RESUMEN
The bias of αß T cells for MHC ligands has been proposed to be intrinsic to the T-cell receptor (TCR). Equally, the CD4 and CD8 coreceptors contribute to ligand restriction by colocalizing Lck with the TCR when MHC ligands are engaged. To determine the importance of intrinsic ligand bias, the germ-line TCR complementarity determining regions were extensively diversified in vivo. We show that engagement with MHC ligands during thymocyte selection and peripheral T-cell activation imposes remarkably little constraint over TCR structure. Such versatility is more consistent with an opportunist, rather than a predetermined, mode of interface formation. This hypothesis was experimentally confirmed by expressing a hybrid TCR containing TCR-γ chain germ-line complementarity determining regions, which engaged efficiently with MHC ligands.
Asunto(s)
Complejo Mayor de Histocompatibilidad/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Linaje de la Célula/inmunología , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Células Germinativas/inmunología , Ligandos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutación/genética , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Recombinación Genética/genética , Selección Genética , Timo/inmunologíaRESUMEN
As thymocytes differentiate, Ag sensitivity declines, with immature CD4-CD8- double-negative (DN) cells being most susceptible to TCR signaling events. We show that expression of alphabetaTCR from the DN3 stage lowers the threshold for activation, allowing recognition of MHC peptides independently of the TCR beta-chain and without either T cell coreceptor. The MHC class I-restricted C6 TCR recognizes the Y-chromosome-derived Ag HYK(k)Smcy. Positive selection in C6 alphabetaTCR females is skewed to the CD8 compartment, whereas transgenic male mice exhibit early clonal deletion of thymocytes. We investigated the effect of the HYK(k)Smcy complex on developing thymocytes expressing the C6 TCR alpha-chain on a TCR-alpha(-/-) background. On the original selecting haplotype, the skew to the CD8 lineage is preserved. This is MHC dependent, as the normal bias to the CD4 subset is seen on an H2b background. In male H2k C6 alpha-only mice, the presence of the HYK(k)Smcy complex leads to a substantial deletion of thymocytes from the DN subset. This phenotype is replicated in H2k C6 alpha-only female mice expressing an Smcy transgene. Deletion is not dependent on the beta variable segment of the C6 TCR or on a restricted TCR-beta repertoire. In contrast, binding of HYK(k)Smcy and Ag-specific activation of mature CD8+ T cells is strictly dependent on the original C6 beta-chain. These data demonstrate that, in comparison with mature T cells, alphabetaTCR+ immature thymocytes can recognize and transduce signals in response to specific MHC-peptide complexes with relaxed binding requirements.
Asunto(s)
Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Femenino , Histona Demetilasas , Masculino , Ratones , Ratones Transgénicos , Proteínas/inmunología , Factores Sexuales , Transducción de Señal/inmunología , Bazo/citología , Timo/citologíaRESUMEN
Thyroid autoimmune disorders comprise more than 30% of all organ-specific autoimmune diseases and are characterized by autoantibodies and infiltrating T cells. The pathologic role of infiltrating T cells is not well defined. To address this issue, we generated transgenic mice expressing a human T-cell receptor derived from the thyroid-infiltrating T cell of a patient with thyroiditis and specific for a cryptic thyroid-peroxidase epitope. Here we show that mouse major histocompatibility complex molecules sustain selection and activation of the transgenic T cells, as coexpression of histocompatibility leukocyte antigen molecules was not needed. Furthermore, the transgenic T cells had an activated phenotype in vivo, and mice spontaneously developed destructive thyroiditis with histological, clinical and hormonal signs comparable with human autoimmune hypothyroidism. These results highlight the pathogenic role of human T cells specific for cryptic self epitopes. This new 'humanized' model will provide a unique tool to investigate how human pathogenic self-reactive T cells initiate autoimmune diseases and to determine how autoimmunity can be modulated in vivo.
Asunto(s)
Modelos Inmunológicos , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Tiroiditis Autoinmune/etiología , Animales , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/inmunología , Radioisótopos de Cromo , Fragmentación del ADN , Epítopos , Citometría de Flujo , Humanos , Etiquetado Corte-Fin in Situ , Yoduro Peroxidasa/metabolismo , Complejo Mayor de Histocompatibilidad/inmunología , Ratones , Ratones Transgénicos , Modelos Moleculares , Radioinmunoensayo , Receptores de Antígenos de Linfocitos T/metabolismo , Estadísticas no Paramétricas , Linfocitos T/metabolismo , Tiroiditis Autoinmune/fisiopatología , Tirotropina/metabolismo , Tiroxina/sangreRESUMEN
Deficiency of C1q, the initiator of the complement classical pathway, is associated with the development of systemic lupus erythematosus (SLE). Explaining this association in terms of abnormalities in the classical pathway alone remains problematic because C3 deficiency does not predispose to SLE. Here, using a mouse model of SLE, we demonstrate that C1q, but not C3, restrains the response to self-antigens by modulating the mitochondrial metabolism of CD8+ T cells, which can themselves propagate autoimmunity. C1q deficiency also triggers an exuberant effector CD8+ T cell response to chronic viral infection leading to lethal immunopathology. These data establish a link between C1q and CD8+ T cell metabolism and may explain how C1q protects against lupus, with implications for the role of viral infections in the perpetuation of autoimmunity.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos T CD8-positivos/metabolismo , Complemento C1q/fisiología , Lupus Eritematoso Sistémico/inmunología , Coriomeningitis Linfocítica/inmunología , Animales , Autoanticuerpos/inmunología , Autoinmunidad/genética , Complemento C1q/genética , Complemento C3/genética , Complemento C3/fisiología , Vía Clásica del Complemento/genética , Vía Clásica del Complemento/inmunología , Modelos Animales de Enfermedad , Inmunoglobulinas/inmunología , Memoria Inmunológica/inmunología , Lupus Eritematoso Sistémico/genética , Coriomeningitis Linfocítica/genética , Ratones , Ratones MutantesRESUMEN
MHC class II molecules are formed from polymorphic alpha and beta chains. While pairing of chains is most efficient within class II isotypes and haplotypes, limited pairing and surface expression of mixed-haplotype and -isotype class II molecules is common. The function of such molecules in antigen presentation has been established by the unique restriction of responses in F1 mice. However, it has not been established whether mixed class II molecules are able to mediate selection of functional T cells and how the reduced avidity of the TCR/MHC interaction influences the repertoire. In this report we have addressed these issues through the production of mice expressing solely mixed-haplotype class II molecules. The mixed class II molecules promote selection of a small CD4+ T cell repertoire with modified TCR use. The selected CD4+ T cells are functional in vivo and in vitro.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Haplotipos/genética , Antígenos de Histocompatibilidad Clase II/genética , Selección Genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Presentación de Antígeno , Linfocitos B/inmunología , Células de la Médula Ósea , Técnicas de Cultivo de Célula , Células Cultivadas , Cruzamientos Genéticos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Citometría de Flujo , Rayos gamma , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Haplotipos/inmunología , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Endogámicos , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T/inmunología , Homología de Secuencia de Aminoácido , Trasplante de Piel/inmunología , Bazo/citologíaRESUMEN
αß T cells respond to peptide epitopes presented by major histocompatibility complex (MHC) molecules. The role of T cell receptor (TCR) germline complementarity determining regions (CDR1 and 2) in MHC restriction is not well understood. Here, we examine T cell development, MHC restriction and antigen recognition where germline CDR loop structure has been modified by multiple glycine/alanine substitutions. Surprisingly, loss of germline structure increases TCR engagement with MHC ligands leading to excessive loss of immature thymocytes. MHC restriction is, however, strictly maintained. The peripheral T cell repertoire is affected similarly, exhibiting elevated cross-reactivity to foreign peptides. Our findings are consistent with germline TCR structure optimising T cell cross-reactivity and immunity by moderating engagement with MHC ligands. This strategy may operate alongside co-receptor imposed MHC restriction, freeing germline TCR structure to adopt this novel role in the TCR-MHC interface.
Asunto(s)
Antígenos de Histocompatibilidad/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Sustitución de Aminoácidos , Animales , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Reacciones Cruzadas , Femenino , Complejo Mayor de Histocompatibilidad/inmunología , Ratones , Modelos Moleculares , Receptores de Antígenos de Linfocitos T alfa-beta/inmunologíaRESUMEN
BACKGROUND: Regulatory T (Treg) cells, generated in vitro by Foxp3 gene transfer into naive CD4+25- T cells, have been shown to inhibit the development of inflammation and autoimmune disease, but it is not known whether they are able to prevent allograft rejection. This study investigated whether Treg cells generated from naive CD4+ T cells by Foxp3 gene transfer could induce transplantation tolerance. METHODS: HY-specific, T-cell receptor (TCR)-transgenic CD4+25- T cells were retrovirally transduced with the Foxp3 gene. The phenotype, function, and cytokine profiles of the transduced cells were examined in vitro by fluorescence-activated cell sorter, T-cell proliferation assays, enzyme-linked immunosorbent assay, and intracellular cytokine staining. Adoptive transfer and skin grafting experiments were conducted to assess whether Foxp3-transduced HY-specific T cells could prevent the rejection of syngeneic male grafts. RESULTS: CD4+25- T cells retrovirally transduced with Foxp3 express a panel of cell surface and intracellular molecules closely associated with Treg activity. This Treg phenotype was stable during in vitro culture with some further maturation. In vitro, Foxp3-transduced cells were functionally anergic and suppressive T cells. In vivo adoptive transfer of Foxp3-transduced HY-specific TCR-transgenic CD4+ T cells protected male skin grafts from rejection by syngeneic females. Retroviral transduction of the Foxp3 gene into non-TCR-transgenic CD4+25- T cells, however, had no influence on male skin graft rejection. CONCLUSION: This study provides the first evidence that Foxp3-transduced T cells can control the rejection of an allogeneic transplant and suggests that T-cell Foxp3 gene transfer may have therapeutic value in clinical transplantation.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Proteínas de Unión al ADN/genética , Técnicas de Transferencia de Gen , Receptores de Interleucina-2/deficiencia , Linfocitos T/trasplante , Tolerancia al Trasplante , Animales , División Celular , Células Cultivadas , Citocinas/metabolismo , Femenino , Factores de Transcripción Forkhead , Vectores Genéticos , Rechazo de Injerto/prevención & control , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Fenotipo , Retroviridae/genética , Trasplante de Piel , Linfocitos T/citología , Linfocitos T/metabolismo , Trasplante IsogénicoRESUMEN
T cell receptor transfer is an attractive strategy for the generation of antigen specific T cells to target infection and malignancy. Cross pairing of the transduced and endogenous TCR chains produces new and potentially auto-reactive specificities and dilutes the therapeutic TCR. This is further complicated as the efficiency of pairing for each alphabeta pair is unpredictable and the factors which influence it are not well characterized. Complementarity determining region 3 (CDR3) loops are the main sources of TCR alpha and beta diversity due to nucleotide insertion and deletion at V(D)J junctions. Given the variability in composition and length of these non-germ line encoded structures, it is likely that structural strain may occur during formation of some TCR hetero-dimers contributing to the observed pairing restrictions. The beta chain of the HY specific T cell receptor C6 is such an example. Despite pairing efficiently with the C6 alpha chain, it pairs poorly with many other alpha chains. To investigate whether the long, C6 beta CDR3 region underlies this effect, it was replaced with a short, artificial CDR3 region that restored efficient pairing with the endogenous alpha chain repertoire. Molecular modelling is consistent with the beta chain CDR3 region causing steric incompatibility. Despite poor pairing and low surface expression, the WT C6 beta chain mediates positive selection in retrogenic mice.
Asunto(s)
Regiones Determinantes de Complementariedad/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Secuencia de Aminoácidos , Animales , Región de Unión de la Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/inmunología , Ratones , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
Studies of human and murine T cells have shown that public TCR beta-chain rearrangements can dominate the Ag-specific and naive repertoires of distinct individuals. We show that mouse T cells responding to the minor histocompatibility Ag HYDbSmcy share an invariant Vbeta8.2-Jbeta2.3 TCR gene rearrangement. The dominance of this rearrangement shows that it successfully negotiated thymic selection and was highly favored during clonal expansion in all animals examined. We hypothesized that such beta-chains are advantaged during thymic and/or peripheral selection and, as a result, may be over-represented in the naive repertoire. A sequencing study was undertaken to examine the diversity of Vbeta8.2-Jbeta2.3 CDR3 loops from naive T cell repertoires of multiple mice. Public TCR beta-chain sequences were identified across different repertoires and MHC haplotypes. To determine whether such public beta-chains are advantaged during thymic selection, individual chains were followed through T cell development in a series of novel bone marrow competition chimeras. We demonstrate that beta-chains were positively selected with similar efficiency regardless of CDR3 loop sequence. Therefore, the establishment and maintenance of public beta-chains in the periphery is predominantly controlled by post-thymic events through modification of the primary, thymus-derived TCR repertoire.
Asunto(s)
Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Selección Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Antígenos CD4/análisis , Antígenos CD8/análisis , Femenino , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/metabolismo , Histona Demetilasas , Humanos , Masculino , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Proteínas/inmunología , Linfocitos T/inmunología , Timo/inmunologíaRESUMEN
How positive selection molds the T cell repertoire has been difficult to examine. In this study, we use TCR-beta-transgenic mice in which MHC shapes TCR-alpha use. Differential AV segment use is directly related to the constraints placed on the composition of the CDR3 loops. Where these constraints are low, efficient selection of alphabeta pairs follows. This mode of selection preferentially uses favored AV-AJ rearrangements and promotes diversity. Increased constraint on the alpha CDR3 loops leads to inefficient selection associated with uncommon recombination events and limited diversity. Further, the two modes of selection favor alternate sets of AJ segments. We discuss the relevance of these findings to the imprint of self-MHC restriction and peripheral T cell activation.
Asunto(s)
Supresión Clonal , Regiones Determinantes de Complementariedad , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Animales , Células Cultivadas , Regiones Determinantes de Complementariedad/genética , Femenino , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Antígenos H-2/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos/genética , Unión Proteica/genética , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
Memory lymphocytes are important mediators of the immune response. These cells are long-lived and undergo clonal expansion upon reexposure to specific antigen, differentiating into effector cells that secrete Ig or cytokines while maintaining a residual pool of memory T and B lymphocytes. Here, the ability of antigen-specific lymphocytes to undergo repeated cycles of antigen-driven clonal expansion and contraction is exploited in a therapeutic protocol aimed at regulating protein delivery. The principle of this strategy is to introduce genes encoding proteins of therapeutic interest into a small number of antigen-specific B lymphocytes. Output of therapeutic protein can then be regulated in vivo by manipulating the size of the responder population by antigen challenge. To evaluate whether such an approach is feasible, we developed a mouse model system in which Emu- and Iglambda-based vectors were used to express human erythropoietin (hEPO) gene in B lymphocytes. These mice were then immunized with the model antigen phycoerythrin (PE), and immune splenocytes (or purified PE-specific B lymphocytes) were adoptively transferred to normal or mutant (EPO-deficient) hosts. High levels of hEPO were detected in the serum of adoptively transferred normal mice after PE administration, and this responsiveness was maintained for several months. Similarly, in EPO-deficient anemic recipients, antigen-driven hEPO expression was shown to restore hematocrit levels to normal. These results show that antigen-mediated regulation of memory lymphocytes can be used as a strategy for delivering therapeutic proteins in vivo.