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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.

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slide1

Summary of Thymic Development

Zuniga-Pflucker, NRI, 2004

slide3

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.

lineage commitment decisions during t c ell 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

slide7

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

slide8

Is the ETP, recently arrived

in the thymus, already committed to the

T cell lineage?

slide9

Alternative Fates of the Early Thymic Progenitor

Yui and Rothernberg, NRI, 2014

A critical role for Notch in T cell lineage commitment

slide11

How would you demonstrate the importance of

Notch in T cell lineage commitment of ETPs?

slide12

OP9 stromal cells transfected with Deltex-1 can support

T Cell Lineage Commitment

Zuniga-Pflucker, NRI, 2004

Schmitt, et al, JEM, 2004

slide13

ab T Cell Development

Yui and Rothernberg, NRI, 2014

slide14

Notch signaling is critical for the induction of multiple

transcription factors

Yui and Rothernberg, NRI, 2014

slide15

Early Notch signaling induces TCF-1 (Tcf7) and Gata3 at

The DN1 -> DN2 transition

Yui and Rothernberg, NRI, 2014

slide16

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
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

slide18

Evolving Transcriptional Networks as Notch Influences Early T Cell Development

Yui and Rothernberg, NRI, 2014

slide19

DN Cells (CD4-/CD8-)

DN1 DN2 DN3 DN4

slide20

Sequential Rearrangement of TCR ab Genes

DN DP

Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003

slide21

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 ligand-

independent 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  chain allelic exclusion

slide24

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

slide25

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)

slide26

Two Lineages of T cells (cont.)

Stochastic Instructive model

Recent data suggest that receptor expression results in stronger signal that

can provides instructional cue for cell to become  lineage

( Reviewed in Ciofani, et al., Nat. Rev. Immunol. 2010)

slide28

Why might the pre-TCR and the TCRgd signal

strengths be differentdifferently?

strength of signal a fundamental mechanism for cell fate specification
Strength of signal: a fundamental mechanism for cell fate specification

Immunological ReviewsVolume 209, Issue 1, pages 170-175, 31 JAN 2006 DOI: 10.1111/j.0105-2896.2006.00356.xhttp://onlinelibrary.wiley.com/doi/10.1111/j.0105-2896.2006.00356.x/full#f3

slide30

CD44-

CD25+

DN3

Reciprocal regulation of Syk and ZAP-70 expression

during thymocyte development

Palacios & Weiss, JEM, 2007

WT DN3

-selection

Development

TCR selection

strength of signal a fundamental mechanism for cell fate specification1
Strength of signal: a fundamental mechanism for cell fate specification

Immunological ReviewsVolume 209, Issue 1, pages 170-175, 31 JAN 2006 DOI: 10.1111/j.0105-2896.2006.00356.xhttp://onlinelibrary.wiley.com/doi/10.1111/j.0105-2896.2006.00356.x/full#f1

slide33

Starting with a TCRgd Transgene:

Increasing signaling strength by elimination of a negative

regulator, CD5, favors gd lineage commitment

Hayes, et al., Immunity, 2005

slide34

Instructing ab vs gd Lineage Commitment via

Strength of Signal

Ciofani and Zuniga-Pflucker, Nat. Rev. Immunol. 2010

slide35

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 thymocytesimpairs 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.

slide37

Checkpoints in Thymocyte Development

Modified from Carpenter and Bosselut, Nature Immunology 2010

Notch Pre-TCR TCRab

slide38

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

Helper T cells regulate the activity of other cells of the immune system that have endocytosed pathogens.

Cytotoxic CD8 T cells can kill invaded host cells before pathogens can replicate and spread.

slide39

Recognition of MHC-1 or MHC-2 during positive selection in the thymus determines the CD4 versus CD8 T cell lineage choice.

slide40

CD4

CD8

Recognition of MHC-1 or MHC-2 during positive selection in the thymus determines the CD4 versus CD8 T cell lineage choice.

MHC-1

MHC-2

CD8

CD4

Early evidence of the link between TCR specificity for MHC-1 vs. MHC-2 and the CD4 vs. CD8 lineage choice:

TCR transgenic mice and MHC gene ko mice.

slide41

Generation of transgenic mice bearing rearranged TCR genes with defined specificity provided important tools for study of positive selection.

slide42

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

slide43

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 TCR receptor

slide44

Increased development of CD4 SP thymocytes and T cells in mice expressing a rearranged class II MHC specific transgene

CD4

CD8

slide45

Generating H-Y TCR transgenic mice

(anti-male peptide TCR)

Male H-2b cells

Rest and restimulate with Ag and cytokines, rest, repeat

9 days

Female

H-2b mouse

Expand anti-male

CD8 T cells

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

slide46

Increased development of CD8 SP thymocytes and T cells in mice expressing a rearranged class I MHC specific transgene

5%

37%

46%

10%

slide47

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

. . . . .

. . . .. .

. . . . .

. . . .. .

… . .

… . .

MHC deficient mice provide evidence for positive selection.

Lack of MHC class II expressionprevents development of CD4 cells

WT

CD4

CD8

MHC Class II o

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

slide49

CD4

CD8

MHC-1

MHC-2

CD8

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?

slide50

CD4

CD8

MHC-1

MHC-2

CD8

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?

slide52

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
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

Block in CD4 T cell development, or “lineage swap”? How could you tell?

CD4

CD8

He et al. Nature 2005

slide54

Uncoupling between positive selection and lineage commitment.

Mutation in ThPOK leads to the development of “mismatched” class II specific, CD8 T cells

CD4

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 ThPOKtg MHC-2 ko MHC-2 ko x ThPOKtg

slide55

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

slide56

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

slide57

TRANSCRIPTION FACTOR NETWORK CONTROLING CD4 VS CD8 FATE:

THE SIMPLE VERSION

MHC-2 positive selection

MHC-1 positive selection

CD8

Mutual antagonism

Auto-

regulation

ThPOK

CD4

RUNX3

slide58

TRANSCRIPTION FACTOR NETWORK CONTROLING CD4 VS CD8 FATE:

MORE PLAYERS

MHC-2 positive selection

MHC-1 positive selection

GATA3

cMyb

Tox

CD8

ThPOK

ThPOK

CD4

CD4

MAZR

MAZR

RUNX3

RUNX3

Would you consider ThPOK to be a “master regulator” of the CD4 fate. Why or why not?

CD8

slide59

CD4

CD8

MHC-1

MHC-2

CD8

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?

slide60

Relating the transcription factor networks that control CD4 versus CD8 to recognition of MHC-1 or MHC-2 during positive selection.

slide62

An “instructive” model?

More transient signal

More prolonged signal

A kinetic model for CD4/CD8 development: duration of TCR signals influence lineage choice.

slide63

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.

slide64

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 co-receptor expression eliminate cells that turned down the wrong co-receptor.

Thymocytes expressing TCR specific for MHC-1

A stochastic/selection model?

slide65

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

slide66

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
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

rag-

CD69+

Long-lived, indep of TCR signal

These changes occur asynchronously over a period of 1-3 days.

Can identify thymocytesexhibiting some, but not all signs of positive selection.

Would you say that a cortical CD4+CD8+ CD69+ rag- thymocyte has been positively selected?

slide68

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)

slide69
Lingeage commitment represents the loss in the ability of a cell to make alternative lineage choices under permissive conditions.

How long does it take for a thymocyte to commit to the

CD4 or CD8 lineage?

slide71

What does TCR “signal strength” really mean?

TCR affinity (signal strength)

CD8 lineage commitment

CD4 lineage commitment

T reg development

(agonist selection)

Death by neglect

Clonal deletion

Positive

selection

TCR affinity for peptide-MHC complex?

Duration, frequency, or intensity of TCR signaling?

Presence/absence of co-stimulatory signals?

slide72

Temporal requirement for TCR signaling during CD8 T cell positive versus negative selection?

intermittent

days

continuous

hours

Temporal pattern of TCR signaling during positive versus negative selection?

How do distinct temporal signaling patterns encode distinct T cell fates?

Why does positive selection take 2-3 days?

positive selection and cd4 versus cd8 lineage development may be lengthy reversible processes
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.

slide74

CD4 loop

CD8 loop

MHC-2 recognition:

stronger, continuous signal

MHC-1 recognition:

weaker, intermittent signal

GATA3

CD8

CD8

ThPOK

ThPOK

Maintain

CD8 expression allowing for late

MHC-1 recognition

Maintain

CD4 expression allowing for late

MHC-2 recognition

CD4

RUNX3

RUNX3

Loops can be initiated by biasing signals, and/ or stochastic fluctuations.

Positive feedback makes lineage choices more robust and allows for re-verification.