RESUMO
The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4+ T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4+ T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4+ T cells in MS.
Assuntos
Subtipos Sorológicos de HLA-DR/imunologia , Esclerose Múltipla/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Alelos , Antígenos/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Reações Cruzadas/imunologia , Feminino , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Peptídeos/imunologia , Proteoma/metabolismo , Adulto JovemRESUMO
Medullary thymic epithelial cells (mTECs) are critical in establishing and maintaining the appropriate microenvironment for negative selection and maturation of immunocompetent T cells with a self-tolerant T cell antigen receptor repertoire. Cues that direct proliferation and maturation of mTECs are provided by members of the tumor necrosis factor (TNF) superfamily expressed on developing thymocytes. Here we demonstrate a negative role of the morphogen TGF-ß in tempering these signals under physiological conditions, limiting both growth and function of the thymic medulla. Eliminating TGF-ß signaling specifically in TECs or by pharmacological means increased the size of the mTEC compartment, enhanced negative selection and functional maturation of medullary thymocytes as well as the production of regulatory T cells, thus reducing the autoreactive potential of peripheral T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T Reguladores/imunologia , Timo/imunologia , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Microambiente Celular/imunologia , Proteínas de Ligação a DNA/genética , Células Epiteliais/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais/imunologia , Timócitos/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Newborn screening (NBS) for severe combined immunodeficiency (SCID) has been introduced in various countries with the aim of reducing morbidity and mortality. However, studies analyzing outcomes before and after the implementation of NBS programs remain limited. This study sought to compare the outcomes of SCID patients identified through Switzerland's national SCID NBS program, introduced in January 2019, with those of a historical cohort diagnosed between 2007 and 2019. The study included seven patients (32%) identified through NBS, and 15 (68%) born before NBS implementation and diagnosed based on clinical signs. Children in the NBS group were younger at diagnosis (median age 9 days vs 9 months, P = .002) and at hematopoietic stem cell transplantation (HSCT, median age 5 months vs 11 months, P = .003) compared to the clinical group. The NBS group had a lower incidence of infections before HSCT (29% vs 93%, P = .004). Although not statistically significant, the overall survival rate on last follow-up was higher in the NBS group (86% vs 67%, P = .62). Importantly, patients with active infections undergoing HSCT had a significantly lower overall survival probability compared to those without (P = .01). In conclusion, the introduction of NBS in Switzerland has led to earlier and often asymptomatic diagnosis of affected children, enabling timely intervention, infection prevention, and prompt treatment. These factors have contributed to higher survival rates in the NBS group. These findings underscore the critical importance of NBS for SCID, offering potential life-saving benefits through early detection and intervention.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Imunodeficiência Combinada Severa , Criança , Recém-Nascido , Humanos , Lactente , Suíça/epidemiologia , Triagem Neonatal , Imunodeficiência Combinada Severa/diagnóstico , Imunodeficiência Combinada Severa/epidemiologia , Imunodeficiência Combinada Severa/terapia , MorbidadeRESUMO
BACKGROUND: Hematopoietic cell transplantation (HCT) is an established curative therapy for transfusion-dependent thalassemia (TDT) and sickle cell disease (SCD). The latest American Society of Hematology guidelines recommend myeloablative preparative regimen in patients under 18 years of age. PROCEDURE: The objective was to demonstrate safety and efficacy of a reduced intensity conditioning (RIC) regimen including high-dose fludarabine, anti-thymocyte globulin, and targeted busulfan as a single alkylator to sub-myeloablative exposures. RESULTS: Between 2012 and 2021, 11 patients with SCD and five patients with TDT and matched related donor (MRD) HCT were included. The median age at transplantation was 8.3 years (range: 3.7-18.8 years). The median administered busulfan AUC was 67.4 mg/L×h (range: 60.7-80 mg/L×h). Overall survival was 93.8% and event-free survival 87.5% with one engrafted SCD patient with pre-existing moyamoya disease succumbing after drainage of a subdural hematoma. One SCD patient developed a secondary graft failure and was treated with a second HCT. Myeloid chimerism was full in all other patients with a median follow-up time of 4.1 years (range: 2.0-11.1 years), whereas T-cell donor chimerism was frequently mixed. CONCLUSION: This RIC conditioning followed by MRD HCT is sufficiently myeloablative to cure pediatric patients with hemoglobinopathies without the need for additional total body irradiation or thiotepa.
Assuntos
Bussulfano , Transplante de Células-Tronco Hematopoéticas , Hemoglobinopatias , Condicionamento Pré-Transplante , Humanos , Bussulfano/administração & dosagem , Bussulfano/uso terapêutico , Condicionamento Pré-Transplante/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Pré-Escolar , Criança , Masculino , Feminino , Adolescente , Hemoglobinopatias/terapia , Seguimentos , Taxa de Sobrevida , Doença Enxerto-Hospedeiro/etiologia , Sobrevivência de Enxerto , Vidarabina/análogos & derivados , Vidarabina/administração & dosagem , Vidarabina/uso terapêutico , Anemia Falciforme/terapia , Doadores de Tecidos , Prognóstico , Talassemia/terapiaRESUMO
Chronic granulomatous disease (CGD) is a primary immunodeficiency resulting in life-threatening infections and inflammatory complications. Allogeneic hematopoietic cell transplantation (allo-HCT) can cure the disease, but the indication to transplant remains controversial. We performed a retrospective multicenter study of 712 patients with CGD who underwent allo-HCT transplantation from March 1993 through December 2018. We studied 635 children (aged <18 years) and 77 adults. Median follow-up was 45 months. Median age at transplantation was 7 years (range, 0.1-48.6). Kaplan-Meier estimates of overall survival (OS) and event-free survival (EFS) at 3 years were 85.7% and 75.8%, respectively. In multivariate analysis, older age was associated with reduced survival and increased chronic graft-versus-host disease. Nevertheless, OS and EFS at 3 years for patients ≥18 years were 76% and 69%, respectively. Use of 1-antigen-mismatched donors was associated with reduced OS and EFS . No significant difference was found in OS, but a significantly reduced EFS was noted in the small group of patients who received a transplant from a donor with a >1 antigen mismatch. Choice of conditioning regimen did not influence OS or EFS. In summary, we report an excellent outcome after allo-HCT in CGD, with low incidence of graft failure and mortality in all ages. Older patients and recipients of 1-antigen-mismatched grafts had a less favorable outcome. Transplantation should be strongly considered at a younger age and particularly in the presence of a well-matched donor.
Assuntos
Doença Granulomatosa Crônica/terapia , Transplante de Células-Tronco Hematopoéticas/mortalidade , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Seguimentos , Doença Granulomatosa Crônica/patologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Condicionamento Pré-Transplante , Transplante Homólogo , Adulto JovemRESUMO
During acute graft-versus-host disease (aGVHD) in mice, autoreactive T cells can be generated de novo in the host thymus implying an impairment in self-tolerance induction. As a possible mechanism, we have previously reported that mature medullary thymic epithelial cells (mTEC(high)) expressing the autoimmune regulator are targets of donor T-cell alloimmunity during aGVHD. A decline in mTEC(high) cell pool size, which purges individual tissue-restricted peripheral self-antigens (TRA) from the total thymic ectopic TRA repertoire, weakens the platform for central tolerance induction. Here we provide evidence in a transgenic mouse system using ovalbumin (OVA) as a model surrogate TRA that the de novo production of OVA-specific CD4(+) T cells during acute GVHD is a direct consequence of impaired thymic ectopic OVA expression in mTEC(high) cells. Our data, therefore, indicate that a functional compromise of the medullary mTEC(high) compartment may link alloimmunity to the development of autoimmunity during chronic GVHD.
Assuntos
Autoimunidade , Linfócitos T CD4-Positivos/patologia , Doença Enxerto-Hospedeiro/patologia , Tolerância a Antígenos Próprios , Timo/patologia , Animais , Autoantígenos/análise , Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Doença Enxerto-Hospedeiro/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologia , Timo/imunologiaRESUMO
Development of acute graft-versus-host disease (aGVHD) predisposes to chronic GVHD with autoimmune manifestations. A characteristic of experimental aGVHD is the de novo generation of autoreactive T cells. Central tolerance is dependent on the intrathymic expression of tissue-restricted peripheral self-antigens (TRA), which is in mature medullary thymic epithelial cells (mTEC(high)) partly controlled by the autoimmune regulator (Aire). Because TECs are targets of donor T-cell alloimmunity, we tested whether murine aGVHD interfered with the capacity of recipient Aire(+)mTEC(high) to sustain TRA diversity. We report that aGVHD weakens the platform for central tolerance induction because individual TRAs are purged from the total repertoire secondary to a decline in the Aire(+)mTEC(high) cell pool. Peritransplant administration of an epithelial cytoprotective agent, fibroblast growth factor-7, maintained a stable pool of Aire(+)mTEC(high), with an improved TRA transcriptome despite aGVHD. Taken together, our data provide a mechanism for how autoimmunity may develop in the context of antecedent alloimmunity.
Assuntos
Antígenos/genética , Células Epiteliais/imunologia , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Timo/metabolismo , Doença Aguda , Animais , Antígenos/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Feminino , Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Timo/patologiaRESUMO
Hassall's corpuscles are concentric clusters of keratinized epithelial cells located within the thymic medulla of humans and guinea pigs but are scant in mouse and rat. They are considered to be the terminally differentiated stages of medullary thymic epithelial cells (mTECs) but the mechanisms of their origin are unclear. We have previously deleted the TGF-ß type II receptor (TGFßRII) specifically in mouse TECs and reported that these mice have mitigated thymic involution and exhibit earlier reconstitution post-irradiation. In this study, we analyzed the differentiation of mTECs in the TGFßRII-knockout mice. Interestingly, the TGFßRII-knockout mice display enhanced development of Hassall's corpuscles. The expression of Aire, stromal-cell-derived factor 1 and thymic stromal lymphopoietin in the thymi of the TGFßRII-knockout mice was similar to that previously reported for the human thymus. In addition, the putative epithelial progenitor markers MTS20 and MTS24 labeled Hassall's corpuscles in normal mice, but the extent and intensity of this staining were greatly enhanced in Hassall's corpuscles of the TGFßRII-knockout mice. The phosphorylated forms of ERK and JNK were also found in Hassall's corpuscles of the TGFßRII-knockout mice. Taken together, we suggest that TGFßRII-mediated signaling in TECs inhibits their development into Hassall's corpuscles in mice.
Assuntos
Células Epiteliais/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Timo/citologia , Animais , Imunofluorescência , Camundongos , Camundongos Knockout , Microscopia Confocal , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/deficiência , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/deficiênciaRESUMO
Allogeneic HCT is a potentially curative treatment strategy for patients with inborn errors of immunities (IEIs). Objective of this study was to assess the optimal busulfan exposure prior to allogeneic HCT for patients with an IEI who received an intravenous busulfan-based conditioning regimen between 2000 and 2023. Patients from 17 international centers were included. Main outcome of interest was event-free survival (EFS). Patients were categorized into 4 IEI subgroups: combined-immunodeficiency (CID), severe combined immunodeficiency (SCID), neutrophil disorders and hemophagocytic lymphohistiocytosis (HLH)-related disorders. Busulfan exposure was calculated by individual centers (AUCCENTER) and was re-estimated using a validated model (AUCNONMEM). Overall, 562 patients were included: 173 (30.8%) CID, 154 (27.4%) SCID, 101 (18.0%) HLH-related disorders, and 134 (23.8%) neutrophil disorders. Median busulfan AUCNONMEM was 69.0 mg×h/L and correlated poorly with AUCCENTER (r2=0.54). Patients with SCID, HLH-related, and neutrophil disorders were analyzed together (n=389), because CID disease subtype was an effect modifier (p=0.03). Estimated 2-year EFS was 78.5%. In patients with the found optimal busulfan AUCNONMEM of 70-90 mg×h/L, 2-year EFS was superior to <70 mg×h/L (adj-HR 1.97, 95% CI 1.11-3.49, p=0.02), and >90 mg×h/L (adj-HR 5.05, 95% CI 2.43-10.49, p<0.0001). Full donor chimerism increased with higher busulfan AUCNONMEM, plateauing at 90 mg×h/L. For CID patients, optimal AUCNONMEM for donor chimerism was found to be >70 mg×h/L. Improved EFS and higher donor chimerism may be achieved by targeting a cumulative busulfan AUCNONMEM of 80 mg×h/L (range 70-90). Our study stresses the importance to uniformly using a validated population PK-model to estimate the AUCNONMEM.
RESUMO
Reactive oxygen species (ROS) are a family of highly reactive molecules with numerous, often pleiotropic functions within the cell and the organism. Due to their potential to destroy biological structures such as membranes, enzymes and organelles, ROS have long been recognized as harmful yet unavoidable by-products of cellular metabolism leading to "oxidative stress" unless counterbalanced by cellular anti-oxidative defense mechanisms. Phagocytes utilize this destructive potential of ROS released in high amounts to defend against invading pathogens. In contrast, a regulated and fine-tuned release of "signaling ROS" (sROS) provides essential intracellular second messengers to modulate central aspects of immunity, including antigen presentation, activation of antigen presenting cells (APC) as well as the APC:T cell interaction during T cell activation. This regulated release of sROS is foremost attributed to the specialized enzyme NADPH-oxidase (NOX) 2 expressed mainly in myeloid cells such as neutrophils, macrophages and dendritic cells (DC). NOX-2-derived sROS are primarily involved in immune regulation and mediate protection against autoimmunity as well as maintenance of self-tolerance. Consequently, deficiencies in NOX2 not only result in primary immune-deficiencies such as Chronic Granulomatous Disease (CGD) but also lead to auto-inflammatory diseases and autoimmunity. A comprehensive understanding of NOX2 activation and regulation will be key for successful pharmaceutical interventions of such ROS-related diseases in the future. In this review, we summarize recent progress regarding immune regulation by NOX2-derived ROS and the consequences of its deregulation on the development of immune disorders.
Assuntos
Doença Granulomatosa Crônica , NADPH Oxidases , Humanos , Espécies Reativas de Oxigênio/metabolismo , NADPH Oxidases/metabolismo , Neutrófilos/metabolismo , Doença Granulomatosa Crônica/metabolismo , Fagócitos/metabolismoRESUMO
Reaching target exposure of busulfan-based conditioning prior to hematopoietic stem cell transplantation is vital for favorable therapy outcomes. Yet, a wide inter-patient and inter-occasion variability in busulfan exposure has been reported, especially in children. We aimed to identify factors associated with the variability of busulfan pharmacokinetics in 124 consecutive patients transplanted at the University Children's Hospital Zurich between October 2010 and February 2020. Clinical data and busulfan plasma levels after twice-daily intravenous administration were analyzed retrospectively by population pharmacokinetic modeling. The volume of distribution correlated with total body water. The elimination rate constant followed an age-dependent maturation function, as previously suggested, and correlated with the levels of serum albumin. Acute lymphoblastic leukemia reduced busulfan clearance by 20%. Clearance significantly decreased by 17% on average from the start to the third day of busulfan administration, in agreement with other studies. An average reduction of 31% was found in patients with hemophagocytic lymphohistiocytosis and X-linked lymphoproliferative disease. In conclusion, we demonstrate that in addition to known factors, underlying disease and serum albumin significantly impact busulfan pharmacokinetics in pediatric patients; yet, substantial unexplained variability in some patients remained. Thus, we consider repeated pharmacokinetic assessment essential to achieve the desired target exposure in twice-daily busulfan administration.
RESUMO
T cell recognition of human leukocyte antigen (HLA)-presented tumor-associated peptides is central for cancer immune surveillance. Mass spectrometry (MS)-based immunopeptidomics represents the only unbiased method for the direct identification and characterization of naturally presented tumor-associated peptides, a key prerequisite for the development of T cell-based immunotherapies. This study reports on the implementation of ion mobility separation-based time-of-flight (TOFIMS) MS for next-generation immunopeptidomics, enabling high-speed and sensitive detection of HLA-presented peptides. Applying TOFIMS-based immunopeptidomics, a novel extensive benignTOFIMS dataset was generated from 94 primary benign samples of solid tissue and hematological origin, which enabled the expansion of benign reference immunopeptidome databases with > 150,000 HLA-presented peptides, the refinement of previously described tumor antigens, as well as the identification of frequently presented self antigens and not yet described tumor antigens comprising low abundant mutation-derived neoepitopes that might serve as targets for future cancer immunotherapy development.
Assuntos
Antígenos de Histocompatibilidade Classe I , Neoplasias , Humanos , Antígenos de Neoplasias , Espectrometria de Massas/métodos , Antígenos HLA , Neoplasias/terapia , Peptídeos/química , Antígenos de Histocompatibilidade Classe IIRESUMO
After allogeneic hematopoietic stem cell transplantation (allo-HSCT), the recurrence of recent thymic emigrants (RTE) and self-tolerant T cells indicate normalized thymic function. From 2008 to 2019, we retrospectively analyzed the RTE-reconstitution rate and the minimal time to reach normal age-specific first percentiles for CD31+CD45RA+CD4+T cells in 199 pediatric patients after allo-HSCT for various malignant and non-malignant diseases. The impact of clinically significant graft-versus-host disease (GvHD), age at transplantation, underlying disease and cumulative area under the curve of busulfan on RTE-reemergence was assessed in multivariable longitudinal analysis. RTE-reconstitution (coefficient -0.24, 95% CI -0.33 to -0.14, p < 0.001) was slowed down by GvHD and the time to reach P1 was significantly longer (Event Time Ratio 1.49, 95% CI 1.25 to 1.78, p < 0.001). Older age at transplantation was also associated with a slower RTE-reconstitution (coefficient -0.028, 95% CI -0.04 to -0.02, p < 0.001) and time to reach P1 was significantly longer (Event Time Ratio 1.03, 95% CI 1.02 to 1.05, p < 0.001). RTE-reconstitution velocity was not influenced by underlying disease or cumulative busulfan exposure. In summary, duration until thymic reactivation was independent of both conditioning intensity and underlying disease and was negatively influenced by older age and GvHD.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Bussulfano/uso terapêutico , Criança , Humanos , Estudos Retrospectivos , Linfócitos T , Timo , Condicionamento Pré-Transplante , Transplante HomólogoRESUMO
X-linked lymphoproliferative disease (XLP) is either caused by loss of the SLAM-associated protein (SAP; XLP-1) or the X-linked inhibitor of apoptosis (XIAP; XLP-2). In both instances, infection with the oncogenic human Epstein Barr virus (EBV) leads to pathology, but EBV-associated lymphomas only emerge in XLP-1 patients. Therefore, we investigated the role of XIAP during B cell transformation by EBV. Using humanized mice, IAP inhibition in EBV-infected mice led to a loss of B cells and a tendency to lower viral titers and lymphomagenesis. Loss of memory B cells was also observed in four newly described patients with XIAP deficiency. EBV was able to transform their B cells into lymphoblastoid cell lines (LCLs) with similar growth characteristics to patient mothers' LCLs in vitro and in vivo. Gene expression analysis revealed modest elevated lytic EBV gene transcription as well as the expression of the tumor suppressor cell adhesion molecule 1 (CADM1). CADM1 expression on EBV-infected B cells might therefore inhibit EBV-associated lymphomagenesis in patients and result in the absence of EBV-associated malignancies in XLP-2 patients.
Assuntos
Infecções por Vírus Epstein-Barr , Transtornos Linfoproliferativos , Animais , Humanos , Camundongos , Molécula 1 de Adesão Celular/genética , Molécula 1 de Adesão Celular/metabolismo , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/metabolismo , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/patologia , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Linfócitos BRESUMO
CONTEXT: Aromatase deficiency in women is a rare 46, XX disorder of sex differentiation characterized by a defect in catalysing oestrogens from androgens. OBJECTIVE: To better understand this rare disorder, we searched for mutations in the CYP19A1 gene of an affected girl and analysed their functional consequences. DESIGN AND PATIENT: We examined a girl presenting with clitoral hypertrophy at birth and mild maternal virilization (acne) during pregnancy. MAIN OUTCOME MEASUREMENT: A genotype-phenotype correlation was found. RESULTS: By direct sequencing of the CYP19A1 gene, we identified a heterozygous A>G mutation (c. A1374G) mutation in exon IX, leading to the missense p.N411S in the P450Aro protein and a heterozygous placenta promoter variant -41 base pairs upstream of exon I.1. Aromatase enzyme activity was completely lost when the mutant p.N411S protein was expressed in COS-1 cells. The placenta promoter variant had a significantly reduced (-50%) transactivation ability compared to wild-type. CONCLUSION: Our data describe a novel loss-of-function missense mutation in CYP19A1 combined with the first-described variant of the placenta promoter with a significant reduction in function, likely to be the molecular basis of this rare 46, XX disorder of sex development. This seems to represent a unique case of aromatase deficiency occurring in utero only.
Assuntos
Transtornos 46, XX do Desenvolvimento Sexual/genética , Aromatase/deficiência , Ginecomastia/genética , Infertilidade Masculina/genética , Erros Inatos do Metabolismo/genética , Transtornos 46, XX do Desenvolvimento Sexual/patologia , Adulto , Aromatase/genética , Criança , Feminino , Genótipo , Ginecomastia/patologia , Humanos , Infertilidade Masculina/patologia , Erros Inatos do Metabolismo/patologia , Mutação de Sentido Incorreto , Fenótipo , Placenta/metabolismo , Gravidez , Regiões Promotoras GenéticasRESUMO
Thymic T cell development is dependent on a specialized epithelial microenvironment mainly composed of cortical and medullary thymic epithelial cells (TECs). The molecular programs governing the differentiation and maintenance of TECs remain largely unknown. Wnt signaling is central to the development and maintenance of several organ systems but a specific role of this pathway for thymus organogenesis has not yet been ascertained. In this report, we demonstrate that activation of the canonical Wnt signaling pathway by a stabilizing mutation of beta-catenin targeted exclusively to TECs changes the initial commitment of endodermal epithelia to a thymic cell fate. Consequently, the formation of a correctly composed and organized thymic microenvironment is prevented, thymic immigration of hematopoietic precursors is restricted, and intrathymic T cell differentiation is arrested at a very early developmental stage causing severe immunodeficiency. These results suggest that a precise regulation of canonical Wnt signaling in thymic epithelia is essential for normal thymus development and function.
Assuntos
Diferenciação Celular/imunologia , Células Epiteliais/metabolismo , Inibidores do Crescimento/genética , Inibidores do Crescimento/metabolismo , Timo/imunologia , Timo/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Endoderma/citologia , Endoderma/imunologia , Endoderma/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/patologia , Fatores de Transcrição Forkhead/genética , Marcação de Genes , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Timo/citologia , Timo/patologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/biossíntese , beta Catenina/fisiologiaRESUMO
Thymic epithelial cells (TECs) are essential in supporting the development of mature T cells from hematopoietic progenitor cells and facilitate their lineage-commitment, proliferation, T-cell receptor repertoire selection and maturation. While animal model systems have greatly aided in elucidating the contribution of stromal cells to these intricate processes, human tissue has been more difficult to study, partly due to a lack of suitable surface markers comprehensively defining human TECs. Here, we conducted a flow cytometry based surface marker screen to reliably identify and quantify human TECs and delineate medullary from cortical subsets. These findings were validated by transcriptomic and histologic means. The combination of EpCAM, podoplanin (pdpn), CD49f and CD200 comprehensively identified human TECs and not only allowed their reliable distinction in medullary and cortical subsets but also their detailed quantitation. Transcriptomic profiling of each subset in comparison to fibroblasts and endothelial cells confirmed the identity of the different stromal cell subsets sorted according to the proposed strategy. Our dataset not only demonstrated transcriptional similarities between TEC and cells of mesenchymal origin but furthermore revealed a subset-specific distribution of a specific set of extracellular matrix-related genes in TECs. This indicates that TECs significantly contribute to the distinct compartmentalization - and thus function - of the human thymus. We applied the strategy to quantify TEC subsets in 31 immunologically healthy children, which revealed sex-specific differences of TEC composition early in life. As the distribution of mature CD4- or CD8-single-positive thymocytes was correspondingly altered, the composition of the thymic epithelial compartment may directly impact on the CD4-CD8-lineage choice of thymocytes. We prove that the plain, reliable strategy proposed here to comprehensively identify human TEC subpopulations by flow cytometry based on surface marker expression is suitable to determine their frequency and phenotype in health and disease and allows sorting of live cells for downstream analysis. Its use reaches from a reliable diagnostic tool for thymic biopsies to improved phenotypic characterization of thymic grafts intended for therapeutic use.
Assuntos
Separação Celular , Células Epiteliais/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Células Estromais/metabolismo , Timo/metabolismo , Transcriptoma , Síndrome da Deleção 22q11/genética , Síndrome da Deleção 22q11/imunologia , Síndrome da Deleção 22q11/metabolismo , Adolescente , Fatores Etários , Biomarcadores/metabolismo , Criança , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos Par 22 , Células Epiteliais/imunologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Miastenia Gravis/genética , Miastenia Gravis/imunologia , Miastenia Gravis/metabolismo , Fenótipo , Fatores Sexuais , Células Estromais/imunologia , Timo/citologia , Timo/imunologiaRESUMO
BACKGROUND: The human leucocyte antigen (HLA) complex controls adaptive immunity by presenting defined fractions of the intracellular and extracellular protein content to immune cells. Understanding the benign HLA ligand repertoire is a prerequisite to define safe T-cell-based immunotherapies against cancer. Due to the poor availability of benign tissues, if available, normal tissue adjacent to the tumor has been used as a benign surrogate when defining tumor-associated antigens. However, this comparison has proven to be insufficient and even resulted in lethal outcomes. In order to match the tumor immunopeptidome with an equivalent counterpart, we created the HLA Ligand Atlas, the first extensive collection of paired HLA-I and HLA-II immunopeptidomes from 227 benign human tissue samples. This dataset facilitates a balanced comparison between tumor and benign tissues on HLA ligand level. METHODS: Human tissue samples were obtained from 16 subjects at autopsy, five thymus samples and two ovary samples originating from living donors. HLA ligands were isolated via immunoaffinity purification and analyzed in over 1200 liquid chromatography mass spectrometry runs. Experimentally and computationally reproducible protocols were employed for data acquisition and processing. RESULTS: The initial release covers 51 HLA-I and 86 HLA-II allotypes presenting 90,428 HLA-I- and 142,625 HLA-II ligands. The HLA allotypes are representative for the world population. We observe that immunopeptidomes differ considerably between tissues and individuals on source protein and HLA-ligand level. Moreover, we discover 1407 HLA-I ligands from non-canonical genomic regions. Such peptides were previously described in tumors, peripheral blood mononuclear cells (PBMCs), healthy lung tissues and cell lines. In a case study in glioblastoma, we show that potential on-target off-tumor adverse events in immunotherapy can be avoided by comparing tumor immunopeptidomes to the provided multi-tissue reference. CONCLUSION: Given that T-cell-based immunotherapies, such as CAR-T cells, affinity-enhanced T cell transfer, cancer vaccines and immune checkpoint inhibition, have significant side effects, the HLA Ligand Atlas is the first step toward defining tumor-associated targets with an improved safety profile. The resource provides insights into basic and applied immune-associated questions in the context of cancer immunotherapy, infection, transplantation, allergy and autoimmunity. It is publicly available and can be browsed in an easy-to-use web interface at https://hla-ligand-atlas.org .
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos HLA/imunologia , Imunoterapia Adotiva , Neoplasias/terapia , Peptídeos/imunologia , Proteoma , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/transplante , Idoso , Idoso de 80 Anos ou mais , Cromatografia Líquida , Bases de Dados de Proteínas , Feminino , Humanos , Lactente , Recém-Nascido , Ligantes , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Neoplasias/imunologia , Proteômica , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Espectrometria de Massas em TandemRESUMO
Signals mediated by the transforming growth factor-beta superfamily of growth factors have been implicated in thymic epithelial cell (TEC) differentiation, homeostasis, and function, but a direct reliance on these signals has not been established. Here we demonstrate that a block in canonical transforming growth factor-beta signaling by the loss of Smad4 expression in TECs leads to qualitative changes in TEC function and a progressively disorganized thymic microenvironment. Moreover, the number of thymus resident early T-lineage progenitors is severely reduced in the absence of Smad4 expression in TECs and directly correlates with extensive thymic and peripheral lymphopenia. Our observations hence place Smad4 within the signaling events in TECs that determine total thymus cellularity by controlling the number of early T-lineage progenitors.
Assuntos
Proteína Smad4/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Timo/citologia , Timo/metabolismo , Animais , Sequência de Bases , Quimiocinas/genética , DNA Complementar/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Homeostase , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Gravidez , Transdução de Sinais , Proteína Smad4/deficiência , Proteína Smad4/genética , Linfócitos T/imunologia , Timo/imunologia , Transcrição GênicaRESUMO
The thymus constitutes the primary lymphoid organ responsible for the generation of naive T cells. Its stromal compartment is largely composed of a scaffold of different subsets of epithelial cells that provide soluble and membrane-bound molecules essential for thymocyte maturation and selection. With senescence, a steady decline in the thymic output of T cells has been observed. Numeric and qualitative changes in the stromal compartment of the thymus resulting in reduced thymopoietic capacity have been suggested to account for this physiologic process. The precise cellular and molecular mechanisms underlying thymic senescence are, however, only incompletely understood. Here, we demonstrate that TGF-beta signaling in thymic epithelial cells exerts a direct influence on the cell's capacity to support thymopoiesis in the aged mouse as the physiologic process of thymic senescence is mitigated in mice deficient for the expression of TGF-beta RII on thymic epithelial cells. Moreover, TGF-beta signaling in these stromal cells transiently hinders the early phase of thymic reconstitution after myeloablative conditioning and hematopoietic stem cell transplantation. Hence, inhibition of TGF-beta signaling decelerates the process of age-related thymic involution and may hasten the reconstitution of regular thymopoiesis after hematopoietic stem cell transplantation.