Identification of CD Marker Expression and …...ii Identification of CD Marker Expression and...
Transcript of Identification of CD Marker Expression and …...ii Identification of CD Marker Expression and...
IdentificationofCDMarkerExpressionandNeutrophilSurfaceMarkerChangesinHealthandDiseaseusingHigh-
throughputscreeningflowcytometry
by
FlaviaS.Lakschevitz
AthesissubmittedinconformitywiththerequirementsforthedegreeofMaster’sofScience
FacultyofDentistryUniversityofToronto
©CopyrightbyFlaviaS.Lakschevitz2016
ii
IdentificationofCDMarkerExpressionandNeutrophilSurfaceMarkerChanges
inHealthandDiseaseusingHigh-throughputscreeningflowcytometry
FlaviaS.Lakschevitz
Master’sofScience
FacultyofDentistryUniversityofToronto
2015
Abstract
Neutrophil hyperactivation can contribute to tissue damage in inflammatory diseases.
Althoughmany cell-surface proteins are known to be expressed on neutrophils there is no
comprehensive study of the surface-markers that can be used to phenotype neutrophils.
Neutrophils subpopulations isolated (blood and oral rinses) from healthy and chronic-
periodontitispatientswerescreenedagainstapanelof374knownClusterofDifferentiation
(CD)antibodies to identify cell-surfacemarkers specific toneutrophils. This screen identified
CD11b+, CD16b+, and CD66c+ as markers that are always expressed on neutrophils. Cell-
sortingwithanantibodyagainstCD11b/CD16b/CD66callowedfor theenrichmentofmature
neutrophils,yieldingpopulationsofupto99%,confirmingthevalidityofthesemarkerswhen
isolating neutrophils. These findings provide a simplemethod for isolating neutrophils from
humans,andtherebyestablishavalidatedmethodthatallowsfortheaccurateidentificationof
neutrophils.Thisknowledgewillbecrucialforidentifyingneutrophilsubtypesassociatedwith
neutrophil-mediatedinflammatorydiseases.
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Acknowledgments
Firstandforemost,IwouldliketothankDr.MichaelGlogauer,hissupportandguidanceduring
mytrainingatUniversityofToronto,hasproventoberelentless.Thankyou!
I am grateful to all past and current members of the Matrix Dynamics Group for their
friendship and scientific collaboration. I am especially grateful to Guy Aboodi, Siavash
HassanpourandChunxiangSunfortheirfriendshipandsupportinthelab.I’dliketothankmy
summerstudents,especiallyAyalaRubinforreadingmymanuscriptandhelpfulsuggestions.
IgivespecialthankstoJoshuaPatersonfromtheUNHAntibodyCoreFacilityforhisassistance
withHTS-FlowCytometryandtoDionneWhitefromFlowCytometryfacilityatDepartmentof
Immunology,UniversityofToronto,Toronto,ONforherassistancewithcellsorting.
Hownot tomention KerryD’Costa and Jason Yee!My inseparable classmates in Perio! You
guysarethebestcolleagues/friendsthatanyonecouldhopeandmore!
I thank my program advisory members Drs. Christopher McCulloch and Dr. Howard
Tenenbaum for their help and insight. Special thanks toDr. Limor Avivi-Arber andDr. Scott
Gray-Owenforkindlyservingasdefenseexaminers.
LastlyIthankmyfriends,familyandlovedoneseverythingispossiblebecauseofyou!
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TableofContents
Abstract...............................................................................................................................................ii
Acknowledgments.............................................................................................................................iii
TableofContents...............................................................................................................................iv
ListofAbbreviations...........................................................................................................................vi
ListofTables.....................................................................................................................................viii
ListofFigures.....................................................................................................................................ix
Chapter1GeneralIntroduction..........................................................................................................1
1. Introduction................................................................................................................................1
NeutrophilIdentification................................................................................................................1
Clusterofdifferentiation(CD).........................................................................................................2
High-throughputscreening(HTS)flowcytometry...........................................................................3
2. CDexpressionandNeutrophilDistribution..................................................................................4
BoneMarrow.................................................................................................................................4
Circulation......................................................................................................................................7
Tissues............................................................................................................................................8
3. CDexpressionandNeutrophilsFunctionality...............................................................................9
NeutrophilRecruitment..................................................................................................................9
BacterialkillingandPhagocytosis.................................................................................................11
Non-traditionalfunctionsofneutrophils......................................................................................14
4. PeriodontalDiseases.................................................................................................................14
ChronicPeriodontitis........................................................................................................................14
TheroleofneutrophilsinthepathogenesisofPeriodontitis.........................................................15
5. Objectivesofthisstudyandhypothesis.....................................................................................17
Objectives....................................................................................................................................17
Hypothesis...................................................................................................................................18
Chapter2..........................................................................................................................................19
Immunophenotypicalcharacterizationofhumanneutrophils.......................................................19
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Abstract............................................................................................................................................19
Introduction......................................................................................................................................20
Materialsandmethods....................................................................................................................21
ResultsandDiscussion.....................................................................................................................29
Conclusions.......................................................................................................................................42
Chapter3..........................................................................................................................................44
ThesisSummaryandFutureDirections.........................................................................................44
Summary..........................................................................................................................................44
FutureDirections..............................................................................................................................46
References........................................................................................................................................48
SUPPLEMENTALFILES...................................................................................................................60
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ListofAbbreviations
AAP-AmericanAcademyofPeriodontology
Ab-Antibody
ADCC–Antibody-DependentCell-mediatedCytotoxicity
APC-Allophycocyanin
CD–ClusterofDifferentiation
CF-CysticFibrosis
COPD-ChronicObstructivePulmonaryDisease
CP–Chronicperiodontitis
FACS-Fluorescence-activatedcellsorting
Fc–Fragmentcrystallizable
FcR–FcReceptor
FITC-Fluoresceinisothiocyanate
FSC-Forward-scatter
HLA-humanleukocyteantigen
HTFC-highthroughputflowcytometry
HTS-High-throughputscreening
ICAM-1-IntercellularAdhesionMolecule1
Ig–Immunoglobulin
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ITAMS-immunoreceptortyrosine-basedactivationmotifs
LFA3-lymphocytefunction-associatedantigen3
MFI-Meanfluorescenceintensity
PAMPs-pathogen-associatedmolecularpatterns
PE-Phycoerythrin
PMN-B–Bloodpolymorphonuclearneutrophilicgranulocyte,
PMN-O–Oralpolymorphonuclearneutrophilicgranulocyte,
PRRs-patternrecognitionreceptors
SSC-Side-scatter
TLRs-Tolllikereceptors
VAP1–Vascularadhesionprotein1
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ListofTables
Chapter1:
Table1–Cellsurfaceexpressionindifferentstagesofgranulocyticdifferentiation
Table 2 – Cell surface markers expressed in neutrophils associated with recruitment andmigratoryfunctions
Table3–NeutrophildeficienciesmanifestedintheoralcavityChapter2:Table1-ListofCDmarkersanditsdistributionTable2-FulllistofantibodiesusedinthisstudyTable3–ComprehensiveliteraturereviewofcellsurfacemarkersexpressioninNeutrophils
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ListofFigures
Chapter1:
Figure 1 - Model of pathogenesis of Periodontitis (adapted from Page & Kornman,
Periodontology2000,1997).Bacteriaandbiofilm insultarecrucial for the initiationof
periodontal disease. However, the presence of bacteria alone does not cause tissue
destruction. The majority of tissue destruction occurs as a consequence of the host
immuno-inflammatory response against this microbial challenge. Both genetic and
environmentalriskfactorsworksasmodulatorsofthehostimmuneresponse.Thefinal
resultwillbetissuebreakdown,whichischaracterizedbybonelossanddestructionof
theperiodontalapparatus.
Chapter2:
Figure 1 - Gating strategy for flow cytometric analysis. Gating was performed based on
fluorescence-minus one controls. Neutrophils were identified based on the following
gating strategy:FSCvs. time,FSCvs. SSC, theneFluor780+, followedbySinglets+.The
imagesarerepresentativeofbloodPMNofhealthypatients.
Figure 2 - Gating strategy for flow cytometric analysis. Gating was performed based on
fluorescence-minus one controls. Neutrophils were identified based on the following
gating strategy:FSCvs. time,FSCvs. SSC, theneFluor780+, followedbySinglets+.The
imagesarerepresentativeoforalPMNofhealthypatients.
Figure 3 - To confirm purity of the samples used for screening with HTS flow cytometry,
neutrophils from blood and oral rinse were isolated by FACs. (A-1) Representative
contourplotsofblood(B-1)andoralrinse.(A-2andB-2)Cytospinpreparationsofeach
sortedcelltypewerevisualizedwithDiff-Quikstainingtoconfirmpurityofthesamples.
(A-3) Contaminatingmonocytes/lymphocyteswere excluded from our analysis by our
gatingstrategy.
Figure4-DiversityofNeutrophilSurfaceMarkersDependsSourceandHealthofTissue.(A)
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The heatmap of the initial screen of all 374 known CD markers using HTS – flow
cytometryofbloodandoralneutrophils isolatesfromhealthyandCPpatients.(B)We
couldconfirmexpressionof145surfacesmarkersinneutrophilseitherincirculationor
oral rinse samples; an extensive literature search reveals 141 CD markers previously
reported to be expressed in neutrophils in different states of activation. (C) Percent
positivemarkerexpressionprofilesareshowninaheatmapformatforarepresentative
set of 20 surfacemarkers on neutrophils from blood and oral rinse in health and CP
patientsrevealsdegreeofdifferencesbetweensourcesofneutrophils.(D)Examplesof
histograms of cell surface markers commonly used to isolate neutrophils. Note
variabilityofexpressionofCD31andCD62LinPMN-OinhealthyandCPpatients.CP–
ChronicPeriodontitis,CTR–Control(Healthy),PMN-B–Bloodneutrophils,PMN-O–Oral
neutrophils.
Figure 5 - Cluster analysis Unsupervised hierarchical clustering of percent-positive marker
expression values generated on 40 sampleswas performed. Colors indicate source of
biologicallyrelatedsamplesintoclustersbasedonsurfacemarkerprofiles.SeeFigureS
for a magnified image of the significant markers. CP – Chronic Periodontitis, CTR –
Control(Healthy),PMN-B–Bloodneutrophils,PMN-O–Oralneutrophils.Red–PMN-B,
Green–PMN-O.
Figure6-Magnifiedimageofthesignificantmarkers.Unsupervisedhierarchicalclusteringof
percent-positive marker expression values generated on 40 samples was performed.
Colors indicate source of biologically related samples into clusters based on surface
marker profiles. CP – Chronic Periodontitis, CTR – Control (Healthy), PMN-B – Blood
neutrophils,PMN-O–Oralneutrophils.Red–PMN-B,Green–PMN-O
Figure 7 – Oral neutrophils in CP patients are characterized by an active phenotype. (A)
Representativeflowhistogramsofcellsurfacemarkers(black)thatareup-regulatedin
PMN-OfromCPpatientsareoverlaidonisotypecontrolmAbs(monoclonalantibodies?)
(inred).(B)QuantificationofflowcytometricassessmentofOralandBloodneutrophils
from Healthy and Chronic periodontitis patients (expressed as MFI). MFI - Mean
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Fluorescentintensity.*P≤0.05
Figure 8 – Cell sortingwith an antibody against CD11b, CD16 and CD66b allowed for the
enrichmentofhighlypurematureneutrophils in theblood(A)Quantificationof flow
cytometric assessment of Blood neutrophils from Healthy and Chronic periodontitis
patients (expressedas%of+ve cells) (B) Flowcytometricplots froma representative
analysisofbloodandoralPMNs fromhealthyandCPpatientsaftercell isolation.The
selected markers are highly expressed in neutrophils independently of inflammatory
condition. CD11b, CD16 and CD66b expression (black) are overlaid on isotype control
mAbs (in red). (C) Cytospin preparations of each sorted cells based on expression of
CD11b+, CD16+ and CD66b+ for each compartment were visualized with Diff-Quik
stainingtoconfirmpurityofthesamples.
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Chapter1 GeneralIntroduction
1. Introduction
Themost abundant type ofwhite blood cells, neutrophils or polymorphonuclear leukocytes
(PMNs), are important modulators of the host innate immune response1. In healthy
individualsthebonemarrowproduces2×1011neutrophilsdailyfrommyeloidprecursorcells2.
Developmentofneutrophils in thebonemarrowhasclassicallybeendivided into fivestages
basedoncellsize,nuclearmorphology,andgranulecontent,andiscontrolledbygranulocyte
colony stimulating factor (G-CSF). This maturation and differentiation from myeloblast to
promyelocyte, myelocyte, metamyelocyte, and finally to a mature polymorphonuclear cell
takes approximately six days. Mature neutrophils are released into circulation, where they
circulatefor10to24hoursbeforemigratingintothetissues3.Onceinthetissues,neutrophils
areknownfortheirprotectiveroleagainstacuteinfections,buthavealsobeenimplicatedin
the pathogenesis of multiple chronic inflammatory diseases4. Proper identification and
characterizationofneutrophilswillplayasignificantroleincharacterizingneutrophilmediated
diseases.
NeutrophilIdentification
Theliteratureisvastindescribingdifferenttechniquestoisolateandcharacterizeneutrophils.
However, without a proper standard it is difficult to interpret disparaging reported results,
likely causedby inconsistences between cell yields, viability, variation in cell surfacemarker
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expression,andincorrectlyidentifiedcellpopulations5-7.Forinstance,inastudythatinduced
endotoxemiainhumans,neutrophilswereidentifiedbasedontheexpressionofCD16(cluster
of differentiation) and CD62L8. Other studies have identified neutrophils after gradient
separation by the expression of CD11b9, CD15/CD1610, CD181/CD11b/CD66 and CD6911.
Studies using negative immunoselection claim to achieve purity of samples of 99.7%,
nevertheless, these techniques might select a subpopulation of neutrophils, by excluding a
subset that might co-express markers that are abundantly expressed in other cell
populations12. The use of visual inspection under a light microscope with May-Grünwald-
Giemsa, or a similar stain, is still the gold standard for neutrophil identification.One of the
most recognizable characteristic of neutrophils is themorphology of their nucleus normally
described as having a distinctmultilobulated appearance. Another defining characteristic of
neutrophils is an abundance of cytoplasmic granules. These granules contain a variety of
enzymes,membraneproteins andmatrixproteins, andaredivided into threemajor groups:
primary (azurophilic), secondary (specific), and tertiary (gelatinase) granules13. The ability to
correctly identify, and isolateneutrophils are critical for furtherunderstandingof both their
biologyandalsotheimmunesystemingeneral.
Clusterofdifferentiation(CD)
Cluster of differentiation (CD) markers are cell surface molecules that act as receptors or
ligands. They can be used to identify cell populations via a process known as
immunophenotyping14. The CD marker system has been developed by the Human Cell
Differentiation Molecules (HCDM) organization since 1982, in a collaborative effort by
scientistsaroundtheworldatHumanLeukocyteDifferentiationAntigens(HLDA)Workshops15.
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TheCDmarkerandantigensystemareused inmanyapplications in researchandmedicine,
including identification, localization, quantification, and isolation of cells, as well as for
therapeuticapplications.ImmunophenotypingusingCDmarkersarebeenusefulforidentifying
cellpopulationsubsets,fromstemcellsandcancercellstoT-cellsubsets.
Flow cytometry has become a popular method for evaluation of neutrophils16. However,
identification and characterization and interpretation of flow cytometry results is still
controversial,sinceadefinitiveCDmarkersetforneutrophilshasyettobeidentified17.CD14
and CD15 are among themarkers that are commonly used to identifyhuman neutrophils.
CD14 is a lipopolysaccharide-binding protein that acts as a co-receptor for bacterial binding
lipopolysaccharide (LPS) andotherpathogen-associatedmolecularpatterns. Monocytesand
mosttissuemacrophagesaretheothercellpopulationsknowntohighlyexpressCD14.CD14is
alsoexpressedingranulocytes,andasmallpercentageofperipheralbloodlymphocytes,albeit
toalesserextent18.CD15,acarbohydrateadhesionmolecule,knowntomediatechemotaxis
andphagocytosis is alsoexpressed in granulocytes.A significantproportionofmonocytes, a
subsetofmacrophages,andepithelialcellsandtheirmalignantcounterpartsmayalsoexpress
CD156.Neutrophilsareofutmostimportanceinalargenumberofclinicaldisorders. Inorder
forustointerpretsurfacemarkerexpressionindisease,itisimperativethatwefirstelucidate
thepatternofexpressionofsurfaceantigensonnormalneutrophils.
High-throughputscreening(HTS)flowcytometry
Cellsurfacemolecules,collectivenamedasthecell‘‘surfaceome’’,areknowntoperformvital
cell functions, acting as receptors, transporters, channels, and enzymes19. High-throughput
screening (HTS) assays, commonly used to test chemical compounds in the pharmaceutical
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industry,arenowwidelyusedtophenotypeandcategorizecells.Flowcytometry (FC)utilize
fluorescently-taggedantibodiestodetectcellsurfaceproteins.ThecombinationofHTSwithFC
allow the evaluation of over 350 known CD markers at a time, using a high-speed sample
loading device for flow cytometers20. This high throughput flow cytometry (HT-FC) assay is
highly reproducible and canbeused to answer awide rangeof researchquestions, such as
characterizationofcellpopulations,identificationofcellsubsets,isolationforfunctionalassays
and molecular profiling, development of therapeutic targets, or biomarkers that can be
prognosticorassociatedwithspecificresponses,andmoreover,theycanbeusedfordisease
detection and classification14,19-21. Understanding the differential expression of surface
markers can be useful to elucidate the role that neutrophils play in immunological and
inflammatoryresponses.
2. CDexpressionandNeutrophilDistribution
BoneMarrow
NeutrophilCellLineage
Neutrophils are produced in the bone marrow by a process know as hematopoiesis22.
Neutrophilprecursorsaredifferentiatedfromhematopoieticstemcellsthataredriventoward
themyeloid lineage, and further intomatureneutrophils 23. Thebonemarrow is a complex
tissuewheremultiplehematopoieticlineagescoexistinvariousmaturationalstages.Knowing
theexpressionlevelsofneutrophilspecificmarkersandotherlineage-specificmarkersduring
normal hematopoietic development gives us a standard by which to recognize abnormal
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patternsofdifferentiation23.
During granulocytic differentiation, myeloblasts, the most immature neutrophil precursor,
cannotbediscriminatedfrommonoblasts,themostimmatureformofthemonocyticlineage,
sincebothshareacommonparentalstemcell(Granulocyte-Macrophagecolonyformingunit–
GM-CFU).Thematurationprocessisregulatedbygrowthfactors,suchasGM-CSF,M-CSFand
G-CSF, aswell as by cytokines and chemokines. The relative size of the cells, togetherwith
expression levels of specific markers such as CD34, CD117, CD45 (membrane-associated
tyrosinephosphatase),CD13(aminopeptidaseN),CD33,CD16,andCD11b,canbeusedstudy
granulocytic differentiation. Themarkers that havebeenused to identify neutrophils during
differentstagesofhematopoiesisaresummarizedinTable1.
Table1–Cellsurfaceexpressionindifferentstagesofgranulocyticdifferentiation
StageofPMNmaturation
Surfaceexpression
Size Characteristics
myelo/monoblasts CD16_CD13
_
CD45intCD11b_
12-20µm Nucleus:round/ovoid
Ratio:6:1
promyelocytes CD117CD13highCD33highCD15,CD34,MHCII
15-21µm Nucleus:round/ovoid
Ratio:4:1
Myelocytes CD13dimCD33dimCD34,CD15,
CD11b
12-18µm Nucleus:round/ovoid/flattenedonone
side
Ratio:2:1
Metamyelocytes CD13CD33dimCD34-CD15,
10-18µm Nucleus:indented/kidney-shaped
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CD11b,CD16Ratio:1.5:1
Bandcell CD13CD33dimCD34-CD15,CD11b,CD16
10-16µm Nucleus:horseshoe
Ratio:1:2
Neutrophils CD13highCD33CD15,CD11bhigh
CD16high
9-16µm Nucleus:segmented
Ratio:1:3
To identifypurifiedpopulationsofneutrophilsand itsprecursors, somestudieshaveutilized
cellularsize,granularity,andexpressionprofileoftheCD13,CD15,CD11b,andCD16surface
markers,afterexcludingCD3,CD19,CD14,glycophorin-A,CD56,andCD61positivecells.CD13
showsdynamicchangesinexpressionduringgranulocyticdifferentiation.Incombinationwith
CD11b and CD16, these changes define the sequential stages of granulopoiesis. CD13
expression is up-regulated on myeloblasts (MB) and promyelocytes (PM), and then down-
regulatedonmyelocytes(MC).CD13expressionisgraduallyup-regulatedagainasneutrophils
reach their final stages of differentiation and develop into segmented neutrophils. On the
otherhand,CD11bandCD16,whichare initiallyexpressedat low levels, showprogressively
increasedexpressionduringthedevelopmentalprocess,particularly inthe last twostagesof
neutrophildifferentiation23,24.CD16comprises the lowaffinityFc receptors, FcγRIIIa (CD16a)
and FcγRIIIb (CD16b). These receptors bind to the Fc portion of Immunoglobulin G (IgG)
antibodies, which then activates the (NK) cell for Antibody-Dependent Cell-mediated
Cytotoxicity (ADCC). A lack of CD16 in a given population of neutrophils may indicate
prematurity, as could be caused by a left-shift (increased ratio of immature to mature
leukocytes)duetoneutrophilicleukocytosisinducedbytissuenecrosisorbacterialinfection.
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Circulation
After being stored in the bone marrow for 5 to 7 days, neutrophils are released in the
circulationandhaveahalf-lifeof6–9hours inthatcompartment25. Intheblood,neutrophils
comprise about 70% of leukocytes and more than 90% of phagocytes. Neutrophils can
reversiblymovefromcirculatingtomarginatingpools,wheretheyare‘stored’inthecapillaries
ofcertaintissues,mostnotablyinthelungs,duetothelargenumberofsmallcapillaries26.
AstudybyKuijpersetal.showedthatexpressionofsurfacemoleculescouldbeinfluencedby
cellpurificationprocedures.Theyidentified7D5(cytochromeb,a-subunit)andCD10as“early
activationantigens,”becausebothantigenswereabsentprior todensity-gradientneutrophil
purification,butpresentonpurifiedrestingneutrophils27.Also, surfaceexpressionofseveral
antigens thatwereexpressedon circulatingneutrophils increased significantly after density-
gradient centrifugation. The isolation method caused increased expression of CD13, CD16,
CD18, CD45, and CD67 and no changes in expression of CD32 (FcRII), CD54 (ICAM-I), CD58
(LFA-3),Leu-8andHLAclassIantigens27.
Systemic diseases can also play a major role in defining the phenotype of circulating
neutrophils. Pillay et al. identified a subset of circulating neutrophils that would display an
alteredphenotypecharacterizedbyCD62Llow,an increasedexpressionofCD11b,CD11c,and
CD54,andanequalexpressionofCD88,afterendotoxemiawasinducedwithLPS8.Othershave
demonstratedasubpopulationofneutrophilscharacterizedbyCD10-/CD16lowthatrepresents
40%ofallcirculatingneutrophils immediatelyaftercardiacandnon-cardiacthoracicsurgery,
representing newly released neutrophils from the bonemarrow during acute inflammatory
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states28.
Tissues
Although little information can be found in the literature regarding CD expression in tissue-
neutrophils, in a study that evaluated the neutrophil phenotype in a model of reverse
endothelial migration, it was demonstrated that patients with rheumatoid arthritis (RA)
present with a subset of neutrophils in the synovial fluid that had a specific phenotype
characterized by CD181low, CD54low 29. In a recent study that compared cell surfacemarkers
expressiononbloodandsputumneutrophilsofpatientswithChronicObstructivePulmonary
Disease (COPD), they found up-regulation of adhesion molecules and neutrophil activation
markers such as CD11b, CD63 and CD66 on sputum neutrophils,while the levels of CD11b,
CD162 and CD62L were significantly reduced on circulating neutrophils in COPD subjects30.
Additionally,whentheycomparecellsurfaceexpressionwithclinicalparametersofCOPD,they
reportedthat lower levelsofCD11b in thebloodcorrelatedwithclinicalexacerbationof the
disease30. In another study that evaluated tissue neutrophils in patientswith Cystic Fibrosis
(CF)reporteddifferencesintheneutrophilphenotypewithup-regulationofCD63,amember
oftetraspaninfamilyandmarkerforazurophilicgranulefusion,loseofexpressionofCD16and
CD14, and expression of CD80, major histocompatibility complex type II, and CD294 a
prostaglandinD2 receptor,which arenormally associatedwithother cell lineages31. Shifting
the paradigm that neutrophils are a unique and homogeneous cell population; similar to
monocytes or T-cells, subsets of neutrophils might indicate a dysfunctional phenotype and
possibletargetfortreatmentofneutrophilmediateddisease.
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3. CDexpressionandNeutrophilsFunctionality
NeutrophilRecruitment
The recruitmentofneutrophils from thevasculature into inflamed tissue is key foraproper
host defense against invading microorganisms 32. This process occurs mainly in the
microvasculature,wherehemodynamicshearforcesareminimized,andinvolvesthefollowing
steps:tethering(orcapture),rolling,slowrolling,arrest,adhesion,crawlingandtransmigration
inacascade-likefashion2.Ineachstepofneutrophilrecruitmentandtransmigration,integrins
andselectinsplayacrucialrole,wheretheycanactasligandsandreceptorsforneutrophilsto
fulfill these important functions. I will explore the role of some of those markers such as
CD11a,CD11b,CD18,CD62L(Table2).
Table2–Cellsurfacemarkersexpressedinneutrophilsassociatedwithrecruitmentandmigratory
functions
Functions CDmarkers
Cellularadhesionmolecules CD44, CD50, CD54, CD56, CD102, CD106, CD146, CD166,
CD321,CD322,CD326
Integrins CD11a,CD11bCD18,CD29,CD49a-f,CD51,CD61,CD104
Selectins CD62E,CD62L,CD62P
Chemokinereceptors CD117, CD119, CD121a, CD123, CD124, CD126, CD127,
CD140a,CD140b
Membrane-bound
receptorsinvolvedin
apoptosisornecrosis
CD95,CD178,CD120a,CD12b
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Neutrophil tethering and rolling on the endothelial surface are initiated in response to
inflammatory mediators such as histamine, leukotrienes or cytokines released by tissue-
resident sentinel cells or by direct stimulation via pathogen-associated molecular patterns
(PAMPs)32.Tetheringandrollingaremediatedbytheup-regulationofE-selectin(CD62E)and
P-selectin (CD62; CD62P) on the vascular endothelial cell surface, which bind to neutrophil
expressedglycosylated ligands,suchasP-selectinglycoprotein ligand-1(PSGL-1)orCD16232.
Rollingneutrophilsareactivatedbycytokinespresentonthesurfaceofendothelialcells,which
promotetheactivationofintegrinssuchasLFA-1(CD11a/CD18)andMAC-1(CD11b/CD18)on
thecell surfaceofneutrophils.These integrinsbindto ICAM1and ICAM2onendotheliumto
facilitate arrest and transition to a crawling regime2.Neutrophil crawling on the endothelial
surface requires dynamic regulation of integrin-ligand interactions as well as associated
intracellular F-actin. In order to undergo productivemigration neutrophilsmust release the
existing integrin-ligand associations at the rear of the cell while forming new bonds at the
leadingedge.Thisallowsthemtomaintainafirmadhesiontothevascularwalluntiltheyfinda
preferential site where they can exit the vasculature in a process called transendothelial
migration2.TransmigrationrequiresintegrinsandCAMs(ICAM1alsoknownasCD54),ICAM2
orCD102andvascularcelladhesionprotein1(VCAM1orCD106)aswellasdifferentjunctional
proteins, including platelet/endothelial cell adhesion molecule 1 (PECAM1; also known as
CD31), CD99, junctional adhesion molecules 1 (JAM-1 or CD321), epithelial cell adhesion
molecule (ECAM; CD326) and some other endothelial cell molecules, for example, vascular
adhesion protein 1 (VAP1) and CD15732. Neutrophil transmigration occurs preferentially
paracellularly (between endothelial cells) but can also occur transcellularly (through an
endothelialcell),howevertranscellulartransmigrationisalessefficientmethod,andcantake
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up to 30 minutes to complete. Once they enter the three-dimensional tissue matrix,
neutrophils undergo directional migration towards a concentration gradient of
chemoattractant,throughaprocessknownaschemotaxis33.
BacterialkillingandPhagocytosis
Neutrophilsarehighlyphagocyticcells.Consistentwiththisistheirhighsurfaceexpressionof
low affinity Fc receptors such as CD16 and CD3234. Neutrophils destroy invading
microorganisms,byexposingthemtooxidative-dependentand/or independentmechanisms.
Thiscanbeaccomplishedbyengulfingpathogensorbythereleaseofneutrophilextracellular
traps (NETs).Neutrophils killphagocytosedmicroorganisms throughenzymaticandoxidative
destruction.Tofinalizetheseprocesses,andeffectivelydisposeengulfedparticles,neutrophils
diebyapoptosisandarethenthemselvesphagocytosedbymacrophages,thuspreventingthe
releaseofcytotoxicproducts35,36.
Whenneutrophilsreachthesiteofinflammation,theyareexposedtoanenvironmentfullof
inflammatory mediators, bacteria, fungi, cell debris, apoptotic cells, etc37. An array of
neutrophilcellsurfacereceptorsrecognizeagonistssuchaspeptidesequences,surfacebinding
proteins, secreted bacterial products, opsonized host proteins, components of the
complement cascade,double strandedRNAandFcdomainsofantibodies37.Neutrophils can
alsorecognizenon-opsonizedligands,suchaslipopolysaccharides(LPS),orparticlesopsonized
with host-derived proteins called opsonins36. Once neutrophil surface receptors recognize a
cognateligand,thereceptoris‘activated’andasignalistransmittedtotheinteriorofthecell
resultingintheinitiationofphagocytosis38.ThisprocessismarkedbyactivationofSrc-tyrosine
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kinases, which trigger the aggregation of CD32 (FCGR2A) and CD16 (FCGR3A) and the
phosphorylation of their cytoplasmic immunoreceptor tyrosine-based activation motifs
(ITAMS),whichtriggerstheactivationofPI3KandRhoproteins39.ActiveRhoproteins induce
extensionofmembraneprotrusionsoverthesurfaceofthetargetparticleandengulfment40.
Tosimplify,themainmechanismbywhichbacterialrecognitionandneutrophilactivationare
regulated, is by the interaction of pathogen-associatedmolecular patterns (PAMPs) and the
pattern recognition receptors (PRRs), among them the Toll like receptors (TLRs). TLRs,
expressed on the cell surface of neutrophils, are up-regulated after stimulation by LPS.
Althoughneutrophilspresentwith selected specificitybetween its componentandmicrobial
byproducts,suchasTLR4(CD284)interactswithlipopolysaccharide(LPS)fromGram-negative
bacteria and TLR2 (CD282) with peptidoglican, lipoteicoic acid from Gram-positive bacteria,
amongothers.Moreover,thegenerationofreactiveoxygenspeciescanbetriggeredthrough
TLRs.Which,inturn,canresultinupregulationofCD64,ahighaffinityFcreceptorexpressed
inneutrophils,resultinginincreasedoxidativeburstandphagocyticpotential41.
Phagocytosis by neutrophils is more efficient when microbes are coated with serum host
proteinscalledopsonins,which includecomplementproteinsandantibodies.Activationofa
complement leads to deposition of complement components on microbial surface.
Complement surface receptors (CRs) expressed on the surface of neutrophils efficiently
recognizemicrobesboundwithcomplementcomponents.CRs includeCIqR,CR1(CD35),CR3
(CD11b/CD18)andCR4(CD11c/CD18)42.
13
The granules in mature neutrophils contain a variety of proteins that contribute to anti-
microbial host defense43.Mobilization and release (degranulation) of their content into the
phagosome, thereby exposing the ingested microbe to toxic agents, embodies the oxygen
independentmechanism40. Neutrophil granules can be classified as primary, secondary, and
tertiary granules, or secretory vesicles. These granules store at least 300 different proteins,
which can be either released into the surrounding environment in a hierarchical order,
incorporated into the cell membrane or remain attached to the membrane upon granule
mobilization44. Protein components of neutrophil granules include: defensins, bactericidal-
permeability-increasing protein and azurocidin that compromise the permeability of the
bacterial membrane; proteases such as neutrophil elastase and cathepsin G that degrade
bacterial products; and the enzyme myeloperoxidase (MPO) that generates toxic ROS. In
general,thesecretoryvesicleshavepreferenceforextracellularrelease.Thesevesiclesprovide
thesecellswithcellsurfacereceptorsandmoleculesthatarevital,althoughinlowlevels,for
neutrophil activation. Secondary granules in neutrophils include a large number of key cell
adhesionmolecules, suchasCD11b/CD18,CD47andSIRPα (CD172a),whichare required for
neutrophiladhesionandchemotaxis44.Uponstimulationneutrophilgranuleswill fusewitha
newly formed phagosome and release their products contributing directly to microbial
killing40,45.
To summarize, neutrophils express Fc receptors that recognize antibodies. These antibodies
have an antigen binding site and an Fc region for the Fc receptors on neutrophils. These
receptorswhichincludeCD23(FcεRI,IgEreceptor),CD89(FcαR,IgAreceptor),CD64(FcγRI,IgG
14
receptor), CD32 (FcγRIIa, low affinity IgG receptor) and CD16 (FcγRIIIb, low affinity IgG
receptor)willplayacriticalroleintheabilityofneutrophilsperformtheirbasicfunctions42.
Non-traditionalfunctionsofneutrophils
Classically neutrophils are viewed as short-lived cells with a main phagocytic role. In more
recent years, theneutrophil have alsobeendescribedashaving an active regulatory role in
angiogenesisandtumoralfate,aswellas,neutrophilscaninfluencetheimmuneresponseby
releasingavarietyofcytokinesandbyactingasantigen-presentingcell(APC)expressingMHC
Class II, and finally by stimulating T cells activation12,46,47. Nowadays, a number of diseases
includingperiodontitis,arthritisandacuterespiratorydistresssyndrome(ARDS)areassociated
with neutrophil dysregulation that may results in significant tissue damage. I will focus in
periodontaldiseaseasamodelforstudyingnuancesoftheneutrophilcellsurfacesignature.
4. PeriodontalDiseases
Periodontaldiseasesencompassavarietyofdiseasesaffectingthehealthoftheperiodontium.
Theyrangeinseverityfromareversibleinflammationofthegingivatermedgingivitis,whichis
highly prevalent and readily reversible, to a more severe form of chronic destruction of
periodontal tissues that includes gingiva, periodontal ligament, and alveolar bone with
eventualtoothloss,knownasperiodontitis48.
ChronicPeriodontitis
ChronicPeriodontitis(CP)isrecognizedasanimmune-mediatedresponsetoaplaquebiofilm
resulting in inflammation within the supporting tissues of the teeth, which leads to a
progressivelossofattachmentandbonedestruction.AccordingtotheAmericanAcademyof
15
Periodontology(AAP)classification,itcanbefurtherclassifiedaslocalizedorgeneralized,and
withoneofthepossibleseverities:slight,moderateorsevere.Environmentalfactorssuchas
smokingandemotionalstresscanmodifytheseverityandprogressionofthedisease,aswell
assystemicdiseasessuchasdiabetes48.
TheroleofneutrophilsinthepathogenesisofPeriodontitis
Neutrophilsarerecognizedaskeyplayerinmediatingperiodontaltissuedestruction.Thereare
twomainmechanismsdescribedbywhichneutrophilswouldmediatetissuedestruction:byan
impairedphenotypeandbyahyperactivephenotype 49.A thirdpossiblemechanismbyhow
neutrophilswouldmediateperiodontaldestructionwouldbeviarecruitmentandactivationof
thenormalneutrophil49,50.
Theexactmechanismofhowperiodontaldestructionoccursandwhysomepatientsaremore
susceptibletotissuedamagethanothersremainsunknown51.However,advancesinthefield
ofcellandmolecularbiologyindicateamultifactorialetiologyforperiodontitis.
16
Figure 1 -Model of pathogenesis of Periodontitis (adapted from Page & Kornman, Periodontology
2000,1997).Bacteriaandbiofilm insultarecrucial for the initiationofperiodontaldisease.However,
the presence of bacteria alone does not cause tissue destruction. Themajority of tissue destruction
occursasaconsequenceofthehost immuno-inflammatoryresponseagainstthismicrobialchallenge.
Bothgeneticandenvironmental risk factorsworksasmodulatorsof thehost immuneresponse. The
final result will be tissue break down, which is characterized by bone loss and destruction of the
periodontalapparatus.
Page and Schroeder, in 1976, published a seminal work describing for the first time the
different stages in the development of the periodontal lesion52. Using histopathologic and
ultrastructural analysis of the diseased gingival tissue, they divided periodontal lesions into
fourstages:1) initial lesion,2)early lesion,3)establishedlesion,and4)advancedlesion.For
the first time, an increased numbers of neutrophilsmigrating into the junctional epithelium
and underlying connective tissuewas described52. The combination of this initial workwith
recentknowledgeofmolecularbiologywerethebasisforthecurrentmodelforpathogenesis
17
of periodontitis proposed by Page & colleagues in 1997 (Figure 1)53,54. The idea is that a
pathogenic dental plaque biofilm would work as an initiator of periodontitis; however, by
itself,isnotenoughtocausedisease.Systemicconditions,environmentalfactorsandthehost
geneticsusceptibility,suchasdiabetesmellitus,smokingandIL-1αpleomorphism,wouldplay
a role inmodulating the host response.Ultimately, and likely themost significant, the host
immune response would determine the clinical outcome. Lipopolysaccharides (LPS) are
released frombacterialbiofilm into theperiodontal tissues.Whichwouldactivatecells from
thejunctionalandpocketepitheliumtoreplicateandrelease inflammatorymediators.These
pro-inflammatory cytokines, IL8 and IL-lα, would recruit and activate neutrophils,which, in
turn,wouldsecretematrixmetalloproteinases(MMPs).Thesemoleculesareabletofacilitate
destruction of the extracellular matrix of the gingiva and periodontal ligament and induce
resorptionofthealveolarbone.Aninflammatoryinfiltrateformedmostlybyneutrophils,but
alsocontainingB&Tlymphocytes,andmonocytes/macrophageswillbepresent.Additionally,
oral inflammation increases production of cytokines, such as interleukin-6, that in turn,
stimulateosteoclastactivityandpromoteboneresorption.
5. Objectivesofthisstudyandhypothesis
Objectives
Inthecurrentstudy,Iusedisolatedneutrophilsfromperipheralbloodandfromtheoralcavity
of healthy individuals and patients with generalized severe chronic periodontitis, to
understandchangesintheneutrophilcell-surfacesignature,astheytransitfromthebloodinto
differentlocationsandactivationstates.
18
Tosummarize,myobjectiveswiththisstudywere:
• To use HTS Flow Cytometry as a tool to identify a unique neutrophil cell-surface
signaturethatcanbeusedtoidentifyneutrophils,whichisindependentofitsstateof
activationand/orlocation.
• To validate a specific set of cell surface markers, by FACS and/or magnetic
immunoselection,thatcanbeusedtoidentifyneutrophils.
Hypothesis
• Theneutrophilhasauniquecell-surfacesignature,whichisindependentofitslocation
andstateofactivation.
Using a novel method, the high-throughput screening (HTS) flow cytometry, I was able to
characterize and compare the neutrophil cell surface profile in the blood and oral
compartments.Additionally,IcouldconfirmthelevelofexpressionofallknownCDmakersin
the cell surface of neutrophils. This screen allowed the identification of CD11b, CD16b, and
CD66casmarkersthatarealwaysexpressedonneutrophils independentofthecell location,
levelofactivationanddiseasestate.
19
Chapter2
Immunophenotypicalcharacterizationofhumanneutrophils
FlaviaS.Lakschevitz,SiavashHassanpour,AyalaRubin&MichaelGlogauer.Identification
ofNeutrophilSurfaceMarkerChangesinHealthandInflammationusingHigh-throughput
ScreeningFlowCytometry(Manuscriptinpreparationforsubmission)
Abstract
Neutrophilsare themost abundantwhite blood cell and are an essential componentof the
innate immune system. A complete cataloguing of cell surface markers has not been
undertakenforneutrophilsisolatedfromcirculationaswellashealthyandinflamedtissues.To
identify cell-surface markers specific to human neutrophils, we used high-throughput flow
cytometrytoscreenneutrophilpopulations isolatedfrombloodandoral rinses fromhealthy
andchronicperiodontitispatientsagainstapanelof374knownclusterofdifferentiation(CD)
antibodies.ThisscreenidentifiedCD11b+,CD16+,andCD66b+asmarkersthatareconsistently
expressedonneutrophilsindependentofthecelllocation,levelofactivationanddiseasestate.
CellsortingagainstCD11b,CD16andCD66ballowedfortheenrichmentofmatureneutrophils,
yieldingneutrophilpopulationswithupto99%purity.Thesefindingssuggestanidealsurface
markersetforisolatingmatureneutrophilsfromhumans.Thescreenalsodemonstratedthat
tissue neutrophils from chronically inflamed tissue display a unique surface marker set
comparedtotissueneutrophilspresentinhealthy,non-inflamedtissues.
20
Introduction
Aneutrophil cellular infiltrate is commonly found in the oral cavity of healthy and diseased
subjects55.Nevertheless,patientsdiagnosedwithchronicperiodontitis,aneutrophil-mediated
inflammatoryconditionthataffectsthesupportingtissuesoftheteethresultinginirreversible
tissuedestruction,havehigher levelsofneutrophils in theoral cavity56-58.Theabundanceof
oral neutrophils in both health and disease provides a non-invasive means of harvesting
transmigratedneutrophilsthroughoralrinses. Isolatedoralneutrophils(PMN-O)canthenbe
examined to determine phenotypical changes of tissue neutrophils in healthy and inflamed
tissuescomparedtothebasalphenotypeofcirculatorybloodneutrophils(PMN-B)56,59.
Well-described and consistent methods of neutrophil isolation, identification, and detailed
characterization are critical for further understanding of neutrophil biology and the innate
immunesystem.Unfortunately,non-standardizedapproachesinneutrophilisolationprotocols
leads to inconsistencies between studies in termsof cell yields, purity and viability 5-7. Flow
cytometry is commonly used to evaluate neutrophil surface markers16. However, the
identificationandcharacterizationofneutrophilsusingthistechnology is far fromuniformin
thefieldsinceadefinitivemarkersetforneutrophilshasyettobeidentified8.CurrentlyCD11b,
CD14,CD15,CD16andCD62Lareusedsolelyorinsomecombinationtoidentifypurehuman
neutrophilpopulations8,10,17,60,61.Likewise,CD11bandLy6Ghavebeenusedtoidentifymouse
neutrophils62,63.Presently,therearereferencesandwebresourcesthatcharacterizeallknown
CDmarkersinallcelltypesbasedonacataloguingofexistingstudies.Despiteover370known
CDmarkers,most studies evaluate one or twomarkers at a timewith non-uniform patient
populationsandcell isolationprotocols.Therefore, conclusionsdrawn fromreportedstudies
21
arenotalwayscomparable64,resultinginconflictingdataaboutthepresenceofmanymarkers
and reports of CD of markers that fail to differentiate between neutrophils and other
leukocytes 15. Neutrophils are key in the pathogenesis and resolution of a variety of clinical
disorders.Therefore, it is imperativethatwefirstelucidatethebaselineexpressionprofileof
surface antigens on neutrophils in health. The use of high-throughput screening (HTS) flow
cytometrygivesustheopportunitytorapidlyevaluateallknownCDmarkerssimultaneouslyin
anunbiasedmanner20,65.Onit’sown,HTSisadiscovery-orientedscreeningtoolthatallowsfor
identificationofsurfaceproteinsincellsof interestbyusingalargepanelofCDmarkersina
highlyreproduciblefashion.Inaddition,HTScanbecombinedwithfluorescentcellbarcoding,
sorting66 and functional assays that ultimately allow to for the identification and
characterizationofauniquesetofbiomarkersinhealthanddisease.Inthisstudy,ourultimate
aimwastoidentifyauniqueanduniversalcell-surfacesignatureinneutrophilsindependentof
its locationandstateofactivation.Aconsistentandwelldefinedneutrophilsignaturewould
permitrapidandreproducibleisolationofcirculatorymatureneutrophils.Inparallelweaimto
demonstrate that the signature we identify does not change significantly as neutrophils
transmigratefromcirculationintohealthyandinflamedtissues.
Materialsandmethods
StudyPopulation
Patients were screened at the University of Toronto, Faculty of Dentistry for periodontal
disease. Periodontal examination was conducted to confirm the diagnosis of periodontal
disease, which is based on the current diagnostic framework67,68. Ten patients without
22
periodontaldisease (Healthy),and tenpatientswithgeneralizedseverechronicperiodontitis
(CP),wereenrolled in this study.All enrolledparticipantswere systemicallyhealthyand self
reportednon-smokers.Afteracompleteintraoralexamination,bloodandoralrinsecollection
wasconducted.Participantsprovidedwritteninformedconsentinordertoparticipate,andthe
ScientificandEthicsReviewBoardsattheUniversityofToronto,FacultyofDentistry,approved
thestudy(Protocol#29410).
Bloodcollectionandprocessing
Bloodsamplesweredrawnfromtheantecubitalveinintoavacutainercontaining0.1volume
ofsodiumcitrateanticoagulant(BectonDickinson,Canada).Samplesweredividedintwo.For
the first set, neutrophils were separated using 1-layer gradient of 1-step polymorphs as
previouslydescribed69.Briefly,gradientswerecentrifugedat527relativecentrifugalforcefor
30minutesat room temperature, the lowerof twobandswas collected.Cellswerewashed
withHanks balanced salt solutionwithout calciumormagnesium (HBSS-/-), and erythrocyte
lysiswasperformedbyincubatingwith1mLofBDPharmLysebuffer.Forthesecondset,only
erythrocytedepletionwasperformedbyincubatingthesamplesat4oCfor5minwith1mLof
BDPharmLysebuffer(BectonDickinson,Canada).Cellcountwasobtainedusinganautomated
counter (CoulterCounter,BeckmanCoulter,Brea,CA), and viabilityof cellswasassessedby
trypanblueexclusiontest.
Oralsamplecollectionandprocessing
Theoralcavityofeachparticipantwasrinsedfor30secondswith0.9%NaClandcollectedinto
a50mLFalcontube,aspreviouslydescribed56.Thissequencewasrepeated5-20times(5–10x
23
forCPpatientsand15–20xforhealthypatients)withthreeminutesintervalsbetweenrinses.
The collectedmaterial was divided in two. The first set was passed sequentially through a
40µm,20µmand11µmnylonmeshtoeliminateepithelialcellcontaminationandwasusedfor
High-throughputscreeningassay.Forthesecondsetonly40µmfiltrationwasperformedand
was used for standard FACS. Similar to the blood samples, cells were counted utilizing an
automated counter (Coulter Counter, Beckman Coulter, Brea, CA), and viability of cells was
assessedby trypanblueexclusion test. Tominimizephenotypic and functional changes as a
resultofcellularactivation,allstepsofthedensitygradientseparationprocedure,sortingand
cell staining procedureswere performed immediately after cell collection at 4°C using non-
pyrogenic reagents and plasticware. oral PMN isolation would start at chairside at 4°C,
simultaneouslytotheoralrinsecollection.
High-throughputscreeningassay
TwentymillionviablecellswereresuspendedinHBSS-/-supplementedwith2mMEDTAand1%
BSA(Flowbuffer), immediatelyaftercollectionandisolationfrombloodandoralcavity.Cells
were blocked with rat and mouse IgG (1 mg ml-1) at 1:100 dilution for 10 min and 500
microlitersofcellswereremovedtoserveascontrols.Allcellswereresuspended instaining
media containing eFluor506 or eFluor780 to identify non-vital cells. Fifty microliters of the
above mixture was added directly to each well, preloaded with 2- 5μl of each individual
antibody based on previous plate set up and validation20 (for a full list of antibodies used,
please see supplement table S1) and incubated for 30 min in the dark. Plates were then
centrifuged, and medium removed by aspiration. Cells were sequentially resuspended and
washed after each step. Finally, cells were fixed with Fixation/Permeabilization solution
24
(Becton Dickinson, Canada) and kept in the dark at 4oC until analysis. In parallel, control
samples were prepared; aliquots were stained in 1.5ml tubes for fluorescence-minus-one
controlsforeachofthefluorophoresused(PE,APCandFITC)andcontrolforviabilitystaining.
The high-throughput flow cytometry analysis was performed using a BD-high-throughput
samplerLSRIIflowcytometer(BectonDickinson,Canada),withdefaultfilterconfigurationand
compensationwassetbyusingBDCompBeads (BectonDickinson,Canada)atUHNAntibody
Core Facility, Toronto, ON, as previously described20, 2 to 5 days after sample preparation.
Fourtosixhourswererequiredtoprepareeachsample.
Dataacquisitionandanalysis
Eachwellof the96-wellplatewas sampledwitha total read timeof30min. Foreachwell,
1,000-10,000cellswereanalyzed;forincreasedreliabilitysampleswithlessthan1,000events
wereexcludedfromouranalysis.Aninitialtemplatefilewasgeneratedusingelectronicgates
withinthesoftwaretocreateahierarchicalpopulationtreeatthebeginningofthescreen,and
using the template, all additional analyses were completed after data acquisition was
complete.Thetemplatefileincludedcompensationadjustment,whichwasuniformlyapplied
to all the data collected in order to minimize fluorescence overlap between detection
channels.Thefollowinggatingstrategywasused:a forwardandsidescatterprofiles (plotof
FSC-Avs.SSC-A)wereusedtovisuallyidentifytheneutrophilpopulationandeliminatedebris.
Gatesweremanuallydrawntoremovecelldoublets(plotsofFSC-Wvs.FSC-HandthenSSC-W
vs.SSC-H)andsubsequently toselectviablecellsbasedonexclusionofeFluor506 (oreFluor
780) positive cells. Next, two-dimensional plots were used (APC vs. PE and/or FITC vs. PE).
25
Finally,one-dimensionalhistogramsforeachfluorescenceparameterwereconstructedforthe
identifiedcells.“Positive”and“negative”gatingofthefluorescencesignalwasdrawnbasedon
fluorescenceintensityofthepositiveandisotopecontrols,andthesegateswereappliedtoall
individualwellsonaperplatebasis(SupplementalFigure1and2).Thistemplatewasapplied
toallsubsequentplates.Ifrequired,amanualadjustmentofeachgatewascompletedbefore
dataanalysis.Alldata (FCS3.0 files)wereexportedand thenanalyzedwithFlowJosoftware
(Treestar).
26
27
28
CellSorting
Cellsweresortedataconcentrationof106cells/mlinPBS/25%BSAusingaFACSAriacellsorter
(BD Bioscience) at the Flow Cytometry facility at Department of Immunology, University of
Toronto,Toronto,ON.Topreventcelldeathduetopressureandsheerstress,all sortswere
performed with a 100μm nozzle. Side scatter and forward scatter profiles were used to
eliminate debris and cell doublets, while non-viable cells were eliminated by excluding
eFluor506(oreFluor780)positivecells.
CytospinPreparationandStaining
Inordertoconfirmthepurityofneutrophils,sortedcellswerecytocentrifugedandmounted
on slides using a Cytospin centrifuge (Shandon, Ramsey, MN) for 5 min at 800 RPM, as
previouslydescribed70.Briefly,cytospinsofsortedpopulationswerefixed inmethanolfor30
seconds at room temperature. The cytospinswere then air dried and stainedwithDiff-Quik
(Fisher Scientific) staining kit according to the manufacturer’s instructions. The cells were
examinedbylightmicroscopyat200and400×finalmagnification.
StatisticalAnalysis
Statistical analysis was performed using one-way ANOVA with Bonferroni's multiple
comparisons test,unless specifiedotherwise.P≤0.05wasconsideredstatistically significant
(GraphPad software). Heatmaps, hierarchical clustering analyses were performed using
MultiExperimentViewer(MeV)software(www.tm4.org).Unsupervisedhierarchicalclustering
wasperformedusingaPearsoncorrelationdistancemetricwithcompletelinkageclustering.
29
ResultsandDiscussion
Cell surfacemarker screen of neutrophils from blood and oral rinses in health and during
chronicoralinflammation.
Neutrophilswereisolatedfrombloodandoralrinsesofhealthyandchronicperiodontitis(CP)
patientsandscreenedagainstapanelof374knownCDantibodies.Thegatingstrategyused
(seemethods) yielded a highly pure neutrophil population (≥99% ofmature neutrophils) as
confirmedbyvisualinspectionwithlightmicroscopyfollowingcellsorting(Figure3).Basedon
thescreens,itwasevidentthatneutrophilsexpress145differentCDmarkersinatleastonof
thefourcompartments(Blood-Health,Blood-Disease,Oral-Health,Oral-Disease).
An extensive literature search was carried out to identify previously described CD marker
expression in neutrophils, which identified 141 CD markers previously reported to be
expressed in neutrophils isolated from different sites and in different states of health and
activation(Figure4andSupplementaltableS2).Thenewlyidentifiedcell-surfacemarkersnot
previouslydescribedwereCDw198,CDw199,CD322andCD328.Further,theexpressionoffive
30
CD markers (CDw12, CD156a, CD156c, CD285, CD361) previously reported in the literature
couldnotbeconfirmed,asthesemarkerswerenotpartofourpanel.Lastly,theexpressionof
CD218a could also not be confirmed,with an average expression of 0.2% (±0.1) of positive
neutrophils across our samples. CDw198 and CDw199 are predicted to be transmembrane
proteins similar to G protein-coupled receptors. CDw198 is also known as C-C chemokine
receptor8(CCR8),withasuggestedroleinregulationofmonocytechemotaxisandthymiccells
apoptosis.CDw199isC-Cchemokinereceptor9(CCR9),andisknowntobeexpressedinthe
lymphoid tissue located in the large intestine and is up-regulated during dextran sulfate
sodiuminducedcolitis71.CD322isamemberoftheimmunoglobulinsuperfamily,knowntobe
expressedinepithelialcellsaswellinmonocytesandplaysaroleinleukocytetransmigration
during inflammation72. CD328or Siglec-7 is primarily found inNK cellswhere itworks as an
inhibitoryreceptor73.Futureresearchwillberequiredtoinvestigatethepossibleroleofthese
moleculesinneutrophils.
31
32
Neutrophilspresentwithauniquesignaturebasedontheirlocation
The currentneutrophil literature acknowledges thatneutrophils arehighly adaptable cells74.
Under certain pathological conditions, neutrophils are able to differentiate into different
subsets, each one with a unique phenotype and functional profile75,76. Using HTS flow
cytometricanalysisfor immunophenotyping,wenarrowedourfocusfrom147markersto90
CDmarkersonneutrophilsisolatedfrombloodandoralrinsesof10healthycontrolsand10CP
patients.The90CDmarkerswerechosenbasedonpreviousknowledgeofreportedexpression
intheliterature.Also,constitutivelyexpressedmarkerswithconstantexpressionindependent
oflocationorunderlyingdiseasewereincludedinthepanelofchosenCDmarkers.Inaddition,
markerswithmarkedlyvariedexpressionfollowingtransmigrationoutofcirculationand into
theoralcavitywerealsoselectedforfurtheranalysis.Lastly,markersreportedlynotexpressed
inneutrophilswereincludedasnegativecontrols.
With the selected 90 CD markers, an unsupervised hierarchical clustering using percent-
positivevalueswaspreformedtodetermineifcellsurfaceprofilingcouldbeusedtodefinea
subtypesignatureforneutrophils(Figure5and6).Cellsurfacephenotypingstratifiedsamples
intoclustersrelatedtotheoriginsuchthatPMN-B,PMN-Oeachformedacluster,irrespective
ofdiseasestatus(Figure5).Furtherexplorationidentifiedasite-specificneutrophilCDmarker
profilethatwasfurtheralteredinthepresenceoflocalinflammation.TheinflammatoryPMN-
Oprofilewascharacterizedby increasedexpressionofmarkersassociatedwithan inhibitory
roleof theneutrophil functionwithup-regulationofCD85a,CD305andCD312. Similar toa
report by Baudhin et al.77, expression of CD85a was almost absent in PMN-B. Conversely,
CD85awashighlyexpressedinthePMN-O,whichmaybeindicativeoftheactivationstateof
33
these cells. The PMN-B cluster also displayed an up-regulation of CD31, CD43, CD44, CD46,
CD50, CD62L, and CD162, which collectively play a role in regulating neutrophil-endothelial
interactions. In addition, CD147 and CD181were also found to be up-regulated in PMN-B,
bothofwhicharereportedtoenhanceneutrophilchemotaxisandfunction59,78.Althoughnot
statisticallysignificant,CD114,CD132andCD182alsodisplayedincreasedexpressioninPMN-B
fractionofhealthypatients.ItwaspreviouslyreportedthatCD116,CD132,CD182andCD217
arecytokinereceptorsthatallowneutrophilstorespondtoextracellularinflammatorycues79.
CD132 alongwith CD122 are functional subunits of IL-15 receptor (IL15R) 80. IL-15 is a pro-
inflammatorycytokinethatplaysaroleinenhancingneutrophilphagocytosis.Periodontitisisa
commonchronicinflammatorydiseasethatistriggeredbypathogenicmicroflorainthebiofilm
ordentalplaquethatformsaroundtotheteeth81,82.Therehasbeenarecentparadigmshiftin
our understanding of the etiology of periodontitis from a biofilm driven condition to a
conditionmediatedby amisdirected immunological response to a bacterial insult. Although
thepresenceofbacteriaisessentialfortheinitiationandprogressionofperiodontaldisease,
thesimplepresenceofbacteriadoesnot leadtotissuebreakdown;ratherneutrophilsplaya
central role in maintaining periodontal health9. Chronic periodontitis is associated with a
hyperactive neutrophil response that includes hyperactive oxidative stress and secretion of
inflammatorymediators aswell as phagocytic abnormalities 82. The ability of neutrophils to
regulatephagocytosis,byalteringtheexpressionofCD44andCD116canplayamajorrolein
balancing health and disease at the biofilm-tissue interface where a multitude of bacterial
speciesandconstantinfluxofPMN-Oconstantlyinteract.Ourdataclearlydemonstratedown-
regulationofbothmarkersinPMN-OofCPpatientswhencomparedwithhealthycontrols.
34
35
36
OralneutrophilsinCPpatientsarecharacterizedbyanactivephenotype.
TheCDmarkerprofileofPMN-OofCPpatientsischaracterizedbyanincreasedexpressionof
CD11b, CD63, CD66, CD66b, CD66c and CD66e (p-value > 0.05, CTR PMN-O vs. CP PMN-O)
(Figure7).CD11b,alsoknownasMac-1α, isan integrinαMchainand ispartofthe integrin
family that pairs with CD18 (integrin Mβ2 chain) to form the C3 complement receptor.
CD11b/CD18 is involved in chemotaxis, adhesion and transmigration of neutrophils.
Additionally, it plays a crucial role in neutrophil phagocytosis83. CD63 is a glycoprotein,
memberofthetetraspaninfamilythatuponactivationisstronglyexpressedonthesurfaceof
neutrophilsand is thereforeconsideredasamarker forgranule release27,84. Similar toother
membersof the tetraspanins family,CD63operates through interactionwith integrins,most
likelyCD11b/CD18.Inadditiontoservingasamarkerofcellactivationandmediatinggranule
release,CD63hasalsobeenreportedtoplayaroleinmediatingmembranefusionevents85.
Neutrophilsareknowntoexpressseveralglycosylatedcarcinoembryonicantigen(CEA)-related
glycoproteins(CD66antigens)86.ThesemarkersplayaroleinadhesiontoE-selectinandtheir
up-regulationisassociatedwithactivationofneutrophils,activationofβ2-integrins,primingof
therespiratoryburstandmediatingcellshapechange87.Takentogetherwespeculatethatas
partofthechronicoral inflammationprocess,PMN-OaltertheirbasalCDmarkerexpression
profile to one that is characterized by an active phenotype. These results likely reflect the
neutrophilactivationsstateinachronicinflamedenvironment.Ofparticularinterestisthelack
ofthesemarkersonoralneutrophilspresentinthetissuesofhealthymouths.Thesecellsare
certainlyrecruitedbythebacterialbiofilmpresent inthemouthsofhealthypatientsbutare
notactivatedtothesameextentastheoralneutrophilsinpatientswithperiodontaldisease.
37
This could be due to the differences in the biofilms in health and disease88 or possibly to
variationsinpatientneutrophilresponsesandsensitivitytothebiofilmspresent.
38
39
CellsortingwithanantibodyagainstCD11b,CD16andCD66ballowedfortheenrichmentof
highlypurematureneutrophilsintheblood
Density gradients used to isolate neutrophils result in neutrophil cell purity of ≥95%, with
contaminatingcellsbeingidentifiedas1-5%foreosinophils,basophils,mastcellsand1–2%for
other mononuclear cells89. Even a 5% cellular contamination can alter phenotypical and
functional assays, which is why researchers are constantly striving for methods that yield
increasedpurity. Cell surfacemarkers havebeenused for the identification and isolationof
neutrophils by FACS and immunoselection8,12,90 . Denny et al. (2010) described amethodof
using antibodies and negative selection to eliminate other leukocyte contaminants from a
density gradient purified neutrophil fraction60. However, until now, there is no specific cell-
surfacemarkersignaturethatcanbeusedto isolateneutrophils,sincemostmarkersareco-
expressed by other cell types that commonly contaminate neutrophil preparations (Table 1
summarizesknownexpressionand functionsof thesemarkers).Among theCDmarkers that
wereexpressedinallanalyzedsamples,independentofthecompartmentoractivationstate,
we found CD11a, CD11b, CD13, CD16, CD18, CD55, CD66b, CD170 and CD172 to be each
consistentlyexpressedbymorethan90%ofneutrophils(Figure8–A).Byselecting3markers
CD11b,CD16andCD66b,wewereabletodetermineauniqueneutrophilsignature(Figure8–
B).CD11bisanintegrinfamilymember,whichpairswithCD18toformtheCR3heterodimer
andisthemostcommonlyusedmarkerinneutrophilbiology.StudieshaveshownCD11btobe
directlyinvolvedincellularadhesion,howevermigrationwillonlytakeplaceifCD18subunitis
present. CD11b is known to be expressed inmany leukocytes subsets includingmonocytes,
neutrophils, natural killer cells, andmacrophages91,92. CD16 is glycosyl phosphatidyl inositol-
40
anchored(GPI)proteinthatactsasareceptorfortheFcregionofimmunoglobulingamma(Fc
gamma RIII). It was also reported that CD16 is involved in neutrophil transendothelial
migration by interacting with integrins during inflammation. It is specifically expressed by
human neutrophils and activated eosinophils90,93. CD66b is a granulocytic specific receptor,
memberofcarcinoembryonicantigenfamily.Itsmainfunctionistomediatephagocytosis27.By
combiningthethreeaforementionedmarkerswouldallowtoselectapurematureneutrophil
population,byexcludingthemaincontaminatingcells,whileselectallthecellsofinterest.We
sortedwholebloodcells,aftererythrocytelysis,andoralrinsesamples,basedonexpressionof
CD11b,CD16andCD66b.Purity andmorphologyof sorted sampleswas furtheranalyzedby
lightmicroscopic examinations of cytospins preparations (Figure 4 - C). After using these 3
markers combined,eosinophilswere theprevalent contaminating cells in theblood samples
(0.4±0.3%of cells),whileepithelial cells constitute themajorityof contaminants in theoral
samples (0.8 ± 0.2%). Monocytes and lymphocytes comprised a negligible amount of
contaminants in both populations. This clearly demonstrates that a combination of these
markers is a simple method to isolate mature neutrophils in healthy and patients with
neutrophilmediateddiseases.
41
42
Conclusions
Flow cytometry is routinely used to determine if a selected cell population is expressing a
particularproteinorreceptorofinterest.Inaddition,flowcytometryhasbeenusedtoquantify
the amount of protein expressed on the basis of intensity of fluorescence94.
Immunophenotyping leukocyte populations using multicolor flow cytometry has been
established and offers the advantage of parallel comparison of surface expression of a
moleculebetweentwoormorecellpopulations95.However,withoutacleardefinitionofcell-
surface expression profile of each cell population, interpretation of data might me
compromised.Theintroductionofhigh-throughputflowcytometrywithit’sabilityforamulti-
parameteranalysisanda rapid functionalprofileof specific cellpopulationshasprovidedus
withanalternativetocurrentmethodsandproducesresultsthatarereliableandreproducible.
We used high-throughput flow cytometry analysis of tissue and blood neutrophils to
investigate simultaneously over 370 cell-surface markers in patients with different
inflammatory conditions.We identifiedauniqueneutrophil signaturewitha combinationof
selectedmarkersincludingCD11b,CD16andCD66.Wewerealsoabletodistinguishbetween
neutrophil phenotypes during chronic inflammatory disease and health. Oral neutrophils of
chronic periodontitis patients are characterized by active neutrophil phenotype with up-
regulationofmembersoftheintegrinsfamilysuchasCD11b.Wealsonotedup-regulationof
CD63 and CD66, which are markers of neutrophil activation. Future screens may reveal
markers that possibly define subpopulations of neutrophils and opens new avenues for
researchersinthefield.
43
Authorship
F.L.designed,conductedexperiments,analyzeddata, interpretedexperimentsandwrotethe
paper. S.HandA.R.wrote thepaper.M.G.designed the study, interpretedexperimentsand
wrotethepaper.
Acknowledgments
TheauthorsgivespecialthankstoJoshuaPatersonfromtheUNHAntibodyCoreFacilityforhis
assistance with HTS-Flow Cytometry and to Dionne White from Flow Cytometry facility at
Department of Immunology, University of Toronto, Toronto,ON for her assistancewith cell
sorting.
Sources of support: This work was funded by The Canadian Institutes of Health Research
(CIHR,Ottawa,ON).
Disclosures:Theauthorsreportnoconflictsofinterestrelatedtothisstudy.
44
Chapter3
ThesisSummaryandFutureDirections
Summary
Periodontitis isacommonneutrophil-mediateddiseasethataffects thesupportingtissuesof
the teeth resulting in irreversible alveolar bone loss9,96. Patients diagnosed with chronic
periodontitishavean increase influxofneutrophils to theoral cavity56,whichallows for the
opportunity to non-invasively collect transmigrated neutrophils and study changes in the
neutrophil phenotype in the context of both inflammatory and non-inflammatory
conditions56,59.
Detailedcharacterizationsofneutrophilsprovetobecriticalforfurtherunderstandingofboth
neutrophilbiologyandalsotheimmunesystemingeneral.Inconsistenciesbetweencellyields,
viability,orcell surfacemarkerexpression,oreven incorrectly identifiedcellpopulationsare
likelytoaccountforsomeofthevariationsamongpreviousstudiesofneutrophilfunctions5-7.
Untilnow,visualinspectionunderalightmicroscopewithMay-Grünwald-Giemsa,orasimilar
stain, has been the standard method for neutrophil identification8. Neutrophils could be
recognized by the morphology of their nucleus and by a pale pink cytoplasmic color after
stainingwithneutraldyes.Theneutrophilnucleushasadistinctmultilobulatedappearance97.
Anotherdefiningcharacteristicofneutrophilsisanabundanceofcytoplasmicgranules.These
granules contain a variety of enzymes, membrane proteins and matrix proteins, and are
45
divided into three major groups: primary (azurophilic), secondary (specific), and tertiary
(gelatinase)granules98.
Although, flowcytometryhasbeencommonlyused forevaluationofneutrophils16.Aproper
identification and characterization and how to interpret flow cytometry results was still
controversial,sinceadefinitivemarkerforneutrophilsneededtobeidentified17.CD14,CD15,
CD16 and CD62L were among the commonly used markers to identifyhuman
neutrophils8,10,60,61.Withover350knownCDmarkers,theuseofreferencesandwebresources
that catalog could be cumbersome to navigate15. Furthermore, these tables often report
conflicting results about the presence of many markers, and do not differentiate between
neutrophilsandothercelltypes15
Theuseofhigh-throughputscreening(HTS)flowcytometrygaveustheopportunitytorapidly
evaluateallknownCDmarkerssimultaneously99.Inthisstudyourultimateaimwastoidentify
auniquecell-surfacesignatureinneutrophils,whichisindependentofitslocationandstateof
activation that would permit isolation of mature neutrophils. In parallel we aim to identify
changes in the neutrophil phenotype as they transmigrate from circulation into the site of
inflammation, characterizing tissue neutrophils in patients diagnosed with a chronic
inflammatory disease. We could identified CD11b, CD16, and CD66b as markers that are
always expressed on neutrophils independent of the cell location, level of activation and
disease state. Furthermore, based on the CD markers expression and using unsupervised
clusteranalysis:1)wewereable todiscriminate the locationof theneutrophils;2)wewere
alsoable todistinguishbetweenneutrophilphenotypesduringchronic inflammatorydisease
and health. Finally, we could demonstrate that oral neutrophils of chronic periodontitis
46
patientsarecharacterizedbyactiveneutrophilphenotypewithup-regulationofmembersof
theintegrinsfamilysuchasCD11bwithup-regulationofCD63andCD66,whicharemarkersof
neutrophilactivation.Futurescreensmayrevealmarkersthatpossiblydefinesubpopulations
ofneutrophilsandopensnewavenuesforresearchersinthefield.
FutureDirections
This study shows a shift in the paradigm from the current gold standard for neutrophils
identificationandcharacterizationbyvisualinspection,underalightmicroscope,totheuseof
CD markers expression in the cell surface of neutrophils. Although, neutrophils can still
berecognized by their well known nuclear morphology. It is important to recognize that
neutrophil subsetscanpresentwithanalteredmorphology,andthus,visual inspection,may
reduce our ability to correctly identify different neutrophil subsets or sub-populations. For
example,whenFridlenderandcolleaguesdescribedanewsubsetofneutrophilsinatumoral
site, each subset presented with a distinct morphological characteristic. N1 neutrophil
presentedwithahypersegmentednucleus,whereasN2hadacircularnuclearmorphology100.
Tsuda and colleagues, also described neutrophils subsets based on different morphological
features101, although, these differences might represent different stages of the neutrophil
maturation.
The use of flow cytometry for identification of neutrophils has become a common
practice8,34,90. With the advent of recently developed technology, we can now use high-
throughputflowcytometrytoidentifysimultaneouslyover350clustersofdifferentiation(CD)
markers expressed on the cell surface of neutrophils. One of themany advantages of this
47
technique is the ability to performmultiple analyses on each cell in a sample, knownmore
commonlyasmultiplexing99.Usingthisapproachwewerebeabletoidentifyspecificmarkers
in theneutrophil cell surface thatwouldallowus to later characterizeneutrophil subsets in
healthanddisease.
Future studies could investigate the role of newly identified markers such as CDw198 and
CDw199 in neutrophil biology. Little is known about its function. CDw198 is also known as
chemokine (C-C motif) receptor 8, and previous studies of this receptor demonstrated its
importanceforthemigrationofvariouscelltypesintotheinflammatorysites102.Alsoitwould
be particularly interesting to further explore the bimodal expression of CD177 that was
confirmed in the present study. CD177, which is a counter-receptor for CD31 or platelet
endothelial cell adhesion molecule-1 (PECAM-1), may play a role on neutrophil activation
during on-going inflammation103. Shift in its expression could potentially be used to identify
patientsatriskofactivationoftheneutrophilmediated-disease.
Finally, Ibelievethatexploringthedifference inregulationofkeycellsurfacemarkers inthe
neutrophils, during health and chronic inflammation will not only allow us to gain further
insightsintoinflammatorydiseasepathogenesisaswellasallowforthesesurfacemarkersto
bepotentiallybeusedasbiomarkers for specificdiseases.Hopefully, in thenear future, the
use of selected surface markers can be used as biomarkers to identify patients at risk of
developingneutrophilmediateddiseases.
48
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60
SUPPLEMENTALFILES
SupplementalTableS1-Antibodiesuseinthisstudy
# Antibody Clone Conjugation
Isotype Supplier Catalogue NCBIGeneName
NCBIOtherNames
NCBIGeneID
1 CD1a HI149 PE MouseIgG1,κ
BD 555807 CD1A CD1 909
2 CD1b M-T101 FITC MouseIgG1,κ
BD 555969 CD1B CD1 910
3 CD1c L161 PE MouseIgG1,κ
BioLegend
331506 CD1C CD1 911
4 CD1d CD1d42 PE MouseIgG1,κ
BD 550255 CD1D none 912
5 CD2 RPA-2.10
PE MouseIgG1,κ
BD 555327 CD2 SRBC;T11
914
6 CD3 HIT3a PE MouseIgG2a,к
BD 555340 CD3E seeCD3D,CD3E,CD3G
916
7 CD3e UCHT1 APC MouseIgG1
R&DSystems
FAB100A CD3E CD3-EPSILON;T3E;TCRE
916
8 CD4 RPA-T4 PE MouseIgG1,κ
BD 555347 CD4 none 920
9 CD5 UCHT2 PE MouseIgG1,κ
BD 555353 CD5 LEU1;T1
921
10 CD6 M-T605 PE MouseIgG1,κ
BD 555358 CD6 TP120 923
11 CD7 M-T701 PE MouseIgG1,κ
BD 555361 CD7 GP40;LEU-9;TP41;Tp40
924
12 CD8 HIT8a PE MouseIgG1,κ
BD 555635 CD8A CD8;Leu2;MAL;p32
925
13 CD8b 2ST8.5H7
PE MouseIgG2a,к
BD 641057 CD8B CD8B;LYT3;Leu2;Ly3
926
14 CD9 M-L13 PE MouseIgG1,κ
BD 555372 CD9 BA2;DRAP-27;MIC3;MRP-1;P24
928
15 CD10 HI10a PE MouseIgG1,κ
BD 557143 MME CALLA;CD10;
4311
61
NEP
16 CD11a HI111 PE MouseIgG1,κ
BD 555384 ITGAL CD11A;LFA-1;LFA1A
3683
17 CD11b ICRF44 PE MouseIgG1,κ
BD 555388 ITGAM CD11B;CR3A;MAC-1;MAC1A;MO1A
3684
18 CD11c B-ly6 PE MouseIgG1,κ
BD 555392 ITGAX CD11C 3687
19 CD13 WM15 PE MouseIgG1,κ
BD 555394 ANPEP CD13;LAP1;PEPN;gp150
290
20 CD14 M5E2 PE MouseIgG2a,к
BD 555398 CD14 none 929
21 CD15 HI98 PE MouseIgM,k
BD 555402 FUT4 CD15;ELFT;FCT3A;FUC-TIV
2526
22 CD16 3G8 PE MouseIgG1,κ
BD 555407 FCGR3A
CD16;FCG3;FCGR3;IGFR3
2214
23 CD16b CLB-gran11.5
PE MouseIgG2a,к
BD 550868 FCGR3B
CD16;FCG3;FCGR3
2215
24 CD17 notgiven
FITC MouseIgM
LifespanBiosciences
LS-C78376
carbohydrate
carbohydrate
N/Anotaprotein
25 CD18 6.7 PE MouseIgG1,κ
BD 555924 ITGB2 CD18;LAD;LCAMB;LFA-1;MF17;MFI7
3689
26 CD19 HIB19 PE MouseIgG1,κ
BD 555413 CD19 B4;MGC12802
930
27 CD20 2H7 PE MouseIgG2b,k
BD 555623 MS4A1
B1;Bp35;CD20;LEU-16;MGC3969;MS4A2;S7
931
28 CD21 B-ly4 PE MouseIgG1,κ
BD 555422 CR2 C3DR;CD21
1380
29 CD22 S-HCL-1 PE Mouse BD 347577 CD22 SIGLEC- 933
62
IgG2b,k 2
30 CD23 M-L233 PE MouseIgG1,κ
BD 555711 FCER2 CD23;CD23A;FCE2;IGEBF
2208
31 CD24 ML5 PE MouseIgG2a,к
BD 555428 CD24 CD24A 100133941
32 CD25 M-A251 PE MouseIgG1,κ
BD 557138 IL2RA CD25;IL2R;TCGFR
3559
33 CD26 M-A261 PE MouseIgG1,κ
BD 555437 DPP4 ADABP;ADCP2;CD26;DPPIV;TP103
1803
34 CD27 M-T271 PE MouseIgG1,κ
BD 555441 CD27 CD27;MGC20393;S152;T14;Tp55
939
35 CD28 CD28.2 PE MouseIgG1,κ
BD 555729 CD28 Tp44 940
36 CD29 MAR4 PE MouseIgG1,κ
BD 555443 ITGB1 CD29;FNRB;GPIIA;MDF2;MSK12;VLAB
3688
37 CD30 BerH8 PE MouseIgG1,κ
BD 550041 TNFRSF8
CD30;D1S166E;KI-1
943
38 CD31 WM59 PE MouseIgG1,κ
BD 555446 PECAM1
CD31 5175
39 CD32 3D3 PE MouseIgG1,κ
BD 552884 FCGR2A
CD32;CDw32;FCG2;FCGR2;FCGR2A1;FcGR;IGFR2;MGC23887;MGC30032
2212
40 CD33 P67.6 PE MouseIgG1,κ
BD 347787 CD33 SIGLEC-3;p67
945
41 CD34 581 PE MouseIgG1,κ
BD 555822 CD34 none 947
42 CD35 E11 PE MouseIgG1,κ
BD 559872 CR1 C3BR;CD35
1378
43 CD36 CB38 PE Mouse BD 555455 CD36 FAT; 948
63
(NL07) IgM,k GP3B;GP4;GPIV;PASIV;SCARB3
44 CD37 M-B371 FITC MouseIgG1,κ
BD 555457 CD37 GP52-40
951
45 CD38 HIT2 PE MouseIgG1,κ
BD 555460 CD38 T10 952
46 CD39 TÜ66 APC MouseIgG2b,k
BD 560239 ENTPD1
ATPDase;CD39;NTPDase-1
953
47 CD40 5C3 PE MouseIgG1,κ
BD 555589 CD40 p50;Bp50;CDW40;MGC9013;TNFRSF5
958
48 CD41a HIP8 PE MouseIgG1,κ
BD 555467 ITGB3 3674
49 CD41b HIP2 FITC MouseIgG3,к
BD 555469 ITGA2B
CD41;CD41B;GP2B;GPIIb;GTA
3690
50 CD42a ALMA.16
PE MouseIgG1,κ
BD 558819 GP9 CD42a 2815
51 CD42b HIP1 PE MouseIgG1,κ
BD 555473 GP1BA BSS;CD42B;CD42b-alpha;GP1B;MGC34595
2811
52 CD43 1G10 PE MouseIgG1,κ
BD 560199 SPN CD43;GPL115;LSN
6693
53 CD44 G44-26 FITC MouseIgG2b,k
BD 555478 CD44 CDW44;ECMR-III;IN;INLU;LHR;MC56;MDU2;MDU3;MGC10468;MIC4;MUTCH-I;Pgp1
960
64
54 CD45 HI30 PE notgiven
BD 555483 PTPRC B220;CD45;GP180;LCA;LY5;T200
5788
55 CD45RA HI100 PE MouseIgG2b,k
BD 555489 PTPRC 5788
56 CD45RB MT4 PE MouseIgG1,κ
BD 555904 PTPRC 5788
57 CD45RO UCHL1 PE MouseIgG2a,к
BD 555493 PTPRC 5788
58 CD46 E4.3 FITC MouseIgG2a,к
BD 555949 CD46 CD46;MGC26544;MIC10;TLX;TRA2.10
4179
59 CD47 B6H12 PE MouseIgG1,κ
BD 556046 CD47 IAP;MER6;OA3
961
60 CD48 TÜ145 PE MouseIgM,k
BD 552855 CD48 BCM1;BLAST;BLAST1;MEM-102;SLAMF2;hCD48;mCD48
962
61 CD49a SR84 PE MouseIgG1,κ
BD 559596 ITGA1 CD49a;VLA1
3672
62 CD49b 12F1 PE MouseIgG2a,к
BD 555669 ITGA2 BR;CD49B;VLAA2
3673
63 CD49c C3II.1 PE MouseIgG1,κ
BD 556025 ITGA3 CD49C;GAP-B3;GAPB3;MSK18;VCA-2;VL3A;VLA3a
3675
64 CD49d 9F10 PE MouseIgG1,κ
BD 555503 ITGA4 CD49D 3676
65 CD49e IIA1 PE MouseIgG1,κ
BD 555617 ITGA5 CD49e;FNRA;VLA5A
3678
66 CD49f GoH3 PE RatIgG2a,к
BD 555736 ITGA6 CD49f 3655
67 CD50 TÜ41 FITC MouseIgG2b,k
BD 555958 ICAM3 CD50;CDW50;ICAM-R
3385
68 CD51/CD 23C6 PE Mouse BD 550037 ITGAV CD51; 3685
65
61 IgG1,κ MSK8;VNRA
69 CD52 HI186 PE MouseIgG2b,k
BioLegend
316006 CD52 CD52 1043
70 CD53 HI29 PE MouseIgG1,κ
BD 555508 CD53 MOX44 963
71 CD54 HA58 PE MouseIgG1,κ
BD 555511 ICAM1 BB2;CD54
3383
72 CD55 IA10 PE MouseIgG2a,к
BD 555694 CD55 CD55;CR;TC
1604
73 CD56 B159 PE MouseIgG1,κ
BD 555516 NCAM1
CD56;MSK39;NCAM
4684
74 CD57 HNK-1 FITC MouseIgM,k
BD 347393 B3GAT1
HNK-1;LEU7;NK-1
27087
75 CD58 1C3 PE MouseIgG2a,к
BD 555921 CD58 LFA3 965
76 CD59 p282(H19)
PE MouseIgG2a,к
BD 555764 CD59 MGC2354;MIC11;MIN1;MIN2;MIN3;MSK21;PROTECTIN
966
77 CD60b NA FITC MouseIgM
LifespanBiosciences
LS-C78711
carbohydrate
carbohydrate
N/Anotaprotein
78 CD61 VI-PL2 PE MouseIgG1,κ
BD 555754 ITGB3 CD61;GP3A;GPIIIa
3690
79 CD62E 68-5H11 PE MouseIgG1,κ
BD 551145 SELE CD62E;ELAM;ELAM1;ESEL;LECAM2
6401
80 CD62L Sk11 PE MouseIgG2a,к
BD 341012 SELL CD62L;LAM-1;LAM1;LECAM1;LNHR;LSEL;LYAM1;Leu-8;Lyam-1;PLNHR;TQ1;hLHRc
6402
81 CD62P AK-1 PE Mouse BD 555524 SELP CD62; 6403
66
IgG1,κ CD62P;GMP140;GRMP;PADGEM;PSEL
82 CD63 H5C6 PE MouseIgG1,κ
BD 556020 CD63 LAMP-3;ME491;MLA1;OMA81H
967
83 CD64 10.1 PE MouseIgG1,κ
BD 558592 FCGR1A
CD64;FCRI;IGFR1
2209
84 CD65 88H7 FITC MouseIgM
BeckmanCoulter
IM1654U carbohydrate
carbohydrate
N/Anotaprotein
85 CD65s VIM-2 FITC MouseIgM
Abcam ab74080 carbohydrate
carbohydrate
N/Anotaprotein
86 CD66 B1.1/CD66
PE MouseIgG2a,к
BD 551480 CEACAM1
BGP;BGP1;BGPI;CD66;CD66A
109770
87 CD66b G10F5 FITC MouseIgM,k
BD 555724 CEACAM8
CD66b;CD67;CGM6;NCA-95
1088
88 CD66c B6.2/CD66c
PE MouseIgG1,κ
BD 551478 CEACAM6
CD66c;CEAL;NCA
4680
89 CD66d CLB-gran/10,IH4Fc
PE MouseIgG1
Abcam ab51598 CEACAM3
CD66D;CGM1
1084
90 CD66e 487618 PE MouseIgG1
R&DSystems
FAB4128P
CEACAM5
CD66e;CEA
1048
91 CD68 Y1/82A PE MouseIgG2b,k
BD 556078 CD68 SCARD1 968
92 CD69 FN50 PE MouseIgG1,κ
BD 555531 CD69 none 969
93 CD70 Ki-24 PE MouseIgG3,к
BD 555835 CD70 CD27L;CD27LG;CD70
970
94 CD71 M-A712 PE MouseIgG2a,к
BD 555537 TFRC CD71;TFR;TRFR
7037
95 CD72 J4-118 FITC MouseIgG2b,k
BD 555918 CD72 LYB2 971
96 CD73 AD2 PE MouseIgG1,κ
BD 550257 NT5E CD73;E5NT;
4907
67
NT5;NTE;eN;eNT
97 CD74 M-B741 FITC MouseIgG2a,к
BD 555540 CD74 DHLAG;HLADG;Ia-GAMMA
972
98 CD75 LN1 FITC MouseIgM,k
BD 555654 carbohydrate
carbohydrate
NA
99 CD77 5B5 FITC MouseIgM,k
BD 551353 carbohydrate
carbohydrate
NA
100 CD79a HM47 PE MouseIgG1,κ
BD 555935 CD79A IGA;MB-1
973
101 CD79b CB3-1 PE MouseIgG1,κ
BD 555679 CD79B B29;IGB
974
102 CD80 L307.4 PE MouseIgG1,κ
BD 557227 CD80 CD28LG;CD28LG1;LAB7
941
103 CD81 JS-81 PE MouseIgG1,κ
BD 555676 CD81 S5.7;TAPA1
975
104 CD82 ASL-24 PE MouseIgG2a,к
BioLegend
342104 CD82 4F9;C33;CD82;GR15;IA4;R2;SAR2;ST6
3732
105 CD83 HB15e PE MouseIgG1,κ
BD 556855 CD83 BL11;HB15
9308
106 CD84 CD84.1.21
PE MouseIgG1
Biolegend
326008 CD84 LY9B;SLAMF5;hCD84;mCD84
8832
107 CD85A MKT5.1 PE MouseIgG1
Biolegend
337704 LILRB3 CD85A;HL9;ILT5;LIR-3;LIR3
11025
108 CD85D 42D1 PE RatIgG2a
Biolegend
338706 LILRB2 CD85D;ILT4;LIR-2;LIR2;MIR-10;MIR10
10288
109 CD85G 17G10.2 PE MouseIgG1
BioLegend
326408 LILRA4 ILT7;CD85g;MGC129597
23547
110 CD85H 24 PE RatIgG2a,к
Biolegend
337904 LILRA2 CD85H;ILT1;
11027
68
LIR-7;LIR7
111 CD85J GHI/75 PE MouseIgG2b,k
BD 551053 LILRB1 CD85;CD85J;ILT2;LIR-1;LIR1;MIR-7;MIR7
10859
112 CD86 2331(FUN-1)
PE MouseIgG1,κ
BD 555658 CD86 B7-2;B70;CD28LG2;LAB72;MGC34413
942
113 CD87 VIM5 PE MouseIgG1,κ
BD 555768 PLAUR CD87;UPAR;URKR
5329
114 CD88 C85-4124
PE RabbitIgG
BD 552993 C5AR1 C5A;C5AR;CD88
728
115 CD89 A59 PE MouseIgG1,κ
BD 555686 FCAR CD89 2204
116 CD90 5E10 PE MouseIgG1,κ
BD 555596 THY1 CD90 7070
117 CD91 A2MR-α2
PE MouseIgG1,κ
BD 550497 LRP1 A2MR;APOER;APR;CD91;LRP
4035
118 CD92 VIM-15b PE MouseIgG2b
Abcam ab66228 SLC44A1
CTL1;CDW92;CHTL1;RP11-287A8.1
23446
119 CDw93 R139 FITC MouseIgG2b,k
BD 551531 CD93 C1QR1;C1qRP;CDw93;MXRA4;C1qR(P);dJ737E23.1
22918
120 CD94 HP-3D9 PE MouseIgG1,κ
BD 555889 KLRD1 CD94 3824
121 CD95 DX2 PE MouseIgG1,κ
e-Bioscience
12-0959-73
FAS APT1;CD95;FAS1;APO-1;FASTM;ALPS1A;TNFRSF6
355
69
122 CD96 NK92.39 PE MouseIgG1
Biolegend
338406 CD96 MGC22596;TACTILE
10225
123 CD97 VIM3b PE MouseIgG1,κ
BD 555774 CD97 TM7LN1 976
124 CD98 UM7F8 PE MouseIgG1,κ
BD 556077 SLC3A2
4F2;4F2HC;4T2HC;CD98;MDU1;NACAE
6520
125 CD99 TÜ12 PE MouseIgG2a,к
BD 555689 CD99 MIC2;MIC2X;MIC2Y
4267
126 CD100 133-1C6 FITC MouseIgM
e-Bioscience
53-1009 SEMA4D
CD100;M-semaG;M-sema-G;SEMAJ;coll-4
10507
127 CD101 BB27 PE MouseIgG1
e-Bioscience
12-1019-73
IGSF2 CD101;V7
9398
128 CD102 CBR-1C2/2.1
PE MouseIgG2a,к
BD 558080 ICAM2 CD102 3384
129 CD103 Ber-ACT8
PE MouseIgG1,κ
BD 550260 ITGAE CD103;HUMINAE
3682
130 CD104 439-9B PE RatIgG2b,к
BD 555720 ITGB4 none 3691
131 CD105 SN6 PE MouseIgG1
e-Bioscience
12-1057-42
ENG CD105;END;HHT1;ORW;ORW1
2022
132 CD106 51-10C9 PE MouseIgG1,κ
BD 555647 VCAM1
INCAM-100
7412
133 CD107a H4A3 PE MouseIgG1,κ
BD 555801 LAMP1
CD107a;LAMPA;LGP120
3916
134 CD107b H4B4 FITC MouseIgG1,κ
BD 555804 LAMP2
CD107b;LAMPB
3920
135 CD108 KS-2 PE MouseIgG2a,к
BD 552830 SEMA7A
CD108;CDw108;H-SEMA-K1;H-SemaK1;H-Sema-L;SEMAK1;SEMAL
8482
136 CD109 TEA PE Mouse BD 556040 CD109 DKFZp76 135228
70
2/16 IgG1,κ 2L1111;FLJ38569
137 CD110 1.6.1 PE MouseIgG2b,к
BD 562159 MPL C-MPL;CD110;MPLV;TPOR
4352
138 CD111 R1.302 PE MouseIgG1,κ
BioLegend
340404 PVRL1 CD111;CLPED1;ED4;HIgR;HVEC;PRR;PRR1;PVRR;PVRR1;SK-12
5818
139 CD112 R2.525 PE MouseIgG1,κ
BD 551057 PVRL2 CD112;HVEB;PRR2;PVRR2
5819
140 CD114 LMM741
PE MouseIgG1,κ
BD 554538 CSF3R CD114;GCSFR
1441
141 CD115 notgiven
PE RatIgG1 R&DSystems
FAB329P CSF1R C-FMS;CD115;CSFR;FIM2;FMS
1436
142 CD116 hGMCSFR-M1
PE MouseIgG1,κ
BD 551373 CSF2RA
CD116;CDw116;CSF2R;CSF2RAX;CSF2RAY;CSF2RX;CSF2RY;GM-CSF-R-alpha;GMCSFR;GMR;MGC3848;MGC4838
1438
143 CD117 YB5.B8 PE MouseIgG1,κ
BD 555714 KIT CD117;PBT;SCFR
3815
144 CD118 32953 PE MouseIgG1
R&DSystems
FAB249P LIFR LIFR;SWS;SJS2;STWS
3977
145 CD119 GIR-208 PE Mouse BD 558934 IFNGR CD119; 3459
71
IgG1,κ 1 IFNGR
146 CD120a 16803 PE MouseIgG1
R&DSystems
FAB225P TNFRSF1A
CD120a;FPF;MGC19588;TBP1;TNF-R;TNF-R-I;TNF-R55;TNFAR;TNFR1;TNFR55;TNFR60;p55;p55-R;p60
7132
147 CD120b hTNFR-M1
PE RatIgG2b,к
BD 552418 TNFRSF1B
CD120b;TBPII;TNF-R-II;TNF-R75;TNFBR;TNFR2;TNFR80;p75;p75TNFR
7133
148 CD121b 34141 PE MouseIgG1
R&DSystems
FAB663P IL1R2 IL1RB;MGC47725
7850
149 CD122 Mik-β3 PE MouseIgG1,κ
BD 554525 IL2RB P70-75 3560
150 CD123 7G3 PE MouseIgG2a,к
BD 554529 IL3RA CD123;IL3R;IL3RAY;IL3RX;IL3RY;MGC34174;hIL-3Ra
3563
151 CD124 hIL4R-M57
PE MouseIgG1,κ
BD 552178 IL4R CD124;IL4RA
3566
152 CD125 26815 PE MouseIgG1
R&DSystems
FAB253P IL5RA CDw125;HSIL5R3;IL5R;MGC26560
3568
153 CD126 M5 PE MouseIgG1,κ
BD 551850 IL6R CD126;IL-6R-1;IL-6R-alpha;IL6RA
3570
72
154 CD127 hIL-7R-M21
PE MouseIgG1,κ
BD 557938 IL7R CD127;CDW127;IL-7R-alpha
3575
155 CD129 AH9R7 PE MouseIgG2b,k
BioLegend
310404 IL9R none 3581
156 CD130 AM64 PE MouseIgG1,κ
BD 555757 IL6ST CD130;CDw130;GP130;GP130-RAPS;IL6R-beta
3572
157 CD131 1C1 PE MouseIgG1,κ
e-Bioscience
12-1319-73
CSF2RB
CD131;CDw131;IL3RB;IL5RB
1439
158 CD132 AG184 PE MouseIgG1,κ
BD 555900 IL2RG CD132;IMD4;SCIDX;SCIDX1
3561
159 CD133 AC133 APC MouseIgG1,κ
Miltenyi
130-090-826
PROM1
AC133;CD133;PROML1
8842
160 CD134 ACT35 PE MouseIgG1,κ
BD 555838 TNFRSF4
ACT35;CD134;OX40;TXGP1L
7293
161 CD135 4G8 PE MouseIgG1,κ
BD 558996 FLT3 CD135;FLK2;STK1
2322
162 CD136 ID1 PE notgiven
BeckmanCoulter
41116015
MST1R
CDw136;RON
4486
163 CD137 4B4-1 PE MouseIgG1,κ
BD 555956 TNFRSF9
4-1BB;CD137;CDw137;ILA;MGC2172
3604
164 CD137L 5F4 PE MouseIgG1,κ
BioLegend
311504 TNFSF9
8744
165 CD138 MI15 PE MouseIgG1,κ
BD 552026 SDC1 CD138;SDC;SYND1
6382
166 CD140a αR1 PE MouseIgG2a,к
BD 556002 PDGFRA
CD140A;PDGFR2
5156
167 CD140b 28D4 PE MouseIgG2a,к
BD 558821 PDGFRB
CD140B;JTK12;PDGF-R-beta;
5159
73
PDGFR;PDGFR1
168 CD141 1A4 PE MouseIgG1,κ
BD 559781 THBD CD141;THRM;TM
7056
169 CD142 HTF-1 PE MouseIgG1,κ
BD 550312 F3 CD142;TF;TFA
2152
170 CD143 171417 PE MouseIgG1
R&DSystems
FAB929P ACE ACE1;CD143;DCP;DCP1;MGC26566
1636
171 CD144 55-7H1 PE MouseIgG1,κ
BD 560410 CDH5 7B4 1003
172 CD146 P1H12 PE MouseIgG1,κ
BD 550315 MCAM
CD146;MUC18
4162
173 CD147 HIM6 FITC MouseIgG1,κ
BD 555962 BSG 5F7;CD147;EMMPRIN;M6;OK;TCSF
682
174 CD148 143-41 PE MouseIgG1
R&DSystems
FAB1934P
PTPRJ CD148;DEP1;HPTPeta;R-PTP-ETA;SCC1
5795
175 CD150 A12 PE MouseIgG1,κ
BD 559592 SLAMF1
CD150;CDw150;SLAM
6504
176 CD151 14A2.H1 PE MouseIgG1,κ
BD 556057 CD151 GP27;PETA-3;SFA1
977
177 CD152 BNI3 PE MouseIgG2a,к
BD 555853 CTLA4 CD152 1493
178 CD153 116614 PE MouseIgG2B
R&DSystems
FAB1028P
TNFSF8
CD153;CD30L;CD30LG
944
179 CD154 TRAP1 PE MouseIgG1,κ
BD 555700 CD40LG
CD154;CD40L;CD40LG;HIGM1;IGM;IMD3;T-BAM;TRAP;gp39;hCD40L
959
180 CD155 2H7CD155
PE MouseIgG1
e-Bioscie
12-1550-71
PVR CD155;HVED;
5817
74
nce NECL5;PVS;TAGE4
181 CD156b 111633 PE MouseIgG1
R&DSystems
FAB9301P
ADAM17
CD156b;TACE;cSVP
6868
182 CD157 RF3 PE MouseIgG1,κ
MBL D036-5 BST1 CD157 683
183 CD158A HP-3E4 PE MouseIgM,κ
BD 556063 KIR2DL1
47.11;CD158A;CL-42;NKAT1;p58.1
3802
184 CD158B1 CH-L PE MouseIgG2b,k
BD 559785 KIR2DL2
CD158B1;CL-43;NKAT6;p58.2
3803
185 CD158B2 DX27 PE MouseIgG2a,к
BD 556071 KIR2DL3
CD158B2;CD158b;CL-6;KIR-023GB;NKAT2;NKAT2A;NKAT2B;p58
3804
186 CD158D 181703 PE MouseIgG2a
R&DSystems
FAB2238P
KIR2DL4
103AS;15.212;CD158D;KIR103;KIR103AS
3805
187 CD158E2 DX9 PE MouseIgG1,κ
BD 555967 KIR3DS1
AMB11;CD158E1;CD158E1/2;CD158E2;CL-11;CL-2;KIR;KIR3DS1;NKAT10;NKAT3;NKB1;NKB1B
3813
188 CD158F UP-R1 PE MouseIgG1
BioLegend
341304 KIR2DL5A
CD158F;KIR2DL5;KIR2DL5.1;KIR2DL5.3
57292
75
189 CD158I JJC11.6 PE MouseIgG1
Miltenyi
130-092-680
KIR2DS4
CD158I;KIR1D;KKA3;NKAT8;PAX;cl-39
3809
190 CD159a 131411 PE MouseIgG2a
R&DSystems
FAB1059P
KLRC1 CD159A;MGC13374;MGC59791;NKG2;NKG2A
3821
191 CD159c 134591 PE MouseIgG1
R&DSystems
FAB138P KLRC2 3822
192 CD160 BY55 APC MouseIgM,κ
BioLegend
341204 CD160 BY55;NK1;NK28
11126
193 CD161 DX12 PE MouseIgG1,κ
BD 556081 KLRB1 CD161;NKR;NKR-P1;NKR-P1A;NKRP1A;hNKR-P1A
3820
194 CD162 KPL-1 PE MouseIgG1,κ
BD 556055 SELPLG
CD162;PSGL-1;PSGL1
6404
195 CD163 GHI/61 PE MouseIgG1,κ
BD 556018 CD163 M130;MM130
9332
196 CD164 N6B6 PE MouseIgG2a,к
BD 551298 CD164 MGC-24;MUC-24;endolyn
8763
197 CD165 SN2N56-D11
PE MouseIgG1
e-Bioscience
12-1659-73
CD165 none 23449
198 CD166 3A6 PE MouseIgG1,κ
BD 559263 ALCAM
CD166;MEMD
214
199 CD167a 51D6 PE MouseIgM,k
BioLegend
334006 DDR1 CAK;CD167;DDR;EDDR1;MCK10;NEP;NTRK4;PTK3;PTK3A;RTK6;TRKE
780
200 CD169 7-239 PE Mouse Biolege 346004 SIGLEC CD169; 6614
76
IgG1 nd 1 FLJ00051;FLJ00055;FLJ00073;FLJ32150;SIGLEC-1;dJ1009E24.1
201 CD170 194128 PE MouseIgG1
R&DSystems
FAB10721P
SIGLEC5
CD33L2;OB-BP2;OBBP2;SIGLEC-5
8778
202 CD171 5G3 PE MouseIgG2a
e-Bioscience
12-1719-73
L1CAM
CAML1;CD171;HSAS;HSAS1;MASA;MIC5;N-CAML1;S10;SPG1
3897
203 CD172a SE5A5 PE MouseIgG1,κ
BioLegend
323806 SIRPA BIT;MFR;MYD-1;P84;SHPS-1;SHPS1;SIRP;SIRP-ALPHA-1;SIRPalpha;SIRPalpha2
140885
204 CD172b B4B6 PE MouseIgG1,κ
BD 552602 SIRPB1
SIRP-BETA-1
10326
205 CD172g LSB2.20 PE MouseIgG1,κ
BioLegend
336606 SIRPG SIRP-B2;bA77C3.1
55423
206 CD175s STn219 FITC MouseIgG1
Abcam ab76756 carbohydrate
carbohydrate
N/Anotaprotein
207 CD177 MEM-166
PE MouseIgG1
Abcam ab69777 CD177 CD177;HNA2A;NB1
57126
208 CD178 NOK-1 PE MouseIgG1,κ
BioLegend
306407 FASLG FASL;CD178;
356
77
CD95L;TNFSF6;APT1LG1
209 CD179a HSL96 PE MouseIgG1,κ
BioLegend
347404 VPREB1
IGI;IGVPB;VPREB
7441
210 CD180 G28-8 PE MouseIgG1,κ
BD 551953 CD180 LY64;Ly78;RP105;MGC126233;MGC126234
4064
211 CD181 5A12 PE MouseIgG2b,k
BD 555940 IL8RA C-CCKR-1;C-C-CKR-1;CD128;CDw128a;CMKAR1;CXCR1;IL8R1;IL8RBA
3577
212 CD182 6C6 PE MouseIgG1,κ
BD 555933 IL8RB CDw128b;CMKAR2;CXCR2;IL8R2;IL8RA
3579
213 CD183 1C6/CXCR3
PE MouseIgG1,κ
BD 557185 CXCR3 CD183;CKR-L2;CMKAR3;GPR9;IP10;IP10-R;Mig-R;MigR
2833
214 CD184 12G5 PE MouseIgG2a,к
BD 555974 CXCR4 D2S201E;HM89;HSY3RR;LAP3;LESTR;NPY3R;NPYR;NPYY3R;WHIM
7852
215 CD185 RF8B2 FITC MouseIgG2b,k
BD 558112 BLR1 BLR1;CXCR5;MDR15
643
216 CD186 TG3/CXCR6
APC MouseIgG2b,k
BioLegend
335101 CXCR6 CXCR6;BONZO;STRL33;
10663
78
TYMSTR
217 CD191 53504 PE MouseIgG2b
R&DSystems
FAB145P CCR1 CKR-1;CMKBR1;HM145;MIP1aR;SCYAR1
1230
218 CD192 48607 APC MouseIgG2b,k
BD 558406 CCR2 CC-CKR-2;CCR2A;CCR2B;CKR2;CKR2A;CKR2B;CMKBR2;MCP-1-R
729230
219 CD193 5E8 PE MouseIgG2b,k
BD 558165 CCR3 CC-CKR-3;CKR3;CMKBR3
1232
220 CD194 TG6/CCR4
APC MouseIgG2b,k
BioLegend
335401 CCR4 CC-CKR-4;CKR4;CMKBR4;ChemR13;HGCN
1233
221 CD195 3A9 PE MouseIgG2a,к
BD 556042 CCR5 CC-CKR-5;CCCKR5;CD195;CKR-5;CKR5;CMKBR5
1234
222 CD196 11A9 PE MouseIgG1,κ
BD 559562 CCR6 CCR6;BN-1;CKR6;DCR2;CKRL3;DRY-6;GPR29;CKR-L3;CMKBR6;GPRCY4;STRL22;GPR-CY4
1235
223 CD197 3D12 PE RatIgG2a,к
BD 552176 CCR7 BLR2;CDw197;CMKBR7;EBI1
1236
79
224 CDw198 191704 PE RatIgG2b
R&DSystems
FAB1429P
CCR8 CKR-L1;CKRL1;CMKBR8;CMKBRL2;CY6;GPR-CY6;TER1
1237
225 CDw199 112509 APC MouseIgG2a,к
BD 557975 CCR9 GPR-9-6;GPR28
10803
226 CD200 MRCOX-104
PE MouseIgG1,κ
BD 552475 CD200 MOX1;MOX2;MRC;OX-2
4345
227 CD201 RCR-252 PE RatIgG1,k
BD 557950 PROCR CCCA;CCD41;EPCR;MGC23024;bA42O4.2
10544
228 CD202b 33.1(Ab33)
PE MouseIgG1,κ
BioLegend
334206 TEK CD202B;TIE-2;TIE2;VMCM;VMCM1
7010
229 CD203c NP4D6 PE MouseIgG1,κ
BioLegend
324606 ENPP3 B10;CD203c;NPP3;PD-IBETA;PDNP3
5169
230 CD204 351615 PE MouseIgG2B
R&DSystems
FAB2708P
MSR1 SCARA1;SR-A;phSR1;phSR2
4481
231 CD205 MG38 PE MouseIgG2b,k
BD 558069 LY75 CLEC13B;DEC-205;GP200-MR6
4065
232 CD206 19.2 PE MouseIgG1,κ
BD 555954 MRC1 CLEC13D
4360
233 CD207 343828 APC MouseIgG1
R&DSystems
FAB2088A
CD207 LANGERIN
50489
234 CD208 I10-1112
PE MouseIgG1,κ
BD 558126 LAMP3
DC-LAMP;DCLAMP;LAMP;TSC403
27074
80
235 CD209 DCN46 PE MouseIgG2b,k
BD 551265 CD209 CDSIGN;DC-SIGN;DC-SIGN1
30835
236 CDw210 3F9 PE RatIgG2a,к
BD 556013 IL10RA IL10R;CDW210A;HIL-10R;IL-10R1;IL10RA
3587
237 CD212 2.4e6 PE MouseIgG1,κ
BD 556065 IL12RB1
IL-12R-BETA1;IL12RB;MGC34454
3594
238 CD213a2 B-D13 PE MouseIgG1
Abcam ab27415 IL13RA2
IL-13R;IL13BP
3598
239 CD215 151303 PE MouseIgG2B
R&DSystems
FAB1471P
IL15RA IL15RA 3601
240 CD217 BG/hIL17AR
APC MouseIgG1
bioLegend
340903 IL17RA IL-17RA;IL17RA;MGC10262;hIL-17R
23765
241 CDw218a H44 PE MouseIgG1,κ
e-Bioscience
12-7183-73
IL18R1 IL18R1;IL1RRP;IL-1Rrp
8809
242 CD218b 132029 PE MouseIgG2b
R&DSystems
FAB118P IL18RAP
IL18RAP;ACPL
8807
243 CD220 notgiven
APC GoatIgG
R&DSystems
FAB1544A
INSR none 3643
244 CD221 1H7 PE MouseIgG1,κ
BD 555999 IGF1R JTK13 3480
245 CD222 MEM-238
FITC MouseIgG1,κ
BioLegend
315904 IGF2R CD222;CIMPR;M6P-R;MPRI
3482
246 CD223 notgiven
PE GoatIgG
R&DSystems
FAB2319P
LAG3 CD223 3902
247 CD226 DX11 PE MouseIgG1,κ
BD 559789 CD226 DNAM-1;DNAM1;PTA1;TLiSA1
10666
248 CD227 HMPV FITC MouseIgG1,κ
BD 559774 MUC1 CD227;EMA;
4582
81
PEM;PUM
249 CD229 249936 PE MouseIgG2a
R&DSystems
FAB1898P
LY9 CD229;SLAMF3;hly9;mLY9
4063
250 CD230 4D5 PE MouseIgG1,κ
e-Bioscience
12-9230-73
PRNP ASCR;CJD;GSS;MGC26679;PRIP;PrP;PrP27-30;PrP33-35C;PrPc
5621
251 CD231 SN1a(M3-3D9)
PE MouseIgG1,κ
BioLegend
329406 TSPAN7
A15;CCG-B7;CD231;DXS1692E;MXS1;TALLA-1;TM4SF2b
7102
252 CD234 358307 PE MouseIgG2A
R&DSystems
FAB4139P
DARC CCBP1;DARC;GPD
2532
253 CD235a GA-R2(HIR2)
PE MouseIgG2b,k
BD 555570 GYPA GPA;MN;MNS
2993
254 CD243(BC)
UIC2 PE MouseIgG2a
BeckmanCoulter
IM2370U ABCB1 ABC20;CD243;CLCS;GP170;MDR1;P-gp;PGY1
5243
255 CD243(BD)
17F9 PE MouseIgG2b,k
BD 557003 ABCB1 ABC20;CD243;CLCS;GP170;MDR1;P-gp;PGY1
5243
256 CD244 2-69 PE MouseIgG2a,к
BD 550816 CD244 2B4;NAIL;NKR2B4;Nmrk;SLAMF4
51744
257 CD245 DY12 PE MouseIgG1,κ
BioLegend
334404 NPAT notlisted
4863
258 CD247 G3 PE Mouse AbD MCA129 CD247 CD3- 919
82
IgG2a Serotech
7PE ZETA;CD3H;CD3Q;TCRZ
259 CD249 notgiven
PE RatIgG1,k
LifespanBioscience
LS-C12169
ENPEP APA;gp160;EAP
2028
260 CD252 Ik-1 PE MouseIgG1,κ
BD 558164 TNFSF4
TNFSF4;GP34;OX4OL;TXGP1;CD134L;OX-40L;OX40L
7292
261 CD253 RIK-2 PE MouseIgG1
BD 550516 TNFSF10
TNFSF10;TL2;APO2L;TRAIL;Apo-2L
8743
262 CD254 MIH24 PE MouseIgG2b,k
bioLegend
347504 TNFSF11
ODF;OPGL;sOdf;CD254;OPTB2;RANKL;TRANCE;hRANKL2
8600
263 CD255 CARL-1 PE MouseIgG3
BD 552831 TNFSF12
APO3L;DR3LG;TWEAK
8742
264 CD256 T3-6 PE MouseIgG2a,к
BioLegend
318506 TNFSF13
APRIL;TALL2;TRDL-1;UNQ383/PRO715
8741
265 CD257 T7-241 PE MouseIgG1,κ
BioLegend
318606 TNFSF13B
BAFF;BLYS;TALL-1;TALL1;THANK;TNFSF20;ZTNF4;deltaBAFF
10673
266 CD258 115520 PE MouseIgG1
R&DSystems
FAB664P TNFSF14
TNFSF14;LTg;TR2;HVEML;LIGHT
8740
267 CD261 DJR1 PE Mouse BioLege 307206 TNFRS APO2; 8797
83
IgG1 nd F10A DR4;MGC9365;TRAILR-1;TRAILR1
268 CD262 71908 PE MouseIgG2b
R&DSystems
FAB6311P
TNFRSF10B
DR5;KILLER;KILLER/DR5;TRAIL-R2;TRAILR2;TRICK2;TRICK2A;TRICK2B;TRICKB;ZTNFR9
8795
269 CD263 90906 PE MouseIgG1
R&DSystems
FAB6302P
TNFRSF10C
DCR1;LIT;TRAILR3;TRID
8794
270 CD264 104918 PE MouseIgG1
R&DSystems
FAB633P TNFRSF10D
DCR2;TRAILR4;TRUNDD
8793
271 CD267 1A1-K21-M22
PE RatIgG2a,к
BD 558414 TNFRSF13B
CVID;TACI;CD267;FLJ39942;MGC39952;MGC133214;TNFRSF14B
23495
272 CD268 11C1 PE MouseIgG1,κ
BD 558097 TNFRSF13C
BAFFR;CD268;BAFF-R;MGC138235
115650
273 CD269 notgiven
PE GoatIgG
R&DSystems
FAB193P TNFRSF17
BCM;BCMA
608
274 CD270 122 PE notgiven
BioLegend
318806 TNFRSF14
TR2;ATAR;HVEA;HVEM;LIGHTR;TNFRSF14
8764
84
275 CD271 C40-1457
PE MouseIgG1,κ
BD 557196 NGFR NGFR;TNFRSF16;p75(NTR)
4804
276 CD272 J168-540.90.22
PE MouseIgG1,κ
BD 558485 BTLA BTLA1;FLJ16065
151888
277 CD273 MIH18 PE MouseIgG1,κ
BD 558066 PDCD1LG2
PDCD1LG2;B7DC;Btdc;PDL2;PD-L2;PDCD1L2;bA574F11.2
80380
278 CD274 MIH1 PE MouseIgG1,κ
BD 557924 CD274 B7-H;B7H1;PD-L1;PDCD1L1;PDL1
29126
279 CD275 2D3/B7-H2
PE MouseIgG2b,k
BD 552502 ICOSLG
B7-H2;B7H2;B7RP-1;B7RP1;GL50;ICOS-L;ICOSLG;KIAA0653;LICOS
23308
280 CD276 DCN.70 PE MouseIgG1,κ
BioLegend
331606 CD276 B7H3 80381
281 CD277 BT3.1 PE MouseIgG1
e-Bioscience
14-2779-71
BTN3A1
BTF5;BT3.1
11119
282 CD278 DX29 PE MouseIgG1
BD 557802 ICOS AILIM;MGC39850
29851
283 CD279 MIH4 PE MouseIgG1,κ
BD 557946 PDCD1 PD1;SLEB2;hPD-l
5133
284 CD281 TLR1.136
PE MouseIgG1,κ
BioLegend
334506 TLR1 TLR1;TIL;rsc786;KIAA0012;DKFZp547I0610;DKFZp564I0682
7096
85
285 CD282 11G7 FITC MouseIgG1,κ
BD 558318 TLR2 TIL4 7097
286 CD283 TLR3.7 PE MouseIgG1,κ
e-Bioscience
12-9039-82
TLR3 TLR3 7098
287 CD284 610015 PE MouseIgG2a
R&DSystems
FAB6248P
TLR4 TOLL;hToll
7099
288 CD286 TLR6.127
PE MouseIgG1,κ
BioLegend
334708 TLR6 CD286 10333
289 CD288 44C143 PE MouseIgG1
Abcam ab45097 TLR8 TLR8 51311
290 CD289 eB72-1665
PE RatIgG2a,к
BD 560425 TLR9 none 54106
291 CD290 3C10C5 PE MouseIgG1,κ
BioLegend
354604 TLR10 TLR10 81793
292 CD292 PolyclonalAntibody
PE GoatIgG
R&DSystems
FAB346F BMPR1A
BMPR1A;ALK3;ACVRLK3
657
293 CD294 BM16 APC RatIgG2a,к
BD 558042 GPR44 CRTH2 11251
294 CD295 52263 PE MouseIgG2b
R&DSystems
FAB867P LEPR LEPR;OBR
3953
295 CD298 4A8 FITC MouseIgG2a
MBL D261-4 ATP1B3
ATP1B3;ATPB-3;FLJ29027
483
296 CD299 120604 PE MouseIgG2b
R&DSystems
FAB162P CLEC4M
DC-SIGN2;DC-SIGNR;DCSIGNR;HP10347;LSIGN;MGC47866
10332
297 CD300a MEM-260
PE MouseIgG1
Abcam ab64675 CD300A
CMRF-35-H9;CMRF35H;CMRF35H9;IRC1;IRC2;IRp60
11314
298 CD300c TX45 PE MouseIgG1,κ
BioLegend
334804 CD300C
CMRF-35A;CMRF35
10871
86
A;CMRF35A1;LIR
299 CD300e UP-H2 PE MouseIgG1,κ
BioLegend
339704 CD300E
342510
300 CD301 125A10.03
FITC MouseIgG1
Imgenex
DDX0010A488
CLEC10A
HML;HML2;CLECSF13;CLECSF14
10462
301 CD303 AC144 PE MouseIgG1
Miltenyi
130-090-511
CLEC4C
BDCA2;CLECSF11;DLEC;HECL;PRO34150;CLECSF7
170482
302 CD304 AD5-17F6
PE MouseIgG1
Miltenyi
130-090-533
NRP1 NRP;VEGF165R
8829
303 CD305 DX26 PE MouseIgG1,κ
BD 550811 LAIR1 LAIR-1 3903
304 CD307e 509f6 PE MouseIgG2a,к
BioLegend
340304 FCRL5 CD307;FCRH5;IRTA2;BXMAS1;PRO820
83416
305 CD309 89106 PE MouseIgG1
R&DSystems
FAB357P KDR KDR;FLK1;VEGFR;VEGFR2
3791
306 CD312 2A1 PE MouseIgG1
AbDSerotech
MCA2330PE
EMR2 none 30817
307 CD314 1D11 PE MouseIgG1,κ
BD 557940 KLRK1 KLRK1;KLR;NKG2D;NKG2-D;D12S2489E
22914
308 CD317 RS38E APC MouseIgG1,κ
BioLegend
348404 BST2 none 684
309 CD318 309121 PE MouseIgG2a
R&DSystems
FAB26662P
CDCP1 CDCP1;FLJ22969;MGC31813
64866
310 CD319 235614 PE MouseIgG2a
R&DSystems
FAB1906P
SLAMF7
19A;CRACC;CS1
57823
87
311 CD321 M.AB.F11
PE MouseIgG1,κ
BD 552556 F11R JAM;KAT;JAM1;JCAM;JAM-1;PAM-1
50848
312 CD322 CRAM-18F26
FITC RatIgG2a
AbDSerotech
MCA2211F
JAM2 C21orf43;VE-JAM;VEJAM
58494
313 CD324 36/E-Cadherin
FITC MouseIgG2a,к
BD 560061 CDH1 Arc-1;CDHE;ECAD;LCAM;UVO
999
314 CD325 8C11 PE MouseIgG1,κ
e-Bioscience
12-3259-73
CDH2 CDHN;NCAD
1000
315 CD326 EBA-1 PE MouseIgG1,κ
BD 347198 TACSTD1
CO17-1A;EGP;EGP40;Ep-CAM;GA733-2;KSA;M4S1;MIC18;MK-1;TROP1;hEGP-2
4072
316 CD328 F023-420
PE MouseIgG1,κ
BD 558372 SIGLEC7
p75;QA79;AIRM1;CDw328;SIGLEC-7;p75/AIRM1
27036
317 CDw329 E10-286 FITC MouseIgG1,κ
BD 550906 SIGLEC9
CDw329;OBBP-LIKE
27180
318 CD332 98725 APC MouseIgG1
R&DSystems
FAB684A FGFR2 FGFR2;BEK;JWS;CEK3;CFD1;ECT1;KGFR;TK14;TK25;BFR-1;K-SAM
2263
319 CD333 136334 PE MouseIgG1
R&DSystem
FAB766P FGFR3 FGFR3;ACH;
2261
88
s CEK2;JTK4;HSFGFR3EX
320 CD334 4FR6D3 PE MouseIgG1,κ
BioLegend
324306 FGFR4 FGFR4;TKF;JTK2;MGC20292
2264
321 CD335 9E2/NKp46
PE MouseIgG1,κ
BD 557991 NCR1 LY94;NK-p46;NKP46
9437
322 CD336 P44-8.1 PE MouseIgG1,κ
BD 558563 NCR2 LY95;NK-p44;NKP44
9436
323 CD337 P30-15 PE MouseIgG1,κ
BD 558407 NCR3 1C7;LY117;NKp30
259197
324 CD338 5D3 PE MouseIgG2b,k
BioLegend
332008 ABCG2 MRX;MXR;ABCP;BCRP;BMDP;MXR1;ABC15;BCRP1;CDw338;EST157481;MGC102821
9429
325 CD339 188331 FITC MouseIgG2b
R&DSystems
FAB1277F
JAG1 JAG1;AGS;AHD;AWS;HJ1;JAGL1
182
326 CD340 Neu24.7
PE MouseIgG1,κ
BD 340552 ERBB2 NEU;NGL;HER2;TKR1;HER-2;c-erbB2;HER-2/neu
2064
327 CD344 CH3A4A7
PE MouseIgG1,κ
BioLegend
326606 FZD4 EVR1;FEVR;Fz-4;FzE4;GPCR;FZD4S;MGC34390
8322
89
328 CD349 W3C4E11
APC MouseIgM,k
BioLegend
326706 FZD9 FZD3 8326
329 CD351 TX61 PE MouseIgG1,κ
BioLegend
137306 FCAMR
FCA/MR;FKSG87;FCAMR
83953
330 CD352 NT-7 PE MouseIgG1,κ
BioLegend
317208 SLAMF6
KALI;NTBA;KALIb;Ly108;NTB-A;SF2000
114836
331 CD354 TREM-26
PE MouseIgG1,κ
BioLegend
314906 TREM1
TREM-1 54210
332 CD355 Cr24.1 PE MouseIgG2a,κ
BioLegend
339106 CRTAM
CRTAM 56253
333 CD357 621 APC MouseIgG1,κ
BioLegend
311610 TNFRSF18
AITR;GITR;GITR-D;TNFRSF18
8784
334 CD358/DR6
DR-6-04-EC
PE MouseIgG1
Abcam ab52513 TNFRSF21
DR6;BM-018;TNFRSF21
27242
335 CD360(BL)
2G1-K12 PE MouseIgG1,κ
BioLegend
347806 IL21R NILR 50615
336 CD360(BD)
17A12 PE MouseIgG1,κ
BD 560264 IL21R NILR 50615
337 CD362 305515 APC notgiven
R&DSystems
FAB2965A
SDC2 HSPG;HSPG1;SYND2;SDC2
6383
338 CD363 218713 PE MouseIgG2b
R&DSystems
FAB2016P
S1PR1 EDG1;S1P1;ECGF1;EDG-1;CHEDG1
1901
339 β2-microglobulin
TÜ99 PE MouseIgM,k
BD 551337 B2M 567
340 BLTR-1 203/14F11
PE MouseIgG1
BD 552836 LTB4R 1241
341 CA9 303123 PE MouseIgG2a
R&DSystems
FAB2188P
CA9 768
342 CLA HECA-452
FITC RatIgM,к
BD 555947 modifiedformofCD162
949
343 CDH3 104805 PE MouseIgG1
R&DSystems
FAB861P
CDH3 1001
90
344 CDH6 427909 APC MouseIgG1
R&DSystems
FAB2715A
CDH6 1004
345 CLIP CerCLIP FITC MouseIgG1,κ
BD 555981 NOTFOUND
346 DCIR I3-612 APC MouseIgG1,κ
BD 558220 CLEC4A 50856
347 EGF-R EGFR1 PE MouseIgG2b,k
BD 555997 EGFR 1956
348 FMC7 FMC7 FITC MouseIgM,k
BD 340919 MS4A1 931
349 fMLP-R 5F1 PE MouseIgG1,κ
BD 556016 FPR1 2357
350 FOXP3 259D/C7
PE MouseIgG1
BD 560046 FOXP3 50943
351 Galectin-3
B2C10 PE MouseIgG1,κ
BD 556909 LGALS3 3958
352 Hematopoieticprogenitorcell
BB9 PE MouseIgG1,κ
BD 557928 NOTFOUND
353 HLA-A2 BB7.2 PE MouseIgG2b,k
BD 558570 HLA-A 3105
354 HLA-ABC
DX17 PE MouseIgG1,κ
BD 560168 ABCA1 19
355 HLA-DM
MaP.DM1
PE MouseIgG1,κ
BD 555983 HFE 3077
356 HLA-DR TU36 PE MouseIgG2b,k
BD 555561 HFE 3077
357 ITGB7 FIB504 PE RatIgG2a,к
BD 555945 ITGB7 3695
358 MICA/B
6D4 PE MouseIgG2a,к
BD 558352 MICA/MICB
100507436/4277
359 Notch1 MHN1-519
APC notgiven
e-Bioscience
17-9889-42
360 Notch2 16F11 PE notgiven
e-Bioscience
12-5786-82
361 Notch3 MHN3-21
APC notgiven
e-Bioscience
17-5787-42
362 NPM-ALK
ALK1 PE MouseIgG3,к
BD 559257 ACVRL1 94
363 PAC-1 PAC-1 FITC MouseIgM,k
BD 340507 DUSP2 1844
364 Podoplanin
NC-08 PE RatIgG2a,λ
BioLegend
337004 PDPN 10630
91
365 SSEA-3 MC631 PE RatIgM BD 560237 FUT4 2526366 SSEA-4 MC813-
70PE MouseI
gG3BD 560128 FUT4 2526
367 Stro-1 STRO-1 APC MouseIgM,λ
BioLegend
340104 NOTFOUND
368 TCRαβ T10B9.1A-31
PE MouseIgM,k
BD 555548 11126
369 TCRγδ B1 PE MouseIgG1,κ
BD 555717 NOTFOUND
370 LTBR hTNFR-RP-M12
PE MouseIgG1,κ
BD 551503 LTBR 4055
371 TPBG 524744 APC MouseIgG1
R&DSystems
FAB49751A
TPBG 7162
372 Vβ8TCR
JR2 PE MouseIgG2b,k
BD 555607 NOTFOUND
373 Vδ2TCR
B6 PE MouseIgG1,κ
BD 555739 NOTFOUND
374 CDH11 667039 FITC notgiven
R&DSystems
FAB17901G
SupplementalTableS2-KnownCDmarkersinneutrophils
CDMarker OtherNames
FamilyandFunction
Knowntobe
ExpressedinPMNs
Confirmedinthecurrentstudy
NotesonExpression Legend
CD4 OKT4,Leu3a,L3T4,T4
IgSF;primaryreceptorforHIV,Tcellmarker
S Y PresentonsomebutnotallcirculatingPMN
UR-Up-regulated
CD10 MME,CALLA,neutralendopeptidase
Zincmetalloprotease;Cleavespeptidesandhydrolyzesotherbiologicalmaterials
C Y DRafterinvivoLPSexposure.IncreasedexpressionafterinvitroLPSexposure
DR-down-regulated
CD11a ITGAL,LFA-1αchain,CR3A
Integrin;playsroleinPMNadhesiontoendothelialwall
C Y S-subset
CD11b ITGAM,α-Mintegrinchain,Mac-1αchain
Integrin;keyleukocyteadhesionmolecule
C Y URwithexposuretoLPSandinflammation.DRfollowing
C-Constitutiveexpression
92
exerciseCD11c ITGAX,alpha
chainofCR4,leukocytesurpaceantigen,p150-95
Integrin;keyleukocyteadhesionmolecule
C Y URininfection
.
CDw12 P90-120 C N URduringapoptosisanduponneutrophilactivation
.
CD13 ANPEP,aminopeptidaseN,APN,gp150,EC3.4.11.2
Cleavesneutralaminoacidsandactsincell-surfaceantigenpresentation
C Y .
CD14 LPSreceptor LRG;actsasacoreceptorinthedetectionofLPS,highaffinityreceptorforLPScomplexes
C Y AlwayspresentandURuponstimulationwithTNF-alpha,G-CSFandGM-CSF
.
CD15 3-fucosyl-N-acetyl-lactosamine
Carbohydrate2;roleinPMNbindingtoendothelium
C Y .
CD16/16a FCGR3A IgSF;playsmajorroleinPMNactivationandapoptosis
C Y DRinapoptoticPMN,DRafterhighintensityexercise
.
CD16b FCGR3B,FcγRIIIb
IgSF;receptorforIgG,signaltransducerandmobilizescalciumstores
C Y ConsideredtobeexpressedonlyonhumanPMN.DRwhenculturedwithIL-8
.
CD17 lactosylceramide
LacCer;bindsPGG-glucan
C Y .
CD18 ITGB2,Integrinβ2
Integrin;leukocyteadhesionmolecule
C Y URwithexposuretoLPS
.
CD23 FCepsilonII IgSF;lowaffinity
S Y Notexpressedin
.
93
receptorforIgF,hascytokine-likeactionswhencleaved
healthyPMN;presenton~50%ofpatientswithrheumatoidarthritis
CD24 BA-1,HAS Glycosylphosphatidylinositol-anchored;InvolvedinsignaltransductionandstimulatesadhesiontoP-selectin
C Y .
CD28 Tp44 IgSF;regulatesPMNmigrationinresponsetoIL-8
C Y URwithPMA .
CD29 ITGBP1,integrinB1chain,VLABchain
Integrin;mediatesPMNadhesiontofibroblasts
C Y .
CD31 PECAM-1,endocam
IgSF;intermediaryinPMNdiapedesis
C Y DRintissueafterextravasationfromblood
.
CD32 FCGR2A,FcγRII
Fcreceptor;regulatesphagocytosisandreleaseofreactiveoxidativespecies
C Y .
CD33 My9,Siglec3,gP67
IgSF;roleincell-celladhesion
S Y Presentinblaststagebutmatureneutrophilsshowweaktonostaining
.
CD35 Complementreceptortype1,CR1,C3b/C4breceptor,immuneadherencereceptor
RCA;bindscellswithC3b/C4bcomplexes
C Y DRaftertrauma,recoveryinexpressionovertime.SignificantDRinexpressionwithexercise
.
CD38 T10,gp45, ADP-ribosyl C Y DRinpatients .
94
ADP-ribosylcyclase
cyclase;actsasaselectinandhasroleinregulatingcelladhesion
withaggressiveperiodontitis
CD43 Leukosialin,sialophorin,gp95,SPN
Sialomucin;anti-adhesive,actsasanegativeregulatorforcelladhesion
C Y DRduringPMNapoptosis;shedfromsurfaceduringPMNactivationandadhesion
.
CD44 Phagocyteglycoprotein1,Hermesantigen,HUTCH-1,H-CAM
Hyaladherin;roleinregulationofphagocytosisofPMNbymacrophages
C Y DRinapoptosis
.
CD45 PTPRC,leukocytecommonantigen,LCA
Proteintyrosinephosphatase;signalmoleculethatregulatesmanycellprocesses
C Y .
CD45RA Bcells,naïveT-cells,monocytes
Proteintyrosinephosphatase
C Y ExpressedtoalowdegreebycontrolPMN(1-15%).URto~30%expressionininfection
.
CD45RB T200,B220,DAKO-LCA
Proteintyrosinephosphatase;modulatesCXCR1andCXCR2expressioninPMN
C Y .
CD45RO UCHL-1 Proteintyrosinephosphatase
C Y .
CD46 Membranecofactorprotein,MCP
RCA;protectstissuefrompathogenicdamage,cofactorthatinactivatesC3bandC4b
C Y DRaftertraumaanddoesnotreturntonormalexpressionevenafter10days
.
95
CD47 Integrinassociatedprotein(IAP),ovariancarcinomaantigenOA3
IgSF;roleinPMNtransmigration
C Y URwithfMLPstimulationandaftertransmigration
.
CD49d α4integrin,VLA-4αchain,ITGA4
Integrin;primaryroleinmonocytemigration
C Y .
CD49f α6,VLA-6αchain,plateletGPIc,ITGA6
Integrin;mediatescellbindingtolaminin
C Y .
CD50 ICAM-3,intercellularadhesionmolecule3
IgSF;roleincell-celladhesionandasatargetrecognitionreceptorforapoptoticcells
C Y DRinapoptosisandactivatedPMN
.
CD53 MOX44,TSPAN25
Tetraspanin/TM4;roleincellgrowthproperties
C Y Higherlevelsofexpressioninlupuspatients.DRwithfMLPstimulation
.
CD54 ICAM-1,intercellularadhesionmolecule1
IgSF;roleinsignaltransductionandsuperoxideproductioninPMN
C Y URwhenstimulatedwithPMA
.
CD55 DAF,decayacceleratingfactor
RCA;inhibitsC3convertaseassemblywhichprotectshosttissue
C Y URaftertraumatobody
.
CD58 LFA-3,lymphocytefunctionassociatedantigen-3
IgSF;CD2ligandandplayroleincelladhesionandsignaling
C Y .
CD59 MACIF,MIRL,P-18,protectin
Ly6;inhibitsMACformationandactsascoreceptorinNKcell
C Y URaftertraumatobodyandURinpatientswithsepsis
.
96
activationCD62L L-selectin,
LAM-1,Mel-14,SELL
Selectin;vitalforPMNtransmigration,mediatestetheringandrollingstagesonendothelium
C Y DRafterapheresisandshedfromcellsurfacewithactivationandapoptosis
.
CD63 LIMP,GP55,LAMP-3,OMA81H,TSPAN30,ME491,granulophysin
Tetraspanin/TM4;involvedinPMNactivationandmigrationthroughroleinadhesion
C Y URinnasaltissueandafterstimulationwithfMLP
.
CD64 FcR1,FcγR1,high-affinityFcγreceptor,FCGR1A
IgSF;bindswithmonomericandaggregatedIgG
S Y VerylowexpressiononrestingPMNbutrapidincreasewithactivation.URinvivowithrhG-CSFandinpatientswithbacterialinfections.Higherexpressioninpatientswithrheumatoidarthritis
.
CD65/CD65s Ceramidedode-casaccharide,4c
Poly-N-acetyllactosamine;currentlyunknown
C Y .
CD66a CEACAM1,NCA-160
IgSF;roleinenhancingneutrophiladhesion
C Y StoredinthesecondarygranulesofPMN,expressedinmorematurePMNs.URwithstimulation/activationofPMN
.
CD66b CEACAM8,NCA-95
IgSF;granulocyteactivationmarker,roleinenhancing
C Y Higherexpressioninpatientswithrheumatoidarthritis.DR
.
97
neutrophiladhesion
asneutrophilmaturesandwithapoptosis.URwithstimulation/activationofPMN
CD66c CEACAM6,NCA-50/90
IgSF;roleinenhancingneutrophiladhesion
C Y MainlylocatedonprimarygranulesinPMN.URwithstimulation/activationofPMN
.
CD66d CGM1,CEACAM3
IgSF;roleinenhancingneutrophiladhesion
C Y URwithstimulation/activationofPMN
.
CD68 Gp110 Sialomucin;actsasascavengerreceptor
C Y URininflamedtissueinCrohn’sdisease
.
CD69 AIM,activatorinducermolecule,CLEC2C,MLR3,EA1,VEA
C-typelectin;inducesneutrophildegranulationandothercellprocesses
S Y VerylowinrestingPMN.URinactivatedPMNandwithfMLPstimulationto~13%
.
CD80 B7,B7-1,CD28LG
IgSF;anantigenpresentingcomplex,inducesTcellactivation
S Y LowonrestingPMN,storedingranules.URwithactivation
.
CD82 R2,IA4,4F9,C33,KAI1,TSPAN27
Tetraspan;rolesinsignaltransductionandinhibitorofcellmigration
C Y .
CD84 LY9B,SLAMF5
SLAM;associatewithSLAM-associatedprotein,promotesT-cellactivation
C Y Presentinsomestudiesbutnotdetectedinotherstudies
.
98
andsecretionofinterferongamma
CD85a LILRB3,ILT5,Immunoglobulin-liketranscripts5.
IgSF;bindsMHCclass1moleculesandinhibitsimmuneresponse
C Y .
CD85d LILRB2,ILT4 IgSF;bindsMHCclass1moleculesandinhibitsimmuneresponse
C Y URinPMNinresponsetoinflammatorystimuliandinsepsis
.
CD85h LILRA2,LIR7,ILT1
shortcytoplasmicdomainandapositivelychargedarginineresiduewithinthetransmembranedomainthatmediatesassociationwiththeimmunoreceptortyrosine-basedactivationmotif-containingFcreceptor ︎ chain(Fc�)
S Y neutrophilshavearestrictedpatternofcell-surfaceLIRexpressionwithLIR3andLIR7beingexpressedinalmostalldonors,withoccasionalexpressionofLIR1and ︎ orLIR2
.
CD85j LILRB1,ILT2 IgSF;bindsMHCclass1moleculesandinhibitsimmuneresponse
C Y .
CD85k LILRB4,ILT3 immunoreceptortyrosine-basedinhibitorymotifs
S Y neutrophilshavearestrictedpatternofcell-surfaceLIRexpressionwithLIR3andLIR7beingexpressedinalmostalldonors,with
.
99
occasionalexpressionofLIR1and�orLIR2
CD86 B7-2,LAB72 IgSF;anantigenpresentingcomplex,roleinmoderatingTcellproliferation
S N LowexpressionincirculationURwithexposuretoIFNgammaandGm-CSF
.
CD87 Urokinase-typeplasminogenactivatorreceptor,PLAUR
GPI-anchored;roleinleukocyteextravasationasregulatorsofintegrin-mediatedadhesion
C Y DRby47%inapoptoticPMN
.
CD88 C5a-receptor,C5aR,C5R1
Rhodopsin;roleintissueinflammationaftertrauma
C N DRbyover50%inapoptoticPMN.DRimmediatelyaftertraumaticinjury
.
CD89 FCAR,FcaRI Fcreceptor;potentcytotoxictriggermolecule
S Y StoredinsecondaryandtertiarygranulesandexpressedonsurfaceinresponsetofMLP,IL-8andC5a
.
CD92 SLC44A1,CHTL
Cholinetransporter,mayhaveroleinIL-10regulation,implicatedinnegativesignalingpathways
C Y .
CD93 C1qR1 O-sialoglycoprotein;roleincell-cellinteractionsandadhesionandapoptotic
Y Y DRandcleavedfromsurfaceinresponsetoinflammation
.
100
cellclean-upCD95 APO-1,Fas,
TNFRSF6TNFR;inducesPMNapoptosis
C Y .
CD97 TM7LN1 EGF-TM7;roleinleukocytetrafficking
C Y Presentinlowconcentration.URexpressionandactivityinpatientsfollowingheartsurgery;URinjointPMNfollowingacutehemoarthrosis
.
CD99 MIC2,E2 Transmembraneglycoprotein;mediatesPMNmigrationacrosstransendothelialmembrane
C Y PresentinlowconcentrationonPMNsurface.URwhenattachedtoendothelialcellsfortransmigration
.
CD101 V7,P126,IGSF2,EWI-101
IgSF;roleinTcellactivation
C Y .
CD107a LAMP-1 LAMP;polylactosaminoglycancarrier
S Y Smallamountpresentonsurface,mainlystoredinsecretoryvesicles.URatsurfaceinresponsetofMLP
.
CD107b LAMP-2 LAMP;polylactosaminoglycancarrier
S Y Smallamountpresentonsurface,mainlystoredinsecretoryvesicles.URatsurfaceinresponsetofMLP
.
CD114 G-CSFR,CSFR,granulocyte
ClassICK-R;initiatescell
C Y DRinresponseto
.
101
colonystimulatingfactorreceptor,CSF3R
proliferationanddifferentiationintomaturePMNandhasaroleindelayingapoptosis
GCSFstimulation
CD116 GM-CSFreceptoralphasubunit,GMRα,CSF2RA
ClassICK-R;roleincellproliferation,augmentsPMNanti-bacteriafunctionsinmultipleways
C Y DRinresponsetoGMCSF.Decreasedexpressionseeninpatientswithinflammatoryboweldisease
.
CD119 IFN-γreceptorαchain,IFNGR1
IgSF;enhancePMNanti-bacterialfunctionsinvariousways
C Y DRwheninfectedwithA.cytophagilum
.
CD120a TNFreceptor1,TNFRS5,TNFRSF1A
TNFR;roleinneutrophilrecruitment
C Y Expressedinlowlevels.RapidDRwhencultureinvitroandinapoptosis
.
CD120b TNFreceptor2,TNFR80,TNFRSF1B
TNFR C Y Expressedinlowlevels.RapidDRwhencultureinvitroandinapoptosis
.
CD121b TypeIIIL-1receptor,IL1R2,IL1RB
IgSF;stimulatesneutrophilactivation
C Y URinpatientswithsepsis
.
CD122 IL-2receptorβchain,p75,IL2RB
ClassICK-R;mediatesIL-2signaltransduction
C Y .
CD123 IL-3receptorαchain,IL3RA
ClassICK-R;roleincellproliferationanddifferentiationandinhibitsapoptosis
S Y Notexpressedinhealthycontrols.URandpresentafterincubationwithGM-CSF
.
CD124 IL-4Rαchain,IL4R
ClassICK-R;mediatesIL-4
C Y .
102
signaltransduction,roleasgrowthfactor
CD130 GP130,IL-6receptorβchain,IL6ST
CRSF;roleinsignaltransduction
C Y .
CD131 Commonβchain,CSF2RB,IL3RB,IL5RB
ClassICK-R;formdimerwithCD123orCD116toactasreceptor
C Y .
CD132 Commonγchain,IL2RG
IgSF;PMNreceptorforcytokinesIL2,IL4,IL7,IL9,IL15andIL21
C Y .
CD138 Syndecan-1,SDC1,SYND1,SDC
Syndecan;roleinPMNmigrationandadhesionbyregulatingchemokinegradients,rolesincellgrowth
C Y IncreasedexpressioninpatientswithType2diabetesandcorrelatedwithBMI
.
CD141 THBD,thrombomodulin,Fetomodulin
C-typelectin;receptorforthrombinandactsasananticoagulant
S Y Expressedverylowonsurface.Unsurewhatstimulatesexpressiononsurface
.
CD147 Basigin,BSG,5F7,EMMPPRIN,M6,OK,TCSF
IgSF;stimulatesproductionofmatrixmetalloproteinasesfromstromalcellsandhasroleincancermetastasis
C Y .
CD148 PTPRJ,DEP-1,HPTPeta
RPTPasetypeIII,phosphatase;similartoCD45,roleininhibitionofFcyRIIafunctions
C Y .
CD151 PETA-3,SFA- Tetraspan C Y .
103
1,TSPAN24 TM4;formscomplexwitha3b1andinfluencescellmigration
CD153 TNFSF8,CD30Ligand,CD30L
TNF;signalregulationofcelldeathandproliferation,inducesURorDRofCD30
C Y .
CD156a ADAM8,MS2 ADAM;metalloproteinase,actsasasheddasetocleaveproteins
C Y .
CD156b TACE(tumornecrosisfactorα-convertingenzyme),ADAM17,snakevenomlikeprotease,CSVP
Zincmetallopeptidase;roleintransmembraneproteincleavage
C Y URwithphagocytosisofB.cepaciaandURduringsevereperiotonitis
.
CD156c ADAM10 ADAM;actsasasheddasesoinvolvedinproteincleavage
C Y DRwithapoptosis
.
CD157 BST-1BP-3/IF7Mo5
ADP-ribosylcyclase;roleinneutrophiladhesionandmigration
C Y URbydoubleafterfMLPstimulation
.
CD162 SELPLG,Pselectinglycoproteinligand1,PSGL-1
Sialomucin;mediatesP-selectinactivity
C Y DRandreleasedintobloodwithPMNactivation
.
CD163 M130 Scavengerreceptor
C Y Presentinlowamounts,elevatedinchildrenandadultswithsepsis
.
CD170 SIGLEC5,OB-BP2
IgSF;primesPMNforenhancedstimulationbyfMLP
C Y URwithfMLPstimulation
.
104
CD172a PTPNS1,SIRPα,SHPS-1signalregulatoryproteinα.
IgSF;roleinPMNmigrationthroughepithelialandendothelialandcollagen-coatedcells,aswellasinadhesion
S Y Storedinintracellularpoolsandmovedtosurfaceaftersimulationbychemoattractants.DRwithapoptosis
.
CD172b CD172β,SIRB1,SIRPβ1
IgSF;stimulatoryinteractionswithITAMmolecules
C Y .
CD177 PRV1(polycythemiaverarubra1),NB1
uPARreceptor;bindsendothelialcellPECAM-1
S Y ExpressedonsubpopulationofPMN,~30-70%.3%expressnoCD177.URwithsevereinfection
.
CD178 Fasligand,FasL,CD95L
TNF;roleinPMNapoptosis
C Y .
CD181 CXCR1;IL-8receptorα,IL8RA
BindsIL-8 C Y DRwithLPSstimulation
.
CD182 CXCR2;IL-8receptorb,IL8RB
GPCRfamily,chemokinereceptor,Rhodopsin;highaffinitytoIL-8,roleinPMNmigrationtoinflammatoryareas
C Y DRinseveresepsisandexposuretoLPS
.
CD183 CXCR3 GPCRfamily,chemokinereceptor;roleincellchemotaxisandcalciummobilization
S Y Marginallypresentincirculation.URatpulmonaryandsynovialsiteofinflammation
.
CD184 CXCR4 GPCRfamily,chemokinereceptor;roleinthebonemarrowinregulationof
S Y Marginallypresentincirculation.URatpulmonaryandsynovial
.
105
hematopoieticcells
siteofinflammation;DRwithincubationwithlithium
CD191 CCR1 GPCRfamily,chemokinereceptor
S N Marginallypresentincirculation.URatpulmonaryandsynovialsitofinflmmation;URwithIFN-gamma
.
CD192 CCR2 GPCRfamily,chemokinereceptor
S Y Marginallypresentincirculation.URatpulmonaryandsynovialsitofinflmmation
.
CD193 CCR3,eosinophileotaxinreceptor,chemokine(C-Cmotif)receptor3,CKR3,CMKBR3
GPCRfamily,chemokinereceptor
S Y Marginallypresentincirculation.URatpulmonaryandsynovialsitofinflmmation;URwithIFN-gamma
.
CD195 CCR5 GPCRfamily,chemokinereceptor
S Y Marginallypresentincirculation.URatpulmonaryandsynovialsitofinflmmation
.
CD200 MRC,OX2 IgSF;roleinregulationofmyeloidcellfunction
C Y .
CD205 LY75,CLEC13B
C-typelectin;largerangeoffunction,amacrophagemannosereceptor
C Y .
CD212 IL12RB1,IL-12R-BETA1,
hemopoietinreceptorSF;
C Y SlightlyURwithLPS
.
106
IL-12RB roleinIL-8production
exposure
CD215 IL-15RA TypeIcytokinereceptorfamily;activatesPMNviaIL-15ligand
C Y .
CD217 IL-17R TypeIcytokinereceptorfamily;proinflammatory,proapoptoticcytokine
C Y .
CD218a IL-18receptoralpha,IL18R1
CK-R;roleinPMNproteinsynthesisandrelease,URCD11b,augmentsreleaseofROS
C N .
CD218b IL-18receptorbeta,IL18RAP
CK-R;roleinPMNproteinsynthesisandrelease,URCD11b,augmentsreleaseofROS
C Y .
CD220 Insulinreceptor,IR,INSR
Tyrosinekinasereceptor;insulinreceptor,moderatesPMNchemotaxis
C Y .
CD221 IGF1Receptor,IGF1R
Tyrosinekinasereceptor;PotentprimerofPMNforenhancedROSsecretion
C Y .
CD222 IGF2R,Man-6preceptor
Lectin;targetsproteintolysosome
C Y URatsiteofinfection
.
107
CD261 TRAIL-R1,DR4,TNFRSF10A
Tumornecrosisfactorreceptorsuperfamily(TNFRSF);deathreceptor,stimulatesapoptosis160andshowntoberesponsibleforPMNcelldeath
S N Presentinsomestudiesbutnotdetectedinotherstudies
.
CD262 TRAIL-R2,DR5,TNFRSF10B
Tumornecrosisfactorreceptorsuperfamily(TNFRSF);deathreceptor,stimulatesapoptosis
C N .
CD263 TRAIL-R3,DcR1,LIT,TRID,TNFRSF10C
Tumornecrosisfactorreceptorsuperfamily(TNFRSF);inhibitsTRAIL-inducedapoptoticsignals
C Y .
CD264 TNFRSF10D,TRAIL-R4,DCR2
Tumornecrosisfactorreceptorsuperfamily(TNFRSF);inhibitsTRAIL-inducedapoptoticsignals
S Y Presentinsomestudiesbutnotdetectedinotherstudies
.
CD265 RANK,TRANCE-R,EOF,TNFRSF11A
TNF/NGFreceptor;keyroleinboneremodelingandosteoclastactivity
S Y WidevarietyofexpressioninhealthyPMN~1-70%.URwithbacterialinfections;UR
.
108
insynovialfluidofarthritispatients
CD274 B7-H1,PD-L1,PDCD1LG1
IgSF;deathligand,inhibitorymolecule
S Y Notexpressedincontrol.Inducedexpressionwithcytokineactivation
.
CD281 TOLL-likereceptor1,TLR1,TIL
TLRF;requiredforimmunerecognitionofmycobacterialipoarabinomannanandtriacylatedllipopeptides
C Y .
CD282 TOLL-likereceptor2,TLR2,TIL4
TLRF;involvedincytokineproduction
S Y URwithGM-CSFincubation;URinbloodofalcoholichepatitispatients;DRinbloodPMNinpatientswithcycsticfibrosis.URinairwayPMN
.
CD284 TOLL-likereceptor4,TLR4
TLRF;receptorforLPS
S Y URsurfaceexpressioninPMNofalcoholichepatitspatients
.
CD285 TLR-5 TLRF;stimulatesIL-8production
S N Expressedintracellularly,surfaceexpressioninducedwithTLRligandsandcytokines.URinairwayPMNinpatientswithcysticfibrosis
.
CD286 TLR-6 TLRF;roleincytokineproduction
C Y .
109
CD287 TLR-7 TLRF;involvedinssRNArecognition
S N Locatedintracellularly
.
CD288 TLR-8 TLRF;involvedinssRNArecognition
S Y Locatedintracellularly
.
CD289 TLR-9 TLRF;inhibitsPMNmigrationthroughDRofCXCR2
C N URinwithGM-CSF
.
CD290 TLR-10 TLRF C N .CD295 LeptinR,
LEPR,OBR,B219
Type1cytokinelikereceptor;leptinreceptor
C Y .
CD298 Na/KATPaseβ3-subunit,ATP1B3
EnzymatictransportofNa/K
C Y .
CD300a CMRF35H,IRC1,IRC2,IRp60
IgSFCMRF;inhibitsROSproductionandinactivatesreceptorsignalling
C Y .
CD302 DCL1,CLEC13A,BIMLEC
Type1TM,Ctypelectinreceptor;roleincelladhesionandmigration
C Y .
CD305 LAIR1 IgSF;inhibitoryreceptor,inhibitsvariouscytokinesignals
S Y .
CD312 EMR2 EGF;involvedinPMNrecruitmentandmaintenanceintotissue
C Y .
CD313 EMR3 EGF-TM7;currentlyunknownfunction
C Y .
CD321 JAM1,JAM-A, IgSF;rolein C Y .
110
F11R adhesionandtransmigrationofPMNandTcells
CD329 SIGLEC9,sialicacid-binding,Ig-likelectin9
SIGLEC;rolesinapoptoticandnonapoptoticcelldeathinneutrophils
C Y .
CD354 TREM1 IgSF;roleinmagnificationoftheinflammatoryresponse
C Y StrongupregulationinPMNwithLPS
.
CD360 IL-21R-alpha IgSF(Nectinfamily);receptorforIL21,enhancesBcellproliferation
C Y .
CD361 EVI2B,EcotropicViralIntigration2B,
Type1transmembraneprotein;poorlydefinedfunctions
C Y .