T-Cell Development: Methods and Protocols

382 Pages·2022·14.436 MB·English

by  Remy Bosselut, Melanie S. Vacchio

Methods in
Molecular Biology 2580
Rémy Bosselut
Melanie S. Vacchio Editors
T-Cell
Development
Methods and Protocols
Second Edition
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire, UK
Forfurther volumes:
http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas
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neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported
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indexedinPubMed.
T-Cell Development
Methods and Protocols
Second Edition
Edited by
Rémy Bosselut and Melanie S. Vacchio
Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National
Institutes of Health, Bethesda, MD, USA
Editors
Re´myBosselut MelanieS.Vacchio
LaboratoryofImmuneCell LaboratoryofImmuneCell
Biology,CenterforCancerResearch Biology,CenterforCancerResearch
NationalCancerInstitute,National NationalCancerInstitute,National
InstitutesofHealth InstitutesofHealth
Bethesda,MD,USA Bethesda,MD,USA
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-2739-6 ISBN978-1-0716-2740-2 (eBook)
https://doi.org/10.1007/978-1-0716-2740-2
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Preface to the Second Edition
It is a pleasure to present the second edition of Methods in Molecular Biology, dedicated to
Tcelldevelopment.EventhoughthekeyconceptsofTcelldevelopmenthaveheldsteadfast
since the initial edition, new emerging technologies, including single-cell analyses of gene
expression and chromatin expression, have shed new light on developing thymocytes and
theirsurroundingstroma,openingnewavenuesofresearch.Inintroducingthefirstedition,
we emphasized the two-faced interest of studies of Tcell development. First, they explore
how the Tcell arm of the immune system builds a suitable repertoire of antigen receptor
specificitiesanddevelopsfunctionsappropriateforresponsestopathogensandforimmune
homeostasis.Second,studiesofTcelldevelopmentcontinuetobringinsightintoprocesses
of broad interest to biologists, including, to name a few, DNA recombination and repair,
mechanismsofcelldeathandsurvival,andcontrolofgeneexpression.
Theobjectiveofthepresentvolumeremainsthesameasintheoriginaledition,namely
tofacilitatesuchstudiesbyovercomingobstaclesassociatedwithexperimentalapproachesof
T cell development. Practically, we sought to provide accessible introductory reviews to
familiarize novice readers with key concepts used in the book, and detailed, practical
experimentalprotocolsforbothfirst-timeandin-depthstudiesofthymusbiology.Although
most chapters of this new edition are new or extensively updated, we have kept the same
organizationinthreepartscoveringbackgroundinformation,corestrategies,andprotocols
for specific experimental approaches. The first part, introducing key basic concepts, was
expanded with new chapters covering innate lymphoid cells (ILCs), antigen receptor-less
cousins of T cells that have attracted immense interest over the past decade, the thymic
stroma, and important technical updates to principles of flow cytometry. The second part
presents experimental strategies and protocols of broad applicability in the field, including
cellpreparation,flowcytometry,preparationofbonemarrowchimeras,andstudiesofTcell
responses. The last part includes new and updated protocols exploring multiple aspects of
thymocytebiology.Inadditiontoupdatingtechniquescoveredinthepreviousedition(e.g.,
retroviraltransduction),wehavetriedtoaddressadditionalneeds,includingstudiesofTcell
andILCbonemarrowprecursors.
The present volume, as the previous edition, largely focuses on mouse Tcell develop-
ment, leveraging the power of mouse genetics and the wealth of reagents targeting or
optimized for mouse cells. However, recent studies, including single-cell analyses of the
transcriptome and chromatin organization, have emphasized the profound similarity
between mouse and human Tcell development. Accordingly, two chapters of the present
volumepresentprotocols,includinguseoforganoidmodels,focusedonthehumanthymus.
Somewhatreciprocally,evenifdistantfromthehumanandmousethymus,thedevelopment
ofzebrafishTcellsprovidesapowerfulexperimentalmodelforquickandpowerfulexperi-
mentalapproaches.
We hope that this new edition will be useful to a broad array of readers, regardless of
whatbroughtthemtothymicbiology.Wewouldliketoexpressourgratitudetothemany
authors who contributed their time and expertise to build this new edition, despite the
multiplechallengesgeneratedbytherecentpandemic.Inadditiontotheircontribution,we
thankthem,andtheserieseditor,JohnWalker,fortheirpatiencewiththeelaborationofthis
volume.Wealsowishtoacknowledgethepreciousinteractionswithourmanycolleaguesat
v
vi PrefacetotheSecondEdition
theUSNationalInstitutesofHealth,which,overtheyears,builtthecollaborativeenviron-
mentwithoutwhichthepresentvolumewouldnothavebeenpossible.Certainlynotleast,
we want to conclude with the invaluable contribution of the myriad of experimental
scientists, especially young students and postdoctoral fellows, who continuously spare no
effort to design, update, and optimize the experiments and techniques included in this
edition.Thisbookproceedsfromtheirlaborandisdedicatedtothem.
Bethesda,MD,USA Re´myBosselut
MelanieS.Vacchio
Contents
PrefacetotheSecondEdition ................................................... v
Contributors................................................................. ix
PART I BACKGROUND INFORMATION
1 ABeginner’sGuidetoTCellDevelopment......... ........ ....... ........ 3
Re´myBosselut
2 RevelationsinThymicEpithelialCellBiologyandHeterogeneity
fromSingle-CellRNASequencingandLineageTracingMethodologies....... 25
AbigailMorales-Sanchez,SusannahC.Shissler,
JenniferE.Cowan,andAvinashBhandoola
3 EarlyDevelopmentofInnateLymphoidCells....... ........ ....... ........ 51
YiDing,ChristelleHarly,ArundhotiDas,
andAvinashBhandoola
4 DevelopmentofγδTCells:SoldiersontheFrontLines
ofImmuneBattles ......... ........ ....... ....... ........ .. ..... ...... .. 71
AlejandraV.ContrerasandDavidL.Wiest
5 PrinciplesofAdvancedFlowCytometry:APracticalGuide .......... ........ 89
ShafiuddinSiddiquiandFerencLiva´k
PART II CORE APPROACHES AND STRATEGIES
6 GeneticStrategiestoStudyTCellDevelopment..... ........ ....... ........ 117
Re´myBosselut
7 ASimplifiedApproachtoEvaluatingTCellDevelopment
byFlowCytometry ........ ........ ....... ....... ........ ....... ........ 131
JanY.M.LeeandPaulE.Love
8 PurificationofThymocyteandTCellSubsets ...... ..... .... ....... ........ 151
AndreaC.Carpenter,LauraB.Chopp,JongKyongKim,
andRe´myBosselut
9 GenerationofBoneMarrowChimeras ...... ....... ........ ....... ........ 165
YayiGaoandRe´myBosselut
10 GenerationofMurineTCellEffectorPopulationsInVitro.......... ........ 173
MelanieS.Vacchio
PART III SPECIFIC TECHNIQUES
11 Large-ScaleIsolationofMouseThymicEpithelialCells....... ....... ........ 189
IzumiOhigashi,MamiMatsuda-Lennikov,
andYousukeTakahama
vii
viii Contents
12 GenerationofRetrogenicMicetoInvestigateTCellDevelopment ........... 199
YayiGaoandRe´myBosselut
13 PurificationofBoneMarrowPrecursorstoTCellsandILCs......... ........ 211
DevinKenneyandChristelleHarly
14 StudyingTCellDevelopmentinNeonatalandAdultThymicSlices........... 233
LauraL.McIntyre,LydiaK.Lutes,andEllenA.Robey
15 InductionofHumanTCellDevelopmentInVitrowithOP9-DL4-7FS
CellsExpressingHumanCytokines ......... ....... ........ ....... ........ 249
MahmoodMohtashami,PatrickM.Brauer,
andJuanCarlosZu´n˜iga-Pflu¨cker
16 MethodsforStudyofMouseTCellReceptorαandβ
GeneRearrangements...... ........ ....... ....... ...... .. ...... ... ...... 261
DanielleJ.Dauphars,GlendonWu,CraigH.Bassing,
andMichaelS.Krangel
17 Ultrasound-GuidedIntra-thymicCellInjection ..... ........ ....... ........ 283
HristoGeorgiev,LauraB.Chopp,andKristinA.Hogquist
18 FetalThymicOrganCultureandNegativeSelection ......... ....... ........ 293
EmmaTeixeiroandMarkA.Daniels
19 InVitroAnalysisofThymocyteSignaling.... ....... ........ ....... ........ 303
EmmaTeixeiroandMarkA.Daniels
20 IdentificationandPurificationofHumanTCellPrecursors..... ..... .. ...... 315
IngeVandeWalle,NinaLambrechts,Ana¨ısDerveeuw,
MariekeLavaert,JulietteRoels,andTomTaghon
21 InVitroModelSystemstoStudyHumanTCellDevelopment....... ........ 335
NinaLambrechts,KaiLingLiang,ImkeVelghe,StevenStrubbe,
Anne-CatherineDolens,andTomTaghon
22 Zebrafish:ATractableModelforAnalysisofTCellDevelopment .... ........ 355
RobertSertori,YongZhang,andDavidL.Wiest
Index ...................................................................... 379
Contributors
CRAIGH.BASSING • ImmunologyGraduateGroup,PerelmanSchoolofMedicineatthe
UniversityofPennsylvania,Philadelphia,PA,USA;DepartmentofPathologyand
LaboratoryMedicine,Center forChildhoodCancerResearch,Children’sHospitalof
Philadelphia,Philadelphia,PA,USA;PerelmanSchoolofMedicineattheUniversityof
Pennsylvania,Philadelphia,PA,USA
AVINASH BHANDOOLA • LaboratoryofGenomeIntegrity,Center forCancerResearch,
NationalCancerInstitute,NationalInstitutesofHealth,Bethesda,MD,USA;TCell
BiologyandDevelopmentUnit,LaboratoryofGenomeIntegrity,Center forCancer
Research,NationalCancerInstitute,NationalInstituteofHealth,Bethesda,MD,USA
RE´MYBOSSELUT • LaboratoryofImmuneCellBiology,CenterforCancerResearch,National
CancerInstitute,NationalInstitutesofHealth,Bethesda,MD,USA
PATRICKM.BRAUER • SunnybrookResearchInstitute,Toronto,ON,Canada
ANDREAC.CARPENTER • InflammationandInnateImmunityUnit,LaboratoryofClinical
ImmunologyandMicrobiology,NationalInstituteofAllergyandInfectiousDiseases,
NationalInstitutesofHealth,Bethesda,MD,USA
LAURA B.CHOPP • LaboratoryofImmuneCellBiology,Center forCancerResearch,
NationalCancerInstitute,NationalInstitutesofHealth,Bethesda,MD,USA
ALEJANDRAV.CONTRERAS • BloodCellDevelopmentandFunctionProgram,FoxChase
CancerCenter,Philadelphia,PA,USA
JENNIFERE.COWAN • LaboratoryofGenomeIntegrity,CenterforCancerResearch,National
CancerInstitute,NationalInstitutesofHealth,Bethesda,MD,USA
MARKA.DANIELS • DepartmentofMolecularMicrobiologyandImmunology,NextGen
PrecisionHealth,UniversityofMissouri,Columbia,MO,USA
ARUNDHOTIDAS • TCellBiologyandDevelopmentUnit,LaboratoryofGenomeIntegrity,
Center forCancerResearch,NationalCancerInstitute,NationalInstituteofHealth,
Bethesda,MD,USA
DANIELLEJ.DAUPHARS • DepartmentofImmunology,DukeUniversitySchoolofMedicine,
Durham,NC,USA
ANA¨ISDERVEEUW • TheDepartmentofDiagnosticSciences,FacultyofMedicineandHealth
Sciences,GhentUniversity,GhentUniversityHospital,Ghent,Belgium
YIDING • TCellBiologyandDevelopmentUnit,LaboratoryofGenomeIntegrity,Centerfor
CancerResearch,NationalCancerInstitute,NationalInstituteofHealth,Bethesda,MD,
USA
ANNE-CATHERINEDOLENS • DepartmentofDiagnosticSciences,FacultyofMedicineand
HealthSciences,GhentUniversity,GhentUniversityHospital,Ghent,Belgium
YAYIGAO • LaboratoryofImmuneCellBiology,Center forCancerResearch,National
CancerInstitute,NationalInstitutesofHealth,Bethesda,MD,USA
HRISTOGEORGIEV • Center forImmunologyandDepartmentofLaboratoryMedicine&
Pathology,UniversityofMinnesota,Minneapolis,MN,USA
CHRISTELLE HARLY • Universite´deNantes,CNRS,Inserm,CRCINA,Nantes,France;
NantesUniversite´,INSERMUMR1307,CNRSUMR6075,Universite´d’Angers,
CRCI2NA,Nantes,France;LabExIGO“Immunotherapy,Graft,Oncology”,Nantes,
France
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