1. T-Cell Development and Selection
Chander Raman, Ph.D.
January 2018

2. Sites of T-cell development
Thymus
Extrathymic T-cell development in the gut
Stages of T-cell development in the thymus
γδ vs αβ T-cell development
T- cell selection
Death by neglect (Lack of Positive Selection)
Negative selection
Positive selection
Agonist selection? (Self-reactive T-cells)

4. Capsule
Cortex
Medulla

6. Klein, L. et al. Nature Reviews Immunology 14:377–391(2014)

7. HSC – Hematopoetic Stem cells
CMP – Common myeloid progenitor
MPP – Multipotent progenitors
LMP –Lymphoid myeloid progenitor
Chi et al, Curr. Opinion Immunol Epub

8. Sites of T-cell development
Thymus
Extrathymic T-cell development in the gut
Stages of T-cell development in the thymus

9. T-cell committed Notch1 dependent Multipotent SCZ – Subcapsular Zone
DN1 – Double negative 1
DN2 – Double negative 2
DN3 – Double Negative 3
DP – Double positive
SP – Single positive
Multipotent
Howard Petrie, Nat. Rev. Immunol. 3:859

10. An overview of regulated migration events during T cell development
Paul E. Love & Avinash Bhandoola
Nature Reviews Immunology 11, (July 2011)

11. ELP – Early lymphoid progenitor
CLP – Common lymphoid progenitor
CTP – Committed T-cell progenitors
TSP –Thymus setting progenitor
DN – Double negative
Chi et al, Curr. Opinion Immunol Epub

12. Overview of T-cell Development
Carpenter, A. C. and Bosselut, R. Nat. Immunol 11: (2010)

13. Yui and Rothenberg, Nature Reviews Immunology, 14:529–545 (2014)

14. Yui and Rothenberg, Nature Reviews Immunology, 14:529–545 (2014)

15. Derk Amsen, Andrey Antov & Richard A. Flavell
Nature Reviews Immunology 9, (2009)

16. Sites of T-cell development
Thymus
Extrathymic T-cell development in the gut
Stages of T-cell development in the thymus
γδ vs αβ T-cell development

18. Narayan and Kang
Current Opinion in Immunology (2007), 19:

19. Sites of T-cell development
Thymus
Extrathymic T-cell development in the gut
Stages of T-cell development in the thymus
γδ vs αβ T-cell development
T- cell selection
Death by neglect (Lack of Positive Selection)
Negative selection
Positive selection
Agonist selection? (Self-reactive T-cells)

20. All T-cells are self-reactive: Selection requires the ability of T-cell to recognize self
Self-reactivity
Immunity

22. Inhibition (attenuation)
A model for the relationship between developmental outcome and TCR affinity for self-peptide–MHC.
A model for the relationship between developmental outcome and TCR affinity for self-peptide–MHC. Cells with TCR that have a low affinity for self die by neglect. Those with an intermediate affinity are positively selected. High-affinity self-reactive clones can die via clonal deletion, and the threshold between positive and negative selection is hypothesized to be steep. Regulatory T cells (Treg) (yellow) have more highly self-reactive TCRs than most conventional T cells (purple). Some Tregs may have very highly self-reactive TCRs and are rescued from deletion via cytokine signaling or by virtue of having a second TCR (dotted line).
Signal Strength
Co-stimulation
Inhibition (attenuation)
Xing Y , Hogquist K A Cold Spring Harb Perspect Biol 2012;4:a006957
©2012 by Cold Spring Harbor Laboratory Press

23. MHC Restriction

24. T-Cell Selection in Thymus
Positive selection -
Selection of T-cells that have functional antigen receptor, i.e. able to initiate signals when engaged.
Deletion of T-cells expressing antigen receptor that reacts strongly when engaged by self-antigen.
Negative selection -
Positive selection -
Selection of T-cells to CD4+ or CD8+ single positive based on reactivity with Ag presented by Class II MHC or Class I MHC, respectively.

25. Major events in thymus cell differentiation.
Miller, J. Nature Rev. Immunol. 11:489 (2011)

26. Klein, Kyewski, Allen and Hogquist
Klein, Kyewski, Allen and Hogquist. Nature Reviews Immunology 14: 377–391 (2014)

27. Klein, Kyewski, Allen and Hogquist
Klein, Kyewski, Allen and Hogquist. Nature Reviews Immunology 14: 377–391 (2014)

28. T-Cell Selection - Overview
Double Negative
(Positive selection for ability to recognize antigen)
Double Positive
CD4+CD8+
(Negative Selection)
Single Positive
CD4+ or
CD8+
(Positive selection based on MHC reactivity)

30. Inhibition (attenuation)
A model for the relationship between developmental outcome and TCR affinity for self-peptide–MHC.
A model for the relationship between developmental outcome and TCR affinity for self-peptide–MHC. Cells with TCR that have a low affinity for self die by neglect. Those with an intermediate affinity are positively selected. High-affinity self-reactive clones can die via clonal deletion, and the threshold between positive and negative selection is hypothesized to be steep. Regulatory T cells (Treg) (yellow) have more highly self-reactive TCRs than most conventional T cells (purple). Some Tregs may have very highly self-reactive TCRs and are rescued from deletion via cytokine signaling or by virtue of having a second TCR (dotted line).
Signal Strength
Co-stimulation
Inhibition (attenuation)
Xing Y , Hogquist K A Cold Spring Harb Perspect Biol 2012;4:a006957
©2012 by Cold Spring Harbor Laboratory Press

31. Signal strength is the major governing factor that dictates the T-cell maturation through the thymus

32. Models for differentiation from Double Positive to Single Positive
Instructive Model
Stochastic Model

33. Class I
Class II

35. CD4 vs CD8 Lineage Commitment
Carpenter, A. C. and Bosselut, R. Nat. Immunol 10: (2010)

36. Thymic and peripheral function of positively selecting peptides
Kai W Wucherpfennig & Etienne Gagnon
Nature Immunology 10, (2009)

37. Model for differentiation into γδ-TCR T-cells development vs αβ-TCR T-cells
Quantitative vs Qualitative
Signal

39. Intracellular signals that drive selection

40. Experimental Evidence for Negative and Positive Selection
Negative selection in Males
Positive selection to CD8

41. CD4 CD4 CD4 CD8 CD8 CD8 Non transgenic Male H-Y Tg Female H-Y Tg 55
4.5
0.5 40
Male H-Y Tg
Female H-Y Tg 12 80 2 50
CD4
CD4 3 5 3 45
CD8
CD8

42. Cortex
Medulla
Positive selection
Negative selection

43. Where does negative selection occur? Cortex?
Medulla?
Both?

44. Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla
McCaughtry et al. J. Exp. Med. 205:257 (2008)

45. Β5t – proteasome subunit
TSSP – thymus specific serine protease
Ctss – cathepsin S
Cell types in central tolerance. (Top) T cells are positively selected in the thymic cortex. Negative selection via clonal deletion can also occur in the cortex, but occurs frequently in the medulla. The thymic medulla is also the site for Treg differentiation. (Bottom) Cortical thymic epithelial cells (cTECs) express several unique genes that relate to proteolysis (cathepsin L, Ctsl; thymus-specific serine protease, TSSP; and β-5t proteasome subunit, β5t), which are essential for positive selection. Tissue-specific antigens (TSAs) can be directly presented by medullary thymic epithelial cells (mTECs) or cross-presented by DC (dotted line with arrow). RANKL, CD40L, and lymphotoxin (LT) expressed by SP thymocytes interact with their receptors on mTEC to promote the development of mTEC.
Xing Y , Hogquist K A Cold Spring Harb Perspect Biol 2012;4:a006957
©2012 by Cold Spring Harbor Laboratory Press

46. Stritesky et al,

47. Immunofluorescence reaction in adrenal cortex section detected with autoimmune Addison’s disease patient serum.
Defects in Central Tolerance leads to Generalized autoimmunity

48. Evidence for Requirement for Central Tolerance
AIRE (autoimmune regulator) Transcription Factor –”Master” Regulator of Ectopic Expression of Peripheral Tissue- Restricted Antigens in stromal cells of the thymic Medulla.
Originally identified as a human autosomal recessive disorder known as APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy).
In human the condition is characterized by (1) chronic mucocutaenous candidiasis, (2) hypoparathyroidism and (3) adrenal insufficiency
AIRE-/- mice exhibit wide spread organ-specific autoimmunity, such as ovary, retina testis, stomach. (Anderson et al, Science)

49. Loss of Thymic Selection lead to Autoimmunity
AIRE-Deficient Mice
Normal Mice
Autoantibodies to different organs shown in GREEN in AIRE-deficient mice

50. Hiroyuki Takaba, Hiroshi Takayanagi
Figure 1
Schematic Diagram of T Cell Selection and TRA Expression in the Thymus. Thymocyte progenitors derived from bone marrow migrate into the cortex, and interact with cortical thymic epithelial cells (cTECs). Thymocytes express αβ T cell receptors (TCRs) with CD4 and CD8, and interact with self-peptide–major histocompatibility complex (MHC) molecule complexes. They differentiate into CD4- or CD8-single-positive T cells (positive selection). CD4 T cells and CD8 T cells migrate into the thymic medulla, and interact with medullary thymic epithelial cells (mTECs), which present the peptide of tissue-restricted antigen (TRA) in the context of MHC. Most autoreactive T cells are eliminated by apoptosis (negative selection), but some differentiate into regulatory T cells (agonist selection). TRA expression is controlled by transcriptional regulators such as Aire and Fezf2 in the mTEC. TRA genes are induced by Aire interacting with histone H3 or directly regulated by Fezf2. TRAs expressed by mTEC are taken up by the dendritic cells (DCs), which also contribute to the TRA presentation to T cells.
Hiroyuki Takaba, Hiroshi Takayanagi
Trends in Immunology  , DOI: ( /j.it )

51. Theofilopoulos et al, Nature Immunology 18:716–724 (2017)

53. Figure 1 Current model of Aire function in medullary thymic epithelial cells (mTECs). Aire is expressed in the nucleus of mTECs where its primary function is to upregulate the expression of tissue specific antigens (TSAs).
Mark S Anderson , Maureen A Su
Aire and T cell development
Current Opinion in Immunology Volume 23, Issue

54. Unanswered questions?
Why do patients with APECED develop candidiasis?
Can AIRE deficiency be overcome by other means of tolerance induction?

55. Selection of High affinity/avidity TCR T-cells

56. Inhibition (attenuation)
A model for the relationship between developmental outcome and TCR affinity for self-peptide–MHC.
A model for the relationship between developmental outcome and TCR affinity for self-peptide–MHC. Cells with TCR that have a low affinity for self die by neglect. Those with an intermediate affinity are positively selected. High-affinity self-reactive clones can die via clonal deletion, and the threshold between positive and negative selection is hypothesized to be steep. Regulatory T cells (Treg) (yellow) have more highly self-reactive TCRs than most conventional T cells (purple). Some Tregs may have very highly self-reactive TCRs and are rescued from deletion via cytokine signaling or by virtue of having a second TCR (dotted line).
Signal Strength
Co-stimulation
Inhibition (attenuation)
Xing Y , Hogquist K A Cold Spring Harb Perspect Biol 2012;4:a006957
©2012 by Cold Spring Harbor Laboratory Press

58. Annual Reviews

59. Outcomes of high-affinity TCR signaling in the thymus
Stritesky and Hogquist, Nature Immunology 13:528–530 (2012)

60. Jochen Huehn, Julia K. Polansky & Alf Hamann
Nature Reviews Immunology 9, (February 2009)