Early Lymphoid Progenitors

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Early Lymphoid Progenitors and the T/B Lineage
Decision
IMM429 Michele K. Anderson, Ph.D. October 21,
2008
A journey through time
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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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Experimental approaches for studying hematopoiesis
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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Origins of the now infamous CLP paradigm
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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
Analyzed blood
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
Analyzed blood
Is this a common lymphoid progenitor? What does
the decrease over time mean?
YES (by this assay)
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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Update CLPs are B-primed precursors that can
still be re-directed to the T cell fate by strong
DL-Notch signals They dont normally leave the
bone marrow and traffic to the thymus
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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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Discovery of now famous ETPs
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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Discovery of now famous ETPs
Isolation of an early thymic subset that can
home to thymus and give rise to T cells
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Kinetic analysis of reconstitution
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Intrathymic injection to assess T lineage
potential
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Ikaros-/- mice lack detectable CLPs in the bone
marrow
Bone marrow CLP
Wildtype
HSC, MPP
Bone marrow CLP
Ikaros-/-
HSC, MPP
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Ikaros-/- mice do have ETPs in the thymus
ETP
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Discovery of ELPs
GFPexpressing Rag-1 Knock-in mouse
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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Gene expression patterns suggest that CLPs are
B-primed whereas ELPs are not
CLP
ELP
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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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Adult ETPs can become macrophages but not B cells
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Revised model of Lymphoid and Myeloid
Specification and Commitment
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Transcriptional Control of Lineage Determination
Even if no precursors are bipotent for T/B
lineages, they still must undergo molecular
changes to restrict to either the T or B lineage
Environment Transcription factors
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Transcriptional Control of Lineage Determination
Even if no precursors are bipotent for T/B
lineages, they still must undergo molecular
changes to restrict to either the T or B lineage
Environment Delta-like Notch interactions IL-7 T
ranscription factors Notch, GATA, E2A, HEB PU.1,
E2A, EBF, Pax5
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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

• ETP production from TSPs is Notch-1-dependent
• ETPs have minimal B cell potential that is not
  augmented by limiting Notch-1 activation (after
  intrafemoral injection)
• TSPs have robust T/B potential - intrathymic
  generation of early B cell progenitors is
  augmented by limiting intrathymic Notch-1
  signaling in L-Fng transgenic thymus
• Production of ETPs and thymic B cells from TSPs
  is differentially sensitive to low-threshold
  Notch-1 signals

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Two step process of T cell specification loss
of B cell potential followed by activation of T
lineage gene expression program
Thymus
TSP
ETP
DN2
T
Loss of B cell potential by low DL-Notch signals
Activation of T cell program by high DL-Notch
signals
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DL-Notch interactions promote T lineage and block
B lineage fates
T
MPP
Notch1-Delta-like 4
B
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EBF and Pax-5 promote B cell fate and block T
cell fate
T
MPP
EBF, Pax-5
B
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Enforced expression of Pax-5 leads to
downregulation of Notch-1
T
MPP
Notch-1
Pax-5
B
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Activated Notch-1 inhibits the ability of EBF to
activate gene expression
T
MPP
activity
Notch-1
EBF
B
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A new player in the T/B lineage choice HEBAlt
AD1
LH
bHLH
E2A
HEBCan
ALT
HEBAlt
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HEBAlt is expressed at the DN stage of T cell
development
RT-PCR of sorted cell populations
Wang et al (2006) The bHLH transcription factor
HEBAlt is expressed in pro-T cells and enhances
the generation of T cell precursors. J. Immunol.
177109.
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System to examine influence of HEBAlt on T cell
development
GFP
MIGR1-control
MIGR1-HEBAlt
MIGR1-HEBCan
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HEBAlt increases the percentage and numbers of
DN2 cells in OP9-DL1 co-culture
Overall cell numbers 3X higher for FL-derived
precursors, same for BM-derived precursors
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HEBAlt accelerates entry into the T cell lineage
MIGR1-HEBAlt
MIGR1-control
Day 7 BM on OP9-DL1
CD44
CD44
CD25
CD25
Day 14 BM on OP9-DL1
CD44
CD44
CD25
CD25
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Early induction of T cell specification by
ectopic HEBAlt
DL-N
LSK
DL-N HEBAlt
Appearance over Time
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HEBAlt restores T cell potential to LK cells in
OP9-DL1 co-culture
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HEBAlt restores T cell potential to LK cells in
FTOC
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DL1-N and HEBAlt synergize to redirect cells from
the B cell to the T cell lineage
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