development of the haematopoietic and immune systems

Development of the Haematopoietic and Immune

Systems

Development and Disease Mechanisms

Oct 24th 2003, Lecture 12

Gerald Crabtree

1. Embryonic origins2. 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

Overview of Environment of Embryo/FetusExtra 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

Death due to anemia,granulocytopenia andthrombocytopenia

Lethal IrradiationSurvival 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 donorLethal Irradiation or Lethal ChemotherapyTo kill all malignant cells

Death of tumor cells And survival of patient

40,000 bone marrow transplantations in 1998General Reference:F. Appelbaum Annu. Rev. Med. 2003. 54:491–512

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)

Pair cell assay: E13.5 cortical culture

P-P

Tuj/LeX (CD15)/DAPI

P-N N-N

Symmetric and Asymmetric Divisions of Neural Stem Cells

Brg Acts Cell-Autonomously to Favor Asymmetric Divisions

Lex (CD15) Stem cell markerTuj Differentiated Marker

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

Possible problems: 1) In vitro creation of a stem cell niche 2) Telemeric shortening with sequential passage in culture;

Under the best of circumstances stem cell reconstitution can only be sustained for 1 or 2 mouse passages

Chromosomal Telemeres Shorten with Passage through the Cell Division Cycle

A possible limitation to the sequential passage of haematopoietic stem cells (HSC)

Elizabeth BlackburnCell 2001

Implies the existence of stem cells for each class of blood cell

The Discovery of Colony Forming Units Demonstrates Self Renewal within Lineages

Sequential Steps of Blood Cell Development are Directed by Cytokines

Sequential Steps of Blood Cell Development are Directed by Cytokines

Cytokine A Cytokine B Cytokine C

CommittedStem Cell

Differentiated and Functional blood cell

Instructive Vs Selective Mechanisms of Receptor Action

• (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

Morrison et al Cell 2002

Death of an Anthropomorphism:The Instructive Hypothesis of Receptor Action

H. LodishAnd 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 kinaseLigand)

SCF (Stem cell factor) the product ofthe White locus effects both neural crestand 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

CommonMyeloid Progenitor

• 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.

Feedback control loop

What regulates Erythropoietin (Epo) Production?

• 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

Semenza G.L.Cell. 2001 Oct 5;107(1):1-3

Hypoxia Prevents Degradation of HIF-1

If HIF-1 Controls Epo, what Controls HIF-1?

PHD = proline hydroxylase

Leadville, CO- The birth defect capital of the

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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

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 discriminationis dependent on decoding signal intensity

Key Points

Pax 5 Repression of Notch Shifts Progenitors into the B Cell

LineageCLP (Common Lymphoid Precursor)

B cell T cell

Pax5

Notch Inactive

Pax5 Inactive

Notch Active

M. Busslinger and colleagues

ThymusBone Marrow

Implies stem cells for each class of blood cellHowever T cell colonies are not found in the spleen

Local Factors Influence the Fate of HSC’s

What defines the field in which T cells develop?

Hox-1.5 essential for thymic development And mice lacking Hox-1.5 have no:ParathyroidThyroidSubmaxillary tissue

WHN (winged Helix Nude or HNF3g) mutant micelack a thymus

DiGeorge Syndrome 22q11.2 microdeletionCongenital heart disease-craniofacial abnormalitiesand 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?

Wnt IL-7 TCRTCR

Thymus

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

Low Avidity Self MHCLow Intensity Signal?

High Avidity Self AntigenBound to self MHCHigh Intensity Signal?

Positive Selection

Differentiation andProliferation of cells able tointeract with self MHC

Negative Selection

Death of self reactive cells

Signal Intensity

DefaultDeath

No Signal

J. Sprent and colleagues

T Cell Development: Selection of CD4 and CD8 Cells by MHC

1

What directs the development of CD4 and CD8 Cells?

CD4 interacts withMHC class IIAnd is requiredFor CD4 Cells

CD8 interacts withMHC class IAnd is requiredFor CD8 Cells

ATP-Dependent Chromatin Remodeling Complexes (BAF) and Runx Transcription Factors Control T

Cell Lineage Committement

CD4 Locus CD8 Locus

Cell. 2002 Nov 27;111(5):621-33.Nature. 2002 Jul 11;418(6894):195-9

BAF complexes requiredFor both silencing and activationof CD4 and CD8 genes.

Present Model for Selection of the Immune Repertoire

Cyclosporin A

Bone Marrow Transplantation as a Paradigm of Therapeutics

Based on Understanding Human Developmental

• 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?

IL-2IL-3IL-4GMCSF, etc, etc, etcB Cell growthand DifferentiationFactorsMacrophage GrowthAnd differentiation factorsFas LigandCD40Ligand

Immunosuppressive Drugs Cyclosporin A and FK506 Block Calcineurin/NFAT Signaling and thereby the

Ability of T Cells to Coordinate the Immune Response

s

s

Foreign Antigen

Cyclosporin

A


CD4 Lymphocytes are the Organizer of the Immune Response

s

s

Foreign Antigen


Cyclosporin A Inhibits NFAT-dependent Transcription of the Genes that “Organize” the

Actions of Cells Involved in the Immune Response

Cyclosporin

A

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 BMP

Positive Selection

Negative Selection

DefaultDeath

MotorNeurons

V1 Interneurons

V2 Interneurons

Dorsalfates

Ventralfates

Interfates

Sensor SensorSensor

Where are the Sensors in the Signaling Pathways?

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, Zap70Lat, Slp76Tec

Sensor

DefaultDeath

Bim:BclBak-BaxNegative Selection

Positive 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, 2002Chi et al Immunity 19, 169, 2003Taniuchi I et al Cell 111, 621, 2002Gebuhr et al J. Exp Med 198, 1937, 2003Reyes 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

Signal Intensity Sensor

Fate1

Fate2

Fate3

Fate4

Lowest CommonMediator

Steps Common to each Cell Fate}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 inside- hence 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

• 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.

E. Goldwasser and colleagues

development of the haematopoietic and immune systems

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