Neutrophil Progenitors and Related Methods and Uses
20230213513 · 2023-07-06
Inventors
- Lai Guan Ng (Singapore, SG)
- Immanuel Weng Han Kwok (Singapore, SG)
- De Li Leonard Tan (Singapore, SG)
- Florent Ginhoux (Singapore, SG)
Cpc classification
A61K35/15
HUMAN NECESSITIES
A61P7/00
HUMAN NECESSITIES
C12N2501/22
CHEMISTRY; METALLURGY
C12N5/0642
CHEMISTRY; METALLURGY
G01N2333/705
PHYSICS
International classification
Abstract
There is provided a method of identifying a neutrophil progenitor, the method comprising: determining an expression of at least one biomarker selected from the group consisting of: CD71, LOX-1, CD164, CD112, CD181, TACSTD2, CD11b and CD49d in a cell. In various embodiments, the cell is identified as a neutrophil progenitor when it is determined to have at least one of the following expression profiles: CD71.sup.hi/+, LOX-1.sup.int/lo/−, CD164.sup.hi/+, CD112.sup.hi/+, CD181.sup.int/lo/−, TACSTD2.sup.hi/+, CD11b.sup.lo/− and/or CD49d.sup.int/hi/+. Also disclosed are a method of sorting and/or separating neutrophil progenitors from a cell population, a composition that is enriched in neutrophil progenitors and related uses and methods.
Claims
1. A method of identifying a neutrophil progenitor, the method comprising: determining an expression of CD71 and at least one biomarker selected from the group consisting of: LOX-1, CD164, CD112, CD181, TACSTD2, CD11b and CD49d in a cell; and identifying the cell as a neutrophil progenitor when it is determined to have an expression profile of: CD71.sup.hi/+, and at least one of the following expression profiles: LOX-1.sup.int/lo/−, CD164.sup.hi/+, CD112.sup.hi/+, CD181.sup.int/lo/−, TACSTD2.sup.hi/+, CD11b.sup.lo/− and/or CD49d.sup.int/hi/+.
2. (canceled)
3. The method according to claim 1, wherein where the cell is identified as a neutrophil progenitor, the method further comprises: determining an expression of a further biomarker selected from CD49d and/or a side-scatter (SSC) property of the neutrophil progenitor; and identifying a subtype of the neutrophil progenitor based on the expression of the further biomarker and/or the side-scatter property.
4. The method according to claim 3, wherein where the cell is determined to be CD49d.sup.hi/+ and/or SSC.sup.lo, the cell is identified as an early committed neutrophil progenitor, and wherein where the cell is determined to be CD49d.sup.hi/+ and/or SSC.sup.hi, the cell is identified as an intermediate neutrophil progenitor that is downstream in neutrophil lineage to the early committed neutrophil progenitor.
5. The method according to claim 1, wherein determination of the expression of the at least one biomarker and/or the further biomarker comprises contacting the cell with one or more antibodies against the biomarker and/or the further biomarker.
6. A method of sorting and/or separating neutrophil progenitors from a cell population, the method comprising: selecting for cells having CD71.sup.hi/+ and at least one of the following expression profiles: LOX-1.sup.int/lo/−, CD164.sup.hi/+, CD112.sup.hi/+, CD181.sup.int/lo/−, TACSTD2.sup.hi/+, CD11b.sup.lo/− and/or CD49d.sup.int/hi/+.
7. (canceled)
8. The method according to claim 6, wherein the cell population is derived from cord blood and/or bone marrow.
9. The method according to claim 6, the method further comprising culturing the neutrophil progenitors to obtain proliferation and/or differentiation of the neutrophil progenitors to obtain progenies thereof.
10. The method according to claim 6, the method further comprising administering the neutrophil progenitors and/or the progenies thereof to a subject in need thereof.
11. The method according to claim 10, wherein the subject has neutropenia.
12. The method according to claim 6, wherein the selecting comprises contacting the cells with one or more antibody against CD71 and with one or more antibodies against one or more of LOX-1, CD164, CD112, CD181, TACSTD2, CD11b and CD49d.
13. A composition that is enriched in neutrophil progenitors having CD71.sup.hi/+ and at least one of the following expression profiles: CD71.sup.hi/+, LOX-1.sup.int/lo/−, CD164.sup.hi/+, CD112.sup.hi/+, CD181.sup.int/lo/−, TACSTD2.sup.hi/+, CD11b.sup.lo/− and/or CD49d.sup.int/hi/+.
14.-17. (canceled)
18. A Tag method of claim 6, further comprising preparing a transfusion composition, wherein the composition is an enriched composition of neutrophil progenitors having CD71.sup.hi/+ and at least one of the following expression profiles: LOX-1.sup.int/lo/−, CD164.sup.hi/+, CD112.sup.hi/+, CD181.sup.int/lo/−, TACSTD2.sup.hi/+, CD11b.sup.lo/− and/or CD49d.sup.int/hi/+.
19.-20. (canceled)
Description
BRIEF DESCRIPTION OF FIGURES
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EXAMPLES
[0099] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It will be appreciated that the example embodiments are illustrative, and that various modifications may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments.
[0100] The granulocyte-monocyte progenitor (GMP) is a Lin.sup.negcKit.sup.+CD34.sup.hiCD16/32.sup.hi lineage-primed progeny derived from common myeloid progenitors (CMPs) that forms characteristic granulocyte-macrophage (GM) colonies in culture. GMPs are known for their potential to generate various myeloid progenies such as neutrophils and monocytes. Since GMPs generate both monocytes and neutrophils, their potential raises important questions during circumstances that demand conflicting needs of these two cell subsets.
[0101] In particular, neutrophils are produced in much larger quantities compared to monocytes and their lineage selection requires the repression of monocyte fate with Gfi1. Furthermore, kinetic labelling studies have demonstrated a much longer transit time of neutrophils in the bone marrow compared to monocytes. It thus remains unclear how GMPs adjust their functional output according to different demands, and whether the disparity in transit time between neutrophils and monocytes is due to distinctions between progenitor characteristics downstream or heterogeneity that already exists within GMPs.
[0102] To address these questions, recent advances in single cell transcriptomics have attempted to determine the developmental cell states of each cell, which has led to a discovery of heterogeneity in myeloid lineages and cell fate decisions. Specifically, it has been proposed that GMPs undergo a mixed-lineage state prior to granulocyte and monocyte specification. While these results provided insights into the lineage priming program within the GMP hierarchy, there is a lack of functional validation on how expression or suppression of lineage-affiliated genes will translate into cellular heterogeneity. Furthermore, although lineage heterogeneity within GMPs has been proposed, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation.
[0103] An attempt was made to resolve this heterogeneity with Ly6C and CD115 (CSF-1R) being utilized to subset GMPs into Ly6C.sup.−GMPs and Ly6C.sup.+CD115.sup.hi monocyte (MPs) and Ly6C.sup.+CD115.sup.lo granulocyte progenitors (GPs). However, these markers alone were not sufficient to fully resolve the strict lineage commitment of each progenitor subset, indicating that better markers are required to evaluate the heterogeneity of these progenitors. Particularly, better markers are required for identifying a neutrophil progenitor.
[0104] Neutrophils are important immune cells which provide protection against bacterial and fungal infections. Due to their short lifespan, neutrophils are continuously produced by specialised bone marrow progenitors to meet the daily demand of 100 million cells per day. Neutrophil development begins with long-lived hematopoietic stem cells, which give rise to highly proliferative progenitors that expand in numbers to generate an adequate supply of mature effector neutrophils for immune surveillance and protection against microbial threats. Despite their importance, the identity and characteristics of a committed neutrophil progenitor are yet to be found.
[0105] The inventors have previously reported the identification of neutrophil precursors known as preNeus. While these precursors developed into neutrophils, they were unable to form colonies in vitro, indicating that they are not the true progenitor cells for neutrophils.
[0106] In this disclosure, through a combination of single-cell transcriptomic and proteomic analyses, the inventors successfully identified an early committed progenitor within the GMPs responsible for the strict production of neutrophils, which they have termed as proNeu1. The comprehensive dissection of GMP hierarchy led to the further identification of a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples.
[0107] The early committed neutrophil progenitor, proNeu1, is already present within the heterogenous population of GMPs. ProNeu1 is shown to give rise to the intermediate progeny proNeu2, which subsequently differentiates into downstream populations. Importantly, it was found that proNeu1 but not proNeu2, expanded extensively and specifically in the early phase of septic inflammation at the expense of monocytic differentiation.
[0108] The two subsets of neutrophil progenitors, proNeu1 and proNeu2, which are responsible for the extensive production of neutrophils in humans, are characterised by distinct protein surface marker expression profiles such as CD71 expression. Based on these surface markers, the inventors were able to isolate these progenitors and demonstrate their development into mature CD16.sup.+CD10.sup.+ neutrophils in vitro.
[0109] Collectively, the findings complete the neutrophil maturation trajectory roadmap and call for a revision of the classical GMP nomenclature. The disclosure further exemplifies the importance of understanding progenitor identities to study their function in health and disease.
Results
GMPs Contain a Heterogeneous Group of Lineage-Committed Progenitors
[0110] GMPs are known to give rise to granulocytes, such as neutrophils, and monocytes. As such, GMPs were commonly assumed to possess oligo-potent differentiation potential. However, contrary to this assumption, recent studies have suggested that the GMPs are instead, a heterogeneous mixture containing lineage-committed precursors. To resolve these two viewpoints and to provide a comprehensive understanding of the true hematopoietic potential of cells within this population of cells, the inventors investigated the GMP population by performing an analysis with published repositories of single-cell transcriptomic datasets (Giladi et al., 2018; Olsson et al., 2016). By extracting the annotated GMPs (based on Lin.sup.−cKit.sup.+Sca-1.sup.−CD34.sup.hiCD16/32.sup.hi), the inventors performed t-Distributed Stochastic Neighbour Embedding (t-SNE) (Maaten and Hinton, 2008) analysis and confirmed that GMPs contained a mixed population of monocytic, neutrophilic, eosinophilic and basophilic progenitor subsets (
[0111] To further look into the various GMP developmental and transitional states leading towards lineage commitment, single-cell RNA-Seq analysis was performed on index-sorted CMPs and total GMPs. The inventors also sorted late precursors, preNeus and TpMos (transitional pre-monocytes), as reference points for neutrophil and monocyte differentiation respectively (
[0112] Upon further analysis with Monocle, the inventors noticed the presence of a specified progenitor population at the terminal end state of the neutrophil branchpoint within the Ly6C.sup.+ fraction of GMPs (
InfinityFlow Resolves GMP Heterogeneity and Identifies Population Discriminating Surface Markers
[0113] Although transcriptomic signatures provide a useful means in determining cell states, they do not confirm a cell's identity or allow further downstream analysis. Therefore, to validate the GMP heterogeneity on a proteomic level, the expression of 261 surface markers (LEGENDScreen™, Biolegend) (Table 1) on total mouse BM cells was evaluated by flow cytometry. The InfinityFlow (Dutertre et al., 2019) pipeline was then used to predict the co-expression of every surface marker tested and this predicted expression information was concatenated into a single analysis file (
[0114] Therefore, to circumvent this challenge, UMAP analysis of the InfinityFlow dataset was performed to understand the developmental relationships between cell types in the BM based on their protein expression (Becht et al., 2018; McInnes et al., 2018). One major strength of UMAP is that it preserves the continuity of cell subsets, which allow for the identification of rare and/or transitional populations that would be masked in a t-SNE analysis (Becht et al., 2018). The UMAP analysis was expanded to total BM cells which discriminated the various cell lineages within the BM (
Identification of an Early Neutrophil Progenitor within the GMP
[0115] Using the newly identified markers CD81 and CD106, the inventors characterised two phenotypically distinct neutrophil progenitors (termed as proNeus). This included a CD34.sup.hiCD106.sup.−CD11b.sup.lo proNeu1 subset and a CD34.sup.loCD106.sup.+CD11b.sup.hi proNeu2 subset (
[0116] Morphological analysis revealed an initial hollowing of the nucleus in the proNeu1 stage, which increases in diameter in the proNeu2 stage (
[0117] It has been shown that a deficiency in C/EBPs leads to a disruption in neutrophil development. Here, the RNA-Seq analysis showed an upregulation of Cebpe from proNeu1 to proNeu2, suggesting that C/EBPs could be critical for the development of early neutrophil progenitors (
[0118] Since proNeu1 s possessed both monocytic and neutrophilic transcription factors (
[0119] As these transfer experiments were performed in separate WT recipients, the inventors wanted to assess the differentiation potential of proNeus within the same microenvironment. To do this, the inventors co-transferred sorted and labelled proNeus (1 &2) and preNeus into WT recipients and tracked their development after three days. The results showed that both proNeu1 s and proNeu2s could only give rise to neutrophils with negligible monocytic differentiation potential (
ProNeu1 and proNeu2 are Functionally Distinct Early Neutrophil Progenitor Populations.
[0120] To dissect the functional heterogeneity and progression of these neutrophil progenitors, the transcriptomic signatures from Ly6C.sup.−GMPs to preNeus was analyzed. By plotting the top variable genes among each subset, the inventors derived three distinct clusters of genes differentially regulated between each stage of neutrophil development (
[0121] As CD34 is a known marker for hematopoietic progenitors, it was wondered if progenitor properties are lost in a CD34.sup.lo proNeu2 subset. To test this, the proliferative potential of proNeu1 and proNeu2 was evaluated. In vitro cultures showed that, unlike the minimal colony-forming activity of preNeus, both proNeu1 and proNeu2 possessed high proliferative potential (
[0122] To understand how these progenitors might play differential roles in inflammatory states, the inventors utilized a model of sepsis and tracked the BM progenitor composition at various timepoints (
G-CSF-Mediated BM Skewing of Myeloid Progenitors can Modulate Neutrophil Expansion
[0123] The ability of BM progenitors to replenish the circulating pool of immune cells is a critical step in infections and diseases when a large demand of mature cells is needed. In particular, neutralization of invading pathogens and microbial insults requires emergency granulopoiesis, which is a mechanism whereby the immune system adapts to produce and mobilize neutrophils in a prompt and efficient manner.
[0124] To determine if the increased proNeu1 frequencies during sepsis are pre-determined in early progenitors, transcriptomic analysis of Ly6C.sup.−GMPs was performed at day 3 of sepsis onset, revealing a down-regulation of multiple lineage associated genes, such as Mcpt8, Ly86, Prss34 and Fcera1. An up-regulation of granule protein genes such as S100a9, S100a8, Chil3 and Lcn2 was also observed (
[0125] To determine the factors and signals that enabled the expansion of proNeu1 and proNeu2, the inventors performed a screen of inflammatory analytes on mice serum at the various time points (
InfinityFlow of Human Cord Blood Cells Reveal a proNeu1 and a proNeu2 Subset
[0126] To determine if such progenitors exist in human, the same workflow (i.e., the InfinityFlow pipeline) was applied to examine whole human cord blood cells to screen for differential surface markers that can help identify these progenitor cells.
[0127] Briefly, Red blood cells (RBC)-lysed cord blood cells are stained with a common backbone panel of surface markers that denote the various cell lineages present in the cord blood. Thereafter, cells are aliquoted into wells, each containing a unique PE-conjugated surface marker (LEGENDScreen™). Samples are acquired and the expression profiles of each PE marker was processed with InfinityFlow, a machine learning application that predicts the co-expression profile of each marker with every tested marker. This allowed for the discovery of new subsets with high dimensional phenotypic characterisation.
[0128] UMAP analysis of total cord blood cells showed distinct populations representing the various lineages (
[0129] The inventors could further subset proNeus into proNeu1 and proNeu2 by their CD49d expression level and side-scatter properties (
[0130] The data suggest a similarity in neutrophil development between mouse and human with the identification of human equivalents of proNeu1 and proNeu2. To further show this correlation, the inventors utilized and compared the protein expression profiles of each neutrophil subset from both the mouse and human InfinityFlow datasets. The MFIs were extracted from each subset and it was observed that each subset had moderate correlation between the mouse and human equivalent neutrophil subset (
[0131] Finally, to show that the identified progenitors proNeu1 and proNeu2 have the potential to generate mature neutrophils, in vitro cultures of sorted proNeu1 and proNeu2 cells were performed. The data showed that both proNeu1 and proNeu2 were capable of generating CD16.sup.+CD10.sup.+ mature neutrophils (
Discussion
[0132] The classical model of haematopoiesis is a hierarchical and step-wise differentiation program, led by instructive transcription factors that govern each cell's lineage fate. The generation of myeloid cells are through GMPs (Lin.sup.−cKit.sup.+Sca-1.sup.−CD34.sup.+CD16/32.sup.hi) as shown by the formation of both granulocytic and monocytic colonies in methylcellulose colony-forming assays (Akashi et al., 2000). Yet, whether GMPs are a bona fide homogenously multipotent subset or a heterogenous blend of lineage-restricted populations remains a long-standing question. To gain better insights into this, the inventors employed a combinatorial approach of transcriptomic, proteomic and bioinformatic tools to develop a phenotyping scheme for identifying putative progenitors within the GMP populations. By subjecting the currently defined GMPs through optimized subset-defining surface markers derived from InfinityFlow (Dutertre et al., 2019) and bioinformatic approaches, the inventors identified a population of neutrophil-committed progenitors (i.e. CD81.sup.+CD106.sup.−proNeu1) that exist within GMP population. Furthermore, with deep surface marker phenotyping and RNA-Seq profiling, the inventors further characterized a downstream CD81.sup.+CD106.sup.+proNeu2 progenitor subset. Together, these data serve as a missing link in the early stages of the neutrophil developmental pathway, which allows for the mapping of their development from proNeu1.fwdarw.proNeu2.fwdarw.preNeu.fwdarw.immature neutrophil.fwdarw.mature neutrophil.
[0133] The ability to delineate early neutrophil progenitors from GMPs allows for the assessment of transcription factors that are involved in instructing myeloid progenitor lineage specification and commitment. Inspection of a list of TFs (Irf8, KIf4, Irf5, Gfi1, Cebpe and Per3) that are known to govern monocytic and granulocytic lineage commitment consistently showed that Ly6C.sup.−GMPs and proNeu1 express similar levels of these TFs, while differential expression of these TFs only became evident at the proNeu2 and cMoP stage. This indicates that a neutrophil-monocyte lineage bifurcation may occur at this hierarchical level. This raises the question about the extent of lineage commitment by proNeu1 population, i.e., whether this subset of progenitor is specified or committed towards the neutrophil lineage. The in vitro and in vivo functional studies revealed that proNeu1 is committed to the neutrophil lineage, as proNeu1 can only give rise to neutrophils even when they were cultured with a strong monocytic lineage promoting cytokine such as M-CSF. Additionally, the RNA-Seq data showed that cMoP, Ly6C-GMP, proNeu1 and proNeu2 possess distinct “TF signatures”, providing an overview of putative TFs that could be important for lineage specification and commitment of these progenitors. For instance, the inventors observed increased levels of Jag1 and Sox13 expression specifically only in proNeu1, suggesting that these two TFs can be developed as markers for tagging proNeu1 and to be examined as key regulators for proNeu1 differentiation. Collectively, the data not only confirmed previous lineage-associated TFs, but also extended the list of putative TFs for myeloid lineage specification and commitment.
[0134] A previous in vivo tracking study demonstrated that granulocytes and monocytes are closely related in terms of their clonal origin. Here, the disclosure describes a transcriptionally and molecularly defined neutrophil progenitor in the GMP hierarchy, allowing for a better understanding of the lineage relationships and the dynamics of neutrophil/monocyte production. Given that myeloid progenitors can be sustained without the input from HSCs, it is conceivable that these progenitors are highly adaptable to varying inflammatory perturbations, according to the demands of the immune response. The study revealed that there is a selective expansion of proNeu1 and disappearance of cMoP during the acute phase of sepsis onset. This phenomenon could be a consequence of skewed differentiation potential of progenitor cells, conceivably at the Ly6C-GMP level, and a preferential proliferation of early neutrophil progenitors to meet the immediate requirements for neutrophils.
[0135] In summary, the disclosure provides new insight into the divergent pathways of myeloid progenitor development towards neutrophils and monocytes from GMPs, and how the balance between neutrophil/monocyte production is important for host homeostasis. Moreover, the identification of early neutrophil progenitors opens up new avenues for therapeutic strategy for the management of neutropenia in hematopoietic stem cell transplantation or high dose chemotherapy, by the infusion of an expanded proNeu subset. This can serve a source for rapid neutrophil repopulation to help confer protection against infection during this critical period of need.
Materials and Methods
Mice
[0136] Eight to twelve-week-old C57BL/6 mice were bred and maintained under specific pathogen-free (SPF) conditions in the Biological Resource Centre (BRC) of A*STAR, Singapore. Both males and females were used for experiments, but animals were sex- and age-matched in each experiment as much as possible. uGFP (C57BL/6-Tg(UBC-GFP)30Scha/J), CD45.1 (B6.SJL-Ptprc.sup.a Pepc.sup.b/BoyJ), MaFIA (C57BL/6-Tg(Csf1 r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J) and Rosa26.sup.mT/mG (STOCK Gt(ROSA).sup.26sotm4(AcTB-tdTomato,-EGFP)Luo/J mice were obtained from The Jackson Laboratory. Fucci-S/G2/M (#474) were obtained from the RIKEN BioResource Center (Ibaraki, Japan; (Tomura et al., 2013)). Cebpe.sup.−/− provided by P. Koeffler (Cancer Science Institute of Singapore, NUS, Singapore) (Yamanaka et al., 1997). All transgenic mice were maintained on a C57BL/6 background and experiments were performed under the approval of the Institutional Animal Care and Use Committee (IACUC), in accordance with the guidelines of the Agri-Food and Veterinary Authority (AVA) and the National Advisory Committee for Laboratory Animal Research (NACLAR) of Singapore.
Human Blood, Cord Blood and Fetal Bone Marrow
[0137] All samples were obtained in accordance with a favorable ethical opinion from SingHealth CIRB or A*STAR, the Singapore Immunology Network. Fresh (<24 hours from collection) human umbilical cord blood (UCB) units were collected through a collaboration with National Cancer Centre Singapore (NCCS). NCCS obtained the UCB units through Singapore Cord Blood Bank (SCBB), from research consented units failing to meet the criteria for public clinical banking. Usage of the samples were approved by the Centralized Institutional Review Board (CIRB) of Singapore Health Services (that covers NCCS) as well as the dedicated Research Advisory Ethics Committee of SCBB.
Treatments
[0138] For 5-Fluorouracil (5-FU) myeloablative treatment, mice were injected once intraperitoneally with 150 mg/kg 5-FU (Sigma-Aldrich) or PBS control. For G-CSF treatment, mice were injected once intraperitoneally with 1.5 μg of G-CSF/anti-G-CSF antibody complex (G-CSFcx) as previously described (Rubinstein et al., 2013). Briefly, G-CSFcx were generated by incubating G-CSF (Neupogen) and anti-G-CSF (BVD11-37G10; Southern-Biotech) at 1:5 cytokine to antibody ratio for 20 min at 37° C., and were next diluted at least 10-fold in PBS before injection.
Tissue Preparation and Data Analysis for Flow Cytometry and Cell Sorting
[0139] Blood was obtained via an incision in the submandibular region and was then lysed in red blood cell lysis buffer (eBioscience). For BM cells, mice femurs were flushed using a 23-gauge needle in PBS containing 2 mM EDTA and 2% fetal bovine serum (FBS) and passed through a 70-μm nylon mesh sieve. Spleens were harvested and homogenized into single-cell suspensions using 70-μm nylon mesh sieves and syringe plungers. Antibodies were purchased from BD, Biolegend, eBioscience or R&D. For the identification of BM myeloid progenitor cell subsets, cells were stained with fluorophore-conjugated anti-mouse antibodies against CD34 (RAM34), CD11b (M1/70), CD16/32 (2.4G2), CD115 (AFS598), cKit (2B8), CXCR2 (SA044G4), CXCR4 (2B111), Gr1 (RB6-8C5), Ly6C (HK1.4) CD106 (429), CD81 (Eat-2), and Flt3 (A2F10), together with exclusion lineage markers that include Ly6G (1A8), CD90.2 (53-2.1), B220 (RA3-6B2), NK.1.1 (PK136), and Sca-1 (D7). After exclusion of cell doublets and dead cells with DAPI, proNeu1 were identified as (Lin/CD115/Flt3)-cKit.sup.hiCD16/32.sup.hiLy6C.sup.+ CD34.sup.hiCD11b.sup.loCD106.sup.−, proNeu2 were identified as (Lin/CD115/Flt3).sup.−cKit.sup.hiCD16/32.sup.hiLy6C.sup.+ CD34.sup.hiCD11b.sup.hiCD106.sup.+, preNeus were identified as (Lin/CD115/Siglec-F).sup.−Gr1.sup.+CD11b.sup.+CXCR4.sup.h ickit.sup.intCXCR2.sup.−, CMPs were identified as Lin.sup.−cKit.sup.+Sca-1.sup.−CD16/32.sup.intCD34.sup.int, Ly6C.sup.−GMPs were identified as Lin.sup.−cKit.sup.+Sca-1.sup.−CD16/32.sup.hiCD34hi, MDPs were identified as Lin.sup.−cKit.sup.+Sca-1.sup.−CD115.sup.+Flt3.sup.+Ly6C.sup.− or and cMoPs were identified as Lin.sup.−cKit.sup.+Sca-1.sup.−CD115.sup.+CD81.sup.−Flt3.sup.−Ly6C.sup.+. Flow cytometry acquisition was performed on a 5-laser BD LSR II (BD) using FACSDiva software, and data was subsequently analyzed with FlowJo software (Tree Star). Cell numbers were quantified with count beads (CountBright; Life Technologies) according to the manufacturer's instructions. Sorting of BM neutrophil subsets were performed using a BD ARIAII (BD) to achieve >98% purity.
LEGENDScreen™ and InfinityFlow Pipeline
[0140] Mice femurs, tibias, pelvic bones, humeri and spinal bone marrow were harvested and crushed in PBS containing 2 mM EDTA and 2% foetal bovine serum (FBS) and passed through a 70-μm nylon mesh sieve. For human cord blood, RBC lysis was performed on one unit of cord blood donor sample for 10 min at room temperature. Cells were spun down at 400 g for 10 min. This process was repeated once to remove most RBC cells. Mouse cells were first stained with fixable live/dead for 30 min before staining with a backbone panel cocktail of mouse antibodies to define the various lineages in the BM. These markers include: CD34 (RAM34), CD11b (M1/70), CD16/32 (2.4G2), CD115 (AFS598), cKit (2B8), CXCR2 (SA044G4), Ly6C (HK1.4), Flt3 (A2F10), Sca-1 (D7). CD48 (HM48-1), CD43 (S11), CD62L (MEL-14) and FITC-conjugated lineage markers (Ly6G (1A8), CD90.2 (53-2.1), B220 (RA3-6B2), NK.1.1 (PK136)). After 90 min of staining at 4° C., cells were lysed in 1×RBC lysis (eBioscience) for 5 min, then spun down at 400 g for 5 min. Cells were then stained with secondary streptavidin for 30 min and washed as before. To enrich the BM for progenitor identification, mature neutrophils, B, T and NK cells were partially depleted using FITC selection kit (Stem Cell Technologies) according to the manufacturer protocol. For cord blood cells, cells were stained with a backbone panel of antibodies for 30 min at 4° C., including: CD3 (UCHT1), CD56 (HCD56), CD19 (HIB19), CD10 (H110a), CD49d (9F10), CD34 (581), CD66b (G10F5), cKit (104D2), CD38 (HIT2), CD15 (H198), CD14 (M5E2), CD101 (BB27), CD45 (H130), CD11b (ICRF44), CD16 (3G8). Cells were then counted and aliquoted into individual wells containing specific PE-conjugated marker. (Tables 1 and 3). After staining for 30 min, plates were washed and fixed before FACS acquisition was performed on a 5-laser BD LSR II (BD) using FACSDiva software, and data was subsequently processed through the InfinityFlow pipeline as described elsewhere (Dutertre et al., 2019).
Cytospin and Wright-Giemsa Staining
[0141] Sorted neutrophil subsets (5×10.sup.4 cells each) were spun onto glass slides using Cytospin 4 Cytocentrifuge (Thermo scientific), dried for 10 minutes, fixed in methanol and stained with the Hema 3 manual staining system (Fisher Diagnostics) according to the manufacturer's protocol. Images were acquired with an Olympus BX43 equipped with a 100× oil immersion objected, and image brightness was adjusted with Photoshop (Adobe).
Transcriptomics
[0142] For single-cell transcriptomic analysis, BM CMPs, GMPs, preNeus and TpMos cells were sorted based on the gating strategy depicted in
[0143] For total-mRNA bulk RNA-seq analysis, BM Ly6C-GMP, proNeu1, proNeu2 and cMoP were sorted based on the gating strategy of
In Vitro Cell Culture
[0144] Sorted cells (1×10.sup.4 for each subset) were plated onto 96-well plates in triplicates and cultured at 37° C., 5% CO.sub.2 in StemSpan™ SFEM II (Stem Cell Technologies) containing penicillin (100 U/ml), streptomycin (100 ug/ml), a combination of cytokines (50 ng/ml SCF, 10 ng/ml LIF, 20 ng/ml IL-3, 20 ng/ml IL-6) and with or without 50 ng/ml CSF-1. For human fetal marrow cultures, frozen samples were thawed and sorted before plating in 96-well plates in StemSpan™ SFEM II containing penicillin (100 U/ml), streptomycin (100 ug/ml) and StemSpan™ myeloid expansion supplement (Stem Cell Technologies). For colony assays, sorted cells (3×10.sup.4) were cultured for in Iscove's modified Dulbecco's medium (Sigma) with 25 mM HEPES and L-Glutamine (Chemtron) containing 10% (vol/vol) FBS, 1 mM sodium pyruvate, penicillin (100 U/ml) and streptomycin (100 ug/ml), 1% (wt/vol) methylcellulose (MethoCult M3234, Stem Cell Technologies) and the same cytokine combination as above. Representative colony images were collected with an Olympus IX-81 microscope (Olympus). Image brightness was adjusted with Photoshop.
Adoptive Cell Transfer
[0145] Sorted uGFP.sup.+ proNeu1, RFP.sup.+ (Rosa26.sup.mT/mG) proNeu2 and CD45.1 preNeus (5×10.sup.4 cells each) were transferred intra-BM into wildtype recipients as described previously (Chong et al., 2016). Briefly, recipient mice were anesthetized with ketamine (150 mg/kg)/xylazine (10 mg/kg), and had their right leg shaved to expose the kneecap. Sorted proNeu1, proNeu2 and preNeus were mixed and resuspended in 1×PBS in equal proportions and a volume of 10 μL was administered into the tibia through the kneecap using a 29-gauge insulin needle. For single population transfers, sorted uGFP.sup.+Ly6C-GMP, proNeu1 and proNeu2 (5×10.sup.4 cells) were used. After 24 hr or 72 hours after cell transfer, tibias were collected, stained and analyzed by flow cytometry.
CLP-Induced Sepsis
[0146] Cecal ligation and puncture was performed as described previously (Rittirsch et al., 2009). Briefly, the peritoneal cavity was exposed under ketamine/xylazine anesthesia and the cecum was exteriorized. 50% (mid-grade) or 80% (high-grade) of the cecum was ligated distal of the ileo-cecal valve using a non-absorbable 7-0 suture. A 26-gauge needle was used to perforate the distal end of the cecum, and a small drop of feces was extruded through the puncture before being relocated into the peritoneal cavity. The peritoneum was closed and mice were subsequently treated with saline and Buprenorphine (5-20 mg/kg) by subcutaneous injection. For sham-operated controls, the peritoneum was exposed and the cecum was exteriorized before closing the peritoneum as mentioned above. Mice were euthanized and harvested 2 weeks after the surgery.
Quantification and Statistical Analysis
[0147] Statistical analyses were done using Prism software (Graphpad). Student's t-test or one-way analysis of variance (ANOVA) with Bonferroni correction were performed.
Tables
[0148]
TABLE-US-00001 TABLE 1 (Related to FIG. 2) List of surface markers tested in mouse BM LEGENDScreen ™ Clone Location Marker (Biolegend) Isotype Plate_1_A1_013 Blank Plate_1_A10_022 CD103 2E7 Armenian Hamster IgG Plate_1_A11_023 Delta_like 4 HMD4-1 Armenian Hamster IgG Plate_1_A12_024 CD195 HM-CCR5 Armenian Hamster IgG Plate_1_A2_014 Isotype_AHIgG HTK888 Plate_1_A3_015 CD3e 145-2C11 Armenian Hamster IgG Plate_1_A4_016 CD80 16-10A1 Armenian Hamster IgG Plate_1_A5_017 CD81 Eat-2 Armenian Hamster IgG Plate_1_A6_018 CD154 MR1 Armenian Hamster IgG Plate_1_A7_019 Notch 1 HMN1-12 Armenian Hamster IgG Plate_1_A8_020 CD30 mCD30.1 Armenian Hamster IgG Plate_1_A9_021 CD178 MFL3 Armenian Hamster IgG Plate_1_B1_025 Notch 4 HMN4-14 Armenian Hamster IgG Plate_1_B10_034 CD11c N418 Armenian Hamster IgG Plate_1_B11_035 Delta_like 1 HMD1-3 Armenian Hamster IgG Plate_1_B12_036 CD196 29-2L17 Armenian Hamster IgG Plate_1_B2_026 CD229 Ly9ab3 Armenian Hamster IgG Plate_1_B3_027 CD69 H1.2F3 Armenian Hamster IgG Plate_1_B4_028 Notch 3 HMN3-133 Armenian Hamster IgG Plate_1_B5_029 JAML 4E10 Armenian Hamster IgG Plate_1_B6_030 Notch 2 HMN2-35 Armenian Hamster IgG Plate_1_B7_031 CD194 2G12 Armenian Hamster IgG Plate_1_B8_032 CD152 UC10-4B9 Armenian Hamster IgG Plate_1_B9_033 CD120a 55R-286 Armenian Hamster IgG Plate_1_C1_037 CD29 HMβ1-1 Armenian Hamster IgG Plate_1_C10_046 CD16.2 mDcR2-1 Armenian Hamster IgG Plate_1_C11_047 CD36 HM36 Armenian Hamster IgG Plate_1_C12_048 DcTRAIL_R1 mDcR1-3 Armenian Hamster IgG Plate_1_C2_038 CD55 RIKO-3 Armenian Hamster IgG Plate_1_C3_039 Jagged 2 HMJ2-1 Armenian Hamster IgG Plate_1_C4_040 CD79b HM79-12 Armenian Hamster IgG Plate_1_C5_041 IFNgR b chain MAR1-5A3 Mouse IgG1, k Plate_1_C6_042 CD61 2C9.G2 Armenian Hamster IgG (HMβ3-1) Plate_1_C7_043 CD121a JAMA-147 Armenian Hamster IgG Plate_1_C8_044 TCRb chain GL3 Hamster IgG Plate_1_C9_045 FceRIa MAR-1 Armenian Hamster IgG Plate_1_D1_049 CD84 mCD84.7 Armenian Hamster IgG Plate_1_D10_058 CD339 HMJ1-29 Armenian Hamster IgG Plate_1_D11_059 CD49a HMa1 Armenian Hamster IgG Plate_1_D12_060 PD1H MH5A Armenian Hamster IgG Plate_1_D2_050 CD48 HM48-1 Armenian Hamster IgG Plate_1_D3_051 CD49b HMa2 Armenian Hamster IgG Plate_1_D4_052 CD120b TR75-89 Armenian Hamster IgG Plate_1_D5_053 CD183 CXCR3-173 Armenian Hamster IgG Plate_1_D6_054 CD262 MD5-1 Armenian Hamster IgG Plate_1_D7_055 HVEM HMHV-1B18 Armenian Hamster IgG Plate_1_D8_056 TCR Vd1.1_1.2 H57-597 Armenian Hamster IgG Plate_1_D9_057 B7H4 HMH4-5G1 Armenian Hamster IgG Plate_1_E1_061 CD85k H1.1 Armenian Hamster IgG Plate_1_E10_070 Ly108 330-AJ Mouse IgG2a, k Plate_1_E11_071 CD207 4C7 Mouse IgG2a, k Plate_1_E12_072 CX3CR1 SA011F11 Mouse IgG2a, k Plate_1_E2_062 Plexin B2 3E7 Armenian Hamster IgG Plate_1_E3_063 CD27 LG.3A10 Armenian Hamster IgG1, Plate_1_E4_064 DR3 4C12 Armenian Hamster IgG1 Plate_1_E5_065 TCR gd 4B2.9 Armenian Hamster IgG Plate_1_E6_066 Isotype_mIgG2a MOPC-173 Mouse IgG2a, k Plate_1_E7_067 CD45.1 A20 Mouse (A.SW) IgG2a, k Plate_1_E8_068 CD45.2 104 Mouse (SJL) IgG2a, k Plate_1_E9_069 NK1.1 PK136 Mouse IgG2a, k Plate_1_F1_073 Isotype_mIgG1 MOPC-21 Mouse IgG1, k Plate_1_F10_082 Trem_like 4 16E5 Mouse IgG1, k Plate_1_F11_083 CD59a mCD59.3 Mouse IgG1, k Plate_1_F12_084 Ly49H 3D10 Mouse IgG1, k Plate_1_F2_074 CD66a MAb-CC1 Mouse IgG1, k Plate_1_F3_075 IFNAR1 MOB-47 Armenian Hamster IgG Plate_1_F4_076 Tim2 F37-2C4 Mouse IgG1, k Plate_1_F5_077 CD272 8F4 Mouse IgG1, k Plate_1_F6_078 CD64 X54-5/7.1 Mouse IgG1, k Plate_1_F7_079 CD351 TX61 Mouse IgG1, k Plate_1_F8_080 LAP TW7-20B9 Mouse IgG1, k Plate_1_F9_081 TIGIT 1G9 Mouse IgG1, k Plate_1_G1_085 CD90.1 OX-7 Mouse IgG1, k Plate_1_G10_094 Siglec H 551 Rat IgG1, K Plate_1_G11_095 CD255 MTW-1 Rat IgG1, k Plate_1_G12_096 CD202b TEK4 Rat IgG1, k Plate_1_G2_086 Isotype_mIgG2b MPC-11 Mouse IgG2b, k Plate_1_G3_087 CD157 BP-3 Mouse IgG2b, k Plate_1_G4_088 CD159a 16A11 Mouse IgG2b, k Plate_1_G5_089 XCR1 ZET Mouse IgG2b, k Plate_1_G6_090 Isotype_mIgM MM-30 Mouse IgM, k Plate_1_G7_091 SSEA_1 MC-480 Mouse IgM, k Plate_1_G8_092 Isotype_rIgG1 RTK 2071 Rat IgG1, k Plate_1_G9_093 Ig light chain κ RMK-45 Rat IgG Plate_1_H1_097 GITR Ligand YGL 386 Rat IgG1, k Plate_1_H10_106 Mac3 M3/84 Rat IgG1, k Plate_1_H11_107 CD223 C9B7W Rat IgG1, k Plate_1_H12_109 CD134 OX-86 Rat IgG1, k Plate_1_H2_098 CD147 OX-114 Rat IgG1, k Plate_1_H3_099 CD73 TY/11.8 Rat IgG1, k Plate_1_H4_100 CD51 RMV-7 Rat IgG1, k Plate_1_H5_101 NKG2D CX5 Rat IgG1, k Plate_1_H6_102 CD96 3.3 Rat IgG1, k Plate_1_H7_103 Integrin b7 FIB27 Rat IgG1, k Plate_1_H8_104 CD210 1B1.3a Rat IgG1, k Plate_1_H9_105 CD83 Michel-19 Rat IgG1, k Plate_2_A12_119 Isotype_rIgG2a RTK2758 Rat IgG2a, k Plate_2_A2_111 CD41 MWReg30 Rat IgG1, k Plate_2_A3_112 CD268 7H22-E16 Rat IgG1, k Plate_2_A4_113 CD144 BV13 Rat IgG1, k Plate_2_A5_114 CD370 7H11 Rat IgG1, k Plate_2_A6_115 CD369 RH1 Rat IgG1, k Plate_2_A7_116 PIR A/B 6C1 Rat IgG1, k Plate_2_A8_117 CD22 OX-97 Rat IgG1, k Plate_2_A9_118 E_Cadherin DECMA-1 Rat IgG1, k Plate_2_B1_120 MAIRV TX70 Rat IgG2a, k Plate_2_B10_129 CD197 4B12 Rat IgG2a, k Plate_2_B11_130 CD47 miap301 Rat IgG2a, k Plate_2_B12_131 CD98 RL388 Rat IgG2a, k Plate_2_B2_121 CD146 ME-9F1 Rat IgG2a, k Plate_2_B3_122 VISTA MIH63 Rat IgG2a, k Plate_2_B4_123 CD8a 53-6.7 Rat IgG2a, k Plate_2_B5_124 CD275 HK5.3 Rat IgG2a, k Plate_2_B6_125 CD34MEC14.7 MEC14.7 Rat IgG2a, k Plate_2_B7_126 Sca_1 D7 Rat IgG2a, k Plate_2_B8_127 CD40 3/23 Rat IgG2a, k Plate_2_B9_128 B220 RA3-6B2 Rat IgG2a, k Plate_2_C1_132 CD14 Sa14-2 Rat IgG2a, k Plate_2_C10_141 Tim4 RMT4-54 Rat IgG2a, k Plate_2_C11_142 CD71 RI7217 Rat IgG2a, k Plate_2_C12_143 H2 M1/42 Rat IgG2a, k Plate_2_C2_133 CD107a 1D4B Rat IgG2a, k Plate_2_C3_134 CD18 M18/2 Rat IgG2a, k Plate_2_C4_135 Ly6G 1A8 Rat IgG2a, k Plate_2_C5_136 CD21, CD35 7E9 Rat IgG2a, k Plate_2_C6_137 Mac2 M3/38 Rat IgG2a, k Plate_2_C7_138 CD199 9B1 Rat IgG2a, k Plate_2_C8_139 Ly51 6C3 Rat IgG2a, k Plate_2_C9_140 IgD 11-26c.2a Rat IgG2a, k Plate_2_D1_144 CD45RB C363-16A Rat IgG2a, k Plate_2_D10_153 CD105 MJ7/18 Rat IgG2a, k Plate_2_D12_154 4_1BB Ligand TKS-1 Rat IgG2a, k Plate_2_D2_145 CD326 G8.8 Rat IgG2a, k Plate_2_D3_146 IgM RMM-1 Rat IgG2a, k Plate_2_D4_147 CD155 TX56 Rat IgG2a, k Plate_2_D5_148 CD200R OX-110 Rat IgG2a, k Plate_2_D6_149 CD254 IK22/5 Rat IgG2a, k Plate_2_D7_150 IL21R 4A9 Rat IgG2a, k Plate_2_D8_151 CD276 RTAA15 Rat IgG2a, k Plate_2_D9_152 CD9 MZ3 Rat IgG2a, k Plate_2_E1_155 CD265 R12-31 Rat IgG2a, k Plate_2_E10_164 F4/80 BM8 Rat IgG2a, k Plate_2_E11_165 CD94 18d3 Rat IgG2a, k Plate_2_E12_166 CD267 8F10 Rat IgG2a, k Plate_2_E2_156 TLR4 MTS510 Rat IgG2a, k Plate_2_E3_157 CD19 6D5 Rat IgG2a, k Plate_2_E4_158 LPAM_1 DATK32 Rat IgG2a, k Plate_2_E5_159 CD62L MEL-14 Rat IgG2a, k Plate_2_E6_160 CD23 B3B4 Rat IgG2a, k Plate_2_E7_161 CD5 53-7.3 Rat IgG2a, k Plate_2_E8_162 CD273 TY25 Rat IgG2a, k Plate_2_E9_163 CD31 390 Rat IgG2a, k Plate_2_F1_167 Ly_49A YE1/48.10.6 Rat IgG2a, k Plate_2_F10_176 PDC_TREM 4A6 Rat IgG2a, k Plate_2_F11_177 CD135 A2F10 Rat IgG2a, k Plate_2_F12_178 CD127 A7R34 Rat IgG2a, k Plate_2_F3_169 CD11a M17/4 Rat IgG2a, k Plate_2_F4_170 LT beta R 5G11 Rat IgG2a, k Plate_2_F6_172 CD106 429 (MVCAM.A) Rat IgG2a, k Plate_2_F7_173 CD365 RMT1-4 Rat IgG2a, k Plate_2_F8_174 CD115 AFS98 Rat IgG2a, k Plate_2_F9_175 CD140a APA5 Rat IgG2a, k Plate_2_G1_179 CD140b APB5 Rat IgG2a, k Plate_2_G10_188 CD200R3 Ba13 Rat IgG2a, k Plate_2_G11_189 MAIR_IV TX69 Rat IgG2a, k Plate_2_G12_190 Ly49D 4E5 Rat IgG2a, k Plate_2_G2_180 ESAM 1G8/ESAM Rat IgG2a, k Plate_2_G3_181 CD200 OX-90 Rat IgG2a, k Plate_2_G4_182 CD309 Avas12 Rat IgG2a, k Plate_2_G5_183 TLT_2 MIH47 Rat IgG2a, k Plate_2_G6_184 CD253 N2B2 Rat IgG2a, k Plate_2_G7_185 CD335 29A1.4 Rat IgG2a, k Plate_2_G8_186 CD205 NLDC-145 Rat IgG2a, k Plate_2_G9_187 Galectin9 108A2 Rat IgG2a, k Plate_2_H1_191 CD123 5B11 Rat IgG2a, k Plate_2_H10_200 CD63 NVG-2 Rat IgG2a, k Plate_2_H11_201 CD49e 5H10-27(MFR5) Rat IgG2a, k Plate_2_H12_202 CD193 J073E5 Rat IgG2a, k Plate_2_H2_192 CD355 11-5/CRTAM Rat IgG2a, k Plate_2_H3_193 CD169 3D6.112 Rat IgG2a, k Plate_2_H4_194 CD138 281-2 Rat IgG2a, k Plate_2_H5_195 CD160 7H1 Rat IgG2a, k Plate_2_H6_196 CD39 Duha59 Rat IgG2a, k Plate_2_H7_197 GARP F011-5 Rat IgG2a, k Plate_2_H8_198 CD179a R3 Rat IgG2a, k Plate_2_H9_199 CD371 5D3/CLEC12A Rat IgG2a, k Plate_3_A10_212 MAdCAM1 MECA-367 Rat IgG2a, k Plate_3_A11_213 MERTK 2B10C42 Rat IgG2a, k Plate_3_A12_214 CD226 TX42.1 Rat IgG2a, k Plate_3_A2_204 CD300LG ZAQ5 Rat IgG2a, k Plate_3_A3_205 CD301 LOM-8.7 Rat IgG2a, k Plate_3_A4_206 IL33Ra DIH9 Rat IgG2a, k Plate_3_A5_207 CD304 3E12 Rat IgG2a, k Plate_3_A6_208 CD6 OX-129 Rat IgG2a, k Plate_3_A7_209 CD100 BMA-12 Rat IgG2a, k Plate_3_A8_210 CD104 346-11A Rat IgG2a, k Plate_3_A9_211 CD182 SA044G4 Rat IgG2a, k Plate_3_B1_215 Ly6K MK34 Rat IgG2a, k Plate_3_B10_224 CD43 S11 Rat IgG2b, k Plate_3_B11_225 FR4 12A5 Rat IgG2b, k Plate_3_B12_226 CD1d 1B1 Rat IgG2b, k Plate_3_B2_216 CD16/32 93 Rat IgG2a, λ Plate_3_B3_217 CD150 TC15-12F12.2 Rat IgG2a, λ Plate_3_B4_218 CD25 PC61 Rat IgG2a, λ Plate_3_B5_219 CD38 90 Rat IgG2a, λ Plate_3_B6_220 CD133 315-2C11 Rat IgG2a, λ Plate_3_B7_221 CD301b URA-1 Rat IgG2a, λ Plate_3_B8_222 CD34_SA376A4 SA376A4 Rat IgG2a, λ Plate_3_B9_223 Isotype_rIgG2b RTK4530 Rat IgG2b, k Plate_3_C1_227 CD70 FR70 Rat IgG2b, k Plate_3_C10_236 CD24 M1/69 Rat IgG2b, k Plate_3_C11_237 Gr1 RB6-8C5 Rat IgG2b, k Plate_3_C12_238 CD86 PO3 Rat IgG2b, k Plate_3_C2_228 CD4 GK1.5 Rat IgG2b, k Plate_3_C3_229 IA/IE M5/114.15.2 Rat IgG2b, k Plate_3_C4_230 CD153 RM153 Rat IgG2b, k Plate_3_C5_231 CD54 YN1/1.7.4 Rat IgG2b, k Plate_3_C6_232 33D1 33D1 Rat IgG2b, k Plate_3_C7_233 CD90.2 30-H12 Rat IgG2b, k Plate_3_C8_234 TER119 TER-119 Rat IgG2b, k Plate_3_C9_235 CD49d R1-2 Rat IgG2b, k Plate_3_D1_239 CD11b M1/70 Rat IgG2b, k Plate_3_D10_248 CD3 17A2 Rat IgG2b, k Plate_3_D11_249 CD274 10F.9G2 Rat IgG2b, k Plate_3_D12_250 CD117 2B8 Rat IgG2b, k Plate_3_D2_240 CD45 30-F11 Rat IgG2b, k Plate_3_D3_241 CD279 RMP1-30 Rat IgG2b, k Plate_3_D4_242 RAE1y CX1 Rat IgG2b, k Plate_3_D5_243 CD8b YTS156.7.7 Rat IgG2b, k Plate_3_D7_245 CD126 D7715A7 Rat IgG2b, k Plate_3_D8_246 CD317 927 Rat IgG2b, k Plate_3_D9_247 CD132 TUGm2 Rat IgG2b, k Plate_3_E1_251 CD88 20/70 Rat IgG2b, k Plate_3_E10_260 CD130 4H1B35 Rat IgG2b, k Plate_3_E11_261 CD198 SA214G2 Rat IgG2b, k Plate_3_E12_262 CD20 SA275A11 Rat IgG2b, k Plate_3_E2_252 CD93 AA4.1 Rat IgG2b, k Plate_3_E3_253 CD252 RM134L Rat IgG2b, k Plate_3_E4_254 MD1 MD-113 Rat IgG2b, k Plate_3_E5_255 CD357 YGITR 765 Rat IgG2b, k Plate_3_E6_256 CD185 L138D7 Rat IgG2b, k Plate_3_E7_257 CD37 Duno85 Rat IgG2b, k Plate_3_E8_258 CD300c/d TX52 Rat IgG2b, k Plate_3_E9_259 CD186 (CXCR6) SA051D1 Rat IgG2b, k Plate_3_F1_263 CD124 I015F8 Rat IgG2b, k Plate_3_F10_272 GL7 GL7 Rat IgM, k Plate_3_F11_273 Isotype_SHIgG SHG-1 Syrian Hamster IgG Plate_3_F12_274 CD28 37.51 Syrian Hamster IgG Plate_3_F2_264 IL23R 12B2B64 Rat IgG2b, k Plate_3_F3_265 CD184 L276F12 Rat IgG2b, k Plate_3_F4_266 CD2 RM2-5 Rat IgG2b, λ Plate_3_F5_267 Isotype_rIgG2c RTK4174 Rat IgG2c, k Plate_3_F6_268 Ly6C HK1.4 Rat IgG2c, k Plate_3_F7_269 Ly6D 49-H4 Rat IgG2c, k Plate_3_F8_270 Isotype_rIgM RTK2118 Rat IgM, k Plate_3_F9_271 CD49b_IgM DX5 Rat IgM, k Plate_3_G1_275 Podoplanin 8.1.1 Syrian Hamster IgG Plate_3_G2_276 CD137 17B5 Syrian Hamster IgG Plate_3_G3_277 CD278 15F9 Syrian Hamster IgG Plate_3_G4_278 KLRG1 2F1/KLRG1 Syrian Hamster IgG Plate_3_G5_279 Ly49CFIH 14B11 Syrian Hamster IgG
TABLE-US-00002 TABLE 2 (related to FIG. 2). Cell type identification of PhenoGraph clusters Phenograph Label Markers 1 Transitional Pre-Monocytes CD184.sup.hi, Ly6C.sup.hi, CD115, (tpMo) CX3CR1, CD62L.sup.hi 2 Pro B cells B220, CD19, CD16/32.sup.lo, CD48.sup.lo 3 cMoP, tpMo CD34, Ly6C, CD115, CD62L 4 IgM+ B Cells IgM, B220 5 Dendritic Cells/preDCs FLT3, CD11c, SiglecH, IFNAR1, CD317 6 Eosinophils SiglecF, F4/80, CD41 7 NK, T cells, NKT cells NK1.1, CD3, NKG2D 8 Megakaryocytes CD41, CD61, SSC.sup.hi 9 Immature Neutrophils CD371, CD63, Ly6G, CD81 10 Mature Neutrophils CD182.sup.hi, Ly6G, Gr1, CD11b 11 Classical monocytes CD115, Ly6C, CD11b (Ly6C.sup.hi Monocytes) 12 Mature Neutrophils CD182.sup.hi, Ly6G, Gr1, CD11b 13 Mature Neutrophils CD182.sup.hi, Ly6G, Gr1, CD11b 14 Recirculating/Aged CD43.sup.lo, Ly6G, CD11b.sup.hi, Neutrophils CD62L.sup.lo, CD182.sup.lo 15 Basophils, Mast cells CD220R, CD49b, IL33R, CD16/32.sup.hi, cKit, CD123 16 Ly6C.sup.hi Monocytes downre- CD115, Ly6C, CD11b gulated in CD115 during harvest 17 Mature Neutrophils CD182.sup.hi, Ly6G, Gr1, CD11b 18 HSPCs cKit, Sca-1, Flt3, CD48, CD11b− 19 Pre-pro B Cells B220.sup.lo, CD2−, CD14, CD19−, CD103 20 Stromal Cells CD140a, CD140b, CD45−, CD144 21 Transitional Pre-Monocytes CD184.sup.hi, Ly6C.sup.hi, CD115, (tpMo) CX3CR1, CD62L.sup.hi 22 Red Blood Cells CD147, Ter119 23 pre-Neutrophils cKit, Gr1, CD11b, CD43, Ly6C 24 Ly6C− Monocytes (non- CD43, CD115, CX3CR1, CD11c classical monocytes) 25 Recirculating Ly6C.sup.hi Ly6C, CX3CR1, CD62L, Classical Monocytes CD16/32, CD115 26 pre B cells CD16/32.sup.hi, B220, CD19 27 IgD+ B cells IgD, B220
TABLE-US-00003 TABLE 3 (Related to FIG. 7). List of surface markers tested in human cord blood LEGENDScreen ™. Location Marker Clone Isotype Plate_1_A1_001 Blank Plate_1_A10_009 CD2 RPA-2.10 mouse IgG1, k Plate_1_A12_011 B7-H4 MIH43 mouse IgG1, k Plate_1_A3_003 CCR10 6588-5 Armenian Hamster IgG Plate_1_A4_004 CD278 C398.4A Armenian Hamster IgG Plate_1_A5_005 IFN-γ R b chain 2HUB-159 Hamster IgG Plate_1_A7_006 CD46 TRA-2-10 Mouse IgG1 Plate_1_A8_007 CD70 113-16 Mouse IgG1 Plate_1_A9_008 CD1a HI149 mouse IgG1, k Plate_1_B1_012 Cadherin 11 16G5 mouse IgG1, k Plate_1_B10_021 CD111 R1.302 mouse IgG1, k Plate_1_B11_022 CD112 TX31 mouse IgG1, k Plate_1_B12_023 CD114 LMM741 mouse IgG1, k Plate_1_B2_013 CD10 HI10a mouse IgG1, k Plate_1_B3_014 CD100 A8 mouse IgG1, k Plate_1_B4_015 CD103 Ber-ACT8 mouse IgG1, k Plate_1_B5_016 CD105 (Endoglin) SN6h mouse IgG1, k Plate_1_B6_017 CD106 STA mouse IgG1, k Plate_1_B7_018 CD107a H4A3 mouse IgG1, k Plate_1_B8_019 CD107b H4B4 mouse IgG1, k Plate_1_B9_020 CD109 W7C5 mouse IgG1, k Plate_1_C1_024 CD116 4H1 mouse IgG1, k Plate_1_C10_033 CD13 WM15 mouse IgG1, k Plate_1_C11_034 CD131 1C1 mouse IgG1, k Plate_1_C12_035 CD134 Ber-ACT35 mouse IgG1, k (ACT35) Plate_1_C2_025 CD117 104D2 mouse IgG1, k Plate_1_C3_026 CD119 GIR-208 mouse IgG1, k Plate_1_C4_027 CD11a HI111 mouse IgG1, k Plate_1_C5_028 CD11b ICRF44 mouse IgG1, k Plate_1_C6_029 CD122 TU27 mouse IgG1, k Plate_1_C7_030 CD123 6H6 mouse IgG1, k Plate_1_C8_031 CD126 UV4 mouse IgG1, k Plate_1_C9_032 CD127 A019D5 mouse IgG1, k Plate_1_D1_036 CD135 BV10A4H2 mouse IgG1, k Plate_1_D10_045 CD143 5-369 mouse IgG1, k Plate_1_D11_046 CD146 P1H12 mouse IgG1, k Plate_1_D12_047 CD148 A3 mouse IgG1, k Plate_1_D2_037 CD137 4B4-1 mouse IgG1, k Plate_1_D3_038 4-1BB Ligand 5F4 mouse IgG1, k Plate_1_D4_039 CD138 MI15 mouse IgG1, k Plate_1_D5_041 CD14 63D3 mouse IgG1, k Plate_1_D6_040 CD140a 16A1 mouse IgG1, k Plate_1_D7_042 CD140b 18A2 mouse IgG1, k Plate_1_D8_043 CD141 M80 mouse IgG1, k Plate_1_D9_044 CD142 NY2 mouse IgG1, k Plate_1_E1_048 CD15 W6D3 mouse IgG1, k Plate_1_E10_057 CD163 GHI/61 mouse IgG1, k Plate_1_E11_058 CD164 67D2 mouse IgG1, k Plate_1_E12_059 CD165 SN2 (N6-D11) mouse IgG1, k Plate_1_E2_049 CD150 A12 (7D4) mouse IgG1, k Plate_1_E3_050 CD151 50-6 mouse IgG1, k Plate_1_E4_051 CD154 24-31 mouse IgG1, k Plate_1_E5_052 CD156c SHM14 mouse IgG1, k Plate_1_E6_053 CD158e1 DX9 mouse IgG1, k Plate_1_E7_054 CD16 3G8 mouse IgG1, k Plate_1_E8_055 CD161 HP-3G10 mouse IgG1, k Plate_1_E9_056 CD162 KPL-1 mouse IgG1, k Plate_1_F1_060 CD166 3A6 mouse IgG1, k Plate_1_F10_069 CD180 MHR73-11 mouse IgG1, k Plate_1_F11_070 CD182 5E8/CXCR2 mouse IgG1, k Plate_1_F12_071 CD183 G025H7 mouse IgG1, k Plate_1_F2_061 CD169 7-239 mouse IgG1, k Plate_1_F3_062 CD170 1A5 mouse IgG1, k Plate_1_F4_063 CD172a/b (SIRPα/β) SE5A5 mouse IgG1, k Plate_1_F5_064 CD172g (SIRPγ) LSB2.20 mouse IgG1, k Plate_1_F6_065 CD178 NOK-1 mouse IgG1, k Plate_1_F7_066 CD179a HSL96 mouse IgG1, k Plate_1_F8_067 CD179b HSL11 mouse IgG1, k Plate_1_F9_068 CD18 TS1/18 mouse IgG1, k Plate_1_G1_072 CD185 J252D4 mouse IgG1, k Plate_1_G10_081 CD203c NP4D6 mouse IgG1, k Plate_1_G11_082 CD205 HD83 mouse IgG1, k Plate_1_G12_083 CD206 15-2 mouse IgG1, k Plate_1_G2_073 CD19 HIB19 mouse IgG1, k Plate_1_G3_074 CD191 5F10B29 mouse IgG1, k Plate_1_G4_075 CD194 L291H4 mouse IgG1, k Plate_1_G5_076 CD1b SN13 (K5-1B8) mouse IgG1, k Plate_1_G6_077 CD1c L161 mouse IgG1, k Plate_1_G7_078 CD200 OX-104 mouse IgG1, k Plate_1_G8_079 CD200R OX-108 mouse IgG1, k Plate_1_G9_080 CD202b 33.1 (Ab33) mouse IgG1, k Plate_1_H10_093 CD229 HLy-9.1.25 mouse IgG1, k Plate_1_H11_094 CD23 EBVCS-5 mouse IgG1, k Plate_1_H2_085 CD21 Bu32 mouse IgG1, k Plate_1_H3_086 CD213α1 SS12B mouse IgG1, k Plate_1_H4_087 CD213α2 SHM38 mouse IgG1, k Plate_1_H5_088 CD218a H44 mouse IgG1, k Plate_1_H6_089 CD221 1H7/CD221 mouse IgG1, k Plate_1_H7_090 CD223 (LAG-3) 11C3C65 mouse IgG1, k Plate_1_H8_091 CD226 11A8 mouse IgG1, k Plate_1_H9_092 CD227 16A mouse IgG1, k Plate_2_A10_009 CD268 11C1 mouse IgG1, k Plate_2_A11_010 CD27 M-T271 mouse IgG1, k Plate_2_A2_001 CD244 (2B4) C1.7 mouse IgG1, k Plate_2_A3_002 CD245 DY12 mouse IgG1, k Plate_2_A4_003 CD25 M-A251 mouse IgG1, k Plate_2_A5_004 CD252 11C3.1 mouse IgG1, k Plate_2_A6_005 CD261 DJR1 mouse IgG1, k Plate_2_A7_006 CD262 DJR2-4 (7-8) mouse IgG1, k Plate_2_A8_007 CD263 DJR3 mouse IgG1, k Plate_2_A9_008 CD266 ITEM-1 mouse IgG1, k Plate_2_B1_012 CD275 9F.8A4 mouse IgG1, k Plate_2_B10_021 CD30 BY88 mouse IgG1, k Plate_2_B11_022 CD300c TX45 mouse IgG1, k Plate_2_B12_023 CD309 7D4-6 mouse IgG1, k Plate_2_B2_013 CD276 MIH42 mouse IgG1, k Plate_2_B3_014 CD277 BT3.1 mouse IgG1, k Plate_2_B4_015 CD279 EH12.2H7 mouse IgG1, k Plate_2_B5_016 CD28 CD28.2 mouse IgG1, k Plate_2_B6_017 CD29 TS2/16 mouse IgG1, k Plate_2_B7_018 CD290 3C10C5 mouse IgG1, k Plate_2_B8_019 CD298 LNH-94 mouse IgG1, k Plate_2_B9_020 CD3 UCHT1 mouse IgG1, k Plate_2_C1_024 CD31 WM59 mouse IgG1, k Plate_2_C10_033 CD336 P44-8 mouse IgG1, k Plate_2_C11_034 CD337 P30-15 mouse IgG1, k Plate_2_C12_035 CD34 581 mouse IgG1, k Plate_2_C2_025 CD314 1D11 mouse IgG1, k Plate_2_C3_026 CD317 RS38E mouse IgG1, k Plate_2_C4_027 CD324 67A4 mouse IgG1, k Plate_2_C5_028 CD325 8C11 mouse IgG1, k Plate_2_C6_029 CD328 6-434 mouse IgG1, k Plate_2_C7_030 CD33 WM53 mouse IgG1, k Plate_2_C8_031 CD334 4FR6D3 mouse IgG1, k Plate_2_C9_032 CD335 9E2 mouse IgG1, k Plate_2_D1_036 CD340 24D2 mouse IgG1, k Plate_2_D10_045 CD38 HIT2 mouse IgG1, k Plate_2_D11_046 CD39 A1 mouse IgG1, k Plate_2_D12_047 CD4 RPA-T4 mouse IgG1, k Plate_2_D2_037 CD344 CH3A4A7 mouse IgG1, k Plate_2_D3_038 CD35 E11 mouse IgG1, k Plate_2_D4_039 CD354 TREM-26 mouse IgG1, k Plate_2_D5_040 CD360 17A12 mouse IgG1, k Plate_2_D6_041 CD365 1D12 mouse IgG1, k Plate_2_D7_042 CD366 F38-2E2 mouse IgG1, k Plate_2_D8_043 CLEC4A 9E8 mouse IgG1, k Plate_2_D9_044 CD36L1 m1b9 mouse IgG1, k Plate_2_E1_048 CD40 5C3 mouse IgG1, k Plate_2_E10_057 CD49b P1E6-C5 mouse IgG1, k Plate_2_E11_058 CD49c ASC-1 mouse IgG1, k Plate_2_E12_059 CD49d 9F10 mouse IgG1, k Plate_2_E2_049 CD41 HIP8 mouse IgG1, k Plate_2_E3_050 CD42b HIP1 mouse IgG1, k Plate_2_E4_051 CD43 CD43-10G7 mouse IgG1, k Plate_2_E5_052 CD44 BJ18 mouse IgG1, k Plate_2_E6_053 CD45 HI30 mouse IgG1, k Plate_2_E7_054 CD47 CC2C6 mouse IgG1, k Plate_2_E8_055 CD48 BJ40 mouse IgG1, k Plate_2_E9_056 CD49a TS2/7 mouse IgG1, k Plate_2_F1_060 CD5 UCHT2 mouse IgG1, k Plate_2_F10_069 CD62L DREG-56 mouse IgG1, k Plate_2_F11_070 CD62P AK4 mouse IgG1, k Plate_2_F2_061 CD50 CBR-IC3/1 mouse IgG1, k Plate_2_F3_062 CD54 HA58 mouse IgG1, k Plate_2_F4_063 CD55 JS11 mouse IgG1, k Plate_2_F5_064 CD56 (NCAM) 5.1H11 mouse IgG1, k Plate_2_F6_065 CD58 TS2/9 mouse IgG1, k Plate_2_F7_066 CD6 BL-CD6 mouse IgG1, k Plate_2_F8_067 CD61 VI-PL2 mouse IgG1, k Plate_2_G10_081 CD83 HB15e mouse IgG1, k Plate_2_G11_082 CD85 17G10.2 mouse IgG1, k Plate_2_G12_083 CD85k ZM4.1 mouse IgG1, k Plate_2_G2_073 CD69 FN50 mouse IgG1, k Plate_2_G4_075 CD74 LN2 mouse IgG1, k Plate_2_G5_076 CD79b CB3-1 mouse IgG1, k Plate_2_G6_077 CD8a SK1 mouse IgG1, k Plate_2_G7_078 CD80 2D10 mouse IgG1, k Plate_2_G8_079 CD81 5A6 mouse IgG1, k Plate_2_G9_080 CD82 ASL-24 mouse IgG1, k Plate_2_H1_084 CD87 VIM5 mouse IgG1, k Plate_2_H10_093 CD97 VIM3b mouse IgG1, k Plate_2_H11_094 CD99 3B2/TA8 mouse IgG1, k Plate_2_H12_095 CXCL16 22-19-12 mouse IgG1, k Plate_2_H2_085 CD89 A59 mouse IgG1, k Plate_2_H3_086 CD8a RPA-T8 mouse IgG1, k Plate_2_H4_087 CD9 HI9a mouse IgG1, k Plate_2_H5_088 CD90 5E10 mouse IgG1, k Plate_2_H6_089 CD93 VIMD2 mouse IgG1, k Plate_2_H7_090 CD94 DX22 mouse IgG1, k Plate_2_H8_091 CD95 DX2 mouse IgG1, k Plate_2_H9_092 CD96 NK92.39 mouse IgG1, k Plate_3_A10_009 HVEM 122 mouse IgG1, k Plate_3_A11_010 Ig light chain κ MHK-49 mouse IgG1, k Plate_3_A12_011 IgM MHM-88 mouse IgG1, k Plate_3_A2_001 DLL1 MHD1-314 mouse IgG1, k Plate_3_A3_002 DLL4 MHD4-46 mouse IgG1, k Plate_3_A4_003 DR3 JD3 mouse IgG1, k Plate_3_A5_004 EGFR AY13 mouse IgG1, k Plate_3_A6_005 CD357 108-17 mouse IgG1, k Plate_3_A7_006 GPR19 K152D10 mouse IgG1, k Plate_3_A8_007 GPR56 CG4 mouse IgG1, k Plate_3_A9_008 HLA-E 3D12 mouse IgG1, k Plate_3_B1_012 CD360 2G1-K12 mouse IgG1, k Plate_3_B10_020 TNAP W8B2 mouse IgG1, k Plate_3_B11_021 MUC-13 TCC16 mouse IgG1, k Plate_3_B12_022 NKp80 5D12 mouse IgG1, k Plate_3_B2_013 Integrin α9β1 Y9A2 mouse IgG1, k Plate_3_B3_014 Jagged 2 MHJ2-523 mouse IgG1, k Plate_3_B4_015 Ksp37 TDA3 mouse IgG1, k Plate_3_B5_016 LAP TW4-2F8 mouse IgG1, k Plate_3_B6_017 LY6G6D 13.8 mouse IgG1, k Plate_3_B7_018 MERTK 590H11G1E3 mouse IgG1, k Plate_3_B8_019 MSC W7C6 mouse IgG1, k Plate_3_C1_023 Notch 1 MHN1-519 mouse IgG1, k Plate_3_C10_032 Siglec-8 7C9 mouse IgG1, k Plate_3_C11_033 Siglec-9 K8 mouse IgG1, k Plate_3_C12_034 SSEA-5 8E11 mouse IgG1, k Plate_3_C2_024 Notch3 MHN3-21 mouse IgG1, k Plate_3_C3_025 Notch 4 MHN4-2 mouse IgG1, k Plate_3_C4_026 NPC 57D2 mouse IgG1, k Plate_3_C5_027 CD352 NT-7 mouse IgG1, k Plate_3_C6_028 PSMA LNI-17 mouse IgG1, k Plate_3_C8_030 Siglec-10 5G6 mouse IgG1, k Plate_3_C9_031 CD328 S7.7 mouse IgG1, k Plate_3_D1_035 SUSD2 W5C5 mouse IgG1, k Plate_3_D10_044 VEGFR-3 9D9F9 mouse IgG1, k Plate_3_D12_045 APCDD1 7.13 mouse IgG2a, k Plate_3_D2_036 TCR α/β IP26 mouse IgG1, k Plate_3_D4_038 Tim-4 9F4 mouse IgG1, k Plate_3_D5_039 TLT-2 MIH61 mouse IgG1, k Plate_3_D6_040 TM4SF20 C9 mouse IgG1, k Plate_3_D7_041 TRA-2-49 TRA-2-49/6E mouse IgG1, k Plate_3_D8_042 TRA-2-54 TRA-2-54/2J mouse IgG1, k Plate_3_D9_043 TSLPR 1B4 mouse IgG1, k Plate_3_E1_046 CD272 MIH26 mouse IgG2a, k Plate_3_E10_055 CD155 TX24 mouse IgG2a, k Plate_3_E11_056 CD158b DX27 mouse IgG2a, k Plate_3_E12_057 CD184 12G5 mouse IgG2a, k Plate_3_E2_047 CD198 L263G8 mouse IgG2a, k Plate_3_E3_048 CCRL2 K097F7 mouse IgG2a, k Plate_3_E4_049 CD102 CBR-IC2/2 mouse IgG2a, k Plate_3_E5_050 CD104 58XB4 mouse IgG2a, k Plate_3_E6_051 CD124 G077F6 mouse IgG2a, k Plate_3_E7_052 CD130 2E1B02 mouse IgG2a, k Plate_3_E8_053 CD144 BV9 mouse IgG2a, k Plate_3_E9_054 CD152 (CTLA-4) BNI3 mouse IgG2a, k Plate_3_F1_058 CD186 K041E5 mouse IgG2a, k Plate_3_F10_067 CD26 BA5b mouse IgG2a, k Plate_3_F11_068 CD269 19F2 mouse IgG2a, k Plate_3_F12_069 CD282 TL2.1 mouse IgG2a, k Plate_3_F2_059 CD192 K036C2 mouse IgG2a, k Plate_3_F3_060 CD197 G043H7 mouse IgG2a, k Plate_3_F4_061 CD199 L053E8 mouse IgG2a, k Plate_3_F5_062 CD209 9E9A8 mouse IgG2a, k Plate_3_F6_063 CD217 W15177A mouse IgG2a, k Plate_3_F7_064 CD230 (Prion) 3F4 mouse IgG2a, k Plate_3_F8_065 CD24 ML5 mouse IgG2a, k Plate_3_F9_066 CD243 UIC2 mouse IgG2a, k Plate_3_G1_070 CD284 HTA125 mouse IgG2a, k Plate_3_G10_078 CD370 8F9 mouse IgG2a, k Plate_3_G11_079 CD371 50C1 mouse IgG2a, k Plate_3_G12_080 CD45RO UCHL1 mouse IgG2a, k Plate_3_G2_071 CD301 H037G3 mouse IgG2a, k Plate_3_G3_072 CD303 201A mouse IgG2a, k Plate_3_G4_073 CD304 12C2 mouse IgG2a, k Plate_3_G5_074 CD307e 509f6 mouse IgG2a, k Plate_3_G6_093 CD323 SHM33 mouse IgG2a, k Plate_3_G7_075 CD357 108-17 mouse IgG2a, k Plate_3_G8_076 CD36 5-271 mouse IgG2a, k Plate_3_G9_077 CD369 15E2 mouse IgG2a, k Plate_3_H1_081 CD51 NKI-M9 mouse IgG2a, k Plate_3_H10_090 Ganglioside GD2 14G2a mouse IgG2a, k Plate_3_H11_091 GPR83 K07JP05 mouse IgG2a, k Plate_3_H12_092 HLA-A, B, C W6/32 mouse IgG2a, k Plate_3_H2_082 CD59 p282 (H19) mouse IgG2a, k Plate_3_H3_083 CD7 CD7-6B7 mouse IgG2a, k Plate_3_H4_084 CD71 CY1G4 mouse IgG2a, k Plate_3_H5_085 CD84 CD84.1.21 mouse IgG2a, k Plate_3_H6_086 CD88 S5/1 mouse IgG2a, k Plate_3_H7_087 CD355 Cr24.1 mouse IgG2a, k Plate_3_H8_088 erbB3 1B4C3 mouse IgG2a, k Plate_3_H9_089 FPR3 K102B9 mouse IgG2a, k Plate_4_A10_010 SUSD2 W3D5 mouse IgG2a, k Plate_4_A11_011 Notch 2 MHN2-25 mouse IgG2a, k Plate_4_A12_012 TACSTD2 NY18 mouse IgG2a, k Plate_4_A2_002 HLA-DR L243 mouse IgG2a, k Plate_4_A3_003 Ig light chain λ MHL-38 mouse IgG2a, k Plate_4_A4_004 IgD IA6-2 mouse IgG2a, k Plate_4_A5_005 IL-28RA MHLICR2a mouse IgG2a, k Plate_4_A6_006 integrin β5 AST-3T mouse IgG2a, k Plate_4_A7_007 KLRG1 SA231A2 mouse IgG2a, k Plate_4_A8_008 LOX-1 15C4 mouse IgG2a, k Plate_4_A9_009 MICA/MICB 6D4 mouse IgG2a, k Plate_4_B1_013 TIGIT (VSTM3) A15153G mouse IgG2a, k Plate_4_B10_021 CD196 G034E3 mouse IgG2b, k Plate_4_B11_022 CD1d 51.1 mouse IgG2b, k Plate_4_B12_023 CD20 2H7 mouse IgG2b, k Plate_4_B3_014 C3aR hC3aRZ8 mouse IgG2b, k Plate_4_B4_015 CCX-CKR (CCRL1) 13E11 mouse IgG2b, k Plate_4_B5_016 CD11c S-HCL-3 mouse IgG2b, k Plate_4_B6_017 CD129 AH9R7 mouse IgG2b, k Plate_4_B7_018 CD158 HP-MA4 mouse IgG2b, k Plate_4_B8_019 CD181 8F1/CXCR1 mouse IgG2b, k Plate_4_B9_020 CD193 5E8 mouse IgG2b, k Plate_4_C1_024 CD22 S-HCL-1 mouse IgG2b, k Plate_4_C10_033 CD368 9B9 mouse IgG2b, k Plate_4_C11_034 CD45RA HI100 mouse IgG2b, k Plate_4_C12_035 CD45RB MEM-55 mouse IgG2b, k Plate_4_C2_025 CD220 B6.220 mouse IgG2b, k Plate_4_C3_026 CD235ab HIR2 mouse IgG2b, k Plate_4_C4_027 CD258 T5-39 mouse IgG2b, k Plate_4_C5_028 CD274 29E.2A3 mouse IgG2b, k Plate_4_C6_029 CD319 162.1 mouse IgG2b, k Plate_4_C7_030 CD32 FUN-2 mouse IgG2b, k Plate_4_C8_031 CD326 9C4 mouse IgG2b, k Plate_4_C9_032 CD338 5D3 mouse IgG2b, k Plate_4_D1_036 CD49e NKI-SAM-1 mouse IgG2b, k Plate_4_D10_045 Dopamine Receptor L205G1 mouse IgG2b, k D1 (DRD1) Plate_4_D11_046 EphA2 SHM16 mouse IgG2b, k Plate_4_D12_047 FcεRIα AER-37 (CRA-1) mouse IgG2b, k Plate_4_D2_037 CD52 HI186 mouse IgG2b, k Plate_4_D3_038 CD66a/c/e ASL-32 mouse IgG2b, k Plate_4_D4_039 CD85h 24 mouse IgG2b, k Plate_4_D5_040 CD85 GHI/75 mouse IgG2b, k Plate_4_D6_041 CD86 IT2.2 mouse IgG2b, k Plate_4_D7_042 CD92 VIM15b mouse IgG2b, k Plate_4_D8_043 CXCR7 8F11-M16 mouse IgG2b, k Plate_4_D9_044 Delta Opioid Receptor DOR7D2A4 mouse IgG2b, k Plate_4_E1_048 GARP 7B11 mouse IgG2b, k Plate_4_E10_056 SSEA-4 MC-813-70 Mouse IgG3, k Plate_4_E12_057 Sialyl Lewis X (dimeric) FH6 Mouse IgM, k Plate_4_E2_049 CD215 JM7A4 mouse IgG2b, k Plate_4_E3_050 Lymphotoxin β Receptor 31G4D8 mouse IgG2b, k Plate_4_E4_051 MRGX2 K125H4 mouse IgG2b, k Plate_4_E5_052 TMEM8A SA065C3 mouse IgG2b, k Plate_4_E6_053 CD254 MIH24 mouse IgG2b, k Plate_4_E7_054 CD318 CUB1 mouse IgG2b, k Plate_4_E9_055 CD255 CARL-1 Mouse IgG3, k Plate_4_F1_058 TRA-1-81 TRA-1-81 Mouse IgM, k Plate_4_F10_066 CD120b 3G7A02 Rat IgG2a, k Plate_4_F11_067 CD210 3F9 Rat IgG2a, k Plate_4_F12_068 CD267 1A1 Rat IgG2a, k Plate_4_F2_059 CD160 BY55 Mouse IgM, k Plate_4_F3_060 CD57 HNK-1 Mouse IgM, k Plate_4_F4_061 CD66b G10F5 Mouse IgM, k Plate_4_F5_062 TRA-1-60-R TRA-1-60-R Mouse IgM, k Plate_4_F7_063 CD115 9-4D2-1E4 Rat IgG1, k Plate_4_F8_064 CD201 RCR-401 Rat IgG1, k Plate_4_G1_069 CD294 BM16 Rat IgG2a, k Plate_4_G10_077 CD132 TUGh4 Rat IgG2b, k Plate_4_G11_078 CD195 J418F1 Rat IgG2b, k Plate_4_G12_079 CX3CR1 2A9-1 Rat IgG2b, k Plate_4_G2_070 CD49f GoH3 Rat IgG2a, k Plate_4_G3_071 CD85 MKT5.1 Rat IgG2a, k Plate_4_G4_072 CD85d 42D1 Rat IgG2a, k Plate_4_G5_073 IgG Fc M1310G05 Rat IgG2a, k Plate_4_G6_074 Integrin β7 FIB504 Rat IgG2a, k Plate_4_G7_075 XCR1 S15046E Rat IgG2a, k Plate_4_G8_076 Podoplanin NC-08 Rat IgG2a, l Plate_4_H2_080 SSEA-3 MC-631 Rat IgM, k
TABLE-US-00004 TABLE 4 Key resources table REAGENT SOURCE IDENTIFIER Antibodies B220 (Clone RA3-6B2) Thermo Fisher Cat# 25-0452; RRID: AB_2341160 CD81 (Clone Eat-2) Biolegend Cat# 104906, RRID: AB_2076266 CD106 (Clone 429 Biolegend Cat# 105716, RRID: AB_1595489 (MVCAM.A)) cKit (Clone 2B8) Thermo Fisher Cat# 62-1171-82, RRID: AB_2637141 CD11b (Clone M1/70) Thermo Fisher Cat# 63-0112-80, RRID: AB_2637407 CD115 (Clone AFS98) Biolegend Cat# 135510, RRID: AB_2085221 Flt3 (Clone A2F10) Biolegend Cat# 135308, RRID: AB_1953267 NK1.1 (Clone PK136) Thermo Fisher Cat# 13-5941-81, RRID: AB_466803 B220 (Clone RA3-6B2) Thermo Fisher Cat# 13-0452-82, RRID: AB_466449 CD90.2 (Clone 53-2.1) Biolegend Cat# 140314, RRID: AB_10643274 F4/80 (Clone BM8) Biolegend Cat# 123118, RRID: AB_893477 CD16/32 (Clone 2.4G2) BD Horizon Cat# 565502, RRID: AB_2739269 CD34 (Clone RAM34) Thermo Fisher Cat# 13-0341-81, RRID: AB_466424 Gr1 (Clone RB6-8C5) Thermo Fisher Cat# 45-5931-80; RRID: AB_906247 Ly6C (Clone HK1.4) Biolegend Cat# 128026; RRID: AB_10640120 Ly6G (Clone 1A8) Biolegend Cat# 127618; RRID: AB_1877261 Sca-1 (Clone D7) Biolegend Cat# 108114; RRID: AB_493596 Siglec-F (Clone E50- BD Biosciences Cat# 562757; RRID: AB_394341 2440) CXCR2 (Clone Biolegend Cat# 149306; RRID: AB_2565694 SA044G4) CXCR4 (Clone 2B11) Thermo Fisher Cat# 13-9991-82; RRID: AB_10609202 CD45.1 (Clone A20) Biolegend Cat#110707; RRID: AB_313496 CD45.2 (Clone 104) Biolegend Cat#109807; RRID: AB_313444 CD3 (Clone UCHT1) Biolegend Cat#300440; RRID: AB_2562046 CD10 (Clone HI10a) Biolegend Cat#312208; RRID: AB_314919 CD19 (Clone HIB19) Biolegend Cat#302205; RRID: AB_314235 CD56 (Clone HCD56) Biolegend Cat#318304; RRID: AB_604100 CD14 (Clone M5E2) Biolegend Cat#301838; RRID: AB_2562909 CD15 (Clone HI98) BD Biosciences Cat#564232; RRID: AB_2738686 CD49d (Clone 9F10) BD Biosciences Cat#563645; RRID: AB_2738344 CD66b (Clone G105F) BD Biosciences Cat#305111; RRID: AB_2563293 CD101 (Clone BB27) Biolegend Cat#331007; RRID: AB_2121761 CD16 (Clone 3G8) BD Biosciences Cat#560248; RRID: AB_1645467 CD34 (Clone 581) Biolegend Cat#343515; RRID: AB_1877252 CD117 (Clone 104D2) Thermo Fisher Cat#64-1178-42; RRID: AB_2734860 CD45 (Clone 2D1) Biolegend Cat368515; RRID: AB_2566375 CD11b (ICRF44) BD Biosciences Cat#563839; RRID: AB_2716869 CD38 (S17015F) Biolegend Cat#303531; RRID: AB_2561527 CD71 (CY1G4) Biolegend Cat#334106; RRID: AB_2201481 Chemicals, Peptides, and Recombinant Proteins 1X RBC Lysis Buffer eBioscience Cat# 00-4333-57 5-Fluorouracil Sigma-Aldrich Cat# F6627 CountBright Absolute Life Cat# C36950 Counting Beads Technologies DAPI Life Cat# D1306 Technologies Hema 3 Manual Thermo Fisher Cat# 22-122911 Staining System EQ Four Element Fluidigm Cat# 201078 Calibration Beads Human G-CSF Amgen Cat# 100696800 (Neupogen) MethoCult ™ M3234 Stem Cell Cat# 03234 Technologies StemSpan ™ SFEM II Stem Cell Cat# 09655 Technologies Critical Commercial Assays ERCC RNA Spike-In Thermo Fisher Cat# 4456740 Mix MILLIPLEX MAP Merck Cat# MCYTMAG-70K-PX32 Mouse Cytokine/Chemokine Magnetic Bead Panel - Premixed 32 Plex - Immunology Multiplex Assay LEGENDScreen ™ Biolegend Cat# 700007 Human Kit LEGENDScreen ™ Biolegend Cat# 70000 Mouse Kit Arcturus PicoPure Thermo Fisher Cat# KIT0214 RNA isolation kit Deposited Data Bulk RNA-seq of This disclosure GEO: myeloid progenitor populations Smart-Seq2 Single- This disclosure GEO: cell Dataset of myeloid precursors Tabula Muris BM 10× (Schaum et GEO: GSE109774 single-cell RNA-seq al., 2018) dataset Single-cell RNA-seq (Olsson et GEO: GSE70240 of BM GMPs al., 2016) Experimental Models: Organisms/Strains Mouse: C57BL/6J The Jackson Stock No: 000664 Laboratory Mouse: Rosa26.sup.mT/mG:STOCK The Jackson Stock No: 007676 Gt(ROSA).sup.26Sortm4(ACTB-tdTomato, −EGFP)Luo/J Laboratory Mouse: Fucci-S/G2/M RIKEN BRC, Stock No: RBRC02704 (#474) Ibaraki, Japan Mouse: C57BL/6-Tg(UBC- The Jackson Stock No: 04353 GFP)30Scha/J; UBI-GFP Laboratory Mouse: B6.SJL-Ptprc.sup.a The Jackson Stock No: 002014 Pepc.sup.b/BoyJ; B6 Cd45.1 Laboratory Mouse: Cebpe.sup.−/− H. P. Koeffler (Kyme et al., 2012) Software and Algorithms GraphPad Prism 7 GraphPad Software http://www.graphpad.com FlowJo 10 TreeStar http://flowjo.com/ t-SNE (Van Der Maaten https://cran.r-project.org/web/packages/Rtsne/index.html and Hinton, 2008) edgeR (Robinson et https://bioconductor.org/packages/release/bioc/html/edgeR.html al., 2009) pheatmap R package https://cran.r-project.org/web/packages/pheatmap/index.html Enrichr (Chen et al., http://amp.pharm.mssm.edu/Enrichr/ 2013) Monocle 2 (Qiu et al., http://cole-trapnell-lab.github.io/monocle-release/docs/ 2017) Seurat v3 (Stuart et https://github.com/satijalab/seurat al., 2018) UMAP (McInnes et https://umap-learn.readthedocs.io/en/latest/ al., 2018) SVM regression used The R foundation https://cran.r-project.org/web/packages/e1071/ in InfinityFlow R 3.6 The R foundation https://www.r-project.org/
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[0174] It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.