Transcript Powerpoint Lineage Commitment

Lineage Commitment During T
Cell Development
Art Weiss & Mark Anderson
10/19/16
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. How
SOX13 is regulated is not clear.
Bcl11b, a zinc finger transcription factor, is preferentially expressed in ab
lineage T cells and is induced in DN2a-DN2b, before the is expressed. 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.
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.
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.
How does a DP decide it is a CD4 or
CD8?
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
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
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.
10000
12
82.3
Medulla
1000
SP (CD4)
100
DP
Cortex
10
DN
CD4
3.4
1
1
2.34
10
100
1000
10000
SP (CD8)
Medulla
CD8
DN
Cortex
DP
SP (CD4)
Medulla
SP (CD8)
Exit to the body
Class II KO mice inject in vivo with antiCD3: Look in the thymus
Kappes et al.
Immunity 2008
Instructive model: A key transition
step CD4+CD8lo
Al Singer Curr Opinion Immunology 2002
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.