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IMMU7020
Haematopoiesis and Immunity
Afshin RaoufPh. 787-2294
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
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-cellsT-Cells (80% of the lymphocytes), Cytotoxic (CD8+), Helper (CD4+), memory T-Cells
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.
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
Haematopoiesis generates immune cells
Hematopoietic stem cells: 1. Self renewal 2. Multipotency3. extensive proliferation
They make immune cells, platelets, and RBCs
In bone marrow 1:10, 000In 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
Where are the undifferentiated haematopoietic cells found?
Immunological Reviews 2010 Vol. 238: 47–62
M. Haggstrom and A. Rad, Wikipedia
Hematopoietic growth factorsProliferation Potential
Differentiation Potential
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
Annu Rev Pathol. Mech Dis, 2009; 4:175-198
Haematopoiesis: Lymphoid differentiation
HSC MPP LMPP CLPLy6D-
CLPLy6D+BM
IL-7R
Kit
Rag
CCR7CCR9
Blood CLPFlt3high
CCR9+
CCR7+
Flt3
Thymus
TSP ETPFlt3+
ETPFlt- DN2a DN2b
Notch
IL-7R
DN3
Kit
Rag
CD4-CD8- CD4-CD8-
T Cell differentiation
Flt3
Role of Notch signaling in T cell Development
Bone marrow
Thymus
Notch signaling does not affect HSC, MPP, or LMPP frequency or functionThey will still home to the thymus
Deletion of Notch1 in ETP cells accumulation of B cells in the thymusDeletion of DLL4 in the Thymus epithelial cells yield similar results
B Cell or T Cell: a series of binary decisions
Immunological Reviews 2010 Vol. 238: 47–62
B Cell differentiation
Oncogene (2002) 21, 3295 ± 3313
Unlike T cell development, B cell development mostly takes place in the bone marrow
Regulation of HSC expansion and stemness
Wnt, Notch, Hox
Apoptosis
Apoptosis of one stem cell can potentially eliminate 106 cellsX
Symmetric vs. asymmetric division
Self-renewal
Quiescence
CRU
Functional definition of primitive haematopoietic cells
Functional definitions of stem cells and progenitors
Current protocols in Immunology, 2008 Unit 22B.2
Competitive Repopulating Assay: CRU
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 neu trophils.
CFU-GM (Colony forming unit-granulocyte, macrophage): Progenitors that give rise to colonies containing a heterogene ous population of macrophages and granulocytes. The mor phology 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
J Seita et al. WIREs Syst Biol Med 2010.
Purification of HSC and progenitor subtypes
Role of haematopoietic stem cells in immunologyChronic 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)
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
Cancer
Tumorstem cells
Regulated and co-ordinated proliferation,
differentiation & death
Normaltissue
Normalstem cells
Normalend cells
Both normal & malignant
stem cells create developmental
hierarchies
Deregulated and/or aberrant proliferation,
differentiation & death
Bulk oftumor cells
Cancers viewed as evolving clonal hierarchies
Genomic instability can alter the phenotype of the tumor
Bjerkvig R., et al., 2005, Nat Rev Cancer 5, 899-904Raouf 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)
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).
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
