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.

iii

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

v

Abstract............................................................................................................................................19

Introduction......................................................................................................................................20

Materialsandmethods....................................................................................................................21

ResultsandDiscussion.....................................................................................................................29

Conclusions.......................................................................................................................................42

Chapter3..........................................................................................................................................44

ThesisSummaryandFutureDirections.........................................................................................44

Summary..........................................................................................................................................44

FutureDirections..............................................................................................................................46

References........................................................................................................................................48

SUPPLEMENTALFILES...................................................................................................................60

vi

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

vii

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

viii

ListofTables

Chapter1:

Table1–Cellsurfaceexpressionindifferentstagesofgranulocyticdifferentiation

Table 2 – Cell surface markers expressed in neutrophils associated with recruitment andmigratoryfunctions

Table3–NeutrophildeficienciesmanifestedintheoralcavityChapter2:Table1-ListofCDmarkersanditsdistributionTable2-FulllistofantibodiesusedinthisstudyTable3–ComprehensiveliteraturereviewofcellsurfacemarkersexpressioninNeutrophils

ix

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)

x

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

xi

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.

1

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

2

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.

3

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

4

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

5

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

6

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.

7

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

8

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.

9

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

10

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

11

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

12

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 .