Thymus and T cell development

Question 1

Which cells express Foxn1?

Answer 1

Thymic epithelial cells (TECs) and also skin cells, however, the function of Foxn1 in TECs is different than in skin cells.

Question 2

What is CCL25?

Answer 2

It is a chemokine secreted by thymic epithelial cells. It binds to CCR9 receptor. It attracts lymphocyte precursors into the thymus.

Question 3

What is DLL4?

Answer 3

Expressed on cortical thymic epithelial cells. It binds to the receptor on the lymphocyte progenitors called Notch1 and this is imposing the T cell lineage on the precursor cell.

Question 4

What is Notch1?

Answer 4

It is a receptor on lymphocyte progenitor cells which binds to DLL4 on cortical thymic epithelial cells which imposes the T cell lineage on the precursor cell.

Question 5

What is KIT-Ligand?

Answer 5

It is a cytokine, produced by thymic epithelial cells, which drives T cell expansion and proliferation in the thymus.

Question 6

Where in the thymus does negative control take place?

Answer 6

In the medulla.

Question 7

Where in the thymus does positive control take place?

Answer 7

In the cortex.

Question 8

What is the role of the thymic cortex?

Answer 8

T cell specification, positive selection.

Question 9

What is the role of the thymic medulla?

Answer 9

Negative selection, Treg development.

Question 10

What is Foxn1?

Answer 10

It is a transcription factor, expressed by thymic epithelial cells. It controls thymic epithelial cell function, it controls chemokines, T cell specification, T cell expansion etc.

Question 11

What drives Foxn1 expression?

Answer 11

Wnt molecules, Bmp4, Bmp7, Shh (sonic hedgehog).

Question 12

What is AIRE?

Answer 12

It is a transcriptional regulator (autoimmune regulator). It allows medullary thymic epithelial cells to express modules of genes normally associated with other organs and tissues in the periphery.

Question 13

What cells express AIRE?

Answer 13

Medullary thymic epithelial cells.

Question 14

What happens when a patient has a mutation in AIRE?

Answer 14

They lose expression of those peripheral tissue antigens which leads to generation of autoreactive T cells which leads to severe autoimmunity.

Question 15

What is Fezf2?

Answer 15

It is another transcriptional regulator like AIRE which allows medullary thymic epithelial cells to express modules of genes normally associated with other organs and tissues in the periphery.

Question 16

What transcriptional regulators are found in medullary thymic epithelial cells?

Answer 16

AIRE and Fezf2.

Question 17

Does TCR recognise only one epitope or antigen?

Answer 17

No, it recognises a range of peptides presented by MHC but with different levels of affinity.

Question 18

What does a very strong affinity of TCR to self-antigen lead to during negative selection?

Answer 18

T cell deletion.

Question 19

What does a very weak affinity of TCR to peptide presented on MHC lead to during positive selection in the thymus?

Answer 19

T cell anergy and cell death as no survival signals are given to the T cell.

Question 20

What is FGF7?

Answer 20

FGF7 (Fibroblast growth factor 7), also known as KGF (Keratinocyte Growth Factor), binds to the FGFR2iiib receptor on thymic epithelial cells and drives their proliferation.

Question 21

What can lead to the loss of thymic tissue (thymic atrophy)?

Answer 21

Inherited primary genetic defects (mutations in Foxn1), acquired acute loss (pregnancy, infection, radiation) or progressive loss (ageing, sex steroids).

Question 22

How can you potentially restore thymus function?

Answer 22

Stimulate residual thymic tissue (if there is still some thymic tissue left), transplant thymus cells and tissues (if there is no thymic tissue left), Interleukin 22 treatment.

Question 23

What can be challenging with restoring or manipulating the thymus?

Answer 23

We need to ensure a proper balance between cortex and medulla. If we restore the cortex but not the medulla it will lead to immune protection, however, it could also lead to autoimmunity. So we need to ensure the control and balance between the cortex and the medulla.

Question 24

What is a Di George Syndrome?

Answer 24

It is a genetic deficiency which is associated with the micro-deletion in chromosome 22. It is characterised by developmental defects like palate formation and parathyroid. Patients with this disease have either a small (hypoplastic) thymus or an absent (aplastic) thymus. Because of the defects of the thymus, these individuals are immunosuppressed.

Question 25

What's a potential treatment for patients with a Di George Syndrome?

Answer 25

Doing an in vitro culture of the thymic tissue from the pediatric donor who underwent cardiac surgery and transplanting the culture into a patient with Di George syndrome. There is a certain recovery in T cell number but also there is a certain level of autoimmunity as MHC is not perfectly genetically matched between the donor and the patient. However, the gain outweighs the risk.

Question 26

What is Lymphotoxin-beta receptor?

Answer 26

It is a receptor on stromal cells in the thymic niche. It controls endothelial cells in the thymus, entry and exit points and other stromal populations which control the development of T cells. There is a study which showed that injecting an antibody stimulating lymphotoxin-beta receptor alongside bone marrow transplant supported the t cell lineage recovery.

Question 27

How does bone marrow transplant affect T cell lineage?

Answer 27

As haematopoietic stem cells (HSC) are firstly irradiated and new HSC are transplanted from the donor, when the thymus is large, it still takes time for new T cells to develop which leads to the window of infectious susceptibility, however, when the thymus is small for example in an elderly patient, there is less clustering space for T cells to develop, there is a window for infectious susceptibility and there is a reduced diversity of newly generated T cells.

Question 28

How can you enhance T cell lineage recovery?

Answer 28

Treatment with IL-22 or ablation of sex steroids, possible use of FGF7, however, it has previously shown positive effects on mice but not on humans.

Question 29

How do the double-negative T cells become double-positive?

Answer 29

Double negative T cells become double positive after the rearrangement of the beta chain of the T cell receptor. This takes place in the cortex of the thymus.

Question 30

How do double-positive T cells become single-positive?

Answer 30

After the rearrangement of the alpha chain of T cell receptor, this takes place in the cortex of the thymus.

Question 31

Do we have a single epithelial cell progenitor that gives rise to both the medulla and cortex, or do we have a separate progenitor for the cortex and a separate one for the medulla?

Answer 31

There is a single progenitor cell, which gives rise to both the medulla and cortex. It was shown in two experiments. One where a single thymic epithelial cell bound to a fluorescent die was injected back into the developing thymus, which was then implanted into the renal capsule and developed both cortical and medullary thymic epithelial cells and then the second experiment used FoxN1 KO mice which did not have the thymus and a single progenitor cell with FoxN1 expression led to the development of fully functional thymus.

Question 32

What happens to mice deficient in Rag2?

Answer 32

No TCR and BCR rearrangement, no T cells and B cells.

Question 33

Are TECs controlling T cells development?

Answer 33

Yes, but there is some sort of symbiotic relationship as it seems like T cells are controlling TECs as well.

Question 34

Do we have specific markers for bipotent thymic epithelial precursors?

Answer 34

Not yet

Question 35

What can be found in the thymic niche?

Answer 35

Thymic epithelial cells, fibroblasts, macrophages, dendritic cells, progenitor cells, chemokines

Question 36

What is a potential stem cell-based treatment strategy for thymic atrophy?

Answer 36

Reprogramming of fibroblasts to the thymic epithelial lineage by switching on FoxN1 expression which has been reported to support development of the thymus as well as support T cell development in vivo.

Question 37

How does IL-23 support thymus regeneration?

Answer 37

The decrease in double-positive thymocytes alerts dendritic cells, which secrete IL-23. IL-23 binds to IL-23 receptor on ILC3 (Innate lymphoid cell type 3) cells, which begin secreting IL-22. IL-22 binds to IL-22 receptor on thymic epithelial cells enhancing thymus recovery

Question 38

What does Foxn1 mutation lead to?

Answer 38

It leads to the absence of the thymus and no development of T cells. Severe immunosuppression.

Question 39

Where does the thymus used for research come from?

Answer 39

From paediatric patients undergoing cardiac surgery. The thymus is located just in front of the heart, covering it, surgeons need to remove part of the thymus to perform cardiac surgery. With parents consent, the thymic tissue is then directed to research.

Question 40

How do progenitor cells enter the thymus?

Answer 40

Lymphoid precursors leave the bone marrow into the bloodstream, chemokine CCL25 recruits progenitors from the blood into the thymus by binding to CCR9 receptor.

Question 41

Is the thymus the same size throughout life?

Answer 41

No, as people age, more medulla is replaced with fat tissue, and fewer T cells are generated. However, T cells are very long-lived, so that does not affect immunity.

Question 42

What are the two main cellular elements of the thymus?

Answer 42

- Haematopoietic (precursor cells) - Non-haematopoietic (stromal cells)

Question 43

What are the stages of T cell development?

Answer 43

- Double negative precursors (CD4-CD8-) - Double positive precursors (CD4+CD8+) after the rearrangement of the beta chain of TCR - Single positive (CD4+ or CD8+) after the rearrangement of the alpha chain of TCR - positive selection (T cell receptors which recognise MHC class I or II with strong enough affinity get selected and receive survival signals (cortex)) - negative selection (T cells with a strong affinity to self-peptides presented are deleted from the repertoire (medulla)) - cells that passed negative control are becoming mature naive T cells and are released to the periphery

Question 44

What growth factors mediate the cellular expansion of double-negative thymocytes in the thymus?

Answer 44

IL-7, IL-15, and SCF (Stem cell factor) are secreted by thymic epithelial cells. This was confirmed by studies on IL-7 receptor and c-Kit KO mice whose thymus contained less than 1 million thymocytes, compared to WT mice with around 120 million thymocytes.

Question 45

How many genes encode T cell receptors?

Answer 45

Only 2 genes (beta chain and alpha chain). However, genes consist of variable, diversity, joining and constant regions which can randomly rearrange, giving rise to many different combinations of T cell receptors.

Question 46

What is a Pre-T cell receptor complex?

Answer 46

It is expressed only on double negative thymocytes, it consists of TCR-beta protein, CD3 complex and pre-T-alpha. Only cells which rearranged their beta chain successfully can progress to rearranging the alpha chain. If the rearrangement of the beta chain was successful it will: - stop TCR-beta rearrangement, - lead to CD4 and CD8 (double positive) expression, - TCR-alpha rearrangement - cellular expansion

Question 47

What are the possible outcomes of TCR rearrangement?

Answer 47

- Does not recognise peptide/MHC at all - cell death - Recognise peptide/MHC of low affinity and avidity - progress to maturation - Recognise peptide/MHC of high affinity and avidity - cell death

Question 48

What does the rearrangement of alpha chain of TCR lead to?

Answer 48

It leads to the generation of mature TCRS, which either recognises MHC II, which leads to the loss of CD8 expression or recognises MHC I, which leads to the loss of CD4 expression. This leads to the creation of single-positive thymocytes.

Question 49

What disease is caused by a mutation in Aire?

Answer 49

APECED

Question 50

What happens with Foxn1 KO mice?

Answer 50

They don't have thymus and no T cells produced, severe immunodeficiency.

Question 51

Do we have a dual epithelial progenitor pool or a single epithelial progenitor pool?

Answer 51

We have a single epithelial cell progenitor, which gives rise to both cortex and medulla thymic epithelial cells.

Question 52

Nude mice

Answer 52

Lacks Foxn1 and lacks thymus

Question 53

Possible stem-cell-based strategies for thymic regeneration?

Answer 53

- Isolate bipotent thymic epithelial precursor and grow them in vitro (no markers identified yet) - Use embryonic stem cells - Use induced pluripotent stem cells (iPSC)