Development of T lymphocytes

Question 1

Briefly answer the following questions regarding lymphopoiesis:

1) What is lymphopoiesis?

2) Where does it occur?

3) What happens once antigen receptor is generated? What is the goal?

Answer 1

1) Lymphopoesis is the production of lymphocytes. It is a stepwise process that is largely defined by the assembly and expression of functional antigen receptor genes.

2) It occurs in central lymphoid tissues: bone marrow for B cells and thymus for T cells

3) Once antigen receptor is generated, lymphocytes undergo a rigorous testing process called SELECTION. The goal is to generate a diverse repertoire antigen receptors that recognize many types of pathogens but that do not react against self.

Question 2

Successful antigen-receptor gene rearrangement signals for ________ to subsequent step of lymphopoiesis.

Answer 2

progression

Question 3

T-cell development can be grouped into 4 sequential steps.

Briefly describe them.

Answer 3

1&2. T-cell progenitors develop in the bone marrow and migrate to the thymus where the cells complete their development by rearranging their antigen-receptor genes and undergoing repertoire selection.

3. Mature T cells encounter foreign antigens in the peripheral lymphoid organs and become activated.
4. Activated T cells proliferate and eliminate infection in sites of infection.

Question 4

1) What are thymocytes?

2) Where is the thymus located?

Answer 4

1) Developing T cells

2) Just above the heart (upper anterior thorax)

Question 5

Fill in the blanks regarding the structure of the thymus:

1) Thymus consists of distinct regions: _______ (outer) and _______ (inner).

2) The thymus contains a network of epithelia known as the _______ _______ which serve to provide thymocytes a specialized ____________ that is essential for their development.

3) The cortex contains tightly packed ________ thymocytes and scattered _______.

4) The medulla contains more of loosely packed _______ thymocytes, ________ and ______ cells.

5) Macrophages and dendritic cells contribute to the _______ of ________ thymocytes.

Answer 5

1) CORTEX; MEDULLA

2) THYMIC STROMA; micro environments

3) immature; macrophages

4) mature; macrophages; dendritic

5) clearance; apoptotic

*in both the cortex and the medulla, there are respective epithelial cells and these are referred to as thymic cells.

Question 6

Mouse 1 has a defect that prevents lymphocyte production.

Mouse 2 has a defect that affects development of the thymus.

T cells do not develop in either mice.

Transplantation of mouse 1 thymus graft to mouse 2 generates T cells.

Translation of mouse 2 bone marrow stem cells to mouse 1 generates T cells.

What does this tell us?

Answer 6

Thymus provides essential microenvironment for T cell development!

It provides signals to the thymocytes to allow them to develop properly.

Question 7

Thymocyte development occurs in ______ within the thymus, which are marked by changes in ____ _____ _____ and the _________ status of the T cell receptor genes.

Answer 7

Stages; cell surface molecules; rearrangement

Question 8

What do the surface changes of thymocytes reflect?

Answer 8

It reflects the state of functional maturation of the cell!

Particular combinations of cell surface proteins are used as markers for T cells at different stages of differentiation.

Question 9

(T/F) Mature T cells express both the CD4 and the CD8 co-receptors.

Answer 9

False!

Mature T cells can express EITHER the CD4 or CD8 co-receptors, NOT BOTH.

Question 10

1) What are double-negative thymocytes?

2) What double-positive thymocytes?

3) What are single-positive thymocytes?

Answer 10

1) thymocytes that do not express CD4 or CD8

2) developing thymocytes that express both CD4 and CD8

3) thymocytes that express either CD4 or CD8, not both

Question 11

Match the stages of thymocyte development:

1) Stage 1
2) Stage 2
3) Stage 3
4) Stage 4
5) Stage 5

A) Large active double-positive thymocytes become SMALL resting cells that EXPRESS LOW LEVELS of the T-cell receptor. Most thymocytes die within the thymus in this stage (SELECTION).

B) Positively selected thymocytes EXPRESS HIGH LEVELS of the T cell receptor and will stop production of one of the two co-receptors, making single-positive thymocytes.

C) Double negative thymocytes enter the thymus and lack most of surface molecules that are characteristic of T cells.

D) Mature T cells exported from the thymus to the periphery.

E) The development of prospective α:β T cells proceeds through stages in which both CD4 and CD8 are expressed (double positive). These ENLARGE and DIVIDE and also express the pre-TCR.

Answer 11

Stage 1: Double negative thymocytes enter the thymus and lack most of surface molecules that are characteristic of T cells.

Stage 2: The development of prospective α:β T cells proceeds through stages in which both CD4 and CD8 are expressed (double positive). These ENLARGE and DIVIDE and also express the pre-TCR.

Stage 3: Large active double-positive thymocytes become SMALL resting cells that EXPRESS LOW LEVELS of the T-cell receptor. Most thymocytes die within the thymus in this stage (SELECTION).

Stage 4: Positively selected thymocytes EXPRESS HIGH LEVELS of the T cell receptor and will stop production of one of the two co-receptors, making single-positive thymocytes.

Stage 5: Mature T cells exported from the thymus to the periphery.

Question 12

_____, ____, and T-cell receptor complex molecules (____, ____ and ___ chains) are important cell-surface molecules for identifying thymocyte subpopulations.

Answer 12

CD4; CD8; CD3, α, β

Question 13

Only 2-5% of thymocytes generated actually leave the thymus as mature T cells while the remained die by apoptosis.

1) What happens to the apoptotic thymocytes?

2) Why is there an extensive loss of thymocytes?

Answer 13

1) Thymocytes that undergo apoptosis in the cortex are INGESTED BY MACROPHAGES.

2) The extensive loss of thymocytes reflects the INTENSIVE screening that each thymocyte experiences to ensure SELF-TOLERANCE.

Question 14

Thymocytes localize to distinct regions of the thymus depending on their development stage.

1) T cell progenitors from the bone marrow migrate through the blood and enter the thymus at the ____-________ junction and then migrate to the ____.

2) In the ____________ region, immature double-negative thymocytes undergo extensive proliferation.

3) As thymocytes proliferate and develop into double-positive cells, they migrate _______ into the _____, where positive selection occurs.

4) After positive selection is complete, double positive thymocytes migrate to the _______ where they undergo negative selection.

Answer 14

1) cortico-medullary; cortex

2) subcapsular

3) deeper; cortex

4) medulla

Question 15

Which one of these statements is true?

1) The cortex contains epithelial cells that express MHC I and II molecules; which play a role in negative selection of double-positive thymocytes.

2) Most T cell development occurs at the cortex.

3) Dendritic cells and epithelial cells in medulla play an important role in the positive selection process.

4) Thymocytes depend more on thymic cortex epithelial cells for their own survival the more they proceed through development.

Answer 15

2!

For 1, the cortex contains epithelial cells that express MHC I and II molecules; which play a role in POSITIVE selection of double-positive thymocytes.

For 3, dendritic cells and epithelial cells in medulla play an important role in the NEGATIVE selection process.

For 4, thymocytes depend LESS on thymic cortex epithelial cells for their own survival the more they proceed through development.

Question 16

(T/F) The co-receptors (CD4 & CD8) and CD3 are the only cell surface molecules that change during T development.

Answer 16

False!!!

The co-receptors (CD4 & CD8) and CD3 are NOT the only cell surface molecules that change during T development.

There are many additional factors expressed such as SIGNALLING proteins, ADHESION molecules, TRANSCRIPTION FACTORS, RECEPTORS during stages of T cell development.

These alter the morphology and behaviour of the cells to allow them to progress forward in a uni-directional manner.

Question 17

1) What is the pre-TCR composed of?

2) What does it do?

3) What would happen if pre-TCR is not present?

Bonus: do you remember in which stage of thymocyte development that the formation of pre-TCR occurs during development? ;)

Answer 17

1) β chain assembles with a surrogate α chain (pTα). This heterodimer forms a complex with the CD3 chains to form a pre-TCR (pTα:β).

2) The pre-TCR provides critical signals that trigger subsequent steps in T cell development.

3) If pre-TCR is not present (either due to defected β chain or defected pTα), there will no be critical signals for T cell development, resulting in T cells developing improperly.

Bonus: it occurs in step 2 when α:β chains are forming, double positive thymocytes are enlarging and dividing!

Question 18

Somatic recombination of which locus begins first for T cells?

This process for this locus occurs in which cells?

Answer 18

Somatic recombination of β locus begins first!

D to J rearrangement is followed by V-to-DJ rearrangement.

Process occurs in DOUBLE NEGATIVE CELLS.

Question 19

Somatic recombination of α chain occurs second. This process occurs in double ________ cells.

Answer 19

positive

*α chain is composed only of V and J gene segments.

Question 20

(T/F) The β protein is expressed with the α protein in the cytoplasm.

Answer 20

False!

The β protein is expressed initially within the cytoplasm.

Somatic recombination of beta chain occurs first. The product is then expressed in the cytoplasm.

Question 21

1) When does the rearrangement (somatic recombination) of the alpha chain begin?

2) When does the surrogate alpha chain get replaced in the cell surface? What does this prepare the thymocyte for?

Answer 21

1) At the end of PROLIFERATIVE BURST, the CD4 and CD8 molecules are expressed, the cell ceases cycling and the alpha chain is now able to undergo rearrangement.

2) When a functional alpha chain is produced that pairs efficiently with the B chain! This prepares the DP thymocyte to undergo selection.

Question 22

What are the four critical signals generated by the pre-TCR?

Answer 22

1) Cell becomes permissive for TCR alpha chain locus rearrangement

2) Stimulates expression of CD4 and CD8 co-receptors

3) Stimulates proliferation

4) Stops additional TCR b-chain locus rearrangements (allelic exclusion)

Question 23

1) What is allelic exclusion?

2) Why is allelic exclusion important?

3) How does allelic exclusion occur?

Answer 23

1) The exclusive expression of only ONE of the two inherited alleles encoding the beta chains of TCR (also occurs for BCR).

2) Successful rearrangements at both loci (paternal & maternal) could result in a T cell with two receptors of different antigen specificities. Allelic exclusion ensures the T cell expresses a single receptor, thus maintaining specificity (same applies for B cells).

3) The pre-TCR promotes allelic exclusion, in part, by temporally regulating gene expression of RAG-1 AND RAG-2.

Question 24

How does temporally regulating gene expression of RAG-1 and RAG-2 promote allelic exclusion?

Answer 24

These endonucleases initiate the receptor gene rearrangement process.

The pre-TRC shuts down RAG 1 and RAG 2 so that there is no more beta chain re-arrangement happening.

*it is almost like a race on which one of the two chromosomes will produce a functional beta chain of TCR first. when one is produced and forms a pre-TCR, the pre-TCR signals the stopping of the second one.

Question 25

1) Why do thymocytes undergo selection in thymus?

2) The selection process occurs in what kind of phases? Briefly describe them.

3) Differentiate positive from negative selection.

Answer 25

1) Thymocytes undergo selection to PRESERVE useful cells and to ELIMINATE harmful cells (self-reactive)

2) The selection process occurs in SEQUENTIAL PHASES. First, negative and positive selection of DP cells in the cortex. Then,
thymocytes positively selected in the cortex migrate to medulla as SP cells where they undergo a second round of negative selection.

3)
Positive selection: T cell receives signals to SURVIVE.

Negative selection: T cell triggered to DIE.

Question 26

Positive and negative selection of a thymocyte depends on the strength of binding the T cell receptor to _______:_______ complexes present on the surface of thymic epithelial cells or bone marrow-derived macrophages and dendritic cells.

This is known as the ________ model.

Answer 26

self-peptide:self-MHC

affinity

Question 27

Thymic epithelial cells express high levels of MHC class I and II (in complex with self peptides). The double positive thymocytes browse these complexes in the cortex.

What are the three outcomes that can occur when double positive thymocytes encounter self peptide-MHC complexes (first round of selection)?

Answer 27

1) No interaction: cells die by NEGLET.

2) High affinity binding: cells are negatively selected aka clonal deletion (apoptosis of the thymocyte)

3) Low/intermediate affinity binding: cells are positively selected to survive and transition to SP stage. they migrate to medulla for second round of negative selection.

Question 28

(T/F) During the first round of selection, most cells are positively selected.

Answer 28

False!

Most cells (90-95%) die by NEGLET. They don’t get the chance to be tested (the body doesn’t want to risk it).

2-5% are positively and 2-5% are negatively selected.

Question 29

What is the purpose of the second round of negative selection that occurs in the medulla?

Answer 29

Many tissue-specific proteins are not expressed in the thymus.

The thymus needs to screen thymocytes to ensure they are NOT SELF-REACTIVE FOR PROTEINS that are restricted to other parts of the body.

Question 30

How does the thymus screen thymocytes to ensure they are NOT SELF-REACTIVE FOR PROTEINS that are restricted to other parts of the body?

Answer 30

Medulla thymic epithelial cells (mTEC) express AIRE (autoimmune regulator) which is a transcriptional regulator that PROMOTES EXPRESSION of many proteins not found in the thymus.

mTECs process these proteins and present them in their MHC I and MHC II molecules to single positive thymocytes. If there is a strong interaction, they are negatively selected. If there is no or weak interaction, the T cells are passed through.

DENDRITIC CELLS and MACROPHAGES also engulf mTECs and present their protein contents in their MHC molecules.

Question 31

(T/F) All self proteins are expressed in the thymus through AIRE.

Answer 31

False!

It is unlikely that all self proteins are expressed in the thymus. Therefore, additional mechanisms function in the periphery to ensure self-tolerance.

Question 32

What happens to the T cells in the absence of AIRE?

Answer 32

In the absence of AIRE, T cells reactive to tissue-specific antigens mature and leave the thymus.