Lecture 5 - MHC class I presenting

Question 1

MHC: what is it, what does it do, and why is it necessary?

Answer 1

Major histocompatibility complex - also known as human leukocyte antigens (HLA) - a set of genes that code for proteins detecting foreign substances

• Recognises foreign material, without it we’d have no immune system activation
• Displays the pathogen peptides for detection by the immune system
• Stably bind to and present peptides for t-cell recognition
• Isolated peptide expression - peptide exchanges should not occur as they may cause inappropriate activation to occur

Question 2

MHC I vs MHC II cells

Answer 2

MHC-I restricted effector cytotoxic CD8+ T cells target cells infected with viruses and other intracellular pathogens, leading to lysis of infected cell

MHC-II restricted effector CD4+ T cells recognise antigens displayed by professional antigen-presenting cells and then “help” other cells of the immune system

(2 x 4 = 1 x 8)

Question 3

Expression of MHC class I: why is it necessary, what cells express it, and are there any exceptions?

Answer 3

Once activated, CD8+ cytotoxic t-cells can be triggered by just signal I (MHC class I) and can exact its function of destroying infected cells

Therefore, since all cells can become infected, almost all cells can express MHC class I

Red blood cells don’t - the key issue with plasmodium (pathogen causing malaria), they hide in RBCs and escape the immune system

Question 4

Expression of MHC class II: why is it necessary and what cells express it?

Answer 4

Allows cells to act as APCs for CD4+ t-cells

APCs

Question 5

Are maternal or paternal MHC genes expressed?

Answer 5

MHC genes are codominantly expressed

Question 6

What are MHC genes called in humans?

Answer 6

Human leukocyte antigen (HLA) genes

Question 7

MHC: how many types are there, are there variations among the population, and is this beneficial?

Answer 7

Polygenic - contains several different MHC class I and class II genes:
* Class II - HLA DP, HLA DQ, and HLA DR
* Class I - HLA A, HLA B, and HLA C

Highly polymorphic - there are multiple alleles of each gene within the population

Beneficial - it is unlikely that any one pathogen will be able to avoid the immune response in an entire population

Question 8

MHC polymorphism: when does it occur?

Answer 8

During “crossing over” during meiosis

Question 9

What chromosome is the MHC found on?

Answer 9

Chromosome 6

Question 10

MHC I structure: what is it, how is it structurally linked, what domains does it have, and what does it result in?

Answer 10

MHC-I is a heterodimeric glycoprotein composed of a transmembrane polymorphic alpha chain (43kDa) + invariant β2-microglobulin chain (12kDa)

The two chains are non-covalently linked

• The alpha chain has 3 distinct domains and the closed peptide-binding groove is formed between the α1 and α2 domains
• α1 and α2 domains are polymorphic, determining the type and sequence of peptide that can bind - specificity

Question 11

MHC-I peptide binding: how large are the peptides it can bind, what allows it to bind to peptides, how does polymorphism affect it, and what are ‘anchor residues?’

Answer 11

MHC-I molecules bind short peptides 8-10 amino acids in length - peptides lie in an elongated conformation, with occasional minor kinks allowing slightly longer peptides to bind

Invariant tyrosine residues found in all MHC class I molecules bind the amino and carboxy termini of the peptides

Polymorphisms occur at key sites in the MHC groove, altering the sequence of the peptide that stably binds to the specific MHC molecule

Amino acids on peptides that bind to these polymorphic residues in MHC

Question 12

Anchor residues: how do they result in millions and millions of different peptides being recognised?

Answer 12

• Anchor residues do not have to be identical in amino acid composition but they must have comparable properties to bind to a given MHC variant -ie hydrophobic, aromatic
• Any given MHC-I molecule can theoretically bind any number of peptides, as long as they are comparable at the anchor residues, meaning the non-anchor residues amino acids are largely variable
• Each peptide-MHCI complex is recognised by a distinct TCR on T cells - Specificity

Question 13

Where are MHC class I binding peptides sourced from?

Answer 13

The job of MHC class I is to signal to CD8+ T cells that the cell is infected:
* Cytosol - this is where peptides from infecting pathogens will be

Question 14

MHC class I: what are the key issues with their function and what mechanisms do cells undergo to counteract these issues?

Answer 14

How are perfectly sized peptides obtained for MHC class I binding - proteasomal degradation

How is the peptide-binding site of MHC I never exposed to the cytosol -

How does MHC class I bind the cytosolic peptides -

Question 15

Proteasome: what is it, what is its structure, what are the types, and what do they do?

Answer 15

Large, multi-catalytic protease complex degrading ubiquitin-labelled proteins

• Cylindrical complex of 28 subunits; 4 stacked rings, each of 7 subunits
• Hollow core lined by active sites of proteolytic subunits

Immunoproteosome - split up pathogen peptides into peptides that can be bound to MHC class I which can then be transported by the TAP complex
26S proteosome - constitutively produced proteosome necessary for cell homeostasis (damaged/unwanted peptide degradation)

Question 16

26S proteasome: when is it made, what is its structure, and what is its function?

Answer 16

Constitutively produced

• 20S Core region - ATPases from 19S cap cause its opening
• Two 19S caps on each end with one side containing ATP binding sites which control the entry of ubiquitin-labelled proteins into the core and the other releasing broken down peptides

Essential for normal homeostasis of cell -
degradation of damaged, unwanted peptides for disposal

Question 17

Immunoproteasome: what is it, what is its structure, when is it formed, how does it take in pathogen peptides, and what does it do?

Answer 17

Proteosome that degrades pathogen peptides into lengths that can interact with MHC class I molecules

• PA28 - regulates entry into the immunoproteasome
• 20S subunit - how it opens

Induced by IFN-γ

• Increased cleavage of polypeptides after hydrophobic residues
• Reduced cleavage after acidic residues
• Produces peptides with carboxy-terminal residues that are preferred anchor residues for class I molecules
• Produces peptides favoured by TAP for transportation and MHC class I loading
• Increased rate at proteins enter and peptides efflux

Question 18

PA28: what is it, where is it located in its organelle, and what does it do?

Answer 18

Proteasome activator 28 - 11S regulatory particle

Replaces the 19S units on constitutive proteasome, seven-membered ring consisting of PA28α and PA28β

• Bind to the ends of the immunoproteasome 20S cylinder

Question 19

Immunoproteosome forming: what is the process?

Answer 19

Immunproteosome forming begins with IFN-γ affecting expression in cells:
* b1i - encoded for in MHC locus
* b2i - not encoded in MHC
* b5i - encoded for in MHC locus

Upon translation, these three subunits displace and substitute for constitutively expressed subunits of the proteasome

The substituted and substitute subunits are the active proteases of the proteasome - this
replacement alters the specificity and function of the proteasome, forming the immunoproteasome

Question 20

TAP: what is it, where is it located, what is its structure, and what does it do?

Answer 20

Transporter associated with antigen processing ATP-binding cassette (ABC) family of proteins

Located in the ER membrane

TAP1 and TAP2 (transporters associated with antigen processing -1 and -2) form a heterodimer across the ER membrane

• Acts as a channel allowing entry of peptides into the ER lumen
• TAP complex prefers to transport peptides between 8 and 16 amino acids with hydrophobic or basic residues at the carboxy terminus - peptides capable of MHC class I binding
• Absence of TAP molecules leads to defective expression of MHC class I peptide complexes

Question 21

ABC proteins: what are they and what do they do?

Answer 21

ATP binding casette proteins

Mediate ATP-dependent transport of ions, sugars, amino acids and peptides across membranes

Question 22

Peptide loading to MHC class I: what is the process of the classical pathway?

Answer 22

• Newly synthesized MHC class I α chains assemble in ER with membrane-bound chaperone protein, calnexin, which stabilises the chain to allow β2m binding
• When the complex binds β2m, calnexin dissociates and MHC I binds to TAP via the TAP-associated protein, tapasin. Chaperones calreticulin and Erp57 also bind at this stage (Erp57: Tapasin complex involved in peptide editing)
• The MHC molecule is stuck at this stage until bound by a peptide (transported from the cytosol by TAP), which completes the folding of the MHC molecule
• If a bound peptide has unstable binding to the MHC class I molecule, further trimming may occur (ERAP1 on the amino end and Tapasin- carboxy end), if more stable binding is established then the MHC class I molecule progresses to the next step but if not it goes back to the previous step
• Fully folded MHC molecule leaves ER, and is transported through the Golgi to the cell surface

Question 23

What affects the amount of MHC I present in the ER?

Answer 23

MHC is produced in excess and forms a rapid expression of pathogenic peptides if a cell becomes infected

Question 24

If only APCs can prime a naive CD8+ cytotoxic t-cell, then how can an immune response be generated if an infected cell is a non-APC (or inhibited APC)?

Answer 24

Cross presentation - DCs take up pathogen peptides on infected cells and display them to CD8+ cytotoxic t-cells

Question 25

Cross presentation of antigens: is the efficiency of the process equal among all APCs and what is the process?

Answer 25

Affected by subsets of dendritic cells (CD141+ better at CP) as well as the specific receptors binding the material (Fc, Mannose, C-type lectin, and Dectin-1 receptors are typically better) - helps to direct antigens to their intended pathway

• Antigens are phagocytosed and join a vesicle containing TAP, TPN, and ERAD (ERAD - opposite to TAP: translates material from the formed vacuole into the cytosol)
• This vesicle has NOX2 added to it which causes slow acidification of the vesicles (fast acidification would promote an MHC class II response)
• MHC class I molecules are recycled from the surface by Rab11a and SNAP23 or sourced from the ER by CD74 and fused with the vesicle
• Proteins can be removed from the vesicle/vacuole by ERAD and moved to the proteasome for degradation
• Proteins are then either transferred through the ER to bind MHC class I molecules and then moved to the cell surface to interact with CD8+ cytotoxic t-cells or move from the proteasome to the vesicle/vacuole which now contains TAP, Tapasin, an MHC class I molecule, and IRAP and can then be trafficked to the cell surface to interact with CD8+ cytotoxic t-cells

Question 26

ER

Answer 26

Janeway’s Immunobiology Edition 10. (Chapters 4/6)

Rock et al Trends Immunol 2016 Sep 7 S1471-4906 (MHC molecules and peptide expression)

Cruz et al Seminars in Immunology 2023 101729 (Cross Presentation)

Blum et al Ann. Rev. Immunol 2013 43-73 (general antigen processing review-great diagrams)

Question 27

Mechanisms of cross-presentation and MHC class I peptide loading

Answer 27

ER

Question 28

ER - MHC and attraction

Answer 28

Instinctively drawn to breeding for a wider MHC - a study (Wedekind (1995)) found that women found shirts worn by men who had different MHCs to them smelled more attractive than those with similar MHCs to them