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MHC: Antigen Processing and Presentation

This module will help you

• see how pathogen antigens are processed and presented to T cells.
• understand the role of MHC in antigen recognition.
• test your knowledge and immunology problem-solving skills.

MHC Structure and Function
Antigen Processing and Presentation
MHC Gene Expression

MHC Structure and Function

Exogenous pathogens include bacteria, their soluble toxins, and extracellular protozoan parasites and fungi. They can often be phagocytosed and destroyed by lysosomal enzymes or oxidative burst in neutrophils and macrophages, especially with the opsonizing activities of complement and antibody. However, some pathogens persist unharmed and protected in phagocytic vesicles or escape into the phagocyte cytosol. Th1 cytokines increase the ability of macrophages to kill vesicular pathogens, while TH2 cytokines stimulate B cells to make neutralizing and opsonizing antibodies. To recognize and respond to exogenous antigen, CD4 Th1 and Th2 cells must use their TCR to "see" (bind) antigen peptides bound to Class II MHC proteins and "presented" on the membranes of professional antigen-presenting cells (APC). B cells, dendritic cells (DC) and macrophages are professional APC.

Endogenous pathogens reproduce in host cell cytosol. They include viruses and intracellular bacteria and protozoan parasites. Since antibody and complement cannot enter infected cells and phagocytes cannot detect that they are infected (or engulf them whole because of their size), cytotoxic T cells (Tc) must be activated to recognize and kill the infected cells. To be activated by endogenous antigen, CD8 Tc cells use their TCR to bind endogenous antigen peptides presented on membrane Class I MHC proteins of target cells (infected cells).

MHC antigen-presenting molecules are identical to cell-surface molecules which determine "self" or "non-self" for transplantation. They are called Human Leukocyte Antigens (HLA) in humans and H-2 antigens in the mouse and are encoded in the Major Histocompatibility Complex (MHC) genes. Class I MHC proteins are expressed on all nucleated cells; however, leukocytes express the most Class I MHC and neural cells the least. Class II MHC proteins are found constitutively (always) on B cells, dendritic cells, and thymic epithelial cells and can be induced on macrophages and human T cells.

Class I MHC is a heterodimer of membrane-bound a chain and non-covalently associated b₂-microglobulin. Both a chain and b₂-microglobulin are members of the Ig superfamily and share with antibody a disulfide-bonded domain structure. Class I a chain is encoded by highly polymorphic genes in the MHC. It has three domains named a1, a2, and a3 and a region adjoining a3 that anchors it in the plasma membrane. b₂-microglobulin is encoded by a gene on another chromosome. b₂-microglobulin molecules are non-covalently associated with Class I a chain and are not polymorphic or attached to the plasma membrane.

Antigen binds Class I a1 and a2 domains; the amino acid sequence of these domains varies from allele to allele. Variability is maximized in the amino acids which contact antigen, but it is less than that for Ig V_H and V_L domains. Class I a chain folds so that its variable region forms a cleft with a-helical sides and a b-pleated sheet floor which will hold an 8-10 amino acid peptide. The cleft is closed at the ends, limiting the size of the peptide which can bind. The a1 and a2 domains also bind TCR. CD8 binds Class I a3 domain, which has a species-specific constant amino acid sequence. Three Class I gene loci encode human HLA-A, HLA-B, and HLA-C or mouse H-2 D, H-2 K, and H-2 L a chains.

Peptide which binds Class I lies extended along the antigen binding cleft, anchored at its amino and carboxyl termini to invariant sites at each end of the cleft. Anchor residues at two or three other positions along the peptide interact with residues in the MHC binding cleft. For a given MHC allele, these anchor residues must be related: for example, all hydrophobic residues for one anchor site, or all acidic amino acids. Anchor residues vary between MHC alleles, and residues not in the anchor positions can vary considerably, allowing many different peptides to be presented by a few Class I alleles. TCR distinguishes different peptides by their conformation and by the Class I conformation induced by their binding.

Class II MHC is a non-covalently bonded heterodimer of a and b chains, called HLA-DP, HLA-DQ, and HLA-DR in humans and IA and IE in mice. Peptide antigen (13-18 residues) binds Class II a1 and b1 domains, which are variable from allele to allele. a1 and b1 domains also bind TCR. The membrane-bound a2 and b2 domains are invariant and a2 binds CD4.

Class II peptide-binding site is similar in structure to that of Class I, except that its ends are more open so that longer peptides can be bound. Peptides which bind Class II are at least 13 amino acids long and are not anchored by their amino and carboxyl termini. They stretch along the binding groove, with residues fitting into binding pockets along the edges of the cleft. Class II-binding peptides also have a small number of anchor residues that must be of certain types to bind a particular allele; other residues in the peptide are less constrained.

Antigen Processing and Presentation

MHC proteins must bind peptide, and Class I must be complexed with b₂-microglobulin in intracellular compartments before MHC can be expressed on the cell surface. Although many MHC alleles have been identified in the human population, each individual has a limited number of MHC proteins with which to present a great many pathogen epitopes to T cells. Peptide binding to MHC is less specific than epitope binding to Ig or TCR; each MHC presents many different epitopes. Peptide must bind MHC with enough affinity to be retained on the plasma membrane and not exchange with soluble peptide. MHC molecules are unstable in the absence of bound peptide and are folded around peptide before transport to the plasma membrane.

A virus-infected cell synthesizes virus proteins on ribosomes in its cytoplasm. In order to be presented, these proteins must be broken down into short peptides and transported into the endoplasmic reticulum (ER) to bind to newly synthesized Class I MHC proteins. In the cytosolic processing pathway, cytosolic proteins are degraded to peptides in proteasomes, cylindrical arrays of proteolytic enzymes with their active sites towards the center of the cylinder. Both pathogen proteins and self cell proteins can be complexed with ubiquitin to target them to the proteasome for processing. Two proteases encoded in the MHC Class II region (LMP2 and LMP7) and a third subunit not encoded in MHC are produced in response to interferon, which is synthesized in response to virus infection. These inducible proteases replace constitutive proteases in the proteasome and produce peptides with basic and hydrophobic carboxyl terminal residues preferred as anchor residues in Class I peptide binding sites and for transport from the cytosol into the ER. Two polypeptide chains, Transporters of Antigen Peptide TAP-1 and TAP-2, are present in the ER membrane with ATP-binding domains on the cytosolic side and hydrophobic transmembrane domains spanning the ER membrane. The TAP-1/TAP-2 complex transports cytosolic peptides into the lumen of the ER with the expenditure of ATP. Both TAP molecules are required for membrane expression of Class I.

Newly synthesized, partly folded Class I MHC a chain binds the chaperone calnexin in the lumen of the ER. Chaperone binding prevents mis-folding that otherwise would occur in the absence of antigen peptide. When b₂-microglobulin binds to Class I a chain, calnexin dissociates and Class I a plus b₂-microglobulin form a complex with calreticulin, tapasin, and TAP transporter. When Class I MHC binds peptide, it is released from TAP transporter and the Class I MHC-peptide complex is transported through the Golgi to the plasma membrane. Unbound peptides are thought to be transported back into the cytoplasm for reprocessing and retransport. In uninfected cells, membrane Class I MHC presents self peptides. Viruses which can interfere with TAP function or with transport of Class I MHC to the plasma membrane can evade destruction by cytotoxic T cells.

Exogenous antigen is processed in the endosomal processing pathway. Bacteria, soluble protein antigens, and antibody-coated viruses which have been taken up by macrophages and B cells, envelope proteins from the plasma membrane of DC, and some bacteria and parasites that live in endosomal vesicles, enter the endosomal processing pathway. The endosomes become increasingly acidic as they move from the plasma membrane farther into the cytoplasm. Increased acidity activates proteases that cut the antigen into peptides.

Class II MHC a and b chains are synthesized on the rough endoplasmic reticulum (ER) and transported into the ER lumen, where they assemble with another polypeptide, invariant chain (Ii = upper case I and lower case i). Ii and Class II MHC form nine-chain trimeric complexes, with part of each Ii molecule occupying the Class II MHC peptide-binding site. Ii allows Class II MHC to assemble in the absence of foreign peptide and blocks association with normal cell ("self") and endogenous peptides present in the ER lumen. Ii also directs the transfer of Class II MHC through the Golgi to a specialized vesicular compartment, MIIC (MHC Class II Compartment), where over several hours Ii is degraded in the low pH . The last part of Ii to dissociate from Class II MHC is CLIP, a short fragment which occupies the peptide binding site. HLA-DM, a Class II MHC ab heterodimer resembling other Class II MHC molecules but not expressed on the cell surface, facilitates CLIP removal and peptide binding in the MIIC compartment. Class II MHC-peptide complex is then transported to the plasma membrane. Class II MHC which does not bind peptide when CLIP dissociates is unstable and is rapidly degraded. In the absence of infection, APC present Class II MHC containing self peptides, including self Class II MHC peptides. Peptide/Class II MHC complexes are very stable, ensuring that the APC presents its own exogenous peptides and not peptides released from another cell. Like other plasma membrane proteins, Class II MHC proteins shuttle between the membrane and endosomal compartments where they can pick up new exogenous antigen peptide or be degraded.

MHC Gene Expression

Human Major Histocompatibility Complex is a cluster of genes on a single chromosome. MHC is highly polygenic and polymorphic, meaning there are several genes (gene loci) and many alleles (gene versions) for Class I and Class II MHC proteins. Three human Class I genes encode the HLA-A, B, and C a chains. Class II genes for DP, DQ, and DR are arranged in pairs encoding a and b chains that comprise Class II proteins. The Class II region also encodes proteins for processing (LMP) and transporting (TAP) cytoplasmic peptides into the endoplasmic reticulum for binding Class I MHC.

The MHC Class III gene region encodes numerous genes, some related to the immune system and some unrelated. Included are genes for complement proteins, cytokines TNFa and TNFb, enzymes required for steroid synthesis, heat shock proteins, and many unidentified proteins.

Numerous HLA alleles have been identified. Since studies of some populations have been limited, the numbers underestimate the polymorphism of the entire human species. For HLA-A, B, and C, 59, 111, and 37 alleles have been identified. The alpha and beta chain alleles of HLA-DP, DQ, and DR a and b chains number 8, 62, 16, 25, 1, and 122, respectively. All alleles are not found with equal frequencies in humans. MHC diversity is a population diversity, since each individual inherits a maximum of two alleles for each locus.

MHC genes are expressed codominantly on cell membranes. All cells which express Class I express two different alleles if the individual is heterozygous at a particular locus. For example, skin cells of a person heterozygous at the Class I A, B, and C alleles will express six different Class I a chains (each with identical b₂-microglobulin chains).The a chains of one Class II allele can combine with b chain of the same or the other allele for that locus, increasing the number of Class II molecules which can be expressed.

The set of MHC genes inherited from each parent is called a haplotype. Siblings have a 1 in 4 probability of inheriting identical haplotypes from both parents and are likely to provide the best match for transplantation (see tables below). MHC polymorphism makes tissue matching between unrelated people very unlikely.

Although Class I and Class II MHC are expressed constitutively in high amounts by a few cell types, especially DC and B cells, they are present in low amounts on most cells. Their expression, along with processing enzymes, transporter subunits, and chaperones to aid in their proper folding and association with peptide, is stimulated by interferon gamma (IFNg) produced early in viral infections.

When inbred mice were used to investigate immune activation, it was discovered that the ability to recognize antigens (particularly haptens) was genetically linked to MHC. Some strains of inbred mice were non-responders to particular haptens; no IgG antibody was produced to that hapten (complexed with carrier) in that strain of mouse, although in other strains the same hapten-carrier complex stimulated IgG synthesis.

Similar systems were used to understand the roles of different cell types in immune responses. When syngeneic (matching at all MHC loci) mouse T cells, B cells, and macrophages were mixed with antigen in vitro, antibody to the antigen was produced; when allogeneic (not matching at MHC loci) cells were used, the immune response was absent even in responder mice.

T cells are MHC-restricted in their ability to see antigen; they only recognize it presented by (in the context of) syngeneic (self) MHC on APC. CD4 Th cells bind exogenous antigen on Class II MHC of professional APC: DC, macrophages, and B cells. TCR binds both peptide plus Class II MHC a1 and b1 domains; CD4 binds to a2. CD8 Tc cells recognize antigen on Class I MHC of cells infected with endogenous pathogens. TCR binds peptide plus the a1 and a2 domains of Class I MHC; CD8 binds the Class I a3 domain.

Developing T cells are educated (selected) in the thymus to recognize self MHC (see T Cell Development). However, up to 5% of T cells can respond to cells bearing allogeneic MHC, compared to a much lower frequency of T cells which can respond to a particular foreign peptide-self MHC complex. Alloreactivity of T cells is responsible for rejection of grafts between individuals mismatched at MHC loci. In general, CD8 T cells respond to foreign Class I MHC and CD4 T cells respond to foreign Class II MHC. The ability of T cells to be activated by allogeneic MHC may be due to several factors. Some of the T cells may be specific for peptides on foreign MHC and may bind tightly enough to become activated even though the MHC differs from self MHC. In other cases, cross-reactive structural epitopes on the foreign MHC may bind to TCR irrespective of the peptides it contains. The high numbers of MHC-TCR interactions between a particular APC and T cell are able to activate the T cells even if the binding affinity is low. Cross-reactivity between self and non-self MHC is much more common than cross-reactivity between any two random peptides, so the allogeneic response can involve a higher proportion of T cells than the peptide-specific response.

A few people have been discovered with bare lymphocyte syndrome, a partial or complete deficiency in Class I or Class II MHC proteins. People with bare lymphocyte syndrome have an increased susceptibility to viral and opportunistic infections. Symptoms range from none to severe combined immune deficiency (SCID, a lack of both humoral and cellular adaptive immune responses), depending on the number of MHC loci that can be expressed.

A genetic linkage exists between HLA type and several diseases. These include insulin-dependent diabetes, which occurs four times as often in people with HLA-DR4 as in people without the DR4 allele; idiopathic hemochromatosis (excessive iron levels), for which people with HLA-A3 have a sevenfold increased risk; and ankylosing spondylitis (arthritis of spinal vertebrae), with a B27-linked risk factor of 87. Many possible explanations for this linkage exist. Proteins encoded by genes closely linked with MHC may be responsible for these diseases, or disease may be caused by viruses which use particular MHC alleles as receptors to enter host cells. Molecular mimicry between a pathogen epitope and an MHC epitope may exist, so that antibodies made against the pathogen bind self MHC and cause autoimmunity. Thymic education in people with certain alleles may fail to delete T cells which could respond to self antigen and cause autoimmunity.

In addition to Class I and Class II MHC, there are minor histocompatibility antigens that induce a weaker graft rejection response. Some of these are tissue-specific (skin) or sex-specific (male). Some are proteins encoded by viral DNA integrated with host cell DNA (Mls antigens of mice). Others are foreign peptides bound to Class I and Class II MHC which occasionally trigger graft rejection episodes between tissues of identical twins.

Practice Quiz

Pick the one BEST answer for each question by clicking on the letter of the correct choice.

1. Exogenous antigen includes all of the following EXCEPT

a. bacterial toxins.
b. extracellular protozoan parasites.
c. most bacteria.
d. ragweed pollen.
e. viruses.

2. Human Class I MHC a chain molecules are

a. b₂-microglobulin.
b. H-2 D, K, and L.
c. H-2 IA and IE
d. HLA-A. -B, and -C.
e. HLA-DR, -DP, and -DQ.

3. Cells which have MHC Class II are _________________, which present _____________ antigen to Th cells.

a. antigen presenting cells, endogenous
b. antigen presenting cells, exogenous
c. infected cells, inflammatory
d. target cells, endogenous
e. target cells, exogenous

4. Signaling to a cytotoxic T cell that a liver cell is infected with hepatitis virus depends on

a. binding of Ii to Class I MHC until the peptide is loaded.
b. binding of TCR on the cytotoxic T cell to Class II MHC on the infected cell.
c. binding of processed antigen to liver cell Class I MHC.
d. processing the hepatitis virus peptides to the correct size and anchor residues in the endosomal pathway.
e. both c and d are correct.

5. Endogenous antigen presentation requires delivery of antigen peptides to the endoplasmic reticulum by

a. Class I MHC and invariant chain.
b. calnexin and tapasin.
c. HLA-DM.
d. leader sequence.
e. TAP-1 and TAP-2.

6. Following virus infection, peptides produced from the proteasome are more likely to be presented on the surface of the target cell because

a. MHC Class I is synthesized in response to virus infection.
b. proteasomal enzymes which produce shorter peptides are synthesized in response to virus infection.
c. TAP-1 and TAP-2 specifically bind virus peptides.
d. virus amino and carboxyl terminal amino acids bind better to Class I MHC than peptides from self proteins.
e. virus infection induces expression of proteases which cut proteins at sites which bind best to TAP-1 and TAP-2.

7. Exogenous antigen is processed

a. after presentation by antigen presenting cells.
b. by nearly every nucleated cell.
c. by the cytosolic processing pathway.
d. in the presence of b₂-microglobulin.
e. in acidified endosomes.

8. Class II MHC does not efficiently present endogenous antigen because

a. antigen synthesized inside the cell never makes it to the endosomal compartment.
b. endogenous antigen cannot be processed into peptides small enough.
c. HLA DM transports Class II to the surface before it can bind endogenous peptide.
d. invariant chain blocks binding of endogenous peptide in the ER.
e. phagocytosed antigen binds Class II as rapidly as Class II is synthesized.

9. MIIC is a specialized intracellular compartment where

a. HLA DM promotes the release of CLIP and peptide binding to Class II MHC.
b. invariant chain binds to Class II MHC a and b chains.
c. peptides are transported into the ER for binding to Class II.
d. proteins are broken down into peptides by proteasomes.
e. some pathogens live protected from lysosomal enzymes.

10. In order to have pathogen peptide plus Class II MHC molecules expressed on the membrane of host cells, all of the following are required EXCEPT

a. b₂-microglobulin.
b. CLIP.
c. HLA-DM.
d. HLA-DR, -DP, and -DQ alpha chains.
e. Ii .

11. Invariant chain (Ii)

a. inhibits binding of endogenous peptide to Class I MHC.
b. is degraded in the MIIC compartment to CLIP.
c. is released from Class II upon binding of b₂-microglobulin.
d. is the constant region of Class I peptide binding site.
e. prevents exogenous peptide binding to Class II MHC in the ER.

12. Antigen binding by Class I MHC molecules

a. accommodates many different peptides.
b. preferentially occurs for peptides 13-18 amino acids in length.
c. occurs at a site on Class I MHC formed by folding of a1 and b₂-microglobulin domains.
d. occurs only on antigen presenting cells.
e. takes place at the plasma membrane of the infected cell.

13. Both Class I and Class II MHC molecules are

a. composed of a and b chains with variable and constant regions.
b. expressed constitutively on all nucleated cells.
c. expressed on the B cell membrane.
d. part of the T cell receptor for antigen.
e. synthesized in response to antigen processing.

14. The major histocompatibility complex has

a. dozens of loci for Class I and Class II proteins.
b. genes that encode proteins associated with antigen processing.
c. only genes encoding Class I and Class II molecules.
d. single loci for Class I and Class II proteins.
e. three regions encoding Class I, Class II, and Class III receptors.

15. MHC polymorphism

a. is generated by recombination of HLA A, B, and C gene segments.
b. is present primarily in the peptide-binding regions of MHC proteins.
c. is the result of random association of many alpha and beta genes.
d. restricts the ability of B cells to bind antigen.
e. results in expression of dozens of MHC alleles on each APC.

16. T cells are MHC-restricted in their ability to respond to antigen because

a. all antigen must be processed and presented to activate lymphocytes.
b. during an infection, all cells in the body present antigen on MHC Class I.
c. MHC binds antigen more specifically than TCR does.
d. TCR must recognize both antigen and MHC molecules.
e. the T cells should not respond to antigen on allogeneic cells.

17. Linkage of a disease to an HLA allele means that

a. everyone with that allele will eventually get the disease.
b. people with that allele have a higher risk for the disease.
c. the MHC protein encoded by that allele is defective.
d. the allele will eventually disappear from the population.
e. None of the above is true.

18. All of the following are associated with the expression of Class I MHC molecules EXCEPT

a. antigen peptide presentation on membrane Class I MHC to Tc.
b. graft rejection.
c. increased risk of certain autoimmune diseases.
d. lysis of virus-infected cells.
e. stimulation of antibody production.

19. Human Class II MHC molecules

a. are encoded by the genes HLA-A, B, and C.
b. are found on all nucleated cells.
c. have an antigen binding site formed from regions of two polypeptide chains.
d. must be associated with b₂-microglobulin molecules to bind peptide.
e. present antigen to CD8 cytotoxic T cells.

20. Humans inherit from each of their parents

a. a random set of MHC Class I, Class II, and Class III genes.
b. enough diversity in MHC to present epitopes from most pathogens.
c. enough diversity in MHC to present every possible antigen epitope.
d. genes for a and b chains that can be recombined to increase their diversity.
e. the same Class I and Class II MHC genes as their siblings.

21. The a chain of HLA-DR

a. can be expressed with the b chain of any MHC molecule.
b. can be expressed with the b chain of any Class II MHC molecule.
c. can be expressed with the b chain of any Class II DR molecule.
d. must be expressed with b₂-microglobulin.
e. must be expressed with the b chain of Class II DR from the same chromosome.

22. Which of the following statements is TRUE?

a. Each individual expresses all the diversity of MHC protein structure.
b. If a family has four children, no two of them will have the same MHC genotype.
c. Someone with bare lymphocyte syndrome who expressed no MHC proteins would die in infancy.
d. TCR on Tc cells binds a1 and b2 domains of Class I MHC protein.
e. The chances of finding a tissue match are much higher between children and their parents than between siblings.

23. Which of the following statements is FALSE?

a. All MHC alleles in the population have been counted.
b. CD4 T cells see antigen on self Class II MHC but not on self Class I MHC.
c. Human Class II MHC proteins are called HLA DP, HLA DQ, and HLA DR.
d. Class I and Class II MHC are less antigen-specific than Ig.
e. Peptides presented by Class I MHC must be 8-10 amino acids long.

24. Which of the following statements is FALSE?

a. A peptide binding to Class I must have certain amino- and carboxyl-terminal amino acids to bind tightly to the ends of the Class I binding cleft.
b. A transplant is most likely to be successful between people who share the same alleles at all Class I and Class II MHC loci.
c. Identical twins share all their Class I and Class II MHC alleles.
d. Peptide binding to TCR is influenced by both its own conformation and the conformation of the MHC protein to which it is bound.
e. The gene for b₂-microglobulin is in the Class I region of the MHC.

Problems

1. What is the maximum number of Class I and Class II proteins expressed on a liver cell (hepatocyte)? on a macrophage?

2. Complete the table about MHC below:

3. Describe antigen processing and presentation of antigens from Epstein-Barr Virus (EBV) which has infected B cells.

4. You are sent a sample of human blood cells to analyze for MHC antigens. The genotype of the human donor is HLA-A 3⁺5⁺ and HLA-DR 1⁺2⁺. You remove the erythrocytes from the sample by Ficoll Hypaque density centrifugation and incubate aliquots of the cells with FITC-anti-DR1 or FITC anti-A5 and PE-anti-A3 or PE-anti-DR2. [FITC is a green fluorochrome and PE is a red fluorochrome.] After washing off unbound antibody, you analyze the surface MHC on your blood cells by flow cytometry. Complete flow cytometry plots for staining with a) FITC anti-DR1 and PE anti-A3, b) FITC anti-DR1 and PE anti-DR2, and c) FITC anti-A5 and PE anti-A3 by writing the names of the cells which will appear in each quadrant. Hint: Think about which cell types are in the blood and whether each cell type has Class I or Class II or both on its membrane. Cells positive for FITC fluorescence are in the top boxes, cells negative for FITC fluorescence in the bottom boxes. Cells positive for PE fluorescence are in the right-hand boxes, cells negative for PE fluorescence are in the left-hand boxes.

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