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1.
Nat Immunol ; 20(4): 514, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30862955

RESUMO

In the version of this article initially published, the first affiliation lacked 'MRC'; the correct name of the institution is 'MRC Weatherall Institute of Molecular Medicine'. Two designations (SP110Y and ST110H) were incorrect in the legend to Fig. 6f,h,i. The correct text is as follows: for panel f, "...loaded with either the CdtB(105-125)SP110Y (DRB4*SP110Y) or the CdtB(105-125)ST110H (DRB4*ST110H) peptide variants..."; for panel h, "...decorated by the DRB4*SP110Y tetramer (lower-right quadrant), the DRB4*ST110H (upper-left quadrant)..."; and for panel i, "...stained ex vivo with DRB4*SP110Y, DRB4*ST110H...". In Fig. 8e, the final six residues (LTEAFF) of the sequence in the far right column of the third row of the table were missing; the correct sequence is 'CASSYRRTPPLTEAFF'. In the legend to Fig. 8d, a designation (HLyE) was incorrect; the correct text is as follows: "(HlyE?)." Portions of the Acknowledgements section were incorrect; the correct text is as follows: "This work was supported by the UK Medical Research Council (MRC) (MR/K021222/1) (G.N., M.A.G., A.S., V.C., A.J.P.),...the Oxford Biomedical Research Centre (A.J.P., V.C.),...and core funding from the Singapore Immunology Network (SIgN) (E.W.N.) and the SIgN immunomonitoring platform (E.W.N.)." Finally, a parenthetical element was phrased incorrectly in the final paragraph of the Methods subsection "T cell cloning and live fluorescence barcoding"; the correct phrasing is as follows: "...(which in all cases included HlyE, CdtB, Ty21a, Quailes, NVGH308, and LT2 strains and in volunteers T5 and T6 included PhoN)...". Also, in Figs. 3c and 4a, the right outlines of the plots were not visible; in the legend to Fig. 3, panel letter 'f' was not bold; and in Fig. 8f, 'ND' should be aligned directly beneath DRB4 in the key and 'ND' should be removed from the diagram at right, and the legend should be revised accordingly as follows: "...colors indicate the HLA class II restriction (gray indicates clones for which restriction was not determined (ND)). Clonotypes are grouped on the basis of pathogen selectivity (continuous line), protein specificity (dashed line) and epitope specificity; for ten HlyE-specific clones (pixilated squares), the epitope specificity was not determined...". The errors have been corrected in the HTML and PDF versions of the article.

2.
Nat Immunol ; 19(7): 742-754, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29925993

RESUMO

To tackle the complexity of cross-reactive and pathogen-specific T cell responses against related Salmonella serovars, we used mass cytometry, unbiased single-cell cloning, live fluorescence barcoding, and T cell-receptor sequencing to reconstruct the Salmonella-specific repertoire of circulating effector CD4+ T cells, isolated from volunteers challenged with Salmonella enterica serovar Typhi (S. Typhi) or Salmonella Paratyphi A (S. Paratyphi). We describe the expansion of cross-reactive responses against distantly related Salmonella serovars and of clonotypes recognizing immunodominant antigens uniquely expressed by S. Typhi or S. Paratyphi A. In addition, single-amino acid variations in two immunodominant proteins, CdtB and PhoN, lead to the accumulation of T cells that do not cross-react against the different serovars, thus demonstrating how minor sequence variations in a complex microorganism shape the pathogen-specific T cell repertoire. Our results identify immune-dominant, serovar-specific, and cross-reactive T cell antigens, which should aid in the design of T cell-vaccination strategies against Salmonella.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Salmonella paratyphi A/imunologia , Salmonella typhi/imunologia , ADP-Ribosil Ciclase 1/análise , Adulto , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Linfócitos T CD4-Positivos/química , Células Clonais , Humanos , Fenótipo , Receptores CCR7/análise , Febre Tifoide/imunologia
3.
Nat Immunol ; 15(12): 1181-9, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25306126

RESUMO

Advances in cell-fate mapping have revealed the complexity in phenotype, ontogeny and tissue distribution of the mammalian myeloid system. To capture this phenotypic diversity, we developed a 38-antibody panel for mass cytometry and used dimensionality reduction with machine learning-aided cluster analysis to build a composite of murine (mouse) myeloid cells in the steady state across lymphoid and nonlymphoid tissues. In addition to identifying all previously described myeloid populations, higher-order analysis allowed objective delineation of otherwise ambiguous subsets, including monocyte-macrophage intermediates and an array of granulocyte variants. Using mice that cannot sense granulocyte macrophage-colony stimulating factor GM-CSF (Csf2rb(-/-)), which have discrete alterations in myeloid development, we confirmed differences in barrier tissue dendritic cells, lung macrophages and eosinophils. The methodology further identified variations in the monocyte and innate lymphoid cell compartment that were unexpected, which confirmed that this approach is a powerful tool for unambiguous and unbiased characterization of the myeloid system.


Assuntos
Citometria de Fluxo/métodos , Células Mieloides/citologia , Animais , Inteligência Artificial , Análise por Conglomerados , Camundongos , Camundongos Endogâmicos C57BL
4.
Immunity ; 45(2): 442-56, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27521270

RESUMO

Depending on the tissue microenvironment, T cells can differentiate into highly diverse subsets expressing unique trafficking receptors and cytokines. Studies of human lymphocytes have primarily focused on a limited number of parameters in blood, representing an incomplete view of the human immune system. Here, we have utilized mass cytometry to simultaneously analyze T cell trafficking and functional markers across eight different human tissues, including blood, lymphoid, and non-lymphoid tissues. These data have revealed that combinatorial expression of trafficking receptors and cytokines better defines tissue specificity. Notably, we identified numerous T helper cell subsets with overlapping cytokine expression, but only specific cytokine combinations are secreted regardless of tissue type. This indicates that T cell lineages defined in mouse models cannot be clearly distinguished in humans. Overall, our data uncover a plethora of tissue immune signatures and provide a systemic map of how T cell phenotypes are altered throughout the human body.


Assuntos
Sangue/imunologia , Movimento Celular , Tecido Linfoide/imunologia , Espectrometria de Massas/métodos , Especificidade de Órgãos , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/fisiologia , Animais , Biodiversidade , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Citocinas/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Receptores de Retorno de Linfócitos/metabolismo , Transcriptoma
5.
Nature ; 557(7706): 575-579, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29769722

RESUMO

Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types1-4. Patients and tumours may respond unpredictably to immunotherapy partly owing to heterogeneity of the immune composition and phenotypic profiles of tumour-infiltrating lymphocytes (TILs) within individual tumours and between patients5,6. Although there is evidence that tumour-mutation-derived neoantigen-specific T cells play a role in tumour control2,4,7-10, in most cases the antigen specificities of phenotypically diverse tumour-infiltrating T cells are largely unknown. Here we show that human lung and colorectal cancer CD8+ TILs can not only be specific for tumour antigens (for example, neoantigens), but also recognize a wide range of epitopes unrelated to cancer (such as those from Epstein-Barr virus, human cytomegalovirus or influenza virus). We found that these bystander CD8+ TILs have diverse phenotypes that overlap with tumour-specific cells, but lack CD39 expression. In colorectal and lung tumours, the absence of CD39 in CD8+ TILs defines populations that lack hallmarks of chronic antigen stimulation at the tumour site, supporting their classification as bystanders. Expression of CD39 varied markedly between patients, with some patients having predominantly CD39- CD8+ TILs. Furthermore, frequencies of CD39 expression among CD8+ TILs correlated with several important clinical parameters, such as the mutation status of lung tumour epidermal growth factor receptors. Our results demonstrate that not all tumour-infiltrating T cells are specific for tumour antigens, and suggest that measuring CD39 expression could be a straightforward way to quantify or isolate bystander T cells.


Assuntos
Efeito Espectador/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Pulmonares/imunologia , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/imunologia , Antígenos de Neoplasias/imunologia , Antígenos Virais/imunologia , Apirase/análise , Apirase/deficiência , Apirase/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Separação Celular , Neoplasias Colorretais/genética , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/genética , Linfócitos do Interstício Tumoral/metabolismo , Fenótipo
6.
Proc Natl Acad Sci U S A ; 116(2): 609-618, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30587582

RESUMO

Anti-CTLA-4 mAb is efficacious in enhancing tumor immunity in humans. CTLA-4 is expressed by conventional T cells upon activation and by naturally occurring FOXP3+CD4+ Treg cells constitutively, raising a question of how anti-CTLA-4 mAb can differentially control these functionally opposing T cell populations in tumor immunity. Here we show that FOXP3high potently suppressive effector Treg cells were abundant in melanoma tissues, expressing CTLA-4 at higher levels than tumor-infiltrating CD8+ T cells. Upon in vitro tumor-antigen stimulation of peripheral blood mononuclear cells from healthy individuals or melanoma patients, Fc-region-modified anti-CTLA-4 mAb with high antibody-dependent cell-mediated cytotoxicity (ADCC) and cellular phagocytosis (ADCP) activity selectively depleted CTLA-4+FOXP3+ Treg cells and consequently expanded tumor-antigen-specific CD8+T cells. Importantly, the expansion occurred only when antigen stimulation was delayed several days from the antibody treatment to spare CTLA-4+ activated effector CD8+T cells from mAb-mediated killing. Similarly, in tumor-bearing mice, high-ADCC/ADCP anti-CTLA-4 mAb treatment with delayed tumor-antigen vaccination significantly prolonged their survival and markedly elevated cytokine production by tumor-infiltrating CD8+ T cells, whereas antibody treatment concurrent with vaccination did not. Anti-CTLA-4 mAb modified to exhibit a lesser or no Fc-binding activity failed to show such timing-dependent in vitro and in vivo immune enhancement. Thus, high ADCC anti-CTLA-4 mAb is able to selectively deplete effector Treg cells and evoke tumor immunity depending on the CTLA-4-expressing status of effector CD8+ T cells. These findings are instrumental in designing cancer immunotherapy with mAbs targeting the molecules commonly expressed by FOXP3+ Treg cells and tumor-reactive effector T cells.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos Imunológicos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Vacinas Anticâncer/farmacologia , Neoplasias/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Antineoplásicos Imunológicos/imunologia , Antígeno CTLA-4/imunologia , Vacinas Anticâncer/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/patologia
7.
Eur J Immunol ; 48(6): 1014-1019, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29510451

RESUMO

Pressure ulcers are a chronic problem for patients or the elderly who require extended periods of bed rest. The formation of ulcers is due to repeated cycles of ischemia-reperfusion (IR), which initiates an inflammatory response. Advanced ulcers disrupt the skin barrier, resulting in further complications. To date, the immunological aspect of skin IR has been understudied, partly due to the complexity of the skin immune cells. Through a combination of mass cytometry, confocal imaging and intravital multiphoton imaging, this study establishes a workflow for multidimensionality single cell analysis of skin myeloid cell responses in the context of IR injury with high spatiotemporal resolution. The data generated has provided us with previously uncharacterized insights into the distinct cellular behavior of resident dendritic cells (DCs) and recruited neutrophils post IR. Of interest, we observed a drop in DDC numbers in the IR region, which was subsequently replenished 48h post IR. More importantly, in these cells, we observe an attenuated response to repeated injuries, which may have implications in the subsequent wound healing process.


Assuntos
Células Dendríticas/imunologia , Neutrófilos/imunologia , Úlcera por Pressão/imunologia , Traumatismo por Reperfusão/imunologia , Pele/patologia , Idoso , Animais , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Análise de Célula Única
8.
J Immunol ; 198(2): 927-936, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27986910

RESUMO

The identification of blood-borne biomarkers correlating with melanoma patient survival remains elusive. Novel techniques such as mass cytometry could help to identify melanoma biomarkers, allowing simultaneous detection of up to 100 parameters. However, the evaluation of multiparametric data generated via time-of-flight mass cytometry requires novel analytical techniques because the application of conventional gating strategies currently used in polychromatic flow cytometry is not feasible. In this study, we have employed 38-channel time-of-flight mass cytometry analysis to generate comprehensive immune cell signatures using matrix boolean analysis in a cohort of 28 stage IV melanoma patients and 17 controls. Clusters of parameters were constructed from the abundance of cellular phenotypes significantly different between patients and controls. This approach identified patient-specific combinatorial immune signatures consisting of high-resolution subsets of the T cell, NK cell, B cell, and myeloid compartments. An association with superior survival was characterized by a balanced distribution of myeloid-derived suppressor cell-like and APC-like myeloid phenotypes and differentiated NK cells. The results of this study in a discovery cohort of melanoma patients suggest that multifactorial immune signatures have the potential to allow more accurate prediction of individual patient outcome. Further investigation of the identified immune signatures in a validation cohort is now warranted.


Assuntos
Biomarcadores Tumorais/sangue , Citometria de Fluxo/métodos , Melanoma/imunologia , Melanoma/patologia , Adulto , Idoso , Biomarcadores Tumorais/imunologia , Análise por Conglomerados , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Melanoma/mortalidade , Pessoa de Meia-Idade
9.
Cytometry A ; 91(1): 48-61, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27798817

RESUMO

The advent of mass cytometry has facilitated highly multi-parametric single-cell analysis allowing for the deep assessment of cellular diversity. While the data and analytical power of this approach are well described, associated technical and experimental hurdles remain. Issues like equipment breakdown and sampling of large-scale batches, which may require multiple days of data acquisition, are minor but critical obstacles that prompt a technical solution, especially when dealing with precious samples. An ability to cryopreserve mass cytometry samples that have already been stained would alleviate numerous technical limitations we face with currently used sample-handling approaches. Here, we evaluated two protocols for freezing of already-stained and fixed cellular samples and compared them with standard sample refrigeration in staining buffer. A comprehensive human T cell staining phenotypic and functional profiling panel was used and the signal intensity and reliability of each marker was assessed over a 4-week period. In general, cellular viability, DNA Ir-Intercalator and barcode staining were minimally affected by freezing compared to refrigeration, and the signal intensities for cell surface markers and receptors were not compromised. Intracellular cytokine staining did show some decreases in signal intensity after freezing, with the decreases more prominent in a methanol-based protocol compared to a protocol involving the use of 10% DMSO in FBS. We conclude that freezing already-stained samples suspended in 10% DMSO in FBS is practical and efficient way to preserve already-stained samples when needed. © 2016 International Society for Advancement of Cytometry.


Assuntos
Criopreservação/métodos , Citometria de Fluxo/métodos , Análise de Célula Única/métodos , Sobrevivência Celular/genética , Humanos , Coloração e Rotulagem , Linfócitos T/ultraestrutura
10.
Biomaterials ; 305: 122460, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38246018

RESUMO

Ex vivo patient-derived tumor slices (PDTS) are currently limited by short-term viability in culture. Here, we show how bioengineered hydrogels enable the identification of key matrix parameters that significantly enhance PDTS viability compared to conventional culture systems. As demonstrated using single-cell RNA sequencing and high-dimensional flow cytometry, hydrogel-embedded PDTS tightly preserved cancer, cancer-associated fibroblast, and various immune cell populations and subpopulations in the corresponding original tumor. Cell-cell communication networks within the tumor microenvironment, including immune checkpoint ligand-receptor interactions, were also maintained. Remarkably, our results from a co-clinical trial suggest hydrogel-embedded PDTS may predict sensitivity to immune checkpoint inhibitors (ICIs) in head and neck cancer patients. Further, we show how these longer term-cultured tumor explants uniquely enable the sampling and detection of temporal evolution in molecular readouts when treated with ICIs. By preserving the compositional heterogeneity and complexity of patient tumors, hydrogel-embedded PDTS provide a valuable tool to facilitate experiments targeting the tumor microenvironment.


Assuntos
Neoplasias de Cabeça e Pescoço , Hidrogéis , Humanos , Hidrogéis/farmacologia , Avaliação de Medicamentos , Microambiente Tumoral
11.
Sci Immunol ; 7(78): eadd3330, 2022 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-36525505

RESUMO

Langerhans cell histiocytosis (LCH) is a potentially fatal neoplasm characterized by the aberrant differentiation of mononuclear phagocytes, driven by mitogen-activated protein kinase (MAPK) pathway activation. LCH cells may trigger destructive pathology yet remain in a precarious state finely balanced between apoptosis and survival, supported by a unique inflammatory milieu. The interactions that maintain this state are not well known and may offer targets for intervention. Here, we used single-cell RNA-seq and protein analysis to dissect LCH lesions, assessing LCH cell heterogeneity and comparing LCH cells with normal mononuclear phagocytes within lesions. We found LCH discriminatory signatures pointing to senescence and escape from tumor immune surveillance. We also uncovered two major lineages of LCH with DC2- and DC3/monocyte-like phenotypes and validated them in multiple pathological tissue sites by high-content imaging. Receptor-ligand analyses and lineage tracing in vitro revealed Notch-dependent cooperativity between DC2 and DC3/monocyte lineages during expression of the pathognomonic LCH program. Our results present a convergent dual origin model of LCH with MAPK pathway activation occurring before fate commitment to DC2 and DC3/monocyte lineages and Notch-dependent cooperativity between lineages driving the development of LCH cells.


Assuntos
Histiocitose de Células de Langerhans , Neoplasias , Humanos , Linhagem da Célula , Histiocitose de Células de Langerhans/metabolismo , Histiocitose de Células de Langerhans/patologia , Diferenciação Celular , Monócitos/metabolismo
12.
Front Immunol ; 12: 710217, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34867943

RESUMO

Severe SARS-CoV-2 infection can trigger uncontrolled innate and adaptive immune responses, which are commonly associated with lymphopenia and increased neutrophil counts. However, whether the immune abnormalities observed in mild to severely infected patients persist into convalescence remains unclear. Herein, comparisons were drawn between the immune responses of COVID-19 infected and convalescent adults. Strikingly, survivors of severe COVID-19 had decreased proportions of NKT and Vδ2 T cells, and increased proportions of low-density neutrophils, IgA+/CD86+/CD123+ non-classical monocytes and hyperactivated HLADR+CD38+ CD8+ T cells, and elevated levels of pro-inflammatory cytokines such as hepatocyte growth factor and vascular endothelial growth factor A, long after virus clearance. Our study suggests potential immune correlates of "long COVID-19", and defines key cells and cytokines that delineate true and quasi-convalescent states.


Assuntos
COVID-19/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , COVID-19/complicações , Estudos de Coortes , Convalescença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome de COVID-19 Pós-Aguda
13.
Nat Commun ; 9(1): 253, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29343684

RESUMO

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can detect bacteria-derived metabolites presented on MR1. Here we show, using a controlled infection of humans with live Salmonella enterica serovar Paratyphi A, that MAIT cells are activated during infection, an effect maintained even after antibiotic treatment. At the peak of infection MAIT cell T-cell receptor (TCR)ß clonotypes that are over-represented prior to infection transiently contract. Select MAIT cell TCRß clonotypes that expand after infection have stronger TCR-dependent activation than do contracted clonotypes. Our results demonstrate that host exposure to antigen may drive clonal expansion of MAIT cells with increased functional avidity, suggesting a role for specific vaccination strategies to increase the frequency and potency of MAIT cells to optimize effector function.


Assuntos
Proliferação de Células , Células T Invariantes Associadas à Mucosa/imunologia , Febre Paratifoide/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Salmonella paratyphi A/imunologia , Adolescente , Adulto , Linhagem Celular Tumoral , Células Clonais/imunologia , Células Clonais/metabolismo , Células Clonais/microbiologia , Voluntários Saudáveis , Interações Hospedeiro-Patógeno/imunologia , Humanos , Células Jurkat , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/microbiologia , Pessoa de Meia-Idade , Células T Invariantes Associadas à Mucosa/metabolismo , Células T Invariantes Associadas à Mucosa/microbiologia , Febre Paratifoide/metabolismo , Febre Paratifoide/microbiologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Salmonella paratyphi A/fisiologia , Adulto Jovem
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