Geoffrey Brown College of Medical and Dental Sciences

Revising textbook accounts of Haematopoiesis

Products of Haematopoiesis

MARROW BLOOD TISSUES

Megakaryoblast platelet

Erythroblast erythrocyte

Mast cell precursor mast cell mucosal and connective tissue mast cells

basophilMyeloblast eosinophil in sites of inflammation neutrophilMonoblast monocyte tissue / lymphoid tissue macrophages; Kuppfer cell; osteoclastPrecursor? dendritic cell tissue dendritic cell / Langerhans’ cell; veiled cell; interdigitating cellPrecursor? natural killer cell lymphoid tissues; sites of inflammation

Pro-B cell type 1 and type 2 B cells in blood and secondary lymphoid tissues;(matures in marrow) plasma cells also in mucosal surfaces and bone marrowT cell precursor and T cells; CD4+ve and CD8+ve T cells; in blood,(matures in thymus) secondary lymphoid tissues and sites of inflammation

Products of Haematopoiesis

Megakaryocyte

Erythrocyte

Lymphoid cells (i) B lymphocyte (ii) T lymphocyte

Granulocytes(i) Neutrophil

(ii) Eosinophil

(iii) Basophil

Monocyte

Macrophage

Dendritic Cell

(iii) NK cell

STEMCELLS

HAEMOPOIETICPROGENITORS

MATURECELLS

Immortal Lineage selectionExpansion

Self renewal?

FunctionalSelf renewal for

lymphocytes

Progressive Stages in Haematopoiesis

NK cell

Hematopoietic Stem Cell

Common Myeloid Progenitor

Common Lymphoid Progenitor

MacrophageGranulocyteB cell

ErythrocytePlatelet

M/E Progenitor G/M Progenitor

The ‘Conventional’ model of haematopoiesis - Weissman

T cell

Myeloid lineages Lymphoid lineages

Haematopoiesis – Principles

(i)Two families of cells – myeloid and lymphoid

(ii)Preferred single route to each cell type

(iii)Immortality confined to stem cells

(iv)Growth factors permissive or instructive?

Two families of cells?

Colony forming assays reveal myeloid potentials

G-CFU M-CFU

Haematopoietic stem cell

Myeloid progenitor (GEMM-CFU) Lymphoid progenitor

BFU-E GM-CFC Eo-CFC Mast-CFCMeg-CFC Pro-B Pro-T

erythrocyte

monocyte

neutrophil

eosinophil

platelets

mast cell

B lymphocyte

T lymphocyte

CONVENTIONAL MODEL OF HAEMATOPOIESIS

A lymphoid/myeloid dichotomy

Dependent on unique antigen-specific receptor

Dependent on receptor for growth factor

Domain of immunologists

Province of hematologists

B and T lymphocytes are exceptional

More mundane myeloid cells

Haematopoiesis – Principles

(i)Are there two families of cells – myeloid and lymphoid?

(ii)Preferred single route to each cell type

(iii)Immortality confined to stem cells

(iv)Growth factors permissive?

Evidence that precursor cells of monocytes and B lymphocytes are closely related

Wong, Bunce, Lord, Salt & Brown – Exp. Hematol. 1989

HL60 cells restricted to neutrophil differentiation (HL60Ast3)

Lines restricted to monocyte differentiation (U937, ML-1, HL60M2 & 15-12)

Pre-B cell lines (Nalm 6 & SMSB)

3D gels of phosphoproteins

IEF

SDS

1982 – Macrophages from pre-B lymphoma cells (5-AZT & transduced CSF1 R (1990))

1988/94 – HAFTL-1 and 702/3 cell lines

There are bi-potent B lymphocyte/monocyte cells

Bi-potent progenitor

B cell Monocyte

1992 – Foetal liver of mice

2001 – Bone marrow of adult mice(Montecino-Rodriguez et al.)

1995 – Tumours in IL-7 transgenic mice(Fisher et al.)

(Cumano et al.)

Progenitors with lymphoid potentials and an incomplete set of myeloid potentials

Pax5 (B cell factor)-/-

Pro-B cell

Myeloid NK cell T cell

(Balciunaite et al., 2005)

Myeloid

M-CSF

NK cell

IL-2 trans. Notch

T cell

IL-7 Notch

B cell

IL-7

Early Progenitors with Lymphoid and Myeloid develop. potential

Lin-veSca-1+veKit+ve

Flt3hiMpl-ve

Meg/Ery potentials

Gran/Mon potentials

Lymphoid priming

Lymphoid-primed MultiPotent Progenitors (Adolfsson et al., 2005)

Lin-veSca-1+veKit+ve

Flt3hiMpl+ve

LMPP

There isn’t a strict myeloid/lymphoid dichotomy

Haematopoiesis – Principles

(i)Two families of cells – myeloid and lymphoid

(ii)Is there a preferred single route to each cell type?

(iii)Immortality confined to stem cells

(iv)Growth factors permissive?

MegEryBas

Eos

Neu

Mon B

NK

TCMP

CLP

Dendritic cell sub-sets are phenotypically and transcriptionally identical (Ishikawa et al. 2007)

Is there just a single route to each cell type?

There are at least two alternative routes to dendritic cells

Multiple routes to DCs, neutrophils and monocytes

B.

C.

T cellPlatelet

Myeloid

Myeloid

Myeloid

Erythroid

HSCT cell

pro T

ErythroidMEP

MEP

B cell B cell (NK?)

HSC

CMP

Myeloid

??

?

Platelet

Granulocyte/MonocytePlatelet/Erythroid

A.

Platelet/Erythroid

HSC

B cell

T cellNK cell

CLP

CMPGranulocyte/Monocyte

More versatility - thymus progenitors have clandestine myeloid potentials

Thymus-Settling Progenitors

Early Thymocyte Progenitors (DN1)

Double Negative-2 (DN2)

DN-3

CCR9+ve, CD135+ve, CD117high, CD44+ve,

CD25-ve

Loss of CCR9 & CD135 Gain of CD25Loss of CD117 & CD44

Gain of cytoplasmic CD3

B cell

NKDC

Myeloid

B cell

X X

NK DC Myeloid

Haematopoiesis – Principles

(i)Two families of cells – myeloid and lymphoid

(ii)Preferred single route to each cell type

(iii)Is immortality confined to stem cells?

(iv)Growth factors permissive?

STEMCELLS

HAEMOPOIETICPROGENITORS

MATURECELLS

Immortal Lineage selectionExpansion

Self renewal?

FunctionalSelf renewal for

lymphocytes

Immortality extends to lineage-biased cells

Myeloid-biased

Lymphoid-biased

Haematopoiesis – Revised Principles

(i)A single family of cells

(ii)Immortality extends to lineage-biased cells

(iii)More than one route to some cell types

(iv)Progenitors have clandestine options

(v)HSC and progenitors are more versatile than previously thought

HSC CLP

T lymphocytePlatelet Erythrocyte

Mast Cell/ Basophil

Eosinophil

NeutrophilMonocyte

B lymphocyte

The Sequential Determination Model (1985)

Meg

Meg

Ery

Ery Bas

Bas

Eos

Eos

Neu

Neu

Mon

Mon

B

B

T

T

?

G GM M

Neutrophil Monocyte

HL60Ast4 Ast3 Sp1

M2 M4

15-12

Ast25 17-6

Ast1

macrophages from pre-B lymphoma lines

sensitivity to DMSO

(i)A single family of cells(ii)Immortality extends to lineage-biased cells (iii)More than one route to some cell types (iv)Progenitors have clandestine options(v)HSC and progenitors are more versatile than previously thought

HSC CLP

T lymphocytePlatelet Erythrocyte

Mast Cell/ Basophil

Eosinophil

NeutrophilMonocyte

B lymphocyte

A pair-wise relationship model

TRANSCRIPTION FACTORS AND PAIR-WISE RELATIONSHIPS

Knock-out mice Gene expression

eosinophil

basophil

mast cell

erythrocyte

megakaryocyte

neutrophil

monocyte

B lymphocyte

T lymphocyte

GATA-1decreasinglevels

EKLF&NF-E2

GATA-2

C/EBP

} EBF

} GATA-3

c-myb

PU.1

ikarus

GATA-1

FOG-1MLLT3

EKLF

Myb

high

EDAG

PU.1

Notch/CSL

E proteinsPax5

C/EBP

Ery NKBas Neu Mon B TEosMeg DC

Ery NKBas Neu Mon B TEosMeg DC

IL-7 IL-21 IL-15

GM-CSF

M-CSFG-CSF

IL-9

EpoTpo

IL-3IL-5

IL-33IL-4

IL-10

Haematopoiesis – Revised Principles

(i)A single family of cells

(ii)Immortality extends to lineage-biased cells

(iii)More than one route to some cell types

(iv)Progenitors have clandestine options

(v)HSC and progenitors are more versatile than previously thought

Lineage – where to draw the lines?

Lymphocytes

B cellNK cell

T cell

CD8 T cell

T follicular helper

CD4 T cell ?

T helper 1

T helper 2

T helper 17T reg

IFNIL-2, Lt

(IL-10)

IL-4, IL-5IL-13, IL-10

IL-25

IL-17a, IL-17fIL-21, IL-22

IL-10TGF, IL-35

IL-10

IL-4?IL-21

Archetypes?

Accessorised?

Versatility – Are leukaemia stem cells as versatile?

Is decision-making growth factor driven?

Pluripotentstemcell

Unipotentprogenitor

A

B

(i) High level cell indeterminacy Mature cells

(ii) Boundary conditionsGrowth factors, cell-cell contacts

Outcome&

Performance

What is the way forward?

Dr G Brown and Prof A Rot - University of Birmingham; Prof R Ceredig - National University of Ireland, Galway; Prof A Rolink - University of Basel; Prof E Marcinkowska - University of Wroclaw; Prof. G Studzinski - University of Medicine and Dentistry, New Jersey; Drs A Zelent and K Petrie - The Institute of Cancer Research, London; Prof A Kutner - The Pharmaceutical Research Institute, Warsaw; Dr S Elliman - Orbsen Therapeutics Ltd., Galway; Prof N Barnes - Celentyx Ltd., Birmingham. Martin Smith - High-Point Rendel Ltd, LondonProf Michael Danilenko, Ben Gurion University of the Negev, IsrealDr Eustace Johnson, University of AstonProf Daniela Finke, University of Basel