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CD34 and EPCR coordinately enrich functional murine hematopoietic stem cells under normal and inflammatory conditions

Open AccessPublished:December 18, 2019DOI:https://doi.org/10.1016/j.exphem.2019.12.003

      Highlights

      • EPCR and CD34 enrich for long-term repopulating SLAM cells in chronic IL-1 conditions.
      • EPCR and CD34 identify molecularly distinct SLAM fractions.
      • EPCR+/CD34 SLAM cells are highly quiescent regardless of IL-1 exposure.
      • EPCR-SLAM cells possess limited repopulating activity and exhibit Mk priming.
      • Fgd5 expression marks long-term HSCs minimally impacted by chronic IL-1.
      Hematopoiesis is dynamically regulated to maintain blood system function under nonhomeostatic conditions such as inflammation and injury. However, common surface marker and hematopoietic stem cell (HSC) reporter systems used for prospective enrichment of HSCs have been less rigorously tested in these contexts. Here, we use two surface markers, EPCR/CD201 and CD34, to re-analyze dynamic changes in the HSC-enriched phenotypic SLAM compartment in a mouse model of chronic interleukin (IL)-1 exposure. EPCR and CD34 coordinately identify four functionally and molecularly distinct compartments within the SLAM fraction, including an EPCR+/CD34 fraction whose long-term serial repopulating activity is only modestly impacted by chronic IL-1 exposure, relative to unfractionated SLAM cells. Notably, the other three fractions expand in frequency following IL-1 treatment and represent actively proliferating, lineage-primed cell states with limited long-term repopulating potential. Importantly, we find that the Fgd5-ZSGreen HSC reporter mouse enriches for molecularly and functionally intact HSCs regardless of IL-1 exposure. Together, our findings provide further evidence of dynamic heterogeneity within a commonly used HSC-enriched phenotypic compartment under stress conditions. Importantly, they also indicate that stringency of prospective isolation approaches can enhance interpretation of findings related to HSC function when studying models of hematopoietic stress.
      Hematopoietic stem cells (HSCs) are tasked with maintaining lifelong blood production, including under nonhomeostatic conditions induced by physiological stresses [
      • King KY
      • Goodell MA
      Inflammatory modulation of HSCs: Viewing the HSC as a foundation for the immune response.
      ]. HSCs must therefore cope with demands brought about by infection, injury, aging, inflammatory disease, and myeloablative therapeutic interventions such as irradiation (IR) and chemotherapy [
      • Pietras EM
      Inflammation: A key regulator of hematopoietic stem cell fate in health and disease.
      ]. Thus, understanding the impact of these stresses on HSC function and the long-term viability of the HSC pool is crucial for identifying mechanisms that drive pathogenic processes such as leukemogenesis, aging, and bone marrow (BM) failure.
      Prospective identification and isolation of HSCs under stress conditions by flow cytometry offer opportunities for detailed studies to address the unique biological features of stress hematopoiesis. Phenotypic definitions such as Lineage (Lin)/cKit+/Sca-1+/Flk2/CD48CD150+ surface marker combination (hereafter referred to as SLAM) are commonly used to enrich for HSCs in these studies [
      • Warr MR
      • Pietras EM
      • Passegue E
      Mechanisms controlling hematopoietic stem cell functions during normal hematopoiesis and hematological malignancies.
      ,
      • Kiel MJ
      • et al.
      SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.
      ,
      • Chen J
      • Ellison FM
      • Keyvanfar K
      • et al.
      Enrichment of hematopoietic stem cells with SLAM and LSK markers for the detection of hematopoietic stem cell function in normal and Trp53 null mice.
      ]. Surface markers including CD34 and endothelial protein C perceptor (EPCR/CD201) have also been used in combination with the SLAM definition to further enrich for HSC activity [
      • Balazs AB
      • Fabian AJ
      • Esmon CT
      • Mulligan RC
      Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow.
      ,
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ]. Other methods, such as Hoescht side-population (SP) and rhodamine dye, have been used for further enrichment in combination with SLAM [
      • Winkler IG
      • Barbier V
      • Wadley R
      • Zannettino AC
      • Williams S
      • Lévesque JP
      Positioning of bone marrow hematopoietic and stromal cells relative to blood flow in vivo: Serially reconstituting hematopoietic stem cells reside in distinct nonperfused niches.
      ,
      • Shin JY
      • Hu W
      • Naramura M
      • Park CY
      High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias.
      ,
      • Challen GA
      • Boles N
      • Lin KK
      • Goodell MA
      Mouse hematopoietic stem cell identification and analysis.
      ]. In addition, a growing body of work has highlighted the functional and molecular heterogeneity at the population and single-cell levels within phenotypic HSC gates [
      • MacLean AL
      • Smith MA
      • Liepe J
      • et al.
      Single cell phenotyping reveals heterogeneity among hematopoietic stem cells following infection.
      ,
      • Haas S
      • Trumpp A
      • Milsom MD
      Causes and consequences of hematopoietic stem cell heterogeneity.
      ,
      • Morita Y
      • Ema H
      • Nakauchi H
      Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.
      ,
      • Nestorowa S
      • Hamey FK
      • Pijuan Sala B
      • et al.
      A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.
      ,
      • Yamamoto R
      • Morita Y
      • Ooehara J
      • et al.
      Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.
      ,
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ], suggesting that markers such as SLAM contain distinct functional compartments within. Moreover, proteins such as CD41 and interferon-γ receptor have been used to distinguish unique functional compartments within the SLAM gate under stress conditions [
      • Haas S
      • Hansson J
      • Klimmeck D
      • et al.
      Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors.
      ,
      • Matatall KA
      • Shen CC
      • Challen GA
      • King KY
      Type II interferon promotes differentiation of myeloid-biased hematopoietic stem cells.
      ]. Outcomes common to most studies of inflammatory stress include loss of long-term repopulating activity as measured by transplantation of SLAM HSC, expansion of the phenotypic SLAM HSC compartment, increased cell proliferation, and activation of unique HSC-like subsets, including megakaryocyte (Mk)-biased CD41+ cells, within the SLAM gate [
      • Haas S
      • Hansson J
      • Klimmeck D
      • et al.
      Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors.
      ,
      • Pietras EM
      • Lakshminarasimhan R
      • Techner JM
      • et al.
      Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons.
      ,
      • Pietras EM
      • Mirantes-Barbeito C
      • Fong S
      • et al.
      Chronic interleukin-1 exposure drives haematopoietic stem cells toward precocious myeloid differentiation at the expense of self-renewal.
      ,
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ,
      • Walter D
      • Lier A
      • Geiselhart A
      • et al.
      Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells.
      ,
      • Essers MA
      • Offner S
      • Blanco-Bose WE
      • et al.
      IFNalpha activates dormant haematopoietic stem cells in vivo.
      ].
      However, a wide variety of stressors including IR, chemotherapy agents such as 5-FU, and inflammatory cytokines can induce phenotypic shifts in key surface markers such as c-Kit and Sca-1 that may lead to contamination of the phenotypic HSC gate with non-HSC cell types [
      • Pietras EM
      • Lakshminarasimhan R
      • Techner JM
      • et al.
      Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons.
      ,
      • Domen J
      • Weissman IL
      Hematopoietic stem cells and other hematopoietic cells show broad resistance to chemotherapeutic agents in vivo when overexpressing bcl-2.
      ,
      • Hérault A
      • Binnewies M
      • Leong S
      • et al.
      Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis.
      ,
      • Vazquez SE
      • Inlay MA
      • Serwold T
      CD201 and CD27 identify hematopoietic stem and progenitor cells across multiple murine strains independently of Kit and Sca-1.
      ] or exclusion of stem/progenitor cells that may transiently express traditionally excluded lineage markers such as Mac-1 under stress conditions [
      • Hidalgo A
      • Peired AJ
      • Weiss LA
      • Katayama Y
      • Frenette PS
      The integrin alphaMbeta2 anchors hematopoietic progenitors in the bone marrow during enforced mobilization.
      ]. In recent years, numerous reporter mouse lines have also been developed to enrich for HSCs, based on expression of fluorescent reporters driven by promoters of genes including Hoxb5, Fgd5, Vwf, Gprc5c, and Krt18 [
      • Chapple RH
      • Tseng YJ
      • Hu T
      • et al.
      Lineage tracing of murine adult hematopoietic stem cells reveals active contribution to steady-state hematopoiesis.
      ,
      • Cabezas-Wallscheid N
      • Buettner F
      • Sommerkamp P
      • et al.
      Vitamin A–retinoic acid signaling regulates hematopoietic stem cell dormancy.
      ,
      • Gazit R
      • Mandal PK
      • Ebina W
      • et al.
      Fgd5 identifies hematopoietic stem cells in the murine bone marrow.
      ,
      • Chen JY
      • Miyanishi M
      • Wang SK
      • et al.
      Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche.
      ,
      • Pinho S
      • Marchand T
      • Yang E
      • Wei Q
      • Nerlov C
      • Frenette PS
      Lineage-biased hematopoietic stem cells are regulated by distinct niches.
      ]. Together, the capacity of surface markers and reporters to identify functional HSCs under nonhomeostatic conditions such as inflammation has not been extensively evaluated. Hence, careful evaluation and enhancement of commonly used HSC marker/reporter definitions can ensure correct interpretation of experimental results in studies of stress hematopoiesis.
      In the present study, we used a model of chronic interleukin (IL)-1β exposure to evaluate changes within the SLAM gate during inflammatory stress. We use the markers EPCR and CD34 to coordinately identify four distinct SLAM fractions with unique functional and molecular properties. Strikingly, we find that the relative abundance of these fractions changes significantly following IL-1 treatment, with a sharp reduction in the EPCR+/CD34 fraction, which enriches for the vast majority of HSC activity. Importantly, we find that chronic IL-1 exposure does not substantially change the molecular or proliferative properties of each fraction. Likewise, we find that Fgd5-ZSGreen expression identifies HSCs with equivalent molecular and functional properties regardless of IL-1 exposure, indicating that a functional long-term HSC pool is retained even under chronic inflammatory stress. This work provides critical insights into the dynamic nature of stress hematopoiesis and provides critical insight into improved strategies to identify functional HSCs under nonhomeostatic conditions.

      Methods

      Mice

      Wild-type C57BL/6, CD45.1+ congenic B6.SJL-PtprcaPepcb/BoyJ (Boy/J) mice and Fgd5-ZSGreen-CreERT mice [
      • Gazit R
      • Mandal PK
      • Ebina W
      • et al.
      Fgd5 identifies hematopoietic stem cells in the murine bone marrow.
      ] were obtained from The Jackson Laboratory and bred in-house. Vwf-GFP mice [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ] were a kind gift of Dr. C. Nerlov, Oxford University (Oxford, UK). Both male and female mice were used for experiments. All procedures were performed in accordance with an Institutional Animal Care and Use Committee (IACUC)-approved University of Colorado Anschutz Medical Campus animal protocol (Protocol No. 00091).

      In vivo experiments

      Recombinant murine IL-1β injections were performed as previously described [
      • Pietras EM
      • Mirantes-Barbeito C
      • Fong S
      • et al.
      Chronic interleukin-1 exposure drives haematopoietic stem cells toward precocious myeloid differentiation at the expense of self-renewal.
      ]. Mice were injected intraperitoneally with 0.5 μg IL-1β (Peprotech) in 100 μL of 0.22-μm sterile-filtered phosphate-buffered saline (PBS)/0.2% bovine serum albumin (BSA), or 100 μL PBS/0.2% BSA alone. Mice were injected daily on alternating sides for 20 days. Transplantation experiments were performed as previously described [
      • Pietras EM
      • Mirantes-Barbeito C
      • Fong S
      • et al.
      Chronic interleukin-1 exposure drives haematopoietic stem cells toward precocious myeloid differentiation at the expense of self-renewal.
      ]. Eight- to twelve-week-old Boy/J mice were lethally irradiated with 11 Gy in a split dose 3 hours apart using a 137Cs fixed-beam source (J. L. Shepherd & Associates, San Fernando, CA) and injected retro-orbitally with 250 donor test cells plus 5 × 105 Sca-1-depleted Boy/J helper BM cells. Mice were maintained on Bactrim in water for 3 weeks following transplantation. Secondary transplantations were performed similarly, but with 500 donor test cells. Donor peripheral blood chimerism was analyzed every 4 weeks by submandibular bleed and collection of blood in 4 mL of ACK buffer (150 mmol/L NH4Cl/10 mmol/L KHCO3) for flow cytometry analyses. 5-Ethynyl-2ʹ-deoxyuridine (EdU) labeling was performed by injecting 2 mg EdU in PBS intraperitoneally at the same time as the final IL-1/PBS injection, and euthanizing mice 24 hours later. At the termination of all experiments, mice were euthanized by CO2 inhalation followed by cervical dislocation according to approved protocols.

      Flow cytometry

      BM cells were isolated by crushing legs, arms, pelves, and spines from mice in staining medium (SM) consisting of Hanks’ buffered saline solution (HBSS) with 2% heat-inactivated fetal bovine serum (FBS). Cells were subsequently incubated on ice in ACK buffer for 3 min to remove erythroid cells, washed with SM, and centrifuged on a Histopaque gradient (Histopaque 1119, Sigma-Aldrich, St. Louis, MO). To enrich c-Kit+ cells for sorting, BM cells were incubated for 20 min with c-Kit microbeads (Miltenyi Biotec, 130-091-224), washed with SM, and enriched on an AutoMACS Pro cell separator (Miltenyi Biotec). For analysis of BM populations, BM cells were flushed with SM from both femurs and tibias from each mouse, treated with ACK, and counted in a ViCell automated counter (Beckman-Coulter) before staining. For hematopoietic stem and progenitor cell analyses, 107 cells were blocked with purified rat IgG (Sigma Aldrich) and stained with PE-Cy5-conjugated lineage antibodies against B220, CD3, CD4, CD5, CD8, Gr1, and Ter119, plus CD34-FITC, EPCR-PE, Mac-1-PE/Cy7, Flk2-Biotin, ESAM-APC, CD48-A700, and c-Kit-APC/Cy7 for 30 min on ice, washed with SM, and stained with Sca-1-BV421, CD41-BV510, strepatavidin-BV605, and CD150-BV785 in a staining buffer composed of 1:3 v/v Brilliant Buffer (BD Biosciences)/SM. Cells were also analyzed for IL-1R (anti-IL-1R-PE) and CD49b (anti-CD49b-PE/Cy7) by substituting EPCR-APC for EPCR-PE. For SLAM chimerism analyses, a variation of this panel was used in which CD41 and Mac-1 were omitted and CD45.1 BV650 and CD45.2-PE/Cy7 were used. For mature BM cell analyses, 5 × 105 cells were stained with Gr-1-Pacific Blue, B220-BV785, CD4-FITC, CD8-PE, Mac-1-PE/Cy7, IgM-APC, CD3-A700, and CD19-A780. Dead cells were excluded by resuspending cells in SM containing 1 µg/mL propidium iodide (PI). Samples were analyzed on a 12-channel, 3-laser FACSCelesta or a 20-channel, 5-laser Fortessa X-20 analyzer running FACSDiva software (Becton-Dickenson). Data were analyzed using FlowJo Version 10 (FlowJo). For cell sorting, anti-CD41 antibodies were not included in the staining cocktail. For sorting experiments, cells were double-sorted to purity on a FACSAria IIu or Aria Fusion cell sorter (Becton-Dickenson). For Ki67/DAPI cell cycle analysis, 107 cells were stained as previously described [
      • Jalbert E
      • Pietras EM
      Analysis of murine hematopoietic stem cell proliferation during inflammation.
      ] using the 12-color panel described above, except using Sca-1-PE/Cy7 and excluding CD41 and Mac-1. After staining, cells were fixed with CytoFix/CytoPerm (BD Biosciences) for 30 min at room temperature (RT), washed with 1 × PermWash buffer (BD Biosciences), permeabilized with Perm Buffer Plus (BD Biosciences), washed with PermWash buffer, re-fixed with CytoFix/CytoPerm for 10 min at RT, and stained with anti-Ki67-PerCP-Cy5.5 for 1 hour at RT before being washed in PermWash. Cells were subsequently incubated with SM containing 2 μg/mL DAPI. For EdU analysis, we found that EPCR was destroyed by the fix/perm regimen. Hence, we double-sorted SLAM fractions from EdU-injected mice and mixed the cells with 1 × 105 B220+ carrier cells harvested from the spleen, and EdU was visualized using the Click-iT Plus EdU Flow Cytometry Assay Kit (Thermo Fisher Scientific) containing the AlexaFluor 488 picolyl azide fluorochrome according to the kit instructions. Fix, perm/wash, and click reagents were diluted according to the manufacturer's instructions, and sorted cells were fixed for 15 min at RT, washed in PBS/1% BSA, permeabilized for 15 min at RT, washed, and incubated with click reagents for 30 min at RT. Cells were then washed in perm/wash before analysis. For all flow cytometry applications, cells were analyzed on a four-laser, 20-channel LSRII analyzer running FACSDiva software (Becton-Dickenson) and analyzed using FlowJo. For all procedures listed above, antibody clone, manufacturer, catalogue number, and dilution information are contained in Supplementary Table E1 (online only, available at www.exphem.org.
      Supplementary Table E1Antibodies used in this study. Antibody information including target, fluor, manufacturer, catalog number, clone and dilution used in this study.
      TargetFluorManufacturerCatalogCloneDilution
      B220APC-Cy7BioLegend105826RA3-6821:200
      B220PE-Cy5BioLegend103201RA3-6821:800
      c-KitAPC-Cy7BioLegend1058262B81:800
      CD150BV786BioLegend115937TC15-12F12.21:100
      CD19APC-Cy7BioLegend1155236D51:400
      CD3PE-Cy5BioLegend15-0031-8117A21:100
      CD34FITCeBioscience11-0341-85RAM341:25
      CD4PE-Cy5eBioscience15-0051-81GK1.51:1600
      CD41BV510BioLegend133923MW/reg301:400
      CD45APC-Cy7BD Biosciences55765930-F111:400
      CD45.2FITCBioLegend1098061041:400
      CD45.1PE-Cy7BD Biosciences560578A201:400
      CD48AlexaFluor 700BioLegend103416HM48-11:100
      CD49bPE-Cy7BioLegend103517HMα21:200
      CD5PE-Cy5BioLegend10061053-7.31:800
      CD8PE-Cy5BioLegend10071053-6.71:800
      ESAMAPCBioLegend1362051G81:200
      FcγRPerCP-eFluor710eBioscience46-0161-82931:1600
      Flk2BiotineBioscience13-1351-82RMV71:400
      Gr-1Pacific BlueBioLegend108430RB6-8C51:800
      Gr-1PE-Cy5BioLegend108410RB6-8C51:800
      IgMAPCeBioscience17-5790-82II/411:200
      IL-1RPEBiolegend113505JAMA-1471:50
      Ki67PerCP-eFluor710eBioscience46-5698-80SolA151:400
      Ly6CBV605BD Biosciences6077610AL211:600
      Mac-1PE-Cy7BioLegend101215M1/701:800
      Sca-1Pacific BlueBioLegend108120D71:400
      Sca-1PE-Cy7BioLegend108113D71:400
      StreptavidinBV605BioLegend405229NA1:100
      Ter119PE-Cy5BioLegend116201ter1191:400

      Gene expression analysis

      For gene expression analyses, pools of 100 cells were sorted into wells of a DNase- and RNase-free 96-well plate (Applied Biosystems) containing 5 μL CellsDirect 2 × reaction buffer (Invitrogen), centrifuged for 5 min at 500g, snap-frozen on dry ice, and stored at –80°C until use. RNA was reverse-transcribed using Superscript III Taq polymerase (Invitrogen) and pre-amplified for 18 rounds with a custom 96-target DeltaGene (Fluidigm) primer panel on a PCR cycler (Eppendorf). Excess primers were removed from the pre-amplified product by incubation with Exonuclease-1 (New England Biolabs), and cDNA samples were diluted in DNA buffer. Primers and cDNAs mixed with SsoFast Sybr Green Master Mix (BioRad) were subsequently loaded onto a Fluidigm 96.96 Dynamic Array IFC and run on a BioMark HD system (Fluidigm). Data were subsequently analyzed using Fluidigm Gene Expression Software and normalized to Gusb. Relative changes were subsequently calculated using the ΔΔCt approach. Unsupervised clustering of Gusb-normalized ΔΔCt values, with Gusb removed along with poorly performing Ebf1 and Hoxa2 primer sets, was performed using average linkage. Clustering and principal component analysis (PCA) and heatmap generation were performed using ClustVis software (biit.cs.ut.ee/clustvis). PCA and PCA loading plots were generated using Prism 8 (Graphpad, San Diego, CA) from data generated by ClustVis.

      Statistical analysis

      Statistical analyses were performed using Prism 8 software (GraphPad). p Values were determined using either a Mann–Whitney U test for bivariate comparisons or two-way analysis of variance (ANOVA) for multivariate comparisons. p Values ≤ 0.05 were considered to indicate statistical significance.

      Results

      EPCR and CD34 coordinately enrich for functional HSCs during chronic IL-1 exposure

      To address the impact of chronic IL-1-driven inflammatory signaling on functional heterogeneity in the HSC-enriched SLAM (LSK/Flk2/CD48/CD150+/ESAM+) HSC faction, we injected mice intraperitoneally each day for 20 days with or without 0.5 μg of recombinant murine IL-1β. Consistent with our prior published results, chronic IL-1 treatment induced expansion of myeloid cells coincident with contraction of B-cell populations in the BM (Supplementary Figure E1A–C, online only, available at www.exphem.org), as well as expansion of SLAM HSC and multipotent progenitor (MPP)-2 and MPP3 populations [
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ,
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ] (Supplementary Figure E1D,E, online only, available at www.exphem.org). To study IL-1-induced changes in the SLAM compartment, we re-analyzed cells in this compartment using EPCR and CD34, which have previously been used to distinguish functional HSCs in the SLAM fraction [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ,
      • Kent DG
      • Copley MR
      • Benz C
      • et al.
      Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential.
      ]. Coordinate use of EPCR and CD34 allowed us to prospectively identify four distinct phenotypic compartments within the SLAM gate (Figure 1A). Strikingly, the relative abundance of these compartments was altered significantly following chronic IL-1 exposure, with a significant decrease in the frequency of EPCR+/CD34 cells, alongside twofold expansion in the frequency of the EPCR/CD34 compartment (Figure 1B). Despite decreased frequency, the absolute number of EPCR+/CD34 cells in the BM was unchanged (Figure 1C), whereas increased absolute numbers of the EPCR/CD34 fraction substantially contributed to the overall numerical expansion of SLAM cells observed after chronic IL-1 exposure (Figure 1C; Supplementary Figure E1D, online only, available at www.exphem.org).
      Supplementary Figure E1
      Supplementary Figure E1Chronic IL-1 drives myeloid expansion. Mice were treated ± IL-1β for 20 days. Absolute number of (A) BM myeloid cells and (B) BM B cells, and (D) immature progenitor cells including SLAM HSC. Representative FACS plots are shown in (C, E). Gr: granulocytes; Pre Gr; pre granulocytes (N = 7-8/grp). * p < 0.05; *** p <0.001 by Mann-Whitney u-test.
      Figure 1
      Figure 1The EPCR+/CD34 SLAM fraction enriches for functional HSCs. (A) Experimental design and representative fluorescence-activated cell sorting (FACS) plots of SLAM cells from mice treated with or without IL-1 for 20 days fractionated by EPCR and CD34 expression. (B,C) Frequency (B) and absolute number (C) of EPCR/CD34 SLAM cell fractions (n = 9 or 10 per group). (D–F) Long-term engraftment of purified SLAM cells fractionated by EPCR and CD34 expression, from mice treated with or without IL-1 for 20 days: (D) experimental design; (E) donor chimerism in peripheral blood (PB); (F) phenotypic SLAM compartment of recipient mice at the indicated time points (N = 18–20/group, compiled from two independent experiments). (G) Donor PB and (H) SLAM chimerism after secondary transplant of donor-derived SLAM cells from primary recipient mice in (D) (n = 18–20/group, compiled from two independent experiments). (I–K) Long-term engraftment of SLAM and EPCR+/CD34 SLAM cells from mice treated with or without IL-1 for 20 days: (I) experimental design; (J) donor chimerism in peripheral blood (PB); (K) phenotypic SLAM compartment of recipient mice at the indicated time points (N = 9 or 10 per group, representative of two independent experiments). °°p < 0.01 versus –IL-1 EPCR+/CD34 SLAM cells by one-way analysis of variance. *p < 0.05, **p < 0.01, ***p < 0.001 versus –IL-1 condition in each fraction by Mann–Whitney U test.
      To interrogate the functional properties of SLAM fractions defined by CD34 and EPCR expression in homeostatic and chronic inflammatory conditions, we first isolated each fraction from mice treated with or without IL-1 for 20 days, transplanted them into lethally irradiated CD45.1+ recipient mice, and analyzed long-term repopulating activity by bleeding recipient animals every 4 weeks (Figure 1D; Supplementary Figure E2A, online only, available at www.exphem.org). Notably, the EPCR+/CD34 fraction contained the majority of long-term repopulating activity, exhibiting high levels of multilineage donor chimerism in the peripheral blood (PB) out to 20 weeks (Figure 1E; Supplementary Figure E2B, online only, available at www.exphem.org). On the other hand, EPCR+/CD34+ cells and both EPCR fractions generated consistently low to absent donor chimerism by 20 weeks (Figure 1E). Along these lines, we recovered donor-derived SLAM cells exclusively in mice transplanted with EPCR+/CD34 cells (Figure 1F; Supplementary Figure E2C, online only, available at www.exphem.org). Notably, chronic IL-1 exposure moderately decreased donor-derived peripheral blood chimerism in mice transplanted with EPCR+/CD34 cells (Figure 1E). However, donor-derived SLAM cell chimerism in the BM was unchanged, suggesting that chronic IL-1 exposure has a limited effect on long-term repopulating activity of cells in the EPCR+/CD34 fraction (Figure 1F). In addition, chronic IL-1 exposure did not impair the capacity of transplanted EPCR+/CD34 cells to give rise to all SLAM fractions including EPCR+/CD34 cells after 20 weeks (Supplementary Figure E2C, online only, available at www.exphem.org). To address the serial repopulating activity of the EPCR+/CD34 fraction, we re-isolated donor-derived EPCR+/CD34 cells from the primary recipient mice and transplanted them into lethally irradiated CD45.1+ secondary recipients (Figure 1G). In line with our primary transplant data, we observed roughly equal myeloid/lymphoid donor PB chimerism, as well as SLAM HSC chimerism, regardless of chronic IL-1 exposure (Figure 1H,I; Supplementary Figure E2D, online only, available at www.exphem.org). Collectively, these experiments establish the EPCR+/CD34 fraction as the major source of long-term HSC activity in the SLAM compartment, consistent with prior studies using this definition [
      • Lazare S
      • Ausema A
      • Reijne AC
      • van Dijk G
      • van Os R
      • de Haan G
      Lifelong dietary intervention does not affect hematopoietic stem cell function.
      ].
      Supplementary Figure E2
      Supplementary Figure E2Lineage distribution of transplanted SLAM fractions. (A) Sorting strategy for prospective isolation of EPCR and CD34-defined SLAM fractions. Recorded sort gates are shown at the far right (sorted cells). Num. = number. (B) Myeloid and lymphoid lineage distribution from mice transplanted with SLAM cell fractions in E. (C) FACS plots showing donor chimerism and frequency of EPCR+/CD34 cells in donor-derived SLAM compartment from mice transplanted with EPCR+/CD34 in E. (D) Myeloid and lymphoid lineage distribution from secondary transplant of EPCR+/CD34 SLAM cells in G.
      Given the relatively minor impact of chronic IL-1 exposure on EPCR+/CD34 cell repopulating activity, we next assessed whether the EPCR+/CD34 marker definition provides superior enrichment for long-term HSC activity relative to the total SLAM fraction following IL-1 exposure (Figure 1I). Notably, SLAM cells from IL-1-exposed mice exhibited significantly reduced long-term donor-derived PB and SLAM chimerism relative to controls, as well as relative to EPCR+/CD34 cells in either condition (Figure 1J,K), suggesting the EPCR+/CD34 SLAM fraction provides superior enrichment for long-term HSCs versus unfractionated SLAM cells. We also analyzed expression of CD49b in each SLAM fraction, as low CD49b expression identifies long-term repopulating cells (LTRCs) with chemoresistant “reserve HSC” (R-HSC) properties [
      • Kiel MJ
      • Radice GL
      • Morrison SJ
      Lack of evidence that hematopoietic stem cells depend on N-cadherin-mediated adhesion to osteoblasts for their maintenance.
      ,
      • Benveniste P
      • Frelin C
      • Janmohamed S
      • et al.
      Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential.
      ]. We found the EPCR+/CD34 fraction was most highly enriched for CD49blo LTRCs (Supplementary Figure E3A,B, online only, available at www.exphem.org). The non-long-term repopulating EPCR/CD34 fraction also exhibited extensive enrichment for CD49blo cells, suggesting that CD49b alone may not completely exclude non-long-term repopulating cells in the SLAM gate. The EPCR+/CD34 fraction also contained CD49bhi cells, indicating this fraction likely contains a mixture of R-HSCs and long-term repopulating but chemosensitive “primed HSCs” (P-HSCs) that can be selectively depleted by 5-FU stress [
      • Zhao M
      • Tao F
      • Venkatraman A
      • et al.
      N-Cadherin-expressing bone and marrow stromal progenitor cells maintain reserve hematopoietic stem cells.
      ].
      Supplementary Figure E3
      Supplementary Figure E3Surface marker gene expression in SLAM fractions. (A) Representative FACS plots and (B) mean fluorescence intensity (MFI) of CD49b in SLAM fractions from mice treated ± IL-1β for 20 days (n = 5/grp). Expression of other HSC-associated surface markers by (C) surface staining (n = 5/grp) or (D) Fluidigm qRT-PCR analysis of surface marker genes in SLAM fractions from mice treated ± IL-1β for 20 days (n = 7-8/grp). Data in (D) are expressed as log10 fold expression vs. -IL-1 EPCR+/CD34 SLAM cells. ° p < 0.05, °° p < 0.01, °°° p < 0.001 vs. -IL-1 EPCR+/CD34 SLAM cells; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. -IL-1 condition within each fraction, as determined by two-way ANOVA.
      Taken together, these findings indicate that EPCR and CD34 coordinately identify a SLAM fraction that effectively enriches for functional long-term HSC regardless of chronic exposure to IL-1. They also suggest that the impaired long-term repopulating activity of IL-1-exposed SLAM cells we previously reported [
      • Pietras EM
      • Mirantes-Barbeito C
      • Fong S
      • et al.
      Chronic interleukin-1 exposure drives haematopoietic stem cells toward precocious myeloid differentiation at the expense of self-renewal.
      ] may be at least partially caused by a decreased proportion of EPCR+/CD34 cells occupying the phenotypic SLAM gate following IL-1 treatment, rather than functional impairment of long-term repopulating HSC themselves (Figure 1A).

      EPCR and CD34 coordinately identify distinct molecular phenotypes in the SLAM gate

      To understand the molecular basis for our transplantation assays, we used a custom 96-gene Fluidigm quantitative reverse transcription polymerase chain reaction (qRT-PCR) array to evaluate the molecular characteristics of each SLAM fraction in mice treated with or without IL-1 for 20 days (Figure 2A). Notably, hierarchical clustering analysis revealed that EPCR expression constitutes the major breakpoint between the four populations, with EPCR+ fractions exhibiting the highest degree of relatedness regardless of IL-1 exposure (Figure 2B). Along these lines, principal component analysis (PCA) revealed two distinctive molecular phenotypes, with the first principal component (PC1) establishing a linear relationship between SLAM fractions that potentially resembles a differentiation gradient, with HSC activity-enriched EPCR+/CD34 cells and EPCR/CD34+ cells, which do not exhibit long-term donor engraftment, at the extrema. On the other hand, the second principal component (PC2) separated populations based on IL-1 exposure (Figure 2C). Interestingly, IL-1 treatment did not substantially alter the order of the fractions in PC space, suggesting that IL-1 does not fundamentally alter the molecular identity of each fraction, but instead activates a discrete gene program(s) within each. Consistent with this model, PC loading analysis identified unique gene sets that define each principal component (Figure 2C). Along these lines we analyzed expression of genes associated with functional HSCs in each SLAM fraction with or without chronic IL-1 exposure. As predicted by our transplantation assays, EPCR+/CD34 cells expressed the highest levels of HSC genes, including Hoxb5, Fgd5, Rarb and Procr, alongside high surface expression of Sca-1 and EPCR (Figure 2D; Supplementary Figure E3C, online only, available at www.exphem.org). Consistently, chronic IL-1 exposure significantly reduced expression of several of these genes in the EPCR/CD34+ fraction (Figure 2D; Supplementary Figure E3C). On the other hand, expression of HSC genes was lowest in the EPCR/CD34+ fraction, with EPCR/CD34+ cells also expressing the highest level of CD48, which serves as a marker of HSC differentiation (Figure 2D; Supplementary Figure E3C,D). Notably, EPCR+/CD34+ cells expressed the highest levels of Flk2, which could be consistent with a proliferative, metabolically active MPP1 phenotype [
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ]. Moreover, increased CD150 expression, which is associated with hematopoietic stress responses [
      • Pietras EM
      • Mirantes-Barbeito C
      • Fong S
      • et al.
      Chronic interleukin-1 exposure drives haematopoietic stem cells toward precocious myeloid differentiation at the expense of self-renewal.
      ,
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ], occurred exclusively in EPCRcell fractions following chronic IL-1 exposure (Supplementary Figure E3C,D). Furthermore, we assayed IL-1R expression at the mRNA and protein levels and found detectable levels in each compartment (Supplementary Figure E4A–C, online only, available at www.exphem.org), with IL-1R surface expression at low levels in at least a fraction of cells, suggesting cells in each compartment can respond to IL-1. Although IL-1 treatment led to increased levels of Il1r1 mRNA in all SLAM fractions, this did not translate to increased surface expression (Supplementary Figure E4A–C). Lastly, given that transplantation of the EPCR+/CD34 SLAM fraction yielded superior enrichment for HSC activity relative to unfractionated SLAM cells after IL-1 treatment, we compared expression of HSC-associated genes between the EPCR+/CD34 fraction and unfractionated SLAM cells. As predicted by our functional analyses, EPCR+/CD34 cells expressed significantly higher levels of Hoxb5, Fgd5, Mecom, Procr, Rarb, and Egr1 than unfractionated SLAM cells (Figure 2E), regardless of IL-1 exposure. Hence, these data indicate that EPCR and CD34 identify molecularly distinct compartments within the SLAM gate, complementing our functional studies. They also provide a molecular basis for the superior repopulating activity in EPCR+/CD34 cells versus unfractionated SLAM cells after IL-1 exposure.
      Figure 2
      Figure 2EPCR and CD34 identify molecularly distinct SLAM compartments. (A) Experimental design of Fluidigm-based molecular analysis of SLAM cells fractionated by EPCR and CD34 expression from mice treated with and without IL-1 for 20 days. (B,C) Heatmap and hierarchical clustering analysis (B) and principal component analysis (C) (PCA: left, PCA loading plot: right) of Fluidigm gene expression data (N = 7 or 8/group). (D) Expression of HSC-associated genes in SLAM cells fractionated by EPCR and CD34 expression, from mice treated with and without IL-1 for 20 days. (E) Expression of HSC-associated genes in total SLAM cells and EPCR+/CD34 SLAM cells from mice treated with and without IL-1 for 20 days. Data in (D) and (E) are expressed as log fold expression versus –IL-1 EPCR+/CD34 SLAM cells. °p < 0.05, °°p < 0.01, °°°p < 0.001 versus –IL-1 EPCR+/CD34 SLAM cells. *p < 0.05, **p < 0.01, ***p < 0.001 versus –IL-1 condition within each fraction, as determined by two-way analysis of variance. Data are representative of two independent experiments.
      Supplementary Figure E4
      Supplementary Figure E4IL-1R expression in SLAM fractions. (A) Fluidigm qRT-PCR analysis of Il1r1 expression in SLAM fractions from mice treated ± IL-1β for 20 days (n = 7-8/grp). Data are expressed as log10 fold expression vs. -IL-1 EPCR+/CD34 SLAM cells. (B) Representative FACS plots and (C) quantification of IL-1R surface expression in SLAM fractions (n = 7/grp). Grey histogram peaks represent fluorescence minus one (FMO) controls. ° p < 0.05, °° p < 0.01, °°° p < 0.001 vs. -IL-1 EPCR+/CD34 SLAM cells; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. -IL-1 condition within each fraction, as determined by two-way ANOVA.

      The EPCR+/CD34 SLAM fraction remains quiescent after chronic IL-1 exposure

      SLAM cells remain largely quiescent in chronic inflammatory conditions such as interferon (IFN) stimulation, obesity, and collagen-induced arthritis (CIA) [
      • Pietras EM
      • Lakshminarasimhan R
      • Techner JM
      • et al.
      Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons.
      ,
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ,
      • Hernandez G
      • Mills TS
      • Rabe JL
      • et al.
      Pro-inflammatory cytokine blockade attenuates myeloid expansion in a murine model of rheumatoid arthritis.
      ,
      • Lee JM
      • Govindarajah V
      • Goddard B
      • et al.
      Obesity alters the long-term fitness of the hematopoietic stem cell compartment through modulation of Gfi1 expression.
      ]. To better understand the biology of the different SLAM fractions after chronic IL-1 exposure, we analyzed their cell cycle distribution by Ki67/DAPI staining in mice treated with or without IL-1 for 20 days. Interestingly, we uncovered a gradient of cell cycle activity in each compartment, with EPCR+/CD34 cells almost entirely in G0, whereas roughly half of EPCR/CD34+ cells were actively cycling and spread between G1 and S/G2/M phases (Figure 3A,B). Consistent with the MPP1 phenotype, CD34 expression marked elevated cell cycle activity regardless of EPCR expression. Strikingly, cell cycle distribution of all SLAM fractions was unchanged after chronic IL-1 exposure, suggesting that chronic IL-1 does not substantially modulate this key cellular characteristic. We confirmed these findings independently using EdU incorporation to measure the proliferative activity in each SLAM fraction (Supplementary Figure E5A, online only, available at www.exphem.org). Consistent with our Ki67/DAPI analyses, we found that EPCR SLAM fractions had a higher proliferative index than EPCR+ cells. Also similar to our Ki67/DAPI analyses, IL-1 exposure did not significantly alter proliferative activity in any fraction (Supplementary Figure E5B,C, online only, available at www.exphem.org). Likewise, our molecular analyses revealed that induction of CD34 expression in EPCR+ SLAM cells, followed by loss of EPCR expression, was associated with increased priming for cell cycle activity, with progressively increasing levels of Ccna2, Ccnb1, Ccne1, and Cdk1 between EPCR+/CD34 and EPCR/CD34+ fractions (Figure 3C). On the other hand, consistent with an HSC identity, EPCR+/CD34 cells had the highest expression of cell cycle inhibitor genes including Foxo3, Cdkn1c/p57, and Rb1 (Figure 3D). Cdkn2c/p18 levels were highest in EPCR/CD34+ fractions, suggesting distinct mechanisms may regulate cell cycle activity in these SLAM fractions. Consistent with minimal induction of cell cycle activity by chronic IL-1, expression levels of cell cycle activator genes did not undergo substantial shifts after IL-1 exposure (Figure 3D). Interestingly, Rb1 and Cdkn2c/p18 levels increased significantly with IL-1 exposure in both EPCR fractions, perhaps indicating activation of an Rb-mediated “braking” mechanism unique to these cells (Figure 3D). On the other hand, although Foxo3 and Cdkn1c/p57 levels decreased in EPCR+/CD34 cells after IL-1 exposure, our analyses identified significant IL-1 treatment-associated decreases in expression of Myc, Mycn, and Ccnd1, which are crucial mediators of HSC cell cycle entry, as well as increased levels of the Cdkn1b/p27 (Figure 3E). Notably, this expression pattern is similar to the cell cycle restriction gene program we recently identified in SLAM cells from mice with collagen-induced arthritis [
      • Hernandez G
      • Mills TS
      • Rabe JL
      • et al.
      Pro-inflammatory cytokine blockade attenuates myeloid expansion in a murine model of rheumatoid arthritis.
      ], suggesting modulation of these genes is a conserved response to multiple inflammatory stimuli.
      Figure 3
      Figure 3EPCR+/CD34 SLAM cells are highly quiescent. (A,B) Representative FACS plots (A) and quantification of cell cycle distribution (B) in SLAM cells fractionated by EPCR and CD34 expression, from mice treated with and without IL-1 for 20 days (n = 5/group). (C–E) Expression of cell cycle activator and inhibitor genes in SLAM cells fractionated by EPCR and CD34 expression from mice treated with and without IL-1 for 20 days (n = 7 or 8/group). Data in (C)–(E) are expressed as log fold expression versus –IL-1 EPCR+/CD34 SLAM cells. °p < 0.05, °°p < 0.01, °°°p < 0.001 versus –IL-1 EPCR+/CD34 SLAM cells. *p < 0.05, **p < 0.01, ***p < 0.001 versus –IL-1 condition within each fraction, as determined by two-way analysis of variance. Data are representative of two independent experiments.
      Supplementary Figure E5
      Supplementary Figure E5EdU incorporation in SLAM fractions. (A) Experimental design; (B) Representative FACS histograms and (C) quantification of EdU incorporation in each SLAM fraction 24h after injection of EdU into mice treated ± IL-1β for 20 days. n = 3-4/grp. °°° p < 0.001 vs. -IL-1 EPCR+/CD34 SLAM cells, as determined by two-way ANOVA.
      We also analyzed our gene expression data to uncover potential regulatory mechanisms promoting quiescence and long-term repopulation capacity in EPCR+/CD34 cells during chronic IL-1. We found that EPCR+/CD34 SLAM cells maintained high relative levels of Nrf2- and Hif1α-regulated genes versus the remaining SLAM fractions. Both pathways are known to be highly active in HSCs, and deletion of key components such as Nrf2 and Hif1a leads to excessive HSC proliferation and/or mobilization and loss of self-renewal [
      • Tsai JJ
      • Dudakov JA
      • Takahashi K
      • et al.
      Nrf2 regulates haematopoietic stem cell function.
      ,
      • Singh RP
      • Franke K
      • Kalucka J
      • et al.
      HIF prolyl hydroxylase 2 (PHD2) is a critical regulator of hematopoietic stem cell maintenance during steady-state and stress.
      ,
      • Ramasz B
      • Krüger A
      • Reinhardt J
      • et al.
      Hematopoietic stem cell response to acute thrombocytopenia requires signaling through distinct receptor tyrosine kinases.
      ]. We also observed high levels of target genes that regulate redox state (Gstt3) and promote quiescence-associated metabolic pathways including fatty acid metabolism (Cpt1a) and glycolysis (Gapdh) [
      • Ito K
      • Carracedo A
      • Weiss D
      • et al.
      A PML–PPAR-delta pathway for fatty acid oxidation regulates hematopoietic stem cell maintenance.
      ,
      • Takubo K
      • Nagamatsu G
      • Kobayashi CI
      • et al.
      Regulation of glycolysis by Pdk functions as a metabolic checkpoint for cell cycle quiescence in hematopoietic stem cells.
      ] (Supplementary Figure E6A,B, online only, available at www.exphem.org). We also observed robust induction of the Hif1α target genes Vldlr and Pdgfrb by IL-1 in multiple SLAM fractions, which could reflect metabolic adaptation to increased hypoxia in the BM or changes in energy source abundance. Hence, EPCR+/CD34 cells retain key metabolic properties associated with their quiescence and self-renewal under chronic IL-1 conditions. Together, these data indicate that the SLAM compartment is composed of cell populations with heterogenous cell cycle states whose distinct proliferative and metabolic activities are maintained during chronic inflammation. They also identify IL-1-driven modulations in key cell cycle and metabolic regulators that may account for minimal cell cycle changes.
      Supplementary Figure E6
      Supplementary Figure E6Nrf2 and Hif pathway gene expression is retained in EPCR+ SLAM fractions. Fluidigm qRT-PCR analysis of (A) Nrf2 and (B) Hif pathway genes in SLAM fractions from mice treated ± IL-1β for 20 days (n = 7-8/grp). Data are expressed as log10 fold expression vs. -IL-1 EPCR+/CD34 SLAM cells. ° p < 0.05, °° p < 0.01, °°° p < 0.001 vs. -IL-1 EPCR+/CD34 SLAM cells; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. -IL-1 condition within each fraction, as determined by two-way ANOVA.

      EPCR expression resolves lineage priming in SLAM compartments

      In addition to changes in cell cycle gene expression, we investigated expression of key lineage-determinant genes in SLAM fractions. Our PC loading analyses identified the megakaryocyte/erythroid (Mk/E)-lineage genes Gata1, Vwf, Itga2b (Cd41) and EpoR as key markers distinguishing EPCR+ and EPCR SLAM fractions (Figure 2C). Along these lines, we observed significant upregulation of these four genes in both EPCR cell fractions, suggesting they may represent Mk/E-biased populations (Figure 4A). Expression of these genes also increased significantly in EPCR fractions after chronic IL-1 exposure. On the other hand, both EPCR+ fractions were enriched for expression of the myeloid lineage determinants Spi1/Pu.1 and Cebpa, as well as the lymphoid gene Ikzf1, consistent with a more extensive myeloid/lymphoid repopulating activity in these fractions relative to EPCR cells (Figure 1E). Notably, the chronic inflammation-mediated increase in Spi1 expression we had previously documented in unfractionated SLAM cells [
      • Pietras EM
      • Mirantes-Barbeito C
      • Fong S
      • et al.
      Chronic interleukin-1 exposure drives haematopoietic stem cells toward precocious myeloid differentiation at the expense of self-renewal.
      ,
      • Hernandez G
      • Mills TS
      • Rabe JL
      • et al.
      Pro-inflammatory cytokine blockade attenuates myeloid expansion in a murine model of rheumatoid arthritis.
      ] was particularly robust in the EPCR+/CD34 SLAM fraction (Figure 4B). Interestingly, expression of Itgam (Mac-1), normally considered a myeloid lineage marker, increased significantly in all SLAM fractions after chronic IL-1 exposure, though expression levels were nearly 10-fold higher in the EPCR+ fractions (Figure 4B). Given our molecular findings, we assessed CD41 and Mac-1 levels on the surface of each SLAM fraction. In line with previous work identifying an inflammation-induced Mk-biased CD41+ SLAM compartment [
      • Haas S
      • Hansson J
      • Klimmeck D
      • et al.
      Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors.
      ,
      • Pietras EM
      • Mirantes-Barbeito C
      • Fong S
      • et al.
      Chronic interleukin-1 exposure drives haematopoietic stem cells toward precocious myeloid differentiation at the expense of self-renewal.
      ], we found that CD41 expression was induced by IL-1 exclusively in EPCR SLAM fractions (Figure 4C,D). Strikingly, on the other hand, Mac-1 surface expression was induced exclusively on EPCR+ SLAM fractions after IL-1 exposure (Figure 4C,D). Collectively, these data suggest that EPCR and EPCR+ SLAM fractions have distinct lineage priming, with EPCR fractions exhibiting a strong Mk/E signature. On the other hand, EPCR+ fractions express myeloid and lymphoid gene markers consistent with robust myeloid/lymphoid repopulating activity, particularly in the EPCR+/CD34 fraction. Our data also indicate that Mac-1, which is often excluded as a mature myeloid lineage marker in HSC staining panels, can be expressed at low levels in SLAM fractions enriched for long-term HSC activity, after chronic inflammatory stimulation.
      Figure 4
      Figure 4EPCR expression distinguishes Mk/E-primed SLAM cells. (A,B) Expression of (A) Mk/E and (B) myeloid/lymphoid lineage genes in SLAM cells fractionated by EPCR and CD34 expression, from mice treated with and without IL-1 for 20 days (n = 7 or 8/group). Data in (A) and (B) are expressed as log fold expression versus –IL-1 EPCR+/CD34 SLAM cells. (C,D) Representative fluorescence-activated cell sorting plots (C) and geometric mean fluorescence intensity (MFI) quantification (D) of CD41 and Mac-1 expression in cells fractionated by EPCR and CD34 expression, from mice treated with and without IL-1 for 20 days (n = 10 or 11/group). °p < 0.05, °°p < 0.01, °°°p < 0.001 versus –IL-1 EPCR+/CD34 SLAM cells; *p < 0.05, **p < 0.01, ***p < 0.001 versus –IL-1 condition within each fraction, as determined by two-way analysis of variance. Data are representative of two independent experiments.

      Fgd5 enriches for functional HSCs in homeostatic and inflammatory conditions

      We next assessed whether two reporter strains found to identify HSCs, Vwf-eGFP [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ] and Fgd5-ZSGreen [
      • Gazit R
      • Mandal PK
      • Ebina W
      • et al.
      Fgd5 identifies hematopoietic stem cells in the murine bone marrow.
      ], faithfully enrich for HSC activity under chronic inflammatory stress. We therefore transplanted reporter-positive SLAM cells from mice treated with and without IL-1β for 20 days into lethally irradiated CD45.1+ recipient mice (Figure 5A,F). Interestingly, although the proportions of Vwf-eGFP+ SLAM cells before and after chronic IL-1 exposure were roughly equal, the proportion of EPCR+/CD34 cells in the Vwf-eGFP+ SLAM gate decreased after chronic IL-1 exposure (Figure 5B). Consistently, donor chimerism as measured by CD45.2 expression in the peripheral blood and BM SLAM compartment was significantly lower in mice transplanted with Vwf-GFP+ SLAM cells after chronic IL-1 exposure (Figure 5C,D). Consistent with a decreased abundance of EPCR+/CD34 cells in the Vwf-eGFP+ SLAM gate, levels of HSC genes, with the exception of Ctnnal1, were significantly lower in Vwf-eGFP+ SLAM cells from chronic IL-1-treated mice (Figure 5E). Likewise, this phenotypic change was reflected in the decreased proportion of Vwf-eGFP+ cells in the EPCR+/CD34 SLAM fraction itself, whereas the abundance of Vwf-eGFP+ cells increased in EPCR SLAM fractions (Supplementary Figure E7A, online only, available at www.exphem.org). This suggests that the Vwf-eGFP+ SLAM gate becomes contaminated with non-long-term engrafting cells after chronic exposure to IL-1.
      Figure 5
      Figure 5Fgd5 enriches for functional EPCR+/CD34 HSCs under inflammatory conditions. (A) Experimental design. (B) Representative FACS plots revealing frequency of Vwf-GFP+ SLAM cells and EPCR+/CD34 cells within the Vwf-GFP+ gate. (C) Donor-derived PB and (D) BM SLAM cell chimerism after transplantation into recipient mice (n = 8/group). (E) Expression of HSC-associated genes in Vwf-GFP+ SLAM cells from mice treated with and without IL-1 for 20 days. (F) Experimental design. (G) Representative fluorescence-activated cell sorting plots revealing frequency of ZSGreen+ SLAM cells and EPCR+/CD34 cells within the Fgd5-ZSGreen+ gate. (H,I) Donor-derived PB (H) and BM SLAM cell (I) chimerism after transplantation into recipient mice (n = 9/group). (J) Expression of HSC-associated genes in Fgd5-ZSGreen+ SLAM cells from mice treated with and without IL-1 for 20 days. Data are representative of two independent experiments. Data in (E) and (J) are expressed as log fold expression versus –IL-1 condition. *p < 0.05, **p < 0.01, ***p < 0.001 versus –IL-1 condition, as determined by Mann–Whitney U test.
      Supplementary Figure E7
      Supplementary Figure E7GFP+ and ZSGreen+ frequency in SLAM fractions in mice treated ± IL-1β. Representative histograms showing the frequency of (A) GFP+ cells in SLAM fractions of Vwf-GFP mice and (B) ZSGreen+ cells in Fgd5-ZSGreen mice treated ± IL-1β for 20 days.
      On the other hand, while the Fgd5-ZSGreen reporter marked a smaller proportion of SLAM cells in chronic IL-1-exposed Fgd5-ZSGreen mice, the Fgd5-ZSGreen+ gate contained roughly equal proportions of EPCR+/CD34 cells regardless of IL-1 exposure (Figure 5G). Consistent with this observation, donor chimerism was essentially equivalent in the PB and BM SLAM compartments (Figure 5H,I). Likewise, expression of HSC genes was equal or even higher in Fgd5-ZSGreen+ SLAM cells from IL-1-treated mice (Figure 5J). On the other hand, while the abundance of EPCR+/CD34 cells in the Fgd5+ SLAM compartment was essentially unchanged, the fraction of Fgd5+ cells in the EPCR+/CD34 SLAM fraction decreased (Supplementary Figure E7B, online only, available at www.exphem.org). This is consistent with decreased Fgd5 mRNA levels (Figure 2D) in the EPCR+/CD34 SLAM fraction after IL-1 treatment and the moderate decrease in their engraftment after IL-1 exposure (Figure 1E). These data suggest that Fgd5 expression can identify a subset of EPCR+/CD34 SLAM cells with essentially unimpaired functional properties during inflammatory challenge. Together, these data indicate that the Fgd5-ZSGreen reporter faithfully marks phenotypic and functional HSCs after chronic IL-1 exposure. These findings are consistent with a report that Fgd5 reporter expression enriches for functional HSCs after acute poly I:C stimulation [
      • Bujanover N
      • Goldstein O
      • Greenshpan Y
      • et al.
      Identification of immune-activated hematopoietic stem cells.
      ]. These data also suggest that the EPCR+/CD34 surface marker combination may have predictive value in assessing the degree to which HSC-specific reporter strains faithfully mark functional HSCs in inflammatory conditions.

      Discussion

      In the present study, we found that the surface markers CD34 and EPCR can be used to prospectively identify a continuum of molecular and functional cell states within the phenotypic SLAM compartment, which include induction of cell cycle activity and distinct lineage programs. Using transplantation, we found that these cell states are associated with varying degrees of serial myeloid/lymphoid repopulating activity, with EPCR+/CD34 SLAM cells enriching for a highly potent HSC activity under normal and inflammatory conditions. We showed that EPCR expression distinguishes CD41+ Mk/E-primed SLAM cells from long-term repopulating HSCs, which express Mac-1 after IL-1 treatment. Lastly, we found that the Fgd5-ZSGreen reporter strain can be used to prospectively enrich for EPCR+/CD34 SLAM cells with equivalent long-term repopulating activity after IL-1 exposure. Together, these data indicate that the phenotypic SLAM compartment is dynamically regulated under nonhomeostatic conditions, while still maintaining a viable long-term repopulating HSC compartment. They also suggest that careful validation of prospective isolation strategies is required to interpret models of stress hematopoiesis and that different HSC definitions (e.g., unfractionated SLAM versus Fgd5+) can impact outcomes of studies addressing the functional status of HSCs after inflammatory stress.
      Numerous markers have been found to enrich for functional long-term HSC activity. The extent to which they faithfully enrich for long-term HSC activity under nonhomeostatic conditions has not been extensively studied. Here, we found that EPCR and CD34 mark a continuum of cell states, with EPCR+/CD34 representing the most immature, quiescent cellular fraction. Interestingly, our data indicate that acquisition of CD34 by EPCR+ cells is associated with induction of cell cycle genes and loss of repopulating activity, consistent with prior work indicating that CD34+ SLAM cells, also termed MPP1, represent a metabolically activated SLAM fraction with poor reconstitution activity [
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ]. Likewise, other studies have demonstrated equivalent levels of HSC reconstitution activity shortly after 5-FU treatment or IR, based on marker systems that exclude c-Kit and Sca-1 while incorporating EPCR and other markers such as CD48/CD150 and CD27 [
      • Vazquez SE
      • Inlay MA
      • Serwold T
      CD201 and CD27 identify hematopoietic stem and progenitor cells across multiple murine strains independently of Kit and Sca-1.
      ,
      • Umemoto T
      • Hashimoto M
      • Matsumura T
      • Nakamura-Ishizu A
      • Suda T
      Ca2+-mitochondria axis drives cell division in hematopoietic stem cells.
      ]. Along these lines, we found that the EPCR+/CD34 fraction is highly enriched for CD49blo cells, which were recently defined as chemoresistant R-HSCs [
      • Zhao M
      • Tao F
      • Venkatraman A
      • et al.
      N-Cadherin-expressing bone and marrow stromal progenitor cells maintain reserve hematopoietic stem cells.
      ]. Hence, it is likely there is a high degree of phenotypic and functional overlap between these two definitions, with the intersection between the two markers likely representing a R-HSC fraction. Along these lines, our data suggest that a viable, functional HSC pool enriched by EPCR+/CD34 is maintained in the BM despite ongoing hematopoietic stress. Combinatorial use of markers such as CD49b and EPCR may thus provide highly stringent enrichment for functional HSC under stress conditions and should be investigated further. As EPCR is also expressed on human HSCs [
      • Fares I
      • Chagraoui J
      • Lehnertz B
      • et al.
      EPCR expression marks UM171-expanded CD34(+) cord blood stem cells.
      ], similar systems may be applicable for enriching human HSCs in disease settings. Hence, future studies can determine the extent to which the EPCR+/CD34 definition identifies functional HSCs under multiple stress conditions. Moreover, studies to assess HSC responses to stress stimuli at the single-cell level may provide insight into how the HSC pool is maintained under stress conditions and which gene(s) most faithfully report functional long-term repopulating HSC in such contexts.
      EPCR is an anti-coagulant, anti-inflammatory endothelial surface protein that has also been characterized based on its capacity to identify long-term HSCs with quiescent cell cycle activity and chemoresistant properties [
      • Balazs AB
      • Fabian AJ
      • Esmon CT
      • Mulligan RC
      Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow.
      ,
      • Gur-Cohen S
      • Itkin T
      • Chakrabarty S
      • et al.
      PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells.
      ]. Notably, maintenance of EPCR expression and retention in the BM niche requires a close partnership between HSCs and endothelial cells. Indeed, endothelial cells facilitate activated protein C (aPC) processing and binding to EPCR, which induces cleavage of protease-activated receptor 1 (PAR1) to promote HSC retention in the BM, via a mechanism of nitric oxide (NO) restriction [
      • Gur-Cohen S
      • Itkin T
      • Chakrabarty S
      • et al.
      PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells.
      ,
      • Gur-Cohen S
      • Kollet O
      • Graf C
      • Esmon CT
      • Ruf W
      • Lapidot T
      Regulation of long-term repopulating hematopoietic stem cells by EPCR/PAR1 signaling.
      ]. Interestingly, activation of the protease thrombin by inflammation and other insults leads to PAR1 cleavage at a distinct site that drives increased NO production, EPCR shedding, and HSC mobilization from the BM niche [
      • Gur-Cohen S
      • Itkin T
      • Chakrabarty S
      • et al.
      PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells.
      ,
      • Gur-Cohen S
      • Kollet O
      • Graf C
      • Esmon CT
      • Ruf W
      • Lapidot T
      Regulation of long-term repopulating hematopoietic stem cells by EPCR/PAR1 signaling.
      ]. Here, although we found a small but significant decrease in EPCR surface expression on EPCR+/CD34 cells that could be related to thrombin activity and EPCR shedding, it does not appear sufficient to mobilize large numbers of HSCs as we do not observe decreased HSC numbers in the BM. Hence, our data imply activation of adaptive mechanism(s) that enforces HSC retention and quiescence despite ongoing inflammatory stress.
      In support of such a model, we found that activation of Nrf2- and Hif-driven gene programs are maintained in EPCR+/CD34 SLAM cells. Notably, Nrf2 and Hif pathways are also required for HSC quiescence, and their deletion leads to HSC mobilization from the BM [
      • Tsai JJ
      • Dudakov JA
      • Takahashi K
      • et al.
      Nrf2 regulates haematopoietic stem cell function.
      ,
      • Wielockx B
      • Grinenko T
      • Mirtschink P
      • Chavakis T
      Hypoxia pathway proteins in normal and malignant hematopoiesis.
      ]. Thus, further studies can assess the degree to which EPCR cooperates and/or converges with metabolic control mechanisms like Nrf2 and Hif to enforce quiescence and localization of EPCR+ HSCs in the BM niche, particularly under inflammatory stress conditions. Along these lines, our data raise an intriguing possibility that increased Mac-1 expression in EPCR+ SLAM fractions following IL-1 exposure could enforce retention of these cells in the BM. This mechanism could serve as a stress-inducible counterpart to VLA-4 expression, which maintains EPCR+ HSC localization in the BM under homeostatic conditions. These data are also similar to prior work indicating increased Mac-1 expression anchors hematopoietic progenitors in the BM after granulocyte colony-stimulating factor (G-CSF) administration [
      • Hidalgo A
      • Peired AJ
      • Weiss LA
      • Katayama Y
      • Frenette PS
      The integrin alphaMbeta2 anchors hematopoietic progenitors in the bone marrow during enforced mobilization.
      ]. The extent to which EPCR regulates surface expression of Mac-1 and other adhesion molecules under inflammatory stress conditions should therefore be more thoroughly investigated. Lastly, because quiescence and BM niche localization also underwrite HSC chemoresistance, such studies will shed further light on the capacity of phenotypic CD49blo R-HSC within the EPCR+/CD34 SLAM fraction to persist under myeloablative stressors such as 5-FU and IR.
      Interestingly, we found that loss of EPCR expression is associated with high levels of cell cycle and Mk/E lineage genes including Gata1 and surface expression of CD41. This is accompanied by minimal long-term myeloid/lymphoid engraftment activity, consistent with prior work indicating Gata1 expression anti-correlates with long-term HSC repopulating activity [
      • Drissen R
      • Buza-Vidas N
      • Woll P
      • et al.
      Distinct myeloid progenitor-differentiation pathways identified through single-cell RNA sequencing.
      ]. Our findings are also consistent with work using H2B-GFP transgenic reporter systems to assess divisional history within the SLAM gate [
      • Foudi A
      • Hochedlinger K
      • Van Buren D
      • et al.
      Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells.
      ,
      • Qiu J
      • Papatsenko D
      • Niu X
      • Schaniel C
      • Moore K
      Divisional history and hematopoietic stem cell function during homeostasis.
      ,
      • Bernitz JM
      • Kim HS
      • MacArthur B
      • Sieburg H
      • Moore K
      Hematopoietic stem cells count and remember self-renewal divisions.
      ]. These studies found that divisional history is associated with loss of EPCR expression and induction of Mk/E genes and is limited to no long-term myeloid/lymphoid reconstitution activity [
      • Foudi A
      • Hochedlinger K
      • Van Buren D
      • et al.
      Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells.
      ,
      • Qiu J
      • Papatsenko D
      • Niu X
      • Schaniel C
      • Moore K
      Divisional history and hematopoietic stem cell function during homeostasis.
      ,
      • Bernitz JM
      • Kim HS
      • MacArthur B
      • Sieburg H
      • Moore K
      Hematopoietic stem cells count and remember self-renewal divisions.
      ]. Likewise, our molecular analyses order SLAM fractions in a linear fashion associated with EPCR and CD34 levels, with acquisition of CD34 in each compartment representing an activated cellular state EPCR+/CD34+ cells may therefore a “decision point” between self-renewal, myeloid/lymphoid differentiation, and/or acquisition of Mk/E priming. As inflammation has been found to induce “emergency” megakaryopoiesis [
      • Haas S
      • Hansson J
      • Klimmeck D
      • et al.
      Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors.
      ], analyses of platelet and erythrocyte reconstitution kinetics using the Vwf-eGFP system can identify whether EPCR SLAM cells are indeed primed to generate Mk and/or erythroid cells and the impact of inflammatory stress on their activity. Additional detailed in vivo lineage tracing studies using single-cell transplants, reporter mice, and/or native barcoding systems will be needed to ascertain how fate(s) and localization in the BM of cells within each fraction are regulated.
      We found that more stringent approaches to enrich for functional HSCs, including the EPCR+/CD34 SLAM and Fgd5-ZsGreen+ SLAM definitions, provided superior enrichment for HSC activity and molecular phenotype relative to unfractionated SLAM cells following chronic IL-1 exposure. Similarly, a recent study reported long-term myeloid/lymphoid reconstitution activity in Fgd5-mCherry+ SLAM cells from poly I:C-treated mice was essentially identical to that of control mice [
      • Bujanover N
      • Goldstein O
      • Greenshpan Y
      • et al.
      Identification of immune-activated hematopoietic stem cells.
      ], whereas prior work had shown that unfractionated IFN-exposed SLAM cells exhibited impaired long-term reconstitution activity [
      • Pietras EM
      • Lakshminarasimhan R
      • Techner JM
      • et al.
      Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons.
      ,
      • Essers MA
      • Offner S
      • Blanco-Bose WE
      • et al.
      IFNalpha activates dormant haematopoietic stem cells in vivo.
      ]. As our current study and previous work both found that Fgd5+ SLAM cells are highly enriched for EPCR+/CD34 cells [
      • Gazit R
      • Mandal PK
      • Ebina W
      • et al.
      Fgd5 identifies hematopoietic stem cells in the murine bone marrow.
      ], we anticipate the EPCR+/CD34 definition may be useful for enriching HSCs under multiple inflammatory contexts.
      Our data suggest that inflammation-induced changes in the relative abundance of SLAM fractions is likely a factor in the reduced reconstitution capacity of unfractionated SLAM cells from IL-1-exposed mice. The frequency of long-term engrafting EPCR+/CD34 decreases by nearly twofold after IL-1 exposure, replaced by EPCR cells with limited to no reconstitution activity. Along similar lines, while the Vwf-eGFP+ SLAM compartment includes abundant long-term HSCs in homeostatic conditions [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ], IL-1 exposure leads to contamination of the Vwf-eGFP+ SLAM compartment with non-reconstituting EPCR cells. Thus, when transplantation assays are used to assess the long-term reconstitution capacity of enriched HSCs under non-homeostatic conditions, changes in the cellular composition of HSC-enriched compartments such as SLAM can potentially lead to “false-positive” readouts of impaired HSC function due to contamination by non-HSC cell types. Likewise, selection of lineage markers may need to be reconsidered, as long-term HSCs can express markers such as Mac-1 [
      • Wang J
      • Liu X
      • Zhang S
      • et al.
      Lineage marker expression on mouse hematopoietic stem cells.
      ]. Hence, it may be important to experimentally re-evaluate long-term HSC reconstitution activity under stress using stringent approaches.
      It is also possible that changes in HSC engraftment represent discrete and transient cellular states within the HSC pool. For instance, transplantation as an assay of self-renewal may provide an incomplete picture of HSC activity, as transient changes in cell cycle and other parameters may affect functions such as homing, lodging, and engraftment that would not otherwise be impaired if the cells were left in the donor animal. Furthermore, the effects of different inflammatory pathways on HSC biology must also be considered. For instance, exposure to bacterial lipopolysaccharide, double-stranded RNA, and live infection with pathogens such as Salmonella and Mycobacterium can induce excessive proliferation, DNA damage, and/or attrition in the HSC pool, largely through TRIF and/or interferon-related mechanisms [
      • Pietras EM
      • Lakshminarasimhan R
      • Techner JM
      • et al.
      Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons.
      ,
      • Walter D
      • Lier A
      • Geiselhart A
      • et al.
      Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells.
      ,
      • Takizawa H
      • Fritsch K
      • Kovtonyuk LV
      • et al.
      Pathogen-induced TLR4-TRIF innate immune signaling in hematopoietic stem cells promotes proliferation but reduces competitive fitness.
      ,
      • Matatall KA
      • Jeong M
      • Chen S
      • et al.
      Chronic infection depletes hematopoietic stem cells through stress-induced terminal differentiation.
      ,
      • Baldridge MT
      • King KY
      • Boles NC
      • Weksberg DC
      • Goodell MA
      Quiescent haematopoietic stem cells are activated by IFN-gamma in response to chronic infection.
      ]. On the other hand, MyD88-dependent signaling, which is conserved between IL-1 signaling and several TLRs, does not contribute to these effects after LPS or Salmonella infection [
      • Takizawa H
      • Fritsch K
      • Kovtonyuk LV
      • et al.
      Pathogen-induced TLR4-TRIF innate immune signaling in hematopoietic stem cells promotes proliferation but reduces competitive fitness.
      ]. Hence, the dose, timing, and signaling pathway(s) activated by inflammatory challenge may dictate the impact on HSC function. Lastly, changes in HSC activity may be transient effects that cease on resolution of the insult, suggesting that inflammation may not always result in HSC “damage,” but instead may play a reparative role in the hematopoietic system. Our study underscores the molecular and phenotypic heterogeneity within the SLAM gate and provides a relevant example of how chronic inflammation can dynamically alter the cellular subsets therein. These data suggest that continual re-evaluation of HSC definitions and the use of prospective and retrospective functional assays is crucial for accurately reporting HSC function in non-homeostatic conditions. Such studies will continue to generate improved approaches to defining HSC activity and cellular identity, particularly in dynamic stress situations.

      Acknowledgments

      This work was supported by National Institutes of Health (NIH) Grants K01 DK098315 and R01 DK119394 and the Cleo Meador and George Ryland Scott Chair of Medicine in Hematology (to EMP); NIH Grant F31 HL138754 (to JLR); and a National Science Foundation Graduate Research Fellowship Program (to TSM). This work was supported in part by the University of Colorado Cancer Center Flow Cytometry Shared Resource, funded by National Cancer Institute Grant P30 CA046934 .

      Conflict of interest disclosure

      The authors declare no competing interests.

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