T cell development, Receptor repertoire selection and CD4/CD8 lineage commitment

Question 1

Where do T cells come from?

Answer 1

1. Multipotent lymphoid.

Progenitors migrate from the bone marrow to the thymus through blood vessels

2. In the thymus, the lymphoid progenitors differentiate to pre-T cells and are educated to differentiate self from non-self
3. Positively selected T cells emigrate from the thymus to mediate and effect the cognate immune response

Question 2

Cells in the thymus produce chemokines

Answer 2

Thymosin

Thymotaxin

Thymopetin

Thymic factors

Chemical attractants which secreted and transported via blood to bm, T cells recognise these and follow the gradient to the thymus

Question 3

Thymus is divided into

Answer 3

cortex

medulla - where late phase of T cell development within thymus occurs

Question 4

What cells are present in the thymus

Answer 4

Cortical epithelial cell

Medullar epithelial cell

Lymphocytes interspersed within the thymus

Other cell types play important role later in T cell development e.g macrophages, dendritic cells

Hassal’s corpuscle is where aggregation of mature lymphocytes occurs

Question 5

Thymocytes at different developmental stages are found in distinct parts of the thymus

Answer 5

immature T cells come via the blood into the thymus they do not express functional TCR or core receptors CD4/8.

Proceed to have interactions with stromal cells and other cell types, double positive lymphocytes will express CD4 and CD8 , and mature CD3 or T cell receptor

Finally proceed towards the final stage- generation of a single positive mature CD4 or CD8 T lymphocyte (not double positive)

Then exit thymus

Question 6

Key players

Answer 6

T cells:

• TCR
• CD4
• CD8

These are on surface of T cell as a T cell receptor complex, they can both bind (TCR and CD4/8) to MHC class molecules.

Non-T cells

• MHC I
• MHC II

On other cells, not T cells

CD = cluster of differentiation

MHC = major histocompatibility complex

Question 7

What cells can you identify on a flow cytometry?

Answer 7

Can identify cells that are either only cd4 positive

Or CD8 - single positive

Neither – double negative

CD4 and CD8 – double positive

DN’s can be further subdivided into DN1 through to DN4

Question 8

Where do double negative cells appear?

What cells are dominant in thymus?

Answer 8

Appear in the fetal thymus before double positive cells

Thoughout further maturation more cells appear DP.

DP in thymus are dominant population. Different from other secondary lymphoids like spleen where majority of cells committed to CD4/8, no DP.

Question 9

Does T cell differentiate and TCR rearrangement occur separately?

Answer 9

T cell differentiation and t cell receptor rearrangement occurs simultaneously so that the double positive CD4/8 cells express mature t cell receptor ready for selection

Gamma delta T cells diverge from the main pathway of T cell development, at the stage of DN2.

These cells undergo little further changes before exiting thymus.

Alpha beta have different pathway.

Question 10

What T cells are favoured during early fetal development?

Answer 10

Gamma delta T cells

Show up during early gestation, subside progressively, numbers go down in adults, only represent small proportion of all T lymphocytes in circulation

Alpha beta T cells other way around, progresisvely increase their numbers, present as main T cell type in adults

Question 11

Antigen recognition by gamma delta T cells is different than alpha beta t cells

How are they different

Answer 11

Gamma delta T cells bearing specific receptors end up in skin (Vg5), gut (Vg2), uterus (Vg6) etc

Gamma delta T cells not MHC restricted, doesn’t recognise MHC presented peptides as conventional T cells do

Instead antigen is recognised directly, more like an antibody

Some cases ligands for the gamma delta TCR are self-proteins upregulated under stress conditions

In humans circulating gamma delta cells recognise a phospholipid antigen from mycobacterium tuberculosis – rather than peptide fragments

Play a role in cancer surveillance

Question 12

What % of T cells are gamma delta

Answer 12

Gamma delta 10% of all generated T cells, alpha beta is 90%

Question 13

Alpha beta T cells are characterised by expression of the alpha beta TCR, what is the process of assembly of alpha beta T cell?

Answer 13

Original germline chain of both alpha and beta are rearranged multiple times

Different fragments combined

Assembly of functional alpha beta t cell receptor is achieved

Progression through development correlates with rearrangement

• Maturation of T cell receptor takes place simultaneously with the transition of the T cell in terms of phenotype from DN to DP to SP cell
• Beta chain recombines during double negative stage
• Alpha chain recombines and rearranges later, during transition to double positive

Question 14

Why do DP thymocytes need to progress to the SP stage?

What signalling supports transition to SP?

Answer 14

Needs functional TCRa chain rearrangement , a cell without a functional TCR can’t serve its purpose

Both CD4 and MHC II – needed to be a CD4+ cell

Or needs CD8, MHC I and TAP to be a CD8+ cell

Needs important molecules involved in cell signalling that support transition to SP

• ERK signalling
• Calcineurin signalling

Question 15

What happens to cells that fail to complete thymocyte maturation (95%)?

Answer 15

95% cells fail either positive or negative selection

Therefore undergo death by neglect, by apoptosis

These cells are removed from tissue by macrophages, phagocytic cells that ingest them and recycle some components

Question 16

Describe apoptosis process of cells

Answer 16

Non-select cells are removed from tissue

Programmed cell death

Controlled and supported by right processes within environment

Doesn’t cause damage to other cells or tissues

Acquires interaction between sFas (soluble Fas), present on surface of apoptotic cell

And Fas ligand, and other cell types that can initiate apoptotic cell death

Following the interaction, signalling pathway will be activated

Ends up with expression of active caspase 8, which will disintegrate DNA or cleave it, and kill the cell

Question 17

DP cells have both CD4 and CD8 on surface

How do DP bind to MHC molecules?

What happens if binding is not strong enough?

Answer 17

DP cells will look for MHC molecules in thymic tissue

MHC molecules may be present on epithelial cells or APC, also present in thymic tissue.

Random event whether CD4 or CD8 will first find either MHC I or MHC II.

Whichever finds the target first will bind to the molecule, e.g DP binding to CD4 to MHC II on another cell, if binding is strong enough it will result in down regulation of the CD8.

Therefore DP cell will emerge as a single positive cd4 cell with very little or no CD8 expression at all.

Vice versa, CD8 on DP cells, first it finds targets or MHC I molecule on another cell, binds to it. If binding is strong enough CD4 is downregulated and DP cell emerges as single cd8 positive cell.

Binding is not strong enough, double positive cells will be killed by apoptosis or neglect. Eventually removed from tissue.

Most cells fail to bind to MHC I or II and will not be selected as CD4 or CD8 cells.

Question 18

What is MHC in humans?

Answer 18

Human leukocyte antigens of MHC I and II type

MHC I = HLA- A, B, C

MHC II = HLA –DR, DQ, DP

HLA-A / MHC I expressed on thymic stromal cells and low level on APC (DC and macrophages)

HLA-DR / MHC II expressed on thymic medullary stromal cells and high level on APC

Question 19

When does positive selection of T cells occur?

Answer 19

Positive selection occurs when DP T cell with a functional TCR binds to HLA molecules

If binding is strong enough, cell will be positively selected and allowed to continue development.

When binding is weak/no binding, the cell will be useless and be removed by apoptosis.

Question 20

What does positive selection ensure?

Answer 20

Positive selection ensures that only T cells that are useful and can engage in recognition are selected. All other cells are removed

DP CD4/CD8 cells bind to MHC-I or MHC-II on thymic epithelial cells – it is a random event which one binds.

Following adequate binding of CD4:MHC-II, CD8 is downregulated and vice versa. Allows transition from DP to SP.

From here on, the SP CD4 or CD8 T cells are ready for negative selection.

Unselected cells die by apoptosis.

Question 21

What is negative selection?

What problem do T cells have with this? how is it overcome?

Answer 21

Exclusion of self-reactive T cells as they would cause autoimmunity

Determined based on affinity of TCR for presented self-peptide: high – kill him, low – keep him

Eensures that remaining T cells are only reactive to foreign peptides

Self-reactive cells are not removed immediately, go through further TCR rearrangements (second chance) - before they are eventually removed if still self-reactive

Problem for T cells: thymus does not represent all self-antigens

But has a transcription activator gene which can induce expression of other tissue specific proteins (kidney, heart, etc)

Gene is called AIRE (autoimmune regulator): this allows negative selection against most bodily self-proteins

Question 22

How does AIRE allow T cells to negatively select against self-antigens not present in thymus?

Answer 22

Transcription factor autoimmune regulator (AIRE) mediates ectopic gene expression in the thymic medullary stroma

other tissue specific genes, kidney, heart, liver, lungs, gut, apart from brain and testes – might be expressed if their transcription is activated by AIRE

Known as promiscuous gene expression – about 10% of all genes in the thymus are expressed this way

Remaining pool of self-reactive cells smaller

Without AIRE, immature thymocytes/t-cells negatively selected against a limited number of self proteins

Question 23

Positive and negative selection summary altogether

Answer 23

Positive selection

• DP CD4/CD8 cells that bind either to MHC I or II
• Depending on strength of binding will have different fates
• Strong binding = useful, self restricted and progress towards SP CD4 or CD8
• Weak binding cells will be killed by apoptosis

SP cells can go to negative selection

• TCR binding to MHC peptide complex which contains self-peptides that can be found in the thymus
• Self-peptides presented by thymic cells, stromal cells and APCs like macrophages and dendritic cells
• APCs will also present some self-peptides found in other tissues due to action of AIRE

Question 24

Antigen presentation and MHC expression control thymocyte slection

What cells present antigen?

Answer 24

• DC
• Medullary epithelial cells
• Cortical epithelial cells
• Endothelial cells

Certain cell types control different T cell developmental fates

• Positive selection
• Negative selection
• Receptor editing

Question 25

How are regulatory T cells formed? What surface antigens do they express?

What is their main role?

Answer 25

Some SP T cells do not immediately exit thymus and go to other tissues and lymphoid organs.

They remain in the thymus for a short period of time, undergo stages of differentiation where they express CD25 and FoxP3 surface antigens

These cells do not proliferate in response to MHC self-peptide complexes

T regs accumulate in Hassall corpuscles and later migrate to different tissues

Main role: dampen T cell response, regulating it when further accumulation of T cells are no longer required

Question 26

What happens once T cells pass both positive and negative selection?

Answer 26

Become conventional T cells

Migrate to secondary lymphoid organs looking for their target antigen

Immunological synapse

If they encounter specific antigen, they get activated, proliferate and become effector T cells

Some become memory T cells

If they don’t find the target they eventually die by apoptosis after period of circulation