Generation of Diversity in The T Cell Repertoire

Question 1

What is an antigen?

Answer 1

Antigen – A combination of ‘antibody’ and ‘generate’.
It is any molecule that can bind specifically to an antibody. They are normally immunogenic, so they will induce an immune response from the host.

However in everyday science, ‘antigen’ usually refers to proteins, carbohydrates and lipids capable of binding to B-cell receptors, T-cell receptors and/or innate immune receptors.

Question 2

What is an epitope?

Answer 2

An epitope is a small portion of the antigen.
It is the target of antibodies, MHC and T cell receptors.

Adaptive immune reactions occur to specific epitopes (portions of the antigen) as opposed to the entire antigen itself.
Infection and vaccination usually induce polyclonal T- and B-cell responses – this means that multiple epitopes can be recognised on a single antigen.

Question 3

What is the biggest difference in what B cells and T cells recognise?

Answer 3

The biggest difference between B cells and T cells is that T cells do not recognise native antigens.

Question 4

How do T cells recognise antigens?

Answer 4

Instead, T cells process antigens in order to recognise them.

An antigen will generate multiple peptides, which can then be presented on the surface of an antigen- presenting cell.
Only when the peptide is presented on the surface will we get a T cell response.

Question 5

How are the antigens taken up by APCs?

Answer 5

It can occur by phagocytosis, or via membrane Ig receptor-mediated uptake. In both instances the antigen will get presented, but they will produce slightly different immune responses.

Question 6

Describe the types of APCs.

Answer 6

There are two main types: myeloid cells (which include monocytes and macrophages) and dendritic cells (the most advanced type of APC.

Dendritic cells are also known as ‘professional’ APCs, as there are other cells that do the same thing, but with a lower efficiency.

B cells can also present certain types of antigens, but they are less efficient in doing so.

Monocytes are blood-circulating cells, while macrophages are found in tissue. Macrophages are essentially terminally differentiated monocytes.

Question 7

Describe macrophages and dendritic cells.

Answer 7

They’re rare in peripheral blood - but enriched in mucosal tissues.

They are highly phagocytic cells – induce strong T-cell responses and inflammation. This is important for protection against Mycobacterium tuberculosis.

Macrophages are better-equipped to kill pathogens (higher NO production); DCs are better at migrating to lymph nodes (via CCR7) and presenting antigen to T-cells.

They are both specialised, but ultimately have overlapping functions.

Question 8

Describe B cells as a type of APC.

Answer 8

They are highly abundant in the blood and mucosal tissues.

They execute receptor-mediated internalisation of antigens, as opposed to phagocytosis.

Their primary function is to make antibody (plasma cell) – but they’re still very good at antigen presentation.

It is possibly the main inducer of the T-cell immune response to pathogens such as
Neisseria meningitidis.

Question 9

Describe the process of endogenous antigen processing.

Answer 9

UPTAKE:
The antigens/ pathogens are already present in the cell.

DEGRADATION:
The antigens synthesised in the cytoplasm undergo limited proteolytic degradation in the cytoplasm.

ANTIGEN-MHC COMPLEX FORMATION:
The loading of peptide antigens onto MHC class I molecules  is different to the loading of MHC class II molecules.

PRESENTATION:
There is transport and expression of antigen-MHC complexes on the surface of cells for recognition by T cells.

Question 10

Is exogenous antigen processing sufficient?

Answer 10

Macrophages have well-developed lysosomal systems. They are specialised for motility, phagocytosis and the introduction of particles to the lysosomal system.

Most cell types do not have lysosomal systems developed as well as macrophages, but viruses can infect most cell types.

A non-lysosomal mechanism to process antigens for presentation to T cells is required.

Question 11

Describe cytosolic protein presentation.

Answer 11

The antigen/viral protein enters the proteasome, which cleaves it into multiple peptides. These are then loaded onto MHC Class I molecules and presented on the cell surface.

Question 12

Describe non-lysosomal antigen processing.

Answer 12

Inactive virus raises a weak cytotoxic T lymphocyte (CTL) response.
The processing of antigens from inactive viruses is sensitive to
lysosomotrophic drugs.
Thus, antigens from inactive viruses are processed via the exogenous pathway.

Infectious virus raise a strong CTL response.
The processing of antigens from infectious viruses is NOT sensitive to lysosomotrophic drugs.
Most CTLs recognise antigens generated via a non-lysosomal pathway. Protein synthesis is required for non-lysosomal antigen processing.
Thus, antigens from infectious viruses are processed via the endogenous pathway.

Question 13

How are antigens processed and presented via the two pathways?

Answer 13

In the endosomal pathway, the antigen is processed in the proteosome, cleaved into multiple peptides and presented on MHC Class I molecules that reside in the cytosol.

In the exogenous pathway, the antigen is endocytosed, then sequestered to lysosomes or endosomes and processed there. The resulting peptides are presented on MHC Class II molecules that reside in the lysosome/endosome. They are then loaded to MHC Class II on the surface of the cell.

Based on the way in which the antigen is acquired, the different pathways will activate different T cells: the exogenous pathway will activate CD8 T cells, and the endogenous pathway will activate CD4 T cells.

Different MHC will activate different T cells: MHC II will activate CD4 cells, and MHC I will activate CD8 cells.

Question 14

How are exogenous and endogenous pathogens eliminated?

Answer 14

EXOGENOUS PATHOGENS:
They are eliminated by antibodies and phagocyte activation by T helper cells that use antigens generated by exogenous processing.

ENDOGENOUS PATHOGENS:
They are eliminated by the killing of infected cells by CTL that use antigens generated by endogenous processing.

Question 15

Describe MHCs.

Answer 15

MHC stands for major histocompatibility complex. There are two types, I and II.

They are quite similar in structure as they both have alpha and beta subunits. The common thing between both of them is that both of them have the peptide binding groove, where the peptide binds.
The groove interacts with many different domains. If you modify that structure, the capacity of the MHC to bind to peptides will be lost.

Question 16

List some differences between MHC I and MHC II.

Answer 16

MHC Class I:

• expressed on all nucleated cells
• binds short peptides (8-10 amino acids)
• presents to CD8+ T-cells
• recognise antigens from the cytosol (+ cross-presentation)

MHC Class II:

• expressed on APCs and activated T-cells
• long peptides (typically 15-24 amino acids)
• presents to CD4+ T-cells
• recognise antigens from phagosomes and ensodomes

Question 17

Describe the TCR.

Answer 17

It binds to peptide-MHC (pMHC) complexes – it cannot recognise peptide alone (B cells can).

There is a huge diversity – with potentially up to 1 x 10^13 different TCRs.

It exists in a TCR complex with accessory molecules such as CD3.

Question 18

List some similarities and differences between the TCR and BCR.

Answer 18

Similarities to B cell receptor/antibody:

• belongs to Ig superfamily
• like Fab fragment of antibody – on the tip of the molecule
• large diversity
• once produced, they have a single specificity

Differences to B cell receptor/antibody:

• antibodies have a very high affinity, TCR has much lower affinity
• TCR cannot be released
• TCR has no Fc fragment, so no cellular functions
• TCR has single binding site rather than two binding sites (antibodies)
• a BCR/Ab has 5 classes, while a TCR hs 2 classes (αβ and γδ)

Question 19

Why do TCRs have a lower affinity that BCRs?

Answer 19

Mechanisms which generate B-cell receptor diversity:

• Before antigen stimulation: Somatic recombination
• After antigen stimulation: Somatic hypermutation

Mechanisms which generate T-cell receptor diversity:

• Before antigen stimulation: Somatic recombination
• After antigen stimulation: None

The missing hypermutations explain the lower affinity of the T cells.

Question 20

What are the the signals that are needed to activate T cells?

Answer 20

1. Peptide-MHC (pMHC)
2. Co-stimulation
3. Cytokines

Question 21

Describe the three signals needed to activate T cells.

Answer 21

The main signal (Signal One) is delivered from the APC by a peptide-MHC complex to the TCR.

The co-stimulatory signal (Signal Two) is delivered from the APC by germline-encoded accessory receptors such as the ‘B7 family’ (CD80 and CD86) – although many of these receptors are not fully characterised or understood.

Lastly, Signal Three is formed of cytokines secreted by the APC to determine the T-cell phenotype.

• IL-12 promotes TH1 cells
• IL-4 promotes TH2 cells
• IL-23 promotes TH17 cells

These have specialised functions in dealing with pathogens.

Question 22

How do we differentiate between helper T cells and cytotoxic T cells?

Answer 22

Helper T cells express CD4, and cytotoxic T ells express CD8.

CD4 T cells are also called helper T cells, mostly from helping other cell types by secreting cytokines. They will kill pathogens indirectly.

Cells that predominantly express CD8 are called cytotoxic T cells, which directly recognise and kill infected cells.

Question 23

Describe the different T cell function based on whether it is a CD8 T cell, CD4 Th1 or Th2 cell.

Answer 23

A CD8 cell will recognise a virus-infected cell by the viral peptides that it presents on its surface. It will attach to it and kill the cell, ultimately killing the virus inside it.

A CD4 Th1 cell will produce cytokines that activate macrophages., and then those macrophages will kill intracellular pathogens more efficiently.

A CD4 Th2 cell will produce cytokines that activate B cells, which will produce antibodies that will neutralise and prevent the colonisation of bacteria.

Question 24

Why do we need negative regulators of antigen presentation?

Give two examples.

Answer 24

An overly-vigorous immune response is harmful to the host

Negative regulators of antigen presentation provide an ‘immune
checkpoint’ to limit T-cell activation, thus achieving HOMEOSTASIS.
The CD8 response should subside, and only the memory should remain.

Two important molecules – CTLA4 (Cytotoxic T-Lymphocyte-Associated Protein 4) and PD-L1 (Programmed Death-Ligand 1) are crucial for dampening the T-cell response.

Question 25

Describe the mechanism of PD-L1 and CTLA-4 inhibition of T-cell function.

Answer 25

The TCR interacts with APCs and that leads to T cell activation.

If the PD-1 receptor is activated by its ligand, it leads to blockade of the TCR activation. This is through multiple signalling pathways, but the end product is that SHP-2 dephosphorylated the TCR signalling molecules.

The CTLA-4 acts on the costimulatory molecule. It competes with the CD38 for APC ‘attention’.

Since both signals are needed, both molecules can be blocked.

This is important because you want to make sure these pathways are not blocked when you want a robust immune response, for e.g. to cancer or to a viral infection.

Question 26

How do T cells ensure that they do not recognise self-antigens?

Answer 26

T-cells arise from the thymus, which is a ‘school’ for T-cells. T-cells are exposed to self-antigens and tested for reactivity.

T-cells that can’t bind to self-antigen MHC are deleted: POSITIVE SELECTION.
These T-cells are useless because they won’t protect against pathogens.

T-cells that bind to self-antigen MHC too strongly are also deleted: NEGATIVE SELECTION.
These T-cells are dangerous because they are too self- reactive.

Question 27

Describe Treg’s role in T cell development.

Answer 27

Tregs are involved in the selection of self-reactive T cells. They do so by inducing their apoptosis or deletion.
These cells are characterised by the presence of FOXP3.

Question 28

List some pathogens that can impede antigen presentation.

Answer 28

Mycobacterium tuberculosis:

• up-regulates PD-L1 on APCs to shut down T-cell activation
• blocks MHC Class II expression via multiple mechanisms

Neisseria meningitidis:

• blocks DC activation – low CD40, CD86 and MHC Class I & II expression.
• the antigens (capsule) have a homology to self- antigen, therefore anergic (lack of reaction from) T-cells

Neisseria gonorrhoeae:
- expresses Opa protein, which binds to T-cells and induces tyrosine phosphatases that ‘switch off’ key molecules involved in TCR signalling

HIV

• up-regulates PD-1 on T-cells, which antagonises TCR signalling
• binds to DC-SIGN to suppress DC activation via Rho-GTPases

Herpes Simplex Virus (HSV):

• produce protein which binds to and inhibits TAP
• prevents viral peptide transfer to ER

Adenovirus:

• produce protein which binds MHC class I molecule
• prevents MHC class I molecule from leaving ER