Single-cell transcriptomics of human T cells reveals tissue and activation signatures in health and disease

Nature Communications, Год журнала: 2019, Номер 10(1)

Опубликована: Окт. 17, 2019

Human T cells coordinate adaptive immunity in diverse anatomic compartments through production of cytokines and effector molecules, but it is unclear how tissue site influences cell persistence function. Here, we use single RNA-sequencing (scRNA-seq) to define the heterogeneity human isolated from lungs, lymph nodes, bone marrow blood, their functional responses following stimulation. Through analysis >50,000 resting activated cells, reveal signatures mucosal lymphoid sites, lineage-specific activation states across all sites including distinct for CD8+ an interferon-response state CD4+ cells. Comparing scRNA-seq profiles tumor-associated our dataset reveals predominant compared within multiple tumor types. Our results therefore establish a high dimensional reference map health analyzing disease.

Язык: Английский

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Peripheral T cell expansion predicts tumour infiltration and clinical response

Nature, Год журнала: 2020, Номер 579(7798), С. 274 - 278

Опубликована: Фев. 26, 2020

Язык: Английский

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Runx3 programs CD8+ T cell residency in non-lymphoid tissues and tumours

Nature, Год журнала: 2017, Номер 552(7684), С. 253 - 257

Опубликована: Дек. 6, 2017

Язык: Английский

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Location, location, location: Tissue resident memory T cells in mice and humans

Science Immunology, Год журнала: 2019, Номер 4(34)

Опубликована: Апрель 5, 2019

The discovery of T cells resident in diverse tissues has altered our understanding adaptive immunity to encompass site-specific responses mediated by tissue-adapted memory throughout the body. Here, we discuss key phenotypic, transcriptional, and functional features these tissue-resident (TRM) as established mouse models infection translated humans novel tissue sampling approaches. Integration findings from human studies may hold unlocking potential TRM for promoting preventing infection.

Язык: Английский

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Tissue-Resident T Cells and Other Resident Leukocytes

Annual Review of Immunology, Год журнала: 2019, Номер 37(1), С. 521 - 546

Опубликована: Фев. 6, 2019

Resident memory T (Trm) cells stably occupy tissues and cannot be sampled in superficial venous blood. Trm are heterogeneous but collectively constitute the most abundant cell subset. form an integral part of immune sensing network, monitor for local perturbations homeostasis throughout body, participate protection from infection cancer, likely promote autoimmunity, allergy, inflammatory diseases impede successful transplantation. Thus major candidates therapeutic manipulation. Here we review CD8

Язык: Английский

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Guidelines for the use of flow cytometry and cell sorting in immunological studies^*

European Journal of Immunology, Год журнала: 2017, Номер 47(10), С. 1584 - 1797

Опубликована: Окт. 1, 2017

The marriage between immunology and cytometry is one of the most stable productive in recent history science. A rapid search PubMed shows that, as July 2017, using "flow immunology" a term yields more than 68 000 articles, first which, interestingly, not about lymphocytes. It might be stated after short engagement, exchange wedding rings officially occurred when idea to link fluorochromes monoclonal antibodies came about. After this, recognizing different types cells became relatively easy feasible only by simple fluorescence microscope, but also complex sometimes esoteric instrument, flow cytometer that able count hundreds single second, can provide repetitive results tireless manner. Given possibility analyse immune phenotypes variety clinical conditions has changed use cytometer, which was incidentally invented late 1960s measure cellular DNA intercalating dyes, such ethidium bromide. epidemics HIV/AIDS 1980s then gave dramatic impulse technology counting specific cells, since it clear quantification number peripheral blood CD4+ T crucial follow course infection, eventually for monitoring therapy. As consequence, development cytometers had easy-to-use all laboratories helped widely disseminate this technology. Nowadays, rare find an immunological paper or read conference abstract authors did main tool dissect system identify its fine functions. Of note, developments have created sophisticated mass cytometry, simultaneously dozens molecules at cell level allows us better understand complexity beauty system. However, moon dark side. strengths technology, i.e. fact often brief training sufficient start producing data, weakness. Indeed, several (too many) papers, eye well-trained cytometrist aspects would need, polite, "little" improvement. Not mention cases technical mistakes are performed, involving, among others, (in)adequate controls, (lack appropriate) compensation, sorting strategies, even description methods used. For reason, editorial team European Journal Immunology feels worthwhile offer our community guidelines correct cytometric techniques field immunology. Thus, starting Congress (ECI 2015) Vienna (Austria) under guidance Professor Andreas Radbruch, we asked colleagues friends, renowned field, contribute sharing their knowledge particular areas expertise, order present collection protocols great interest. Such information includes, suggestions tricks regarding how study phenotypes, type amount produced secreted stimulation population interest, signalling processes, differentiation, proliferation death, cytotoxic activities, cell-cell interactions, activity intracellular organelles mitochondria, response induced against tumours anticancer immunosuppressive drugs, transcription factor activity, soluble molecules, drug uptake, events. Today's challenges involve choice reagents, preparation eventual storage analysis, overall experimental plan and, last least, data analyses. We no longer limited instrumentation, creativity ask critical questions. These "Guidelines studies" thus represent effort collect currently accepted best variation major players (along with functionality) include standards interpretation, well cautions issues. One aspect concerns reproducibility, topic recently attracted considerable attention. Therefore, meant help researchers avoid potential pitfalls could drastically alter interpretation data. While preparing guidelines, feedback received feel should highlighted Introduction. Firstly, "FACS" (fluorescence activated sorting) used Becton Dickinson (BD) technologies BD trademark (FACSTM); general sorting" company agnostic. Secondly, CD mAbs anti-CD (in other words CD1 mAb anti-CD1 mAb, example) This because nomenclature primarily cluster/characterize later describe respective molecules. Thirdly, although comprehensive possible, there naturally limitations e.g. subset antigens shown times, certain reagents/companies examples. opinion efforts must improved—this science works! glad receive from readers comments, new ideas, articles possible future updates Guidelines. Before closing, like thank four people who played role ensuring Radbruch's Andrea Cossarizza's vision reality. Hyun-Dong Chang Ute Hoffman, both DRFZ, Karen Chu, former Associate Editor, Cate Livingstone, Managing Editor Immunology. Together core coordinated author invitations, submission, peer review revision sections proofs, sought incorporated. full invaluable work on project. To accurately optical properties pass through uniformly bright center focused laser beams. Light optics intersection point beams pick up scattered light cells. sensing zone here measurements parameters taken. In stream-in-air sorters, located around 0.3 mm nozzle tip, inside cuvette. purpose fluidic move precisely region liquid stream way each illuminated same lasers. sort capabilities establish break off small uniform droplets. Droplets containing interest charged deflected electric sorting. kind technique Mack J. Fulwyler 1965 Los Alamos National Laboratory 1. needed machine testing performance Coulter counters, so particle separator particles volumes. Len Herzenberg interested living basis fluorescence, he got design plans found little group Stanford University build FACS (see video Inventing Cell Sorter, Lab, https://www.youtube.com/watch?v=Ro8P3w9BPhg). precise positioning jet hydrodynamic focusing counters 2. suspension injected thin tubing laminar sheath fluid enters wide into narrow orifice. speeds diameter sample (sample core) decreased (Fig. 1). Crosland-Taylor described Nature 1953 3 device suspended fluid. Some years before 1947, F.T. Gucker similar detecting bacteria air 4. takes place so-called chamber cytometer. detailed optimized sorter patent applications Gerrit van den Engh 5, 6 cuvette another application 7. addition chambers based cytometers, arc lamp source were developed. early standard microscope epi-fluorescence setup. Here lens bring excitation take emission Excitation separated dichroic mirror special filters. With immersion high numerical aperture, stabilized staining protocol, histograms coefficient variations (CVs) lower 1% (0.50–0.7%) achieved 8, 9. technique, aligned precision micrometer. rates increased, however, out focus laser. get illumination. means accuracy lost. loss measurement increases maintain intensity, elliptical spots. Typical sizes spot 60–150 micrometers horizontally 5–20 vertically. Recently, beam shaping flat top introduced manufacturer. intensity profile gaussian 60 100, 150 micrometer diameters Fig. approximation calculation ten times concentration three bigger necessary keep rate. fluid, PBS (phosphate buffered saline) filtered 0.22 0.1 filter compatible species sorted. An acoustic developed Gregory Kaduchak co-workers 2001 11, 12. implemented support focusing. helps increase if cores According manufacturer, acoustic-assisted run samples low concentrations 10 faster compared without still measurements. fundamentals given 13. Based invention Richard Sweet 14, droplet formation vibrations ultrasonic transducer. Little disturbances surface exit orifice generated grow exponentially lead droplets 2, 10. sorted measured moves down breakoff point. During separation jet, voltage pulse jet. So electrons caught cannot go back shut off. static two deflection plates 3). important process right charge droplet. delay charging determined operator electronics. done beads plates. illuminates streams un-deflected direction, camera, observed monitor. observation spots drop brightness maximized minimized. distance controlled held constant. setting fixed during kept constant operator. If velocity works fine, practice always case. Small changes pressure example due partial clogging Timothy Petersen examined problem showed disturb handle 16. Toralf Kaiser temperature alters gives solution stabilizing 17. schematic typical From view detection capable photons wavelengths over dynamic range. achieve range, optics, signal detection, processing units carefully designed. lenses emitted spatial resolution they Furthermore, make collected parallel direct bench detectors. employs collimation lenses. Collection (convex lenses) interrogation either end fiber directly (e.g. aspheric condenser lenses). instruments fibers route detected detectors installed octagon. case ensure routed Inside octagon placed front detector onto photocathode. photocathode lens. photodetectors spectrally broadband therefore unable generate exclusively markers. add specificity, filters mirrors defined manner Optical designed band (BP), long (LP), (SP) mostly common property transmit within spectral BP transmits example, named 660/20, 650 670 nm will photomultiplier tube reflected next set specified configuration. SP block ones whereas LP do opposite meaning below above (LP) wavelength. 660 nm. Due aging, quality coating, contamination, actual parameter differ description. recommended check transmission spectra provided manufacturer dust free. Sometimes (usually silver mirrors) deflect geometrical constructive reasons. >99%, reflective range wavelengths. contrast, deflects wavelength while rest pass-through. effect dependent operating angle. some instruments, dichroics employed working angle 45° others 12.5°. commercial become available spatially dispersing elements instead combination array. rationale behind entire Section I.3: Flow including sorting). element dispersive prism grating. Prisms higher efficiency gratings sensitive polarized light. maybe reason why Sony. Lasers mainly solid-state, continuous wave lasers footprint output power 20 100 mW. coherent sources allow photon density illumination point, efficient energy transfer fluorochrome. Modern equipped seven line ranging 355 flexibility choosing fluorophores. measures biological cell) via photons, needs converted processed amplifier, filter, analog digital converter (ADC), baseline restorer visualize store particles. section, components electronics briefly described. view, related difficult (i) level, (ii) analysis rate, (iii) level. Photomultiplier tubes (PMTs) meet these requirements almost cytometers. PMTs vacuum photocathode, electron electrodes, series dynodes multiplication. converts photoelectrons multiplied driven 5). Photocathodes made bialkali material determines quantum η PMT, ratio incident photons. 0< <1 function (h*f). PMT (R9220, Hamamatsu) = 0.2 500 0.09 700 reduction sensitivity 7 dB. PE-Cy7 less FITC, example. many applications, increasingly being replaced, avalanche photodiodes efficiency. instrument (CytoFlex, Beckman Coulter) APDs improve >700 18. All amplifiers analogue hardware devices very optimal noise ratios (SNRs). SNR >86 Once signals pre-amplifiers, amplifier suitable ADC modern conversion discrete values ADCs sampling frequency resolution. required (DNR) stained unstained brighter DNR 4 log 80 dB (DNR[dB]=20log(104)). ideal by: 6.02*N + 1.76 19. theory N 14 bit 86.04 practice, effective bits is, distortion circuit, decibels theoretical value AD9240AS Diva electronic 78.5 20). limits decades importantly, shrinks dim signals. MHz 30 per speed (pulse length μs). peak error 1–2% 21. 16 250 >4 <0.1%. domain filters, restorer, height, width algorithms, trigger Filtering smoothen raw SNR. resulting consists unwanted DC part scatter (among others) AC part. Hence, subtracted restorers attempts zero. practise restoring perfect negative histogram axis introduce slight increased CV restoring, (height, width, area) extracted *.fcs file. Taken together, algorithms need adjusted maximize DNR. Since prototype Fluorescence Activated Sorter 1968 University, powerful analyze individual functional status. Moreover, provides robust statistic thousands detect events 10–4 uptake multi-well acquisition rate second. decreases increasing cell. Alternatively, AttuneNXT (ThermoFisher) uses keeping tight accurate much throughput. serial multiple automated high-throughput platforms (HyperCyt®). Today, 27 bio-markers Typically markers fluorescently tagged antibodies, molecular sensors, genetically encoded reporters. instance, FACSymphony™ (Becton Dickinson) technically 50 achievable moment appropriate fluorescent dyes limited. Technical maximum detectable overlap tags, channel correlated marker. overcome tags Currently, 325 tags. tunable life time (FLIMs). photomultipliers diodes convert amplified electrical pulses extract area, length, time. consist basically preamp restoration (ADC). post-processing (i.e. integration, log-transformation) computer software. together SNR) (Q) background (B) detection. Avalanche PMTs. open possibilities long-wave spectra. CytoFLEX (Beckman Within divided division multiplexer (WDM) integrated array enables PE-Cy7. itself unsuitable hence mirrors. chosen multiparameter experiment, experiment (markers) analyzed, requires fluorophores simultaneously; consequence spillover III.1: Compensation). Conventional circumvent compensation Compensation) correlate physical Following analyzed multivariate fashion hierarchical gating strategy VI.1: Data analysis—An overview, VI.2: analysis—Automated analysis: Automated identification visualization). essential adapt optical, laser, setup minimize spillover, Q, B reagent IV.2: Titration—Determining concentration), fluorochrome contributes ideally nothing instrument. (the means) blank Q B. characterization Mostly polystyrene (beads) software MACSQuant, Fortessa, Yeti, Cytoflex name just few. Beads say "cell dummies" optimization, setup, counting, scale calibration on. Scale especially useful approach absolute binding photoelectrons) relative mean intensities (MFIs) leads quantitative VII: Cytometric parameters). Beside beads, LED pulses. quantiFlash™ (APE) ultra tool, labs values. Up analytical been can, addition, marker expression downstream (molecular biology, sequ

Язык: Английский

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Tissue-resident memory features are linked to the magnitude of cytotoxic T cell responses in human lung cancer

Nature Immunology, Год журнала: 2017, Номер 18(8), С. 940 - 950

Опубликована: Июнь 19, 2017

Язык: Английский

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Understanding Subset Diversity in T Cell Memory

Immunity, Год журнала: 2018, Номер 48(2), С. 214 - 226

Опубликована: Фев. 1, 2018

Язык: Английский

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CXCR5+ follicular cytotoxic T cells control viral infection in B cell follicles

Nature Immunology, Год журнала: 2016, Номер 17(10), С. 1187 - 1196

Опубликована: Авг. 3, 2016

Язык: Английский

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Programs for the persistence, vigilance and control of human CD8+ lung-resident memory T cells

Nature Immunology, Год журнала: 2016, Номер 17(12), С. 1467 - 1478

Опубликована: Окт. 24, 2016

Язык: Английский

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