Development of the Haematopoietic and Immune Systems

1. Development of the Haematopoietic and Immune Systems Embryonic origins Bone marrow transplantation as a paridigm for generating an organ from stem cells Mechanisms of stem cell renewal and differentiation Specific examples of erythrocyte and lymphocyte development Development and Disease Mechanisms Oct 24th 2003, Lecture 12 Gerald Crabtree

3. Overview of Environment of Embryo/FetusExtra embryonic membranes

4. The Developmental Origin of Blood and Immune Cells • Earliest Site of Haematopoiesis is the Yolk Sac (2-3 weeks) and Dorsal Aorta (AGM region) around 3-5 weeks after conception. • Yolk sac stem cells are not able to supply all the blood cell type. • True haematopoietic stem cells appear in the liver at about 6 weeks post conception

5. Yolk sac, transient extra-embryonic structure – initiation of blood/Hb synthesis

7. Bone Marrow Transplantation:Creating an Organ from a Stem Cell • 20,000 bone marrow transplantations per year in the US • Most commonly used for treatment of malignancy • Also used for treatment of aplastic anemia, autoimmune disorders, myleodysplastic syndromes (bone marrow failure) and exposure to toxins or radiation. • Rely on the ability of a small number of Haematopoietic Stem Cells (HSC) to repopulate the immune and hematopoietic systems

8. Bone Marrow Transplantation • Stanford student- Found to have acute myelogenous leukemia after blood donation • Treated twice with routine chemotherapy-both attempts failed • Immunologically partially compatible cousin identified • Given lethal chemotherapy, followed by transplantation of 108 marrow cells from cousin. • Severe infections for 10 days • Platelet counts and white cell counts began to rise and reached near normal levels within months • Continued on Cyclosporin A to suppress graft rejection for 1 year • Alive and well today- 11 years later with the immune and haematopoietic system of his cousin

9. The Atomic Age dawned at 5:29:45 am on July 16, 1945, at Trinity Site, New Mexico

10. The Discovery of Stem Cells Death due to anemia, granulocytopenia and thrombocytopenia Lethal Irradiation Transfusion of blood or bone marrow from a normal donor Survival of a significant number of irradiated individuals Lethal Irradiation What does blood or bone marrow have that allows the survival of irradiated individuals and the appearance Of white cells, red cells and platalets?

11. Reconstitution of the Entire Haematopoietic System by Bone Marrow Transplantation Transfusion of blood or bone marrow from a normal donor Lethal Irradiation or Lethal Chemotherapy To kill all malignant cells Death of tumor cells And survival of patient Donor Provides: Red cells, platelets, white cells, pulmonary alveolar macrophages, Kupffer cells of the liver, osteoclasts, Langerhans cells of the skin, and microglial cells of the brain 40,000 bone marrow transplantations in 1998 General Reference: F. Appelbaum Annu. Rev. Med. 2003. 54:491–512

12. Can HSCs give rise to other cell types? • Early reports indicated that muscle, neurons, hepatocytes and cardiac muscle might derive from adult HSC. • More recent reports suggests that HSC fuse with other cell types and hence acquired their markers • Science 297, 2256, 2003 A A

13. Experimental Paradigm for Study of Haematopoietic Stem Cells

14. Many types of cells originate from a single type of haematopoietic stem cell (HSC)

15. Possible Mechanisms for Maintaining a Stem Cell Population A. Asymmetric Divisions B. Symmetric Divisions C. Locally Directed Divisions (Niche directs differentiation after a symmetrical division)

16. Symmetric and Asymmetric Divisions of Neural Stem Cells Tuj/LeX (CD15)/DAPI P-P P-N N-N Lex (CD15) Stem cell marker Tuj Differentiated Marker Brg Acts Cell-Autonomously to Favor Asymmetric Divisions Pair cell assay: E13.5 cortical culture

17. Maintaining Long Term Haematopoietic Stem Cells in Culture: A Major Unsolved Therapeutic Goal • Soluble factors that maintain HSCs: • SIF, Flt3L, Tpo, IL-3 • Wnt, Notch and Sonic Hedgehog (Shh) • Transcription factors that increase the replication of HSC • HoxB4 and A9 Under the best of circumstances stem cell reconstitution can only be sustained for 1 or 2 mouse passages Possible problems: In vitro creation of a stem cell niche Telemeric shortening with sequential passage in culture;

18. Chromosomal Telemeres Shorten with Passage through the Cell Division Cycle Elizabeth Blackburn Cell 2001 A possible limitation to the sequential passage of haematopoietic stem cells (HSC)

19. The Discovery of Colony Forming Units Demonstrates Self Renewal within Lineages Implies the existence of stem cells for each class of blood cell

20. Sequential Steps of Blood Cell Development are Directed by Cytokines

21. Sequential Steps of Blood Cell Development are Directed by Cytokines Cytokine A Cytokine B Cytokine C Committed Stem Cell Differentiated and Functional blood cell

22. Instructive Vs Selective Mechanisms of Receptor Action Morrison et al Cell 2002 • (A and B) Selective mechanism in which two different factors (F1 and F2) allow the survival and maturation of lineage-committed progenitors generated by a cell-autonomous mechanism; “X” indicates death of the other progenitors. Erythropoietin • (C and D) Instructive mechanism in which the factors cause the stem cell to adopt one fate at the expense of others. Glial growth factor and BMP2

23. Death of an Anthropomorphism:The Instructive Hypothesis of Receptor Action H. Lodish And colleagues

24. If Cytokines Do not Give Instructive Signals… Cytokines probably provide permissive signals that are dependent on the developmental history of a cell _______ Developmental history is reflected by the expression of receptors, signaling molecules, transcription factors and chromatin accessibility

25. The Development of T Lymphocytes and Red Cells IL# (interleukin general name for haematopoietic growth factors SDF-1 (stomal cell Derived factor) FLT-3 or Flk2 (Fems like tryosine kinase Ligand) SCF (Stem cell factor) the product of the White locus effects both neural crest and haematopoietic cell development. Binds C-kit, mutation of which has near identical Phenotype as SCF mutations. Epo- Erthropoietin Tpo- thrombopoietic factor GM-CSF granulocyte macrophage stimulating factor G-CSF granuloctye stimulating factor

26. Common Myeloid Progenitor Development of Red Blood Cells Feedback control loop • First red cells are produced in the yolk sac. Later red cell production shifts to the liver, spleen and then the bone marrow. • Feedback control of RBC Production is through Erythropoietin (Epo). • Necessary to prevent death and promote proliferation of committed precursors • Shifts non-committed progenitor cells into the erythroid lineage • Produced in renal tubular epithelial cells and more widely in the growing embryo • Feedback control targets the first committed cell in the erythroid lineage.

27. What regulates Erythropoietin (Epo) Production? Semenza G.L.Cell. 2001 Oct 5;107(1):1-3 • Epo is regulated transcriptionally by an regulatory region near the gene • This regulatory region binds HIF (Hypoxia Induced Factor) • Hypoxia regulates HIF • HIF also activates VEGF and induces vasculogenesis- a problem in pregnancy

28. If HIF-1 Controls Epo, what Controls HIF-1? Hypoxia Prevents Degradation of HIF-1 PHD = proline hydroxylase

29. Leadville, CO- The birth defect capital of the United States • It is with great pride that we invite you to share Leadville's spectacular scenery with majestic mountains, rushing streams, alpine lakes, wildflowers in vivid colors, and sky so blue it appears retouched. And that doesn't include winter thrills.Leadville was incorporated in 1878 and is the highest incorporated city in the continental United States at 10,152 feet above sea level. • From the Leadville Chamber of Commerce • Anemia stimulates HIF and HIF stimulates VEGF and VEGF induces inappropriate angiogenesis and other patterning defects.

30. Erythropoietin: The Drug • Erythropoietin is given for intractable anemia • Best for chronic renal disease • Ineffective in some cases of aplastic anemia • Also effective for increasing blood production for preoperative storage of autologous blood.

31. Lymphocyte Development Key Points 1) The role of a developmental field in lymphocyte specification. 2) Lineage specification in T cells is dependent on chromatin control. 3) Self vs Non-self discrimination is dependent on decoding signal intensity

32. CLP (Common Lymphoid Precursor) Pax5 Notch Inactive Pax5 Inactive Notch Active B cell T cell Pax 5 Repression of Notch Shifts Progenitors into the B Cell Lineage M. Busslinger and colleagues Bone Marrow Thymus

33. Local Factors Influence the Fate of HSC’s Implies stem cells for each class of blood cell However T cell colonies are not found in the spleen

34. What defines the field in which T cells develop? Hox-1.5 essential for thymic development And mice lacking Hox-1.5 have no: Parathyroid Thyroid Submaxillary tissue WHN (winged Helix Nude or HNF3g) mutant mice lack a thymus DiGeorge Syndrome 22q11.2 microdeletion Congenital heart disease-craniofacial abnormalities and thymic aplasia

35. Molecular Anatomy of the Microdeletion in DiGeorge Syndrome • Microdeletion of 22q11.2 occurs in 1/4000 births • Tbx gene implicated in congenital heart defects • Basis for thymic aplasia is still unknown

36. T Cell Development:How do lymphocytes tolerate self antigens yet respond to foreign antigens? Thymus TCR Wnt TCR IL-7 If we can make new organs from embryonic stem cells they will still be rejected unless we can also control lymphocyte development.

37. Current View of Selection of the Immune Repertoire J. Sprent and colleagues High Avidity Self Antigen Bound to self MHC High Intensity Signal? Low Avidity Self MHC Low Intensity Signal? No Signal Signal Intensity Default Death Negative Selection Death of self reactive cells Positive Selection Differentiation and Proliferation of cells able to interact with self MHC

38. T Cell Development: Selection of CD4 and CD8 Cells by MHC CD4 interacts with MHC class II And is required For CD4 Cells CD8 interacts with MHC class I And is required For CD8 Cells 1 What directs the development of CD4 and CD8 Cells?

39. ATP-Dependent Chromatin Remodeling Complexes (BAF) and Runx Transcription Factors Control T Cell Lineage Committement Cell. 2002 Nov 27;111(5):621-33. Nature. 2002 Jul 11;418(6894):195-9 BAF complexes required For both silencing and activation of CD4 and CD8 genes. CD8 Locus CD4 Locus

40. Cyclosporin A Present Model for Selection of the Immune Repertoire

41. Bone Marrow Transplantation as a Paradigm of Therapeutics Based on Understanding Human Developmental • Endocrine pancreas • Skin • Bone • Joint surface and articular cartilage • Kidney • Liver • Lung • Heart • Eye • Brain???

42. T Cell Development: Selection of CD4 and CD8 Cells by MHC 1 How do lymphocytes come to be self tolerant, yet react with foreign antigen?

43. Immunosuppressive Drugs Cyclosporin A and FK506 Block Calcineurin/NFAT Signaling and thereby the Ability of T Cells to Coordinate the Immune Response IL-2 IL-3 IL-4 GMCSF, etc, etc, etc B Cell growth and Differentiation Factors Macrophage Growth And differentiation factors Fas Ligand CD40Ligand Foreign Antigen Cyclosporin A s s

44. CD4 Lymphocytes are the Organizer of the Immune Response IL-2 IL-3 IL-4 GMCSF, etc, etc, etc B Cell growth and Differentiation Factors Macrophage Growth And differentiation factors Fas Ligand CD40Ligand Foreign Antigen s s

45. Cyclosporin A Inhibits NFAT-dependent Transcription of the Genes that “Organize” the Actions of Cells Involved in the Immune Response IL-2 IL-3 IL-4 GMCSF, etc, etc, etc B Cell growth and Differentiation Factors Macrophage Growth And differentiation factors Fas Ligand CD40Ligand Cyclosporin A

46. Signal Strength Theory of Self vs Non-Self Discrimination • High intensity signals generated by abundant self antigens kill self reactive T cells (Negative Selection) • Low intensity signals generated by MHC interactions select CD4 and CD8 T cells (Positive Selection) • Lympocytes the generate a receptor unable to bind antigen or MHC die by neglect

47. Analogue to Digital Switches in Development TCR Hedgehog BMP Sensor Sensor Sensor V1 Inter neurons Motor Neurons V2 Inter neurons Dorsal fates Ventral fates Inter fates Default Death Positive Selection Negative Selection Where are the Sensors in the Signaling Pathways?

48. If we can make new organs from embryonic stem cells they will be rejected unless we can also control lymphocyte development.

49. The Lineages of Blood Cells are Marked by Cell Surface Proteins

50. Calcineurin-NFAT Signaling in Shaping the Immune Repertoire CD3, Zap70 Lat, Slp76 Tec Sensor Bim:Bcl Bak-Bax Negative Selection DefaultDeath Positive Selection