heptamer and a semiconserved nonamer separated by 12 or 23 variable nucleotides, targets the activity of the rearrangement machinery to the adjoining V, D, and J genes. Following the rearrangement of V, D, or J genes, their respective recombination signals are ligated together. Although these signal joints are allegedly invariant, created by the head-to-head abuttal of the heptamers, some do exhibit junctional diversity. Recombination signals were initially identified by comparison and alignment of germ-line sequences with the sequence of rearranged genes. However, their overall low level of sequence conservation makes their characterization solely from sequence data difficult. Recently, computational analysis unraveled correlations between nucleotides at several positions scattered within the spacer and recombination activity, so that it is now possible to identify putative recombination signals and determine and predict their recombination efficiency. In this paper, we analyzed the variability introduced in signal joints generated after rearrangement of the TRDD1 and TRDD2 genes in murine thymocytes. The recurrent presence of identical nucleotides inserted in these signal joints led us to reconsider the location and sequence of the TRDD1 recombination signal. By combining molecular characterization and computational analysis, we show that the functional TRDD1 recombination signal is shifted inside the putative coding sequence of the TRDD1 gene and, consequently, that this gene is shorter than indicated in the databases.
Asunto(s)
Reordenamiento Génico de Linfocito T/genética , Proteínas/genética , Animales , Secuencia de Bases , Ratones , Datos de Secuencia Molecular , Recombinación Genética/genética , Análisis de Secuencia de ADN , Timo/inmunologíaAsunto(s)
Enfermedades Desmielinizantes/virología , Retrovirus Endógenos/inmunología , Productos del Gen env/inmunología , Esclerosis Múltiple/virología , Proteínas Gestacionales/inmunología , Animales , Enfermedades Desmielinizantes/inmunología , Humanos , Esclerosis Múltiple/inmunología , Superantígenos/inmunologíaRESUMEN
A retroviral element (multiple sclerosis-associated retrovirus, MSRV) defining a family of genetically inherited endogenous retroviruses (human endogenous retrovirus type W, HERV-W) has been characterized in cell cultures from patients with multiple sclerosis. Recently, MSRV retroviral particles or the envelope recombinant protein were shown to display superantigen activity in vitro, but no animal model has yet been set up for studying the pathogenicity of this retrovirus. In the present study, the pathogenicity of different sources of MSRV retroviral particles has been evaluated in a hybrid animal model: severe combined immunodeficiency (SCID) mice grafted with human lymphocytes and injected intraperitoneally with MSRV virion or mock controls. MSRV-injected mice presented with acute neurological symptoms and died within 5 to 10 days post injection. Necropsy revealed disseminated and major brain hemorrhages, whereas control animals did not show abnormalities (P <.001). In ill animals, reverse transcriptase-polymerase chain reaction (RT-PCR) analyses showed circulating MSRV RNA in serum, whereas overexpression of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma was evidenced in spleen RNA. Neuropathological examination confirmed that hemorrhages occurred prior to death in multifocal areas of brain parenchyma and meninges. Further series addressed the question of immune-mediated pathogenicity, by inoculating virion to SCID mice grafted with total and T lymphocyte-depleted cells in parallel: dramatic and statistically significant reduction in the number of affected mice was observed in T-depleted series (P <.001). This in vivo study suggests that MSRV retroviral particles from MS cultures have potent immunopathogenic properties mediated by T cells compatible with the previously reported superantigen activity in vitro, which appear to be mediated by an overexpression of proinflammatory cytokines.
Asunto(s)
Hemorragia Cerebral/virología , Retrovirus Endógenos/aislamiento & purificación , Esclerosis Múltiple/virología , Linfocitos T/virología , Animales , Linfocitos B/citología , Linfocitos B/virología , Barrera Hematoencefálica/inmunología , Muerte Celular/inmunología , Células Cultivadas , Hemorragia Cerebral/inmunología , Plexo Coroideo/citología , Plexo Coroideo/virología , Citocinas/genética , Modelos Animales de Enfermedad , Retrovirus Endógenos/patogenicidad , Expresión Génica , Humanos , Ratones , Ratones SCID , Esclerosis Múltiple/inmunología , Bazo/fisiología , Bazo/virología , Superantígenos/inmunología , Linfocitos T/citología , Virión , VirulenciaRESUMEN
ABBREVIATIONS: Intrahepatic lymphocytes are believed to be involved in the immunopathogenesis of hepatitis C virus (HCV) infection and the evolution of HCV-induced hepatitis. In the present study, we examined the three main intrahepatic lymphocyte subsets, namely CD3+CD56- conventional T lymphocytes, CD3+CD56+ natural T (NT) lymphocytes and CD3-CD56+ natural killer (NK) lymphocytes in HCV-infected patients. The proportion of each lymphocyte subset was evaluated both in liver biopsies and in samples of peripheral blood lymphocytes (PBL) by flow cytometry in 21 patients with histologically proven chronic hepatitis C. Simultaneously, alanine aminotransferase (ALT) levels, viral load and histological lesions were assessed. Neither NT nor NK populations correlated with any biochemical, viral or histological parameters. Furthermore, Valpha24+ NT lymphocytes showed no preferential enrichment in the liver of HCV-infected patients. Regarding conventional T lymphocytes, a highly significant linear correlation was found between intrahepatic CD3+CD56- T lymphocytes and the Knodell score, a numerical score for assessing histological activity and fibrosis (r = 0.715, P < 0.0001) and more specifically with the periportal necrosis parameter, which is the main lesion of chronic hepatitis C. In addition, analysis of the peripheral compartment revealed a high correlation between values of CD3+CD56- lymphocytes and both Knodell score (r = 0.624, P = 0.003) and serum ALT levels and again with periportal necrosis. The strong correlation between the proportion of peripheral CD3+CD56- conventional T lymphocytes and the severity of hepatic lesions leads us to propose that evaluation of this accessible peripheral population could be used as an indicator test for the severity of histological lesions in chronic hepatitis C.
Asunto(s)
Hepacivirus , Hepatitis C Crónica/inmunología , Hígado/inmunología , Subgrupos Linfocitarios/inmunología , Adulto , Anciano , Alanina Transaminasa/sangre , Complejo CD3/análisis , Antígeno CD56/análisis , Femenino , Citometría de Flujo , Hepatitis C Crónica/patología , Hepatitis C Crónica/virología , Humanos , Células Asesinas Naturales/inmunología , Hígado/patología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Linfocitos T/inmunología , Carga ViralRESUMEN
During thymocyte differentiation, TCRA genes are massively rearranged only after productively rearranged TCRB genes are expressed in association with pTalpha and CD3 complex molecules within a pre-TCR. Signaling from the pre-TCR via the CD3 complex is thought to be required to promote TCRA gene accessibility and recombination. However, alphabeta(+) thymocytes do develop in pTalpha-deficient mice, showing that TCRalpha-chain genes are rearranged, either in CD4(-)CD8(-) or CD4(+)CD8(+) thymocytes, in the absence of pre-TCR expression. In this study, we analyzed the TCRA gene recombination status of early immature thymocytes in mutant mice with arrested thymocyte development, deficient for either CD3 or pTalpha and gammac expression. ADV genes belonging to different families were found rearranged to multiple AJ segments in both cases. Thus, TCRA gene rearrangement is independent of CD3 and gammac signaling. However, CD3 expression was found to play a role in transcription of rearranged TCRalpha-chain genes in CD4(-)CD8(-) thymocytes. Taken together, these results provide new insights into the molecular control of early T cell differentiation.
Asunto(s)
Complejo CD3/metabolismo , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Células Madre Hematopoyéticas/citología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/citología , Timo/citología , Animales , Diferenciación Celular , Células Madre Hematopoyéticas/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Mutantes , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Recombinación Genética , Transducción de Señal , Linfocitos T/inmunología , Timo/inmunologíaRESUMEN
A retroviral element (MSRV) defining a family of genetically inherited endogenous retroviruses (HERV-W) has recently been characterized in cell cultures from patients with multiple sclerosis (MS). To address the possible relationship with MS, direct detection of circulating virion RNA was proposed but revealed technically difficult to perform in standardized conditions, in the face of multiple endogenous HERV-W copies. A parallel approach has evaluated MSRV potential pathogenicity in relation to characteristic features of multiple sclerosis, in particular, T-lymphocyte-mediated immunopathology. We report here that MSRV particles induce T-lymphocyte response with a bias in the Vbeta16 chain usage in surface receptor, whatever the HLA DR of the donor. A recombinant MSRV envelope-but not core-protein reproduced similar nonconventional activation. Molecular analysis of Vbeta CDR3 showed that Vbeta16 expansions are polyclonal. Our results thus provide evidence that MSRV envelope protein can trigger an abnormal immune response with similar characteristics to that of superantigens.
Asunto(s)
Retrovirus Endógenos/inmunología , Activación de Linfocitos/inmunología , Esclerosis Múltiple/virología , Infecciones por Retroviridae/inmunología , Linfocitos T/inmunología , Proteínas del Envoltorio Viral/inmunología , Antígenos Virales/inmunología , Células Cultivadas , Citocinas/metabolismo , Retrovirus Endógenos/genética , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Esclerosis Múltiple/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Proteínas Recombinantes/inmunología , Infecciones por Retroviridae/virología , Linfocitos T/metabolismo , Células Tumorales Cultivadas , Virión/inmunologíaRESUMEN
TCRalpha and TCRdelta chains are coded by a common genetic locus using a single set of V gene segments (ADV segments). This article addresses the question of regulation of the use of the ADV segments by the TCRalpha and TCRdelta chains. Using both qualitative and quantitative analyses we have studied the use of 23 ADV gene families as part of TCRalpha and TCRdelta transcripts. A number of previously undetected rearrangement and transcription events are described, indicating that the intrathymic TCRdelta repertoire is much more diverse than previously supposed. Repertoire analysis at several developmental time points allowed the description of regulated waves of ADV gene use, not only for TCRdelta chains, but also for TCRalpha chains, during thymic ontogeny. Control of these waves appears to be linked directly to the ADV segments and their local chromatin environment, which may change over the course of T cell differentiation.
Asunto(s)
Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T/inmunología , Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Timo/citología , Timo/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Frecuencia de los Genes/inmunología , Ratones , Ratones Endogámicos BALB C , Familia de Multigenes/inmunología , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/aislamiento & purificación , Receptores de Antígenos de Linfocitos T gamma-delta/biosíntesis , Receptores de Antígenos de Linfocitos T gamma-delta/aislamiento & purificación , Timo/inmunología , Transcripción Genética/inmunologíaRESUMEN
The gene coding for a T-cell receptor (TCR) alpha chain is assembled from variable (ADV) and joining (AJ) genes located on Chromosome 14. Each of the 90 ADV genes can rearrange with any one of the 61 AJ genes. We have previously demonstrated that ADV and AJ gene segment use evolves with time, with a progressive opening of ADV and AJ regions of the locus. To define the rules governing the use of AJ genes by ADV genes belonging to one family, we carried out a detailed analysis of 268 combinations of ADV2 BALB/c transcripts. We found that the different ADV2 members use different sets of AJ genes depending on their location within the ADV locus: ADV2S7 (the most AJ proximal ADV2 member) rearranges mainly with the AJ genes located close to the TEA element, whereas 50% of the sequences for ADV2S8, which is distal to the AJ locus, use the most distal AJ genes. ADV2S5, an ADV2 member located in the middle of the ADV locus, is associated with a wider set of AJ genes, located in the center of the AJ locus. Taken together, our results indicate that, in addition to the progressive opening of the ADV and AJ loci, the chromosomal location of ADV and AJ genes is a factor affecting AJ use in BALB/c mice.
Asunto(s)
Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T/genética , Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T/genética , Recombinación Genética/genética , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Clonación Molecular , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia de ADN , Timo/citología , Timo/metabolismoRESUMEN
Superantigens (SAg) are proteins of bacterial or viral origin able to activate T cells by forming a trimolecular complex with both MHC class II molecules and the T cell receptor (TCR), leading to clonal deletion of reactive T cells in the thymus. SAg interact with the TCR through the beta chain variable region (Vbeta), but the TCR alpha chain has been shown to have an influence on the T cell reactivity. We have investigated here the role of the TCR alpha chain in the modulation of T cell reactivity to Mtv-7 SAg by comparing the peripheral usage of Valpha2 in Vbeta6(+) (SAg-reactive) and Vbeta8.2(+) (SAg non-reactive) T cells, in either BALB/D2 (Mtv-7(+)) or BALB/c (Mtv-7(-)) mice. The results show, first, that pairing of Vbeta6 with certain Valpha2 family members prevents T cell deletion by Mtv-7 SAg. Second, there is a strikingly different distribution of the Valpha2 family members in CD4 and CD8 populations of Vbeta6 but not of Vbeta8.2 T cells, irrespective of the presence of Mtv-7 SAg. Third, the alpha chain may play a role in the overall stability of the TCR/SAg/MHC complex. Taken together, these results suggest that the Valpha domain contributes to the selective process by its role in the TCR reactivity to SAg/MHC class II complexes, most likely by influencing the orientation of the Vbeta domain in the TCR alphabeta heterodimer.
Asunto(s)
Supresión Clonal , Glicoproteínas de Membrana/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Superantígenos/inmunología , Linfocitos T/inmunología , Animales , Antígenos Virales , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , 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 II/inmunología , Ratones , Ratones Endogámicos BALB C , Modelos MolecularesRESUMEN
INTRODUCTION: Non-myeloablative peripheral stem cell transplantation has been shown to induce tumour rejection in patients with acute leukaemia. However, the immunological mechanisms involved and the immune reconstitution achieved have not been investigated. MATERIALS AND METHODS: We describe the cases of two patients for whom we have studied the lymphocyte reconstitution achieved, using both phenotypic and genetic analyses of the T-cell repertoire, after peripheral stem cell transplantation. RESULTS: : In both cases we observed immune reconstitution with T-cell repertoire evolution and presence of activated CD8(+) T cells. In one of the patients an activated clone expressing Vbeta8 represents 46% of the CD8(+) cells. Expansion of this clone occurred in the absence of graft vs host disease symptoms. In the second case a skin lesion typical of graft vs host disease appeared after complete remission had been achieved. The T-cell repertoire in a biopsy of the lesion was distinct from that observed in the blood. CONCLUSION: Our study indicates that peripheral donor cells can effectively reconstitute a grafted patient while inducing an immune response against antigens expressed by the leukaemic/myeloma cells. Our data provide arguments for different populations of T cells associated with graft vs leukaemia/lymphoma and GVH effects.
Asunto(s)
Sangre/inmunología , Trasplante de Células Madre Hematopoyéticas , Inmunoterapia/métodos , Piel/citología , Enfermedad Aguda , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Células Sanguíneas/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Carmustina/administración & dosificación , Células Clonales/inmunología , Ciclofosfamida/administración & dosificación , Citarabina/administración & dosificación , Daunorrubicina/administración & dosificación , Dexametasona/administración & dosificación , Doxorrubicina/administración & dosificación , Femenino , Supervivencia de Injerto , Reacción Injerto-Huésped/inmunología , Efecto Injerto vs Leucemia/inmunología , Humanos , Inmunofenotipificación , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/terapia , Masculino , Persona de Mediana Edad , Repeticiones de Minisatélite , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/terapia , Prednisona/administración & dosificación , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Piel/inmunología , Subgrupos de Linfocitos T/citología , Acondicionamiento Pretrasplante , Trasplante Homólogo/inmunología , Vincristina/administración & dosificación , Irradiación Corporal TotalRESUMEN
Pre-TCR expression on developing thymocytes allows cells with productive TCRB gene rearrangements to further differentiate. In wild-type mice, most TCRA gene rearrangements are initiated after pre-TCR expression. However, in pTalpha-deficient mice, a substantial number of alphabeta+ thymocytes are still produced, in part because early TCR alpha-chain expression can rescue immature thymocytes from cell death. In this study, the nature of these TCR alpha-chains, produced and expressed in the absence of pre-TCR expression, have been analyzed. We show, by FACS analysis and sequencing of rearranged transcripts, that the TCRA repertoire is diverse in pTalpha-/- mice and that the developmental regulation of AJ segment use is maintained, yet slightly delayed around birth when compared with wild-type mice. We also found that T cell differentiation is more affected by pTalpha inactivation during late gestation than later in life. These data suggest that the pre-TCR is not functionally required for the initiation and regulation of TCRA gene rearrangement and that fetal thymocytes are more dependent than adult cells on pTalpha-derived signals for their differentiation.
Asunto(s)
Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T , Glicoproteínas de Membrana/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/deficiencia , Timo/crecimiento & desarrollo , Animales , Diferenciación Celular , Clonación Molecular , Citometría de Flujo , Ratones , Ratones Mutantes , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Transducción de Señal , Subgrupos de Linfocitos T/citología , Timo/citología , Timo/embriologíaRESUMEN
The CD3 signaling complex is required for cell surface expression and selection of both alphabeta and gammadelta TCR. In this study we analyzed TCRD transcripts in both wild-type and CD3-epsilon-deficient mice. We show that the repertoire of ADV segments used by the delta chain is unchanged in the latter. Not all ADV genes participate in making up the TCRD repertoire. However, their use does not depend on their distance from the other TCRD-forming segments. For example ADV12, situated at more than 870 kb from the DD region, is expressed as part of TCRD transcripts, whereas ADV8, members of which are proximal to the DD region, is not. These data suggest that the accessibility of ADV8 gene segments is differentially regulated during T cell development in the thymus. Taken together, our results suggest that TCRA and TCRD rearrangements are independently controlled, and that the absence of TCRA expression in CD3-epsilon-deficient mice is not due to a lack of accessibility of the ADV gene segments but rather to inaccessibility of the AJ gene region.
Asunto(s)
Complejo CD3/fisiología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Linfocitos T/fisiología , Animales , Mapeo Cromosómico , Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Familia de MultigenesRESUMEN
The genes coding for TCR alpha and delta chains share the same genetic locus (TCRA/D). The rules governing the utilization of a V gene with the alpha and delta chains have not been established. More specifically, it is not known whether the position of a gene within the locus influences its utilization in alpha and delta TCR. To elucidate these points, we mapped ADV2 genes in the TCRA/D locus of BALB/c mice and analyzed their utilization in TCR alpha and delta transcripts from thymi isolated from mice of different ages. Our results show that all ADV2 genes can be used by the two chains, but with strikingly different patterns. Moreover, ADV2 utilization by the alpha chain proceeds in successive concentric waves during development, suggesting a progressive regulation of gene accessibility and utilization. These results support independent control of TCRA and TCRD gene assembly.
Asunto(s)
Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T/genética , Genes Codificadores de la Cadena delta de los Receptores de Linfocito T/genética , Animales , Southern Blotting , Mapeo Cromosómico , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
The number of mouse Tcra-V gene segments varies from one individual to another and is estimated to be about 100. Southern blot analysis revealed that most of the Tcra-V are organized in clusters composed of copies of Tcra-V belonging to different subfamilies. We analyzed in detail a Tcra-V subfamily and looked for new Tcra-V in order to improve the knowledge of the mouse Tcra locus organization. A series of genomic clones derived from the B10.A mouse strain enclosing these clusters was used to determined the structure of all the Tcra-V2. We were able to identify ten Tcra-V2. This study showed that the Tcra-V2 can be organized into three structural subgroups. The distribution of the genes along the Tcra locus, plus their structural organization, indicates that successive duplications occurred during the processes of expansion and contraction of the Tcra-V gene subfamilies. Several Tcra-V2 are also identical, indicating recent duplications. The most divergent Tcra-V2 differ by 7.4% nucleotides, leading to 5.2% differences in amino acid contents.
Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Mapeo RestrictivoRESUMEN
When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) V beta segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, -7, -8 and -9) and 38CH (Mtv-) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, -7, -8 or -9. The TcR V beta chain (TcR V beta) usage in these mice was analyzed using monoclonal antibodies specific for TcR V beta 2, V beta 3, V beta 4, V beta 5, V beta 6, V beta 7, V beta 8, V beta 11, V beta 12 and V beta 14 segments. Both Mtv-8+ mice and Mtv-9+ mice deleted TcR V beta 5+ and V beta 11+ T cells. Moreover, we also observed the deletion of TcR V beta 12+ cells by Mtv-8 and Mtv-9 products. Mtv-6+ and Mtv-7+ animals deleted TcR V beta 3+ and V beta 5+ cells, and TcR V beta 6+, V beta 7+ and V beta 8.1+ cells, respectively. Unexpectedly, TcR V beta 8.2+ cells were also deleted in some backcross mice expressing Mtv-7. TcR V beta 8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn-->Asp) in position 19 on the TcR V beta 8.2 fragment. Reactivities of BALB.D2 TcR V beta 8.2 and 38CH TcR V beta 8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR V beta 2+, V beta 4+ and V beta 8+ CD4+ T cell subsets in Mtv-8+ and Mtv-9+ mice, and TcR V beta 4+ CD4+ T cells in Mtv-6+ and Mtv-7+ mice, when compared with the T cell repertoire of Mtv- mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.
Asunto(s)
Virus del Tumor Mamario del Ratón/inmunología , Provirus/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Alelos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Southern Blotting , Clonación Molecular , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Ratones Mutantes , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Superantígenos/inmunología , Linfocitos T/inmunologíaRESUMEN
The nucleocapsid (NC) of the rabies virus behaves as an exogenous superantigen (SAg) in humans. In the present report, we analyzed whether it is also a SAg in mice by studying the effect of NC on T cell receptor (TCR) V beta expression in BALB/c mice. Repeated injection of NC in newborn BALB/c mice led to a marked reduction by two- to sixfold of V beta 6 expressing CD4+ T cells in spleen and in peripheral blood. Decrease of V beta 6-expressing CD3+ mature T cells was also observed in thymus. Single NC injection in footpad resulted in a three- to sixfold expansion of V beta 6 CD4+ T cells, but not of CD8+ T cells, in the draining lymph nodes of BALB/c mice. The intensity of the stimulation was dose dependent and was maximal 3 d after the NC injection. The clonal deletion of T cells bearing a particular V beta demonstrates that NC is a SAg in mice. T cells, especially CD4+ T cells, are an essential factor in host resistance to rabies virus and also in the pathophysiology of paralysis; thus, we postulate that a rabies virus component, which stimulates T cells, such as a SAg, may increase virus immunopathogenicity. To evaluate this hypothesis, we compared the course of rabies in adult BALB/c lacking V beta 6, 7, 8.1, and 9 T cells and in normal BALB/c. Immune-related paralysis was decreased in BALB/c missing the NC target V beta T cells. Transfer of V beta 6 but not of V beta 8.1-3 T cells into recipient mice lacking V beta 6, 7, 8.1, and 9 allowed the immune-related paralysis to evolve. Taken together, these results strongly support the hypothesis that T cells expressing rabies SAg-specific V beta 6 T cells, are involved in the genesis of the immunopathology that is characteristic of paralytic rabies.
Asunto(s)
Cápside/inmunología , Supresión Clonal , Virus de la Rabia/inmunología , Superantígenos/inmunología , Linfocitos T/fisiología , Animales , Animales Recién Nacidos , Cricetinae , Femenino , Ratones , Ratones Endogámicos BALB C , Ratas , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Receptores de Antígenos de Linfocitos T alfa-beta/fisiologíaRESUMEN
The locus encoding mouse TCR-alpha chain includes approximately 100 V alpha gene segments that can be organized in about 20 structural subfamilies. Southern blot analysis of a T cell line derived from the BALB/c strain, M5T, has indicated that both alpha loci were rearranged, as assessed by the deletion of the delta locus, and that the V alpha gene segment involved in one of the rearrangements did not belong to any of the V alpha subfamilies already described. Transcripts of TCR-alpha chains from the M5T line were cloned after cDNA synthesis and anchored-polymerase chain reaction, revealing a V alpha gene segment of an as yet unidentified subfamily, V alpha 5T. Molecular cloning of germ-line V alpha 5T gene segments has shown that this subfamily contained two members, one of them being a pseudogene. The two members were located to each extremity of the alpha locus associated with a member of the V alpha 13 and V alpha BWB subfamilies. Analysis of transcripts bearing the V alpha 5T gene segment in the M5T line as well as in thymocytes has revealed that J alpha are frequently absent. This is due to an alternate donor splice site generated at the V alpha 5T-J alpha junction that leads to a splicing from the end of V alpha 5T to C alpha instead of the J alpha to C alpha conventional splicing. The impact of J alpha spliced-out transcripts on the allelic exclusion process is discussed.
Asunto(s)
Empalme Alternativo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia MolecularRESUMEN
Expression of V beta (beta-chain variable region) gene segments was investigated in the Mus m. domesticus DDO strain, which possesses a large genomic deletion encompassing 20 of the 29 V beta gene segments known in BALB/c. Stainings using V beta-specific monoclonal antibodies revealed that up to 60% of the peripheral T cells use 3 V beta gene segments. Variable frequencies of V beta 2 T cells were observed among DDO individuals. Segregation analyses of F2 crosses between V beta 2-deletor mice and mammary tumor virus (Mtv)-free mice led to the identification of a new endogenous Mtv, named Mtv-DDO, mediating V beta 2 T cell clonal deletion. Mtv-DDO structure is conserved with the exception of the carboxy-terminal region as compared to other Mtv. Comparison between Mtv sharing the same V beta specificity and isolated from laboratory or wild mice confirms that a stretch of 11 amino acids, defined as the V beta-specific region, is required for the V beta-specific interaction. Limited substitutions in this region account for the shift of the Mtv specificity towards different V beta.
Asunto(s)
Virus del Tumor Mamario del Ratón/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Clonales/inmunología , Células Clonales/microbiología , ADN Viral/genética , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Superantígenos/genética , Linfocitos T/microbiología , Proteínas Virales/genéticaRESUMEN
DBA/2 (H-2d) HLA-B7 x human beta 2-microglobulin transgenic and DBA/2 nontransgenic mice stimulated by DBA/2 HLA-A3 x human beta 2-microglobulin transgenic mouse spleen cells developed potent H-2Kd restricted cytolytic responses with recognition of a peptide from the second domain of the HLA-A3 H chain. These H-2Kd-restricted responses obliterated, as a rule, cytolytic responses with direct recognition of the HLA-A3 molecules, even in HLA-B7 transgenic mice. These immunodominant H-2Kd-restricted responses could be abrogated in DBA/2 HLA-A24 mice because of cross-tolerance, the HLA-A3 derived-H-2Kd presented peptide being shared by several (including A24) HLA class I H chain allelic variants. Under such experimental circumstances, strong CTL responses with exclusive direct recognition of HLA-A3 molecules constantly developed. Further analysis of these responses in six DBA/2 HLA-A24 responder mice indicated that a large fraction of the mouse V beta and V alpha genes could be used to mount such CTL responses. Thus, by combining classical HLA class I transgenesis and selective destruction of H-2K and H-2D genes, it should be possible to derive useful strains of mice for the study of HLA class I-restricted CTL responses.