MHC

Question 1

Describe what MHC is?

Answer 1

MHC = Major Histocompatibility Complex (Histocompatibility = tissue compatibility)

They are two types: MHC-I and MHC-II

Immune responses to transplant are caused by genetic differences between the donor and the recipient. For transplant compatibility, most important genetic differences are MHCs (either 1 or 2) so transplants were fairly unsuccessful until Human MHC was disvovered in 1967

Human MHC proteins = Human Leukocyte Antigen (HLA)

MHC is set of genes that code for cell surface proteins essential for the acquired immune system to recognize foreign molecules in vertebrates, which in turn determines histocompatibility.
The main function of MHC molecules is to bind to antigens derived from pathogens and display them on the cell surface for recognition by the appropriate T-cells

Question 2

Why is MHC matching so important in transplants?

Answer 2

There is a major correlation between transplant patient outcome and MHC matching.

The more differences between MHC between donor and recpient the shorter life for transplanted organ and therefore the patient

Question 3

What is the structure and function of MHC class 1?

Answer 3

• MHC Class one is expressed on all nucleated cells
The overall structure consists of a glycosylated alpha heavy chain ~45kD which is non-covelanely bound with a beta 2 microglobulin chain

The alpha chain consists of 3 extracellular domains (a1 N terminal, a2 and a3)
• A transmembrane tail
• A cytoplasmic tail
• In humans, MHC class 1 is coded from HLA-A, -B, -C gene loci

MHC class 1 molecules binds short peptides (8-10aas) and present these to CD8 cytotoxic T cells which are important in controlling viral infections by lysing infected cells

They recognise endogenous (intrinsic) antigens rom the cytosol and cross presentation

Question 4

What is the structure and function of MHC class 2?

Answer 4

MHC class 2 is present on all professional antigen presenting cells (dendritic cells, macrophages and B cells)

Has 2 polypeptide chains with 4 domains -> a1 and b1 (antigen/peptide binding cleft), a2 and b2 which are glycosylated (apart from b2) and inserted into the membrane

B2 domain contains a binding site for CD4 T cells like MHC class 1 binding to CD8 – it is also nonglycoslyated

The a chains are 30-34kDa
The B chains range from 26-29kDa depending on locus

MHC class 2 is encoded by the HLA gene complex. This includes:
• HLA-DP, -DM, -DOA, -DOB, -DQ and -DR in humans
• H-2A and E in mice

MHC-class 2 recognises antigens from phagosomes and endosomes and other fragments taken up endocytosis (from outside the cell) -> exogenous pathway

Question 5

What is the relevance of polymorphism?

Answer 5

There are multiple variants of each gene within the population as a whole. The MHC genes are, in fact, the most polymorphic genes known and the more MHC, the less chance the pathogens have to evade the immune system. MHCs are central to anti-viral immune responses

The MHC polymorphism is found in the upper peptide-binding region. It is here amino acids create pockets where the bound peptide can “anchor” – at the top of alpha helices or inside the lining of the peptide binding groove
The repertoire of peptides bound by any MHC allele is determined by these pockets and each allele binds a different repertoire of peptides

Question 6

What are the peptide pockets?

Answer 6

The peptide pockets are little grooves where peptides (antigens) anchor themselves
Different amino acids between different alleles affects both size and charge of the pockets . Variations in the different MHC alleles include variations in size, charge and location of pockets

Question 7

What are the differences between peptide binding grooves in the different classes of MHC?

Answer 7

MHC Class 1 -> bind short peptides that are 8-10 amino acids with more closed ends. It is easier to predict the peptides that will bind here so it is better for vaccine design, particularly for cancer vaccines.

MHC class 2-> It has a more open structure than class 1 and can accommodate longer peptides, usually around 13-17 amino acids long.

We cannot therefore predict which petides/foreign antigens are going to bind and there

Question 8

What are the main molecules involved in MHC class 1 antigen processing?

Answer 8

• Chaperone proteins such as Calnexin and Calreticulin and ERp57
• The proteasome -> a cytoplasmic structure which digests poorly folded or damaged proteins to prevent them from being presented. The ones to be destroyed are tagged with ubiquitin.
• TAP transporters -> these are transporters proteins which are encoded alongside MHC genes. They stand for Transporter associated with antigen processing - TAP and they are ER membrane proteins which transport peptides from cytoplasm into ER lumen and associates with MHC class 1 molecule to help load peptides into their groove

ERAPP -> endoplasmic reticulum aminopeptidase

This protease trims peptide fragments further so they can be presented.

Also needed is the B2microglobulin molecule that hasn’t been attach dot the “mature” MHC molecule yet.

Question 9

How does antigen processing work in MHC class 1?

Answer 9

1. Partly folded MHC I Heavy chain (alpha chain) binds to chaperone Calnexin
2. Partially folded chain is transported to a second chaperone Calreticulin which further aids folding of the chain and associated of Beta 2 M
3. Other proteins such as EPp57 and Tapasin associate with TAP transporter to form a peptide loading complex
4. Cytosolic proteins and defective ribosomal products (DRiPs) are degraded by proteasome into peptide fragments
5. TAP delivers the peptides to the ER
6. ERAAP then trims the terminus of the peptides so they can bind the MHC molecule
7. Some molecules are too unstable to bind to the MHC molecule so are unbound – Peptide Editing process)
8. Stable complex with high affinity binding so the peptide loading complex dissociates
9. The MHC with peptide is then transported from the ER via the Golgi apparatus to attach on cell surface of T cells

Question 10

What are the ways that pathogens can evade MHC class 1 antigen processing?

Answer 10

Human Cytomegalovirus (HCMV):

• It has these 3 stages of replication, where it can inhibit different proteins in the antigen processing part in MHC 1 - cascading fashion of immediate-early (IE), early (E), and late (L) genes
• IE > US3, a short lived protein which can bind and inhibit tapasin and also prevent MHC from exiting the ER (retention)
• E and E/L -> has US2 and US11 degrade the MHC heavy chains
• E/L -> US6 which doesn’t bind directly to TAP proteins but does inhibit the action of it

Other viruses which can have this effect including; Cow pox, Epstein Barr Virus and HSV.

HLA B27 misfolding:
• HLA B27 (allele of MHC class one) has a tendency to misfold with itself rather than b2-microglobulin
• Misfold and still be stable to move to the surface
• Stability of properly folded complex is influenced by ERAAP
• Leads to retention in the ER, triggering a stress response in ER
• Some misfolded HLA B27 reaches the cell surface where it can be recognised by immune cells and have inappropriate responses

Question 11

Which molecules are necessary for MHC class 2 antigen processing?

Answer 11

• Appropriate endocytosis of the antigen fragment by one of 3 methods
  1. Clathrin-mediated endocytosis with clathrin inside the cell and Lectin and endocytic receptors
  2. Macropinocytosis with actin and soluble antigen
  3. Phagocytosis with phagocytic receptors and TLRs

To make a stable complex for the MHC before getting an antigen outside the ER:

• Invariant chain (Li) protein - > which fits inside the peptide binding group, acts as a chaperone and directs MHC class II complexes towards the endosomal compartment of the cell. This chain gives the MHC class 2 stability and blocks the binding until the MHC is in the right place

CLIP (class II-associated invariant-chain peptide) – this takes over after the Invariant chain has disintegrated and blocks the bidning groove. CLIP is made in ER whilst MHC folds

HLA-DM -> acts as a catalyst to remove CLIP when binding of antigen is needed.

Question 12

What happens during MHC class 2 antigen processing

Answer 12

1. MHC class 2 molecule assembles in the ER of some cells (phagocytes)
2. 3rd chain the invariant chain binds in the binding groove of MHC 2 and prevents peptide or unfolded proteins within the ER
3. The invariant chain guides the MHC class 2 out of the ER into the Golgi apparatus as a vesicle into the endosomal pathway
4. Progressive acidification of the vesicle activates proteases which cleave the In chain in 2 places and leaving the small CLIP (Class 2 associated invariant chain peptide) peptide bound to the class 2 molecule
5. Engulfed pathogens or their proteins are also degraded by acids activated proteases but they can’t immediately bind due to clip
6. Removal of CLIP by HLA-DM functions as a catalyst to remove CLIP and allow binding of foreign peptides
7. Transported to the cell membrane where it can be recognised by CD4 cells

Question 13

How do T cells recognise the MHC?

Answer 13

MHC class 1 is recognised by CD8 cytotoxic T cells whereas MHC class2 is recognised by CD4 T cells.

The TCR of a T cell will recognise 2 things: 1. the MHC protein itself (self allele) and the foreign peptide presented by the MHC

The TCR binds to distant binding sites on the lower region of the MHC. The T cells act as co-receptors for the MHCs and this is required for the T cell to make an effective response as we don’t want to interfere with binding.

Question 14

What is MHC restriction?

Answer 14

MHC-restricted antigen recognition is when a T cell can interact with a self-MHC molecule and a foreign peptide bound to it, but will only respond to the antigen when it is bound to a particular MHC molecule.

T cells recognise both peptide and the MHC allele presenting it but the TCR will only recognise antigen if it is presented by self MHC .

If this process goes whong and we don’t get dual antigen/MHC recognition we can get something called Toxic shock syndrome

Toxic shock syndrome e.g. caused by the staphylococcal syndrome toxin-1 which acts as a superantigen
• The superantigen binds directly to both MHC class II and T cell receptor, triggering multiple T cells to produce cytokines
• Losing specificity and every protein is a binding target so non-specific t cells are produced.

Question 15

What is Cross Presentation.

Answer 15

Cross presentation is the phenomenon which may occur when exogenous peptides are presented by MHC-1.
Certain APCs such as Dendritic cells can take up, process and present extracellular antigens with MHC-1 to CD8 cytotoxic T cells

There are many factors that determine cross presentation function such as:
- antigen uptake and processing mechanism,
- environmental signals
- activation of cross presenting dendritic cells.
o This is dependent on stimulation by CD4 T helper cells.
o The co-stimulatory molecule CD40/CD40L along and the danger presence of an exogenous antigen are catalysts for dendritic cell licensing so cross presentation and activation of naive CD8+ cells

TAP transporter is also vital for this to work

Cross presentation is also required for the induction of cytotoxic immunity by vaccination with protein antigens, e.g. tumour vaccination

Question 16

What are the 2 pathways to cross presentation?

Answer 16

1. Cytosolic Pathway

- typically for smaller peptides and involves some form of structural leakage
o	Antigens are taken into the cell through endocytosis. 
o	Antigen proteins are transported out of the phagolysosome  into the cytosol  by unknown mechanisms. 
o	Within the cytoplasm, exogenous antigens are processed by the proteasome and degraded into peptides.
o	These processed peptides can either be transported by the TAP transporter into the ER or back into the same endosome for loading onto MHC class I complexes

2. Vacuolar Pathway -> typically for larger peptides which might also require recruitment of ‘extra’ membrane from ER, bringing ER proteins with it

• It is initiated through the endocytosis of an extracellular antigen by a dendritic cell.
• This results in the formation of a phagocytic vesicle, where an increasingly acidic environment along with the activation of enzymes such as lysosomal proteases triggers the degradation of antigen into peptides.
• The peptides can then be loaded onto MHC I binding grooves within the phagosome.
• It is unclear whether the MHC I molecule is being exported from the endoplasmic reticulum before peptide loading, or is being recycled from the cell membrane prior to peptide loading.
• Once the exogenous antigen peptide is loaded onto the MHC class I molecule, the complex is exported to the cell surface for antigen cross presentation.

Question 17

What is the link with TLR4 and cross presentation?

Answer 17

TLR4 is a transmembrane protein and a toll-like receptor which belongs to the pattern recognition receptor (PRR) family. Its activation leads to an intracellular signalling pathway NF-κB and inflammatory cytokine production which is responsible for activating the innate immunity
It is most well-known for recognizing LPS), a component present in many Gram-negative bacteria (e.g. Neisseria spp.) and some Gram-positive bacteria. Its ligands also include several viral proteins, polysaccharide, and a variety of endogenous proteins such as low-density lipoprotein, beta-defensins, and heat shock protein.
LPS acts as danger signal to the TLR4 and causes DC maturation and slows down antigen degradation allowing for more efficient cross presentation onto MHC 1

Question 18

Describe the process of autophagy

Answer 18

Autophagy is the natural, regulated mechanism of the cell that removes unnecessary or dysfunctional components. It allows the orderly degradation and recycling of cellular components. Three forms of autophagy are commonly described: macroautophagy, microautophagy, and chaperone-mediated autophagy.

Degredation in the autophgosome (lysosome) feeds into the MHC class 2 processing compartment, helping present this antigen to the cell surface.

Examples in MHC 2 include up regulation of autophagosomes in a intracellular infection like mycobacteria (seen in LC3 marker under luminescence). We can also see this in Rheumatoid arthritis

Question 19

How can MHC also be related to the innate immune system?

Answer 19

• MHC-I and MHC-II are central to the adaptive immune response but they also play an important role in the innate immune system
• MHC disease associations may also be explained by innate immune receptor recognition
• There are 2 receptors which allow this to happen:

• Killer Ig-like receptors (KIR) - regulate the activity of Natural Killer cells
• Leukocyte Ig-like receptors (LILR) - regulate the functions of NK cells and antigen presenting cells
- KIR and LILR are encoded in a gene complex (the leukocyte receptor complex or LRC) on chromosome 19

Question 20

Explain KIR receptors and what they do.

Answer 20

KIR = killer-like receptors which are expressed on the majority of CD56 (low) NK cells. KIRS are split into 2 types:

• Long cytoplasmic tail = inhibitory – with ITIMS in the cytoplasm
• Short cytoplasmic tail = activating with a charged Lysine residue on the end on the transmembrane domain > activate

KIR receptors regulate the killing function of NK cells as they can interact with MHC class 1. The receptors can distinguish between MHC 1 allelic variants allowing them to detect virally infected or transformed (cancer) cells.
• When KIR recognise MHC-I, they inhibit release of lytic granules, suppressing the cytotoxic activity of their NK cell
• Some viruses down-regulate MHC-I as a means to evade cytotoxic T cells, loss of MHC-I is also a common feature of tumour cells
• If a target cell does not express MHC-I then there is no KIR inhibition, lytic granules from the NK cells will be released to lyse the target – killing the cell
Only a limited number of KIRs are activating, meaning that their recognition of MHC molecules activates the cytotoxic activity of their cell
As a result of KIR’s role in killing unhealthy self-cells and not killing healthy self-cells, KIRs are involved in protection against and propensity to viral infection, autoimmune disease, and cancer

Question 21

What is the function of LILRs?

Answer 21

• Expressed on variety of cells e.g. antigen presenting cells
• Functions not fully determined, seem to be important in pregnancy
• Binding strength shown to correlate with viral load in HIV Infection
• The leukocyte immunoglobulin-like receptors (LILR) are a receptor family possessing extracellular Ig domains They are also known as CD85 and can exert immunomodulatory effects on a wide range of immune cells

Question 22

What is non-classical MHC-1 and give some examples.

Answer 22

• Non-classical MHC class I (MHC-Ib) are exhibit limited polymorphism, expression patterns and presented antigens and tend to show a more restricted pattern of expression than their classical counterparts
• Some MHC-Ib act as antigen presenting structures, whilst others are recognised as direct ligands for immune receptors.
• Some are encoded alongside classical MHC in the Major Histocompatibility Complex, others are encoded outside the MHC
• this group is subdivided into a group encoded within MHC loci (e.g., HLA-E, -F, -G), as well as those not (e.g., stress ligands such as ULBPs, Rae1, and H60);
• the antigen/ligand for many of these molecules remain unknown, but they can interact with each of CD8+ T cells, NKT cells, and NK cells.

Question 23

Describe some examples of non-classical MHC

Answer 23

HLA-G:
• Encoded in MHC
• Powerful immune inhibitory protein
• Expressed by placental trophoblast cells during pregnancy (foetal derived placental cells)
• Don’t want mother’s immune system to kill the foetus so you need to express different MHC molecules
• Placental cells express HLA-G as a decoy when the turn off classical MHC -> enough to inhibit NK cells
• High affinity ligand for inhibitory LILR

CD1:
- • Encoded outside MHC gene sequence but are related to MHC 1
• There are 2 groups
• Group 1
• been shown to present foreign lipid antigens, and specifically a number of mycobacterial cell wall components, to CD1-specific T cells.
• Group 2
• molecules activate NK cells because of their expression of NK surface markers e.g. CD161. By presenting antigens and rapidly producing TH1 and TH2 cytokines (IFNg and IL-4)
• Specialised binding groove presents glycolipids or phospholipids

MICA:
- • MICA (MHC class 1 polypeptide-related sequence A) is a non-classical MHC
• a highly polymorphic surface glycoproteins encoded by MICA gene on MHC locus
• Similar to MHC 1 with similar domain structure but it’s not associated with B2m
• It functions as a stress-induced ligand (danger signal) for integral membrane receptor, NK group 2-member D (NKG2D)
• Upregulated expression is induced by stress, shock, inflammation
• Recognised by NK cells, γδ T cells, and CD8+ αβ T cells which carry NKG2D receptor on their cell surface and which are activated via this
• Does not present antigen

Question 24

How does MHC work in pregnancy?

Answer 24

With a neonatal Fc receptor. It is an Fc receptor (Ig) which is similar in structure to MHC-1 and also associates with beta2M – it shuttles IgG across the placenta
Further studies revealed a similar receptor in humans, leading to the naming as a neonatal Fc receptor. In humans, however, it is found in the placenta to help facilitate transport of mother’s IgG to the growing foetus. It has also been shown to play a role in monitoring IgG and serum albumin turnover.
Neonatal Fc receptor expression is up-regulated by the proinflammatory cytokine, TNF-a and down-regulated by IFN-g

Shuttles IgG across the placenta – has MHC class 1 structure