L2 - Antigen Processing and Presentation

Question 1

Describe the structure of MHC class I

Answer 1

1 transmembrane domains (alpha-3), 1 associated beta-2 microglobulin, alpha-2 and alpha-1 create peptide-binding cleft (preferentially binds 8-10 amino acids)

Question 2

Describe the structure of MHC class II

Answer 2

2 transmembrane domains (beta-2 and alpha-2), beta-1 and alpha-2 create peptide binding cleft (preferentially binds 13-17 amino acids)

Question 3

Describe the basis of the two routes of antigen processing and presentation

Answer 3

Intracellular antigens degraded in the cytosol and peptides presented by MHC I to CD8+ T cells

Extracellular antigens endocytosed and degraded in endosomes and peptides presented by MHC II to CD4+ T cells

Question 4

Describe how MHC and peptide combinations drive thymic education

Answer 4

• Each thymus displays a unique ‘peptidome’
• A person’s individual T cell repertoire arises from clonal selection
• By defining self, the immune system can then identify novel pathogens and respond to them

Question 5

Describe the main components involved in MHC-1 processing

Answer 5

Effector molecules involved in producing peptide antigen (polypeptide, polyubiquitin, proteasome, ERAP (ER aminopeptidase))

Chaperone proteins (calnexin, calreticulin, ERp57) - help assemble, stabilise and load peptides onto MHC-1

Transporters e.g. TAP - transporter associated with antigen processing (TAP1/2 heterodimer that uses ATP to transport peptides across ER membrane), Tapasin (bridges TAP + MHC-1 to increase TAP levels, facilitate peptide loading and influence peptide selection)

Question 6

Describe the main process of antigen presentation via MHC-1

Answer 6

A polypeptide is ubiquitinated in the cytosol which targets it for degradation by the proteasome. the peptide antigen is then processed at the ER and transported across the membrane before being loaded onto the MHC-1 molecule. The peptide-loaded MHC-1 complex is then transported to the plasma membrane, first via an ER-Golgi intermediate compartment that then forms a vesicle.

Viral proteins often target this process at various points.

Question 7

Describe the main components involved in MHC-2 processing

Answer 7

HLA-DO/H2-O and HLA-DM/H2-DM (regulate peptide loading)

Invariant chain, li (prevents premature peptide binding)

CLIP = class II associated invariant peptide chain (prevents premature degradation of MHC II before antigen binding)

Proteolytic enzymes (prepare peptides for binding)

Question 8

Describe the main process of antigen presentation via MHC-2

Answer 8

1) HLA-DO binds HLA-DM to inhibit exchange of CLIP for peptide
2) li is proteolysed by proteolytic enzymes, leaving behind the residual li fragment called CLIP in the peptide binding groove
3) HLA-DM binds MHC-II and displaces CLIP, allowing an antigen peptide to bind
4) The peptide:MHC-II complex is transported to the cell surface in a vesicle

Question 9

Which cells present MHC-II peptides?

Answer 9

Professional APCs present MHC-II all the time (dendritic cells, macrophages and B cells)

Cells of other tissues can present MHC-II peptides under inflammatory drive, leading to an increase in activation of CD4+ T cells

Question 10

Describe the production of peptides for MHC-I and MHC-II complexes

Answer 10

Class I - the proteasome and associated enzymes e.g. ERAP (ER aminopeptidase)

Class II - late endosome/lysosome enzymes - cysteine proteases e.g. cathepsins B, D, S and L and asparaginyl endopeptidase

Question 11

Describe how peptides are edited for high affinity binding to MHC-I and MHC-II and why it is needed

Answer 11

Class I - Tapasin competes with high abundance / low affinity endosomal peptides but is expelled by high affinity peptides that allow groove to close

Class II - HLA-DM edits repertoire in favour of high affinity / stable binding peptides

High affinity binding is needed so that the peptide:MHC complexes don’t dissociate when they reach the more harsh extracellular environment and can activate T cells

Question 12

What are the 3 pathways for extracellular class II antigen acquisition?

Answer 12

Clathrin-mediated endocytosis (receptor-mediated endocytosis)

Phagocytosis

Macropinocytosis

Question 13

Describe clathrin-mediated endocytosis as a pathway for class II antigen acquisition

Answer 13

Also known as receptor-mediated endocytosis.

There is a specific uptake of material bound to receptors on APC e.g. BCR, FcR, lectin R, complement R. Material enters cell via clathrin-coated vesicles. MHC-II biosynthetic pathway can also go through these vesicles so the MHC + antigens are in the ‘same space’.

B cells rely almost completely on this mode of antigen uptake.

Question 14

Describe phagocytosis as a pathway for class II antigen acquisition

Answer 14

Actin dependent, requires a lot of membrane and involves receptors e.g. C-type lectins, FcR, complement and scavenging. Results in uptake of larger molecules than clathrin-mediated endocytosis which uses similar receptors. Process forms a phagosome (low proteolytic activity) that fuses with a lysosome to generate a phagolysosome (highly destructive).

Most significant mechanism of antigen uptake for dendritic cells and macrophages.

Question 15

Describe macropinocytosis as a pathway for class II antigen acquisition

Answer 15

Plasma membrane ruffles entrap external material. This is actin-dependent and results in a non-specific uptake of material which varies in size from small molecules to complete cells (e.g. proteins, viruses, bacteria).

Common in dendritic cells (particularly immature ones) and macrophages but rare in B cells.

Question 16

What is cross-presentation in terms of antigen presentation?

Answer 16

When there is extracellular antigen acquisition for MHC-I (cytosolic or vacuolar pathways) or intracellular antigen acquisition for MHC-II (autophagy).

Cross-presentation of extracellular antigens on MHC-I allows CD8+ T cells to respond to components that would not be found normally in the cytoplasm of dendritic cells e.g. some viruses and tumour antigens.

Question 17

Describe the cytosolic pathway in terms of MHC-I cross presentation

Answer 17

The exogenous antigen is endocytosed and exported to the cytosol via channels/translocators. The vesicle ruptures and the antigen enters the usual cytosolic pathway where it is degraded by the proteasome and then either transported to the ER via TAP for MHC-I loading in the ER or re-imported to a phagosome via TAP and loaded onto MHC-I in the phagosome.

Degradation via the proteasome is sensitive to proteasome inhibitors

Question 18

Describe the vacuolar pathway in terms of MHC-I cross presentation

Answer 18

The exogenous antigen is endocytosed and degraded in the phagosome. Peptide is loaded onto MHC-I in the endosome (loading is therefore independent of ER transporters like TAP).

Degradation is therefore resistant to proteasome inhibitors (TAP-independent) but sensitive to inhibitors of lysosomal proteolysis (especially Cathepsin S inhibitors).

Question 19

How do the MHC-I molecules for cross-presentation get into the endosomes?

Answer 19

Newly synthesised MHC-I transported into endosomes (evidence - machinery critical for trafficking MHC-I from ER to endosomes is essential for cross-presentation).

Pre-existing MHC-I recycled from plasma membrane into endosomes and peptide is then exchanged or loaded in the endosome (evidence - tyrosine residue at cytosolic end of MHC-I molecule is essential for internalisation from cell surface and for cross-presentation).

Question 20

Describe the autophagy in terms of MHC-II cross presentation

Answer 20

20-30% of MHC-II peptides are from cytosolic or nucleic proteins

Autophagy generates citrullinated proteins and peptides and microtubule-associated light chain 3 (LC-3) labels autophagy.

Mutation of autophagy gene 5 (ATG5) significantly disrupts thymic selection which causes quantitative alteration of MHC-II peptidome in cortical thymic epithelial cells. ATG5-deficient dendritic cells are also unable to activate herpes simplex virus-specific CD4+ T cells.

Question 21

Describe changes of state in antigen processing

Answer 21

Basal state vs stimulated state (aka activated and is driven by cytokines).

Stimulation increases class I and II expression and alters antigen processing, changing the peptidome.

Class I - IFN-gamma is produced in response to infection and modified the 20S subunit to form the immunoproteasome which contains inducible proteolytic units).

Class II - GILT (IFN-gamma induced lysosomal thiol reductase) reduces intramolecular disulphide bonds, facilitating proteolysis

Therefore in the presence of inflammation, the production of peptides increases.

Question 22

Describe changes of state in dendritic cells

Answer 22

In immature DCs, there is a high MHC-II turnover which is regulated by ubiquitination of a conserved lysine in the beta-chain.

Stimulation of CD11+ conventional DCs e.g. by TLR signalling, there is a short term increase in MHC II synthesis and then MHC-II turnover is reduced, increasing the half-life of surface MHC-II antigen complexes to allow an adaptive immune response to develop.

Question 23

Describe changes of state in macrophages

Answer 23

Steady state macrophages internalise the equivalent of their whole plasma membrane in 30 minutes and the rapid turnover of peptide:MHC-II complexes and CD86 is regulated by the E3 ubiquitin ligase MARCH1.

During stimulation, MARCH1 is down-regulated which stabilises MHC and co-stimulatory molecules on the cell surface. IL-10 (an anti-inflammatory cytokine) upregulates MARCH1.

Question 23

How do APCs and relevant T cells meet?

Answer 23

• Immunosurveillance is constrained to secondary lymphoid tissue, concentrating DCs and T cells
• Blood and lymph flow is upregulated by innate immune signalling to increase the rate of native T cell entry and retention time through upregulation of CD69
• Lymphocyte searching favours detection of rare populations, switching between a scanning and a motion phase
• Co-localisation of different rare cell types is driven by chemokine/chemokine receptor expression
• High sensitivity: T cells can respond to fewer than 10 pMHC per APC