Early Lymphoid Progenitors

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Early Lymphoid Progenitors and the T/B Lineage
Decision
IMM429 Lecture 8 Michele K. Anderson,
Ph.D. November 6, 2007
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Classical Hematopoiesis Lymphoid, Myeloid, and
Erythroid/Megakaryocyte Lineages
Are there common intermediates shared between
these different lineages, and can they be
isolated? How?
Katsura, Y. (2002) Redefinition of lymphoid
progenitors. Nature Reviews Immunology. 2, 1-6.
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Strategies for Identification/Isolation of LP
(lymphoid progenitors) 1) Isolate prospective
LP subsets based on cell surface markers 2)
Test lineage potential in clonal and non-clonal
assays Assays for measuring lineage
potential 1) Bone marrow reconstitution of
irradiated host (usually Ly5 markers) 2) In
vitro differentiation -stromal co-culture, OP9
vs. OP9-DL1 -colony forming assays
(methylcellulose) -fetal thymic organ
culture -multilineage potential culture (high
oxygen submersion FTOC)
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Model stepwise progression of development
with early division between lymphoid and myeloid
progenitors
Macrophage
Granulocyte
Eryth/Megakaryocyte
Why do T and B cells appear more closely related?
Bhandoola and Sambandam, 2006, Nat Rev Imm
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Isolation of a common lymphoid progenitor
Isolation of Sca1low ckitlow IL7RThy1- population
from bone marrow What is its lineage potential?
Kondo Weissman, Cell 1997
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Bone marrow chimera assay for lineage potential
Kondo Weissman, Cell 1997
2000 CLPs plus 200,000 bone marrow cells injected
into irradiated mice
Is this a common lymphoid progenitor? What does
the decrease over time mean?
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Bone marrow chimera assay for lineage potential
Kondo Weissman, Cell 1997
2000 CLPs plus 200,000 bone marrow cells injected
into irradiated mice
Is this a common lymphoid progenitor? What does
the decrease over time mean?
YES
Lack of self-renewal ability
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Clonal assay for bipotent T/B precursors
Start methylcellulose to isolate progeny of
single cells Intrathymic injection vs. continued
methylcellulose culture
Kondo Weissman, Cell 1997
Why are clonal assays important? What is an
alternative explanation for the development of
different lineages from a bulk culture?
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The MLP assay tests for T cell, B cell, and
myeloid potential at the single cell level
(clonal assay)
Fetal liver HSCs
Submerged lobe plus cytokines under high oxygen
(70) conditions
Katsura, Y. (2002) Redefinition of lymphoid
progenitors. Nature Reviews Immunology. 2, 1-6.
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Evidence for unipotent and multipotent cells
Katsura, Y. (2002) Redefinition of lymphoid
progenitors. Nature Reviews Immunology. 2, 1-6.
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Evidence for T cell/myeloid and B cell/myeloid
potential But...no evidence for common lymphoid
progenitor!
How might you account for these different results?
Katsura, Y. (2002) Redefinition of lymphoid
progenitors. Nature Reviews Immunology. 2, 1-6.
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Different pathways to lineage commitment
Fetal?
Adult?
Katsura, Y. (2002) Redefinition of lymphoid
progenitors. Nature Reviews Immunology. 2, 1-6.
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TSP thymus seeding progenitors
What is the Nature of Thymus-Seeding Progenitors
(TSPs)? Are they HSCs, CLPs or T-committed?
Initial Colonization of the Fetal Thymus
T-progenitors emerge in FL prior to emergence of
B-progenitors (MLP assay) HSC and T-progenitors
but not B-progenitors are found in fetal blood
at time of colonization E12-13 Fetal thymus
contains T-progenitors, not HSC Collectively,
the data suggest colonization by Tp (or T/NKp)
Ongoing Colonization of the Neonatal and Adult
Thymus LT-HSC ST-HSC (aka MPP) found in
circulation DN1 thymocytes can generate T, B,
NK, and DC after IV adoptive transfer
Uncommitted HSC or CLP will adopt T cell fate
after intrathymic injection Both HSC and CLP
have T cell potential, but which cells actually
colonize the thymus?
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CLP as the direct progenitor of the earliest
thymic immigrants?
T
HSC
MPP
CLP
B
Addressed by studies of bone marrow and thymic
population assays of T cell potential using
stromal cell culture and bone marrow chimeras
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What do the earliest thymic progenitors look like
in terms of cell surface markers?
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Culture each subset six days on OP9-DL1 stroma,
assess proliferative capacity
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Culture each subset six days on OP9-DL1 stroma,
assess proliferative capacity
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Analysis of NK cell and B cell potential of DN1
subsets by OP9 co-culture
All subsets have some T cell potential but
different kinetics and proliferative capacity
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Analysis of NK cell and B cell potential of DN1
subsets by OP9 co-culture
Highest proliferative burst
ckit-high, CD24-low
ckit-high, CD24-int
higher B cell potential
All subsets have some T cell potential but
different kinetics and proliferative capacity
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Are CLP the thymus seeding progenitors? They can
become T cells, but do they in vivo? Earliest
thymic progenitors do not resemble CLPs by
phenotype (Sca-1, c-kit, IL-7R), but look more
like HSCs. Strategy by Porritt et al Separate
DN1 populations and assess their lineage
potential in vitro using two stromal systems.
They also did intravenous injection of DN1a and
DN1b and showed that they can home to the
thymus. Another paper compared bone marrow CLP
to thymic ETP to see whether ETP are direct
descendants of CLP (Allman et al)
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Isolation of an early thymic subset that can
home to thymus and give rise to T cells
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Ikaros a pivotal regulator of hematopoiesis
Loss of Ikaros selectively blocks fetal but not
adult T cell development
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Ikaros-/- mice contain ETPs but lack detectable
BM CLPs.
Bone marrow CLP
Wildtype
HSC, MPP
Bone marrow CLP
Ikaros-/-
HSC, MPP
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ETP
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GFPexpressing Rag-1
ELP
CLP
no GFP
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Kinetics of development reveal which precursor is
"earlier" more developmental steps necessary to
reach the same developmental stage
"ELP"
"CLP"
7 days
14 days
Mac-1
Mac-1
B cell development
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Kinetics of development reveal which precursor is
"earlier" more developmental steps necessary to
reach the same developmental stage
T cell development
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CLP
ELP
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TSPs and ETPs are Functionally Distinct Tan et
al., Nature Immunology, 2005
Thymus
Thymic stromal cells
?
DN2
DN3
CD8
DN4
DP
ETP
?
V ?DJb
CD4
Survival Positive Selection
DC
gd T
B
Active Notch Signaling
NK
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B cell-centric view of lymphocyte development
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Transcriptional Regulation of the T/B lineage
decision
T
MPP
B
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Transcriptional Regulation of the T/B lineage
decision
T
MPP
Notch1-Delta1
B
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Transcriptional Regulation of the T/B lineage
decision
T
MPP
EBF, Pax-5
B
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Transcriptional Regulation of the T/B lineage
decision
T
MPP
Notch-1
Pax-5
B
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Transcriptional Regulation of the T/B lineage
decision
T
LRF
signaling
transcr
MPP
Notch-1
Pax-5
B
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Transcriptional Regulation of the T/B lineage
decision
T
MPP
activity
Notch-1
EBF
B
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A regulatory network for B cell commitment
E.V. Rothenberg (2000) Stepwise specification of
lymphocyte developmental lineages. Curr Opin
Genet Dev. 10(4)370-9.
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A regulatory network for B cell commitment
Notch-1
E.V. Rothenberg (2000) Stepwise specification of
lymphocyte developmental lineages. Curr Opin
Genet Dev. 10(4)370-9.
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CLASS DISCUSSION