T cell Development

Question 1

How many T cells are in 1ml of blood?

Answer 1

1 e6 (1 milion) in blood
There is lymphocytes under skin and lining the gut

Question 2

Where do T cells develop?

Answer 2

Thymus

Question 3

What defines a T cell?

Answer 3

The presence of a T cell receptor.

Question 4

Describe the pathway of development and differentiation in the thymus

Answer 4

Development: First stem cell is called CD4-8-
Later in development, the cell downregulates one of CD4/8.

Differeniation: In periphery antigen stimulates naive T cells to become effector cells to clear infection.
Effector cells die after resolution; some are memory cells.

Question 5

What is he ratio of the different types of T cells?

Answer 5

2 CD4+: 1 CD8+

Question 6

Describe the pathway of development and differentiation in the thymus

Answer 6

Development: First stem cell is called CD4-8-
Later in development, the cell downregulates one of CD4/8.

Differentiation: In periphery antigen stimulates naive T cells to become effector cells to clear infection.
Effector cells die after resolution; some are memory cells.

Question 7

Where do T cells develop?

Answer 7

Thymus; this shrinks from heart-size to very small; more T cells needed early on.

Question 8

What defines a T cell?

Answer 8

The presence of a T cell receptor.

It does not see bacteria or virus; it only sees processes pathogenic peptides via MHC that will cause a response. presentation.

Question 9

In periphery, CD4+ have various roles

Answer 9

he differentiation determined by cytokines.

• Regulatory T cells which control the immune system.
• Th1, respond to Tb disease and makes IFN-gamma.
• Th17, which makes IL17
• Th2, mediates allergies making IL4.

Question 10

What benefit to T cells does recognition of self peptides have?

Answer 10

All MHC molecules must have peptide; only way it can be on surface. Lot of different self peptides presentated

T cells recognise self peptide-MHC and this triggers T cell survival.

Question 11

What are the two classes of T cell

Answer 11

This is determine by TRC-protein complex on surface

1) alpha/beta TCR due to the alpha/beta dimer to recognise the peptide. These are in blood and lymphatic circulation
2) gamma/delta TCR due to the gamma/delta dimer used for recognition. These are in gut, and skin.

Question 12

gamma-delta T cells

Answer 12

Primary specific immune responses, monitoring tumours, wound healing.

These are not dependant on specialised antigen presenting cells for activation. Ligand unknown.

Question 13

alpha/beta T cells

Answer 13

These recognise small peptides-MHC 1/2 and mainly mediate antigen-specific

Two types of alpha/beta cells: CD4+, CD8+

Question 14

Briefly describe the function of CD4+ and CD8+ ?

Answer 14

CD4+: They produce cytokines and growthfactors to regulate other immune cells

CD8+: They differentiate to make cytotoxic cells that can kill virus infected cells

Question 15

gamma-delta T cells

Answer 15

Primary specific immune responses, monitoring tumours, wound healing.

These are not dependant on specialised antigen presenting cells for activation. Ligand unknown.

Question 16

What makes the TCR so variable?

Answer 16

alpha/beta subunits are highly varible; the other subunits which are responsible for signal transduction from membrane into cell are invariable

the genes encoding alpha/beta subunits undergo protein rearrangements which increases protein diversity

Each cell will have thousands of the same TCR molecule

Question 17

alpha/beta T cells

Answer 17

Alpha and beta are disulphide linked heterdimer.

These recognize small peptides-MHC 1/2 and mainly mediate antigen-specific

Two types of alpha/beta cells: CD4+, CD8+

Question 18

Describe the TCR gene beta loci

Answer 18

from right-left: There is variable regions, exons of diveristy regions, joining regions and constant regions

Recombinase genes called RAG1 and RAG2 randomly rearrange these exons during T cell development when the Beta subunit is forming to make V-D-J.

Question 19

What processes happen during V-D-J recombination?

Answer 19

The double stranded DNA is broken and DNA is cut out so certain exon regions can join.
DNA repair enzymes rejoin the free ends.

1) recombination from germline DNA to form rearranged DNA of joined V-J in alpha and V-D-J in beta chain.
2) Transcription and splicing translation leads to the final T cell receptor protein.

Question 20

What two selections are used to check all of the completely random gene rearragments

Answer 20

1) Positive selection: That it can recognise self peptides

2) Negative selection: that it does not have a high reactivity to self peptides.

Question 21

What is the diversity of V-D-J recombination

Answer 21

1 e18.

Question 22

Describe how to analyse a thymocyte? What is shown?

Answer 22

Pull thymus apart with needle, and stain the thymocytes with anti-CD4+ and anti-CD8+. Measure using a flow cytometer to get a fax-plot.

There are few double negative, but most cells double positive: 80%. These will differentiate into single positive.

Question 23

What controls the development of DP T cells into single positive cell?

Answer 23

• antigen receptor
• cytokines
• cell itself

IL7 controls survival of DN cell stage. They then express the preTCR. If this is successful, the cell becomes double positive. If they express alpha/beta, it is this that determines which single positive cell.

Question 24

What is the preTCR composed of?

Answer 24

Beta chain, preT-alpha, and signalling subunits of CD3 antigen.

Question 25

Describe the T cell development and ALLELIC EXCLUSION

Answer 25

Enter Thymus: 1) rearrangement of ONLY ONE BETA TCR allele in cell precursor from bone marrow (DN) 2) If successfully rearranged BETA allele the thymocyte will express the pre-TCR 3) If this is expressed on the membrane it promotes cell survival and entry into the cell cycle. It downregulates RAG gene expression so the second beta gene is not rearranged. 4) Therefore only one beta chain, only one specificity 5) Cells with preTCR, proliferate and differentiate into cells that are DP 6) DP cells REXPRESS RAG genes which starts rearrangment of the alpha chain.

Question 26

What is allelic exclusion

Answer 26

Where only one allele is rearranged. (the second one may serve as a backup)

Question 27

What is the preTCR composed of? What is it function?

Answer 27

Beta chain, preT-alpha, and signalling subunits of CD3 antigen. It induces: - survival - differentiation - proliferation - upregulation to SP It does not need a ligand to signal, it simply expresses all subunits at the membrane in order to signal to next step and stabilize the complex.

Question 28

Describe the T cell development and ALLELIC EXCLUSION

Answer 28

Enter Thymus: 1) rearrangement of ONLY ONE BETA TCR allele in cell precursor from bone marrow (DN) 2) If successfully rearranged BETA allele the thymocyte will express the pre-TCR. If no B chain from either allele then cell will die. 3) If this is expressed on the membrane it promotes cell survival and entry into the cell cycle. It downregulates RAG gene expression so the second beta gene is not rearranged. 4) Therefore only one beta chain, only one specificity 5) Cells with preTCR, proliferate and differentiate into cells that are DP 6) DP cells REXPRESS RAG genes which starts rearrangment of the alpha chain.

Question 29

What is the preTCR composed of? What is it function?

Answer 29

Signaling complex: Beta chain, preT-alpha (invariant), and signalling subunits of CD3 antigen. It induces: - survival - differentiation - proliferation - upregulation to SP It does not need a ligand to signal, it simply expresses all subunits at the membrane in order to signal to next step and stabilize the complex.

Question 30

What two selections are used to check all of the completely random gene rearragments and that there is a useful TCR?

Answer 30

1) Positive selection: That it can recognise self peptides and MHC 2) Negative selection: that it does not have a high reactivity to self peptides and MHC; it becomes cross-reactive T cell and death signal triggered. A moderate binding and presence of MHC triggers a survival signal. It needs to recognise self but not strongly to cause autoimmunity.

Question 31

What is allelic exclusion?

Answer 31

Where only one allele is recombined. (the second allele may be used as a backup)

Question 32

What does 'death by neglect' refer to? When does the alpha chain recombine.

Answer 32

double positive cells express preTCR allowing the DP cell to survive as it is dimerised to the B chain. At this point (where cells are DP) the alpha chain is rearranged and simultaneous down regulation of the the preTCR. If there is no successful alpha rearrangement by the time the preTRC is losT the cell will die; it has to replace dimer in time.

Question 33

What is the ligand in the thymus, which allow the TCR to signal

Answer 33

The self peptides

Question 34

Describe thymic epithelial cells and what is the significance of this?

Answer 34

They have unique patterns of expression which means it express lots of different protein not usually expressed by one cell. This enables T cells to 'see' lots of different self peptide antigens from all around the body.

Question 35

What is AIRE and

Answer 35

Autoimmune disease where the gene AIRE has mutated. This gene contros the tissue specific antigens in thymus and so no AIRE means T cells are not selected for self-recognizing T cell. T cells cannot be educated; need a new thymus. Reacts with self insulin and destroys pancreas.

Question 36

The TCR

Answer 36

It regulates a cascade of signalling cascade that control T cell differentiation. TCR cannot signal itself, (no intrinsic activity) but it can couple to intracellular tyrosine kinases such as Lck, Fyn and ZAP70 which are activated as the TCR is activated. This coupling is through ITAM

Question 37

How does The TCR signal its activation?

Answer 37

It regulates a cascade of signalling cascade that control T cell differentiation. TCR cannot signal itself, (no intrinsic activity) but it can couple to intracellular tyrosine kinases such as Lck, Fyn and ZAP70 which are activated as the TCR is activated. Signals cause T cell to proliferate and become an effector cell.. This coupling is through ITAM

Question 38

What is ITAM

Answer 38

Immune tyrosine based Activation motif: an intracellular signalling motif CD3 invariant subunits have 1 ITAM; dimer a/b has 3 ITAMs.

Question 39

What happens in terms of signalling if a ligand bind to TCR?

Answer 39

- If ligand binds, co receptor (CD8+/CD4+) on T cell binds to ligand as well, stabilising complex. - ITAM become phosphorylated by kinase Lck. - ZAP70 (with a high affinity recognising domain for phosphorylating tyrosines) then binds to tyrosines of phosphorylated ITAMS of a/b dimer. - This means ZAP70 is activated and now phosphorylates proteins

Question 40

What is the function of ZAP70?

Answer 40

It phosphorylates adaptor proteins like: - LAT: which links activated T cells - SLP76

Question 41

What do ZAP70 phosphorylated adaptor proteins do?

Answer 41

They recruit SH2 domain containing enzymes to the membrane where the act to regulate the lipid metabolism. LAT adaptor protein recruits regulatory proteins for RasGTPases for signalling pathways. Cascade begins.

Question 42

Summary of TCR and signalling.

Answer 42

TCR is a signalling unit, it sees peptide-MHC it rapidly triggers biochemical changes inside the cell which indicate to the T cell to develop further into effector cells.

Question 43

What is used to measure how many phosphorylation occur inside the cell.

Answer 43

Mass spectrometer Thousands of phosphorylations which change as soon as a ligand binds. Overall causes changes in gene expression.