Lecture 6 - MHC class II presenting

Question 1

MHC class II: what is it, what is its structure, what is its peptide binding cleft, how is variation achieved, and how many forms are there?

Answer 1

MHC-II is a dimeric glycoprotein composed of two transmembrane, two domain, glycoprotein chains:
* Alpha chain (34kDa)
* Beta chain (29kDa)

Peptide binding cleft - open at each end and is generated between the β1 and α1 subunits

Found primarily in the β1 and α1 subunits, specifically the areas forming the cleft

HLA-DP, HLA-DQ, HLA-DR:
* Alpha and beta chains from the different chromosomes can pair up - i.e. Up to 4X DP, 4X DQ, 4X DR (N/B DR alpha chain is monomorphic-so 4, not 8 DRs)
* (M)DRα - (M)DRβ
* (M)DRβ - (P)DRα
* (M)DRα - (P)DRβ
* (P)DRα - (P)DRβ

I DON’T UNDERSTAND THIS

Question 2

MHC class II-peptide binding: what peptides can bind, is this binding different from MHC-I binding, and how do peptides bind?

Answer 2

Open-ended MHC-II molecules bind longer peptides of between 10 and 30 amino acids

The ends of the peptide are not bound in the groove - different than MHC-I

• Peptide lies in an extended conformation and is held by hydrogen bonds distributed along the length of the peptide
• Peptide held by interactions with polymorphic amino acids (within groove pockets) and conserved side chains of invariant amino acids that line the peptide-binding groove

Question 3

MHC-II-peptide binding: what restrictions are there for peptide binding?

Answer 3

• Since peptide is not bound at the ends and protrudes out of the MHC molecule any length of peptide is theoretically possible BUT long peptides appear to be trimmed by peptidases, to make peptides of 13-17 amino acids typically
• Peptides binding to HLA-DR3 display varying lengths but core structures have similar properties, if not sequences
• Anchor residues of peptide bind to polymorphic residues in MHC, giving specificity of binding - same principle as MHC class I

Question 4

MHC binding: process in a nutshell

Answer 4

MHC interacts with peptide backbone and amino acid side chains that insert into the pockets

Question 5

Difference between MHC class I and II molecules: what is the peptide binding domain, the nature of peptide-binding cleft, the general size of bound peptides, the peptide motifs involved in MHC binding, and the nature of bound peptide?

Answer 5

MHC class I:
* α1/α2
* closed at both ends
* anchor residues at both ends of peptide - typically hydrophobic carboxyl-terminal anchors
* extended structure - both ends interact with MHC cleft but middle arches up away from MHC molecule

MHC class II:
* α1/β1
* open at both ends
* anchor residues distributed equally along the peptides
* extended structure - held at a constant elevation above MHC cleft

Question 6

MHC class II cells: what do they do?

Answer 6

The job of MHC class II is to instruct CD4+ T cells to help other immune cells to mediate their functions (i.e. macrophages, B cells)

CD4+ T cells are (normally) required to deal with extracellular pathogens

Thus, MHC class II typically displays peptides originating from extracellular proteins

Question 7

As MHC class II molecules are assembled in the lumen of the endoplasmic reticulum, how does the cell prevent peptides destined for display by MHC class I from being presented by MHC class II molecules?

Answer 7

CLIP - the invariant chain blocks the MHC-II binding site and prevents unwanted peptides from binding

Question 8

Does CLIP stay in the MHC-II peptide binding groove once it has left the ER?

Answer 8

Yes, until it reaches the acidic vesicles where the peptides that MHC-II wants to bind are located

Once in these vesicles, HLA-DM removes CLIP and stabilises MHC-II for peptide binding and removes any weak affinity peptides

Question 9

MHC class II variation

Answer 9

Genes encoding for MHC pair up with their respective counterpart from the maternal/paternal side (cis) but may also move around and pair with others (trans)

Question 10

Peptide processing for MHC-binding: what is the standard process for each type?

Answer 10

MHC class I:
* Cytosol proteins - taken to proteasome, cleaved, and moved to ER for association with MHC-I

MHC class II:
* Extracellular proteins/pathogens are internalised into phagosomes and endosomes, internalised by endocytosis
* Internalised proteins are degraded by acid proteases as the endo/phagosome acidified and ultimately fuses with the lysosome
* This acidification and lysosomal fusion becomes more efficient after macrophage and DC activation

Question 11

Cathepsins: what are they, what do they do, and what are their effects in MHC?

Answer 11

Cysteine proteases - one of the proteases used in the endosomal/lysosomal degradation of peptides for MHC-II binding

Cut in the middle - endoproteases

S and L cathepsin-deficient mice display deficiencies in antigen processing in the MHC-II pathway

Question 12

GILT: what is it, where is it found, why is it necessary, and what is the evidence for this function?

Answer 12

Gamma interferon-induced lysosomal thiol reductase

Localises in endosomal (Lamp2+) compartments

Disulfide bonds may need to be reduced before proteins are digested in endosomes - disulfide bonds are strong (ER?), so cleaving a protein with lots of them often requires GILT action

• CD4+ T cell response to proteins containing disulphide bonds is reduced in GILT-/- mice compared with intact mice
• The response to proteins not containing disulphide bonds (i.e. casein) is intact in GILT-/- mice

Question 13

The invariant chain (li): what is it, what is it stabilised by, why is it necessary, and what form does it take?

Answer 13

Inhibits peptide binding by sitting in the peptide binding groove

Calnexin stabilises the assembly of the li-MHC complex

• MHC class II is synthesised in ER, but it must be blocked from binding peptides (destined for MHC-I) until it is in the correct endosomal compartment
• li chain (via signals from the transmembrane segment) also targets the MHC complex for delivery to low-pH endosomal compartments
• In the MIIC (MHC class II compartment) the li is cleaved by proteases to form the class II-associated invariant chain peptide (CLIP)

CLIP

Question 14

CLIP: what is it, what does it do, and what proteases causes their formation?

Answer 14

Class II-associated invariant chain peptide

• Prevent MHC II dimers from degrading before antigenic peptides bind
• Prevent autoimmunity
• Prevents peptides intended to bind MHC-I from binding to the MHC-II

Cathepsin S - APC
Cathespin L - thymic epithelial cells

Question 15

HLA-DM: what is it, what does it do, and what is it inhibited by?

Answer 15

H-2M - closely resembles MHC-II with α and β chains, but its peptide binding groove is closed so that incorrect binding doesn’t occur

• Catalyses the release of CLIP in MIIC, enabling peptides to bind to MHC class II molecules once the MHC-II is in the acidic vesicles
• Stabilises empty MHC class II molecules in MIIC before peptides bind
• Performs “peptide editing” removing unstably bound peptides from the MHC-II complex - enables stable long-lasting expression of MHC II-peptide complexes on the cell surface

HLA-DO - prevents peptide loading

Question 16

MARCH-1: what is it, what does it do, when are they downregulated, and how are they downregulated?

Answer 16

Membrane-associated RING-CH 1

Promotes degradation of MHC-II in immature DCs - ubiquitination for them to be taken to the proteasome for degradation

Activated DCs - want to express MHC-II so MARCH-1 activity is suppressed through the gene, it is inhibited and, since MARCH-1 has a short life span anyways (t-30mins), MARCH-1-derived MHC-II degradation decreases

Activation of PRRs results in their normal immune responses as well as the downregulation of anti-immune responses, such as MARCH-1

Question 17

MHC class II peptide presentation: what is the process?

Answer 17

• Exogenous proteins are internalised
• Proteins then acidified and degraded in lysosomes
• MHC-II made in ER and transported through to Golgi and then to acidic vesicles
• MHC-II is released from CLIP, allowing peptide binding
• High affinity peptides bind and cause transportation to the cell surface

Question 18

Do cells also need to present endogenous peptides on MHC class II despite endogenous proteins usually being targeted MHC-I?

Answer 18

YES!

The whole point of MHC-I presentation is the destruction and release of pathogens ‘hiding’ in cells so that they can be taken up by APCs and used to generate adaptive immune responses - the primary focus of MHC-II

These pathogens may also infect MHC-II cells so there needs to be a mechanism for using these proteins to generate an adaptive immune response

Question 19

Endogenous antigens: what are their role in promoting CD4+ responses, why is it useful, what is the process, and how frequently does it occur?

Answer 19

Typically promote an MHC-I response, but may also be used to generate MHC-II responses

• Pathogens can invade APCs (i.e. B cells, macrophages, DCs)
• Antibodies and CD4+ T cells are often required for protection - inhibit invasion, activate intracellular killing mechanisms, etc
• Presentation of endogenous material helps broaden the repertoire of CD4+ T cell response against pathogens

Autophagy process - autophagosome made containing endogenous antigens, the formed vesicle intersects with endosomes and phagosomes, then follows the typical process

10-30% of peptides eluted from MHC class II in B cells and DCs are from intracellular proteins

Question 20

Autophagy: what is it, what is it primarily used for, what can it also be used for, what does it do, what cells does it mainly occur in, what cytokine increases its rate of occurrence, and when is it important?

Answer 20

Self-eating:
* Waste recycling system of all eukaryotic cells in multivesicular bodies
* Also used as a mechanism enabling expression of cytosolic antigens by MHC class II, through the spontaneous uptake of intracellular pathogens in vesicles, forming a phagosome and following the typical MHC-II pathway and preventing pathogen escape as well as enabling adaptive responses

Occurs in B cells, DCs and macrophages

Increased by IFN-γ

• MHC II presentation of cytoplasmic proteins to enable CD4+ T cell response
• Extremely important in thymic selection

Question 21

Antigen presentation with MHC-I and MHC-II: what are the types of APCs, what are the responsive t-cells, what is the composition of the stable peptide-MHC complex, what is the cellular location of peptide binding, where are the peptides sourced from, what are the enzymes responsible for peptide generation, and what are the molecules involved in MHC and peptide preparation and transportation?

Answer 21

MHC-I:
* All nucleated cells
* CD8+ T-cells
* Polymorphic α chain, β2 microglobulin, and peptide
* ER
* Cytosolic (endogenous) mainly - (exogenous during cross-presentation in APCs)
* Cytosolic proteasome proteases
* Calnexin, calreticulin, TAP, and Tapasin

MHC-II:
* DCs, mononuclear phagocytes, B lymphocytes, endothelial cells, and thymic epithelium
* CD4+ T-cells
* Polymorphic α and β chains, and peptide
* Specialised endothelial compartments
* Exogenous mainly - (endogenous in infected APCs)
* Endosome/lysosome proteases
* Calnexin, invariant chain, Golgi, MIIC, CIIV, and HLA-DM

Question 22

Self-restriction: what is it, what does it mean, and what are some consequences of it?

Answer 22

The process by which T-cells are tested to ensure they can bind self-MHC molecules with a bound foreign peptide

In the periphery, T-cells that cannot functionally bind to self-MHC complexes are wasteful and essentially useless - the body makes sure only T-cells that can exact a function are produced

T-cells and MHC complexes from two different people will not bind

Question 23

MHC-T-cell interactions: what are the costimulatory molecules and how does their signalling work?

Answer 23

• CD4 and CD8 molecules associated with the TCR bind to invariant (conserved) regions of the MHC - this binding does not inhibit TCR-peptide-MHC interaction
• CD4 and CD8 molecules through binding to MHC localise intracellular signalling kinases to the TCR to amplify the T cell response

Question 24

Non-classical MHC molecules: what are they, how variable are they, what does they result in, and what is the point?

Answer 24

Non-polymorphic MHC class I-type molecules - termed MHC class Iβ genes which encode β2-microglobulin-associated cell-surface molecules

• 50 or more MHC class 1B genes found in mouse
• Non-polymorphic in humans, may have many types but they are the same among all species in a population

Each molecule may have a specific role in antigen presentation, by recognition and display of particular pathogen associated-antigens:
* All bacteria initiate protein synthesis with N-formylmethionine
* MR1 presents riboflavin metabolites, to activate mucosal-associated invariant T cells (MAIT cells)
* Riboflavin metabolites are produced by most bacteria and yeast
* H-2M3 (mouse) presents peptides with N-formylated amino termini, driving strong cytotoxic CD8+ T cell responses.

Allows for essentially always a response against pathogens even while there are ‘holes’ in MHC binding that are present in everyone - a ‘fallback’ mechanism