Lecture 7 - Adaptive recognition and immunological tolerance

Question 1

T-cell generation: how is their diversity formed and what happens when a T-cell recognises an epitope in a dangerous situation?

Answer 1

Somatic recombination in a small number of receptor genes during lymphocyte development results in a large number of naïve circulating lymphocytes, but each with a different specificity

It is a useful clone so it is expanded - the majority respond to combat the threat while others generate memory, ready to respond faster and better if the same threat is encountered again

Question 2

TCR vs BCR: what do they bind, what is their structure, and what is their signalling molecule?

Answer 2

TCR:
* Peptide-MHC complex
* 1 α and β chain
* Bind to ITAMs

BCR:
* Antigens
* 2 heavy and light chains
* Bound to Igα and Igαβ which activate ITAMs

Question 3

Multiple tolerance mechanisms: what is it and why is it needed?

Answer 3

Employing a strategy of multiple layers of checkpoints, deleting the most dangerous ‘reactivities’ and then controlling, or in-effect tuning-down, others to retain the breadth of recognition that may be needed

Self-reactivity is a normal component of any healthy immune system, but it is kept in check by various mechanisms of regulation

Question 4

Tolerance mechanism: what does it aim to do?

Answer 4

• Limit the production of self-reactive T and B cell clones during lymphocyte development
• Prevent unwanted destructive responses by any clones that enter the circulating pool

Question 5

Central tolerance: what is it, what does it do, and is it an absolute process?

Answer 5

Tolerance in primary lymphoid organs (bone marrow for B cells, thymus for T cells)

• Removal of highly self-reactive clones during lymphocyte development (deletion, conversion to tTreg, etc)

Central tolerance is not an absolute process
- ie some self-reactive cells will enter the periphery

Question 6

Peripheral tolerance: what is it and what does it do?

Answer 6

Tolerance in peripheral organs & tissues and secondary lymphoid organs (LNs, spleen)

• Multiple mechanisms limit reactivity against self & harmless antigens in the periphery
• Suppression by tTreg
• Ignorance (eg sequestered inaccessible or low-affinity antigen)
• Deletion (activation-induced cell death; immune privilege)
• Anergy (functional unresponsiveness)
• Induction of iTreg (functional deviation)
• Lack of T cell help for B cells - neglect

Question 7

TCR: what does it need to do to be active?

Answer 7

Make contacts with our produced MHC and a peptide (self-restriction - it should recognise our own MHC-peptide complex)

Question 8

Lineage commitment: what is it, what requires it, and what does it require?

Answer 8

Development of a t-cell into a specific subset (ie CD4+ TH or a CD8+ CTL)

Binding to MHC-peptide complexes - CD4 and CD8 co-receptors bind to invariant sites on MHC-II and MHC-I respectively

Silencing of expression of one of the co-receptors (CD4 or CD8) and initiation of a gene expression program characteristic of T helper (expression of cytokines) or cytotoxic T cells (genes for targeting cell killing)

Question 9

TCR germline gene loci: what are the different gene loci and how do they develop diversity?

Answer 9

The TCR α locus:
* VJ = V exon
* CDR1 & 2 provided by the V gene segment, CDR3 (part that contacts antigens) spans join

The TCR β locus:
* Two gene arrangements; D to J and V to DJ
* VDJ = V exon
* V, D and J all contribute to CDR3

Question 10

What aspect of TCR is diversity focussed on?

Answer 10

TCR diversity is focused on CDR3, which contacts the antigenic peptide

Question 11

Comparison of diversity in BCRs and αβ TCRs

Answer 11

BCR:
* Somatic hypermutation
* Assembled by somatic recombination of multiple gene segments (VDJ (?))
* Similar levels of combinatorial diversity
* Lower levels of junctional diversity than in TCR

αβ TCRs:
* No somatic hypermutation
* Assembled by somatic recombination of multiple gene segments (VDJ (?))
* Similar levels of combinatorial diversity
* Greater level of junctional diversity - D segment read in all reading framed, more J gene segments, and there is TdT activity at all junctions

Question 12

RAG genes: what are they, what do they do, and when are they turned off?

Answer 12

Recombination activating genes

Allow rearrangement of antigen receptor genes - the reason that T-cells are able to recombine and generate varied antigen receptors

Once they pass checkpoints for their function - they should no longer undergo recombination as they have chains that are able to do the intended functions (β chains able to form the pre-TCR, chains that can bind self-MHC-peptide, etc)

Question 13

Common leukocyte progenitos: where do they develop and where do T cells develop?

Answer 13

CLP progenitors originate in the bone marrow, but commit to the T cell lineage and develop into T cells in the Thymus

Question 14

T cell forms: what are they and what is their prevalence?

Answer 14

αβ - 95%
γδ - 5%

Question 15

T cell development: what is the process, what happens at each step and what percentage of DP cells progress enough to survive and escape the thymus?

Answer 15

Antigen-independent quality checkpoints:

• DN - β chain gene rearrangement
• DP - α chain gene rearrangement
• DP (PS) - Expressing both CD4 and CD8 TCRs, positive selection occurs, only allowing cells to progress if they react to self-antigens
  DP (NS) - Expressing both CD4 and CD8 TCRs, negative selection occurs, only allowing cells to progress if they don’t react too strongly to self-antigens
  SP - differences in signalling through the TCR result in either Runx3 (CD8+) or ThPOK (CD4+) expression, causing lineage commitment

Only ~2% of DP cells survive and escape the thymus

Question 16

Positive selection: what is it, what happens in it, and what happens to cells that fail it?

Answer 16

The stage of t-cell development that removes cells that have the potential to become autoimmune

Developing T-cells are now DP and bind to self-antigens

If cells fail to recognise, they’re subjected to death by neglect within the next 3-4 days or undergo gene editing to try and fix the issue

Question 17

T-cell development: how do they develop and commit to a lineage?

Answer 17

Positive Selection
binding MHC turns RAG expression off
- ‘self-restriction’

Question 18

αβ versus γδ lineage choice: what is the process and why are αβ T-cells more frequently produced?

Answer 18

During DN2 - genes for γ, δ and β chains begin to rearrange consecutively
The first to correctly rearrange and associate with the membrane becomes the lineage which then also downregulates the other lineage

αβ cells lineages only have to rearrange one chain instead of two - most likely to occur first

Question 19

γδ T-cells: at what point do they leave the thymus, what do they do, and how do they differ from αβ T cells?

Answer 19

γδ T cells leave the thymus as mature DN T-cells after differentiating

Leave with the ability to secrete cytokines in a process that is not MHC-restricted

• αβ T cells only gain the capacity to secrete cytokines after encountering an antigen in the periphery
• Non-MHC restricted; more innate-like recognition while αβ T-cells depend on MHC-peptide binding

Question 20

The thymus and its framework of epithelial cells: are the areas different at all and what does this mean for T-cell development?

Answer 20

There are different ‘environmental niches’ which allow for distinct areas to produce signals that help to drive cells into different stages specifically - T-cell development does not occur all in one place but instead involves the movements of cells around the Thymus

Question 21

If MHC-I and MHC-II are molecules found within both the Thymus Cortex and Medulla, then how are positive and negative selection occurring in different places?

Answer 21

The repertoire of peptides presented differs in the Thymic Cortex and Medulla, allowing for the Cortex to cause positive selection and the Medulla to cause negative selection:
* Cortex proteins - display ‘private’ peptides that we want to react to
* Medulla proteins - display ‘public’ peptides that we do not want to mount an immune response to

Question 22

Thymus cortex vs medulla: what cells are present, and what MHCs, MHC-I peptides, and MHC-II peptides do they express?

Answer 22

Cortex - Cortical Thymus Epithelial Cells (cTEC):
* Constitutive MHC I and II expression
* MHC I peptides - Thymo-proteasome
* MHC II peptides:
- Cathepsin L
- Thymus-specific serine protease (TSSP) [endosomal/lysosomal]
- Constitutive macroautophagy [delivers self-ag to MHCII]

Medulla - MedullaryThymus Epithelial Cells (mTEC) & Dendritic cells (DC):
* Constitutive MHC I and II expression
* MHC I peptides: Housekeeping-proteasome & Immuno-proteasomes
* MHC II peptides:
- Cathepsin S
- Macroautophagy (mTEC)
Co-stimulation

Question 23

Medulla: how does it differ in colour from the cortex and why?

Answer 23

Paler as there are fewer cells as many have failed the positive selection and so comparatively fewer cells are present

Question 24

How does the thymus know non-thymus antigens to direct negative selection?

Answer 24

The Medulla (mTEC) is inefficient at uptake and presentation of exogenous antigens - it needs aid for sources of self-antigens for tolerance induction:
* Express AIRE (Autoimmune Regulator)
* Express Fezf2

Dendritic cells sample antigens from mTECs and the periphery via the blood

These allow for PGE which prevents autoimmunity

Question 25

PGE: what is it, what does it do, and where is it present?

Answer 25

Promiscuous gene expression - a process in which medulla thymic epithelial cells (mTECs) express tissue-restricted antigens (TRAs) in a non-stochastic (not random) manner using AIRE and Fezf2

In the mTECs in the Thymus

Question 26

AIRE: what is it, what does it do, and what happens if it doesn’t express properly?

Answer 26

Autoimmune regulator

• Interacts with many proteins involved with transcription, promoting lengthening of ‘stalled’ transcripts
• Essential in negative selection and promiscuous gene expression

Genetic deficiency of AIRE results in an autoimmune syndrome - APECED

Question 27

Fezf2: what is it and what does it do?

Answer 27

A Zn finger transcription factor which uses zinc to stabilise its DNA binding structure

• AIRE independent and non-redundant role
• Promiscuous Expression of genes not normally expressed in the thymus, TRAs

Question 28

Mimetic cells: what are they and what do they do?

Answer 28

Small numbers of mTECs expressing lineage defining transcription factors and co-expressing lineage related TRAs

• ie express genes that define ciliated cells in the lung

Question 29

Dendritic cells: how are they used in the Thymus for negative selection?

Answer 29

Display antigens for negative selection:
* Some subsets sample antigen from mTECs and from the periphery via the blood - uptake of TRAs shed from mTECs and mTEC cell debris
* Uptake of blood-bourne antigens
* Cross-presentation of ingested antigens
* Some DCs may come from the periphery and display any antigens they contain

Question 30

What determines if a clone survives to exit into the circulation, or is deleted during negative selection? (or develops into a CD4+ T reg?)

Answer 30

No selection - death by neglect

Too highly - death because dangerous, maybe either gene editing or Treg conversion

Intermediate affinity - Optimal

Question 31

Newly generated SP thermocytes: how do they develop, when do they develop, and how do they join circulation?

Answer 31

Newly generated SP thymocytes mature further to gain functional competency before exiting the thymus

SP thymocytes - approx 4 days further maturation after becoming SP

Thymocytes exit via blood vessels (not lymphatics) through upregulation of the shingosine 1 phosphate receptor (S1P1) which promotes exit of mature thymocytes toward high concentration of S1P in blood

Question 32

RTEs: what are they, what do they do, and why?

Answer 32

Recent thymic emigrants

• Not as mature as circulating peripheral naïve T cells - do not proliferate or secrete cytokines in response to antigens
• Express the lymph node homing receptors CD62L and CCR7, causing localisation to peripheral lymphoid organs for further mature within secondary lymphoid tissue