1. Lineage Commitment During T Cell Development
Art Weiss & Mark Anderson
10/19/16

2. Summary of Thymic Development
Zuniga-Pflucker, NRI, 2004

3. What do we mean by the term “Lineage Commitment”?

4. 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.

5. Name the Lineage Commitment Choices that are made during T cell Development

6. 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

7. First Lineage Commitment Decision in T Cell Development To be, or not to be, a T cell

8. Seeding the Thymus from Precursors in
the Blood
Bhandoola, et al., Immunity, 26: , 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

9. Is the ETP, recently arrived in the thymus, already committed to the
T cell lineage?

10. Alternative Fates of the Early Thymic Progenitor
Yui and Rothernberg, NRI, 2014
A critical role for Notch in T cell lineage commitment

11. Zuniga-Pflucker, NRI, 2004

12. How would you demonstrate the importance of
Notch in T cell lineage commitment of ETPs?

13. OP9 stromal cells transfected with Deltex-1 can support
T Cell Lineage Commitment
Zuniga-Pflucker, NRI, 2004
Schmitt, et al, JEM, 2004

14. ab T Cell Development
Yui and Rothernberg, NRI, 2014

15. Notch signaling is critical for the induction of multiple
transcription factors
Yui and Rothernberg, NRI, 2014

16. Early Notch signaling induces TCF-1 (Tcf7) and Gata3 at
The DN1 -> DN2 transition
Yui and Rothernberg, NRI, 2014

17. 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?

18. Impaired development of TCF-1-deficient BM HSC into CD25+ (i. e
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

19. Evolving Transcriptional Networks as Notch Influences Early T Cell Development
Yui and Rothernberg, NRI, 2014

20. DN Cells (CD4-/CD8-)
DN DN DN DN4

21. Sequential Rearrangement of TCR ab Genes
DN DP
Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003

22. The pre-TCR is Expressed in DN cellsg
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

23. How would you test the ligand-independency of
signaling by the pre-TCR?

24. Reconstitution of rag1-/- mice with various forms of the
pre-TCR
Irving, et al., Science, 1998

25. 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

26. 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)

27. 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 )

29. Why might the pre-TCR and the TCRgd signal
strengths be different differently?

30. Strength of signal: a fundamental mechanism for cell fate specification
Comparison of pre‐T‐cell receptor (preTCR) and γδTCR surface expression on immature thymocytes. Histograms show CD3 levels on gated TCRβ+ (purple histogram) or TCRγδ+ (blue histogram) CD25+CD4–CD8– thymocytes from TCRα–/– mice. Shaded histogram depicts CD3 staining of CD25+CD4–CD8– thymocytes from CD3ɛ–/– mice.
© IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSION' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER.
Immunological Reviews Volume 209, Issue 1, pages , 31 JAN 2006 DOI: /j x

31. Reciprocal regulation of Syk and ZAP-70 expression
during thymocyte development
Palacios & Weiss, JEM, 2007
WT DN3
-selection
Development
TCR selection
CD44-
CD25+
DN3

32. Strength of signal: a fundamental mechanism for cell fate specification
Signal strength model for αβ/γδ lineage choice. Quantitative differences in T‐cell receptor (TCR) signal strength specify cell fate, such that an immature double negative (DN) thymocyte expressing the preTCR receives a ‘weak’ signal and chooses the αβ lineage, whereas one expressing the γδTCR receives a ‘strong’ signal and chooses the γδ lineage.
© IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSION' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER.
Immunological Reviews Volume 209, Issue 1, pages , 31 JAN 2006 DOI: /j x

33. How might you test the signaling hypothesis?

34. Starting with a TCRgd Transgene:
Increasing signaling strength by elimination of a negative
regulator, CD5, favors gd lineage commitment
Hayes, et al., Immunity, 2005

35. Instructing ab vs gd Lineage Commitment via
Strength of Signal
Ciofani and Zuniga-Pflucker, Nat. Rev. Immunol. 2010

36. 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.

38. Checkpoints in Thymocyte Development
Modified from Carpenter and Bosselut, Nature Immunology 2010
Notch Pre-TCR TCRab

39. 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.

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

41. TCR transgenic mice and MHC gene ko mice.
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-2
MHC-1
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.

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

43. TCR specificity for MHC I or II determines CD4 versus CD8 lineage commitment
OT1, HY, F5, etc
OT2, DO11.10, AND, etc

44. MHC deficient mice provide evidence for positive selection.WT
CD4
CD8
… . .
MHC Class II o
Lack of MHC class II expression prevents development of CD4 cells
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.

45. How does a DP decide it is a CD4 or 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?

46. 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?

47. How would you identify transcription factors involved in CD4 versus CD8 lineage commitment?

48. 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

49. 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?
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
Transgene caused “redirection of Class I restricted T cells” to the CD4 lineage.
This is the first transcription factor shown to directly regulate lineage commitment, I.e., act as a master regulator
Gata-3 is necessary but not sufficient for complete development of positively selected thymocytes to the CD4 lineage.
Runx factors regulate coreceptor expression; an additional proposed role in lineage determination remains to be proven.
Now we just need to figure out how TCR/co-receptor signaling talks to these Txn factors!
CD4
CD8
He et al. Nature 2005

50. Uncoupling between positive selection and lineage commitment.
Mutation in ThPOK leads to the development of “mismatched” class II specific, CD8 T cells
CD4
CD8
OT1 TCR tg OT1 x ThPOK tg MHC-2 ko MHC-2 ko x ThPOK tg
Over-expression of ThPOK in thymocytes lead to reciprocal phenotype:
CD4 T cells with TCR specific for MHC-1.

51. TRANSCRIPTION FACTOR NETWORK CONTROLING CD4 VS CD8 FATE:
THE SIMPLE VERSION
MHC-2 positive selection
MHC-1 positive selection
ThPOK
RUNX3
Mutual antagonism
Auto-
regulation
CD4
CD8

52. 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?

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

56. An “instructive” model?
More prolonged signal
More transient signal
A kinetic model for CD4/CD8 development: duration of TCR signals influence lineage choice.

57. Medulla
SP (CD4) DP
Cortex DN
SP (CD8)
Medulla
SP (CD4)
Medulla
Exit to the body DN DP
Cortex
SP (CD8)

59. Class II KO mice inject in vivo with anti-CD3: Look in the thymus
Kappes et al. Immunity 2008

60. Instructive model: A key transition step CD4+CD8lo
Al Singer Curr Opinion Immunology 2002

61. CD4 loop
CD8 loop
MHC-1 recognition:
weaker, intermittent signal
MHC-2 recognition:
stronger, continuous signal
Maintain
CD4 expression allowing for late
MHC-2 recognition
Maintain
CD8 expression allowing for late
MHC-1 recognition
GATA3
RUNX3
RUNX3
CD8
ThPOK
ThPOK
CD8
CD4
Loops can be initiated by biasing signals, and/ or stochastic fluctuations.
Positive feedback makes lineage choices more robust and allows for re-verification.