Identification of CD Marker Expression and Neutrophil Surface Marker Changes in Health and Disease Using High- Throughput Screening Flow Cytometry

Identification of CD Marker Expression and Neutrophil Surface Marker Changes in Health and Disease using High- throughput screening flow cytometry

Flavia S. Lakschevitz

A thesis submitted in conformity with the requirements for the degree of Master’s of Science

Faculty of Dentistry University of Toronto

Identification of CD Marker Expression and Neutrophil Surface Marker Changes in Health and Disease using High-throughput screening flow cytometry

Flavia S. Lakschevitz

Master’s of Science

Faculty of Dentistry University of Toronto

2015

Abstract

Neutrophil hyperactivation can contribute to tissue damage in inflammatory diseases. Although many 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- periodontitis patients were screened against a panel of 374 known Cluster of Differentiation (CD) antibodies to identify cell-surface markers specific to neutrophils. This screen identified CD11b+, CD16b+, and CD66c+ as markers that are always expressed on neutrophils. Cell- sorting with an antibody against CD11b/CD16b/CD66c allowed for the enrichment of mature neutrophils, yielding populations of up to 99%, confirming the validity of these markers when isolating neutrophils. These findings provide a simple method for isolating neutrophils from humans, and thereby establish a validated method that allows for the accurate identification of neutrophils. This knowledge will be crucial for identifying neutrophil subtypes associated with neutrophil-mediated inflammatory diseases.

Acknowledgments

First and foremost, I would like to thank Dr. Michael Glogauer, his support and guidance during my training at University of Toronto, has proven to be relentless. Thank you!

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 Hassanpour and Chunxiang Sun for their friendship and support in the lab. I’d like to thank my summer students, especially Ayala Rubin for reading my manuscript and helpful suggestions.

I give special thanks to Joshua Paterson from the UNH Antibody Core Facility for his assistance with HTS-Flow Cytometry and to Dionne White from Flow Cytometry facility at Department of Immunology, University of Toronto, Toronto, ON for her assistance with cell sorting.

How not to mention Kerry D’Costa and Jason Yee! My inseparable classmates in Perio! You guys are the best colleagues/friends that anyone could hope and more!

I thank my program advisory members Drs. Christopher McCulloch and Dr. Howard Tenenbaum for their help and insight. Special thanks to Dr. Limor Avivi-Arber and Dr. Scott Gray-Owen for kindly serving as defense examiners.

Lastly I thank my friends, family and loved ones everything is possible because of you!

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Table of Contents

Abstract...... ii

Acknowledgments ...... iii

Table of Contents ...... iv

List of Abbreviations ...... vi

List of Tables ...... viii

List of Figures ...... ix

Chapter 1 General Introduction ...... 1

1. Introduction ...... 1 Neutrophil Identification ...... 1 Cluster of differentiation (CD) ...... 2 High-throughput screening (HTS) flow cytometry ...... 3

2. CD expression and Neutrophil Distribution ...... 4 Bone Marrow ...... 4 Circulation ...... 7 Tissues ...... 8

3. CD expression and Neutrophils Functionality ...... 9 Neutrophil Recruitment ...... 9 Bacterial killing and Phagocytosis ...... 11 Non-traditional functions of neutrophils ...... 14

4. Periodontal Diseases ...... 14 Chronic Periodontitis ...... 14 The role of neutrophils in the pathogenesis of Periodontitis ...... 15

5. Objectives of this study and hypothesis ...... 17 Objectives ...... 17 Hypothesis ...... 18

Chapter 2 ...... 19 Immunophenotypical characterization of human neutrophils ...... 19

Abstract ...... 19 Introduction ...... 20 Materials and methods ...... 21 Results and Discussion ...... 29 Conclusions ...... 42

Chapter 3 ...... 44 Thesis Summary and Future Directions ...... 44 Summary ...... 44 Future Directions ...... 46

References ...... 48 SUPPLEMENTAL FILES ...... 60

List of Abbreviations

AAP - American Academy of Periodontology

Ab - Antibody

ADCC – Antibody-Dependent Cell-mediated Cytotoxicity

APC - Allophycocyanin

CD – Cluster of Differentiation

CF - Cystic Fibrosis

COPD - Chronic Obstructive Pulmonary Disease

CP – Chronic periodontitis

FACS - Fluorescence-activated cell sorting

Fc – Fragment crystallizable

Fc R – Fc Receptor

FITC - Fluorescein isothiocyanate

FSC - Forward- scatter

HLA - human leukocyte antigen

HTFC - high throughput flow cytometry

HTS - High-throughput screening

ICAM-1 - Intercellular Adhesion Molecule 1

Ig – Immunoglobulin

ITAMS - immunoreceptor tyrosine-based activation motifs

LFA 3 - lymphocyte function-associated antigen 3

MFI - Mean fluorescence intensity

PAMPs - pathogen-associated molecular patterns

PE - Phycoerythrin

PMN-B – Blood polymorphonuclear neutrophilic granulocyte,

PMN-O – Oral polymorphonuclear neutrophilic granulocyte,

PRRs - pattern recognition receptors

SSC - Side-scatter

TLRs - Toll like receptors

VAP 1 – Vascular adhesion protein 1

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List of Tables

Chapter 1:

Table 1 – Cell surface expression in different stages of granulocytic differentiation Table 2 – Cell surface markers expressed in neutrophils associated with recruitment and migratory functions

Table 3 – Neutrophil deficiencies manifested in the oral cavity

Chapter 2:

Table 1 - List of CD markers and its distribution

Table 2 - Full list of antibodies used in this study

Table 3 – Comprehensive literature review of cell surface markers expression in Neutrophils

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List of Figures

Chapter 1:

Figure 1 - Model of pathogenesis of Periodontitis (adapted from Page & Kornman, Periodontology 2000, 1997). Bacteria and biofilm insult are crucial for the initiation of 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 environmental risk factors works as modulators of the host immune response. The final result will be tissue break down, which is characterized by bone loss and destruction of the periodontal apparatus.

Chapter 2:

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: FSC vs. time, FSC vs. SSC, then eFluor 780+, followed by Singlets+. The images are representative of blood PMN of healthy patients.

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: FSC vs. time, FSC vs. SSC, then eFluor 780+, followed by Singlets+. The images are representative of oral PMN of healthy patients.

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 contour plots of blood (B-1) and oral rinse. (A-2 and B-2) Cytospin preparations of each sorted cell type were visualized with Diff-Quik staining to confirm purity of the samples. (A-3) Contaminating monocytes/lymphocytes were excluded from our analysis by our gating strategy.

Figure 4 - Diversity of Neutrophil Surface Markers Depends Source and Health of Tissue. (A)

The heatmap of the initial screen of all 374 known CD markers using HTS – flow cytometry of blood and oral neutrophils isolates from healthy and CP patients. (B) We could confirm expression of 145 surfaces markers in neutrophils either in circulation or 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 positive marker expression profiles are shown in a heatmap format for a representative set of 20 surface markers on neutrophils from blood and oral rinse in health and CP patients reveals degree of differences between sources of neutrophils. (D) Examples of histograms of cell surface markers commonly used to isolate neutrophils. Note variability of expression of CD31 and CD62L in PMN-O in healthy and CP patients. CP – Chronic Periodontitis, CTR – Control (Healthy), PMN-B – Blood neutrophils, PMN-O – Oral neutrophils.

Figure 5 - Cluster analysis Unsupervised hierarchical clustering of 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. See Figure S for a magnified image of the significant markers. CP – Chronic Periodontitis, CTR – Control (Healthy), PMN-B – Blood neutrophils, PMN-O – Oral neutrophils. Red – PMN-B, Green – PMN-O.

Figure 6 - Magnified image of the significant markers. Unsupervised hierarchical clustering of 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 – Oral neutrophils. Red – PMN-B, Green – PMN-O

Figure 7 – Oral neutrophils in CP patients are characterized by an active phenotype. (A) Representative flow histograms of cell surface markers (black) that are up-regulated in PMN-O from CP patients are overlaid on isotype control mAbs (mono clonal antibodies?) (in red). (B) Quantification of flow cytometric assessment of Oral and Blood neutrophils from Healthy and Chronic periodontitis patients (expressed as MFI). MFI - Mean

Fluorescent intensity. * P≤ 0.05

Figure 8 – Cell sorting with an antibody against CD11b, CD16 and CD66b allowed for the enrichment of highly pure mature neutrophils in the blood (A) Quantification of flow cytometric assessment of Blood neutrophils from Healthy and Chronic periodontitis patients (expressed as % of +ve cells) (B) Flow cytometric plots from a representative analysis of blood and oral PMNs from healthy and CP patients after cell 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 staining to confirm purity of the samples.

Chapter 1 General Introduction

1. Introduction

The most abundant type of white blood cells, neutrophils or polymorphonuclear leukocytes

(PMNs), are important modulators of the host innate immune response1. In healthy individuals the bone marrow produces 2 × 1011 neutrophils daily from myeloid precursor cells2.

Development of neutrophils in the bone marrow has classically been divided into five stages based on cell size, nuclear morphology, and granule content, and is controlled by granulocyte 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 circulate for 10 to 24 hours before migrating into the tissues3. Once in the tissues, neutrophils are known for their protective role against acute infections, but have also been implicated in the pathogenesis of multiple chronic inflammatory diseases4. Proper identification and characterization of neutrophils will play a significant role in characterizing neutrophil mediated diseases.

Neutrophil Identification

The literature is vast in describing different techniques to isolate and characterize neutrophils.

However, without a proper standard it is difficult to interpret disparaging reported results, likely caused by inconsistences between cell yields, viability, variation in cell surface marker

expression, and incorrectly identified cell populations 5-7. For instance, in a study that induced endotoxemia in humans, neutrophils were identified based on the expression of CD16 (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 the morphology of their nucleus normally described as having a distinct multilobulated appearance. Another defining characteristic of neutrophils is an abundance of cytoplasmic granules. These granules contain a variety of enzymes, membrane proteins and matrix proteins, and are divided into three major groups: primary (azurophilic), secondary (specific), and tertiary (gelatinase) granules13. The ability to correctly identify, and isolate neutrophils are critical for further understanding of both their biology and also the immune system in general.

Cluster of differentiation (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 scientists around the world at Human Leukocyte Differentiation Antigens (HLDA) Workshops15.

The CD marker and antigen system are used in many applications in research and medicine, including identification, localization, quantification, and isolation of cells, as well as for therapeutic applications. Immunophenotyping using CD markers are been useful for identifying cell population subsets, from stem cells and cancer cells to T-cell subsets.

Flow cytometry has become a popular method for evaluation of neutrophils16. However, identification and characterization and interpretation of flow cytometry results is still controversial, since a definitive CD marker set for neutrophils has yet to be identified17. CD14 and CD15 are among the markers that are commonly used to identify human neutrophils.

CD14 is a lipopolysaccharide-binding protein that acts as a co-receptor for bacterial binding lipopolysaccharide (LPS) and other pathogen-associated molecular patterns. Monocytes and most tissue macrophages are the other cell populations known to highly express CD14. CD14 is also expressed in granulocytes, and a small percentage of peripheral blood lymphocytes, albeit to a lesser extent18. CD15, a carbohydrate adhesion molecule, known to mediate chemotaxis and phagocytosis is also expressed in granulocytes. A significant proportion of monocytes, a subset of macrophages, and epithelial cells and their malignant counterparts may also express

CD156. Neutrophils are of utmost importance in a large number of clinical disorders. In order for us to interpret surface marker expression in disease, it is imperative that we first elucidate the pattern of expression of surface antigens on normal neutrophils.

High-throughput screening (HTS) flow cytometry

Cell surface molecules, collective named as the cell ‘‘surfaceome’’, are known to perform vital cell functions, acting as receptors, transporters, channels, and enzymes19. High-throughput screening (HTS) assays, commonly used to test chemical compounds in the pharmaceutical

industry, are now widely used to phenotype and categorize cells. Flow cytometry (FC) utilize fluorescently-tagged antibodies to detect cell surface proteins. The combination of HTS with FC 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 can be used to answer a wide range of research questions, such as characterization of cell populations, identification of cell subsets, isolation for functional assays and molecular profiling, development of therapeutic targets, or biomarkers that can be prognostic or associated with specific responses, and moreover, they can be used for disease 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 inflammatory responses.

2. CD expression and Neutrophil Distribution

Bone Marrow

Neutrophil Cell Lineage

Neutrophils are produced in the bone marrow by a process know as hematopoiesis22.

Neutrophil precursors are differentiated from hematopoietic stem cells that are driven toward the myeloid lineage, and further into mature neutrophils 23. The bone marrow is a complex tissue where multiple hematopoietic lineages coexist in various maturational stages. Knowing the expression levels of neutrophil specific markers and other lineage-specific markers during normal hematopoietic development gives us a standard by which to recognize abnormal

patterns of differentiation 23.

During granulocytic differentiation, myeloblasts, the most immature neutrophil precursor, cannot be discriminated from monoblasts, the most immature form of the monocytic lineage, since both share a common parental stem cell (Granulocyte-Macrophage colony forming unit –

GM-CFU). The maturation process is regulated by growth factors, such as GM-CSF, M-CSF and

G-CSF, as well as by cytokines and chemokines. The relative size of the cells, together with expression levels of specific markers such as CD34, CD117, CD45 (membrane-associated tyrosine phosphatase), CD13 (aminopeptidase N), CD33, CD16, and CD11b, can be used study granulocytic differentiation. The markers that have been used to identify neutrophils during different stages of hematopoiesis are summarized in Table 1.

Table 1 – Cell surface expression in different stages of granulocytic differentiation

Stage of PMN Surface Size Characteristics maturation expression

_ _ myelo/monoblasts CD16 CD13 12-20 µm Nucleus: round /ovoid _ int CD45 CD11b Ratio: 6:1

promyelocytes CD117 CD13high 15-21 µm Nucleus: round /ovoid CD33high CD15, CD34, MHCII Ratio: 4:1

Myelocytes CD13dim CD33dim 12-18 µm Nucleus: round CD34, CD15, /ovoid/flattened on one CD11b side

Ratio: 2:1

Metamyelocytes CD13 CD33dim 10-18 µm Nucleus: indented / kidney- CD34- CD15, shaped

CD11b, CD16 Ratio: 1.5:1

Band cell CD13 CD33dim 10-16 µm Nucleus: horseshoe CD34- CD15, CD11b, CD16 Ratio: 1: 2

Neutrophils CD13high CD33 9-16 µm Nucleus: segmented CD15, CD11bhigh CD16high Ratio: 1: 3

To identify purified populations of neutrophils and its precursors, some studies have utilized cellular size, granularity, and expression profile of the CD13, CD15, CD11b, and CD16 surface markers, after excluding CD3, CD19, CD14, glycophorin-A, CD56, and CD61 positive cells. CD13 shows dynamic changes in expression during granulocytic differentiation. In combination with

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- regulated on myelocytes (MC). CD13 expression is gradually up-regulated again as neutrophils reach their final stages of differentiation and develop into segmented neutrophils. On the other hand, CD11b and CD16, which are initially expressed at low levels, show progressively increased expression during the developmental process, particularly in the last two stages of neutrophil differentiation23,24. CD16 comprises the low affinity Fc 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) due to neutrophilic leukocytosis induced by tissue necrosis or bacterial infection.

Circulation

After being stored in the bone marrow for 5 to 7 days, neutrophils are released in the circulation and have a half-life of 6–9 hours in that compartment25. In the blood, neutrophils comprise about 70% of leukocytes and more than 90% of phagocytes. Neutrophils can reversibly move from circulating to marginating pools, where they are ‘stored’ in the capillaries of certain tissues, most notably in the lungs, due to the large number of small capillaries26.

A study by Kuijpers et al. showed that expression of surface molecules could be influenced by cell purification procedures. They identified 7D5 (cytochrome b, a-subunit) and CD10 as “early activation antigens,” because both antigens were absent prior to density-gradient neutrophil purification, but present on purified resting neutrophils27. Also, surface expression of several antigens that were expressed on circulating neutrophils 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-8 and HLA class I antigens27.

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 altered phenotype characterized by CD62Llow, an increased expression of CD11b, CD11c, and

CD54, and an equal expression of CD88, after endotoxemia was induced with LPS8. Others have demonstrated a subpopulation of neutrophils characterized by CD10-/CD16low that represents

40% of all circulating neutrophils immediately after cardiac and non-cardiac thoracic surgery, representing newly released neutrophils from the bone marrow during acute inflammatory

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 surface markers expression on blood and sputum neutrophils of patients with Chronic Obstructive Pulmonary

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, when they compare cell surface expression with clinical parameters of COPD, they reported that lower levels of CD11b in the blood correlated with clinical exacerbation of the disease30. In another study that evaluated tissue neutrophils in patients with Cystic Fibrosis

(CF) reported differences in the neutrophil phenotype with up-regulation of CD63, a member of tetraspanin family and marker for azurophilic granule fusion, lose of expression of CD16 and

CD14, and expression of CD80, major histocompatibility complex type II, and CD294 a prostaglandin D2 receptor, which are normally associated with other 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 possible target for treatment of neutrophil mediated disease.

3. CD expression and Neutrophils Functionality

Neutrophil Recruitment

The recruitment of neutrophils from the vasculature into inflamed tissue is key for a proper host defense against invading microorganisms 32. This process occurs mainly in the microvasculature, where hemodynamic shear forces are minimized, and involves the following steps: tethering (or capture), rolling, slow rolling, arrest, adhesion, crawling and transmigration in a cascade-like fashion 2. In each step of neutrophil recruitment and transmigration, integrins and selectins play a crucial role, where they can act as ligands and receptors for neutrophils to fulfill these important functions. I will explore the role of some of those markers such as

CD11a, CD11b, CD18, CD62L (Table 2).

Table 2 – Cell surface markers expressed in neutrophils associated with recruitment and migratory functions Functions CD markers Cellular adhesion molecules CD44, CD50, CD54, CD56, CD102, CD106, CD146, CD166, CD321, CD322, CD326 Integrins CD11a, CD11b CD18, CD29, CD49a-f, CD51, CD61, CD104 Selectins CD62E, CD62L, CD62P Chemokine receptors CD117, CD119, CD121a, CD123, CD124, CD126, CD127, CD140a, CD140b Membrane-bound CD95, CD178, CD120a, CD12b receptors involved in apoptosis or necrosis

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. Tethering and rolling are mediated by the up-regulation of E-selectin (CD62E) and

P-selectin (CD62; CD62P) on the vascular endothelial cell surface, which bind to neutrophil expressed glycosylated ligands, such as P-selectin glycoprotein ligand-1 (PSGL-1) or CD162 32.

Rolling neutrophils are activated by cytokines present on the surface of endothelial cells, which promote the activation of integrins such as LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) on the cell surface of neutrophils. These integrins bind to ICAM1 and ICAM2 on endothelium to 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 productive migration neutrophils must release the existing integrin-ligand associations at the rear of the cell while forming new bonds at the leading edge. This allows them to maintain a firm adhesion to the vascular wall until they find a preferential site where they can exit the vasculature in a process called transendothelial migration 2. Transmigration requires integrins and CAMs (ICAM1 also known as CD54), ICAM2 or CD102 and vascular cell adhesion protein 1 (VCAM1 or CD106) as well as different junctional 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 endothelial cell), however transcellular transmigration is a less efficient method, and can take

up to 30 minutes to complete. Once they enter the three-dimensional tissue matrix, neutrophils undergo directional migration towards a concentration gradient of chemoattractant, through a process known as chemotaxis33.

Bacterial killing and Phagocytosis

Neutrophils are highly phagocytic cells. Consistent with this is their high surface expression of low affinity Fc receptors such as CD16 and CD3234. Neutrophils destroy invading microorganisms, by exposing them to oxidative-dependent and/or independent mechanisms.

This can be accomplished by engulfing pathogens or by the release of neutrophil extracellular traps (NETs). Neutrophils kill phagocytosed microorganisms through enzymatic and oxidative destruction. To finalize these processes, and effectively dispose engulfed particles, neutrophils die by apoptosis and are then themselves phagocytosed by macrophages, thus preventing the release of cytotoxic products35,36.

When neutrophils reach the site of inflammation, they are exposed to an environment full of inflammatory mediators, bacteria, fungi, cell debris, apoptotic cells, etc37. An array of neutrophil cell surface receptors recognize agonists such as peptide sequences, surface binding proteins, secreted bacterial products, opsonized host proteins, components of the complement cascade, double stranded RNA and Fc domains of antibodies37. Neutrophils can also recognize non-opsonized ligands, such as lipopolysaccharides (LPS), or particles opsonized with host-derived proteins called opsonins36. Once neutrophil surface receptors recognize a cognate ligand, the receptor is ‘activated’ and a signal is transmitted to the interior of the cell resulting in the initiation of phagocytosis38. This process is marked by activation of Src-tyrosine

kinases, which trigger the aggregation of CD32 (FCGR2A) and CD16 (FCGR3A) and the phosphorylation of their cytoplasmic immunoreceptor tyrosine-based activation motifs

(ITAMS), which triggers the activation of PI3K and Rho proteins39. Active Rho proteins induce extension of membrane protrusions over the surface of the target particle and engulfment 40.

To simplify, the main mechanism by which bacterial recognition and neutrophil activation are regulated, is by the interaction of pathogen-associated molecular 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.

Although neutrophils present with selected specificity between its component and microbial byproducts, such as TLR4 (CD284) interacts with lipopolysaccharide (LPS) from Gram-negative bacteria and TLR2 (CD282) with peptidoglican, lipoteicoic acid from Gram-positive bacteria, among others. Moreover, the generation of reactive oxygen species can be triggered through

TLRs. Which, in turn, can result in up regulation of CD64, a high affinity Fc receptor expressed in neutrophils, resulting in increased oxidative burst and phagocytic potential 41.

Phagocytosis by neutrophils is more efficient when microbes are coated with serum host proteins called opsonins, which include complement proteins and antibodies. Activation of a complement leads to deposition of complement components on microbial surface.

Complement surface receptors (CRs) expressed on the surface of neutrophils efficiently recognize microbes bound with complement components. CRs include CIqR, CR1 (CD35), CR3

(CD11b/CD18) and CR4 (CD11c/CD18)42.

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 independent mechanism40. 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, the secretory vesicles have preference for extracellular release. These vesicles provide these cells with cell surface receptors and molecules that are vital, although in low levels, for neutrophil activation. Secondary granules in neutrophils include a large number of key cell adhesion molecules, such as CD11b/CD18, CD47 and SIRPα (CD172a), which are required for neutrophil adhesion and chemotaxis44. Upon stimulation neutrophil granules will fuse with a 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 receptors which include CD23 (FcεRI, IgE receptor), CD89 (FcαR, IgA receptor), CD64 (FcγRI, IgG

receptor), CD32 (FcγRIIa, low affinity IgG receptor) and CD16 (FcγRIIIb, low affinity IgG receptor) will play a critical role in the ability of neutrophils perform their basic functions42.

Non-traditional functions of neutrophils

Classically neutrophils are viewed as short-lived cells with a main phagocytic role. In more recent years, the neutrophil have also been described as having an active regulatory role in angiogenesis and tumoral fate, as well as, neutrophils can influence the immune response by releasing a variety of cytokines and by acting as antigen-presenting cell (APC) expressing MHC

Class II, and finally by stimulating T cells activation12,46,47. Nowadays, a number of diseases including periodontitis, arthritis and acute respiratory distress syndrome (ARDS) are associated with neutrophil dysregulation that may results in significant tissue damage. I will focus in periodontal disease as a model for studying nuances of the neutrophil cell surface signature.

4. Periodontal Diseases

Periodontal diseases encompass a variety of diseases affecting the health of the periodontium.

They range in severity from a reversible inflammation of the gingiva termed gingivitis, which is 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 eventual tooth loss, known as periodontitis48.

Chronic Periodontitis

Chronic Periodontitis (CP) is recognized as an immune-mediated response to a plaque biofilm resulting in inflammation within the supporting tissues of the teeth, which leads to a progressive loss of attachment and bone destruction. According to the American Academy of

Periodontology (AAP) classification, it can be further classified as localized or generalized, and with one of the possible severities: slight, moderate or severe. Environmental factors such as smoking and emotional stress can modify the severity and progression of the disease, as well as systemic diseases such as diabetes48.

The role of neutrophils in the pathogenesis of Periodontitis

Neutrophils are recognized as key player in mediating periodontal tissue destruction. There are two main mechanisms described by which neutrophils would mediate tissue destruction: by an impaired phenotype and by a hyperactive phenotype 49. A third possible mechanism by how neutrophils would mediate periodontal destruction would be via recruitment and activation of the normal neutrophil49,50.

The exact mechanism of how periodontal destruction occurs and why some patients are more susceptible to tissue damage than others remains unknown51. However, advances in the field of cell and molecular biology indicate a multifactorial etiology for periodontitis.

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 four stages: 1) initial lesion, 2) early lesion, 3) established lesion, and 4) advanced lesion. For the first time, an increased numbers of neutrophils migrating into the junctional epithelium and underlying connective tissue was described52. The combination of this initial work with recent knowledge of molecular biology were the basis for the current model for pathogenesis

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, is not enough to cause disease. Systemic conditions, environmental factors and the host genetic susceptibility, such as diabetes mellitus, smoking and IL-1α pleomorphism, would play a role in modulating the host response. Ultimately, and likely the most significant, the host immune response would determine the clinical outcome. Lipopolysaccharides (LPS) are released from bacterial biofilm into the periodontal tissues. Which would activate cells from the junctional and pocket epithelium to replicate and release inflammatory mediators. These pro-inflammatory cytokines, IL8 and IL-lα, would recruit and activate neutrophils, which, in turn, would secrete matrix metalloproteinases (MMPs). These molecules are able to facilitate destruction of the extracellular matrix of the gingiva and periodontal ligament and induce resorption of the alveolar bone. An inflammatory infiltrate formed mostly by neutrophils, but also containing B & T lymphocytes, and monocytes/macrophages will be present. Additionally, oral inflammation increases production of cytokines, such as interleukin-6, that in turn, stimulate osteoclast activity and promote bone resorption.

5. Objectives of this study and hypothesis

Objectives

In the current study, I used isolated neutrophils from peripheral blood and from the oral cavity of healthy individuals and patients with generalized severe chronic periodontitis, to understand changes in the neutrophil cell-surface signature, as they transit from the blood into different locations and activation states.

To summarize, my objectives with this study were:

• To use HTS Flow Cytometry as a tool to identify a unique neutrophil cell-surface

signature that can be used to identify neutrophils, which is independent of its state of

activation and/or location.

• To validate a specific set of cell surface markers, by FACS and/or magnetic

immunoselection, that can be used to identify neutrophils.

Hypothesis

• The neutrophil has a unique cell-surface signature, which is independent of its location

and state of activation.

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, I could confirm the level of expression of all known CD makers in the cell surface of neutrophils. This screen allowed the identification of CD11b, CD16b, and

CD66c as markers that are always expressed on neutrophils independent of the cell location, level of activation and disease state.

Chapter 2 Immunophenotypical characterization of human neutrophils

Flavia S. Lakschevitz, Siavash Hassanpour, Ayala Rubin & Michael Glogauer. Identification of Neutrophil Surface Marker Changes in Health and Inflammation using High-throughput Screening Flow Cytometry (Manuscript in preparation for submission)

Abstract

Neutrophils are the most abundant white blood cell and are an essential component of the innate immune system. A complete cataloguing of cell surface markers has not been undertaken for neutrophils isolated from circulation as well as healthy and inflamed tissues. To identify cell-surface markers specific to human neutrophils, we used high-throughput flow cytometry to screen neutrophil populations isolated from blood and oral rinses from healthy and chronic periodontitis patients against a panel of 374 known cluster of differentiation (CD) antibodies. This screen identified CD11b+, CD16+, and CD66b+ as markers that are consistently expressed on neutrophils independent of the cell location, level of activation and disease state.

Cell sorting against CD11b, CD16 and CD66b allowed for the enrichment of mature neutrophils, yielding neutrophil populations with up to 99% purity. These findings suggest an ideal surface marker set for isolating mature neutrophils from humans. The screen also demonstrated that tissue neutrophils from chronically inflamed tissue display a unique surface marker set compared to tissue neutrophils present in healthy, non-inflamed tissues.

Introduction

A neutrophil cellular infiltrate is commonly found in the oral cavity of healthy and diseased subjects55. Nevertheless, patients diagnosed with chronic periodontitis, a neutrophil-mediated inflammatory condition that affects the supporting tissues of the teeth resulting in irreversible tissue destruction, have higher levels of neutrophils in the oral cavity56-58. The abundance of oral neutrophils in both health and disease provides a non-invasive means of harvesting transmigrated neutrophils through oral rinses. Isolated oral neutrophils (PMN-O) can then be examined to determine phenotypical changes of tissue neutrophils in healthy and inflamed tissues compared to the basal phenotype of circulatory blood neutrophils (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 immune system. Unfortunately, non-standardized approaches in neutrophil isolation protocols leads to inconsistencies between studies in terms of cell yields, purity and viability 5-7. Flow cytometry is commonly used to evaluate neutrophil surface markers16. However, the identification and characterization of neutrophils using this technology is far from uniform in the field since a definitive marker set for neutrophils has yet to be identified8. Currently CD11b,

CD14, CD15, CD16 and CD62L are used solely or in some combination to identify pure human neutrophil populations 8,10,17,60,61. Likewise, CD11b and Ly6G have been used to identify mouse neutrophils62,63. Presently, there are references and web resources that characterize all known

CD markers in all cell types based on a cataloguing of existing studies. Despite over 370 known

CD markers, most studies evaluate one or two markers at a time with non-uniform patient populations and cell isolation protocols. Therefore, conclusions drawn from reported studies

are not always comparable 64, resulting in conflicting data about the presence of many markers 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 imperative that we first elucidate the baseline expression profile of surface antigens on neutrophils in health. The use of high-throughput screening (HTS) flow cytometry gives us the opportunity to rapidly evaluate all known CD markers simultaneously in an unbiased manner 20,65. On it’s own, HTS is a discovery-oriented screening tool that allows for identification of surface proteins in cells of interest by using a large panel of CD markers in a highly reproducible fashion. In addition, HTS can be combined with fluorescent cell barcoding, sorting66 and functional assays that ultimately allow to for the identification and characterization of a unique set of biomarkers in health and disease. In this study, our ultimate aim was to identify a unique and universal cell-surface signature in neutrophils independent of its location and state of activation. A consistent and well defined neutrophil signature would permit rapid and reproducible isolation of circulatory mature neutrophils. In parallel we aim to demonstrate that the signature we identify does not change significantly as neutrophils transmigrate from circulation into healthy and inflamed tissues.

Materials and methods

Study Population

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

periodontal disease (Healthy), and ten patients with generalized severe chronic periodontitis

(CP), were enrolled in this study. All enrolled participants were systemically healthy and self reported non-smokers. After a complete intraoral examination, blood and oral rinse collection was conducted. Participants provided written informed consent in order to participate, and the

Scientific and Ethics Review Boards at the University of Toronto, Faculty of Dentistry, approved the study (Protocol # 29410).

Blood collection and processing

Blood samples were drawn from the antecubital vein into a vacutainer containing 0.1 volume of sodium citrate anticoagulant (Becton Dickinson, Canada). Samples were divided in two. For the first set, neutrophils were separated using 1-layer gradient of 1-step polymorphs as previously described69. Briefly, gradients were centrifuged at 527 relative centrifugal force for

30 minutes at room temperature, the lower of two bands was collected. Cells were washed with Hanks balanced salt solution without calcium or magnesium (HBSS-/-), and erythrocyte lysis was performed by incubating with 1mL of BD Pharm Lyse buffer. For the second set, only erythrocyte depletion was performed by incubating the samples at 4oC for 5 min with 1mL of

BD Pharm Lyse buffer (Becton Dickinson, Canada). Cell count was obtained using an automated counter (Coulter Counter, Beckman Coulter, Brea, CA), and viability of cells was assessed by trypan blue exclusion test.

Oral sample collection and processing

The oral cavity of each participant was rinsed for 30 seconds with 0.9%NaCl and collected into a 50 mL Falcon tube, as previously described56. This sequence was repeated 5-20 times (5 – 10x

for CP patients and 15 – 20x for healthy patients) with three minutes intervals between rinses.

The collected material was divided in two. The first set was passed sequentially through a

40µm, 20µm and 11µm nylon mesh to eliminate epithelial cell contamination and was used for

High-throughput screening assay. For the second set only 40µm filtration was performed and 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 assessed by trypan blue exclusion test. To minimize phenotypic and functional changes as a result of cellular activation, all steps of the density gradient separation procedure, sorting and cell staining procedures were performed immediately after cell collection at 4°C using non- pyrogenic reagents and plasticware. oral PMN isolation would start at chairside at 4°C, simultaneously to the oral rinse collection.

High-throughput screening assay

Twenty million viable cells were resuspended in HBSS-/- supplemented with 2mM EDTA and 1%

BSA (Flow buffer), immediately after collection and isolation from blood and oral cavity. Cells were blocked with rat and mouse IgG (1 mg ml-1) at 1:100 dilution for 10 min and 500 microliters of cells were removed to serve as controls. All cells were resuspended in staining 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

(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 controls for each of the fluorophores used (PE, APC and FITC) and control for viability staining.

The high-throughput flow cytometry analysis was performed using a BD-high-throughput sampler LSRII flow cytometer (Becton Dickinson, Canada), with default filter configuration and compensation was set by using BD CompBeads (Becton Dickinson, Canada) at UHN Antibody

Core Facility, Toronto, ON, as previously described20, 2 to 5 days after sample preparation.

Four to six hours were required to prepare each sample.

Data acquisition and analysis

Each well of the 96-well plate was sampled with a total read time of 30 min. For each well,

1,000-10,000 cells were analyzed; for increased reliability samples with less than 1,000 events were excluded from our analysis. An initial template file was generated using electronic gates within the software to create a hierarchical population tree at the beginning of the screen, and using the template, all additional analyses were completed after data acquisition was complete. The template file included compensation adjustment, which was uniformly applied to all the data collected in order to minimize fluorescence overlap between detection channels. The following gating strategy was used: a forward and side scatter profiles (plot of

FSC-A vs. SSC-A) were used to visually identify the neutrophil population and eliminate debris.

Gates were manually drawn to remove cell doublets (plots of FSC-W vs. FSC-H and then SSC-W vs. SSC-H) and subsequently to select viable cells based on exclusion of eFluor506 (or eFluor

780) positive cells. Next, two-dimensional plots were used (APC vs. PE and/or FITC vs. PE).

Finally, one-dimensional histograms for each fluorescence parameter were constructed for the identified cells. “Positive” and “negative” gating of the fluorescence signal was drawn based on fluorescence intensity of the positive and isotope controls, and these gates were applied to all individual wells on a per plate basis (Supplemental Figure 1 and 2). This template was applied to all subsequent plates. If required, a manual adjustment of each gate was completed before data analysis. All data (FCS 3.0 files) were exported and then analyzed with FlowJo software

(Treestar).

Cell Sorting

Cells were sorted at a concentration of 106 cells/ml in PBS/25% BSA using a FACSAria cell sorter

(BD Bioscience) at the Flow Cytometry facility at Department of Immunology, University of

Toronto, Toronto, ON. To prevent cell death due to pressure and sheer stress, all sorts were 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 (or eFluor 780) positive cells.

Cytospin Preparation and Staining

In order to confirm the purity of neutrophils, sorted cells were cytocentrifuged and mounted on slides using a Cytospin centrifuge (Shandon, Ramsey, MN) for 5 min at 800 RPM, as previously described70. Briefly, cytospins of sorted populations were fixed in methanol for 30 seconds at room temperature. The cytospins were then air dried and stained with Diff-Quik

(Fisher Scientific) staining kit according to the manufacturer’s instructions. The cells were examined by light microscopy at 200 and 400× final magnification.

Statistical Analysis

Statistical analysis was performed using one-way ANOVA with Bonferroni's multiple comparisons test, unless specified otherwise. P ≤ 0.05 was considered statistically significant

(GraphPad software). Heatmaps, hierarchical clustering analyses were performed using

MultiExperiment Viewer (MeV) software (www.tm4.org). Unsupervised hierarchical clustering was performed using a Pearson correlation distance metric with complete linkage clustering.

Results and Discussion

Cell surface marker screen of neutrophils from blood and oral rinses in health and during chronic oral inflammation.

Neutrophils were isolated from blood and oral rinses of healthy and chronic periodontitis (CP) patients and screened against a panel of 374 known CD antibodies. The gating strategy used

(see methods) yielded a highly pure neutrophil population (≥99% of mature neutrophils) as confirmed by visual inspection with light microscopy following cell sorting (Figure 3). Based on the screens, it was evident that neutrophils express 145 different CD markers in at least on of the four compartments (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 (Figure 4 and Supplemental table S2). The newly identified cell-surface markers not previously described were CDw198, CDw199, CD322 and CD328. Further, the expression of five

CD markers (CDw12, CD156a, CD156c, CD285, CD361) previously reported in the literature could not be confirmed, as these markers were not part of our panel. Lastly, the expression of

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 receptor 8 (CCR8), with a suggested role in regulation of monocyte chemotaxis and thymic cells apoptosis. CDw199 is C-C chemokine receptor 9 (CCR9), and is known to be expressed in the lymphoid tissue located in the large intestine and is up-regulated during dextran sulfate sodium induced colitis 71. CD322 is a member of the immunoglobulin superfamily, known to be expressed in epithelial cells as well in monocytes and plays a role in leukocyte transmigration during inflammation72. CD328 or Siglec-7 is primarily found in NK cells where it works as an inhibitory receptor73. Future research will be required to investigate the possible role of these molecules in neutrophils.

Neutrophils present with a unique signature based on their location

The current neutrophil literature acknowledges that neutrophils are highly 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 cytometric analysis for immunophenotyping, we narrowed our focus from 147 markers to 90

CD markers on neutrophils isolated from blood and oral rinses of 10 healthy controls and 10 CP patients. The 90 CD markers were chosen based on previous knowledge of reported expression in the literature. Also, constitutively expressed markers with constant expression independent of location or underlying disease were included in the panel of chosen CD markers. In addition, markers with markedly varied expression following transmigration out of circulation and into the oral cavity were also selected for further analysis. Lastly, markers reportedly not expressed in neutrophils were included as negative controls.

With the selected 90 CD markers, an unsupervised hierarchical clustering using percent- positive values was preformed to determine if cell surface profiling could be used to define a subtype signature for neutrophils (Figure 5 and 6). Cell surface phenotyping stratified samples into clusters related to the origin such that PMN-B, PMN-O each formed a cluster, irrespective of disease status (Figure 5). Further exploration identified a site-specific neutrophil CD marker profile that was further altered in the presence of local inflammation. The inflammatory PMN-

O profile was characterized by increased expression of markers associated with an inhibitory role of the neutrophil function with up-regulation of CD85a, CD305 and CD312. Similar to a report by Baudhin et al.77, expression of CD85a was almost absent in PMN-B. Conversely,

CD85a was highly expressed in the PMN-O, which may be indicative of the activation state of

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 CD181 were also found to be up-regulated in PMN-B, both of which are reported to enhance neutrophil chemotaxis and function 59,78. Although not statistically significant, CD114, CD132 and CD182 also displayed increased expression in PMN-B fraction of healthy patients. It was previously reported that CD116, CD132, CD182 and CD217 are cytokine receptors that allow neutrophils to respond to extracellular inflammatory cues 79.

CD132 along with CD122 are functional subunits of IL-15 receptor (IL15R) 80. IL-15 is a proinflammatory cytokine that plays a role in enhancing neutrophil phagocytosis. Periodontitis is a common chronic inflammatory disease that is triggered by pathogenic microflora in the biofilm or dental plaque that forms around to the teeth81,82. There has been a recent paradigm shift in our understanding of the etiology of periodontitis from a biofilm driven condition to a condition mediated by a misdirected immunological response to a bacterial insult. Although the presence of bacteria is essential for the initiation and progression of periodontal disease, the simple presence of bacteria does not lead to tissue breakdown; rather neutrophils play a central role in maintaining periodontal health9. Chronic periodontitis is associated with a hyperactive neutrophil response that includes hyperactive oxidative stress and secretion of inflammatory mediators as well as phagocytic abnormalities 82. The ability of neutrophils to regulate phagocytosis, by altering the expression of CD44 and CD116 can play a major role in balancing health and disease at the biofilm-tissue interface where a multitude of bacterial species and constant influx of PMN-O constantly interact. Our data clearly demonstrate down- regulation of both markers in PMN-O of CP patients when compared with healthy controls.

Oral neutrophils in CP patients are characterized by an active phenotype.

The CD marker profile of PMN-O of CP patients is characterized by an increased expression of

CD11b, CD63, CD66, CD66b, CD66c and CD66e (p-value > 0.05, CTR PMN-O vs. CP PMN-O)

(Figure 7). CD11b, also known as Mac-1 α, is an integrin αM chain and is part of the 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, member of the tetraspanin family that upon activation is strongly expressed on the surface of neutrophils and is therefore considered as a marker for granule release27,84. Similar to other members of the tetraspanins family, CD63 operates through interaction with integrins, most likely CD11b/CD18. In addition to serving as a marker of cell activation and mediating granule release, CD63 has also been reported to play a role in mediating membrane fusion events 85.

Neutrophils are known to express several glycosylated carcinoembryonic antigen (CEA)-related glycoproteins (CD66 antigens)86. These markers play a role in adhesion to E-selectin and their up-regulation is associated with activation of neutrophils, activation of β2-integrins, priming of the respiratory burst and mediating cell shape change 87. Taken together we speculate that as part of the chronic oral inflammation process, PMN-O alter their basal CD marker expression profile to one that is characterized by an active phenotype. These results likely reflect the neutrophil activations state in a chronic inflamed environment. Of particular interest is the lack of these markers on oral neutrophils present in the tissues of healthy mouths. These cells are certainly recruited by the bacterial biofilm present in the mouths of healthy patients but are not activated to the same extent as the oral neutrophils in patients with periodontal disease.

This could be due to the differences in the biofilms in health and disease88 or possibly to variations in patient neutrophil responses and sensitivity to the biofilms present.

Cell sorting with an antibody against CD11b, CD16 and CD66b allowed for the enrichment of highly pure mature neutrophils in the blood

Density gradients used to isolate neutrophils result in neutrophil cell purity of ≥95%, with contaminating cells being identified as 1-5% for eosinophils, basophils, mast cells and 1–2% 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 increased purity. Cell surface markers have been used for the identification and isolation of neutrophils by FACS and immunoselection8,12,90 . Denny et al. (2010) described a method of 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- surface marker signature that can be used to isolate neutrophils, since most markers are co- expressed by other cell types that commonly contaminate neutrophil preparations (Table 1 summarizes known expression and functions of these markers). Among the CD markers that were expressed in all analyzed samples, independent of the compartment or activation state, we found CD11a, CD11b, CD13, CD16, CD18, CD55, CD66b, CD170 and CD172 to be each consistently expressed by more than 90% of neutrophils (Figure 8 – A). By selecting 3 markers

CD11b, CD16 and CD66b, we were able to determine a unique neutrophil signature (Figure 8 –

B). CD11b is an integrin family member, which pairs with CD18 to form the CR3 heterodimer and is the most commonly used marker in neutrophil biology. Studies have shown CD11b to be directly involved in cellular adhesion, however migration will only take place if CD18 subunit is present. CD11b is known to be expressed in many leukocytes subsets including monocytes, neutrophils, natural killer cells, and macrophages91,92. CD16 is glycosyl phosphatidyl inositol-

anchored (GPI) protein that acts as a receptor for the Fc region of immunoglobulin gamma (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, member of carcinoembryonic antigen family. Its main function is to mediate phagocytosis27. By combining the three aforementioned markers would allow to select a pure mature neutrophil population, by excluding the main contaminating cells, while select all the cells of interest. We sorted whole blood cells, after erythrocyte lysis, and oral rinse samples, based on expression of

CD11b, CD16 and CD66b. Purity and morphology of sorted samples was further analyzed by light microscopic examinations of cytospins preparations (Figure 4 - C). After using these 3 markers combined, eosinophils were the prevalent contaminating cells in the blood samples

(0.4± 0.3% of cells), while epithelial cells constitute the majority of contaminants in the oral 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 neutrophil mediated diseases.

Conclusions

Flow cytometry is routinely used to determine if a selected cell population is expressing a particular protein or receptor of interest. In addition, flow cytometry has been used to quantify 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 molecule between two or more cell populations 95. However, without a clear definition of cell- surface expression profile of each cell population, interpretation of data might me compromised. The introduction of high-throughput flow cytometry with it’s ability for a multi- parameter analysis and a rapid functional profile of specific cell populations has provided us with an alternative to current methods and produces results that are reliable and reproducible.

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 identified a unique neutrophil signature with a combination of selected markers including CD11b, CD16 and CD66. We were also able to distinguish between neutrophil phenotypes during chronic inflammatory disease and health. Oral neutrophils of chronic periodontitis patients are characterized by active neutrophil phenotype with up- regulation of members of the integrins family such as CD11b. We also noted up-regulation of

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 researchers in the field.

Authorship

F.L. designed, conducted experiments, analyzed data, interpreted experiments and wrote the paper. S.H and A.R. wrote the paper. M.G. designed the study, interpreted experiments and wrote the paper.

Acknowledgments

The authors give special thanks to Joshua Paterson from the UNH Antibody Core Facility for his assistance with HTS-Flow Cytometry and to Dionne White from Flow Cytometry facility at

Department of Immunology, University of Toronto, Toronto, ON for her assistance with cell sorting.

Sources of support: This work was funded by The Canadian Institutes of Health Research

(CIHR, Ottawa, ON).

Disclosures: The authors report no conflicts of interest related to this study.

Chapter 3 Thesis Summary and Future Directions

Summary

Periodontitis is a common neutrophil-mediated disease that affects the supporting tissues of the teeth resulting in irreversible alveolar bone loss9,96. Patients diagnosed with chronic periodontitis have an increase influx of neutrophils to the oral cavity56, which allows 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.

Detailed characterizations of neutrophils prove to be critical for further understanding of both neutrophil biology and also the immune system in general. Inconsistencies between cell yields, viability, or cell surface marker expression, or even incorrectly identified cell populations are likely to account for some of the variations among previous studies of neutrophil functions5-7.

Until now, visual inspection under a light microscope with May-Grünwald-Giemsa, or a similar 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 staining with neutral dyes. The neutrophil nucleus has a distinct multilobulated appearance97.

Another defining characteristic of neutrophils is an abundance of cytoplasmic granules. These granules contain a variety of enzymes, membrane proteins and matrix proteins, and are

divided into three major groups: primary (azurophilic), secondary (specific), and tertiary

(gelatinase) granules98.

Although, flow cytometry has been commonly used for evaluation of neutrophils16. A proper identification and characterization and how to interpret flow cytometry results was still controversial, since a definitive marker for neutrophils needed to be identified17. CD14, CD15,

CD16 and CD62L were among the commonly used markers to identify human neutrophils8,10,60,61. With over 350 known CD markers, the use of references and web resources 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 neutrophils and other cell types15

The use of high-throughput screening (HTS) flow cytometry gave us the opportunity to rapidly evaluate all known CD markers simultaneously99. In this study our ultimate aim was to identify a unique cell-surface signature in neutrophils, which is independent of its location and state of 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 cluster analysis: 1) we were able to discriminate the location of the neutrophils; 2) we were also able to distinguish between neutrophil phenotypes during chronic inflammatory disease and health. Finally, we could demonstrate that oral neutrophils of chronic periodontitis

patients are characterized by active neutrophil phenotype with up-regulation of members of the integrins family such as CD11b with up-regulation of 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 researchers in the field.

Future Directions

This study shows a shift in the paradigm from the current gold standard for neutrophils identification and characterization by visual inspection, under a light microscope, to the use of

CD markers expression in the cell surface of neutrophils. Although, neutrophils can still be recognized by their well known nuclear morphology. It is important to recognize that neutrophil subsets can present with an altered morphology, and thus, visual inspection, may reduce our ability to correctly identify different neutrophil subsets or sub-populations. For example, when Fridlender and colleagues described a new subset of neutrophils in a tumoral site, each subset presented with a distinct morphological characteristic. N1 neutrophil presented with a hypersegmented nucleus, whereas N2 had a circular nuclear morphology100.

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- throughput flow cytometry to identify simultaneously over 350 clusters of differentiation (CD) markers expressed on the cell surface of neutrophils. One of the many advantages of this

technique is the ability to perform multiple analyses on each cell in a sample, known more commonly as multiplexing 99. Using this approach we were be able to identify specific markers in the neutrophil cell surface that would allow us to later characterize neutrophil subsets in health and disease.

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 importance for the migration of various cell types into the inflammatory sites102. Also it would 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 patients at risk of activation of the neutrophil mediated-disease.

Finally, I believe that exploring the difference in regulation of key cell surface markers in the neutrophils, during health and chronic inflammation will not only allow us to gain further insights into inflammatory disease pathogenesis as well as allow for these surface markers to be potentially be used as biomarkers for specific diseases. Hopefully, in the near future, the use of selected surface markers can be used as biomarkers to identify patients at risk of developing neutrophil mediated diseases.

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SUPPLEMENTAL FILES

Supplemental Table S1 - Antibodies use in this study # Antibody Clone Conjuga Isotype Supplier Catalogue NCBI NCBI NCBI tion Gene Other Gene ID Name Names 1 CD1a HI149 PE Mouse BD 555807 CD1A CD1 909 IgG1, κ 2 CD1b M-T101 FITC Mouse BD 555969 CD1B CD1 910 IgG1, κ 3 CD1c L161 PE Mouse BioLege 331506 CD1C CD1 911 IgG1, κ nd 4 CD1d CD1d42 PE Mouse BD 550255 CD1D none 912 IgG1, κ 5 CD2 RPA- PE Mouse BD 555327 CD2 SRBC; 914 2.10 IgG1, κ T11 6 CD3 HIT3a PE Mouse BD 555340 CD3E see 916 IgG2a, к CD3D, CD3E, CD3G 7 CD3e UCHT1 APC Mouse R&D FAB100A CD3E CD3- 916 IgG1 System EPSILON s ; T3E; TCRE 8 CD4 RPA-T4 PE Mouse BD 555347 CD4 none 920 IgG1, κ 9 CD5 UCHT2 PE Mouse BD 555353 CD5 LEU1; 921 IgG1, κ T1 10 CD6 M-T605 PE Mouse BD 555358 CD6 TP120 923 IgG1, κ 11 CD7 M-T701 PE Mouse BD 555361 CD7 GP40; 924 IgG1, κ LEU-9; TP41; Tp40 12 CD8 HIT8a PE Mouse BD 555635 CD8A CD8; 925 IgG1, κ Leu2; MAL; p32 13 CD8b 2ST8.5H PE Mouse BD 641057 CD8B CD8B; 926 7 IgG2a, к LYT3; Leu2; Ly3 14 CD9 M-L13 PE Mouse BD 555372 CD9 BA2; 928 IgG1, κ DRAP- 27; MIC3; MRP-1; P24 15 CD10 HI10a PE Mouse BD 557143 MME CALLA; 4311 IgG1, κ CD10;

NEP 16 CD11a HI111 PE Mouse BD 555384 ITGAL CD11A; 3683 IgG1, κ LFA-1; LFA1A 17 CD11b ICRF44 PE Mouse BD 555388 ITGAM CD11B; 3684 IgG1, κ CR3A; MAC-1; MAC1A; MO1A 18 CD11c B-ly6 PE Mouse BD 555392 ITGAX CD11C 3687 IgG1, κ 19 CD13 WM15 PE Mouse BD 555394 ANPEP CD13; 290 IgG1, κ LAP1; PEPN; gp150 20 CD14 M5E2 PE Mouse BD 555398 CD14 none 929 IgG2a, к 21 CD15 HI98 PE Mouse BD 555402 FUT4 CD15; 2526 IgM, k ELFT; FCT3A; FUC-TIV 22 CD16 3G8 PE Mouse BD 555407 FCGR3 CD16; 2214 IgG1, κ A FCG3; FCGR3; IGFR3 23 CD16b CLB- PE Mouse BD 550868 FCGR3 CD16; 2215 gran11. IgG2a, к B FCG3; 5 FCGR3 24 CD17 not FITC Mouse Lifespa LS- carboh carbohy N/A not given IgM n C78376 ydrate drate a Bioscie protein nces 25 CD18 6.7 PE Mouse BD 555924 ITGB2 CD18; 3689 IgG1, κ LAD; LCAMB; LFA-1; MF17; MFI7 26 CD19 HIB19 PE Mouse BD 555413 CD19 B4; 930 IgG1, κ MGC128 02 27 CD20 2H7 PE Mouse BD 555623 MS4A B1; 931 IgG2b, k 1 Bp35; CD20; LEU-16; MGC396 9; MS4A2; S7 28 CD21 B-ly4 PE Mouse BD 555422 CR2 C3DR; 1380 IgG1, κ CD21 29 CD22 S-HCL-1 PE Mouse BD 347577 CD22 SIGLEC- 933

IgG2b, k 2 30 CD23 M-L233 PE Mouse BD 555711 FCER2 CD23; 2208 IgG1, κ CD23A; FCE2; IGEBF 31 CD24 ML5 PE Mouse BD 555428 CD24 CD24A 100133 IgG2a, к 941 32 CD25 M-A251 PE Mouse BD 557138 IL2RA CD25; 3559 IgG1, κ IL2R; TCGFR 33 CD26 M-A261 PE Mouse BD 555437 DPP4 ADABP; 1803 IgG1, κ ADCP2; CD26; DPPIV; TP103 34 CD27 M-T271 PE Mouse BD 555441 CD27 CD27; 939 IgG1, κ MGC203 93; S152; T14; Tp55 35 CD28 CD28.2 PE Mouse BD 555729 CD28 Tp44 940 IgG1, κ 36 CD29 MAR4 PE Mouse BD 555443 ITGB1 CD29; 3688 IgG1, κ FNRB; GPIIA; MDF2; MSK12; VLAB 37 CD30 BerH8 PE Mouse BD 550041 TNFRS CD30; 943 IgG1, κ F8 D1S166E ; KI-1 38 CD31 WM59 PE Mouse BD 555446 PECA CD31 5175 IgG1, κ M1 39 CD32 3D3 PE Mouse BD 552884 FCGR2 CD32; 2212 IgG1, κ A CDw32; FCG2; FCGR2; FCGR2A 1; FcGR; IGFR2; MGC238 87; MGC300 32 40 CD33 P67.6 PE Mouse BD 347787 CD33 SIGLEC- 945 IgG1, κ 3; p67 41 CD34 581 PE Mouse BD 555822 CD34 none 947 IgG1, κ 42 CD35 E11 PE Mouse BD 559872 CR1 C3BR; 1378 IgG1, κ CD35 43 CD36 CB38 PE Mouse BD 555455 CD36 FAT; 948

(NL07) IgM, k GP3B; GP4; GPIV; PASIV; SCARB3 44 CD37 M-B371 FITC Mouse BD 555457 CD37 GP52- 951 IgG1, κ 40 45 CD38 HIT2 PE Mouse BD 555460 CD38 T10 952 IgG1, κ 46 CD39 TÜ66 APC Mouse BD 560239 ENTPD ATPDase 953 IgG2b, k 1 ; CD39; NTPDase -1 47 CD40 5C3 PE Mouse BD 555589 CD40 p50; 958 IgG1, κ Bp50; CDW40; MGC901 3; TNFRSF5

48 CD41a HIP8 PE Mouse BD 555467 ITGB3 3674 IgG1, κ 49 CD41b HIP2 FITC Mouse BD 555469 ITGA2 CD41; 3690 IgG3, к B CD41B; GP2B; GPIIb; GTA 50 CD42a ALMA.1 PE Mouse BD 558819 GP9 CD42a 2815 6 IgG1, κ 51 CD42b HIP1 PE Mouse BD 555473 GP1BA BSS; 2811 IgG1, κ CD42B; CD42b- alpha; GP1B; MGC345 95 52 CD43 1G10 PE Mouse BD 560199 SPN CD43; 6693 IgG1, κ GPL115; LSN 53 CD44 G44-26 FITC Mouse BD 555478 CD44 CDW44; 960 IgG2b, k ECMR- III; IN; INLU; LHR; MC56; MDU2; MDU3; MGC104 68; MIC4; MUTCH- I; Pgp1

54 CD45 HI30 PE not BD 555483 PTPRC B220; 5788 given CD45; GP180; LCA; LY5; T200 55 CD45RA HI100 PE Mouse BD 555489 PTPRC 5788 IgG2b, k 56 CD45RB MT4 PE Mouse BD 555904 PTPRC 5788 IgG1, κ 57 CD45RO UCHL1 PE Mouse BD 555493 PTPRC 5788 IgG2a, к 58 CD46 E4.3 FITC Mouse BD 555949 CD46 CD46; 4179 IgG2a, к MGC265 44; MIC10; TLX; TRA2.10 59 CD47 B6H12 PE Mouse BD 556046 CD47 IAP; 961 IgG1, κ MER6; OA3 60 CD48 TÜ145 PE Mouse BD 552855 CD48 BCM1; 962 IgM, k BLAST; BLAST1; MEM- 102; SLAMF2; hCD48; mCD48 61 CD49a SR84 PE Mouse BD 559596 ITGA1 CD49a; 3672 IgG1, κ VLA1 62 CD49b 12F1 PE Mouse BD 555669 ITGA2 BR; 3673 IgG2a, к CD49B; VLAA2 63 CD49c C3 II.1 PE Mouse BD 556025 ITGA3 CD49C; 3675 IgG1, κ GAP-B3; GAPB3; MSK18; VCA-2; VL3A; VLA3a 64 CD49d 9F10 PE Mouse BD 555503 ITGA4 CD49D 3676 IgG1, κ 65 CD49e IIA1 PE Mouse BD 555617 ITGA5 CD49e; 3678 IgG1, κ FNRA; VLA5A 66 CD49f GoH3 PE Rat BD 555736 ITGA6 CD49f 3655 IgG2a, к 67 CD50 TÜ41 FITC Mouse BD 555958 ICAM3 CD50; 3385 IgG2b, k CDW50; ICAM-R 68 CD51/CD 23C6 PE Mouse BD 550037 ITGAV CD51; 3685

61 IgG1, κ MSK8; VNRA 69 CD52 HI186 PE Mouse BioLege 316006 CD52 CD52 1043 IgG2b, k nd 70 CD53 HI29 PE Mouse BD 555508 CD53 MOX44 963 IgG1, κ 71 CD54 HA58 PE Mouse BD 555511 ICAM1 BB2; 3383 IgG1, κ CD54 72 CD55 IA10 PE Mouse BD 555694 CD55 CD55; 1604 IgG2a, к CR; TC 73 CD56 B159 PE Mouse BD 555516 NCAM CD56; 4684 IgG1, κ 1 MSK39; NCAM 74 CD57 HNK-1 FITC Mouse BD 347393 B3GAT HNK-1; 27087 IgM, k 1 LEU7; NK-1 75 CD58 1C3 PE Mouse BD 555921 CD58 LFA3 965 IgG2a, к 76 CD59 p282 PE Mouse BD 555764 CD59 MGC235 966 (H19) IgG2a, к 4; MIC11; MIN1; MIN2; MIN3; MSK21; PROTEC TIN 77 CD60b NA FITC Mouse Lifespa LS- carbo carbohy N/A not IgM n C78711 hydrat drate a Bioscie e protein nces 78 CD61 VI-PL2 PE Mouse BD 555754 ITGB3 CD61; 3690 IgG1, κ GP3A; GPIIIa 79 CD62E 68-5H11 PE Mouse BD 551145 SELE CD62E; 6401 IgG1, κ ELAM; ELAM1; ESEL; LECAM2 80 CD62L Sk11 PE Mouse BD 341012 SELL CD62L; 6402 IgG2a, к LAM-1; LAM1; LECAM1; LNHR; LSEL; LYAM1; Leu-8; Lyam-1; PLNHR; TQ1; hLHRc 81 CD62P AK-1 PE Mouse BD 555524 SELP CD62; 6403

IgG1, κ CD62P; GMP140 ; GRMP; PADGE M; PSEL 82 CD63 H5C6 PE Mouse BD 556020 CD63 LAMP-3; 967 IgG1, κ ME491; MLA1; OMA81 H 83 CD64 10.1 PE Mouse BD 558592 FCGR1 CD64; 2209 IgG1, κ A FCRI; IGFR1 84 CD65 88H7 FITC Mouse Beckm IM1654U carboh carbohy N/A not IgM an ydrate drate a Coulter protein 85 CD65s VIM-2 FITC Mouse Abcam ab74080 carboh carbohy N/A not IgM ydrate drate a protein 86 CD66 B1.1/CD PE Mouse BD 551480 CEACA BGP; 109770 66 IgG2a, к M1 BGP1; BGPI; CD66; CD66A 87 CD66b G10F5 FITC Mouse BD 555724 CEACA CD66b; 1088 IgM, k M8 CD67; CGM6; NCA-95 88 CD66c B6.2/CD PE Mouse BD 551478 CEACA CD66c; 4680 66c IgG1, κ M6 CEAL; NCA 89 CD66d CLB- PE Mouse Abcam ab51598 CEACA CD66D; 1084 gran/10, IgG1 M3 CGM1 IH4Fc 90 CD66e 487618 PE Mouse R&D FAB4128 CEACA CD66e; 1048 IgG1 System P M5 CEA s 91 CD68 Y1/82A PE Mouse BD 556078 CD68 SCARD1 968 IgG2b, k 92 CD69 FN50 PE Mouse BD 555531 CD69 none 969 IgG1, κ 93 CD70 Ki-24 PE Mouse BD 555835 CD70 CD27L; 970 IgG3, к CD27LG; CD70 94 CD71 M-A712 PE Mouse BD 555537 TFRC CD71; 7037 IgG2a, к TFR; TRFR 95 CD72 J4-118 FITC Mouse BD 555918 CD72 LYB2 971 IgG2b, k 96 CD73 AD2 PE Mouse BD 550257 NT5E CD73; 4907 IgG1, κ E5NT;

NT5; NTE; eN; eNT 97 CD74 M-B741 FITC Mouse BD 555540 CD74 DHLAG; 972 IgG2a, к HLADG; Ia- GAMMA

98 CD75 LN1 FITC Mouse BD 555654 carbo carbohy NA IgM, k hydrat drate e 99 CD77 5B5 FITC Mouse BD 551353 carboh carbohy NA IgM, k ydrate drate 100 CD79a HM47 PE Mouse BD 555935 CD79A IGA; MB- 973 IgG1, κ 1 101 CD79b CB3-1 PE Mouse BD 555679 CD79B B29; 974 IgG1, κ IGB 102 CD80 L307.4 PE Mouse BD 557227 CD80 CD28LG; 941 IgG1, κ CD28LG 1; LAB7 103 CD81 JS-81 PE Mouse BD 555676 CD81 S5.7; 975 IgG1, κ TAPA1 104 CD82 ASL-24 PE Mouse BioLege 342104 CD82 4F9; 3732 IgG2a, к nd C33; CD82; GR15; IA4; R2; SAR2; ST6 105 CD83 HB15e PE Mouse BD 556855 CD83 BL11; 9308 IgG1, κ HB15 106 CD84 CD84.1. PE Mouse Biolege 326008 CD84 LY9B; 8832 21 IgG1 nd SLAMF5; hCD84; mCD84 107 CD85A MKT5.1 PE Mouse Biolege 337704 LILRB3 CD85A; 11025 IgG1 nd HL9; ILT5; LIR-3; LIR3 108 CD85D 42D1 PE Rat Biolege 338706 LILRB2 CD85D; 10288 IgG2a nd ILT4; LIR-2; LIR2; MIR-10; MIR10 109 CD85G 17G10.2 PE Mouse BioLege 326408 LILRA4 ILT7; 23547 IgG1 nd CD85g; MGC129 597 110 CD85H 24 PE Rat Biolege 337904 LILRA2 CD85H; 11027 IgG2a, к nd ILT1;

LIR-7; LIR7 111 CD85J GHI/75 PE Mouse BD 551053 LILRB1 CD85; 10859 IgG2b, k CD85J; ILT2; LIR-1; LIR1; MIR-7; MIR7 112 CD86 2331 PE Mouse BD 555658 CD86 B7-2; 942 (FUN-1) IgG1, κ B70; CD28LG 2; LAB72; MGC344 13 113 CD87 VIM5 PE Mouse BD 555768 PLAUR CD87; 5329 IgG1, κ UPAR; URKR 114 CD88 C85- PE Rabbit BD 552993 C5AR1 C5A; 728 4124 IgG C5AR; CD88 115 CD89 A59 PE Mouse BD 555686 FCAR CD89 2204 IgG1, κ 116 CD90 5E10 PE Mouse BD 555596 THY1 CD90 7070 IgG1, κ 117 CD91 A2MR- PE Mouse BD 550497 LRP1 A2MR; 4035 α2 IgG1, κ APOER; APR; CD91; LRP 118 CD92 VIM-15b PE Mouse Abcam ab66228 SLC44 CTL1; 23446 IgG2b A1 CDW92; CHTL1; RP11- 287A8.1 119 CDw93 R139 FITC Mouse BD 551531 CD93 C1QR1; 22918 IgG2b, k C1qRP; CDw93; MXRA4; C1qR(P); dJ737E2 3.1 120 CD94 HP-3D9 PE Mouse BD 555889 KLRD1 CD94 3824 IgG1, κ 121 CD95 DX2 PE Mouse e- 12-0959- FAS APT1; 355 IgG1, κ Bioscie 73 CD95; nce FAS1; APO-1; FASTM; ALPS1A; TNFRSF6

122 CD96 NK92.39 PE Mouse Biolege 338406 CD96 MGC225 10225 IgG1 nd 96; TACTILE 123 CD97 VIM3b PE Mouse BD 555774 CD97 TM7LN1 976 IgG1, κ 124 CD98 UM7F8 PE Mouse BD 556077 SLC3A 4F2; 6520 IgG1, κ 2 4F2HC; 4T2HC; CD98; MDU1; NACAE 125 CD99 TÜ12 PE Mouse BD 555689 CD99 MIC2; 4267 IgG2a, к MIC2X; MIC2Y 126 CD100 133-1C6 FITC Mouse e- 53-1009 SEMA CD100; 10507 IgM Bioscie 4D M-sema nce G; M- sema-G; SEMAJ; coll-4 127 CD101 BB27 PE Mouse e- 12-1019- IGSF2 CD101; 9398 IgG1 Bioscie 73 V7 nce 128 CD102 CBR- PE Mouse BD 558080 ICAM2 CD102 3384 1C2/2.1 IgG2a, к 129 CD103 Ber- PE Mouse BD 550260 ITGAE CD103; 3682 ACT8 IgG1, κ HUMINA E 130 CD104 439-9B PE Rat BD 555720 ITGB4 none 3691 IgG2b, к 131 CD105 SN6 PE Mouse e- 12-1057- ENG CD105; 2022 IgG1 Bioscie 42 END; nce HHT1; ORW; ORW1 132 CD106 51-10C9 PE Mouse BD 555647 VCAM INCAM- 7412 IgG1, κ 1 100 133 CD107a H4A3 PE Mouse BD 555801 LAMP CD107a; 3916 IgG1, κ 1 LAMPA; LGP120 134 CD107b H4B4 FITC Mouse BD 555804 LAMP CD107b; 3920 IgG1, κ 2 LAMPB 135 CD108 KS-2 PE Mouse BD 552830 SEMA CD108; 8482 IgG2a, к 7A CDw108; H-SEMA- K1; H- Sema K1; H- Sema-L; SEMAK1 ; SEMAL 136 CD109 TEA PE Mouse BD 556040 CD109 DKFZp76 135228

2/16 IgG1, κ 2L1111; FLJ3856 9 137 CD110 1.6.1 PE Mouse BD 562159 MPL C-MPL; 4352 IgG2b, к CD110; MPLV; TPOR 138 CD111 R1.302 PE Mouse BioLege 340404 PVRL1 CD111; 5818 IgG1, κ nd CLPED1; ED4; HIgR; HVEC; PRR; PRR1; PVRR; PVRR1; SK-12 139 CD112 R2.525 PE Mouse BD 551057 PVRL2 CD112; 5819 IgG1, κ HVEB; PRR2; PVRR2 140 CD114 LMM74 PE Mouse BD 554538 CSF3R CD114; 1441 1 IgG1, κ GCSFR 141 CD115 not PE Rat IgG1 R&D FAB329P CSF1R C-FMS; 1436 given Systems CD115; CSFR; FIM2; FMS 142 CD116 hGMCSF PE Mouse BD 551373 CSF2R CD116; 1438 R-M1 IgG1, κ A CDw116; CSF2R; CSF2RAX ; CSF2RAY ; CSF2RX; CSF2RY; GM-CSF- R-alpha; GMCSFR ; GMR; MGC384 8; MGC483 8 143 CD117 YB5.B8 PE Mouse BD 555714 KIT CD117; 3815 IgG1, κ PBT; SCFR 144 CD118 32953 PE Mouse R&D FAB249P LIFR LIFR; 3977 IgG1 System SWS; s SJS2; STWS 145 CD119 GIR-208 PE Mouse BD 558934 IFNGR CD119; 3459

IgG1, κ 1 IFNGR 146 CD120a 16803 PE Mouse R&D FAB225P TNFRS CD120a; 7132 IgG1 System F1A FPF; s MGC195 88; TBP1; TNF-R; TNF-R-I; TNF- R55; TNFAR; TNFR1; TNFR55; TNFR60; p55; p55-R; p60 147 CD120b hTNFR- PE Rat BD 552418 TNFRS CD120b; 7133 M1 IgG2b, к F1B TBPII; TNF-R-II; TNF- R75; TNFBR; TNFR2; TNFR80; p75; p75TNFR

148 CD121b 34141 PE Mouse R&D FAB663P IL1R2 IL1RB; 7850 IgG1 System MGC477 s 25 149 CD122 Mik-β3 PE Mouse BD 554525 IL2RB P70-75 3560 IgG1, κ 150 CD123 7G3 PE Mouse BD 554529 IL3RA CD123; 3563 IgG2a, к IL3R; IL3RAY; IL3RX; IL3RY; MGC341 74; hIL- 3Ra 151 CD124 hIL4R- PE Mouse BD 552178 IL4R CD124; 3566 M57 IgG1, κ IL4RA 152 CD125 26815 PE Mouse R&D FAB253P IL5RA CDw125; 3568 IgG1 System HSIL5R3; s IL5R; MGC265 60 153 CD126 M5 PE Mouse BD 551850 IL6R CD126; 3570 IgG1, κ IL-6R-1; IL-6R- alpha; IL6RA

154 CD127 hIL-7R- PE Mouse BD 557938 IL7R CD127; 3575 M21 IgG1, κ CDW127 ; IL-7R- alpha 155 CD129 AH9R7 PE Mouse BioLege 310404 IL9R none 3581 IgG2b, k nd 156 CD130 AM64 PE Mouse BD 555757 IL6ST CD130; 3572 IgG1, κ CDw130; GP130; GP130- RAPS; IL6R- beta 157 CD131 1C1 PE Mouse e- 12-1319- CSF2R CD131; 1439 IgG1, κ Bioscie 73 B CDw131; nce IL3RB; IL5RB 158 CD132 AG184 PE Mouse BD 555900 IL2RG CD132; 3561 IgG1, κ IMD4; SCIDX; SCIDX1 159 CD133 AC133 APC Mouse Milteny 130-090- PROM AC133; 8842 IgG1, κ i 826 1 CD133; PROML1

160 CD134 ACT35 PE Mouse BD 555838 TNFRS ACT35; 7293 IgG1, κ F4 CD134; OX40; TXGP1L 161 CD135 4G8 PE Mouse BD 558996 FLT3 CD135; 2322 IgG1, κ FLK2; STK1 162 CD136 ID1 PE not Beckm 4111601 MST1 CDw136; 4486 given an 5 R RON Coulter 163 CD137 4B4-1 PE Mouse BD 555956 TNFRS 4-1BB; 3604 IgG1, κ F9 CD137; CDw137; ILA; MGC217 2 164 CD137L 5F4 PE Mouse BioLege 311504 TNFSF 8744 IgG1, κ nd 9 165 CD138 MI15 PE Mouse BD 552026 SDC1 CD138; 6382 IgG1, κ SDC; SYND1 166 CD140a αR1 PE Mouse BD 556002 PDGFR CD140A; 5156 IgG2a, к A PDGFR2 167 CD140b 28D4 PE Mouse BD 558821 PDGFR CD140B; 5159 IgG2a, к B JTK12; PDGF-R- beta;

PDGFR; PDGFR1 168 CD141 1A4 PE Mouse BD 559781 THBD CD141; 7056 IgG1, κ THRM; TM 169 CD142 HTF-1 PE Mouse BD 550312 F3 CD142; 2152 IgG1, κ TF; TFA 170 CD143 171417 PE Mouse R&D FAB929P ACE ACE1; 1636 IgG1 System CD143; s DCP; DCP1; MGC265 66 171 CD144 55-7H1 PE Mouse BD 560410 CDH5 7B4 1003 IgG1, κ 172 CD146 P1H12 PE Mouse BD 550315 MCA CD146; 4162 IgG1, κ M MUC18 173 CD147 HIM6 FITC Mouse BD 555962 BSG 5F7; 682 IgG1, κ CD147; EMMPRI N; M6; OK; TCSF 174 CD148 143-41 PE Mouse R&D FAB1934 PTPRJ CD148; 5795 IgG1 System P DEP1; s HPTPeta ; R-PTP- ETA; SCC1 175 CD150 A12 PE Mouse BD 559592 SLAMF CD150; 6504 IgG1, κ 1 CDw150; SLAM 176 CD151 14A2.H1 PE Mouse BD 556057 CD151 GP27; 977 IgG1, κ PETA-3; SFA1 177 CD152 BNI3 PE Mouse BD 555853 CTLA4 CD152 1493 IgG2a, к 178 CD153 116614 PE Mouse I R&D FAB1028 TNFSF CD153; 944 gG2B System P 8 CD30L; s CD30LG 179 CD154 TRAP1 PE Mouse BD 555700 CD40L CD154; 959 IgG1, κ G CD40L; CD40LG; HIGM1; IGM; IMD3; T- BAM; TRAP; gp39; hCD40L 180 CD155 2H7CD1 PE Mouse e- 12-1550- PVR CD155; 5817 55 IgG1 Bioscie 71 HVED;

nce NECL5; PVS; TAGE4 181 CD156b 111633 PE Mouse R&D FAB9301 ADAM CD156b; 6868 IgG1 System P 17 TACE; s cSVP 182 CD157 RF3 PE Mouse I MBL D036-5 BST1 CD157 683 gG1, κ 183 CD158A HP-3E4 PE Mouse I BD 556063 KIR2D 47.11; 3802 gM, κ L1 CD158A; CL-42; NKAT1; p58.1 184 CD158B1 CH-L PE Mouse BD 559785 KIR2D CD158B 3803 IgG2b, k L2 1; CL-43; NKAT6; p58.2 185 CD158B2 DX27 PE Mouse BD 556071 KIR2D CD158B 3804 IgG2a, к L3 2; CD158b; CL-6; KIR- 023GB; NKAT2; NKAT2A; NKAT2B; p58 186 CD158D 181703 PE Mouse R&D FAB2238 KIR2D 103AS; 3805 IgG2a System P L4 15.212; s CD158D; KIR103; KIR103A S 187 CD158E2 DX9 PE Mouse BD 555967 KIR3D AMB11; 3813 IgG1, κ S1 CD158E1 ; CD158E1 /2; CD158E2 ; CL-11; CL-2; KIR; KIR3DS1; NKAT10; NKAT3; NKB1; NKB1B 188 CD158F UP-R1 PE Mouse BioLege 341304 KIR2D CD158F; 57292 IgG1 nd L5A KIR2DL5; KIR2DL5. 1; KIR2DL5. 3

189 CD158I JJC11.6 PE Mouse Milteny 130-092- KIR2D CD158I; 3809 IgG1 i 680 S4 KIR1D; KKA3; NKAT8; PAX; cl- 39 190 CD159a 131411 PE Mouse R&D FAB1059 KLRC1 CD159A; 3821 IgG2a System P MGC133 s 74; MGC597 91; NKG2; NKG2A 191 CD159c 134591 PE Mouse R&D FAB138P KLRC2 3822 IgG1 System s 192 CD160 BY55 APC Mouse BioLege 341204 CD160 BY55; 11126 IgM, κ nd NK1; NK28 193 CD161 DX12 PE Mouse BD 556081 KLRB1 CD161; 3820 IgG1, κ NKR; NKR-P1; NKR- P1A; NKRP1A; hNKR- P1A 194 CD162 KPL-1 PE Mouse BD 556055 SELPL CD162; 6404 IgG1, κ G PSGL-1; PSGL1 195 CD163 GHI/61 PE Mouse BD 556018 CD163 M130; 9332 IgG1, κ MM130 196 CD164 N6B6 PE Mouse BD 551298 CD164 MGC-24; 8763 IgG2a, к MUC-24; endolyn 197 CD165 SN2 PE Mouse e- 12-1659- CD165 none 23449 N56- IgG1 Bioscie 73 D11 nce 198 CD166 3A6 PE Mouse BD 559263 ALCA CD166; 214 IgG1, κ M MEMD 199 CD167a 51D6 PE Mouse BioLege 334006 DDR1 CAK; 780 IgM, k nd CD167; DDR; EDDR1; MCK10; NEP; NTRK4; PTK3; PTK3A; RTK6; TRKE 200 CD169 7-239 PE Mouse Biolege 346004 SIGLEC CD169; 6614

IgG1 nd 1 FLJ0005 1; FLJ0005 5; FLJ0007 3; FLJ3215 0; SIGLEC- 1; dJ1009E 24.1 201 CD170 194128 PE Mouse R&D FAB1072 SIGLEC CD33L2; 8778 IgG1 System 1P 5 OB-BP2; s OBBP2; SIGLEC- 5 202 CD171 5G3 PE Mouse e- 12-1719- L1CA CAML1; 3897 IgG2a Bioscie 73 M CD171; nce HSAS; HSAS1; MASA; MIC5; N- CAML1; S10; SPG1 203 CD172a SE5A5 PE Mouse BioLege 323806 SIRPA BIT; 140885 IgG1, κ nd MFR; MYD-1; P84; SHPS-1; SHPS1; SIRP; SIRP- ALPHA- 1; SIRPalph a; SIRPalph a2 204 CD172b B4B6 PE Mouse BD 552602 SIRPB SIRP- 10326 IgG1, κ 1 BETA-1 205 CD172g LSB2.20 PE Mouse BioLege 336606 SIRPG SIRP-B2; 55423 IgG1, κ nd bA77C3. 1 206 CD175s STn 219 FITC Mouse Abcam ab76756 carboh carbohy N/A not IgG1 ydrate drate a protein 207 CD177 MEM- PE Mouse Abcam ab69777 CD177 CD177; 57126 166 IgG1 HNA2A; NB1 208 CD178 NOK-1 PE Mouse BioLege 306407 FASLG FASL; 356 IgG1, κ nd CD178;

CD95L; TNFSF6; APT1LG1

209 CD179a HSL96 PE Mouse BioLege 347404 VPREB IGI; 7441 IgG1, κ nd 1 IGVPB; VPREB 210 CD180 G28-8 PE Mouse BD 551953 CD180 LY64; 4064 IgG1, κ Ly78; RP105; MGC126 233; MGC126 234 211 CD181 5A12 PE Mouse BD 555940 IL8RA C-C CKR- 3577 IgG2b, k 1; C-C- CKR-1; CD128; CDw128 a; CMKAR1 ; CXCR1; IL8R1; IL8RBA 212 CD182 6C6 PE Mouse BD 555933 IL8RB CDw128 3579 IgG1, κ b; CMKAR2 ; CXCR2; IL8R2; IL8RA 213 CD183 1C6/CXC PE Mouse BD 557185 CXCR3 CD183; 2833 R3 IgG1, κ CKR-L2; CMKAR3 ; GPR9; IP10; IP10-R; Mig-R; MigR 214 CD184 12G5 PE Mouse BD 555974 CXCR4 D2S201E 7852 IgG2a, к ; HM89; HSY3RR; LAP3; LESTR; NPY3R; NPYR; NPYY3R; WHIM 215 CD185 RF8B2 FITC Mouse BD 558112 BLR1 BLR1; 643 IgG2b, k CXCR5; MDR15 216 CD186 TG3/CX APC Mouse BioLege 335101 CXCR6 CXCR6; 10663 CR6 IgG2b, k nd BONZO; STRL33;

TYMSTR 217 CD191 53504 PE Mouse R&D FAB145P CCR1 CKR-1; 1230 IgG2b System CMKBR1 s ; HM145; MIP1aR; SCYAR1 218 CD192 48607 APC Mouse BD 558406 CCR2 CC-CKR- 729230 IgG2b, k 2; CCR2A; CCR2B; CKR2; CKR2A; CKR2B; CMKBR2 ; MCP-1- R 219 CD193 5E8 PE Mouse BD 558165 CCR3 CC-CKR- 1232 IgG2b, k 3; CKR3; CMKBR3

220 CD194 TG6/CC APC Mouse BioLege 335401 CCR4 CC-CKR- 1233 R4 IgG2b, k nd 4; CKR4; CMKBR4 ; ChemR1 3; HGCN 221 CD195 3A9 PE Mouse BD 556042 CCR5 CC-CKR- 1234 IgG2a, к 5; CCCKR5; CD195; CKR-5; CKR5; CMKBR5

222 CD196 11A9 PE Mouse BD 559562 CCR6 CCR6; 1235 IgG1, κ BN-1; CKR6; DCR2; CKRL3; DRY-6; GPR29; CKR-L3; CMKBR6 ; GPRCY4; STRL22; GPR- CY4 223 CD197 3D12 PE Rat BD 552176 CCR7 BLR2; 1236 IgG2a, к CDw197; CMKBR7 ; EBI1

224 CDw198 191704 PE Rat R&D FAB1429 CCR8 CKR-L1; 1237 IgG2b System P CKRL1; s CMKBR8 ; CMKBRL 2; CY6; GPR- CY6; TER1 225 CDw199 112509 APC Mouse BD 557975 CCR9 GPR-9-6; 10803 IgG2a, к GPR28 226 CD200 MRC PE Mouse BD 552475 CD200 MOX1; 4345 OX-104 IgG1, κ MOX2; MRC; OX-2 227 CD201 RCR-252 PE Rat BD 557950 PROCR CCCA; 10544 IgG1, k CCD41; EPCR; MGC230 24; bA42O4. 2 228 CD202b 33.1 PE Mouse BioLege 334206 TEK CD202B; 7010 (Ab33) IgG1, κ nd TIE-2; TIE2; VMCM; VMCM1 229 CD203c NP4D6 PE Mouse BioLege 324606 ENPP3 B10; 5169 IgG1, κ nd CD203c; NPP3; PD- IBETA; PDNP3 230 CD204 351615 PE Mouse R&D FAB2708 MSR1 SCARA1; 4481 IgG2B System P SR-A; s phSR1; phSR2 231 CD205 MG38 PE Mouse BD 558069 LY75 CLEC13B 4065 IgG2b, k ; DEC- 205; GP200- MR6 232 CD206 19.2 PE Mouse BD 555954 MRC1 CLEC13D 4360 IgG1, κ 233 CD207 343828 APC Mouse R&D FAB2088 CD207 LANGERI 50489 IgG1 System A N s 234 CD208 I10- PE Mouse BD 558126 LAMP DC- 27074 1112 IgG1, κ 3 LAMP; DCLAMP ; LAMP; TSC403

235 CD209 DCN46 PE Mouse BD 551265 CD209 CDSIGN; 30835 IgG2b, k DC- SIGN; DC- SIGN1 236 CDw210 3F9 PE Rat BD 556013 IL10RA IL10R; 3587 IgG2a, к CDW210 A; HIL- 10R; IL- 10R1; IL10RA 237 CD212 2.4e6 PE Mouse BD 556065 IL12RB IL-12R- 3594 IgG1, κ 1 BETA1; IL12RB; MGC344 54 238 CD213a2 B-D13 PE Mouse Abcam ab27415 IL13RA IL-13R; 3598 IgG1 2 IL13BP 239 CD215 151303 PE Mouse R&D FAB1471 IL15RA IL15RA 3601 IgG2B System P s 240 CD217 BG/hIL1 APC Mouse bioLege 340903 IL17RA IL-17RA; 23765 7AR IgG1 nd IL17RA; MGC102 62; hIL- 17R 241 CDw218a H44 PE Mouse e- 12-7183- IL18R1 IL18R1; 8809 IgG1, κ Bioscie 73 IL1RRP; nce IL-1Rrp 242 CD218b 132029 PE Mouse R&D FAB118P IL18RA IL18RAP; 8807 IgG2b System P ACPL s 243 CD220 not APC Goat R&D FAB1544 INSR none 3643 given IgG System A s 244 CD221 1H7 PE Mouse BD 555999 IGF1R JTK13 3480 IgG1, κ 245 CD222 MEM- FITC Mouse BioLege 315904 IGF2R CD222; 3482 238 IgG1, κ nd CIMPR; M6P-R; MPRI 246 CD223 not PE Goat R&D FAB2319 LAG3 CD223 3902 given IgG System P s 247 CD226 DX11 PE Mouse BD 559789 CD226 DNAM- 10666 IgG1, κ 1; DNAM1; PTA1; TLiSA1 248 CD227 HMPV FITC Mouse BD 559774 MUC1 CD227; 4582 IgG1, κ EMA;

PEM; PUM 249 CD229 249936 PE Mouse R&D FAB1898 LY9 CD229; 4063 IgG2a System P SLAMF3; s hly9; mLY9 250 CD230 4D5 PE Mouse e- 12-9230- PRNP ASCR; 5621 IgG1, κ Bioscie 73 CJD; nce GSS; MGC266 79; PRIP; PrP; PrP27- 30; PrP33- 35C; PrPc 251 CD231 SN1a PE Mouse BioLege 329406 TSPAN A15; 7102 (M3- IgG1, κ nd 7 CCG-B7; 3D9) CD231; DXS1692 E; MXS1; TALLA-1; TM4SF2 b 252 CD234 358307 PE Mouse R&D FAB4139 DARC CCBP1; 2532 IgG2A System P DARC; s GPD 253 CD235a GA-R2 PE Mouse BD 555570 GYPA GPA; 2993 (HIR2) IgG2b, k MN; MNS 254 CD243 UIC2 PE Mouse Beckm IM2370U ABCB1 ABC20; 5243 (BC) IgG2a an CD243; Coulter CLCS; GP170; MDR1; P-gp; PGY1 255 CD243 17F9 PE Mouse BD 557003 ABCB1 ABC20; 5243 (BD) IgG2b, k CD243; CLCS; GP170; MDR1; P-gp; PGY1 256 CD244 2-69 PE Mouse BD 550816 CD244 2B4; 51744 IgG2a, к NAIL; NKR2B4; Nmrk; SLAMF4 257 CD245 DY12 PE Mouse BioLege 334404 NPAT not 4863 IgG1, κ nd listed 258 CD247 G3 PE Mouse AbD MCA129 CD247 CD3- 919

IgG2a Serotec 7PE ZETA; h CD3H; CD3Q; TCRZ 259 CD249 not PE Rat Lifespa LS- ENPEP APA; 2028 given IgG1, k n C12169 gp160; Bioscie EAP nce 260 CD252 Ik-1 PE Mouse BD 558164 TNFSF TNFSF4; 7292 IgG1, κ 4 GP34; OX4OL; TXGP1; CD134L; OX-40L; OX40L 261 CD253 RIK-2 PE Mouse BD 550516 TNFSF TNFSF10 8743 IgG1 10 ; TL2; APO2L; TRAIL; Apo-2L 262 CD254 MIH24 PE Mouse bioLege 347504 TNFSF ODF; 8600 IgG2b, k nd 11 OPGL; sOdf; CD254; OPTB2; RANKL; TRANCE; hRANKL 2 263 CD255 CARL-1 PE Mouse I BD 552831 TNFSF APO3L; 8742 gG3 12 DR3LG; TWEAK 264 CD256 T3-6 PE Mouse BioLege 318506 TNFSF APRIL; 8741 IgG2a, к nd 13 TALL2; TRDL-1; UNQ383 /PRO715

265 CD257 T7-241 PE Mouse BioLege 318606 TNFSF BAFF; 10673 IgG1, κ nd 13B BLYS; TALL-1; TALL1; THANK; TNFSF20 ; ZTNF4; delta BAFF 266 CD258 115520 PE Mouse R&D FAB664P TNFSF TNFSF14 8740 IgG1 System 14 ; LTg; s TR2; HVEML; LIGHT 267 CD261 DJR1 PE Mouse BioLege 307206 TNFRS APO2; 8797

IgG1 nd F10A DR4; MGC936 5; TRAILR- 1; TRAILR1 268 CD262 71908 PE Mouse R&D FAB6311 TNFRS DR5; 8795 IgG2b System P F10B KILLER; s KILLER/D R5; TRAIL- R2; TRAILR2; TRICK2; TRICK2A ; TRICK2B; TRICKB; ZTNFR9 269 CD263 90906 PE Mouse R&D FAB6302 TNFRS DCR1; 8794 IgG1 System P F10C LIT; s TRAILR3; TRID 270 CD264 104918 PE Mouse R&D FAB633P TNFRS DCR2; 8793 IgG1 System F10D TRAILR4; s TRUNDD

271 CD267 1A1- PE Rat BD 558414 TNFRS CVID; 23495 K21- IgG2a, к F13B TACI; M22 CD267; FLJ3994 2; MGC399 52; MGC133 214; TNFRSF1 4B 272 CD268 11C1 PE Mouse BD 558097 TNFRS BAFFR; 115650 IgG1, κ F13C CD268; BAFF-R; MGC138 235 273 CD269 not PE Goat R&D FAB193P TNFRS BCM; 608 given IgG System F17 BCMA s 274 CD270 122 PE not BioLege 318806 TNFRS TR2; 8764 given nd F14 ATAR; HVEA; HVEM; LIGHTR; TNFRSF1 4

275 CD271 C40- PE Mouse BD 557196 NGFR NGFR; 4804 1457 IgG1, κ TNFRSF1 6; p75(NTR ) 276 CD272 J168- PE Mouse BD 558485 BTLA BTLA1; 151888 540.90. IgG1, κ FLJ1606 22 5 277 CD273 MIH18 PE Mouse BD 558066 PDCD1 PDCD1L 80380 IgG1, κ LG2 G2; B7DC; Btdc; PDL2; PD-L2; PDCD1L 2; bA574F1 1.2 278 CD274 MIH1 PE Mouse BD 557924 CD274 B7-H; 29126 IgG1, κ B7H1; PD-L1; PDCD1L 1; PDL1 279 CD275 2D3/B7- PE Mouse BD 552502 ICOSL B7-H2; 23308 H2 IgG2b, k G B7H2; B7RP-1; B7RP1; GL50; ICOS-L; ICOSLG; KIAA065 3; LICOS 280 CD276 DCN.70 PE Mouse BioLege 331606 CD276 B7H3 80381 IgG1, κ nd 281 CD277 BT3.1 PE Mouse e- 14-2779- BTN3A BTF5; 11119 IgG1 Bioscie 71 1 BT3.1 nce 282 CD278 DX29 PE Mouse BD 557802 ICOS AILIM; 29851 IgG1 MGC398 50 283 CD279 MIH4 PE Mouse BD 557946 PDCD1 PD1; 5133 IgG1, κ SLEB2; hPD-l 284 CD281 TLR1.13 PE Mouse BioLege 334506 TLR1 TLR1; 7096 6 IgG1, κ nd TIL; rsc786; KIAA001 2; DKFZp54 7I0610; DKFZp56 4I0682

285 CD282 11G7 FITC Mouse BD 558318 TLR2 TIL4 7097 IgG1, κ 286 CD283 TLR3.7 PE Mouse e- 12-9039- TLR3 TLR3 7098 IgG1, κ Bioscie 82 nce 287 CD284 610015 PE Mouse R&D FAB6248 TLR4 TOLL; 7099 IgG2a System P hToll s 288 CD286 TLR6.12 PE Mouse BioLege 334708 TLR6 CD286 10333 7 IgG1, κ nd 289 CD288 44C143 PE Mouse Abcam ab45097 TLR8 TLR8 51311 IgG1 290 CD289 eB72- PE Rat BD 560425 TLR9 none 54106 1665 IgG2a, к 291 CD290 3C10C5 PE Mouse BioLege 354604 TLR10 TLR10 81793 IgG1, κ nd 292 CD292 Polyclon PE Goat R&D FAB346F BMPR BMPR1A 657 al IgG System 1A ; ALK3; Antibod s ACVRLK3 y 293 CD294 BM16 APC Rat BD 558042 GPR44 CRTH2 11251 IgG2a, к 294 CD295 52263 PE Mouse R&D FAB867P LEPR LEPR; 3953 IgG2b System OBR s 295 CD298 4A8 FITC Mouse MBL D261-4 ATP1B ATP1B3; 483 IgG2a 3 ATPB-3; FLJ2902 7 296 CD299 120604 PE Mouse R&D FAB162P CLEC4 DC- 10332 IgG2b System M SIGN2; s DC- SIGNR; DCSIGN R; HP10347 ; LSIGN; MGC478 66 297 CD300a MEM- PE Mouse Abcam ab64675 CD300 CMRF- 11314 260 IgG1 A 35-H9; CMRF35 H; CMRF35 H9; IRC1; IRC2; IRp60 298 CD300c TX45 PE Mouse BioLege 334804 CD300 CMRF- 10871 IgG1, κ nd C 35A; CMRF35

A; CMRF35 A1; LIR 299 CD300e UP-H2 PE Mouse BioLege 339704 CD300 342510 IgG1, κ nd E 300 CD301 125A10. FITC Mouse Imgene DDX0010 CLEC1 HML; 10462 03 IgG1 x A488 0A HML2; CLECSF1 3; CLECSF1 4 301 CD303 AC144 PE Mouse Milteny 130-090- CLEC4 BDCA2; 170482 IgG1 i 511 C CLECSF1 1; DLEC; HECL; PRO341 50; CLECSF7 302 CD304 AD5- PE Mouse Milteny 130-090- NRP1 NRP; 8829 17F6 IgG1 i 533 VEGF165 R 303 CD305 DX26 PE Mouse BD 550811 LAIR1 LAIR-1 3903 IgG1, κ 304 CD307e 509f6 PE Mouse BioLege 340304 FCRL5 CD307; 83416 IgG2a, к nd FCRH5; IRTA2; BXMAS1 ; PRO820 305 CD309 89106 PE Mouse R&D FAB357P KDR KDR; 3791 IgG1 System FLK1; s VEGFR; VEGFR2 306 CD312 2A1 PE Mouse AbD MCA233 EMR2 none 30817 IgG1 Serotec 0PE h 307 CD314 1D11 PE Mouse BD 557940 KLRK1 KLRK1; 22914 IgG1, κ KLR; NKG2D; NKG2-D; D12S248 9E 308 CD317 RS38E APC Mouse BioLege 348404 BST2 none 684 IgG1, κ nd 309 CD318 309121 PE Mouse R&D FAB2666 CDCP1 CDCP1; 64866 IgG2a System 2P FLJ2296 s 9; MGC318 13 310 CD319 235614 PE Mouse R&D FAB1906 SLAMF 19A; 57823 IgG2a System P 7 CRACC; s CS1

311 CD321 M.AB.F1 PE Mouse BD 552556 F11R JAM; 50848 1 IgG1, κ KAT; JAM1; JCAM; JAM-1; PAM-1 312 CD322 CRAM- FITC Rat AbD MCA221 JAM2 C21orf4 58494 18 F26 IgG2a Serotec 1F 3; VE- h JAM; VEJAM 313 CD324 36/E- FITC Mouse BD 560061 CDH1 Arc-1; 999 Cadheri IgG2a, к CDHE; n ECAD; LCAM; UVO 314 CD325 8C11 PE Mouse e- 12-3259- CDH2 CDHN; 1000 IgG1, κ Bioscie 73 NCAD nce 315 CD326 EBA-1 PE Mouse BD 347198 TACST CO17- 4072 IgG1, κ D1 1A; EGP; EGP40; Ep-CAM; GA733- 2; KSA; M4S1; MIC18; MK-1; TROP1; hEGP-2 316 CD328 F023- PE Mouse BD 558372 SIGLEC p75; 27036 420 IgG1, κ 7 QA79; AIRM1; CDw328; SIGLEC- 7; p75/AIR M1 317 CDw329 E10-286 FITC Mouse BD 550906 SIGLEC CDw329; 27180 IgG1, κ 9 OBBP- LIKE 318 CD332 98725 APC Mouse R&D FAB684A FGFR2 FGFR2; 2263 IgG1 System BEK; s JWS; CEK3; CFD1; ECT1; KGFR; TK14; TK25; BFR-1; K-SAM 319 CD333 136334 PE Mouse R&D FAB766P FGFR3 FGFR3; 2261 IgG1 System ACH;

s CEK2; JTK4; HSFGFR 3EX 320 CD334 4FR6D3 PE Mouse BioLege 324306 FGFR4 FGFR4; 2264 IgG1, κ nd TKF; JTK2; MGC202 92 321 CD335 9E2/NK PE Mouse BD 557991 NCR1 LY94; 9437 p46 IgG1, κ NK-p46; NKP46 322 CD336 P44-8.1 PE Mouse BD 558563 NCR2 LY95; 9436 IgG1, κ NK-p44; NKP44 323 CD337 P30-15 PE Mouse BD 558407 NCR3 1C7; 259197 IgG1, κ LY117; NKp30 324 CD338 5D3 PE Mouse BioLege 332008 ABCG2 MRX; 9429 IgG2b, k nd MXR; ABCP; BCRP; BMDP; MXR1; ABC15; BCRP1; CDw338; EST1574 81; MGC102 821 325 CD339 188331 FITC Mouse R&D FAB1277 JAG1 JAG1; 182 IgG2b System F AGS; s AHD; AWS; HJ1; JAGL1 326 CD340 Neu PE Mouse BD 340552 ERBB2 NEU; 2064 24.7 IgG1, κ NGL; HER2; TKR1; HER-2; c-erb B2; HER- 2/neu 327 CD344 CH3A4A PE Mouse BioLege 326606 FZD4 EVR1; 8322 7 IgG1, κ nd FEVR; Fz-4; FzE4; GPCR; FZD4S; MGC343 90

328 CD349 W3C4E1 APC Mouse BioLege 326706 FZD9 FZD3 8326 1 IgM, k nd 329 CD351 TX61 PE Mouse BioLege 137306 FCAM 83953 IgG1, κ nd R FCA/MR; FKSG87; FCAMR 330 CD352 NT-7 PE Mouse BioLege 317208 SLAMF KALI; 114836 IgG1, κ nd 6 NTBA; KALIb; Ly108; NTB-A; SF2000 331 CD354 TREM- PE Mouse BioLege 314906 TREM TREM-1 54210 26 IgG1, κ nd 1 332 CD355 Cr24.1 PE Mouse BioLege 339106 CRTA CRTAM 56253 IgG2a, κ nd M 333 CD357 621 APC Mouse BioLege 311610 TNFRS AITR; 8784 IgG1, κ nd F18 GITR; GITR-D; TNFRSF1 8 334 CD358/D DR-6- PE Mouse Abcam ab52513 TNFRS DR6; 27242 R6 04-EC IgG1 F21 BM-018; TNFRSF2 1 335 CD360 2G1-K12 PE Mouse BioLege 347806 IL21R NILR 50615 (BL) IgG1, κ nd 336 CD360 17A12 PE Mouse BD 560264 IL21R NILR 50615 (BD) IgG1, κ 337 CD362 305515 APC not R&D FAB2965 SDC2 HSPG; 6383 given System A HSPG1; s SYND2; SDC2 338 CD363 218713 PE Mouse R&D FAB2016 S1PR1 EDG1; 1901 IgG2b System P S1P1; s ECGF1; EDG-1; CHEDG1 339 β2- TÜ99 PE Mouse BD 551337 B2M 567 microglob IgM, k ulin 340 BLTR-1 203/14F PE Mouse BD 552836 LTB4R 1241 11 IgG1 341 CA9 303123 PE Mouse R&D FAB2188 CA9 768 IgG2a System P s 342 CLA HECA- FITC Rat IgM, BD 555947 modified 949 452 к form of CD162 343 CDH3 104805 PE Mouse R&D FAB861 CDH3 1001 IgG1 Systems P

344 CDH6 427909 APC Mouse R&D FAB2715 CDH6 1004 IgG1 Systems A 345 CLIP CerCLIP FITC Mouse BD 555981 NOT IgG1, κ FOUND 346 DCIR I3-612 APC Mouse BD 558220 CLEC4A 50856 IgG1, κ 347 EGF-R EGFR1 PE Mouse BD 555997 EGFR 1956 IgG2b, k 348 FMC7 FMC7 FITC Mouse BD 340919 MS4A1 931 IgM, k 349 fMLP-R 5F1 PE Mouse BD 556016 FPR1 2357 IgG1, κ 350 FOXP3 259D/C PE Mouse BD 560046 FOXP3 50943 7 IgG1 351 Galecti B2C10 PE Mouse BD 556909 LGALS3 3958 n-3 IgG1, κ 352 Hemato BB9 PE Mouse BD 557928 NOT poietic IgG1, κ FOUND progeni tor cell 353 HLA-A2 BB7.2 PE Mouse BD 558570 HLA-A 3105 IgG2b, k 354 HLA- DX17 PE Mouse BD 560168 ABCA1 19 ABC IgG1, κ 355 HLA- MaP.D PE Mouse BD 555983 HFE 3077 DM M1 IgG1, κ 356 HLA-DR TU36 PE Mouse BD 555561 HFE 3077 IgG2b, k 357 ITGB7 FIB504 PE Rat BD 555945 ITGB7 3695 IgG2a, к 358 MIC 6D4 PE Mouse BD 558352 MICA/ 100507 A/B IgG2a, к MICB 436/42 77 359 Notch1 MHN1- APC not e- 17- 519 given Bioscie 9889- nce 42 360 Notch2 16F11 PE not e- 12- given Bioscie 5786- nce 82 361 Notch3 MHN3- APC not e- 17- 21 given Bioscie 5787- nce 42 362 NPM- ALK1 PE Mouse BD 559257 ACVRL1 94 ALK IgG3, к 363 PAC-1 PAC-1 FITC Mouse BD 340507 DUSP2 1844 IgM, k 364 Podopla NC-08 PE Rat BioLege 337004 PDPN 10630 nin IgG2a, λ nd

365 SSEA-3 MC631 PE Rat IgM BD 560237 FUT4 2526 366 SSEA-4 MC813- PE Mouse I BD 560128 FUT4 2526 70 gG3 367 Stro-1 STRO-1 APC Mouse BioLege 340104 NOT IgM, λ nd FOUND 368 TCR αβ T10B9.1 PE Mouse BD 555548 11126 A-31 IgM, k 369 TCR γδ B1 PE Mouse BD 555717 NOT IgG1, κ FOUND 370 LTBR hTNFR- PE Mouse BD 551503 LTBR 4055 RP-M12 IgG1, κ 371 TPBG 524744 APC Mouse R&D FAB497 TPBG 7162 IgG1 Systems 51A 372 Vβ8 JR2 PE Mouse BD 555607 NOT TCR IgG2b, k FOUND 373 Vδ2 B6 PE Mouse BD 555739 NOT TCR IgG1, κ FOUND 374 CDH11 667039 FITC not R&D FAB1790 given Systems 1G

Supplemental Table S2 - Known CD markers in neutrophils Known to Confirmed Other Family and be in the Notes on CD Marker Legend Names Function Expressed in current Expression PMNs study CD4 OKT4, Leu 3a, IgSF; primary S Y Present on UR - Up- L3T4, T4 receptor for some but not regulated HIV, T cell all circulating marker PMN CD10 MME, CALLA, Zinc C Y DR after in DR - down- neutral metalloprote vivo LPS regulated endopeptidas ase; Cleaves exposure. e peptides and Increased hydrolyzes expression other after in vitro biological LPS exposure materials CD11a ITGAL, LFA-1 Integrin; C Y S - subset α chain, CR3A plays role in PMN adhesion to endothelial wall CD11b ITGAM, α-M Integrin; key C Y UR with C - integrin leukocyte exposure to Constitutive chain, Mac-1 adhesion LPS and expression α chain molecule inflammation . DR following

exercise CD11c ITGAX, alpha Integrin; key C Y UR in . chain of CR4, leukocyte infection leukocyte adhesion surpace molecule antigen, p150-95 CDw12 P90-120 C N UR during . apoptosis and upon neutrophil activation CD13 ANPEP, Cleaves C Y . aminopeptida neutral se N, APN, amino acids gp150, EC and acts in 3.4.11.2 cell-surface antigen presentation CD14 LPS receptor LRG; acts as a C Y Always . coreceptor in present and the detection UR upon of LPS, high stimulation affinity with TNF- receptor for alpha, G-CSF LPS and GM-CSF complexes CD15 3-fucosyl-N- Carbohydrate C Y . acetyl- 2; role in lactosamine PMN binding to endothelium CD16/16a FCGR3A IgSF; plays C Y DR in . major role in apoptotic PMN PMN, DR activation after high and apoptosis intensity exercise CD16b FCGR3B, FcγR IgSF; receptor C Y Considered to . IIIb for IgG, signal be expressed transducer only on and mobilizes human PMN. calcium DR when stores cultured with IL-8 CD17 lactosylceram LacCer; binds C Y . ide PGG-glucan CD18 ITGB2, Integrin; C Y UR with . Integrin β2 leukocyte exposure to adhesion LPS molecule CD23 FCepsilonII IgSF; low S Y Not . affinity expressed in

receptor for healthy PMN; IgF, has present on cytokine-like ~50% of actions when patients with cleaved rheumatoid arthritis CD24 BA-1, HAS Glycosylphos C Y . phatidylinosit ol-anchored; Involved in signal transduction and stimulates adhesion to P-selectin CD28 Tp44 IgSF; C Y UR with PMA . regulates PMN migration in response to IL-8 CD29 ITGBP1, Integrin; C Y . integrin B1 mediates chain, VLA B PMN chain adhesion to fibroblasts CD31 PECAM-1, IgSF; C Y DR in tissue . endocam intermediary after in PMN extravasation diapedesis from blood CD32 FCGR2A, FcγR Fc receptor; C Y . II regulates phagocytosis and release of reactive oxidative species CD33 My9, Siglec3, IgSF; role in S Y Present in . gP67 cell-cell blast stage adhesion but mature neutrophils show weak to no staining CD35 Complement RCA; binds C Y DR after . receptor type cells with trauma, 1, CR1, C3b/C4b recovery in C3b/C4b complexes expression receptor, im over time. mune Significant DR adherence in expression receptor with exercise CD38 T10, gp45, ADP-ribosyl C Y DR in patients .

ADP-ribosyl cyclase; acts with cyclase as a selectin aggressive and has role periodontitis in regulating cell adhesion CD43 Leukosialin, Sialomucin; C Y DR during . sialophorin, anti- PMN gp95, SPN adhesive, acts apoptosis; as a negative shed from regulator for surface cell adhesion during PMN activation and adhesion CD44 Phagocyte Hyaladherin; C Y DR in . glycoprotein role in apoptosis 1, Hermes regulation of antigen, phagocytosis HUTCH-1, H- of PMN by CAM macrophages CD45 PTPRC, Protein C Y . leukocyte tyrosine common phosphatase; antigen, LCA signal molecule that regulates many cell processes CD45RA B cells, naïve Protein C Y Expressed to . T-cells, tyrosine a low degree monocytes phosphatase by control PMN (1-15%). UR to ~30% expression in infection CD45RB T200, B220, Protein C Y . DAKO-LCA tyrosine phosphatase; modulates CXCR1 and CXCR2 expression in PMN CD45RO UCHL-1 Protein C Y . tyrosine phosphatase CD46 Membrane RCA; protects C Y DR after . cofactor tissue from trauma and protein, MCP pathogenic does not damage, return to cofactor that normal inactivates expression C3b and C4b even after 10 days

CD47 Integrin IgSF; role in C Y UR with fMLP . associated PMN stimulation protein (IAP), transmigratio and after ovarian n transmigratio carcinoma n antigen OA3 CD49d α4 integrin, Integrin; C Y . VLA-4 α primary role chain, ITGA4 in monocyte migration CD49f α6, VLA-6 α Integrin; C Y . chain, mediates cell platelet GPIc, binding to ITGA6 laminin CD50 ICAM-3, IgSF; role in C Y DR in . intercellular cell-cell apoptosis and adhesion adhesion and activated molecule 3 as a target PMN recognition receptor for apoptotic cells CD53 MOX44, Tetraspanin/T C Y Higher levels . TSPAN25 M4; role in of expression cell growth in lupus properties patients. DR with fMLP stimulation CD54 ICAM-1, IgSF; role in C Y UR when . intercellular signal stimulated adhesion transduction with PMA molecule 1 and superoxide production in PMN CD55 DAF, decay RCA; inhibits C Y UR after . accelerating C3 trauma to factor convertase body assembly which protects host tissue CD58 LFA-3, IgSF; CD2 C Y . lymphocyte ligand and function play role in associated cell adhesion antigen-3 and signaling CD59 MACIF, MIRL, Ly6; inhibits C Y UR after . P-18, MAC trauma to protectin formation body and UR and acts as in patients coreceptor in with sepsis NK cell

activation CD62L L-selectin, Selectin; vital C Y DR after . LAM-1, Mel- for PMN apheresis and 14, SELL transmigratio shed from n, mediates cell surface tethering and with rolling stages activation on and apoptosis endothelium CD63 LIMP, GP55, Tetraspanin/T C Y UR in nasal . LAMP-3, M4; involved tissue and OMA81H, in PMN after TSPAN30, activation stimulation ME491, and migration with fMLP granulophysi through role n in adhesion CD64 FcR1, FcγR1, IgSF; binds S Y Very low . high-affinity with expression on Fcγreceptor, monomeric resting PMN FCGR1A and but rapid aggregated increase with IgG activation. UR in vivo with rhG-CSF and in patients with bacterial infections. Higher expression in patients with rheumatoid arthritis CD65/CD65s Ceramide Poly-N- C Y . dode- acetyllactosa casaccharide, mine; 4c currently unknown CD66a CEACAM1, IgSF; role in C Y Stored in the . NCA-160 enhancing secondary neutrophil granules of adhesion PMN, expressed in more mature PMNs. UR with stimulation/a ctivation of PMN CD66b CEACAM8, IgSF; C Y Higher . NCA-95 granulocyte expression in activation patients with marker, role rheumatoid in enhancing arthritis. DR

neutrophil as neutrophil adhesion matures and with apoptosis. UR with stimulation/a ctivation of PMN CD66c CEACAM6, IgSF; role in C Y Mainly . NCA-50/90 enhancing located on neutrophil primary adhesion granules in PMN. UR with stimulation/a ctivation of PMN CD66d CGM1, IgSF; role in C Y UR with . CEACAM3 enhancing stimulation/a neutrophil ctivation of adhesion PMN CD68 Gp110 Sialomucin; C Y UR in . acts as a inflamed scavenger tissue in receptor Crohn’s disease CD69 AIM, C-type lectin; S Y Very low in . activator induces resting PMN. inducer neutrophil UR in molecule, degranulation activated CLEC2C, and other cell PMN and MLR3, EA1, processes with fMLP VEA stimulation to ~13% CD80 B7, B7-1, IgSF; an S Y Low on . CD28LG antigen resting PMN, presenting stored in complex, granules. UR induces T cell with activation activation CD82 R2, IA4, 4F9, Tetraspan; C Y . C33, KAI1, roles in signal TSPAN27 transduction and inhibitor of cell migration CD84 LY9B, SLAM; C Y Present in . SLAMF5 associate some studies with SLAM- but not associated detected in protein, other studies promotes T- cell activation

and secretion of interferon gamma CD85a LILRB3, ILT5, IgSF; binds C Y . Immunoglobu MHC class 1 lin-like molecules transcripts 5. and inhibits immune response CD85d LILRB2, ILT4 IgSF; binds C Y UR in PMN in . MHC class 1 response to molecules inflammatory and inhibits stimuli and in immune sepsis response CD85h LILRA2, LIR7, short S Y neutrophils . ILT1 cytoplasmic have a domain and a restricted positively pattern of charged cell-surface arginine LIR residue expression within the with LIR3 and transmembra LIR7 being ne domain expressed in that mediates almost all association donors, with with the occasional immunorecep expression of tor tyrosine- LIR1 and︎ or based LIR2 activation motif- containing Fc receptor ︎ chain (Fc) CD85j LILRB1, ILT2 IgSF; binds C Y . MHC class 1 molecules and inhibits immune response CD85k LILRB4, ILT3 immunorecep S Y neutrophils . tor tyrosine- have a based restricted inhibitory pattern of motifs cell-surface LIR expression with LIR3 and LIR7 being expressed in almost all donors, with

occasional expression of LIR1 andor LIR2 CD86 B7-2, LAB72 IgSF; an S N Low . antigen expression in presenting circulation UR complex, role with in exposure to moderating T IFNgamma cell and Gm-CSF proliferation CD87 Urokinase- GPI- C Y DR by 47% in . type anchored; apoptotic plasminogen role in PMN activator leukocyte receptor, extravasation PLAUR as regulators of integrin- mediated adhesion CD88 C5a-receptor, Rhodopsin; C N DR by over . C5aR, C5R1 role in tissue 50% in inflammation apoptotic after trauma PMN. DR immediately after traumatic injury CD89 FCAR, FcaRI Fc receptor; S Y Stored in . potent secondary cytotoxic and tertiary trigger granules and molecule expressed on surface in response to fMLP, IL-8 and C5a CD92 SLC44A1, Choline C Y . CHTL transporter, may have role in IL-10 regulation, implicated in negative signaling pathways CD93 C1qR1 O- Y Y DR and . sialoglycoprot cleaved from ein; role in surface in cell-cell response to interactions inflammation and adhesion and apoptotic

cell clean-up CD95 APO-1, Fas, TNFR; C Y . TNFRSF6 induces PMN apoptosis CD97 TM7LN1 EGF-TM7; C Y Present in . role in low leukocyte concentration trafficking . UR expression and activity in patients following heart surgery; UR in joint PMN following acute hemoarthrosi s CD99 MIC2, E2 Transmembra C Y Present in . ne low glycoprotein; concentration mediates on PMN PMN surface. UR migration when across attached to transendothe endothelial lial cells for membrane transmigratio n CD101 V7, P126, IgSF; role in T C Y . IGSF2, EWI- cell activation 101 CD107a LAMP-1 LAMP; S Y Small amount . polylactosami present on noglycan surface, carrier mainly stored in secretory vesicles. UR at surface in response to fMLP CD107b LAMP-2 LAMP; S Y Small amount . polylactosami present on noglycan surface, carrier mainly stored in secretory vesicles. UR at surface in response to fMLP CD114 G-CSFR, CSFR, Class I CK-R; C Y DR in . granulocyte initiates cell response to

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colony proliferation GCSF stimulating and stimulation factor differentiatio receptor, n into mature CSF3R PMN and has a role in delaying apoptosis CD116 GM-CSF Class I CK-R; C Y DR in . receptor role in cell response to alpha proliferation, GMCSF. subunit, GMR augments Decreased α, CSF2RA PMN anti- expression bacteria seen in functions in patients with multiple ways inflammatory bowel disease CD119 IFN-γ IgSF; enhance C Y DR when . receptor α PMN anti- infected with chain, IFNGR1 bacterial A. functions in cytophagilum various ways CD120a TNF receptor TNFR; role in C Y Expressed in . 1, TNFRS5, neutrophil low levels. TNFRSF1A recruitment Rapid DR when culture in vitro and in apoptosis CD120b TNF receptor TNFR C Y Expressed in . 2, TNFR80, low levels. TNFRSF1B Rapid DR when culture in vitro and in apoptosis CD121b Type II IL-1 IgSF; C Y UR in patients . receptor, stimulates with sepsis IL1R2, IL1RB neutrophil activation CD122 IL-2 receptor Class I CK-R; C Y . β chain, p75, mediates IL-2 IL2RB signal transduction CD123 IL-3 receptor Class I CK-R; S Y Not . α chain, role in cell expressed in IL3RA proliferation healthy and controls. UR differentiatio and present n and inhibits after apoptosis incubation with GM-CSF CD124 IL-4 R α chain, Class I CK-R; C Y . IL4R mediates IL-4

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signal transduction, role as growth factor CD130 GP 130, IL-6 CRSF; role in C Y . receptor β signal chain, IL6ST transduction CD131 Common β Class I CK-R; C Y . chain, form dimer CSF2RB, with CD123 IL3RB, IL5RB or CD116 to act as receptor CD132 Common γ IgSF; PMN C Y . chain, IL2RG receptor for cytokines IL2, IL4, IL7, IL9, IL15 and IL21 CD138 Syndecan-1, Syndecan; C Y Increased . SDC1, SYND1, role in PMN expression in SDC migration and patients with adhesion by Type 2 regulating diabetes and chemokine correlated gradients, with BMI roles in cell growth CD141 THBD, C-type lectin; S Y Expressed . thrombomod receptor for very low on ulin, thrombin and surface. Fetomodulin acts as an Unsure what anticoagulant stimulates expression on surface CD147 Basigin, BSG, IgSF; C Y . 5F7, stimulates EMMPPRIN, production of M6, OK, TCSF matrix metalloprotei nases from stromal cells and has role in cancer metastasis CD148 PTPRJ, DEP-1, RPTPase type C Y . HPTPeta III, phosphatase; similar to CD45, role in inhibition of FcyRIIa functions CD151 PETA-3, SFA- Tetraspan C Y .

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1, TSPAN24 TM4; forms complex with a3b1 and influences cell migration CD153 TNFSF8, CD30 TNF; signal C Y . Ligand, CD30L regulation of cell death and proliferation, induces UR or DR of CD30 CD156a ADAM8, MS 2 ADAM; C Y . metalloprotei nase, acts as a sheddase to cleave proteins CD156b TACE (tumor Zinc C Y UR with . necrosis metallopepti phagocytosis factor α- dase; role in of B.cepacia converting transmembra and UR enzyme), ne protein during severe ADAM17, cleavage periotonitis snake venom like protease, CSVP CD156c ADAM10 ADAM; acts C Y DR with . as a sheddase apoptosis so involved in protein cleavage CD157 BST-1 BP- ADP-ribosyl C Y UR by double . 3/IF7 Mo5 cyclase; role after fMLP in neutrophil stimulation adhesion and migration CD162 SELPLG, P Sialomucin; C Y DR and . selectin mediates P- released into glycoprotein selectin blood with ligand 1, activity PMN PSGL-1 activation CD163 M130 Scavenger C Y Present in . receptor low amounts, elevated in children and adults with sepsis CD170 SIGLEC5, OB- IgSF; primes C Y UR with fMLP . BP2 PMN for stimulation enhanced stimulation by fMLP

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CD172a PTPNS1, IgSF; role in S Y Stored in . SIRPα, SHPS-1 PMN intracellular signal migration pools and regulatory through moved to protein α. epithelial and surface after endothelial simulation by and collagen- chemoattract coated cells, ants. DR with as well as in apoptosis adhesion CD172b CD172β, IgSF; C Y . SIRB1, SIRPβ1 stimulatory interactions with ITAM molecules CD177 PRV1 uPAR S Y Expressed on . (polycythemi receptor; subpopulatio a vera rubra binds n of PMN, 1), NB1 endothelial ~30-70%. 3% cell PECAM-1 express no CD177. UR with severe infection CD178 Fas ligand, TNF; role in C Y . FasL, CD95L PMN apoptosis CD181 CXCR1; IL-8 Binds IL-8 C Y DR with LPS . receptor α, stimulation IL8RA CD182 CXCR2; IL-8 GPCR family, C Y DR in severe . receptor b, chemokine sepsis and IL8RB receptor, exposure to Rhodopsin; LPS high affinity to IL-8, role in PMN migration to inflammatory areas CD183 CXCR3 GPCR family, S Y Marginally . chemokine present in receptor; role circulation. in cell UR at chemotaxis pulmonary and calcium and synovial mobilization site of inflammation CD184 CXCR4 GPCR family, S Y Marginally . chemokine present in receptor; role circulation. in the bone UR at marrow in pulmonary regulation of and synovial

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hematopoieti site of c cells inflammation ; DR with incubation with lithium CD191 CCR1 GPCR family, S N Marginally . chemokine present in receptor circulation. UR at pulmonary and synovial sit of inflmmation; UR with IFN- gamma CD192 CCR2 GPCR family, S Y Marginally . chemokine present in receptor circulation. UR at pulmonary and synovial sit of inflmmation CD193 CCR3, GPCR family, S Y Marginally . eosinophil chemokine present in eotaxin receptor circulation. receptor, UR at chemokine pulmonary (C-C motif) and synovial receptor 3, sit of CKR3, inflmmation; CMKBR3 UR with IFN- gamma CD195 CCR5 GPCR family, S Y Marginally . chemokine present in receptor circulation. UR at pulmonary and synovial sit of inflmmation CD200 MRC, OX 2 IgSF; role in C Y . regulation of myeloid cell function CD205 LY75, C-type lectin; C Y . CLEC13B large range of function, a macrophage mannose receptor CD212 IL12RB1, IL- hemopoietin C Y Slightly UR . 12R-BETA1, receptor SF; with LPS

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IL-12RB role in IL-8 exposure production CD215 IL-15RA Type I C Y . cytokine receptor family; activates PMN via IL-15 ligand CD217 IL-17R Type I C Y . cytokine receptor family; proinflammat ory, proapoptotic cytokine CD218a IL-18 CK-R; role in C N . receptor PMN protein alpha, IL18R1 synthesis and release, UR CD11b, augments release of ROS CD218b IL-18 CK-R; role in C Y . receptor PMN protein beta, IL18RAP synthesis and release, UR CD11b, augments release of ROS CD220 Insulin Tyrosine C Y . receptor, IR, kinase INSR receptor; insulin receptor, moderates PMN chemotaxis CD221 IGF1 Tyrosine C Y . Receptor, kinase IGF1R receptor; Potent primer of PMN for enhanced ROS secretion CD222 IGF2R, Man- Lectin; C Y UR at site of . 6p receptor targets infection protein to lysosome

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CD261 TRAIL-R1, Tumor S N Present in . DR4, necrosis some studies TNFRSF10A factor but not receptor detected in superfamily other studies (TNFRSF); death receptor, stimulates apoptosis160 and shown to be responsible for PMN cell death CD262 TRAIL-R2, Tumor C N . DR5, necrosis TNFRSF10B factor receptor superfamily (TNFRSF); death receptor, stimulates apoptosis CD263 TRAIL-R3, Tumor C Y . DcR1, LIT, necrosis TRID, factor TNFRSF10C receptor superfamily (TNFRSF); inhibits TRAIL- induced apoptotic signals CD264 TNFRSF10D, Tumor S Y Present in . TRAIL-R4, necrosis some studies DCR2 factor but not receptor detected in superfamily other studies (TNFRSF); inhibits TRAIL- induced apoptotic signals CD265 RANK, TNF/NGF S Y Wide variety . TRANCE-R, receptor; key of expression EOF, role in bone in healthy TNFRSF11A remodeling PMN ~1- and 70%. UR with osteoclast bacterial activity infections; UR

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in synovial fluid of arthritis patients CD274 B7-H1, PD-L1, IgSF; death S Y Not . PDCD1LG1 ligand, expressed in inhibitory control. molecule Induced expression with cytokine activation CD281 TOLL-like TLRF; C Y . receptor 1, required for TLR1, TIL immune recognition of mycobacteria lipoarabinom annan and triacylated llipopeptides CD282 TOLL-like TLRF; S Y UR with GM- . receptor 2, involved in CSF TLR2, TIL4 cytokine incubation; production UR in blood of alcoholic hepatitis patients; DR in blood PMN in patients with cycstic fibrosis. UR in airway PMN CD284 TOLL-like TLRF; S Y UR surface . receptor 4, receptor for expression in TLR4 LPS PMN of alcoholic hepatits patients CD285 TLR-5 TLRF; S N Expressed . stimulates IL- intracellularly 8 production , surface expression induced with TLR ligands and cytokines. UR in airway PMN in patients with cystic fibrosis CD286 TLR-6 TLRF; role in C Y . cytokine production

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CD287 TLR-7 TLRF; S N Located . involved in intracellularly ssRNA recognition CD288 TLR-8 TLRF; S Y Located . involved in ss intracellularly RNA recognition CD289 TLR-9 TLRF; inhibits C N UR in with . PMN GM-CSF migration through DR of CXCR2 CD290 TLR-10 TLRF C N . CD295 Leptin R, Type 1 C Y . LEPR, OBR, cytokine like B219 receptor; leptin receptor CD298 Na/K ATPase Enzymatic C Y . β3-subunit, transport of ATP1B3 Na/K CD300a CMRF35H, IgSF CMRF; C Y . IRC1, IRC2, inhibits ROS IRp60 production and inactivates receptor signalling CD302 DCL1, Type 1 TM, C C Y . CLEC13A, type lectin BIMLEC receptor; role in cell adhesion and migration CD305 LAIR1 IgSF; S Y . inhibitory receptor, inhibits various cytokine signals CD312 EMR2 EGF; involved C Y . in PMN recruitment and maintenance into tissue CD313 EMR3 EGF-TM7; C Y . currently unknown function CD321 JAM1, JAM-A, IgSF; role in C Y .

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F11R adhesion and transmigratio n of PMN and T cells CD329 SIGLEC9, SIGLEC; roles C Y . sialic acid- in apoptotic binding, Ig- and like lectin 9 nonapoptotic cell death in neutrophils CD354 TREM1 IgSF; role in C Y Strong . magnification upregulation of the in PMN with inflammatory LPS response CD360 IL-21R-alpha IgSF (Nectin C Y . family); receptor for IL21, enhances B cell proliferation CD361 EVI2B, Type 1 C Y . Ecotropic transmembra Viral ne protein; Intigration poorly 2B, defined functions

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