Transcript Lineage Commitment During T cell Development

Summary of Thymic Development
Zuniga-Pflucker, NRI, 2004
What do we mean by the term
“Lineage Commitment”?
What do we mean by the term
“Lineage Commitment”?
Commitment represents the loss in the ability of a cell to
make alternative lineage choices under permissive conditions.
Name the Lineage Commitment
Choices that are made during T cell
Development
Lineage Commitment Decisions During
T Cell Development
•
•
•
•
To become a T cell
To become an TCRab cell vs a TCRgd T cell
To become a CD4 or CD8 T cell
To become one of the minor T cells subset
NK T cell
CDaa T cell
nTreg
First Lineage Commitment Decision in
T Cell Development
To be, or not to be, a T cell
Seeding the Thymus from Precursors in
the Blood
Bhandoola, et al., Immunity, 26:678-689, 2007
HSC Hematopoietic Stem Cell
MPP Multipotent Progenitor
ELP Early Lymphoid Progenitor
CLP Common Lymphoid Progenitor
CMP Common Myeloid Progenitor
CTP Circulating T cell Progenitor
TSP Thymus Settling Progenitors
ETP Early Thymic Progenitor
Is the ETP, recently arrived
in the thymus, already committed to the
T cell lineage?
Alternative Fates of the Early Thymic Progenitor
Yui and Rothernberg, NRI, 2014
A critical role for Notch in T cell lineage commitment
Zuniga-Pflucker, NRI, 2004
How would you demonstrate the importance of
Notch in T cell lineage commitment of ETPs?
OP9 stromal cells transfected with Deltex-1 can support
T Cell Lineage Commitment
Zuniga-Pflucker, NRI, 2004
Schmitt, et al, JEM, 2004
ab T Cell Development
Yui and Rothernberg, NRI, 2014
Notch signaling is critical for the induction of multiple
transcription factors
Yui and Rothernberg, NRI, 2014
Early Notch signaling induces TCF-1 (Tcf7) and Gata3 at
The DN1 -> DN2 transition
Yui and Rothernberg, NRI, 2014
How would you test the importance of one of
the transcription factors (for instance, TCF-1)
that is upregulated during early Notch signaling
for T cell lineage development?
Impaired development of TCF-1-deficient BM HSC
into CD25+ (i.e.,DN2) thymocytes in OP9 cultures
Weber, et al., Nature, 2011
Defects also observed in vivo using competitive repopulation studies of WT vs TCF-1 KO BM
Evolving Transcriptional Networks as Notch Influences Early T Cell Development
Yui and Rothernberg, NRI, 2014
DN Cells (CD4-/CD8-)
DN1
DN2
DN3
DN4
Sequential Rearrangement of TCR ab Genes
DN
DP
Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003
The pre-TCR is Expressed in DN cells
g
Pre-Ta functions as a surrogate for the a chain during thymic development
Expressed in DN cell and heterodimerizes with a functional b chain - assists in quality
control for b chain rearrangement
The pre-Ta/b chain dimer promotes increased CD3 expression and induces a ligandindependent signal, perhaps because of constitutive localization to lipid rafts or
constitutive dimerization (unusual preTalpha structure), that is responsible for
maturation and probably shut off RAG expression and further rearrangement,
resulting in b chain allelic exclusion
How would you test the ligand-independency of
signaling by the pre-TCR?
Reconstitution of rag1-/- mice with various forms of the
pre-TCR
Irving, et al., Science, 1998
Ligand-Independent Dimerization of the pre-TCR
Pang, et al, Nature 2010
Extended structure of Pre-Ta compared to TCR Ca
Dimer of Heterodimers of pre-Ta and TCR b
Two Lineages of Cells Expressing Distinct TCRs Develop
in the Thymus:
Stages of gd and ab T Cell Development
Modified from Ciofani and Zuniga-Pflucker, Nature Rev. Immunol., 2010
(C-Kit)
Two Lineages of T cells (cont.)
Stochastic Instructive model
Recent data suggest that gd receptor expression results in stronger signal that
can provides instructional cue for cell to become gd lineage
( Reviewed in Ciofani, et al., Nat. Rev. Immunol. 2010)
Why might the pre-TCR and the TCRgd signal
strengths be different differently?
Strength of signal: a fundamental mechanism
for cell fate specification
Immunological Reviews
Volume 209, Issue 1, pages 170-175, 31 JAN 2006 DOI: 10.1111/j.0105-2896.2006.00356.x
http://onlinelibrary.wiley.com/doi/10.1111/j.0105-2896.2006.00356.x/full#f3
Reciprocal regulation of Syk and ZAP-70 expression
during thymocyte development
Palacios & Weiss, JEM, 2007
WT DN3
Development
b-selection
TCR selection
CD44CD25+
DN3
Strength of signal: a fundamental mechanism
for cell fate specification
Immunological Reviews
Volume 209, Issue 1, pages 170-175, 31 JAN 2006 DOI: 10.1111/j.0105-2896.2006.00356.x
http://onlinelibrary.wiley.com/doi/10.1111/j.0105-2896.2006.00356.x/full#f1
How might you test the signaling
hypothesis?
Starting with a TCRgd Transgene:
Increasing signaling strength by elimination of a negative
regulator, CD5, favors gd lineage commitment
Hayes, et al., Immunity, 2005
Instructing ab vs gd Lineage Commitment via
Strength of Signal
Ciofani and Zuniga-Pflucker, Nat. Rev. Immunol. 2010
Some caveats to the strength of signaling
Stochastic Instructive model
SOX13, an SRY-related HMG-box transcription factor, is preferentially expressed
in gd T cells. Its KO, decreases gd development. Its over-expression in DN
thymocytes impairs DN -> DP transition and ab T cell development.
Bcl11b, a zinc finger transcription factor, is preferentially expressed in ab
lineage T cells and is induced in DN2a-DN2b. It is low in the gd lineage. The KO
of Bcl11b has little effect on gd T cell development but completely impairs ab
lineage T cell development.
There’s more to come.
Checkpoints in Thymocyte Development
Modified from Carpenter and Bosselut, Nature Immunology 2010
Notch
Pre-TCR
TCRab
Linking CD4 (helper) to CD8 (killer) T cell lineage commitment to the recognition of
class I versus class II MHC ensures that T cell effector functions are directed
appropriately.
MHC class I pathway samples
intracellular antigens (e.g. viruses,
intracellular bacteria).
MHC class II pathway samples
extracellular antigens
Cytotoxic CD8 T cells can kill invaded
host cells before pathogens can
replicate and spread.
Helper T cells regulate the activity of
other cells of the immune system
that have endocytosed pathogens.
Recognition of MHC-1 or MHC-2 during positive selection in the thymus
determines the CD4 versus CD8 T cell lineage choice.
Recognition of MHC-1 or
MHC-2 during positive
selection in the thymus
determines the CD4 versus
CD8 T cell lineage choice.
CD4
CD8
MHC-1
CD8
MHC-2
CD4
Early evidence of the link between TCR specificity for MHC1 vs. MHC-2 and the CD4 vs. CD8 lineage choice:
TCR transgenic mice and MHC gene ko mice.
Generation of transgenic mice bearing rearranged TCR genes with defined
specificity provided important tools for study of positive selection.
T cell receptor transgenic mice:
Antigen (e.g., ovalbumin)
Rest and restimulate
with Ag and
cytokines, rest,
repeat
9 days
T cells + DCs + ovalbumin
plate at 1 cell/well
Test for antigen
specificity
grow and expand
clone the TCR alpha and beta chain genes
from the T cell clone
T cell receptor
alpha- & beta-chain genes
specific for MHC class I or
MHC class II
OT-1 - CD8 T cells
AND - CD4 T cells
All T cells will express the same TCRab receptor
Increased development of CD4 SP thymocytes and T cells
in mice expressing a rearranged class II MHC specific
transgene
57.4
30.6
9.6
CD4
CD8
0.69
Generating H-Y TCR transgenic mice
(anti-male peptide TCR)
Male H-2b cells
9 days
Female
H-2b mouse
Expand anti-male
CD8 T cells
Rest and restimulate
with Ag and
cytokines, rest,
repeat
plate at 1 cell/well
Test for antigen
specificity
grow and expand
clone the TCR alpha and beta chain genes
from the T cell clone
Increased development of
CD8 SP thymocytes and T
cells in mice expressing a
rearranged class I MHC
specific transgene
5%
37%
10%
46%
TCR specificity for MHC I or II determines CD4
versus CD8 lineage commitment
OT1, HY, F5, etc
OT2, DO11.10, AND, etc
MHC deficient mice provide evidence for positive selection.
Lack of MHC class II expression prevents development of CD4 cells
WT
MHC Class II o
CD4
CD4
. . . .. .. . . .
……. .. .
. .. .. ...
.. . .
CD8
CD8
And lack of MHC class I expression (b2-microglobulin deficient
mice)
prevents development of CD8 T cells.
MHC class I and II double deficient mice lack both CD4 and CD8
mature T cells, but have normal numbers of DP thymocytes.
CD4
CD8
MHC-1
CD8
MHC-2
CD4
What are the gene expression programs that
determine the CD4 or CD8 T cell fate?
How are distinct gene expression programs linked
to TCR recognition of MHC class I or class II?
CD4
CD8
MHC-1
CD8
MHC-2
CD4
What are the gene expression programs that
determine the CD4 or CD8 T cell fate?
How are distinct gene expression programs linked
to TCR recognition of MHC class I or class II?
How would you identify transcription factors
involved in CD4 versus CD8 lineage commitment?
How would you identify transcription factors
involved in CD4 versus CD8 lineage commitment?
Gene profiling mature CD4 vs CD8 T cells to identify
differentially expressed TF.
Identify TF that regulate key CD4 vs CD8 specific target
genes (CD4, CD8, perforin, CD40L, etc)
Gene KO of candidate TF and assess impact on CD4, CD8 T
cell development. (embryonic lethality, blocks at earlier
stages of development (T commitment, TCRb checkpoint,
etc)
Serendipity
Th-POK (c-KROX)--a “master regulator” of
CD4 T cell lineage commitment
1997: “HD” mouse strain: lacks CD4 T cells
spontaneous mutation in D. Kappes’
animal colony
Autosomal recessive
Not due to defect in MHC Class II
Genetic mapping and BAC complementation used
to discover the mutant gene: Th-POK
The “HD” allele has a single point mutation in a
zinc-finger domain
CD4
Block in CD4 T cell development, or “lineage swap”? How could you tell?
CD8
He et al. Nature 2005
Mutation in ThPOK
leads to the
development of
“mismatched” class II
specific, CD8 T cells
CD4
Uncoupling between positive selection and lineage commitment.
CD8
Over-expression of ThPOK in thymocytes lead to reciprocal phenotype:
CD4 T cells with TCR specific for MHC-1.
OT1 TCR tg
OT1 x ThPOK tg
MHC-2 ko
MHC-2 ko x ThPOK tg
ThPOK acts together with other
transcription factors in a network
that specifies the CD4 fate.
GATA-3 is required for ThPOK to induce the CD4 fate (but not to repress CD8 fate).
GATA-3 promotes ThPOK expression.
Loss of GATA-3 can (sometimes) divert thymocytes with TCR specific for MHC-2 into
the CD8 lineage.
Wang et al Nat Immunol. 2008 Oct;9(10):1122
ThPOK reinforces its own expression and acts together with other
transcription factors in a network that specifies the CD4 fate.
ThPOK opposes Runx repression of
CD4 and ThPOK expression.
Mutation of both Runx and ThPOK
rescues CD4 development.
Mutant ThPOK allele than cannot
undergo positive autoregulation leads
to “trans-differentiation” of CD4 cells
to the CD8 lineage.
Egawa and Littman and Miroi et al Nat
Immunol. 2008
TRANSCRIPTION FACTOR NETWORK CONTROLING CD4 VS CD8 FATE:
THE SIMPLE VERSION
MHC-2 positive
selection
Autoregulation
ThPOK
CD4
MHC-1 positive
selection
Mutual antagonism
RUNX3
CD8
TRANSCRIPTION FACTOR NETWORK CONTROLING CD4 VS CD8 FATE:
MORE PLAYERS
MHC-2 positive
selection
MHC-1 positive
selection
cMyb
MAZR
GATA3
ThPOK
Tox
RUNX3
MAZR
ThPOK
CD8
CD4
RUNX3
CD4
CD8
Would you consider ThPOK to be a
“master regulator” of the CD4 fate. Why
or why not?
CD4
CD8
MHC-1
CD8
MHC-2
CD4
What are the gene expression programs that
determine the CD4 or CD8 T cell fate?
How are distinct gene expression programs linked
to TCR recognition of MHC class I or class II?
Relating the transcription factor networks that control CD4 versus
CD8 to recognition of MHC-1 or MHC-2 during positive selection.
An “instructive” model?
More
prolonged
signal
More
transient
signal
A kinetic model for CD4/CD8 development: duration of TCR signals influence lineage choice.
But clearly not the whole story. Quantitative model cannot adequately account for the
stringent relationship between MHC-1 vs MHC-2 specificity and positive selection.
Another mechanism to ensure that thymocytes adopt the CD4/CD8
fate appropriate for their TCR specificity:
Some thymocytes make the “wrong” lineage choice, but a late check for coreceptor expression eliminate cells that turned down the wrong co-receptor.
Thymocytes expressing
TCR specific for MHC-1
A stochastic/selection model?
Constitutive expression of CD8 (or CD4) leads to the (inefficient)
development of “mismatched” T cells
Davis et al ‘93, Itano et al ‘94, Robey et al ‘94, Baron et al ‘94, Corbella et al ‘94 Paterson et al
‘94, Chan et al ‘94, Rathemtulla et al, ‘02
How long does a thymocyte need to engage MHC and receive
TCR signals in order to complete positive selection?
How long does it take for a thymocyte to commit to the
CD4 or CD8 lineage?
Typical time required from initial encounter with
extracellular ligand to changes in gene expression?
Multiple changes accompany positive
selection
Pre selection thymocytes are:
In cortex
CD4+CD8+
TCRneg-low
rag+
low expression of activation
markers (CD69-)
Short-lived, dep on TCR signal
post selection thymocytes are:
In medulla
CD4+CD8- or CD4-CD8+
TCRhigh
ragCD69+
Long-lived, indep of TCR signal
These changes occur asynchronously over a period of 1-3 days.
Can identify thymocytes exhibiting some, but not all signs of positive
selection.
Would you say that a cortical CD4+CD8+ CD69+ rag- thymocyte has
been positively selected?
How long does a thymocyte need to engage MHC and receive TCR signals in order to
complete positive selection?
“Antigen-experienced” CD4+CD8+ thymocytes isolated from TCR trangenic positive
selecting mice (HY TCR, H2b) and intrathymically injected into mice with or without
the positive selecting ligand.
A single hit is not sufficient for
positive selection of CD8 T cells:
Recent data from Art and Ellen’s lab showing that 2-3 days of continuous TCR
signaling needed for efficient positive selection of CD8 T cells
(Au-Yeong et al, Ross et al 2014)
How long does it take for a thymocyte to commit to the
CD4 or CD8 lineage?
Lingeage commitment represents the loss in the ability
of a cell to make alternative lineage choices
under permissive conditions.
Impact of removing ThPOK after
CD4 lineage commitment:
What does TCR “signal strength” really mean?
TCR affinity (signal strength)
Death by
neglect
Positive
selection
CD4 lineage
CD8 lineage
commitment commitment
TCR affinity for peptide-MHC complex?
Duration, frequency, or intensity of TCR signaling?
Presence/absence of co-stimulatory signals?
Clonal
deletion
T reg
development
(agonist
selection)
Temporal requirement for TCR signaling during CD8 T cell
positive versus negative selection?
days
hours
Temporal pattern of TCR signaling during positive versus
negative selection?
intermittent
Why does positive
selection take 2-3 days?
continuous
How do distinct temporal
signaling patterns encode
distinct T cell fates?
Positive selection
and CD4 versus CD8
lineage development
may be lengthy,
reversible processes.
Interpretation of TCR
signaling difference and
establishment of stable
gene regulatory network for
CD4 or CD8 fate likely occur
at the same time and may
be mechanistically linked.
CD4 loop
CD8 loop
MHC-2 recognition:
stronger, continuous signal
MHC-1 recognition:
weaker, intermittent signal
GATA3
RUNX3
RUNX3
ThPOK
ThPOK
CD8
CD8
CD4
Maintain
CD4 expression
allowing for late
MHC-2 recognition
Maintain
CD8 expression
allowing for late
MHC-1 recognition
Loops can be initiated by biasing signals, and/ or stochastic fluctuations.
Positive feedback makes lineage choices more robust and allows for re-verification.