1. IMMU7020 Haematopoiesis and Immunity Afshin Raouf Ph. 787-2294 raouf@cc.umanitoba.ca

2. Outline What is immunity? What is haematopoiesis? How does haematopoiesis maintain immunity? in vivo and in vitro assays to study haematopoiesis Leukemia: normal haematopoiesis gone awry

3. What is immunity? Immunity is a state in which the body is protected from infectious diseases and potentially harmful substances. The immune system is a complex network of many different cells and chemicals that act in concert to fight, eliminate, contain and prevent infections and disease. How does the body keep up the with the relentless task of maintaining our immune system? Secondary organs are involved for a complete and long-term immune response: - Spleen (immunologic filter), Lymph Nodes (mostly B-cells macrphages, and dendritic cells), Peyer’s Patches (patches of lymphocytes in the small intestine), Appendix (T-cells and B-cells ), Thymus (T-cell priming) clones of T-Cells and B-Cells resides in these secondary organs to provide rapid response to repeat infections/antigens There are two types of immune responses: i) Innate: is a rapid and non-specific response to a pathogen/antigen - Involves: Phagocytes such as Macrophages (long-lived), Natural Killer Cells, Neutrophils (1-4 day lifespan), Granulocytes such as Eosinophils (8-12 days) and basophils, Antigen presenting cells such as Dendritic cells ii) Acquired (adaptive): is a slow (several days) but very specific response. It can lead to immunological memmory - Involves: lymphocytes that circulate in the peripheral blood (20-50%) and the lymphatic system B-cells (15% of the lymphocytes), antibody producing, memory B-cells T-Cells (80% of the lymphocytes), Cytotoxic (CD8+), Helper (CD4+), memory T-Cells

4. What is haematopoiesis? Hematopoiesis is the process of production, multiplication, and specialization of blood cells in the bone marrow. This process can take place in adults bone marrow. What about fetus (no viable long bones)? The ontology of haematopoietic system Haematopoiesis appears in the liver at approximately 5 weeks' gestation and remains the primary site of haematopoiesis until mid-gestation, when bone marrow haematopoiesis exceeds that of the liver. Unlike in mice, the spleen in humans is never a major hematopoietic organ.

5. Maintenance of the haematopoietic system The haematopoietic cells (WBC and RBC) need constant renewal — the production of millions of new blood cells each day. - As early as 1945 is was evident that haematopoietic system can replenish itself - radiation-induced sickness - Animal studies revealed that sub-lethally irradiated mice can be rescued using bone marrow transplants - 1960’s Till and McCullough identified the cells in the bone marrow have the ability to regenerate the entire haematopoietic system  Haematopoietic Stem cells or HSCs Till, J.E. and McCullough, E.A. (1961), Radiat. Res. 14, 213–222. - HSCs possess the following characteristics: multi-lineage differentiation, extensive proliferation, and life-long self-renewal potentials, apoptosis - Sources of HSCs: Bone marrow, peripheral blood, and umbilical cord blood

6. Haematopoiesis generates immune cells Hematopoietic stem cells: 1. Self renewal 2. Multipotency 3. extensive proliferation They make immune cells, platelets, and RBCs In bone marrow 1:10, 000 In PB: 1: 100, 000 Each HSC is thought to under go 17- 19 divisions at the end of which it would produce 720, 000 progeny! MacKey MC, 2001, Cell. Prolif. 34:71 - 83

7. Where are the undifferentiated haematopoietic cells found? Immunological Reviews 2010 Vol. 238: 47–62

8. M. Haggstrom and A. Rad, Wikipedia Hematopoietic growth factors Proliferation Potential Differentiation Potential

9. Haematopoiesis: Myeloid differentiation Oncogene (2002) 21, 3295 ± 3313 Gata1+ MPPs are functional CMPs (myelo-erythroid) PU.1+ MMPs are functional GMLPs (myelo-lymphoid) PU.1 suppresses Gata1 expression

10. Annu Rev Pathol. Mech Dis, 2009; 4:175-198 Haematopoiesis: Lymphoid differentiation

11. HSC MPP LMPP CLP Ly6D- CLP Ly6D- CLP Ly6D+ CLP Ly6D+ BM IL-7R Kit Rag CCR7 CCR9 Blood CLP Flt3 high CLP Flt3 high CCR9 + CCR7 + Flt3 Thymus TSP ETP Flt3 + ETP Flt3 + ETP Flt - ETP Flt - DN2a DN2b Notch IL-7R DN3 Kit Rag CD4 - CD8 - T Cell differentiation Flt3

12. Role of Notch signaling in T cell Development Bone marrow Thymus Notch signaling does not affect HSC, MPP, or LMPP frequency or function They will still home to the thymus Deletion of Notch1 in ETP cells  accumulation of B cells in the thymus Deletion of DLL4 in the Thymus epithelial cells yield similar results

13. B Cell or T Cell: a series of binary decisions Immunological Reviews 2010 Vol. 238: 47–62

14. B Cell differentiation Oncogene (2002) 21, 3295 ± 3313 Unlike T cell development, B cell development mostly takes place in the bone marrow

15. Regulation of HSC expansion and stemness Wnt, Notch, Hox Apoptosis Apoptosis of one stem cell can potentially eliminate 106 cells X Symmetric vs. asymmetric division Self-renewal Quiescence

16. CRU Functional definition of primitive haematopoietic cells Functional definitions of stem cells and progenitors

17. Current protocols in Immunology, 2008 Unit 22B.2 Competitive Repopulating Assay: CRU

18. The colony forming cell (CFC) or colony forming unit (CFU) assay, also referred to as the methylcellulose assay, is an in vitro assay used in the study of hematopoietic stem cells. The assay is based on the ability of hematopoietic progenitors to proliferate and differentiate into colonies in a semi-solid media in response to cytokine stimulation. The colonies formed can be enumerated and characterized according to their unique morphology. In vitro progenitor differentiation detection It is based on the assumption that each colony arises from a single progenitor subtype CFU-E (Colony forming unit- erythroid): Clonogenic progenitors that produce only one or two clusters. It represents the more mature erythroid progenitors that have less proliferative capacity. CFU-G (Colony forming unit- granulocyte): Clonogenic progenitors of granulocytes that give rise to a homogeneous population of eosinophils, basophils or neu­trophils. CFU-GM (Colony forming unit- granulocyte, macrophage): Progenitors that give rise to colonies containing a heterogene­ous population of macrophages and granulocytes. The mor­phology is similar to the CFU-M and CFU-G descriptions. BFU-E (Burst forming unit- erythroid): These are primitive erythroid progenitors that have high proliferative capacity. CFU-M (Colony forming unit- macrophage): Clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages. CFU-GEMM (Colony forming unit- granulocyte, erythrocyte, macrophage, megakaryocyte): Multi-lineage progenitors that give rise to erythroid, granulocyte, macrophage and megakaryocyte lineages. http://www.rndsystems.com/literature_CFC.aspx

19. J Seita et al. WIREs Syst Biol Med 2010. Purification of HSC and progenitor subtypes

20. Role of haematopoietic stem cells in immunology Chronic Inflammation Clinical uses of Haematopoietic stem cells? Inherited blood disorders: Aplastic anemia, sickle-cell anemia, severe combined immunodeficiency Haematopoietic stem cell rescue after chemotherapy Leukemia treatment (bone marrow transplantation)

21. Stochastic VS Cancer stem cell concept of leukemogenesis Only CD34 + CD38 - subpopulation of acute myelogenous leukemia (AML) cells are capable of initiating and sustaining leukemic clone in immunodeficient mice (SCID & NOD/SCID) serial transplantation assays demonstrated their self-renewal capacity

22. Cancer Tumor stem cells Regulated and co-ordinated proliferation, differentiation & death Normal tissue Normal stem cells Normal end cells Both normal & malignant stem cells create developmental hierarchies Deregulated and/or aberrant proliferation, differentiation & death Bulk of tumor cells Cancers viewed as evolving clonal hierarchies Genomic instability can alter the phenotype of the tumor

23. Bjerkvig R., et al., 2005, Nat Rev Cancer 5, 899-904 Raouf A., 2010, Breast Cancer Res. 12(6):316 Cancer initiating cell: Normal VS tumor stem cells Cancer-initiating cells are referred to the normal cells in the adult tissues that can acquire enough mutations to transform in to cancer stem cells cancer stem cells arise from cancer-initiating cells and are responsible tumor recurrence (i.e. proliferation and self- renewal potentials) and the tumor heterogeneity (i.e. multi-lineage differentiation potential)

24. Cancer stem cell concept in cancer research Stochastic model Every cell has equal probability of proliferating extensively and form new tumors The genetic changes leading to development and progression are operative within all tumor cells Current therapies aimed at the bulk of the tumor are based on this model Tumor stem cell model Only a small subset has the ability to initiate new tumors cancer stem cells are biologically and functionally distinct from the bulk of tumor cells This rare subpopulation must be the target of cancer treatment to achieve permanent cure Wang J.C.Y., et al, Trends in cell biol. Vol. 15, 2005 Perturbations in expression and function of genes involved in the normal regulation of stem and progenitor cell can cause them to become tumor stem cells. Working Hypothesis Corollary hypothesis: Perturbations in expression and function of genes involved in the normal regulation of stem and progenitor cell can cause them to become tumor stem cells (e.g. NOTCH, WNT, and integrin signaling pathways).

25. Further readings Cancer stem cells: an evolving concept. Nat Rev Cancer, 2012 Jan 12 Stem cell concepts cancer research. Blood, 2008, Vol 112(13):4793 – 807 Biology of normal and acute myeloid leukemia stem cells. Int J Hematol 2005, 82(5):389-96