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Cytof Mass Cytometry Reveals Phenotypically Distinct Human Blood Neutrophil Populations Differentially Correlated with Melanoma Stage

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Cytof Mass Cytometry Reveals Phenotypically Distinct Human Blood Neutrophil Populations Differentially Correlated with Melanoma Stage Open access Original research J Immunother Cancer: first published as 10.1136/jitc-2019-000473 on 9 September 2020. Downloaded from CyTOF mass cytometry reveals phenotypically distinct human blood neutrophil populations differentially correlated with melanoma stage 1 1 1 Yanfang Peipei Zhu , Tobias Eggert, Daniel J Araujo, 2 3 Pandurangan Vijayanand, Christian Hermann Ottensmeier , Catherine C Hedrick1 To cite: Zhu YP, Eggert T, ABSTRACT populations, each with opposing actions in Araujo DJ, et al. CyTOF Background Understanding neutrophil heterogeneity immune regulation and cancer progression.6 mass cytometry reveals and its relationship to disease progression has become a phenotypically distinct human Marini and coauthors employed flow cytom- recent focus of cancer research. Indeed, several studies + − blood neutrophil populations etry to show that CD10 and CD10 neutro- have identified neutrophil subpopulations associated with differentially correlated with phils represent populations with opposing protumoral or antitumoral functions. However, this work melanoma stage. Journal for effects on T- cell proliferation.7 Pillay and has been hindered by a lack of widely accepted markers ImmunoTherapy of Cancer colleagues identified three neutrophil 2020;8:e000473. doi:10.1136/ with which to define neutrophil subpopulations. jitc-2019-000473 Methods To identify markers of neutrophil heterogeneity subpopulations, based on their differential in cancer, we used single-cell cytometry by time-of- flight expression of CD16 and CD62L, with each ► Additional material is (CyTOF) coupled with high-dimensional analysis on blood exhibiting specific maturation and activa- 8 published online only. To view, samples from treatment- naïve patients with melanoma. tion statuses. CD45RA, CD63, and CD11b please visit the journal online Results Our efforts allowed us to identify seven blood also indicate activation statuses in certain (http:// dx. doi. org/ 10. 1136/ jitc- neutrophil clusters, including two previously identified neutrophil subsets.9 10 Singhal and collabo- 2019- 000473). individual populations. Interrogation of these neutrophil rators isolated a CD14+ neutrophil subpop- subpopulations revealed a positive trend between specific ulation with antitumor functions, including Accepted 19 June 2020 clusters and disease stage. Finally, we recapitulated these enhancement of effector T cell-based seven blood neutrophil populations via flow cytometry 11 and found that they exhibited diverse capacities for production of interferon- g and granzyme B. phagocytosis and reactive oxygen species production in Evrard and colleagues have demonstrated a + + − vitro. CD15 CD49 CD101 neutrophil precursor http://jitc.bmj.com/ Conclusions Our data provide a refined consensus on (preNeu).12 Our group has identified a neutrophil heterogeneity markers, enabling a prospective CD117+CD66b+CD38+ human neutrophil functional evaluation in patients with solid tumors. progenitor (hNeP), which was also found in the blood of tumor-bearing animals.13 Addi- INTRODUCTION tional work in this area is summarized in 14 15 Neutrophils are bone marrow (BM)-derived two excellent review articles. Neverthe- on October 4, 2021 by guest. Protected copyright. © Author(s) (or their myeloid cells that play pivotal roles in anti- less, a lack of widely accepted subpopulation employer(s)) 2020. Re- use cancer immunity.1 Neutrophils are produced markers has hindered our understanding permitted under CC BY. 11 2 3 of neutrophil heterogeneity. Indeed, the Published by BMJ. at a rate of 10 per day and comprise neutrophil subpopulations thus far described 1Inflammation Biology, La Jolla 50%–70% of blood leukocytes. Due to this Institute for Immunology, La rapid turnover in the body, neutrophils have likely represent intersecting populations. For Jolla, California, USA traditionally been viewed as a homogeneous example, flow cytometry analysis suggests that + − 2Division of Vaccine Discovery, population. However, recent work has shown CD10 and CD10 neutrophil subpopulations La Jolla Institute for that they exhibit a longer life cycle than previ- are fractionated into both LDN and HDN Immunology, La Jolla, California, 7 + ously thought,4 reviving interest in the possi- layers. Furthermore, the CD10 expression USA 3 5 7 3School of Cancer Sciences, bility of distinct neutrophil populations. demonstrated by Marini et al is shared by the bright University of Southampton Spurred on by such findings, several CD16 subpopulation reported by Pillay Faculty of Medicine, groups have since identified and charac- et al.8 Specific CD14+ neutrophils present a Southampton, UK terized several neutrophil subpopulations. CD10− phenotype,11 suggesting this subpop- For example, use of density gradient sepa- ulation overlaps with the CD10− neutrophil Correspondence to 7 + Professor Catherine C Hedrick; ration has uncovered low-density neutrophil population. The CD14 neutrophil subpop- hedrick@ lji. org (LDN) and high-density neutrophil (HDN) ulation also present a CD49d+ phenotype, Zhu YP, et al. J Immunother Cancer 2020;8:e000473. doi:10.1136/jitc-2019-000473 1 Open access J Immunother Cancer: first published as 10.1136/jitc-2019-000473 on 9 September 2020. Downloaded from indicating that it overlaps with the CD49d+ preNeu pipetting to reach final concentration of 3×106 cells per demonstrated by Evrard et al,12 as well as a CD49d+C- 100 µL buffer. D62Llo neutrophil subpopulation (‘aged neutrophil’) reported by Casanova- Acebes et al.16 To determine the Mass cytometry antibodies extent to which previously reported neutrophil subpopu- Metal- conjugated antibodies were purchased directly lations intersect, high- dimensional analysis of neutrophil from Fluidigm for available targets. For all other targets, heterogeneity on a single- cell basis is imperative. purified antibodies were purchased as described before.20 We and others have employed high-dimensional Antibody conjugations were prepared using the Maxpar approaches such as single-cell cytometry by time-of- flight Antibody Labeling Kit (Fluidigm) according to the (CyTOF) and single- cell RNA sequencing (scRNA-seq) manufacturer’s recommendations. Afterwards, Maxpar- to address neutrophil heterogeneity. These endeavors conjugated antibodies were stored in phosphate-buffered demonstrate that the neutrophil lineage comprises a saline- based antibody stabilization solution (Candor heterogeneous pool in mouse and human BM.12 13 Addi- Biosciences) supplemented with 0.05% sodium azide at tionally, scRNA- seq analyses reveal six neutrophil clusters 4°C. All antibodies were titrated before use. with distinct transcriptional signatures in human lung tumors, but the surface markers needed to classify these Mass cytometry (CyTOF) populations were not identified.17 Interestingly, work in CyTOF was performed following previously described this field has also suggested differential involvement of protocols.20 For viability staining, cells were washed in neutrophil subpopulations in cancer.18 19 Thus, the devel- phosphate- buffered saline and stained with Cisplatin opment of consensus neutrophil markers is required for (Fluidigm) at a final concentration of 5 µM. Prior to improving our understanding of neutrophil biology and surface staining, RBC- lysed WB cells were resuspended in its relationship to disease progression. staining buffer for 15 min at RT to block Fc receptors. The Here, we use a CyTOF panel of the most commonly used surface antibody cocktail listed in table 1 was added into surface markers of neutrophil maturation, activation, cell suspensions for 1 hour at 4°C. The cells were then and function to comprehensively investigate neutrophil washed with staining buffer and fixed with 1.6% para- heterogeneity in whole blood (WB) from treatment- naïve formaldehyde (Thermo Fisher) for 15 min at RT. After- patients with melanoma. High-dimensional analysis of wards, 1 mL of intercalation solution for each sample was this dataset revealed seven neutrophil subpopulations prepared by adding Cell-ID Intercalator- Ir (Fluidigm) associated with disease stage and which are reproduc- into Maxpar Fix and Perm Buffer (Fluidigm) to a final ible during manual gating in flow cytometry. Finally, we concentration of 125 nM (a 1000× dilution of the 125 µM found that these seven neutrophil subpopulations harbor stock solution) and vortex to mix. After fixation, the cells distinctive functions, demonstrated by their differential were resuspended with the intercalation solution and capacities for phagocytosis and reactive oxygen species incubated overnight at 4°C. Cells were then washed in (ROS) production. staining buffer and then with subsequent washes in Cell Acquisition Solution (CAS) (Fluidigm) to remove buffer salts. Next, the cells were resuspended in CAS with a 1:10 http://jitc.bmj.com/ METHODS dilution of EQ Four Element Calibration beads (Flui- Melanoma patient blood collection digm) and filtered through a 35 µm nylon mesh filter cap Blood samples from patients with melanoma who were (Corning, Falcon). Samples were analyzed on a Helios treatment- naïve after surgical resection were collected in 2 CyTOF Mass Cytometer (Fluidigm) equipped with a EDTA- coated tubes by the Biospecimen Repository Core Super Sampler (Victorian Airship & Scientific Apparatus) Facility at the University of Kansas Cancer Center and at an event rate ≤500 events/s. Mass cytometry data files on October 4, 2021 by guest. Protected copyright. 21 delivered to La Jolla Institute for Immunology (LJI) via were normalized
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