Exploration of Myeloid Cell Biology Using the Hoxb8 Technology Pui Y. Lee, MD PhD Boston Children’s Hospital BWH / Nigrovic Laboratory I. Introduction of the Hoxb8 system II. Example of studies using murine Hoxb8 cells III. The JBC Hoxb8 Core Schematic of Hematopoietic stem cell differentiation Hematopoietic stem cell Common lymphocyte progenitor Common myeloid Erythrocytes progenitor Megakaryocytes Eosinophils Granulocyte-monocyte progenitor Basophils Monocyte-dendritic cell progenitor Neutrophils Common monocyte Common DC progenitor progenitor monocytes dendritic cells Myeloid cells: immunity and inflammation • Integral components of the innate immune system • Maintain tissue homeostasis at steady-state • First wave responders during infection and inflammation • Arsenal of pro-inflammatory mediators • Bridge to adaptive immunity • Tissue repair and wound healing Myeloid cells and inflammatory diseases • Rheumatoid arthritis • Systemic lupus erythematosus • Crystal arthropathy • Autoinflammatory syndromes Challenges in studying myeloid cells 1. Difficult to maintain ex-vivo 2. Limitation in cell numbers 3. Limited capacity for manipulation (KI, KO, KD) 4. Confounding effects of oncogenes in cell lines HoxB8-ER Myeloid Progenitor Cells - HoxB8: nuclear homeobox protein that suppresses myeloid cell development - immortalization of bone marrow myeloid progenitors by HoxB8 overexpression - Retrovirus transduction of HoxB8 gene driven by estrogen receptor followed by selection in b-estradiol and SCF (Wang et.al, Nature Methods, 2006) - Spontaneous differentiation after estrogen withdrawal - Quantitative production of monocytes and neutrophils HoxB8 Myeloid Progenitor + estrogen Lin Sca-1 CD117 CD34 - estrogen 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FcγRII/III CD135 Ly6C CD11b 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Granulocytes Monocytes Differentiation of HoxB8 cells upon Estradiol Removal - maintenance: RPMI with 10% FCS, 500nM b-estradiol, 20nm SCF - differentiation: RPMI with 10% FCS, 2 nM SCF Day 0 Day 4 Day 8 100 CD11b 80 CD117 60 40 20 % of maximum MFImaximum of % 0 0 2 4 6 8 Day Generation of other myeloid lineages using HoxB8 progenitors Neutrophils (GCSF, D4) Macs/neutrophils (GMCSF, D6) Mast Cells (IL-3 + SCF, Day 23) Osteoclasts (MCSF + RANKL, D6) Neutrophils Monocytes Macrophages Basophils Mast cells Dendritic cells Osteoclasts Lymphocytes Advantages of the Hoxb8 system • Easy to establish, store, and maintain • Versatile and strain independent • Low cost and minimizes need for mice • Rapid expansion for large-scale studies • Compatible with genetic manipulation – shRNA, CRISPR, Cre-mediated deletion • In vivo differentiation upon i.v. transfer Limitations • No parallel system for human stem cells (yet) • ? differences compared to primary cells • Difficult to transfect (requires viral vectors) • Requires confirmation using primary cells and in vivo studies I. Introduction of the Hoxb8 system II. Example of studies using murine Hoxb8 cells III. The JBC Hoxb8 Core Monocyte differentiation in colors: Double Reporter HoxB8 cells - Derived from BM of CCR2-RFP / CX3CR1-GFP mice high + - - Immature monocytes: Ly6C CCR2 CX3CR1 - Mature monocytes: Ly6Clow CCR2- CX3CR1high Day 0 Day 2 Day 4 Day 6 Day 8 104 104 104 104 104 103 103 103 103 103 102 102 102 102 102 CX3CR1-GFP FITC-A CX3CR1-GFP FITC-A CX3CR1-GFP FITC-A CX3CR1-GFP FITC-A CX3CR1-GFP FITC-A CX3CR1-GFP 101 101 101 101 101 CX3CR1 0 0 0 0 0 CD115- 10 10 10 10 10 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CCR2-RFP PerCP-Cy5-5-A CCR2-RFP PerCP-Cy5-5-A CCR2-RFP PerCP-Cy5-5-A CCR2-RFP PerCP-Cy5-5-A CCR2-RFP PerCP-Cy5-5-A CCR2 CD115+ 104 104 104 104 104 103 103 103 103 103 102 102 102 102 102 CX3CR1-GFP FITC-A CX3CR1-GFP FITC-A CX3CR1-GFP FITC-A CX3CR1-GFP FITC-A CX3CR1-GFP FITC-A CX3CR1-GFP 101 101 101 101 101 CX3CR1 100 100 100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Ly6CLy6C PE-Cy7-A Ly6C PE-Cy7-A Ly6C PE-Cy7-A Ly6C PE-Cy7-A Ly6C PE-Cy7-A 100 CCR2 80 CX3CR1 60 Ly6C 40 20 % of maximumMFI of % 0 0 2 4 6 8 10 Day Lee et al. Sci Immunol (in press) Small molecule library screening using DR-HoxB8 cells -150+ compounds (Caymen Biochem) targeting major signaling pathways including Akt, PKC, PKA, NFkB, JNK, ERK, MAPK, Ras, PI3K, mTOR, RIP, JAK/STAT - each compound initially tested at 10uM and 1uM, then titrated if necessary - DR-HoxB8 cells cultured with SCF + individual compounds; FACS analysis on day 4 Lee et al. Sci Immunol (in press) In vivo confirmation using Raptor conditional KO mice Raptor is a critical component of mTOR Complex 1 Peripheral blood Bone marrow Lee et al. Sci Immunol (in press) RNAseq: mTORC1 is required for physiologic down- regulation of c-Myc during myeloid differentiation Primary Myeloid progenitors (GMP) Hoxb8 cells +/- Rapamycin Raptor KO : mTORC1 deficient Rapamycin : mTORC1 inhibitor Tsc2 KO : mTOR overactivation Broad Smart Seq2 platform – 96 well format Lee et al. Sci Immunol (in press) mTORC1 is required for physiologic down-regulation of c-Myc during myeloid differentiation Myc-GFP Hoxb8 cells Wild-type Hoxb8 cells Rapamycin : mTORC1 inhibitor 10058-F4 : Myc Inhibitor SL0101-01 : S6 Kinase inhibitor Lee et al. Sci Immunol (in press) Generation of Hoxb8 cells from KO mice Wild-type RANKL -/- Defective osteoclast production in RankL -/- Hoxb8 cells - Cell lines can be generated from live or frozen bone marrow cells and fetal liver cells (for embryonically lethal KO strains) - 3-4 weeks from receiving primary cells to sufficient Hoxb8 cells for experiments CRISPR/Cas9 Genomic Editing using Hoxb8 cells http://www.doublexscience.org - Cas9-Hoxb8 cells generated from Cas9-KI mice (R. Platt and F. Zhang) - gRNA lentivirus synthesized by the Broad Institute (96 well format) I. Introduction of the Hoxb8 system II. Example of studies using murine Hoxb8 cells III. The JBC Hoxb8 Core The JBC Hoxb8 Core Consultation and Project planning Existing line Library stock Reestablish line Analysis by New line end-user Stem cell expansion User-supplied bone Retroviral infection Establish new line marrow cells / femur and selection Consultation, order Genetic Manipulation CRISPR gRNA or shRNA in bulk format, infection and selection *Hoxb8 vectors are under MTA with UCSD / MGH Hoxb8 Core Library of established Hoxb8 cell lines Wildtype strains: Reporter strains: Transgenics / KO strains: C57BL/6 CX3CR1 +/GFP CX3CR1 GFP/GFP PAD2 -/- Balb/C CCR2 +/RFP CCR2 RFP/RFP PTPN22 -/- SJL (CD45.1) LysM-GFP IRF8 -/- RANKL -/- MRL mpJ RFP-LC3-GFP IRF5 -/- MNK1/2 -/- Myc-GFP MyD88 -/- KLF4 -/- Dock2 -/- PAD4 -/- Raptor fl/fl MRL lpr Raptor fl/+ Ubc-Cre B6 lpr Rictor fl/fl BXSB-YAA RagA fl/f Hif1a fl/fl RagA fl/fl Ubc-Cre VHL fl/fl TSC-1 fl/fl Nlpr3 -/- TSC-1 LysM-Cre Caspase1 -/- TSC-1 -/- CD18 -/- Rbpj -/- Cas9-EGFP-KI Acknowledgement Nigrovic Lab BWH • Peter Nigrovic • Julia Charles • Sarah Ameri • Kevin Wei • Ally Morris • Adam Chicoine • Nate Nelson-Maney • Pierre Cunin MGH • Gang Li • David Sykes • Anais Levescot • Demetrios Kalaitizidis • Marta Martinez • David Scadden • Minah Iqbal Broad Institute • David Root Funding • NIH T32 (BCH Allergy / Immunology) • RRF Scientist Development Award.