15. Thymocyte Development 2 Flashcards
What needs to happen before positive and negative selection can happen?
- Seeding of the thymus by DN cells
- Rearrangement of ß chain of the TCR and expression of the pre-TCR.
- At the DP stage they rearrange the TCR a chain and start to express the TCR
- They can then recognise something for positive and negative selection
How is a diverse repertoire of TCRs generated?
- Somatic V(D)J recombination of separate Tcrb and Tcra gene segments.
- This leads to around 10^15 different TCRs
What CD4+CD8+ thymocytes are positively selected?
Those that express an aß TCR that recognise with low avidity a self-peptide/MHC complex express on cortical epithelial cells.
What does positive selection of thymocytes involve?
- Rescue from apoptosis
- Initiation of T cell differentiation
- Commitment to CD4+ and CD8+ T cell lineage
Positive selection: Rescue from apoptosis
- Up-regulation of Bcl-2 anti-apoptotic survival marker
- Unless this happens T cells will die at this stage
Positive selection: Initiation of T cell differentiation
- Upregulation of TCR levels
- Expression of CD69, CD2 and CD5 which are expression markers for this process
- Down-regulation of heat stable antigen (HSA) which is a stem cell marker
- Expression of CCR7
What is CCR7 important for?
The movement of cells from the thymus cortex to the medulla
How was positive selection discovered by mice animal models?
- They irradiated MHCa or MHCb recipient mice to eliminate the lymphocytes and stem cells.
- They were then reconstituted with bone marrow from an F1 mouse that expresses MHCa and MHCb.
- The bone marrow cells then developed in the environment of each mouse.
- They were then tested to see if their T cells could recognise MHCa or MHCb, and they could only recognise whatever was self for them.
- MHCa mouse could recognise MHCa antigen presenting cells.
- MHCb mouse could recognise MHCb antigen presenting cells.
How was positive selection shown to occur in the thymus?
- Use of nude mice and ATx mice that have a surgically removed thymus
- They were then given an MHCa thymus graft
- Then irradiated to remove all lymphocytes and stem cells in the thymus and circulation.
- Then an F1 bone marrow graft with MHCa and MHCb as a source of stem cells that could mature.
- The thymocytes that mature could only recognise antigen presentation on MHCa.
- MHCb were elsewhere in the mouse by only MHCa were presented in the thymus. This means the MHCb were not selected and the MHCa were.
- This showed the thymus is responsible for positive selection T cell
What are TCR transgenic mice?
- A transgenic mouse that only produces 1 TCR
- This recognises a specific antigen of choice
How are TCR transgenic mice generated?
- You clone rearranged TCR genes from a T cell for a specific antigen
- Then inject the cloned DNA into the male pronucleus of a fertilised egg
- Inject eggs into a pseudo-pregnant female
- With luck, some offspring will carry the transgene
- Take the male TCR transgenic offspring and breed with wild-type females to produce more mice in most or all T cells express the TCR transgene
Example of specific TCR transgenic mice: 2B4
- This recognises pigeon cytochrome C
- It is presented by the MHC2 IEk
- This selects CD4+ T cells
Example of specific TCR transgenic mice: HY
- This recognises HY which is a male pigeon antigen.
- It is presented by MHC1 Db
- This selects CD8+ T cells
What happens with positive selection in 2B4 transgenic mice?
- When p.cytc is not presented on Ek there is no positive selection as the T cell cannot recognise the antigen.
- This means the T cell die by neglect
- When p.cytc is presented on Ek the T cells can be positively selected as they can recognise the antigen.
- This means the T cells survive and become CD4+ cells
What do the specific TCR transgenic mice tell us about positive selection?
- For thymocytes to recognise an antigen and be positively selected they need to be presented on a self MHC.
- The self MHC is essential for development and presentation
What happens with positive selection in HY transgenic mice?
- Both tested were female mice so it is not about antigen specificity.
- In the absence of Db the cells cannot be positively selected.
- With Db you get positive selection and appearance of CD8 T cells.
- This shows that correct MHC is important.
What does positive selection determine?
Coreceptor expression
What happens to a thymocyte that recognises MHC2?
They will move onto the CD4 pathway and down regulate CD8.
What happens to a thymocyte that recognises MHC1?
They move onto the CD8 pathway and downregulate CD4.
What is ß2-microglobulin?
A soluble protein that associates with MHC1 to stabilise it.
How can you generate a ß-2 microglobulin knockout?
- Insert a neomycin resistance gene into the ß2-microglobulin gene.
- Then transfect this into embryonic stem cells.
- Allow homologous recombination to make homozygous cells.
- The neomycin resistance gene allows for selection of the homozygous phenotype by G418 or ganciclovir.
- These embryonic stem cells are injected into blastocysts.
- Then implanted into a female
- Some of the progeny are hopefully chimeric for the knockout cells
- These are then interbred to get homozygous ß2-microglobulin deficient strain
What is the consequence of the ß2-microglobulin knockout?
- Unstable MHC1 that are rapidly degraded
- No CD8+ T cells are generated
What other defects can cause a lack of MHC1 and CD8+ T cells?
- TAP-1
- MHC1 peptides.
- Whole MHC1 molecules
What are Aß knockout mice?
- They already have a defect in the ß chain promoter.
- Knockout the A chain also.
- This results in no expression of MHC2
- This results in no CD4+ T cells.
What are human bare lymphocyte syndromes?
These lack expression of either MHC1 or MHC2.
What defect causes lack of MHC1 expression in humans?
- This includes HLA-A, HLA-B, HLA-C.
- This is due to TAP defects.
- Causes no CD8+ expression
What defect causes lack of MHC2 expression in humans?
- This includes HLA-DR, HLA-DP, HLA-DQ.
- This is due to CIITA defects (CIITA induce MHC2 transcription)
- Causes no CD4+ T cells
Where do immature thymocytes enter the thymus?
Through venules in the medulla
Where do thymocytes migrate in the thymus during development and what interaction do they have?
- As they go through DN2,3,4 they are recruited out to the subcapsular region of the thymus.
- They then begin to express the TCRaß and CD4 and CD8.
- They interact with cortical epithelial cells in positive selection.
- Selected thymocytes are then recruited back to the medulla through chemokines.
- Then they run past dendritic cells, macrophages and medullary epithelial cells for negative selection.
What is critical for CD4+ thymocyte selection?
MHC class 2
What is also critical for thymocyte selection?
Which epithelium the MHC are expressed within the thymus
Where does MHC need to be expressed for positive selection?
- On the cortical epithelium
- It makes little nurse bags of 50 thymocytes
- MHC expression on the medullary epithelium doesn’t lead to positive selection
How did FTOC culture of mutant thymuses show the role of peptides in positive selection?
- MHC1 lacks ß-2 microglobulin
- Addition of human ß2-m and synthetic peptides causes the stabilisation of MHC1 and allows peptides to bind.
- When you increase peptide diversity, you get a higher yield of CD8+ thymocytes as more thymocytes can be positively selected.
- If the peptide causes the binding affinity to increase massively, then it will be negatively selected.
What is avidity?
Accumulated strength of multiple binding affinities like between a MHC/peptide with a TCR.
What is the avidity hypothesis for thymocyte selection?
- Interaction strength is important for selection.
- No interactions = death by neglect
- Low avidity recognition with the MHC on the cortical epithelium = survival and maturation (positive selection)
- High avidity recognition = deletion by apoptosis in negative selection
Where does MHC recognition in positive selection occur?
On the cortical epithlium
What does positive selection result in?
- Coordinated TCR expression and co receptor expression
- Generation of mature CD4+ or CD8+ T cells by down regulating the irrelevant coreceptor.
- Up regulation of TCR levels
- Up regulation of Bcl-2 to promote survival
- RAG1 and RAG2 expression turned off to prevent any rearrangement of the TCR genes.
- Tcra gene segment rearrangement ceases.
- Expression of CD60, CD2, CD5 and CCR7
- Rapid migration to the medulla due to CCL19 and CCL21
What is CCL19 and CCL21?
- They are the ligands for CCR7
- They are expressed in the thymus medulla
- they recruit the thymocytes to the medulla
What is negative selection of aß T cells?
- The avidity hypothesis
- Strongly self-reactive thymocytes are eliminated in the thymus. This is central tolerance.
- They are negatively selected by apoptosis.
What cells mediated negative selection?
- Bone marrow-derived cells
- Medullary thymic epithelial cells which express Aire the autoimmune regulator.
- Can also be mediated by super antigens
What percentage of T cells die by neglect?
Most at around 90%
What percentage of T cells have low avidity recognition and are positively selected?
<5%
What percentage of T cells have high avidity recognition and are negatively selected?
~1%
What other variable is important in negative selection?
- Ligand density
- If the ligand is expressed on multiple MHC and recognised by many TCRs then they are more likely to be deleted.
What is the other option for high avidity T cells?
- Agonist selection
- They will become regulatory cells like Tregs
What regulatory cell types can high avidity T cells become?
- CD25+CD4+ regulatory T cells (Tregs)
- CD8aa intestinal intraepithelial lymphocytes.
- iNKT cells
What are Tregs?
They are very important for controlling self-reactive T cells that escape the thymus.
What are CD8aa intestinal intraepithelial lymphocytes?
- These help maintain the balance in the gut.
- They do this by clearing bacteria, antigen presentation to T cells and immune regulation.
- Plays a role in various intestinal diseases
What are iNKT?
- Invariant NK T cells
- T cells that express NK cell markers
What is the main mechanism of tolerance of T cells?
Central tolerance
What is the result of the critical importance of central tolerance?
A large number of back up mechanisms in the periphery
What is apoptosis?
- Programmed cell death
- Membrane alterations and blebbing.
- cytoplasmic and nuclear shrinkage
- Activation of the caspase cascade and caspase-dependent DNAse
- This results in internucleosomal DNA cleavage.
What can be used to detect apoptosis?
TUNEL
What is TUNEL?
- TdT-mediated dUTP-biotin nick end labelling.
- Tdt normally adds nucleotides in VDJ recombination, but here, it is used to tag the ends of DNA that have been cleaved.
- It adds the dUTP-biotin as the label.
- This is then detected with streptavidin staining.
What does TUNEL staining show?
- Shows a lot of apoptotic thymocytes in the thymus
- Counter staining with antibodies for macrophages shows lots of macrophages.
- The macrophages quickly mop up and degrade the apoptotic thymocytes.
How were mice models used to show negative selection is mediated by bone marrow-derived cells?
- Irradiated MHCa mice so there are no stem cells
- Then give them a bone marrow transplant from an F1 mouse that express MHCa and b.
- These develop in the thymus and enter the periphery.
- Then give a skin graft from an MHCb mouse is tolerated and not rejected.
- As the T cells develop in the mouse, they must have been tolerised by bone marrow cells to be able to tolerate the MHCb cells.
- As the stem cells come from the bone marrow were the only thing to contain MHCb, they must be responsible for negative selection.
What cells mediate negative selection?
- Bone marrow derived cells
- This included dendritic cells and medullary epithelial cells.
What is dendritic cells role in negative selection?
They efficiently mediate negative selection at the cortico-medullary junction.
What is the role of medullary thymic epithelial cells in negative selection?
They play an important role in expressing certain tissue-restricted antigens and eliminating potentially destructive auto-reactive thymocytes.
What are mTECs?
Medullary thymic epithelial cells
What markers do mTECs express?
- Different keratin markers like keratin 5+
- UEA1
- CCL21, which attracts positively selected cells.
- Aire expression which is an autoimmune regulator
What tissue-restricted antigens are expressed on mTECs?
- MS autoantigens like myelin components proteolipid protein and myelin oligodendrocyte glycoprotein
- Insulin produced by ß-cells in type 1 diabetes
- They transiently express up to 18,000 different antigens
Why is it important that mTECs express tissue restricted antigens?
They help tolerate T cells to antigens that are expressed:
1. In particular parts of the body
2. At limited times in life.
What is Aire?
- Autoimmune regulator
- It allows the induction of tolerance mechanisms on mTEC.
- Especially the expression of tissue-restricted antigens
How do thymocytes encounter lots of antigens in negative selection?
- Positively selected thymocytes move around the medulla for a few days before leaving the thymus.
- This gives them time to encounter all the tissue-specific antigens that are expressed at low frequency
What do mutations in Aire cause?
- a rare inherited form of autoimmunity
- Called autoimmune polyendocrinopathy-candidiasis ectodermal dystrophy (APECED)
- These patients cannot express self antigens on mTEC.
- This causes destructive autoimmunity in many endocrine tissues.
- Also leads to recurrent candidiasis due to autoantibodies against IL-17F and IL-22 which are critical in controlling candida infection
What is APECED?
- Autoimmune polyendocrinopathy-candidiasis ectodermal dystrophy
- It is a rare autosomal recessive disorder.
- Also known as autoimmune polyglandular syndrome type 1 (APS-1)
- More frequent amongst Finns, Sardinians, and Iranian Jews
What are some tissues that are effected by APECED?
- Thyroid causing hypoparathyroidism or hypothyroidism.
- Adrenal failure
- Ovarian failure
- Insulin-dependent diabetes mellitus.
- Testicular atrophy
What happens in Aire knock-out mice?
- They fail to delete T cells reactive to some tissue restricted antigens in the thymus.
- This means they develop organ-specific autoimmunity.
What does the importance of Aire highlight?
Highlights the importance of central tolerance mechanism induction that operates in the thymus to delete self-reactive cells.
What are the mechanisms of antigen presentation in mTEC?
- They express tissue specific antigens and MHC directly to thymocytes.
- They engage in antigen handover
- Thymocytes randomly travel around the thymic medulla for 4-5 days to allow them to engage with 1000s of different mTECs and Dendritic cells.
What is antigen handover?
- mTECs can give the transient antigens they express to dendritic cells
- This is very effective for negative selection as dendritic cells move within the medulla/
How was negative selection shown in HY transgenic mice?
- In the female the interactions are competely with the MHC as there is no antigen
- This leads to the selection and appearance of CD8 T cells.
- In male mice the antigen is also present and no CD8+ T cells are formed
- This is because the T cells are eliminated due to the strength of the interactions so are negatively selected.
What is the definition of negative selection?
Thymocytes expressing an aß TCR that recognises a self-peptide/MHC complex with a dangerously high avidity are negatively selected and die by apoptosis
What are some other mechanisms of negative selection?
- Anergy
- Receptor editing
What is central tolerance induction?
The deletion of the most dangerous self-reactive thymocytes
Where does positive selection occur?
on the cortical epithelium in the thymic cortex
Where does negative selection take place?
In the thymic medulla, mediated through dendritic cells and medullary thymic epithelial cells
How do we create the immune diversity from a limited number of genes?
- VDJ recombination with random combinations of different gene segments.
- Junctional diversity with random addition or removal of nucleotides.
- Pairing of different light/heavy chain or different a/ß chains
