OMIP‐100: A flow cytometry panel to investigate human neutrophil subsets

This 14‐color, 13‐antibody optimized multicolor immunofluorescence panel (OMIP) was designed for deep profiling of neutrophil subsets in various types of human samples to contextualize neutrophil plasticity in a range of healthy and diseased states. Markers present in the OMIP allow the profiling of neutrophil subsets associated with ontogeny, migration, phagocytosis capacity, granule release, and immune modulation. For panel design, we ensured that the commonly available fluorophores FITC/AF488, PE, and APC were assigned to the intracellular subset marker Olfactomedin 4, the maturity and activation marker CD10, and whole blood subset marker CD177, respectively. These markers can be easily replaced without affecting the core identification of neutrophils, enabling antibodies to new neutrophil antigens of interest or for fluorescent substrates to assess different neutrophil functions to be easily explored. Panel optimization was performed on whole blood and purified neutrophils. We demonstrate applications on clinical samples (whole blood and saliva) and experimental endpoints (purified neutrophils stimulated through an in vitro transmigration assay). We hope that providing a uniform platform to analyze neutrophil plasticity in various sample types will facilitate the future understanding of neutrophil subsets in health and disease.


| BACKGROUND
Neutrophils are phagocytic, polymorphonuclear leukocytes, and represent the most abundant white blood cell type in the human bone marrow and peripheral blood.Neutrophils are involved in the first line of host defense against injury and pathogens, responding to inflammatory cues through priming, transmigration, and activation [1].Upon transmigration to the inflammatory site by following a chemotactic gradient, neutrophils will typically engage in the clearance of damaged tissue or microorganism killing through phagocytosis, degranulation, reactive oxygen species production and in certain biological situations, the release of neutrophil extracellular traps (NETs) [2].
Traditionally considered to be just short-lived cells that are preprogrammed to carry out the above functions before undergoing apoptotic clearance, neutrophils have recently been recognized for their capacity to extend their lifespan [3] and display profound transcriptional plasticity, leading to the acquisition of novel functions such as the modulation of adaptive immunity [4][5][6][7].Key studies have identified novel activation marker expression or preferential activity of neutrophil subsets in response to infection [8][9][10][11][12], sepsis [13][14][15], autoimmune disease [16,17], and cancer [18][19][20], with sepsis being perhaps the most extreme pathological case for changes and creation of circulating neutrophil subsets that can be linked to sepsis outcomes [14].However, changes in conventional neutrophil maturity/ ontogeny markers [21,22] are also seen during priming, transmigration, and activation, which have been linked to functional responses or disease states [23,24].The current state of understanding of human neutrophil subsets and their putative roles in disease has been recently reviewed and debated [3,7,[25][26][27] but many questions remain.
One issue in interpreting the literature to date and relating findings between diseases is the inconsistency in flow cytometry approaches to characterize neutrophils.With no published panel allowing for the systematic differentiation of human neutrophil subsets, there is a clear need for one that encompasses a robust set of markers for routine application across clinical and laboratory samples.In designing this optimized multicolor immunofluorescence panel (OMIP) [28], we utilized a common set of markers identified in the literature to characterize neutrophil activation and to delineate multiple neutrophil subsets in various biological conditions.

| Implementation
Following typical singlet and live/dead gates, neutrophils can be robustly identified in blood by using CD45, the pan leukocyte marker, and CD15 (also known as Sialyl Lewis X ) (Figure 1A) [22,29].Following delineation, two major neutrophil subsets have been identified in peripheral blood to date based on CD177 and olfactomedin 4 (OLFM-4) expression.CD177 is bimodally expressed by the majority of the human population [30,31] and has associations with an increased migration capacity, although this remains controversial [32,33].OLFM-4 is expressed intracellularly and approximately 20%-25% of peripheral neutrophils feature high expression [34].Released extracellularly upon NETosis, OLFM-4 has some reported associations with neutrophil function, especially during sepsis [35].Neutrophil ontogeny is also informed by the expression of the markers CD10 and CD184 (also known as CXCR4).Considered a neutrophil maturation and activation marker, CD10 is present on most peripheral neutrophils at baseline, but an increased proportion of CD10-negative immature neutrophils can be released in the systemic circulation due to active recruitment from the bone marrow upon acute inflammation [36][37][38].
In addition to becoming expressed by tissue neutrophils following their activation, CD184 is expressed by aged neutrophils in a circadian manner and mediates their retention in and return to the bone marrow [39][40][41].The maturation state of a neutrophil has been associated with functional consequences in multiple different conditions in health and disease [21,23,24,42].
Upon activation, neutrophils undergo priming for transmigration into the injured or infected tissue.Classical rolling/adhesion markers of neutrophil migration CD11b (also known as MAC-1) and CD62L (also known as L-selectin) are included in the panel to assess the migratory state [43,44].In addition, primary and secondary granule exocytosis following migration can be determined by measuring the surface expression of CD63 and CD66b, respectively [45][46][47][48].For determining neutrophil capacity to uptake opsonized particles for phagocytosis and killing, we used the most abundant neutrophil Fc receptor CD16b (also known as FcγRIIIb) [49].From these markers, it could be possible to identify several neutrophil subsets beyond CD177 and OLFM-4 that arise in acute and chronic inflammation, including band, segmented and hypersegmented neutrophils (CD16 and CD62L), immature immunosuppressive neutrophils (CD10), and GRIM neutrophils (CD16 and CD63) [5,8,9,50,51].Finally, markers CD54 (also known as ICAM-1) and CD49d (also known as integrin α4) are included to identify neutrophils that have reverse-transmigrated back to the periphery and individuals with atopy, respectively [52][53][54].
For maximum applicability across the research community, the panel was designed for the five-laser BD LSRFortessa cytometer (Table S1), perhaps the most widely available five-laser cytometer.
Detailed methods of panel development can be found in the Appendix S1.Briefly, fluorophore-antigen selection was informed by antigen density, fluorophore brightness, and conjugate commercial availability (Table 1).We also generated a Spillover Spread Matrix (SSM), which was integral for minimizing spectral overlap in a panel that focuses on a single cell lineage (Figure S1) [55].Care was taken to minimize spectral spillover into the B-510 and YG-650 channels (FITC/AF488 and PE, respectively) as these provide an opportunity to adapt the panel by rapid incorporation of alternate antibodies labeled F I G U R E 1 Example gating strategy and UMAP analysis for the visualization of neutrophil populations using OMIP-100.(A) Example gating strategy on 20 μL of whole blood taken from a healthy adult donor.Samples were gated with time vs SSC-A to exclude acquisition inconsistencies, then gated with FSC-A versus SSC-A gate to exclude non-cell events, then single-cell events were included using FSC-H versus FSC-A.Viable leukocytes were determined via CD45 + R718 versus FVS780 À , and finally pure neutrophils were determined as CD15 HI BV786 and CD66b HI PE/Fire640.Downstream populations on the pure neutrophil populations are delineated, including blood subsets, migration capacity, granule release, immune modulation, rare subsets, granule releasing immunoregulatory and metabolically enhanced (GRIM), and maturation.(B) Uniform Manifold Approximation and Projection (UMAP) analysis upon the neutrophil gate for the visualization of 3 donors' peripheral whole blood neutrophils, purified neutrophils from the same 3 donors migrated toward an uninfected stimulus or a Pseudomonas aeruginosa-infected stimulated (as per Laucirica et al [59]), and healthy human saliva.(C) UMAP clusters are colored based on their sample of origin, purple for whole blood (WB), teal for uninfected migration stimulus (Uninf_mig), red for Pseudomonas aeruginosa-infected migration stimulus (Psa_mig) and green for saliva.(D) Heat map and dendrograms showing the relative expression of each of the marker in the panel per cluster.Individual expression plots are shown in Figure S13.[Color figure can be viewed at wileyonlinelibrary.com] with these common fluorophores or fluorescent probes with peak emission characteristics in these channels.
To ensure this OMIP was suitable for a broad range of cohorts and clinical samples, particularly those where available blood volumes may be low, we first optimized it using a small volume (20 μL) of adult peripheral blood.The panel was subsequently tested on purified blood neutrophils stimulated in an in vitro model of tissue transmigration toward an inflammatory stimulus (supernatant generated by infecting primary air-liquid interface cultures of airway epithelium, as per Laucirica et al) [56][57][58][59], as well as neutrophils from a healthy human saliva sample [60,61].To illustrate individual differences and nuances in neutrophil phenotypes, neutrophil populations from these samples were pooled, downsampled to 10,000 events per experimental condition, and subjected to Uniform Manifold Approximation and Projection by utilizing the Specter R package [62] (Figure 1B).The sample of origin is shown in Figure 1C and Figure 1D shows the combined heat map and dendrogram plot of each marker's relative expression per cluster.
In summary, we report here a panel allowing deep phenotyping of human neutrophils in both clinical and laboratory samples.This panel will complement existing OMIPs that phenotype other innate and adaptive immune cells in clinical samples but do not achieve equally deep characterization of the neutrophil cell population (Table 2).It may also serve use in phenotyping the neutrophil compartment of blood samples prior to isolation and cryopreservation of peripheral blood mononuclear cells, since neutrophils are not conducive to cryopreservation.Iterative application of this panel with different FITC and PE-conjugated antibodies and/or probes to experimental samples will enable an improved understanding of neutrophil functions across subsets, for example, recently identified DEspR+CD11b+/CD66b + "rogue" neutrophils [63].We believe that the flexible application of this panel combined with the capability for widespread adaptation to different human samples will drive much-needed contextualization of neutrophil subsets in human health and disease.

| SIMILARITY TO OTHER OMIPs
No prior OMIP interrogates neutrophils beyond initial classification.
OMIPs related to work in this publication (Table 2) include OMIP-023 and OMIP-038 for the classification of peripheral blood leukocytes and myeloid cells, respectively, and OMIP-049 for the identification of myeloid ontogeny in the bone marrow.OMIP-062 also identifies leukocytes present in low volumes of whole blood, as well as nasal or tracheal aspirate.OMIP-094 is a broad human immunophenotyping panel that can discriminate the CD177 neutrophil subset.While not an OMIP, this year data were published on whole blood neutrophils in sepsis utilizing a multicolor panel for the BD FACS Symphony cytometer with several markers in common to this OMIP [14].
All samples were collected from healthy adult subjects after gaining written consent from participants through the Pediatric Epithelial and Airway Research Surrogate Sample (PEARSS) program, at Perth Children's Hospital, Western Australia.The use of samples was approved by the Children and Adolescent Health Service Ethics Committee (RGS1470) and in accordance with the Declaration of Helsinki 1964.

T A B L E 1
Reagent table.
Summary table.