T cell and B cell activation, MHC lecture I

Question 1

What are the differences between B cell and T cell activation?

Answer 1

B cell receptors and antibodies recognise native protein antigens.

T cells- peptide antigen fragment must be processed and presented to the T cell in context of MHC (eg. CD4 T cell and MHCII).

Question 2

What is MHC?

Answer 2

Major histocompatibility complex.

Peptide binding proteins that bind and present antigens.

The T lymphocytes antigen receptor is programmed to recognise MHC molecules (+ a peptide).

MHC molecules are highly polymorphic- most polymorphic genes and proteins that we express.

These differences look ‘foreign’ to T cells.

Question 3

What classes of MHC interact with which classes of T cells?

Answer 3

CD8 T cells interact with MHCI- bind the a3 domain.

CD4 T cells interact with MHCII- bind the B2 domain.

Question 4

What does MHC I alert T cells to?

Answer 4

Intracellular infections such as viruses.

Virus infects cell. Viral proteins are synthesised in the cytoplasm. Peptide fragments of viral proteins bound by MHCI in ER. Transported to cell surface- follows normal secretory pathway. Cytotoxic T cell recognises complex of viral peptide with MHCI and kills infected cell.

Class I MHC is expressed on almost all cells- in case any cell becomes virally infected.

Captures peptides from endogenous antigens.

Displays them to T cells that express the CD8 co-receptor.

Question 5

What does MHC II alert T cells to?

Answer 5

Extracellular infections such as bacteria.

Macrophage engulfs and degrades bacterium in phagosome or endosome, producing peptides. Bacterial peptides bound to MHCII in vesicles. Transported to surface. Helper T cell recognises complex of antigen with MHCII and activates the macrophage- T cell doesn’t kill directly.

Class II MHC is expressed only on B cells, dendritic cells, & some macrophages.

Captures peptides from exogenous antigens.

Displays them to T cells which express the CD4 co-receptor.

Question 6

What are some key effector functions of T cells?

Answer 6

CD8 + virally infected cell- cell contact- killing of infected cell.

CD4 + macrophage- cell contact and cytokines- activated macrophage which produces cytokines.

CD4 + B cell- cell contact and cytokines- B cell becomes a plasma cell and secretes antibodies.

CD8 directly kills, CD4 activates other things that can kill.

CD4 helps B cell to become an antibody secreting plasma cell.

Question 7

What is the difference in antigen recognition between T and B cells?

Answer 7

B cells and the antibodies they make can recognise virtually any chemical structure-protein, carbohydrate, lipid, nucleic acid.

T cells can recognise internal peptides i.e. no requirement for surface exposure, unlike antibodies.

MHC involvement means T cells recognise other cells: antigen presenting cells (APC) not free antigen, unlike antibodies.

For good antibody responses to a given antigen, the part recognised by B cells must be physically linked to the part (after processing) recognised by T cells.

Question 8

What are the structures of MHC class I and II molecules?

Answer 8

Although the chain composition of 
class I and class II MHC is different
the structure is remarkably similar.

MHCI- 4 subunits- a2 and a1 make up the peptide binding groove, a3 (below a2) is a transmembrane domain, folded into 3 subunits, and B2 (below a1) is a microglobulin.

MHCII- 4 subunits- B1 and a1 make up the peptide binding groove, B2 (below B1) and a2 (below a1) are both transmembrane domains, folded into 2 subunits each.

Question 9

How do antigenic peptides interact with the grooves of MHCI and II?

Answer 9

Grooves are almost the same structure.

Peptides held stably in grooves by hydrogen bonds and charge-charge interactions.

1 interacts with peptide termini (NH1 and COOH)- 2 doesn’t, so 2 can bind much larger peptides as there are no termini restrictions.

Precise interaction.

Can also sometimes use little pockets to interact with peptide side chains.

Question 10

MHC and peptide binding?

Answer 10

MHC molecules display diverse peptides to T cells:

• class I MHC 8-10 amino acids.
• class II MHC usually 12-24 amino acids but no real length limit.

MHC molecules are promiscuous peptide binders i.e. thousands of different peptides can be bound by an individual MHC molecule.

Key hydrogen bond interactions occur between peptide backbone (C=O; N-H) and MHC side chains.

But there are some constraints:

• class I MHC (but not class II) engages peptide termini i.e COOH and NH2 hence length limit.
• C-terminal amino acids in class I MHC peptides are usually either basic or hydrophobic.
• some peptide side chains fit into MHC pockets i.e some positions require certain amino acids to be present.

Question 11

What are the two main cellular compartments?

Answer 11

Cytosol and the vacuolar system.

Cytosol and nucleus are connected- continuous system, so no nuclear pores.

Question 12

Which compartments do MHCI and II capture peptides from?

Answer 12

MHCII captures peptides from vacuolar system (after phagocytosis o pathogen).

MHCI captures peptides from the cytosol (intracellular antigen).

Question 13

For MHCI sampling, where are peptides produced and transported to?

Answer 13

Peptides produced in the cytosol and transported into ER.

The proteasome (multi-subunit, multicatalytic) generates a ‘first draft’ of peptides for class I MHC sampling.

Interferon gamma drives
expression of alternative
proteasome sub-units to create an ‘immunoproteasome’ better able to produce peptides whose C-termini meet class I MHC binding requirements.

Peptides are delivered to the ER lumen by the Transporter Associated with antigen Processing(TAP).

Question 14

What is the peptide loading complex and what does it do?

Answer 14

Orchestrates class I MHC assembly.

Class I heavy chain is stabilised by calnexin until B2 microglobulin binds.

Calnexin is released and the heterodimer of class I heavy chain and B2m forms the peptide-loading complex with calreticulin, tapasin, TAP, ERp57, and PDI.

A peptide delivered by TAP binds to the class I heavy chain, forming the mature MHCI molecule.

The class I molecule dissociates from the peptide-loading complex and is exported from the ER.

Chaperones are involved. Eg. calnexin, calreticulin.
Some are exclusive to class1 system, some are general to peptide processing in ER.
Class 1 only leaves complex if correct tight binding occurs after sampling peptides.
Peptides are sampled first to get the correct fit.

Question 15

What is the role of the ERAP aminopeptidase?

Answer 15

This edits the first draft peptide.

MHCI is loaded with a peptide that is too long at the N terminus.

ERAP removes N terminal amino acids to give a peptide of 8-10 residues.

MHCI travels to cell surface.

Needs a bit of trimming at amino terminus to get a tight fit.

Question 16

What are the MHCI assembly points?

Answer 16

The PLC features generic chaperones that guide folding of many proteins in the ER as well as dedicated proteins:

• generic: calnexin, calreticulin, ERp57, PDI.
• dedicated: TAP, tapasin, ERAP.

Binding a tight-fitting peptide may take several attempts.

Once this occurs, class I MHC is released from the PLC and moves from ER to Golgi.

Genetic deficiency in any of these components leads to defects in peptide loading (e.g weak binding or no peptides) and abnormal CD8 T cell responses:
-e.g bare lymphocyte syndrome results from absence of TAP, only 1% of normal surface class I MHC and poor CD8 responses to viruses.

Some viruses make proteins that block elements of the PLC e.g the TAP transporter.

Question 17

WHere does MHCII peptide loading take place?

Answer 17

In endosomes and lysosomes.

Invariant chain blocks binding of peptides to MHCII molecules in ER

In vesicles, invariant chain is cleaved, leaving the CLIP fragment bound.

CLIP bloks binding of peptides to MHCII in vesicles.

HLA-DM facilitates release of CLIP, allowing peptides to bind.

The class II MHC peptide binding site needs to be protected from peptide binding in the ER.

This is the job of the invariant chain.

Incoming antigen and class II/invariant chain are targeted to the same endosome sites.

Proteolytic enzymes (e.g cathepsins, AEP) and another chaperone DM are also targeted here.

In this ‘reaction vessel’ peptide generation, invariant chain removal and peptide capture occur.

Stop it capturing endogenous peptides- chaperoned by invariant chain (these are same for everyone), acts as dummy peptide to block site, and ensures targeting of MHC to endocytic pathway.
HLA thing stablises 2 when its empty, allows peptide sampling like in class1. Also kicks CLIP out of site.

Question 18

What are the MHCII assembly points?

Answer 18

Phagocytosis (e.g of microorganisms), macropinocytosis and receptor mediated endocytosis (e.g via B cell antigen receptor) drives antigen capture:

• the invariant (yours and mine are the same) chain (Ii):
• controls class II MHC export from ER.
• protects the peptide binding site from premature loading.
• targets class II to the endocytic pathway.

Proteases perform 2 key roles:

• generate peptides from antigen.
• initiate removal of Ii.

DM (in humans HLA-DM) catalyses ejection of last fragment of Ii (Class II associated invariant chain peptide: CLIP) and like tapasin, aids optimal peptide selection.

Successful peptide capture triggers export to cell surface.

Question 19

How to T and B cells collaborate to produce antibodies?

Answer 19

Antigen recognition induces expression of CD40 ligand and cytokines by the Th2 cell, which activates the B cell.

B cell proliferation and differentiation to antibody secreting plasma cells.

B cell is presenting antibodies at cell surface- not secreting antibodies.

When activated to plasma cell, secretes antibodies with same specificity as those on its surface.

Interaction of CD40/CD40L is important. CD40L only expressed when it recognises a peptide that is presented by the B cell.

Question 20

How do T and B cells recognise vaccines?

Answer 20

Linked or cognate recognition.

The Hif vaccine against Haemophilus influenzae B is a conjugate of H.influenzae polysaccaride and tetanus toxoid.

B cell binds polysaccharide component of vaccine conjugate.

B cells recognise the polysaccharide, endocytose the conjugate and generate peptides from the toxoid.

T cells don’t recognise polysaccharides- the conjugate in vaccine is how it can be recognised by T cell.
Toxoid is the peptide part that T cell can recognise.

Display of tetanus toxoid peptides attracts tetanus-specific CD4 T cells to H.influenzae specific B cells.

Tetanus-specific T cells engage not only class II MHC  but also CD40 on the B cell and also secrete cytokines 
e.g IL-4 that drive B cell differentiation & expansion. B cell is activated by the T cell.

Since most people make good responses to tetanus toxoid, this serves as a good source of ‘helper’ T cell epitopes but the actual protein component
is not critical.

Once activated by T cells, the B cell differentiates into plasma cells. Produces anti-polysaccharide antibodies that binds to bacteria.

Antigen-specific B cells present antigen 10^3 to 10^4 x more efficiently than non-specific B cells so T cell ‘help’ is focussed on the B cells that recognise the same antigen. Specific B cells better at taking up and processing vaccine than general B cells.