Haematopoiesis
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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

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Immu7020

Haematopoiesis and Immunity

IMMU7020

AfshinRaouf

Ph. 787-2294

[email protected]


Immu7020

  • 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


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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 asEosinophils (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?


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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.


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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


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Haematopoiesisgenerates 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


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Where are the undifferentiated haematopoietic cells found?

Immunological Reviews 2010 Vol. 238: 47–62


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Hematopoietic growth factors

Differentiation Potential

Proliferation Potential

M. Haggstrom and A. Rad, Wikipedia


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Haematopoiesis: Myeloid differentiation

Gata1+ MPPs are functional CMPs (myelo-erythroid)

PU.1+ MMPs are functional GMLPs (myelo-lymphoid)

Oncogene (2002) 21, 3295 ± 3313

PU.1 suppresses Gata1 expression


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Haematopoiesis: Lymphoid differentiation

Annu Rev Pathol. MechDis, 2009; 4:175-198


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T Cell differentiation

HSC

MPP

LMPP

CLP

Ly6D-

CLP

Ly6D+

BM

Flt3

IL-7R

Kit

Rag

CCR7

CCR9

CLP

Flt3high

CCR9+

CCR7+

Blood

Thymus

CD4-CD8-

CD4-CD8-

TSP

ETP

Flt3+

ETP

Flt-

DN2a

DN2b

DN3

Notch

Flt3

IL-7R

Kit

Rag


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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


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B Cell or T Cell: a series of binary decisions

Immunological Reviews 2010 Vol. 238: 47–62


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B Cell differentiation

Unlike T cell development, B cell development mostly takes place in the bone marrow

Oncogene (2002) 21, 3295 ± 3313


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Regulation of HSC expansion and stemness

Self-renewal

Symmetric vs. asymmetric division

Apoptosis

Quiescence

Wnt, Notch, Hox

Apoptosis of one stem cell can potentially eliminate 106 cells

X


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Functional definition of primitive haematopoietic cells

CRU

Functional definitions of stem cells and progenitors


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Competitive Repopulating Assay: CRU

Current protocols in Immunology, 2008 Unit 22B.2


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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 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


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Purification of HSC and progenitor subtypes

J Seitaet al.WIREsSystBiol Med 2010.


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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)


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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


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Genomic

instability can alter the phenotype of the tumor

Cancers viewed as evolving clonal hierarchies

Normal

tissue

Cancer

Tumor

stem

cells

Normal

stem

cells

Bulk of

tumor

cells

Normal

end

cells

Both normal &

malignant

stem cells create

developmental

hierarchies

Deregulated and/or aberrant

proliferation,

differentiation & death

Regulated and co-ordinated

proliferation,

differentiation & death


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Cancer initiating cell: Normal VS tumor 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-initiating cells are referred to the normal cells in the adult tissues that can acquire enough mutations to transform in to cancer stem cells

Bjerkvig R., et al., 2005, Nat Rev Cancer 5, 899-904

Raouf A., 2010, Breast Cancer Res. 12(6):316


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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

  • 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

  • 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

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).


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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


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