Lecture 4: T-cell and B-cell development

Question 1

Describe the origin of B and T-cell lineages (include names of transcription factors expressed)

Answer 1

All start from multipotent haematopoietic stem cell in bone marrow (and foetal liver), which differentiates into a Common Lymphoid progenitor (CLP)
- If CLP expresses transcription factors EBF, E2A and Pax5, they will become pro-B cells, which can eventually differentiate into follicular (FO) B cells, marginal zone (MZ) B cells, and B-1 cells.
- otherwise, lack of expression by the CLP will differentiate into a T-cell/ILC precursor
- T-cell/ILC precursors that express transcription factor Id2 become innate lymphoid cells
- T-cell/ILC precursors expressing Notch1 and GATA3 become pro-T-cells, which may commit to either the αβ or γδ T cell lineages

Question 2

What is the overall purpose of T-cell development?

Answer 2

produce a large population of diverse T-cells that can interact with MHC for antigen presentation but do not respond to self-antigens

Question 3

What are the three main types of mature naive T cells?

Answer 3

αβ CD4+ T cells (pre-cursors of T-helper cells)
αβ CD8+ T cells (precursors of cytotoxic T-cells)
γδ T cells

Question 4

Where are mature naive T cells produced?

Answer 4

Thymus

Question 5

Describe the T-cell development from CLP to DP thymocyte

Answer 5

• Uncommitted CLP enters thymus from bone marrow
• Those that express Notch receptor become T-cells and interact with Notch ligand expressed by cells in thymus - become committed to T-cell lineage and develop into thymocytes (immature T cells)
• Thymocytes are double negative (express neither CD4 or CD8 co-receptor) that undergo TCR gene rearrangement
• Thymocytes that successfully re-arrange the γ and δ genes mature to the TCRγδ lineage
• Thymocytes rearrange β receptor chain that undergoes β-selection where it is presented on surface with invariant surrogate α chain and CD3 complex to give a pre-TCR –> pre-TCR arrangement results in commitment to the TCRαβ lineage, another burst of proliferation, maturation to DP stage and initiation of TCRα rearrangement
• If successful, the signal is received via the pre-TCR to turn on gene rearrangement of the α chain and express CD4 and CD8, becoming a double positive thymocyte

Question 6

What are the most abundant subpopulation in the thymus and first to express a mature TCRαβ receptor?

Answer 6

DP thymocytes

Question 7

Explain positive and negative selection in T-cell development.

Answer 7

Occurs in the cortex:
MHC/self peptide complexes are expressed by the cortical thymic epithelial cells (cTECs) which are used to assess the affinity of the DP thymocyte TCR.
–> high affinity = negative selection typically by apoptosis (clonal deletion) - some (<1%) become Treg cells
–> low/intermediate affinity = positive selection - initiate maturation programme to the helper CD4+ or cytotoxic CD8+ SP lineages
–> affinity too low (~96% of thymocytes) = don’t recognise MHC/self-peptide complexes and die by neglect (fail to be positively selected)

Question 8

How do positively selected DP thymocytes become committed to CD4 or CD8 lineage?

Answer 8

Commitment to CD4 lineage:
- TCR preferentially interacts with MHC class II = continuous signal via TCR that initiates CD4+ helper T-cell development programme

Commitment to CD8 lineage:
- TCR preferentially interacts with MHC class I = no continuous signal generated (because CD8 surface levels are downregulated in response to positive selection) - interruption in signalling, followed by further stimulation, initiates CD8 development programme

Question 9

What is the role of AIRE+ mTECs in T-cell development?

Answer 9

mTECs are medullary thymic epithelial cells that express AIRE transcription factor.
- mTECs display tissue specific antigens from other organs on MHC and mediate negative selection directly or indirectly via dendritic cells (tests for autoimmunity)
Following this negative selection in the thymus medulla, the mature single positive thymocytes exit the thymus and undergo final functional maturation in the peripheral lymphoid organs.

Question 10

Where does each stage of T-cell development occur?

Answer 10

Bone marrow (HSC to CLP)
CLP to DN thymocyte in thymus
DN thymocyte to DP thymocyte in thymus cortex (positive and negative selection via TCR affinity)
DP thymocyte to mature SP thymocyte in cortex by browsing MHCI/II complexes
SP thymocyte to fully mature thymocyte in medulla follow negative selection by mTECs.

Question 11

What other lineages may DP thymocytes commit to (other than helper and cytotoxic cells)?

Answer 11

• NKT cells (express TCR with invariant TCRα chain, interact with CD1 molecules to present non-peptide antigens)
• Intraepithelial lymphocytes (IELs) = usually CD8+ but also have features of innate immune cells
• Regulatory T cells = CD4+ subset - roles in adaptive immunity

Question 12

What is the difference between natural and induced Tregs?

Answer 12

Natural Tregs: develop in the thymus

Induced Tregs: develop in the periphery from conventional T cells (high levels of growth hormones, such as IL10 can turn a T cell to become regulatory)

Question 13

What are the functions of Tregs in regulating T cell activation?

Answer 13

natural Tregs express transcription factor FoxP3 and IL2 receptor:
- deprives T cells of cytokine stimulation by binding to IL-2 to turn them off
- produces inhibitory cytokines, such as TGFbeta, that turn off T cells
- change APC to express co-inhibitory molecules (instead of co-stimulatory molecules) that inhibits T cell activation upon binding
- can also cause T cells to die by cytotoxity

Question 14

What is the positive and negative selection of T-cells?

Answer 14

Positive selection (recognition of MHC - cTECs)
Negative selection (deletion of high affinity self reactive clones - cTECs and mTECs)

Question 15

What are the major linages of B cells?

Answer 15

Follicular (B2) B cells
B1 B cells
Marginal zone B cells

Question 16

What are B1 B cells?

Answer 16

originate during foetal development where haematopoiesis occurs in the foetal liver
- have relatively limited receptor repertoire
- receptors tend to bind to microbial carbohydrate antigens (with relatively low affinity, much more similar to PRRs of innate immunity)

Question 17

Where does B2 B cell development begin and end?

Answer 17

begins: bone marrow
completed in the spleen

Question 18

Describe the stages of B2 B-cell development in the bone marrow

Answer 18

HSCs and B-cell progenitors make contact with stromal cells within the bone marrow

Pro-B cell:
- heavy chain recombination, presentation with surrogate µ light chain, if successful = receives survival signals and positive selection to Pre-B cell (mediated by IL7)

Pre-B cell:
- pre-BCR, several rounds of cell division, kappa light chain rearrangement (and lambda if kappa light chain insufficient)

Immature B-cell:
- negative selection (deletion and receptor editing - if found to recognise self, turn on RAG genes to rearrange again and change receptor specificity)
–> immature B-cells emerge from the bone marrow as transitional 1 (T1) B cells and circulate to the spleen

Question 19

Describe the maturation of T1 B2 B-cells after leaving the bone marrow

Answer 19

enter the follicles of the spleen where the level of IgD increases and they become T2 cells which then mature into:
- Follicular B2 cells (can exit the spleen, circulate between and populate secondary lymphoid tissues throughout the body, mediating the main B cell response to infection)
- Marginal Zone B cells (remain in the spleen and are specialised for detection of blood-borne antigens, including protein and carbohydrates, and mainly produce IgM).

Question 20

How do B-cells undergo positive and negative selection?

Answer 20

Positive selection: ability to form a functional BCR during development in the bone marrow (those that cannot form a BCR die)

Negative selection: immature B-cells expressing a BCR specific for self-antigens present in the bone marrow are deleted by apoptosis. Interaction with self-antigens in the spleen is another chance for negative selection and can also give rise to apoptosis.

Question 21

What is the equivalent of the AIRE+ mTECs involved in negative selection during B-cell development?

Answer 21

There isn’t an equivalent of the AIRE+ mTECs that has been discovered for ectopic expression of antigens in the bone marrow in order to facilitate clonal deletion of self antigen-specific B cells.

Question 22

What are the four main characteristics shared between the B- and T- cell development al pathways?

Answer 22

1. rearrangement of gene segments
2. Screening processes to avoid self-reactivity
3. Production of small subsets with discrete function
4. Production of larger “general purpose” subsets.