RESUMEN
Autoreactive T cells are eliminated in the thymus to prevent autoimmunity by promiscuous expression of tissue-restricted self-antigens in medullary thymic epithelial cells. This expression is dependent on the transcription factor Fezf2, as well as the transcriptional regulator Aire, but the entire picture of the transcriptional program has been obscure. Here, we found that the chromatin remodeler Chd4, also called Mi-2ß, plays a key role in the self-antigen expression in medullary thymic epithelial cells. To maximize the diversity of self-antigen expression, Fezf2 and Aire utilized completely distinct transcriptional mechanisms, both of which were under the control of Chd4. Chd4 organized the promoter regions of Fezf2-dependent genes, while contributing to the Aire-mediated induction of self-antigens via super-enhancers. Mice deficient in Chd4 specifically in thymic epithelial cells exhibited autoimmune phenotypes, including T cell infiltration. Thus, Chd4 plays a critical role in integrating Fezf2- and Aire-mediated gene induction to establish central immune tolerance.
Asunto(s)
Autoantígenos/inmunología , Tolerancia Central/fisiología , Regulación de la Expresión Génica/inmunología , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/inmunología , Animales , Autoantígenos/biosíntesis , ADN Helicasas/inmunología , ADN Helicasas/metabolismo , Células HEK293 , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Proteína AIRERESUMEN
Aire mediates the expression of tissue-specific antigens in thymic epithelial cells to promote tolerance against self-reactive T lymphocytes. However, the mechanism that allows expression of tissue-specific genes at levels that prevent harm is unknown. Here we show that Brg1 generates accessibility at tissue-specific loci to impose central tolerance. We found that Aire has an intrinsic repressive function that restricts chromatin accessibility and opposes Brg1 across the genome. Aire exerted this repressive influence within minutes after recruitment to chromatin and restrained the amplitude of active transcription. Disease-causing mutations that impair Aire-induced activation also impair the protein's repressive function, which indicates dual roles for Aire. Together, Brg1 and Aire fine-tune the expression of tissue-specific genes at levels that prevent toxicity yet promote immune tolerance.
Asunto(s)
Tolerancia Central/inmunología , ADN Helicasas/inmunología , Regulación de la Expresión Génica/inmunología , Proteínas Nucleares/inmunología , Timo/inmunología , Factores de Transcripción/inmunología , Animales , Cromatina , Ratones , Ratones Transgénicos , Proteína AIRERESUMEN
Efforts to understand autoimmunity have been pursued relentlessly for several decades. It has become apparent that the immune system evolved multiple mechanisms for controlling self-reactivity, and defects in one or more of these mechanisms can lead to a breakdown of tolerance. Among the multitude of lesions associated with disease, the most common seem to affect peripheral tolerance rather than central tolerance. The initial trigger for both systemic autoimmune disorders and organ-specific autoimmune disorders probably involves the recognition of self or foreign molecules, especially nucleic acids, by innate sensors. Such recognition, in turn, triggers inflammatory responses and the engagement of previously quiescent autoreactive T cells and B cells. Here we summarize the most prominent autoimmune pathways and identify key issues that require resolution for full understanding of pathogenic autoimmunity.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Autoinmunidad/inmunología , Linfocitos B/inmunología , Autotolerancia/inmunología , Linfocitos T/inmunología , Animales , Tolerancia Central/inmunología , Humanos , Tolerancia Periférica/inmunologíaRESUMEN
Studies of individual T cell antigen receptors (TCRs) have shed some light on structural features that underlie self-reactivity. However, the general rules that can be used to predict whether TCRs are self-reactive have not been fully elucidated. Here we found that the interfacial hydrophobicity of amino acids at positions 6 and 7 of the complementarity-determining region CDR3ß robustly promoted the development of self-reactive TCRs. This property was found irrespective of the member of the ß-chain variable region (Vß) family present in the TCR or the length of the CDR3ß. An index based on these findings distinguished Vß2(+), Vß6(+) and Vß8.2(+) regulatory T cells from conventional T cells and also distinguished CD4(+) T cells selected by the major histocompatibility complex (MHC) class II molecule I-A(g7) (associated with the development of type 1 diabetes in NOD mice) from those selected by a non-autoimmunity-promoting MHC class II molecule I-A(b). Our results provide a means for distinguishing normal T cell repertoires versus autoimmunity-prone T cell repertoires.
Asunto(s)
Autoinmunidad , Regiones Determinantes de Complementariedad/genética , Diabetes Mellitus Tipo 1/inmunología , Subgrupos de Linfocitos T/fisiología , Linfocitos T Reguladores/fisiología , Animales , Autoantígenos/inmunología , Autoantígenos/metabolismo , Diferenciación Celular , Tolerancia Central , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones NoqueadosRESUMEN
Aire is a transcriptional regulator that induces the promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the induction of immunological self-tolerance. Studies have offered molecular insights into how Aire operates, but more comprehensive understanding of this process still remains elusive. Here we found abundant expression of the protein deacetylase Sirtuin-1 (Sirt1) in mature Aire(+) mTECs, wherein it was required for the expression of Aire-dependent TRA-encoding genes and the subsequent induction of immunological self-tolerance. Our study elucidates a previously unknown molecular mechanism for Aire-mediated transcriptional regulation and identifies a unique function for Sirt1 in preventing organ-specific autoimmunity.
Asunto(s)
Tolerancia Central/inmunología , Sirtuina 1/inmunología , Factores de Transcripción/inmunología , Activación Transcripcional/inmunología , Acetilación , Animales , Antígenos/inmunología , Tolerancia Central/genética , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Citometría de Flujo , Células HEK293 , Humanos , Immunoblotting , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos/inmunología , Unión Proteica/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 1/genética , Sirtuina 1/metabolismo , Timo/citología , Timo/inmunología , Timo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma/inmunología , Proteína AIRERESUMEN
Viral mimicry of host cell structures has been postulated to curtail the B cell receptor (BCR) repertoire against persisting viruses through tolerance mechanisms. This concept awaits, however, experimental testing in a setting of natural virus-host relationship. We engineered mouse models expressing a monoclonal BCR specific for the envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV), a naturally persisting mouse pathogen. When the heavy chain of the LCMV-neutralizing antibody KL25 was paired with its unmutated ancestor light chain, most B cells underwent receptor editing, a behavior reminiscent of autoreactive clones. In contrast, monoclonal B cells expressing the same heavy chain in conjunction with the hypermutated KL25 light chain did not undergo receptor editing but exhibited low levels of surface IgM, suggesting that light chain hypermutation had lessened KL25 autoreactivity. Upon viral challenge, these IgMlow cells were not anergic but up-regulated IgM, participated in germinal center reactions, produced antiviral antibodies, and underwent immunoglobulin class switch as well as further affinity maturation. These studies on a persisting virus in its natural host species suggest that central tolerance mechanisms prune the protective antiviral B cell repertoire.
Asunto(s)
Linfocitos B , Tolerancia Central , Animales , Ratones , Anticuerpos Antivirales , Virus de la Coriomeningitis Linfocítica , Antivirales , Inmunoglobulina MRESUMEN
The maintenance of immunological tolerance requires the deletion of self-reactive T cells in the thymus. The expression of genes encoding tissue-specific antigens (TSAs) by thymic epithelial cells is critical for this process and depends on activity of the transcriptional regulator Aire; however, the molecular mechanisms Aire uses to target loci encoding TSAs are unknown. Here we identified two Aire-interacting proteins known to be involved in gene repression, ATF7ip and MBD1, that were required for Aire's targeting of loci encoding TSAs. Moreover, Mbd1(-/-) mice developed pathological autoimmunity and had a defect in Aire-dependent thymic expression of genes encoding TSAs, which underscores the importance of Aire's interaction with the ATF7ip-MBD1 protein complex in maintaining central tolerance.
Asunto(s)
Tolerancia Central/inmunología , Proteínas de Unión al ADN/inmunología , Regulación de la Expresión Génica/inmunología , Tolerancia Inmunológica , Proteínas Represoras/inmunología , Factores de Transcripción/inmunología , Animales , Autoantígenos/inmunología , Tolerancia Central/genética , Proteínas de Unión al ADN/genética , Citometría de Flujo , Células HEK293 , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Transfección , Técnicas del Sistema de Dos Híbridos , Proteína AIRERESUMEN
Highly self-reactive T cells are censored from the repertoire by both central and peripheral tolerance mechanisms upon receipt of high-affinity TCR signals. Clonal deletion is considered a major driver of central tolerance; however, other mechanisms such as induction of regulatory T cells and functional impairment have been described. An understanding of the interplay between these different central tolerance mechanisms is still lacking. We previously showed that impaired clonal deletion to a model tissue-restricted Ag did not compromise tolerance. In this study, we determined that murine T cells that failed clonal deletion were rendered functionally impaired in the thymus. Programmed cell death protein 1 (PD-1) was induced in the thymus and was required to establish cell-intrinsic tolerance to tissue-restricted Ag in CD8+ thymocytes independently of clonal deletion. In bone marrow chimeras, tolerance was not observed in PD-L1-deficient recipients, but tolerance was largely maintained following adoptive transfer of tolerant thymocytes or T cells to PD-L1-deficient recipients. However, CRISPR-mediated ablation of PD-1 in tolerant T cells resulted in broken tolerance, suggesting different PD-1 signaling requirements for establishing versus maintaining tolerance. Finally, we showed that chronic exposure to high-affinity Ag supported the long-term maintenance of tolerance. Taken together, our study identifies a critical role for PD-1 in establishing central tolerance in autoreactive T cells that escape clonal deletion. It also sheds light on potential mechanisms of action of anti-PD-1 pathway immune checkpoint blockade and the development of immune-related adverse events.
Asunto(s)
Antígeno B7-H1 , Receptor de Muerte Celular Programada 1 , Ratones , Animales , Receptor de Muerte Celular Programada 1/genética , Tolerancia Central , Linfocitos T CD8-positivos , Timo , Antígenos , Tolerancia InmunológicaRESUMEN
The random recombination of immunoglobulin V(D)J gene segments produces unique IgM antibodies that serve as the antigen receptor for each developing B cell. Hence, the newly formed B cell repertoire is comprised of a variety of specificities that display a range of reactivity with self-antigens. Newly generated IgM+ immature B cells that are non-autoreactive or that bind self-antigen with low avidity are licensed to leave the bone marrow with their intact antigen receptor and to travel via the blood to the peripheral lymphoid tissue for further selection and maturation. In contrast, clones with medium to high avidity for self-antigen remain within the marrow and undergo central tolerance, a process that revises their antigen receptor or eliminates the autoreactive B cell altogether. Thus, central B cell tolerance is critical for reducing the autoreactive capacity and avidity for self-antigen of our circulating B cell repertoire. Bone marrow cultures and mouse models have been instrumental for understanding the mechanisms that regulate the selection of bone marrow B cells. Here, we review recent studies that have shed new light on the contribution of the ERK, PI3K, and CXCR4 signaling pathways in the selection of mouse and human immature B cells that either bind or do not bind self-antigen.
Asunto(s)
Tolerancia Central , Receptores de Antígenos de Linfocitos B , Autoinmunidad , Linfocitos B , Células de la Médula Ósea , Humanos , Células Precursoras de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismoRESUMEN
Dendritic cells (DCs) are instrumental in the initiation of T cell responses, but how thymic and peripheral tolerogenic DCs differ globally from Toll-like receptor (TLR)-induced immunogenic DCs remains unclear. Here, we show that thymic XCR1(+) DCs undergo a high rate of maturation, accompanied by profound gene-expression changes that are essential for central tolerance and also happen in germ-free mice. Those changes largely overlap those occurring during tolerogenic and, more unexpectedly, TLR-induced maturation of peripheral XCR1(+) DCs, arguing against the commonly held view that tolerogenic DCs undergo incomplete maturation. Interferon-stimulated gene (ISG) expression was among the few discriminators of immunogenic and tolerogenic XCR1(+) DCs. Tolerogenic XCR1(+) thymic DCs were, however, unique in expressing ISGs known to restrain virus replication. Therefore, a broad functional convergence characterizes tolerogenic and immunogenic XCR1(+) DC maturation in the thymus and periphery, maximizing antigen presentation and signal delivery to developing and to conventional and regulatory mature T cells.
Asunto(s)
Tolerancia Central , Células Dendríticas/inmunología , Tolerancia Periférica , Linfocitos T Reguladores/inmunología , Timo/inmunología , Animales , Presentación de Antígeno , Diferenciación Celular , Células Cultivadas , Factores Reguladores del Interferón/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Quimiocina/metabolismo , Receptores Toll-Like/inmunología , Transcriptoma , Replicación ViralRESUMEN
Medullary thymic epithelial cells (mTECs) are essential for the establishment of T cell central tolerance. The transcription factor Aire plays a key role in this process, but other factors remain understudied. We found that a small population of mTECs expressed the coinhibitory receptor cytotoxic T lymphocyte-associated protein 4 (CTLA-4). These CTLA-4+ cells were detectable in perinates, peaked around young adulthood and expanded sixfold in the absence of Aire. Single-cell transcriptomics revealed CTLA-4+ mTECs to express a distinct gene signature encoding molecules associated with antigen presentation and interferon-gamma signaling. Mice conditionally lacking CTLA-4 in thymic epithelial cells had no major immunological deficiencies but displayed a mildly increased inflammatory tone and a partial defect in the generation of Foxp3+CD4+ regulatory T cells. Consequently, these mice developed modest levels of autoantibodies and lymphocytic infiltration of peripheral tissues. Thus, CTLA-4 expression in mTECs complements Aire to establish T cell central tolerance.
Asunto(s)
Tolerancia Central , Proteínas del Sistema Complemento , Animales , Ratones , Tolerancia Central/genética , Antígeno CTLA-4/genética , Células Epiteliales , Factores de Transcripción/genética , Linfocitos T/inmunologíaRESUMEN
Historically, successful allotransplantation was only achieved by utilizing powerful immunosuppressive drugs that were exposing the patient to severe opportunistic infections. The thymus of the transplant recipient renders such therapy obligatory as it constitutively blocks self-reactive T cells while allowing alloreactive T cells to mature and populate the periphery. In 1992, a follow-up study revealed the presence of donor leukocytes in long-term transplant survivors. The stable persistence of recipient and donor leukocytes in the transplanted patient, referred to as "chimerism", was considered the reason why in some cases it was even possible to stop immunosuppressive treatment without damaging the transplanted organ. Unfortunately, it quickly became evident that stable, persistent allogeneic chimerism was not easily achievable by design. Recently, a novel approach has been identified to help address this clinical gap in knowledge: Cotransplantation of a donor graft with a thymic organoid populated with donor precursor cells generates stable, long-term chimerism in the recipient. In humanized mice, the implantation of thymic organoids, populated with human donor inducible pluripotent stem cell (iPSC)-derived thymic epithelial cells (TECs) and the same donor CD34+ bone marrow precursors, induces tolerance to human leukocyte antigen (HLA)-matched donor tissues/organs. This technology will allow successful allotransplantation of cells/organs even between Major Histocompatibility Complex (MHC)-noncompatible individuals and allow getting rid of immunosuppressive treatments reducing recipient morbidity.
Asunto(s)
Trasplante de Médula Ósea , Tolerancia Central , Humanos , Ratones , Animales , Estudios de Seguimiento , Tolerancia Inmunológica , Linfocitos T , Inmunosupresores , Antígenos de HistocompatibilidadRESUMEN
Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells.
Asunto(s)
Tolerancia Central/genética , Tolerancia Central/inmunología , Citidina Desaminasa/genética , Activación de Linfocitos/inmunología , Células Precursoras de Linfocitos B/inmunología , Adolescente , Adulto , Anciano , Animales , Apoptosis/genética , Apoptosis/inmunología , Estudios de Casos y Controles , Niño , Preescolar , Proteínas de Unión al ADN/genética , Femenino , Genes de Inmunoglobulinas/genética , Genes de Inmunoglobulinas/inmunología , Humanos , Activación de Linfocitos/genética , Masculino , Ratones , Persona de Mediana Edad , Proteínas Nucleares/genética , Recombinación Genética/genética , Recombinación Genética/inmunología , Hipermutación Somática de Inmunoglobulina/genética , Hipermutación Somática de Inmunoglobulina/inmunología , Adulto JovenRESUMEN
Thymic antigen-presenting cells (APCs) such as dendritic cells and medullary thymic epithelial cells (mTECs) use distinct strategies of self-antigen expression and presentation to mediate central tolerance. The thymus also harbors B cells; whether they also display unique tolerogenic features and how they genealogically relate to peripheral B cells is unclear. Here, we found that Aire is expressed in thymic but not peripheral B cells. Aire expression in thymic B cells coincided with major histocompatibility class II (MHCII) and CD80 upregulation and immunoglobulin class-switching. These features were recapitulated upon immigration of naive peripheral B cells into the thymus, whereby this intrathymic licensing required CD40 signaling in the context of cognate interactions with autoreactive CD4(+) thymocytes. Moreover, a licensing-dependent neo-antigen selectively upregulated in immigrating B cells mediated negative selection through direct presentation. Thus, autoreactivity within the nascent T cell repertoire fuels a feed forward loop that endows thymic B cells with tolerogenic features.
Asunto(s)
Linfocitos B/fisiología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD40/metabolismo , Timo/inmunología , Factores de Transcripción/metabolismo , Animales , Presentación de Antígeno/genética , Autoantígenos/inmunología , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Antígenos CD40/genética , Diferenciación Celular/genética , Células Cultivadas , Tolerancia Central/genética , Selección Clonal Mediada por Antígenos/genética , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Cambio de Clase de Inmunoglobulina/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Transducción de Señal , Factores de Transcripción/genética , Proteína AIRERESUMEN
Central B cell tolerance, the process restricting the development of many newly generated autoreactive B cells, has been intensely investigated in mouse cells while studies in humans have been hampered by the inability to phenotypically distinguish autoreactive and nonautoreactive immature B cell clones and the difficulty in accessing fresh human bone marrow samples. Using a human immune system mouse model in which all human Igκ+ B cells undergo central tolerance, we discovered that human autoreactive immature B cells exhibit a distinctive phenotype that includes lower activation of ERK and differential expression of CD69, CD81, CXCR4, and other glycoproteins. Human B cells exhibiting these characteristics were observed in fresh human bone marrow tissue biopsy specimens, although differences in marker expression were smaller than in the humanized mouse model. Furthermore, the expression of these markers was slightly altered in autoreactive B cells of humanized mice engrafted with some human immune systems genetically predisposed to autoimmunity. Finally, by treating mice and human immune system mice with a pharmacologic antagonist, we show that signaling by CXCR4 is necessary to prevent both human and mouse autoreactive B cell clones from egressing the bone marrow, indicating that CXCR4 functionally contributes to central B cell tolerance.
Asunto(s)
Tolerancia Central/fisiología , Células Precursoras de Linfocitos B/metabolismo , Receptores CXCR4/metabolismo , Animales , Autoanticuerpos/metabolismo , Autoantígenos/inmunología , Autoinmunidad/inmunología , Linfocitos B/inmunología , Médula Ósea/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Tolerancia Central/inmunología , Femenino , Humanos , Tolerancia Inmunológica/genética , Recién Nacido , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Fenotipo , Células Precursoras de Linfocitos B/fisiología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores CXCR4/inmunología , Receptores CXCR4/fisiología , Transducción de Señal/genéticaRESUMEN
Butyrophilins are surface receptors belonging to the immunoglobulin superfamily. While several members of the butyrophilin family have been implicated in the development of unconventional T cells, butyrophilin 2a2 (Btn2a2) has been shown to inhibit conventional T cell activation. Here, we demonstrate that in steady state, the primary source of Btn2a2 are thymic epithelial cells (TEC). Absence of Btn2a2 alters thymic T cell maturation and bypasses central tolerance mechanisms. Furthermore, Btn2a2-/- mice develop spontaneous autoimmunity resembling human primary Sjögren's Syndrome (pSS), including formation of tertiary lymphoid structures (TLS) in target organs. Ligation of Btn2a2 on developing thymocytes is associated with reduced TCR signaling and CD5 levels, while absence of Btn2a2 results in increased TCR signaling and CD5 levels. These results define a novel role for Btn2a2 in promoting central tolerance by modulating TCR signaling strength and indicate a potential mechanism of pSS development.
Asunto(s)
Enfermedades Autoinmunes , Tolerancia Central , Ratones , Humanos , Animales , Butirofilinas/genética , Timo , Células Epiteliales , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
The discovery of the autoimmune regulator (AIRE) protein and the delineation of its critical contributions in the establishment of central immune tolerance has significantly expanded our understanding of the immunological mechanisms that protect from the development of autoimmune disease. The parallel identification and characterization of patient cohorts with the monogenic disorder autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), which is typically caused by biallelic AIRE mutations, has underscored the critical contribution of AIRE in fungal immune surveillance at mucosal surfaces and in prevention of multiorgan autoimmunity in humans. In this review, we synthesize the current clinical, genetic, molecular and immunological knowledge derived from basic studies in Aire-deficient animals and from APECED patient cohorts. We also outline major advances and research endeavors that show promise for informing improved diagnostic and therapeutic approaches for patients with APECED.
Asunto(s)
Síndromes de Inmunodeficiencia/genética , Mutación/genética , Poliendocrinopatías Autoinmunes/genética , Factores de Transcripción/genética , Animales , Autoinmunidad , Tolerancia Central , Humanos , Proteína AIRERESUMEN
A T-cell receptor (TCR) with optimal avidity to a tumor antigen can be used to redirect T cells to eradicate cancer cells via adoptive cell transfer. Cancer testis antigens (CTAs) are attractive targets because they are expressed in the testis, which is immune-privileged, and in the tumor. However, CTAs are self-antigens and natural TCRs to CTAs have low affinity/avidity due to central tolerance. We previously described a method of directed evolution of TCR avidity using somatic hypermutation. In this study, we made several improvements to this method and enhanced the avidity of the hT27 TCR, which is specific for the cancer testis antigen HLA-A2-MAGE-A1278-286 . We identified eight point mutations with varying degrees of improved avidity. Human T cells transduced with TCRs containing these mutations displayed enhanced tetramer binding, IFN-γ and IL2 production, and cytotoxicity. Most of the mutations have retained specificity, except for one mutant with extremely high avidity. We demonstrate that somatic hypermutation is capable of optimizing avidity of clinically relevant TCRs for immunotherapy.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Inmunoterapia Adoptiva/métodos , Proteínas de Neoplasias/inmunología , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Células Cultivadas , Tolerancia Central , Citotoxicidad Inmunológica , Antígeno HLA-A2/metabolismo , Humanos , Interferón gamma/metabolismo , Activación de Linfocitos , Mutación Puntual/genética , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Hipermutación Somática de Inmunoglobulina , Linfocitos T/trasplanteRESUMEN
Medullary thymic epithelial cells (mTECs) induce T cell tolerance in the thymus through the elimination of self-reactive thymocytes. Commensal bacteria are also critical for shaping T cell responses in the gut and distal organs. We previously showed that mice depleted of mTECs (Traf6ΔTEC) generated autoreactive T cells and developed autoimmune hepatitis (AIH). In this report, we found that Toll-like receptor (TLR)-mediated microbial sensing on liver hematopoietic cells and the gut microbiota contributed to AIH development in Traf6ΔTEC mice. While adoptive transfer of thymic Traf6ΔTEC T cells in immune-deficient mice was sufficient for AIH development, colonization of germ-free mice with Traf6ΔTEC microbiota failed to induce AIH, suggesting that the gut microbiota contributes to but is not sufficient for AIH development. Microbiota-mediated exacerbation of AIH associated with increased numbers of hepatic Foxp3+ T cells and their increase was proportional to the degree of inflammation. The contribution of the gut microbiota to AIH development associated with an altered microbial signature whose composition was influenced by the qualitative nature of the thymic T cell compartment. These results suggest that aberrant selection of T cells in the thymus can induce changes in the gut microbiota that lead to exacerbation of organ-specific autoimmunity and AIH. Our results add to our understanding of the mechanisms of AIH development and create a platform towards developing novel therapeutic approaches for treating this disease.