RESUMO
An open-label, first-in-human phase 1/2 study is being conducted to evaluate the safety and efficacy of pancreatic endoderm cells (PECs) implanted in non-immunoprotective macroencapsulation devices for the treatment of type 1 diabetes. We report an analysis on 1 year of data from the first cohort of 15 patients from a single trial site that received subcutaneous implantation of cell products combined with an immunosuppressive regimen. Implants were well tolerated with no teratoma formation or severe graft-related adverse events. After implantation, patients had increased fasting C-peptide levels and increased glucose-responsive C-peptide levels and developed mixed meal-stimulated C-peptide secretion. There were immunosuppression-related transient increases in circulating regulatory T cells, PD1high T cells, and IL17A+CD4+ T cells. Explanted grafts contained cells with a mature ß cell phenotype that were immunoreactive for insulin, islet amyloid polypeptide, and MAFA. These data, and associated findings (Shapiro et al., 2021), are the first reported evidence of meal-regulated insulin secretion by differentiated stem cells in patients.
Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Peptídeo C , Diferenciação Celular , Diabetes Mellitus Tipo 1/terapia , Endoderma , Glucose , Humanos , InsulinaRESUMO
BACKGROUND & AIMS: Bacterial flagellin is an important antigen in inflammatory bowel disease, but the role of flagellin-specific CD4+ T cells in disease pathogenesis remains unclear. Also unknown is how changes in intestinal microbiome intersect with those in microbiota-specific CD4+ T cells. We aimed to quantify and characterize flagellin-specific CD4+ T cells in Crohn's disease (CD) and ulcerative colitis (UC) patients and study their relationship with intestinal microbiome diversity. METHODS: Blood was collected from 3 cohorts that included CD patients, UC patients, and healthy controls. Flow cytometry analyzed CD4+ T cells specific for Lachnospiraceae-derived A4-Fla2 and Escherichia coli H18 FliC flagellins, or control vaccine antigens. Serum antiflagellin IgG and IgA antibodies were detected by enzyme-linked immunosorbent assay and stool samples were collected and subjected to 16S ribosomal DNA sequencing. RESULTS: Compared with healthy controls, CD and UC patients had lower frequencies of vaccine-antigen-specific CD4+ T cells and, as a proportion of vaccine-specific cells, higher frequencies of flagellin-specific CD4+ T cells. The proportion of flagellin-specific CD4+ T cells that were CXCR3negCCR4+CCR6+ Th17 cells was reduced in CD and UC patients, with increased proportions of CD39+, PD-1+, and integrin ß7+ cells. Microbiome analysis showed differentially abundant bacterial species in patient groups that correlated with immune responses to flagellin. CONCLUSIONS: Both CD and UC patients have relative increases in the proportion of circulating Fla2-specific CD4+ T cells, which may be associated with changes in the intestinal microbiome. Evidence that the phenotype of these cells strongly correlate with disease severity provides insight into the potential roles of flagellin-specific CD4+ T cells in inflammatory bowel disease.
Assuntos
Anticorpos Antibacterianos/sangue , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Disbiose/complicações , Proteínas de Escherichia coli/imunologia , Flagelina/imunologia , Imunidade Adaptativa , Adolescente , Adulto , Idoso , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Clostridiales/genética , Clostridiales/imunologia , Colite Ulcerativa/sangue , Colite Ulcerativa/microbiologia , Doença de Crohn/sangue , Doença de Crohn/microbiologia , Estudos Transversais , DNA Bacteriano/isolamento & purificação , Disbiose/diagnóstico , Disbiose/imunologia , Disbiose/microbiologia , Ensaio de Imunoadsorção Enzimática , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/imunologia , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Subpopulações de Linfócitos T/imunologia , Adulto JovemRESUMO
The analysis and validation of flow cytometry-based biomarkers in clinical studies are limited by the lack of standardized protocols that are reproducible across multiple centers and suitable for use with either unfractionated blood or cryopreserved PBMCs. Here we report the development of a platform that standardizes a set of flow cytometry panels across multiple centers, with high reproducibility in blood or PBMCs from either healthy subjects or patients 100 days after hematopoietic stem cell transplantation. Inter-center comparisons of replicate samples showed low variation, with interindividual variation exceeding inter-center variation for most populations (coefficients of variability <20% and interclass correlation coefficients >0.75). Exceptions included low-abundance populations defined by markers with indistinct expression boundaries (e.g., plasmablasts, monocyte subsets) or populations defined by markers sensitive to cryopreservation, such as CD62L and CD45RA. Automated gating pipelines were developed and validated on an independent data set, revealing high Spearman's correlations (rs >0.9) with manual analyses. This workflow, which includes pre-formatted antibody cocktails, standardized protocols for acquisition, and validated automated analysis pipelines, can be readily implemented in multicenter clinical trials. This approach facilitates the collection of robust immune phenotyping data and comparison of data from independent studies.
Assuntos
Biomarcadores/sangue , Criopreservação/normas , Análise de Dados , Citometria de Fluxo/normas , Imunofenotipagem/normas , Imunidade Adaptativa , Criopreservação/métodos , Processamento Eletrônico de Dados , Citometria de Fluxo/métodos , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunidade Inata , Imunofenotipagem/métodos , Selectina L , Antígenos Comuns de Leucócito , Leucócitos Mononucleares/imunologia , Monócitos , Reprodutibilidade dos TestesRESUMO
FOXP3-expressing T regulatory cells (Tregs) can be divided into two distinct subsets: naturally occurring Tregs (nTregs) that develop in the thymus, and induced Tregs (iTregs) that differentiate in peripheral tissues upon exposure to Ag in a tolerogenic environment. Recently it has been proposed that expression of Helios, an Ikaros family transcription factor, may specifically identify nTregs, allowing specific tracking of Tregs from different origins in health and disease. Surprisingly, we found that Helios(-) cells can be readily identified within naive (CD45RA(+)CD31(+)CCR7(+)CD62L(+)) FOXP3(+) Tregs, a finding inconsistent with the notion that lack of Helios expression identifies Ag-experienced iTregs that should express memory markers. To investigate the phenotype and function of naive Helios(+) and Helios(-) Tregs within the nTreg population, we isolated single-cell clones from each subset. We found that both Helios(+) and Helios(-) nTreg clones have a similar suppressive capacity, as well as expression of FOXP3 and cell surface proteins, including CD39 and CTLA-4. Helios(-) nTregs, however, produced significantly more CCL3 and IFN-γ compared with Helios(+) nTregs. Despite increased cytokine/chemokine production, Helios(-) FOXP3(+) nTreg clones were demethylated at the FOXP3 Treg-specific demethylated region, indicative of Treg lineage stability. When cultured under Th1-polarizing conditions, Helios(+) and Helios(-) nTreg clones had an equal ability to produce IFN-γ. Collectively, these data show that a lack of Helios expression does not exclusively identify human iTregs, and, to our knowledge, the data provide the first evidence for the coexistence of Helios(+) and Helios(-) nTregs in human peripheral blood.
Assuntos
Linhagem da Célula/imunologia , Fatores de Transcrição Forkhead/genética , Fator de Transcrição Ikaros/genética , Linfócitos T Reguladores/citologia , Timo/citologia , Antígenos CD/genética , Antígenos CD/imunologia , Apirase/genética , Apirase/imunologia , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Diferenciação Celular , Quimiocina CCL3/genética , Quimiocina CCL3/imunologia , Células Clonais , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Expressão Gênica , Humanos , Fator de Transcrição Ikaros/imunologia , Imunofenotipagem , Interferon gama/genética , Interferon gama/imunologia , Especificidade de Órgãos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Timo/imunologia , Timo/metabolismoRESUMO
T regulatory (Treg) cells have a fundamental role in the establishment and maintenance of peripheral tolerance. It is well established that Treg cells have a phenotype and function that is distinct from conventional T effector cells, although how these two T cell subsets differ in terms of molecular signaling cascades remains largely unknown. Analysis of signaling events in Treg cells using classical biochemistry has been hampered due to difficulties in isolating homogeneous populations and limited cell numbers. In order to overcome these challenges, we defined the optimal conditions for culture, in vitro expansion, and stimulation of human CD4(+)CD25(+) Treg and T effector cells to study intracellular signaling events by flow cytometry. In order to avoid the pitfalls associated with cell isolation based on CD25 expression, we developed methodology to analyze subpopulations of FOXP3 positive and negative cells from ex vivo CD4(+) T cells. In addition to examination of ex vivo cells, we optimized expansion conditions for analysis of signaling in Treg and T effector cell lines. Using these methods, we found that human FOXP3(+) Treg cells displayed a greater capacity to phosphorylate the extracellular regulated kinase (ERK) compared to T effector cells, upon TCR-mediated activation. In contrast, FOXP3(+) Treg cells showed a significantly diminished capacity to phosphorylate AKT. This methodology provides a foundation for future investigation into the molecular events that regulate the phenotype and function of Treg cells, and may ultimately lead to the identification of Treg-cell specific therapeutic targets.
Assuntos
Citometria de Fluxo/métodos , Fatores de Transcrição Forkhead/biossíntese , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Humanos , Imunofenotipagem , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Ativação Linfocitária/imunologia , Fosfoproteínas/análise , Fosfoproteínas/biossíntese , Fosfoproteínas/metabolismo , Fosforilação , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismoRESUMO
Suppression by T regulatory cells (Treg cells) is a major mechanism by which the immune system controls responses to self and nonharmful foreign proteins. Although there are many different types of Treg cells, the best characterized are those that constitutively express cell-surface IL-2Ralpha (CD25). We investigated whether altered T-cell-receptor (TCR)-mediated signaling in pure populations of ex vivo human CD4+CD25+ Treg cells might underlie their unique phenotype, including hyporesponsiveness to TCR-mediated activation and lack of cytokine production. CD4+CD25+ Treg cells displayed a consistent defect in phosphorylation of AKT at serine 473 and reduced phosphorylation of the AKT substrates FOXO and S6. Restoration of AKT activity via lentiviral-mediated expression of an inducibly active form of the kinase revealed that reduced activity of this pathway was necessary for the suppressive function of CD4+CD25+ Treg cells. These data represent the first demonstration of a causal association between altered signaling and the function of CD4+CD25+ Treg cells. Moreover, we have created the first system allowing inducible abrogation of suppression through manipulation of the suppressor cells. This system will be a powerful tool to further study the mechanism(s) of suppression by CD4+CD25+ Treg cells.