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Haematopoiesis and Immunity
IMMU7020
Afshin Raouf
Ph. 787-2294
[email protected]
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.
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
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
How does the body keep up the with the relentless task of maintaining our immune system?
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. 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 1719 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
Hematopoietic growth factors
Proliferation Potential
Differentiation Potential
M. Haggstrom and A. Rad, Wikipedia
Haematopoiesis: Myeloid differentiation
Gata1+ MPPs are functional CMPs (myelo-erythroid)
PU.1+ MMPs are functional GMLPs (myelo-lymphoid)
PU.1 suppresses Gata1 expression
Oncogene (2002) 21, 3295 ± 3313
Haematopoiesis: Lymphoid differentiation
Annu Rev Pathol. Mech Dis, 2009; 4:175-198
T Cell differentiation
HSC
BM
MPP
CLP
Ly6D-
LMPP
CLP
Ly6D+
Flt3
IL-7R
Kit
Rag
CCR7
CCR9
CLP
Blood
Flt3high
CCR9+
CCR7+
Thymus
TSP
Notch
Flt3
IL-7R
Kit
Rag
ETP
Flt3+
ETP
Flt-
CD4-CD8-
CD4-CD8-
DN2a
DN2b
DN3
Role of Notch signaling in T cell Development
Bone marrow
Notch signaling does not affect HSC, MPP, or LMPP frequency or function
They will still home to the thymus
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
B Cell or T Cell: a series of binary decisions
Immunological Reviews 2010 Vol. 238: 47–62
B Cell differentiation
Unlike T cell development, B cell development mostly takes place in the bone marrow
Oncogene (2002) 21, 3295 ± 3313
Regulation of HSC expansion and stemness
Self-renewal
Symmetric vs. asymmetric division
Apoptosis
Quiescence
Wnt, Notch, Hox
X
Apoptosis of one stem
cell can potentially
eliminate 106 cells
Functional definition of primitive
haematopoietic cells
CRU
Functional definitions of stem cells and progenitors
Competitive Repopulating Assay: CRU
Current protocols in Immunology, 2008 Unit 22B.2
In vitro progenitor differentiation detection
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.
It is based on the assumption that each colony arises from a single progenitor subtype
CFU-E (Colony forming uniterythroid): 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 unitgranulocyte): Clonogenic
progenitors of granulocytes that
give rise to a homogeneous
population of eosinophils,
basophils or neutrophils.
CFU-GM (Colony forming unitgranulocyte, 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 uniterythroid): These are primitive
erythroid progenitors that have
high proliferative capacity.
CFU-M (Colony forming unitmacrophage): Clonogenic
progenitors of macrophages that
give rise to a homogenous
population of macrophages.
CFU-GEMM (Colony forming unitgranulocyte, erythrocyte,
macrophage, megakaryocyte):
Multi-lineage progenitors that
give rise to erythroid, granulocyte,
macrophage and megakaryocyte
lineages.
http://www.rndsystems.com/literature_CFC.aspx
Purification of HSC and progenitor subtypes
J Seita et al. WIREs Syst Biol Med 2010.
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)
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
Cancers viewed as evolving clonal hierarchies
Normal
tissue
Normal
end
cells
Cancer
Normal
stem
cells
Bulk of
tumor
cells
Both normal &
malignant
stem cells create
developmental
hierarchies
Regulated and co-ordinated
proliferation,
differentiation & death
Tumor
stem
cells
Genomic
instability can alter
the phenotype of
the tumor
Deregulated and/or aberrant
proliferation,
differentiation & death
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 selfrenewal potentials) and the
tumor heterogeneity (i.e.
multi-lineage
differentiation potential)
Bjerkvig R., et al., 2005, Nat Rev Cancer 5, 899-904
Raouf A., 2010, Breast Cancer Res. 12(6):316
Cancer stem cell concept in cancer research
Stochastic model
Tumor stem cell model
•Every cell has equal probability of proliferating
extensively and form new tumors
•Only a small subset has the ability to
initiate new tumors
•The genetic changes leading to development
and progression are operative within all tumor
cells
•cancer stem cells are biologically and
functionally distinct from the bulk of tumor
cells
•Current therapies aimed at the bulk of the
tumor are based on this model
•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
Working 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.
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