Selection of T cell receptor repertoire and CD4/CD8 lineage commitment

Question 1

For context, where do T cells come from?

Answer 1

1. Multipotent Lymphoid: Progenitors Migrate from the Bone Marrow to the Thymus
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

Describe how T cell migration occurs from BM to thymus?

Answer 2

• Thymus secretes a series of cytokines : thymosin, thymotaxin, thympoietin, thymic factors
• These travel to the BM, where T cells recognise these and follow trail along blood vessel to thymus along concentration gradient (high in thymus)

Question 3

What is hassall’s corpuscle?

Answer 3

Site of aggregation of mature lymphocytes found within the medulla

Question 4

State the different development stages of the thymocytes in the parts of the thymus?

Answer 4

Thymocytes at different developmental stages are found in distinct parts of the thymus
Step 1: Immature CD3- 4- 8- double negative thymocytes
Step 2: Immature CD3+ 4+ 8+double positive thymocytes
Step 3: Mature CD4+ 8- and CD8+ 4- thymocytes

Question 5

How can T cell development be characterised? VD

Answer 5

• Flow cytometry
• Allows to see sub-populations with larger cells based on the expression of cell surface markers

Question 6

Using flow cytometry, what can Double Negative’s be further subdivided into? VD

Answer 6

• DN’s can be subdivided into DN1 through to DN4
  alpha chain of IL2R

Question 7

What type of T cell appears in the fetal thymus first?

Answer 7

Double negative cells appear in the fetal thymus before double positive cells

Question 8

Describe the T cell populations during development - thymus and spleen in flow cytometry?

Answer 8

VD
- Spleen only contains a single positive CD4+ or CD8+ cells

Question 9

Lineage commitment
~ Diagram showing T cell development and migration - key concept to understand here is gamma:delta has barely any steps in differentiation to become a mature T cell

Answer 9

T cell development and migration
VD

Question 10

Between gamma:delta and alpha:beta thymocytes, which are favoured during early fetal development? VD for graph

Answer 10

Gamma:delta T cells are favored during early fetal development

Question 11

Describe how antigen recognition by gamma:delta T cells is different to alpha:beta?

Answer 11

• Gamma:delta T cells bearing specific receptors end up in skin (Vg5), gut (Vg2), uterus (Vg6), etc.
• Gamma:delta T cells are not MHC restricted
• Antigen is recognized directly, more like an antibody
• In some cases ligands for the gamma:delta TCR are self proteins upregulated under stress conditions
• In humans, circulating gamma:delta cells recognize a phospholipid antigen from Mycobacterium tuberculosis
• Play a role in cancer surveillance

Question 12

Descrbe the proportion of T cells between TCR alpha:beta and TCR:gamma:delta lineage?

Answer 12

• TCR alpha:beta lineage comprises the majority of T cells
• A:B = 90%
• Gamma:delta = 10%

Question 13

What factor in T cell development correlates with progression through development?

Answer 13

Rearragement: First is beta chain during Development, then alpha chain during Development progression

Question 14

What does a Double positive thymocyte require to progress to SP (single positive) stage?

Answer 14

Functional TCR-alpha chain rearrangement
• CD4 and MHC II (To be a CD4+ cell)
• CD8, MHC I and TAP (To be a CD8+ cell)
• ERK signaling
• Calcineurin signaling

Question 15

State the proportion of cells that fail to complete thymocyte maturation and briefly describe the enzyme required within the process of removing it?

Answer 15

• Most cells fail to complete thymocyte maturation (95%)
• These don’t enter + ve or -ve selection
• These cells undergo apoptosis programmed cell death (DNA fragmentation)
• via enzyme caspase 8
• Causes chromatin disintegration, DNA break up -> cell death
• Removed by macrophages

Question 16

What are the possible fates awaiting alpha/beta T cell selection (DP Thymocyte)? VD

Answer 16

• Dependent on signal intensity and abiltiy to bind to MHC
• Either Negative selection, positive selection CD4 SP, death by neglect or positive selection CD8 SP

Question 17

State what cell and level of APC is MHC-I and MHC_II cells in the thymus expressed on?

Answer 17

• MHC-1: Expressed on thymic stromal cells and low level on APC (DC and macrophages)
• MHC-II: Expressed on thymic medullary stromal cells and high level on APC

Question 18

Positive selection of CD4 or CD8 SP T cells - self restriction
Describe the purpose of positive selection?

Answer 18

• Positive selection ensures that only T cells which are useful and can engage in recognition, are selected
• Specifically DP cells that are able to bind to MHC/HLA
• 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
• From here on, the SP CD4 or CD8 T cells are ready for negative selection
• Unselected cells die by apoptosis

Question 19

Negative Selection
Describe the purpose of negative selection?

Answer 19

• Negative selection ensures that self-reactive cells are removed, as they would cause autoimmunity •
• This is determined based on affinity of TCR for presented self-peptide: high - kill him, low - keep him
• Self-peptide is presented via thymus - remember they don’t represent all self antigens
• This ensures that remaining T cells are only reactive to foreign peptides
• Self-reactive cells are not removed immediately but go through further TCR rearrangements (second chance) - before they are eventually removed if still self-reactive

Question 20

How are T cells negatively selected against self-antigens not present in thymus?

Answer 20

• The Transcription Factor Autoimmune Regulator (AIRE)
• Mediates Ectopic Gene Expression in the Thymic Medullary Stroma of other tissue specific genes… kidney, heart, liver, lungs, gut, … apart from brain and testes
• Allows -ve selection against most bodily self proteins
• This is known as promiscuous gene expression - about 10% of all genes in thymus are expressed this way
• This eliminates many self-reactive T cells

Question 21

What 2 factors control thymocyte selection?

Answer 21

• Antigen presentation
• MHC expression

Question 22

Describe the role of cell types

Answer 22

• Certain cell types control different cell development fates e.g. + ve, -ve selection, receptor editing
• Examples include:
• DC
• Medullary Epithelial cells
• Cortical Epithelial cells
• Endothelial cells

Question 23

What type of cells will develop in the thymus if there is high expression of CD25 and Foxp3 and state its role?

Answer 23

• Regulatory T 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

Question 24

What occurs once the T cells leave the thymus? (PART 1)

Answer 24

• T cells that pass both positive and negative selection become conventional T cells
• They migrate to secondary lymphoid organs looking for their target antigen
• ‘Immunological synapse’

Question 25

What occurs once the T cells leave the thymus? (PART 2)

Answer 25

Possible scenarios:
- 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

Question 26

Take home messages

Answer 26

• TC chains undergo V (D)J recombination to generate diversity; they also exhibit allelic exclusion
• TCRbeta chain is selected with an invariant pTalpha chain at the DN3 stage
• TCRalpha chain is selected with pre-existing TCRbeta chain at the DP stage
• DP Thymocytes Undergo Positive and Negative Selection to Generate a Population of Mature T Lymphocytes that can Recognize Self MHC with Intermediate Affinity
• Recent Evidence Indicates that Positive and Negative Selection Are Mediated by Distinct Pathways
• APC in the thymus may present self antigen that effect negative selection

Question 27

Questions you should be able to answer by the end of the lecture
How do you generate a T cell population that is self-MHC restricted?

Answer 27

T cells that are compatible with the hosts genetic makeup

Question 28

Questions you should be able to answer by the end of the lecture
How do you ensure that those diverse T cell receptors are not- self reactive?

Answer 28

Don’t target the host

Question 29

Questions you should be able to answer by the end of the lecture
How do you coordinate lineage specification with MH specificity and coreceptor expression?

Answer 29

• alpha/beta vs. gamma/delta T cell
• CD4 vs. CD8