Transcript DB206Crabtree

Development of the
Haematopoietic and Immune
Systems
1. Embryonic origins
2. Bone marrow transplantation as a paridigm for
generating an organ from stem cells
3. Mechanisms of stem cell renewal and
differentiation
4. Specific examples of erythrocyte and lymphocyte
development
Development and Disease
Mechanisms
Oct 24th 2003, Lecture 12
Gerald Crabtree
Overview of Environment of Embryo/Fetus
Extra embryonic membranes
The Developmental
Origin of Blood and
Immune Cells
• Earliest Site of Haematopoiesis
is the Yolk Sac (2-3 weeks) and
Dorsal Aorta (AGM region)
around 3-5 weeks after
conception.
• Yolk sac stem cells are not able
to supply all the blood cell type.
• True haematopoietic stem cells
appear in the liver at about 6
weeks post conception
Yolk sac, transient extra-embryonic structure –
initiation of blood/Hb synthesis
Bone Marrow Transplantation:
Creating an Organ from a Stem Cell
• 20,000 bone marrow transplantations per year in
the US
• Most commonly used for treatment of malignancy
• Also used for treatment of aplastic anemia,
autoimmune disorders, myleodysplastic
syndromes (bone marrow failure) and exposure to
toxins or radiation.
• Rely on the ability of a small number of
Haematopoietic Stem Cells (HSC) to repopulate
the immune and hematopoietic systems
Bone Marrow Transplantation
• Stanford student- Found to have acute myelogenous
leukemia after blood donation
• Treated twice with routine chemotherapy-both attempts
failed
• Immunologically partially compatible cousin identified
• Given lethal chemotherapy, followed by transplantation of
108 marrow cells from cousin.
• Severe infections for 10 days
• Platelet counts and white cell counts began to rise and
reached near normal levels within months
• Continued on Cyclosporin A to suppress graft rejection for
1 year
• Alive and well today- 11 years later with the immune and
haematopoietic system of his cousin
The Atomic Age dawned at 5:29:45 am on July 16,
1945, at Trinity Site, New Mexico
The Discovery of Stem Cells
Lethal Irradiation
Transfusion of blood
or bone marrow from a
normal donor
Lethal Irradiation
Death due to anemia,
granulocytopenia and
thrombocytopenia
Survival of a significant
number of irradiated
individuals
What does blood or bone marrow have that allows
the survival of irradiated individuals and the appearance
Of white cells, red cells and platalets?
Reconstitution of the Entire
Haematopoietic System by Bone
Marrow Transplantation
Transfusion of blood
or bone marrow from a
normal donor
Lethal Irradiation or
Lethal Chemotherapy
To kill all malignant cells
40,000 bone marrow
transplantations in 1998
General Reference:
F. Appelbaum
Annu. Rev. Med. 2003. 54:491–512
Death of tumor cells
And survival of patient
Donor Provides:
Red cells,
platelets,
white cells,
pulmonary alveolar macrophages,
Kupffer cells of the liver,
osteoclasts,
Langerhans cells of the skin,
and microglial cells of the brain
Can HSCs give rise to other cell
types?
• Early reports indicated
that muscle, neurons,
hepatocytes and cardiac
muscle might derive from
adult HSC.
• More recent reports
suggests that HSC fuse
with other cell types and
hence acquired their
markers
– Science 297, 2256, 2003
A
A
Experimental Paradigm for Study
of Haematopoietic Stem Cells
Many types of cells originate from a single
type of haematopoietic stem cell (HSC)
Possible Mechanisms for
Maintaining a Stem Cell Population
A. Asymmetric Divisions
B. Symmetric Divisions
C. Locally Directed Divisions (Niche directs
differentiation after a symmetrical division)
Symmetric and Asymmetric Divisions of Neural Stem Cells
Tuj/LeX (CD15)/DAPI
P-P
P-N
N-N
Lex (CD15) Stem cell marker
Tuj Differentiated Marker
Brg Acts Cell-Autonomously to Favor Asymmetric Divisions
Pair cell assay: E13.5 cortical culture
Maintaining Long Term Haematopoietic
Stem Cells in Culture: A Major
Unsolved Therapeutic Goal
• Soluble factors that maintain HSCs:
– SIF, Flt3L, Tpo, IL-3
– Wnt, Notch and Sonic Hedgehog (Shh)
• Transcription factors that increase the replication of HSC
– HoxB4 and A9
Under the best of circumstances stem cell reconstitution
can only be sustained for 1 or 2 mouse passages
Possible problems:
1) In vitro creation of a stem cell niche
2) Telemeric shortening with sequential passage in culture;
Chromosomal Telemeres Shorten with
Passage through the Cell Division Cycle
Elizabeth Blackburn
Cell 2001
A possible limitation to the sequential passage of
haematopoietic stem cells (HSC)
The Discovery of Colony Forming
Units Demonstrates Self Renewal
within Lineages
Implies the existence of stem cells for each class of blood cell
Sequential Steps of Blood Cell
Development are Directed by Cytokines
Sequential Steps of Blood Cell
Development are Directed by Cytokines
Cytokine A
Committed
Stem Cell
Cytokine B
Cytokine C
Differentiated and
Functional blood cell
Instructive Vs Selective
Mechanisms of Receptor Action
Morrison et al Cell 2002
•
(A and B) Selective mechanism in
which two different factors (F1 and F2)
allow the survival and maturation of
lineage-committed progenitors
generated by a cell-autonomous
mechanism; “X” indicates death of the
other progenitors. Erythropoietin
•
(C and D) Instructive mechanism in
which the factors cause the stem cell to
adopt one fate at the expense of others.
Glial growth factor and BMP2
Death of an Anthropomorphism:
The Instructive Hypothesis of Receptor Action
H. Lodish
And colleagues
If Cytokines Do not Give
Instructive Signals…
Cytokines probably provide permissive
signals that are dependent on the
developmental history of a cell
_______
Developmental history is reflected by the
expression of receptors, signaling
molecules, transcription factors and
chromatin accessibility
The Development of T Lymphocytes and
Red Cells
IL# (interleukin general name for
haematopoietic growth factors
SDF-1 (stomal cell Derived factor)
FLT-3 or Flk2 (Fems like tryosine kinase
Ligand)
SCF (Stem cell factor) the product of
the White locus effects both neural crest
and haematopoietic cell development. Binds
C-kit, mutation of which has near identical
Phenotype as SCF mutations.
Epo- Erthropoietin
Tpo- thrombopoietic factor
GM-CSF granulocyte macrophage
stimulating factor
G-CSF granuloctye stimulating factor
Development of Red Blood Cells
Common
Myeloid
Progenitor
Feedback control loop
• First red cells are produced in the yolk sac. Later red cell
production shifts to the liver, spleen and then the bone
marrow.
• Feedback control of RBC Production is through
Erythropoietin (Epo).
– Necessary to prevent death and promote proliferation of committed
precursors
– Shifts non-committed progenitor cells into the erythroid lineage
– Produced in renal tubular epithelial cells and more widely in the
growing embryo
– Feedback control targets the first committed cell in the erythroid
lineage.
What regulates Erythropoietin (Epo)
Production?
Semenza G.L.Cell. 2001 Oct 5;107(1):1-3
• Epo is regulated
transcriptionally by an
regulatory region near the
gene
• This regulatory region
binds HIF (Hypoxia
Induced Factor)
• Hypoxia regulates HIF
• HIF also activates VEGF
and induces
vasculogenesis- a problem
in pregnancy
If HIF-1 Controls Epo,
what Controls HIF-1?
Hypoxia Prevents Degradation of
HIF-1
PHD = proline hydroxylase
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Anemia stimulates HIF and HIF stimulates
VEGF and VEGF induces inappropriate
angiogenesis and other patterning defects.
Erythropoietin: The Drug
•
•
•
•
Erythropoietin is given for intractable anemia
Best for chronic renal disease
Ineffective in some cases of aplastic anemia
Also effective for increasing blood production for
preoperative storage of autologous blood.
Lymphocyte Development
Key Points
1) The role of a developmental field in
lymphocyte specification.
2) Lineage specification in T cells
is dependent on chromatin control.
3) Self vs Non-self discrimination
is dependent on decoding signal intensity
Pax 5 Repression of Notch Shifts
Progenitors into the B Cell
Lineage
M. Busslinger and colleagues
CLP (Common Lymphoid Precursor)
Pax5
Pax5 Inactive
Notch Inactive
Notch Active
B cell
T cell
Bone Marrow
Thymus
Local Factors Influence the Fate
of HSC’s
Implies stem cells for each class of blood cell
However T cell colonies are not found in the spleen
What defines the field in which T
cells develop?
Hox-1.5 essential for thymic development
And mice lacking Hox-1.5 have no:
Parathyroid
Thyroid
Submaxillary tissue
WHN (winged Helix Nude or HNF3g) mutant mice
lack a thymus
DiGeorge Syndrome 22q11.2 microdeletion
Congenital heart disease-craniofacial abnormalities
and thymic aplasia
Molecular Anatomy of the Microdeletion
in DiGeorge Syndrome
• Microdeletion of 22q11.2 occurs in 1/4000 births
• Tbx gene implicated in congenital heart defects
• Basis for thymic aplasia is still unknown
T Cell Development:
How do lymphocytes tolerate self antigens yet
respond to foreign antigens?
Thymus
Wnt
IL-7
TCR
TCR
If we can make new organs from
embryonic stem cells they will still be rejected unless
we can also control lymphocyte development.
Current View of Selection of the Immune
Repertoire
J. Sprent and colleagues
No Signal
High Avidity Self Antigen
Bound to self MHC
High Intensity Signal?
Low Avidity Self MHC
Low Intensity Signal?
Signal Intensity
Default
Death
Positive Selection
Negative Selection
Death of self reactive cells
Differentiation and
Proliferation of cells able to
interact with self MHC
T Cell Development: Selection of
CD4 and CD8 Cells by MHC
CD4 interacts with
MHC class II
And is required
For CD4 Cells
CD8 interacts with
MHC class I
And is required
For CD8 Cells
1
What directs the development of CD4 and CD8 Cells?
ATP-Dependent Chromatin Remodeling Complexes
(BAF) and Runx Transcription Factors Control T
Cell Lineage Committement
Cell. 2002 Nov 27;111(5):621-33.
Nature. 2002 Jul 11;418(6894):195-9
BAF complexes required
For both silencing and activation
of CD4 and CD8 genes.
CD4 Locus
CD8 Locus
Present Model for Selection of the
Immune Repertoire
Bone Marrow Transplantation as a Paradigm
of Therapeutics
Based on Understanding Human Developmental
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•
•
•
•
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Endocrine pancreas
Skin
Bone
Joint surface and articular cartilage
Kidney
Liver
Lung
Heart
Eye
Brain???
T Cell Development: Selection of
CD4 and CD8 Cells by MHC
1
How do lymphocytes come to be self tolerant,
yet react with foreign antigen?
Immunosuppressive Drugs Cyclosporin A and FK506
Block Calcineurin/NFAT Signaling and thereby the
Ability of T Cells to Coordinate the Immune Response
Foreign Antigen
s
s
IL-2
IL-3
IL-4
GMCSF, etc, etc, etc
B Cell growth
and Differentiation
Factors
Macrophage Growth
And differentiation factors
Fas Ligand
CD40Ligand
CD4 Lymphocytes are the Organizer of the
Immune Response
Foreign Antigen
s
s
IL-2
IL-3
IL-4
GMCSF, etc, etc, etc
B Cell growth
and Differentiation
Factors
Macrophage Growth
And differentiation factors
Fas Ligand
CD40Ligand
Cyclosporin A Inhibits NFAT-dependent
Transcription of the Genes that “Organize” the
Actions of Cells Involved in the Immune Response
IL-2
IL-3
IL-4
GMCSF, etc, etc, etc
B Cell growth
and Differentiation
Factors
Macrophage Growth
And differentiation factors
Fas Ligand
CD40Ligand
Signal Strength Theory of Self vs
Non-Self Discrimination
• High intensity signals generated by abundant self antigens
kill self reactive T cells (Negative Selection)
• Low intensity signals generated by MHC interactions
select CD4 and CD8 T cells (Positive Selection)
• Lympocytes the generate a receptor unable to bind antigen
or MHC die by neglect
Analogue to Digital Switches in
Development
TCR
Hedgehog
Sensor
Default
Death
Positive
Selection
Negative
Selection
BMP
Sensor
V1 Inter Motor
neurons Neurons
V2 Inter
neurons
Sensor
Dorsal
fates
Inter
fates
Where are the Sensors in the Signaling Pathways?
Ventral
fates
If we can make new organs from
embryonic stem cells they will be
rejected unless we can also
control lymphocyte development.
The Lineages of Blood Cells are
Marked by Cell Surface Proteins
Calcineurin-NFAT Signaling in
Shaping the Immune Repertoire
CD3, Zap70
Lat, Slp76
Tec
Sensor
Default
Death
Positive Selection
Bim:Bcl
Bak-Bax
Negative
Selection
Bone Marrow Transplantation:
Creating both the Haematopoietic
and Immune Systems from a Stem
Cell
ATP dependent BAF Chromatin Remodeling Complexes
Bind to the CD4 Silencer to Regulate T Lymphoctye
Lineage Determination
Chi et al Nature 418, 195, 2002
Chi et al Immunity 19, 169, 2003
Taniuchi I et al Cell 111, 621, 2002
Gebuhr et al J. Exp Med 198, 1937, 2003
Reyes et al EMBO 23, 6979, 2002
•BAF Subunits are Haploinsufficient for both CD4 Silencing and CD8 Activation
•Silencing requires the CD4 Silencer
•BAF complexes bind directly to the silencer
•Later the same complexes are required for activation of CD4
An Approach to Understanding
Analogue-to-Digital Signaling Switches
}
Steps Common to each Cell Fate
Signal Intensity Sensor
Lowest
Common
Mediator
}
Fate
1
Fate
2
Fate
3
Fate
4
Dedicated Steps
The Origins of Blood Forming Cells
•
•
•
Yolk Sac Stem cells are unable to rescue lethally irradiated
individuals and give rise mostly to primitive nucleated red cells.
True stem cells appear as the Yolk sac cells and cells from the AGM
(aorta/gonad/mesonephros) past to the liver.
Still later blood development moves to the bone marrow.
Prolactin Can Direct Breast
Development using
Erythropoietin Signals
Receptor Switch Experiments Indicate that
Cytokine Signals are Permissive not
Instructive
• Introduced a chimeric
receptor that bound
Prolactin (Prl) on the
outside of the cell, but
used the erythropoietin
receptor on the insidehence it gave
Erythropoietin signals in
response to Prolactin in an
Prolactin Receptor
knockout mouse
Macrophages are Derived from a Myeloid Progenitor
Cell
M-CSF- Macrophage/monocyte Colony
Stimulating Factor
Polymorphonuclear Leukocytes: Eosinophils,
Basophils and Neurotrophils from a Myeloid
Progenitor Cell
Megakaryocytes from a Myeloid Progenitor Cell
Osteoclasts are Derived from the Common Myeloid
Progenitor Cell
Differentiation of Osteoclasts
from CFU-CM Progenitors
Lymphocytes Originate from a HSC through the
Common Lymphoid Percusor
Erythropoietin (Epo): The
Developmental Regulator
E. Goldwasser and colleagues
• Necessary to prevent cell death of committed precursors
• Shifts non-committed progenitor cells into the erythroid
lineage
• Probably also favors proliferation of committed erythroid
precursor cells
• Produced in renal tubular epithelial cells and more widely
in the growing embryo.