14. Thymocyte development 1 Flashcards
What is the thymus?
The site of T cell development
Where is the thymus derived from?
The 3rd pharyngeal pouch endoderm
When does the thymus develop?
about half way during development
What mice are used to study the importance of the thymus?
- Nude mice have a mutation in the Foxn1 transcription factor.
- Foxn1 is required for the development and homeostasis of thymic epithelial cells.
- These mice do not have a thymus.
What is the parathyroid?
- A gland that secretes parathyroid hormone.
- This regulates calcium levels in the blood.
- Develops from the same place as the thymus.
How was the thymus shown to be essential for T cell development?
- Using nude mice and SCID mice.
- When using staining and flow cytometry you can see nude mice have no T cells.
- If you take a thymus from a SCID mouse, it contains all the stem cells needed to make T cells.
- These can develop into mature T cells
- This shows that all that is needed to make mature T cells is a functioning thymus.
What is a SCID mouse?
A mouse that cannot rearrange its TCR genes to make a TCR
What happens when you reverse the experiment and put bone marrow cells from nude mice into SCID mice?
- A SCID mouse has a thymus but cannot make mature T cells due to mutated progenitors.
- If you inject the bone marrow from nude mice it contains the haemopoietic stem cells needed.
- The SCID mouse can then develop mature T cells.
What is Pignata Guarino syndrome?
- Foxn1 deficiency in humans
- Lack of a thymus and very immune impaired.
- Very rare discovered in 2 Italian siblings
What is DiGeorge Syndrome?
- A deletion of chromosome 22 (22q11.2).
- This includes the TBX1 gene.
- This causes deficient migration of neural crest cells.
- This results in a developmental disorder of the 3rd and 4th pharyngeal pouches.
- These patients cause a lack of thymus and no peripheral T cells.
- Also, parathyroid insufficiency, congenital heart disease and abnormalities of the face and ears.
What is the thymic rudiment?
- The thymus precursor
- It recruits the 1st lymphoid progenitors to seed the thymus.
What early lymphoid cells are recruited to the thymus?
- Early lymphoid progenitors seed the thymus express CD34 and CD44.
- This happens before vascularisation
What is CD34?
A stem cell marker
what is CD44?
An adhesion molecule
What do early lymphoid progenitor cells come from?
- It depends on the developmental stage
- Initially from the foetal liver.
- Then from the bone marrow
How do lymphocyte progenitors develop in the bone marrow?
- the haemopoietic stem cells give rise to the early lymphoid progenitor.
- The ELP leaves the bone marrow.
- It goes through further development to give rise to the thymus settling progenitor, then the early thymic progenitor.
What is the early thymic progenitor?
- A very early thymic cell
- Also known as the DN1 cell
- There is no co receptor expression.
- CD44 is expressed for adhesion.
- CD25 is not expressed
How are early lymphoid progenitor recruited to the thymus?
Via chemokines made by thymic epithelial cells.
What chemokine is made by thymic epithelial cells to recruit early lymphoid progenitors?
CCL25
What receptor is expressed by early lymphoid progenitors to detect chemokine released in the thymus?
CCR9
What chemokine binding do early lymphoid progenitors do?
- They express CCR9
- This detects CCL25 in the thymus
- Causes migration of progenitors to the thymus.
- This is essential for T cell development.
How was chemokine signalling shown to be essential in thymus development?
- Pertussis toxin is an inhibitor of G-protein mediated chemokine receptor
- This blocks the CCR9 receptor.
- This blocks thymus colonisation.
In vivo methods to study thymic colonisation: following appearance of lymphocyte subsets
- Follow the different lymphoid subsets across development
- Through development or after transplantation in experiments.
- you can sort specific subset of cells using flow cytometry and see how each cell type affects development.
In vivo methods to study thymic colonisation: Labelled cells.
- Radio labelled or fluorescent
- follow cells
In vivo methods to study thymic colonisation: thymidine analogues
- Using bromodeoxyuridine (BrdU) and putting it in drinking water.
- This incorporates in the DNA.
- You can then use specific antibodies to detect its presence.
In vitro methods to study thymic colonisation: thymocytes in dispersion cultures
- Making single cell suspension from the thymus.
- Dispersion in a flask or rotation flask.
- Limits mean you can only see as far as negative selection.
In vitro methods to study thymic colonisation: Monolayer cultures
- Single layer of cells in a flask
- Doesn’t show good development
In vitro methods to study thymic colonisation: Reconstitution of lobes
- Empty the lobes of the thymus by preventing replication of the cells.
- Then injecting other cell populations into the thymus and seeing what they do or how they develop.
In vitro methods to study thymic colonisation: Foetal thymic organ culture (FTOC)
- Take a foetal thymus
- Separate the two lobes and culture for about 12 days.
- Chop up the lobe, and you get all the cells you would get in an in vivo model.
- Positive and negative selection can occur.
- Functional and mature T cell develop.
In vitro methods to study thymic colonisation: Reaggregate thymic organ culture (RTOC)
- Take a foetal thymus
- Separate the 2 lobes and culture for 12 days.
- Use enzymes to break down the ECM in the lobes and make a single cell suspension
- Drop onto a filter
- The thymic cells reform the lobe again.
- You can introduce other steps like cell sorting and selectively adding them back to see what population of cells are essential for development.
What enzymes are used to digest the ECM in RTOC?
- Collagenase
- Dispase
How is Notch signalling used in thymocyte development?
- Thymocytes express Notch
- Thymocyte epithelial cells express Notch ligand.
- Notch has an extracellular and cytoplasmic domain.
- The cytoplasmic domain is cleaved and acts as a transcription factor.
How can Notch signalling in thymocytes be studied in vitro?
- Cultured in bone marrow stromal cells.
- They express the Notch ligand Delta-like 1.
- You can stain the cells and observe the development of thymocytes.
- You can see development all the way through
What is the key method of analysis of thymocyte development?
Flow cytometry
How does flow cytometry work?
- A mixture of labelled cells that are focused into a single cell stream.
- The laser crosses the stream of cells, and the light is scattered.
- The forward scatter shows the size of the cells.
- The side scatter shows the granularity of the cells.
How does TCR expression change over development?
- Early development cells don’t express the TCR.
- Double positive T cells express the TCR intermediately
- Single positive mature T cells highly express the TCR
How is expression of markers measured in thymocyte development?
Flow cytometry
What is the expression profile of immature T cells?
- TCRab-
- CD4-
- CD8-
What is the expression profile of DP T cells?
- TCRab int
- CD4+
- CD8+
What is the expression profile of mature T cells?
- TCRab high
- CD4+ or CD8+
What are the subdivisions of the double negative thymocytes?
- DN1
- DN2
- DN3
- DN4
What is CD44?
an ECM receptor
What is CD25?
IL-2Ra chain
What do DN1 cells express?
CD44+ CD25-
What do DN2 cells express?
CD44+ CD25+
What do DN3 cells express?
CD44- CD25+
What is the progression of double negative thymocytes?
- DN1 cells express CD44.
- DN2 cells turn on CD25 and express it along with CD44.
- DN3 turns off CD44
- DN4 turns off CD25
What do DN4 cells express?
CD44- CD25-
How do early thymocytes bind to the ECM?
via CD44 or VLA-5
What other cells are needed for early thymocyte development?
- Mesenchymal fibroblasts.
- These make ECM
Why is ECM important for thymocyte development?
- ECM components like fibronectin and collagen are found in the thymus.
- ECM can concentrate and localise growth factors for presentation to haemopoietic cells.
- These growth factors are essential for development of thymocytes.
What growth factor is crucial for thymocyte development?
- IL-7
- It is held and presented to early thymocytes by the ECM
- Held by heparan sulphate
- It signals development of DN1 to DN2
What happens to thymocyte without IL-7?
they cannot develop further then DN1
What molecules in the IL-7 signalling pathway stalling thymocyte development when absent?
- IL7
- IL7Ra
- JAK3
- Rho
What does Notch signalling do in thymocyte development?
- Delta like 4 is a notch ligand expressed on the thymic epithelium.
- Notch 1 on the developing thymocyte binds to delta-like 4.
- This causes cleavage of the cytoplasmic domain and activation of transcription.
- This leads to the V(D)J recombination of the Tcrb segments
What transcription in thymocyte development is activated by Notch signalling?
- pre-TCR a chain
- GATA3
- Tcf1
What processes does notch signalling control in thymocyte development?
- It is involved in cell fate decisions.
- Critical for T versus B lineage commitment in the bone marrow
- Critical through early thymocyte development
- Influences Tcrb gene rearrangement
- Critical for ß-selection checkpoint
- Involved in the alpha-beta vs gamma-delta TCR commitment
When does Tcrb rearrangement occur?
Once the thymocyte has enter the DN2 stage
When does Tcra rearrangement happen?
once the thymocyte has entered the double positive stage
How does somatic recombination of the ß chains genes (Tcrb) occur?
- Lots of VDJ segments and one of each is chosen.
- One of the D segments is bought to the J, and the DNA in between is removed.
- The same happens with the V segment next to the D.
- If the gene is in the correct reading frame and encodes a functional protein then it will be expressed on the cell surface.
- Initially, it is expressed with the pre-t cell, a chain
How does somatic recombination of the a chains genes (Tcra) occur?
- There is no D segments.
- A V is joined to a J segment and the DNA in between is removed.
- It is then transcribed to make a function protein and then form the TCR
What controls somatic recombination?
- The Rag enzymes which are lymphoid specific recombinases.
- Rag 1 and 2 which forms a heterodimer.
- These bring the gene segments together and splice them.
- These are tightly controlled so they are only active when rearrangement is needed.
What is Tdt?
- The enzyme that controls N-nucleotide addition.
- Expressed throughout development
What is the pre-t alpha chain?
- A substitute alpha chain that associates with the ß chain during development.
- Used before the TCRa chain is made
- Associates with the CD3 signalling elements on the immature thymocytes
What happen as soon as the TCRß segment is made?
- It is functionally expressed on the surface of the cell.
- Expressed with the pre-T cell a receptor
- This signalling from the Pre-T cell receptor is required for further development.
- It triggers the down regulation of CD25 and upregulation of CD4 and CD8 to make a double positive T cell.
- This allows the cell to make the TCRa chain and start to express it.
What mutations cause a block in development at the ß selection stage (DN3)?
- Mutations in any of the genes involved in rearrangement or any of the signalling from the Pre-t cell receptor.
- Rag 1 or 2
- TCRß
- PreTa
- CD3
- Lck/fyn
- ZAP70/Sck
Why do mutations in Lck or Fyn prevent T cell development?
- They are required for signalling from the Pre-TCR and the TCR.
- If only one is knocked out then the other can compensate.
- If both are knocked out then T cell development stops.
What do thymuses in Lck and Fyn deficient mice look like?
- Small thymus
- Fewer thymocytes this is due to a lack of DP cells that make up the bulk of cells in the thymus.
What is the pre-TCR receptor essential for in thymocyte development?
- TCRß and preTa associate to from the pre-TCR.
- This presence of a Pre-TCR indicates a functional ß chain.
- Thymocytes that lack a preTCR are blocked from developing past the DN3 stage.
- the Pre-TCR controls the ß-selection check point.
What does the PreTCR control in ß-selection?
- CD4-CD8- to CD4+CD8+ transition.
- Allelic exclusion at the TCRß locus to prevent further rearrangement of the genes.
- Proliferation
- Trigger TCRa gene segment rearrangement
What happens to most thymocytes in the thymus?
They die
How long do CD4+CD8+ T cell survive in the thymus?
- About 3 days
- Most die in selection
How long do single positive mature T cells exist in the thymus?
- They leave once developed to enter the circulation
Why do most T cells die in the thymus?
- To make a diverse repertoire of T cells tcra and tcrb genes undergo somatic recombination.
- This generates random receptors and only thymocytes expressing a TCR that recognises the self-peptide/self-MHC complex can be positively selected.
- If these thymocytes recognise the self-peptide/self-MHC complex with dangerously high affinity, they are negatively selected and die by apoptosis
What is the purpose of thymic selection?
- To allow only self-MHC restricted and self tolerant T cells leave the thymus
- To ensure the mature naive T cells are useful in recognising foreign antigens presented on MHC1/2
When does negative selection occur?
Once the T cells express either CD4 or CD8 and a functional TCR as it needs to be able to recognise a ligand to be selected.
