RESUMO
Mislabeling samples or data with the wrong participant information can affect study integrity and lead investigators to draw inaccurate conclusions. Quality control to prevent these types of errors is commonly embedded into the analysis of genomic datasets, but a similar identification strategy is not standard for cytometric data. Here, we present a method for detecting sample identification errors in cytometric data using expression of human leukocyte antigen (HLA) class I alleles. We measured HLA-A*02 and HLA-B*07 expression in three longitudinal samples from 41 participants using a 33-marker CyTOF panel designed to identify major immune cell types. 3/123 samples (2.4%) showed HLA allele expression that did not match their longitudinal pairs. Furthermore, these same three samples' cytometric signature did not match qPCR HLA class I allele data, suggesting that they were accurately identified as mismatches. We conclude that this technique is useful for detecting sample-labeling errors in cytometric analyses of longitudinal data. This technique could also be used in conjunction with another method, like GWAS or PCR, to detect errors in cross-sectional data. We suggest widespread adoption of this or similar techniques will improve the quality of clinical studies that utilize cytometry.
Assuntos
Estudos Transversais , Alelos , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: The PALISADE study, an international, phase 3 trial of peanut oral immunotherapy (POIT) with AR101, resulted in desensitization in children and adolescents who were highly allergic to peanut. An improved understanding of the immune mechanism induced in response to food allergen immunotherapy would enable more informed and effective therapeutic strategies. Our main purpose was to examine the immunological changes in blood samples from a subset of peanut-allergic individuals undergoing oral desensitization immunotherapy with AR101. METHODS: Blood samples obtained as part of enrollment screening and at multiple time points during PALISADE study were used to assess basophil and CD4+ T-cell reactivity to peanut. RESULTS: The absence of clinical reactivity to the entry double-blinded placebo-controlled peanut challenge (DBPCFC) was accompanied by a significantly lower basophil sensitivity and T-cell reactivity to peanut compared with DBPCFC reactors. At baseline, peanut-reactive TH2A cells were observed in many but not all peanut-allergic patients and their level in peripheral blood correlates with T-cell reactivity to peanut and with serum peanut-specific IgE and IgG4 levels. POIT reshaped circulating peanut-reactive T-cell responses in a subset-dependent manner. Changes in basophil and T-cell responses to peanut closely paralleled clinical benefits to AR101 therapy and resemble responses in those with lower clinical sensitivity to peanut. However, no difference in peanut-reactive Treg cell frequency was observed between groups. CONCLUSION: Oral desensitization therapy with AR101 leads to decreased basophil sensitivity to peanut and reshapes peanut-reactive T effector cell responses supporting its potential as an immunomodulatory therapy.
Assuntos
Hipersensibilidade a Amendoim , Administração Oral , Adolescente , Alérgenos , Arachis , Criança , Dessensibilização Imunológica/métodos , Humanos , Imunidade , Hipersensibilidade a Amendoim/terapiaRESUMO
The significance of islet Ag-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear, partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior Ag exposure, inferred from expanded TCR clonotypes, and essential phenotypic properties in their transcriptomes. To test this, we developed single-cell RNA sequencing procedures for identifying TCR clonotypes and transcript phenotypes in individual T cells. We applied these procedures to analysis of islet Ag-reactive CD4+ memory T cells from the blood of T1D and HC individuals after activation with pooled immunodominant islet peptides. We found extensive TCR clonotype sharing in Ag-activated cells, especially from individual T1D subjects, consistent with in vivo T cell expansion during disease progression. The expanded clonotype from one T1D subject was detected at repeat visits spanning >15 mo, demonstrating clonotype stability. Notably, we found no clonotype sharing between subjects, indicating a predominance of "private" TCR specificities. Expanded clones from two T1D subjects recognized distinct IGRP peptides, implicating this molecule as a trigger for CD4+ T cell expansion. Although overall transcript profiles of cells from HC and T1D subjects were similar, profiles from the most expanded clones were distinctive. Our findings demonstrate that islet Ag-reactive CD4+ memory T cells with unique Ag specificities and phenotypes are expanded during disease progression and can be detected by single-cell analysis of peripheral blood.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Ilhotas Pancreáticas/imunologia , Ativação Linfocitária , Adulto , Células Clonais , Diabetes Mellitus Tipo 1/sangue , Feminino , Perfilação da Expressão Gênica , Humanos , Memória Imunológica , Masculino , Peptídeos/imunologia , Fenótipo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
Most West Nile virus (WNV) infections are asymptomatic, but some lead to neuroinvasive disease with symptoms ranging from disorientation to paralysis and death. Evidence from animal models suggests that neuroinvasive infections may arise as a consequence of impaired immune protection. However, other data suggest that neurologic symptoms may arise as a consequence of immune mediated damage. We demonstrate that elevated immune responses are present in neuroinvasive disease by directly characterizing WNV-specific T cells in subjects with laboratory documented infections using human histocompatibility leukocyte antigen (HLA) class II tetramers. Subjects with neuroinvasive infections had higher overall numbers of WNV-specific T cells than those with asymptomatic infections. Independent of this, we also observed age related increases in WNV-specific T cell responses. Further analysis revealed that WNV-specific T cell responses included a population of atypically polarized CXCR3+CCR4+CCR6- T cells, whose presence was highly correlated with neuroinvasive disease. Moreover, a higher proportion of WNV-specific T cells in these subjects co-produced interferon-γ and interleukin 4 than those from asymptomatic subjects. More globally, subjects with neuroinvasive infections had reduced numbers of CD4+FoxP3+ Tregs that were CTLA4 positive and exhibited a distinct upregulated transcript profile that was absent in subjects with asymptomatic infections. Thus, subjects with neuroinvasive WNV infections exhibited elevated, dysregulated, and atypically polarized responses, suggesting that immune mediated damage may indeed contribute to pathogenic outcomes.
Assuntos
Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Febre do Nilo Ocidental/imunologia , Adulto , Idoso , Epitopos de Linfócito T/imunologia , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The loss of tolerance and the presence of circulating autoantibodies directed against nuclear Ags is the hallmark of systemic lupus erythematosus (SLE). Many of these Ags are complexed with short, noncoding RNAs, such as U1 and Y1. The amount of U1 and Y1 RNA complexed with SLE patient Abs and immune complexes was measured in a cross-section of 228 SLE patients to evaluate the role of these RNA molecules within the known biochemical framework of SLE. The study revealed that SLE patients had significantly elevated levels of circulating U1 and/or Y1 RNA compared with healthy volunteers. In addition, the blood-borne RNA molecules were correlated with SLE disease activity and increased expression of IFN-inducible genes. To our knowledge, this study provides the first systematic examination of the role of circulating RNA in a large group of SLE patients and provides an important link with IFN dysregulation.
Assuntos
Regulação da Expressão Gênica , Interferons/imunologia , Lúpus Eritematoso Sistêmico/genética , RNA/sangue , Adulto , Reações Antígeno-Anticorpo , Autoanticorpos/imunologia , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Pessoa de Meia-Idade , RNA/imunologia , RNA Citoplasmático Pequeno/sangue , RNA Nuclear Pequeno/sangueRESUMO
BACKGROUND: Differentiation of systemic juvenile idiopathic arthritis (SJIA) fever from other childhood fevers is often delayed due to the lack of reliable, specific biomarkers. We hypothesized that PD-L1 expression is dysregulated in SJIA monocytes and compared it to other candidate SJIA biomarkers. METHODS: This pilot study enrolled children with fever without source and compared PD-L1 expression on myeloid cells to C-reactive protein, erythrocyte sedimentation rate, leukocyte counts, S100A12, S100A8, S100A9, calprotectin, and procalcitonin. Logistic regression models were fit to test SJIA diagnosis with each marker used as an independent predictor. Receiver operating characteristic curves and area under curve were calculated. Gene expression profiling on a subset of samples was performed. RESULTS: Twenty subjects (10 active SJIA, 10 febrile non-SJIA) were enrolled. S100 proteins were significantly elevated in SJIA with >80% sensitivity and >90% specificity. PD-L1 expression was significantly lower in SJIA. Other markers were not specific for SJIA. On exploratory gene analysis, 106 genes were significant for SJIA association, and several of these are associated with immune response pathways. CONCLUSION: In this small cohort, S100 proteins were specific diagnostic biomarkers for SJIA in children with fever. Decreased PD-L1 surface expression on circulating myeloid cells in SJIA suggests possible mechanism for loss of peripheral immune regulation.
Assuntos
Artrite Juvenil/sangue , Artrite Juvenil/diagnóstico , Antígeno B7-H1/sangue , Proteínas S100/sangue , Área Sob a Curva , Artrite Juvenil/genética , Biomarcadores/sangue , Análise Química do Sangue , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Masculino , Projetos Piloto , Valor Preditivo dos Testes , Prognóstico , Curva ROCRESUMO
Distinct Natural Killer (NK)-like CD57+ and PD-1+ CD8+ exhausted-like T cell populations (Tex) have both been linked to beneficial immunotherapy response in autoimmune type 1 diabetes (T1D) patients. The origins and relationships between these cell types are poorly understood. Here we show that while PD-1+ and CD57+ Tex populations are epigenetically similar, CD57+ Tex cells display unique increased chromatin accessibility of inhibitory Killer Cell Immunoglobulin-like Receptor (iKIR) and other NK cell genes. PD-1+ and CD57+ Tex also show reciprocal expression of Inhibitory Receptors (IRs) and iKIRs accompanied by chromatin accessibility of Tcf1 and Tbet transcription factor target sites, respectively. CD57+ Tex show unappreciated gene expression heterogeneity and share clonal relationships with PD-1+ Tex, with these cells differentiating along four interconnected lineage trajectories: Tex-PD-1+, Tex-CD57+, Tex-Branching, and Tex-Fluid. Our findings demonstrate new relationships between Tex-like populations in human autoimmune disease and suggest that modulating common precursor populations may enhance response to autoimmune disease treatment.
Assuntos
Linfócitos T CD8-Positivos , Diabetes Mellitus Tipo 1 , Células Matadoras Naturais , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/genética , Humanos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/genética , Antígenos CD57/metabolismo , Linhagem da Célula/genética , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Feminino , Masculino , AdultoRESUMO
Variation in the preservation of ß cell function in clinical trials in type 1 diabetes (T1D) has emphasized the need to define biomarkers to predict treatment response. The T1DAL trial targeted T cells with alefacept (LFA-3-Ig) and demonstrated C-peptide preservation in approximately 30% of new-onset T1D individuals. We analyzed islet antigen-reactive (IAR) CD4+ T cells in PBMC samples collected prior to treatment from alefacept- and placebo-treated individuals using flow cytometry and single-cell RNA sequencing. IAR CD4+ T cells at baseline had heterogeneous phenotypes. Transcript profiles formed phenotypic clusters of cells along a trajectory based on increasing maturation and activation, and T cell receptor (TCR) chains showed clonal expansion. Notably, the frequency of IAR CD4+ T cells with a memory phenotype and a unique transcript profile (cluster 3) were inversely correlated with C-peptide preservation in alefacept-treated, but not placebo-treated, individuals. Cluster 3 cells had a proinflammatory phenotype characterized by expression of the transcription factor BHLHE40 and the cytokines GM-CSF and TNF-α, and shared TCR chains with effector memory-like clusters. Our results suggest IAR CD4+ T cells as a potential baseline biomarker of response to therapies targeting the CD2 pathway and warrant investigation for other T cell-related therapies.
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Diabetes Mellitus Tipo 1 , Humanos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Alefacept/uso terapêutico , Peptídeo C , Leucócitos Mononucleares/metabolismo , Biomarcadores , Receptores de Antígenos de Linfócitos T/uso terapêuticoRESUMO
BACKGROUNDLow-dose anti-thymocyte globulin (ATG) transiently preserves C-peptide and lowers HbA1c in individuals with recent-onset type 1 diabetes (T1D); however, the mechanisms of action and features of the response remain unclear. Here, we characterized the post hoc immunological outcomes of ATG administration and their potential use as biomarkers of metabolic response to therapy (i.e., improved preservation of endogenous insulin production).METHODSWe assessed gene and protein expression, targeted gene methylation, and cytokine concentrations in peripheral blood following treatment with ATG (n = 29), ATG plus granulocyte colony-stimulating factor (ATG/G-CSF, n = 28), or placebo (n = 31).RESULTSTreatment with low-dose ATG preserved regulatory T cells (Tregs), as measured by stable methylation of FOXP3 Treg-specific demethylation region (TSDR) and increased proportions of CD4+FOXP3+ Tregs (P < 0.001) identified by flow cytometry. While treatment effects were consistent across participants, not all maintained C-peptide. Responders exhibited a transient rise in IL-6, IP-10, and TNF-α (P < 0.05 for all) 2 weeks after treatment and a durable CD4+ exhaustion phenotype (increased PD-1+KLRG1+CD57- on CD4+ T cells [P = 0.011] and PD1+CD4+ Temra MFI [P < 0.001] at 12 weeks, following ATG and ATG/G-CSF, respectively). ATG nonresponders displayed higher proportions of senescent T cells (at baseline and after treatment) and increased methylation of EOMES (i.e., less expression of this exhaustion marker).CONCLUSIONAltogether in these exploratory analyses, Th1 inflammation-associated serum and CD4+ exhaustion transcript and cellular phenotyping profiles may be useful for identifying signatures of clinical response to ATG in T1D.TRIAL REGISTRATIONClinicalTrials.gov NCT02215200.FUNDINGThe Leona M. and Harry B. Helmsley Charitable Trust (2019PG-T1D011), the NIH (R01 DK106191 Supplement, K08 DK128628), NIH TrialNet (U01 DK085461), and the NIH NIAID (P01 AI042288).
Assuntos
Soro Antilinfocitário , Diabetes Mellitus Tipo 1 , Humanos , Soro Antilinfocitário/uso terapêutico , Linfócitos T CD4-Positivos/metabolismo , Exaustão das Células T , Peptídeo C , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismoRESUMO
The transcriptional and phenotypic characteristics that define alveolar monocyte and macrophage subsets in acute hypoxemic respiratory failure (AHRF) are poorly understood. Here, we apply CITE-seq (single-cell RNA-sequencing and cell-surface protein quantification) to bronchoalveolar lavage and blood specimens longitudinally collected from participants with AHRF to identify alveolar myeloid subsets, and then validate their identity in an external cohort using flow cytometry. We identify alveolar myeloid subsets with transcriptional profiles that differ from other lung diseases as well as several subsets with similar transcriptional profiles as reported in healthy participants (Metallothionein) or patients with COVID-19 (CD163/LGMN). We use information from CITE-seq to determine cell-surface proteins that distinguish transcriptional subsets (CD14, CD163, CD123, CD71, CD48, CD86 and CD44). In the external cohort, we find a higher proportion of CD163/LGMN alveolar macrophages are associated with mortality in AHRF. We report a parsimonious set of cell-surface proteins that distinguish alveolar myeloid subsets using scalable approaches that can be applied to clinical cohorts.
Assuntos
Pneumopatias , Insuficiência Respiratória , Humanos , Macrófagos Alveolares/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Pneumopatias/metabolismo , Insuficiência Respiratória/genéticaRESUMO
PURPOSE: Early accurate diagnosis of infection ± organ dysfunction (sepsis) remains a major challenge in clinical practice. Utilizing effective biomarkers to identify infection and impending organ dysfunction before the onset of clinical signs and symptoms would enable earlier investigation and intervention. To our knowledge, no prior study has specifically examined the possibility of pre-symptomatic detection of sepsis. METHODS: Blood samples and clinical/laboratory data were collected daily from 4385 patients undergoing elective surgery. An adjudication panel identified 154 patients with definite postoperative infection, of whom 98 developed sepsis. Transcriptomic profiling and subsequent RT-qPCR were undertaken on sequential blood samples taken postoperatively from these patients in the three days prior to the onset of symptoms. Comparison was made against postoperative day-, age-, sex- and procedure- matched patients who had an uncomplicated recovery (n =151) or postoperative inflammation without infection (n =148). RESULTS: Specific gene signatures optimized to predict infection or sepsis in the three days prior to clinical presentation were identified in initial discovery cohorts. Subsequent classification using machine learning with cross-validation with separate patient cohorts and their matched controls gave high Area Under the Receiver Operator Curve (AUC) values. These allowed discrimination of infection from uncomplicated recovery (AUC 0.871), infectious from non-infectious systemic inflammation (0.897), sepsis from other postoperative presentations (0.843), and sepsis from uncomplicated infection (0.703). CONCLUSION: Host biomarker signatures may be able to identify postoperative infection or sepsis up to three days in advance of clinical recognition. If validated in future studies, these signatures offer potential diagnostic utility for postoperative management of deteriorating or high-risk surgical patients and, potentially, other patient populations.
Assuntos
Sepse , Transcriptoma , Biomarcadores , Humanos , Inflamação/complicações , Insuficiência de Múltiplos Órgãos , Complicações Pós-Operatórias/diagnósticoRESUMO
High levels of the inflammatory cytokine IL-6 in the bone marrow are associated with poor outcomes in pediatric acute myeloid leukemia (pAML), but its etiology remains unknown. Using RNA-seq data from pre-treatment bone marrows of 1489 children with pAML, we show that > 20% of patients have concurrent IL-6, IL-1, IFNα/ß, and TNFα signaling activity and poorer outcomes. Targeted sequencing of pre-treatment bone marrow samples from affected patients (n = 181) revealed 5 highly recurrent patterns of somatic mutation. Using differential expression analyses of the most common genomic subtypes (~60% of total), we identify high expression of multiple potential drivers of inflammation-related treatment resistance. Regardless of genomic subtype, we show that JAK1/2 inhibition reduces receptor-mediated inflammatory signaling by leukemic cells in-vitro. The large number of high-risk pAML genomic subtypes presents an obstacle to the development of mutation-specific therapies. Our findings suggest that therapies targeting inflammatory signaling may be effective across multiple genomic subtypes of pAML.
Assuntos
Medula Óssea , Leucemia Mieloide Aguda , Humanos , Criança , Medula Óssea/metabolismo , Interleucina-6/metabolismo , Transdução de Sinais/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismoRESUMO
Individuals with Down syndrome show cellular and clinical features of dysregulated aging of the immune system, including a shift from naïve to memory T cells and increased incidence of autoimmunity. However, a quantitative understanding of how various immune compartments change with age in Down syndrome remains lacking. Here, we performed deep immunophenotyping of a cohort of individuals with Down syndrome across the life span, selecting for autoimmunity-free individuals. We simultaneously interrogated age- and sex-matched healthy controls and people with type 1 diabetes as a representative autoimmune disease. We built an analytical software, IMPACD (Iterative Machine-assisted Permutational Analysis of Cytometry Data), that enabled us to rapidly identify many features of immune dysregulation in Down syndrome shared with other autoimmune diseases. We found quantitative and qualitative dysregulation of naïve CD4+ and CD8+ T cells in individuals with Down syndrome and identified interleukin-6 as a candidate driver of some of these changes, thus extending the consideration of immunopathologic cytokines in Down syndrome beyond interferons. We used immune cellular composition to generate three linear models of aging (immune clocks) trained on control participants. All three immune clocks demonstrated advanced immune aging in individuals with Down syndrome. One of these clocks, informed by Down syndromerelevant biology, also showed advanced immune aging in individuals with type 1 diabetes. Orthologous RNA sequencingderived immune clocks also demonstrated advanced immune aging in individuals with Down syndrome. Together, our findings demonstrate an approach to studying immune aging in Down syndrome that may have implications in other autoimmune diseases.
Assuntos
Doenças Autoimunes , Diabetes Mellitus Tipo 1 , Síndrome de Down , Envelhecimento , Autoimunidade/genética , Linfócitos T CD8-Positivos , Síndrome de Down/genética , Humanos , ImunofenotipagemRESUMO
OBJECTIVE: We previously observed the association of the co-occurrence of the HLA-DRB1 shared epitope (SE) and RANKL single-nucleotide polymorphisms (SNPs) with younger age at the onset of rheumatoid arthritis (RA) in 182 rheumatoid factor (RF)-positive European American patients with early-onset RA. The aim of this study was to fine-map the 48-kb RANKL region in the extended cohort of 210 European American RF-positive patients with early RA, to seek replication of RA-associated SNPs in additional RA cohorts of 501 European Americans and 298 African Americans, and to explore the functional consequences of RA-associated SNPs. METHODS: SNP genotyping was conducted using pyrosequencing or TaqMan polymerase chain reaction (PCR) assays. Associations of rs7984870 with RANKL expression in plasma, peripheral blood mononuclear cells, and isolated T cells were quantified using enzyme-linked immunosorbent assay and reverse transcription-PCR. Site-directed mutagenesis of rs7984870 within the 2-kb RANKL promoter was performed to drive the luciferase reporter gene in osteoblast and stromal cell lines. Interaction of DNA and protein was determined by electrophoretic mobility shift assay. RESULTS: A single promoter SNP, rs7984870, was consistently significantly associated with earlier age at the onset of RA in 3 independent seropositive (RF or anti-cyclic citrullinated peptide antibody) RA cohorts but not in seronegative RA patients. The C risk allele of rs7984870 conferred 2-fold higher plasma RANKL levels in RF-positive patients with RA, significantly elevated RANKL messenger RNA expression in activated normal T cells, and increased promoter activity after stimulation in vitro via differential binding to the transcription factor SOX5. CONCLUSION: The RANKL promoter allele that increased transcription levels upon stimulation might promote interaction between activated T cells and dendritic cells, predisposing to a younger age at the onset of RA in seropositive European American and African American patients.
Assuntos
Artrite Reumatoide/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Ligante RANK/genética , Adulto , Negro ou Afro-Americano/genética , Idade de Início , Artrite Reumatoide/etnologia , Feminino , Predisposição Genética para Doença/etnologia , Predisposição Genética para Doença/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Ligante RANK/sangue , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXD/fisiologia , População Branca/genéticaRESUMO
Immune health requires innate and adaptive immune cells to engage precisely balanced pro- and anti-inflammatory forces. We employ the concept of chemical immunophenotypes to classify small molecules functionally or mechanistically according to their patterns of effects on primary innate and adaptive immune cells. The high-specificity, low-toxicity cyclin-dependent kinase 8 (CDK8) inhibitor 16-didehydro-cortistatin A (DCA) exerts a distinct tolerogenic profile in both innate and adaptive immune cells. DCA promotes regulatory T cells (Treg) and Th2 differentiation while inhibiting Th1 and Th17 differentiation in both murine and human cells. This unique chemical immunophenotype led to mechanistic studies showing that DCA promotes Treg differentiation in part by regulating a previously undescribed CDK8-GATA3-FOXP3 pathway that regulates early pathways of Foxp3 expression. These results highlight previously unappreciated links between Treg and Th2 differentiation and extend our understanding of the transcription factors that regulate Treg differentiation and their temporal sequencing. These findings have significant implications for future mechanistic and translational studies of CDK8 and CDK8 inhibitors.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Fatores de Transcrição Forkhead/metabolismo , Fator de Transcrição GATA3/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Imunofenotipagem , Isoquinolinas/farmacologia , Adolescente , Adulto , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Quinase 8 Dependente de Ciclina/metabolismo , Humanos , Imunidade Inata/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto JovemRESUMO
Human islet antigen reactive CD4+ memory T cells (IAR T cells) play a key role in the pathogenesis of autoimmune type 1 diabetes (T1D). Using single-cell RNA sequencing (scRNA-Seq) to identify T cell receptors (TCRs) in IAR T cells, we have identified a class of TCRs that share TCRα chains between individuals ("public" chains). We isolated IAR T cells from blood of healthy, new-onset T1D and established T1D donors using multiplexed CD154 enrichment and identified paired TCRαß sequences from 2767 individual cells. More than a quarter of cells shared TCR junctions between 2 or more cells ("expanded"), and 29/47 (~62%) of expanded TCRs tested showed specificity for islet antigen epitopes. Public TCRs sharing TCRα junctions were most prominent in new-onset T1D. Public TCR sequences were more germline like than expanded unique, or "private," TCRs, and had shorter junction sequences, suggestive of fewer random nucleotide insertions. Public TCRα junctions were often paired with mismatched TCRß junctions in TCRs; remarkably, a subset of these TCRs exhibited cross-reactivity toward distinct islet antigen peptides. Our findings demonstrate a prevalent population of IAR T cells with diverse specificities determined by TCRs with restricted TCRα junctions and germline-constrained antigen recognition properties. Since these "innate-like" TCRs differ from previously described immunodominant TCRß chains in autoimmunity, they have implications for fundamental studies of disease mechanisms. Self-reactive restricted TCRα chains and their associated epitopes should be considered in fundamental and translational investigations of TCRs in T1D.
Assuntos
Diabetes Mellitus Tipo 1/genética , Células Germinativas/metabolismo , Cadeias alfa de Imunoglobulina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Adolescente , Adulto , Feminino , Humanos , Masculino , Adulto JovemRESUMO
STUDY OBJECTIVES: To investigate associations between HLA-DQB1*0602 allele status and measures of narcolepsy symptom severity. DESIGN: Cross-sectional study of population-based narcolepsy patients. SETTING: King County, Washington. PARTICIPANTS: All prevalent cases (n = 279) of physician-diagnosed narcolepsy ascertained from 2001-2005. INTERVENTIONS: N/A. MEASUREMENTS: Narcolepsy diagnosis was based on cataplexy status, diagnostic sleep study results, and chart review. The number of HLA-DQB1 alleles was determined from buccal genomic DNA. Symptom severity instruments included the Epworth Sleepiness Scale (ESS), the Ullanlinna Narcolepsy Scale (UNS), age of symptom onset, subjective sleep latency and duration, and various clinical sleep parameters. We used linear regression adjusted for African American race and an extended chi-square test of trend to assess relationships across ordered groups defined by allele number (0, 1, or 2). RESULTS: Narcolepsy patients were 63% female and 82% Caucasian, with a mean age of 47.6 years (SD = 17.1). One hundred forty-one (51%) patients had no DQB1*0602 alleles; 117 (42%) had one; and 21 (7%) had two. In the complete narcolepsy sample after adjustment for African American race, we observed a linear relationship between HLA-DQB1*0602 frequency and sleepiness as defined by the ESS (P < 0.01), narcolepsy severity as defined by UNS (P < 0.001), age of symptom onset (P < 0.05), and sleep latency (P < 0.001). In univariate analyses, HLA-DQB1*0602 frequency was also associated with napping (P < 0.05) and increased car and work accidents or near accidents (both P < 0.01). Habitual sleep duration was not associated with HLA status. These race-adjusted associations remained for the ESS (P < 0.05), UNS (P < 0.01), and sleep latency (P < 0.001) when restricting to narcolepsy with cataplexy. CONCLUSIONS: Narcolepsy symptom severity varies in a linear manner according to HLA-DQB1*0602 allele status. These findings support the notion that HLA-DQ is a disease-modifying gene.
Assuntos
Alelos , Genótipo , Antígenos HLA-DQ/genética , Glicoproteínas de Membrana/genética , Narcolepsia/genética , Adulto , Idoso , Cataplexia/diagnóstico , Cataplexia/genética , Estudos Transversais , Feminino , Frequência do Gene/genética , Triagem de Portadores Genéticos , Cadeias beta de HLA-DQ , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Narcolepsia/diagnósticoRESUMO
The activation threshold for antigen-specific T cell responses is dependent on the avidity of the trimolecular interaction between TCR, antigen, and MHC. We compared CD4+ T cell avidities for the diabetes-associated autoantigen glutamic acid decarboxylase 555-567 (GAD 555) among serial samples from autoantibody-positive subjects at high risk of progression to type 1 diabetes (T1D). T cells from three at-risk subjects demonstrated significant avidity increases (p<0.05 by F test) over time. This avidity shift correlated with the outgrowth of T cells expressing TCR BV 9, 15, 17 or 20 that demonstrated higher GAD 555 tetramer-binding levels compared to cells expressing other TCR BV genes. Similar analysis of autoantibody-negative, first-degree relatives and T1D patients did not demonstrate similar changes in avidity. These data implicate the outgrowth of T cells expressing higher affinity TCR in a process of antigen-specific T cell avidity maturation during the pre-clinical stage of T1D.
Assuntos
Autoimunidade/imunologia , Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/imunologia , Adolescente , Adulto , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Insulina/imunologia , Interferon gama/metabolismo , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Adulto JovemRESUMO
BACKGROUND: Relatively few epidemiologic studies have focused on narcolepsy, a disabling sleep disorder with a strong association with HLA-DQB1 *0602. METHODS: We sought to estimate the prevalence of narcolepsy using multiple overlapping techniques to identify residents of King County, WA who were 18 years or older with physician-diagnosed narcolepsy. Patients were entered into a registry and recruited into an epidemiologic study entailing interview and buccal scrapings to determine HLA-DQB1 *0602 status. Missing values were imputed to allow prevalence to be estimated based on all 425 patients entered into the registry between 2001 and 2005, whether they were recruited into the epidemiologic study (n=279) or not (n=146). RESULTS: As of July 01, 2001, estimated prevalence per 100,000 of physician-diagnosed narcolepsy with cataplexy was 21.8 (95% confidence interval (CI): 18.8-24.8), similar to prior studies. The median age of onset was 14 (interquartile range: 10-18). For narcolepsy with HLA-DQB1 *0602, prevalence was 15.3 (95% CI: 12.8-17.9). Estimated prevalence was higher in women than men and in African-Americans than other racial groups. CONCLUSIONS: These differences could reflect problems in identification and recruitment or may provide etiologic clues about narcolepsy. This study illustrates the challenges in performing population-based studies of narcolepsy.
Assuntos
Etnicidade/estatística & dados numéricos , Narcolepsia/epidemiologia , População Branca/estatística & dados numéricos , Adolescente , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Feminino , Antígenos HLA-DQ/genética , Cadeias beta de HLA-DQ , Teste de Histocompatibilidade , Humanos , Masculino , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Narcolepsia/diagnóstico , Narcolepsia/etnologia , Prevalência , Fatores de Risco , Distribuição por Sexo , Washington/epidemiologia , Adulto JovemRESUMO
Globally, diarrheal diseases are a leading cause of death in children under five and disproportionately affect children in developing countries. Children who contract diarrheal diseases are rarely screened to identify the etiologic agent due to time and cost considerations associated with pathogen-specific screening and hence pathogen-directed therapy is uncommon. The development of biomarkers to rapidly identify underlying pathogens could improve treatment options and clinical outcomes in childhood diarrheal diseases. Here, we perform RNA sequencing on blood samples collected from children evaluated in an emergency room setting with diarrheal disease where the pathogen(s) present are known. We determine host response gene signatures specific to Salmonella, Shigella and rotavirus, but not E. coli, infections that distinguish them from each other and from healthy controls. Specifically, we observed differential expression of genes related to chemokine receptors or inflammasome signaling in Shigella cases, such as CCR3, CXCR8, and NLRC4, and interferon response genes, such as IFI44 and OASL, in rotavirus cases. Our findings add insight into the host peripheral immune response to these pathogens, and suggest strategies and limitations for the use host response transcript signatures for diagnosing the etiologic agent of childhood diarrheal diseases.