Immuno 6

Question 1

What chromosome are the genes for MHC encoded on?

Answer 1

6

Question 2

T or F. B cells can recognize particulate or soluble antigens, and the composition of those antigens can be protein, carbohydrate, lipid, nucleic acid…almost anything.

Answer 2

T. In contrast, T cells only recognize peptides in the context of MHC class I or MHC class II molecules. The MHC molecule binds to a protein fragment and “presents” it to the T cell.

Question 3

Describe the composition of a MHC class I molecule.

Answer 3

MHC class I molecules are heterodimers that are composed of a single MHC encoded protein chain, the alpha chain, and a protein known as beta-2-microglobulin that is not encoded within the MHC complex. The alpha chain has three domains (alpha 1, 2, and 3). The alpha 1 and alpha 2 domains fold in a way that creates a peptide binding cleft (they each have an extended alpha helical region.). The bottom of the cleft is formed by a series of alternating beta-sheet secondary structure. The alpha 3 domain is an immunoglobulin-like domain that has a transmembrane region that anchors the chain into the cell membrane.

Question 4

Describe the composition of a MHC class II molecule.

Answer 4

MHC class II molecules are heterodimers that are composed of an alpha and a beta chain. Both of these chains are encoded by the MHC locus. Both of these chains have two domains. The alpha 1 and beta 1 domains come together to form the peptide binding cleft. The alpha 2 and beta 2 domains are both immunoglobulin-like domains that have transmembrane regions that anchor the chains into host cell membranes.

The organization of MHC class II molecules is very similar to that of MHC class I molecules. The alpha 1 and beta 1 domains each have an extended alpha helical structure that forms one side of the peptide binding groove, and the “floor” of the peptide binding groove is composed of alternating beta sheet.

Question 5

T or F. The most important function of MHC molecules is to bind to peptides and present them to T cells. How do they do this?

Answer 5

T. Immunocompetent individuals have a tremendous repertoire of T cells that recognize more than 10^18 different peptide determinants with high specificity. Therefore, MHC molecules must be able to present a large number of different peptides to T cells. They can do this because they bind to peptides in a promiscuous fashion. They do not bind to peptides with the same type of specificity that we have discussed for B cell receptors and will soon discuss for T cell receptors.

Question 6

In contrast to antibodies or T cell receptors (that bind to a highly specific antigenic determinant), each MHC molecule can bind to many different peptides.

Answer 6

In contrast to antibodies or T cell receptors (that bind to a highly specific antigenic determinant), each MHC molecule can bind to many different peptides. MHC class I and II molecules do have slightly different peptide-binding characteristics

Question 7

What kinds of peptides will MHC class I molecules bind?

Answer 7

The peptide binding groove of MHC class I molecules is closed on the ends. Their binding groove will only accommodate peptides that are between 8 and 10 amino acids in length. In fact, peptide binding in the groove is stabilized by contacts with the aminoterminal and carboxyterminal ends of the peptide with invariant sites near the ends of the binding groove.

Question 8

How do MHC class I molecules bind to peptides in a promiscuous fashion?

Answer 8

Ex. only They all must be eight amino acids long, but the sequences of all three can be very different. The only similarity requirement between these peptides are at residues 5 and 8. At position 8, each peptide has a leucine residue. At position 5, each peptide has an amino acid that has a large hydrophobic side chain that has a ring structure (i.e. phenylalanine, tyrosine, etc.). Positions 5 and 8 are referred to as anchor residues. Any 8-10 residue peptide that has amino acids with a large hydrophobic side chain with a ring structure at position 5 and a leucine at position 8 will likely be able to bind to this MHC class I molecule.

There are many different isoforms of MHC class I molecules that are expressed within the human population, each of which has different peptide binding characteristics, mostly based on their requirements for the positions of anchor residues within those peptides.

Question 9

What are the two primary differences between MHC class I and MHC class II molecules?

Answer 9

1. The ends of the binding cleft of class II molecules are open, compared to class I molecules that have closed ends. It is sometimes called the “hot dog in a bun model” because the peptides hang over the edges of the binding groove
2. The open-ended binding groove allows for longer peptides to bind to MHC class II molecules. In fact, peptides that bind to class II molecules must be at least 13 amino acids long, and can be up to at least 35 residues long (maybe longer).

Question 10

How do MHC class II molecules bind to peptides in a promiscuous fashion?

Answer 10

Ex. Here you can see the sequences of three peptides that were extracted from three identical MHC class II molecules. The central 14 amino acid stretch of each has an identical sequence, but they are all of different lengths. What this shows is that MHC class II molecules can bind to peptides of varying lengths that are all too long to fit completely within its binding groove.

(Bottom Panel): Here you see can a set of peptides that were all extracted from identical copies of a different MHC class II molecule isoform. They are all of different lengths, and they all have very different amino acid sequences. The only commonality of these peptides is that they have anchor residues at 4 different positions that allow them to bind tightly within the binding groove of this particular MHC class II molecule.

(Both Panels): Together, these two panels show that the promiscuity of peptide binding to class II molecules is based on a similar feature to that we discussed for MHC class I molecules. Both MHC class I and MHC class II molecules have pockets within their binding grooves that accommodate particularly shaped amino acid side chains. The interactions between the binding grooves and these anchor residues are responsible for the bulk of the affinity between the peptide and the MHC molecule.

Question 11

One of the key decisions made by the immune system is whether to respond with an immune response that is ideal for clearing an intracellular infection vs. one that is ideal for clearance of an extracellular infection. These two processing and presentation pathways are designed to help the immune system to determine what type of infection is ongoing.

Answer 11

The MHC class I processing and presentation pathway primarily presents antigens that are derived from intracellular pathogens, while the MHC class II processing and presentation pathway primarily presents antigens derived from extracellular pathogens. It is critical that you remember and understand this.

Question 12

T or F. The MHC class I processing and presentation pathway primarily presents antigens that are derived from extracellular pathogens

Answer 12

F. They are derived from INTRAcellular pathogens

Question 13

How are antigens brought into a cell?

Answer 13

Some antigens are brought into the cell via endocytic vesicles via phagocytosis, macropinocytosis, endocytosis, etc. This will be important as we discuss the MHC class II processing and presentation pathway.

Question 14

Notes on cellular components that are important in antigen presenting

Answer 14

The endoplasmic reticulum is a structure that surrounds the nucleus of the cell that is an important part of the secretory pathway of the cell. All proteins that are destined to become membrane proteins or secreted proteins are translated on ribosomes that are associated with the ER and are trafficked through the ER for modifications and trafficking eventually to the cell surface.

The golgi apparatus is an organelle that carries out the next phase of shipping newly produced membrane or secreted proteins to the cell surface. It gives rise to secretory vesicles that deliver these proteins to the cell surface.

Lysosomes are vesicle-like organelles that have acid hydrolases that are used to break down macromolecules and invading pathogens that are endocytosed by a cell.

Question 15

T or F. One of the processes that occurs all the time in all cells in each person’s body is recycling of old proteins found in the cytosol of the cell.

Answer 15

T.

Question 16

Proteins that are targeted for destruction are taken up and degraded by a protein complex known as the _______.

Answer 16

proteosome. This complex digests proteins into smaller and smaller peptide pieces, all the way down to single amino acids that can then be used for new protein synthesis.

Question 17

Some of the digestion products are peptides. Some of these peptides are transported from the cytosol into the lumen of the endoplasmic reticulum by what is known as the ______.

Answer 17

TAP transporter complex.

Question 18

What is the TAP transporter complex composed of?

Answer 18

This complex is composed of two proteins: TAP-1 and TAP-2. As the TAP transporter moves peptides from the cytosol to the ER lumen it participates in the process of loading these proteins onto newly synthesized MHC class I molecules.

Question 19

How are MHC class I molecules synthesized?

Answer 19

Because it is a membrane anchored protein, MHC class I (and MHC class II, for that matter) is targeted for transport through the ER and golgi on its way to the cell surface.

1. A class I heavy chain (composed of a1, a2, and a3) is stabilized by calnexin until B2-microglobulin binds
2. Calnexin is released and the heterodimer of class I heavy chain and B2m forms the peptide loading complex with calreticulin, tapasin, TAP, ERp57, and PDI
3. A peptide delivered by TAP binds to the class I heavy chain, forming the mature MHC class I molecule
4. The class I molecules dissociates from the peptide-loading complex, and is exported from the ER to the golgi and ultimately to the surface of the cell

These events that are occurring all of the time in all host cells, whether an infection has occurred on not.

Question 20

What happens if a peptide is not loaded into the newly synthesized MHC class I binding groove?

Answer 20

MHC class I molecules are unstable if they have not been loaded with a peptide. If a peptide is not loaded into its binding groove fairly rapidly, that MHC class I molecule will begin to degrade and will be recycled. This is evidenced by the fact that people that have an inherited deficiency of one or both of the TAP transporter proteins, and therefore cannot load peptides onto MHC class I molecules, have very few cells that bear MHC class I molecules on their surface.

Question 21

What happens in regards to MHC class I synthesis when a host cell becomes infected with an intracellular pathogen?

Answer 21

During an infection with an intracellular pathogen, pathogen-derived proteins will be generated during its replicative cycle and will be available for degradation by the proteosome. Pathogen- derived peptides are therefore going to be among the peptides transported to the ER lumen by the TAP transporter and loaded onto newly synthesized MHC class I molecules. Once the MHC class I molecule bearing a pathogen-derived peptide is transported to the surface of the cell, it can now be presented to T cells. If this is an antigen-presenting cell, the peptide will be presented to naïve T cells with the goal of activating a pathogen-specific T cell response .

If the cell is not an antigen presenting cell, an effector T cell with specificity for that peptide can now potentially recognize the cell as infected and effect its destruction.

So, the immune system takes advantage of a normal housekeeping function of all cells for activation of T cell responses and for recognition of infected cells by effector T cells.

One caveat that you should be aware of: the vast majority of the peptides that are loaded onto MHC class I molecules are derived from self proteins. Therefore, it is critical that all self- reactive T cells be removed from the T cell repertoire.

Question 22

How are MHC class II molecules synthesized?

Answer 22

MHC class II molecules are also shuttled through the ER soon after they are synthesized. The design of the immune system requires that these MHC class II molecules do not get loaded with cytosol-derived peptides as they are trafficked through the ER. Therefore, soon after they are synthesized, MHC class II proteins are associated with a protein known as the invariant chain. Part of this protein fits into the peptide binding groove of the MHC class II molecule. This chain remains associated with the class II molecule until it has completed its passage through the ER and Golgi apparatus and it resides in cytosolic vesicles.

Once in the vesicle, the invariant chain is cleaved, leaving only a short peptide (that is know as the CLIP fragment) in the binding groove of the MHC class II molecule, where it prevents other peptides from having access to the binding groove.

If this vesicle fuses with a phagolysosome, which contains materials collected from the extracellular milieu that are being processed, a protein known as HLA-DM interacts with the MHC class II molecule and dislodges the CLIP peptide so that a processed peptide can be loaded into the binding groove of the MHC class II molecule before it is transported to the cell surface for display to naïve T cells.

Only antigen-presenting cells (with one exception) express MHC class II proteins. It is also important for you to remember that the vast majority of all peptides ever loaded onto MHC class II molecules in a persons body are derived from self-proteins. Only during an infection will that be a source of non-self proteins for loading onto MHC class II molecules. Again, it is critical that self-reactive T cells be removed from the repertoire.

Question 23

MHC class II processing and presentation pathway from start to finish

Answer 23

Extracellular antigens are taken up by phagocytosis/ endocytosis constantly in all antigen presenting cells. These cells are also always producing new MHC class II proteins that are trafficked through the ER and Golgi apparatus. The invariant chain blocks the binding groove of the class II molecule until it completes its journey to a cytoplasmic vesicle. This prevents peptides transported into the ER by the TAP transporter from loading into its binding groove. When the vesicle fuses with a phagolysosome, the CLIP peptide is removed, and a peptide derived from the extracellular milieu is then loaded into the binding groove of the MHC class II molecule. It is now transported to the surface of the cell for display for presentation to naïve T cells.

***These two pathways are not mutually exclusive. Some peptides derived from intracellular infections will be loaded onto MHC class II molecules, and some peptides derived from extracellular pathogens will be loaded onto MHC class I molecules. However, these loading efficiencies will be much lower than for the two pathways that I just described.

Question 24

Which enucleated cells do not express MHC class I?

Answer 24

erythrocytes. there would be no reason for them too because T cells are not able to kill cells that do not have a nucleus.

All other hematopoietic cells do (i.e T cells, B cells, macrophages, dendritic cells, neutrophils)

Question 25

What other cells express some MHC class I on their surface?

Answer 25

thymic epithelium, liver hepatocytes, kidney epithelium, neural tissue

Because neuronal cells are not regenerated very well and because their function is so vital to the host, they are designed to be very refractory to immune effector mechanisms. It would not be useful for the immune system to kill infected cells of the brain (for instance) because those cells function may be critical to survival. I suppose the same could be said of the kidneys and liver.

Question 26

T or F. MHC class II expression is restricted to professional antigen presenting cells (dendritic cells, macrophages, and B cells)

Answer 26

T. because their role is to present peptide antigens to naïve T cells.

Question 27

MHC class II is also found on _____.

Answer 27

thymic epithelium

Question 28

CD4 T cells only recognize peptides bound to MHC class ___ proteins

Answer 28

class II proteins.

Question 29

CD8 T cells only recognize peptides bound to MHC class ___ proteins.

Answer 29

class I proteins

Question 30

T or F. Antibody-mediated immune responses are usually the best for clearance of extracellular infections

Answer 30

T. Effector CD4+ T cells are required for priming acquired antibody responses.

Therefore, we have an antigen processing and presentation pathway that primarily presents antigens from extracellular sources to naïve CD4 T cells (the MHC class II processing and presentation pathway), because the primary role of CD4 effector T cells is to drive development of antibody responses

Question 31

T or F. Clearance of intracellular pathogens is mediated primary by a T cell-mediated immune response

Answer 31

T. and specifically an effector CD8 T cell response, there is an antigen processing and presentation pathway that efficiently presents antigens to CD8 T cells, namely the MHC class I processing and presentation pathway.

Question 32

Describe what polygenic nature of MHC expression means

Answer 32

the term polygeny refers to the existence of multiple genetic loci within the genome that encode proteins that are structurally similar and have identical (or nearly identical) function. Both MHC class I and MHC class II expression is polygenic.

Question 33

Describe what polymorphic nature of MHC expression means

Answer 33

refers to the existence of two or more forms of a given gene (alleles) within a population. In this case, we are talking about within the human population. For each MHC class I or class II isoform, there are many alleles that are expressed within the human population.

Variant alleles occur at a frequency of at least 1%

Question 34

On each copy of chromosome __ that a person inherits, there are three loci that encode MHC class I molecules. These genes are designated __, __, and ___.

Answer 34

6

HLA-A, HLA-B, and HLA-C.

Question 35

There are also 3 loci that encode the different isoforms of both the alpha and beta chains of MHC class II molecules on each copy of chromosome 6. These are designated __, __, and __.

Answer 35

HLA-DP, HLA-DQ, and HLA-DR.

Note that the HLA-DR locus has two different genes (or can have) that encode HLA-DR beta chains.

Question 36

MHC genes are the most polymorphic genes in the human genome. What exactly does this mean?

Answer 36

In regards to MHC class I molecules, there are at least 414 different alleles of HLA-A that have been identified within the human population. What this means is that when you are in class with all of your classmates, it is possible that every single one of you express a different allele of HLA-A. Likewise, there are 728 alleles of HLA-B and 210 alleles of HLA-C

Question 37

If you were to sequence each of those proteins and map where the differences lie along the entire MHC class I alpha chain, you could determine where the differences are in the fully folded protein. This has been done and is shown in the lower panel. You can see some red bars that represent amino acid positions where lots of changes are observed between the different alleles. The two upper panels show that these amino acid differences map to the binding groove of the MHC molecule. This indicates that the different alleles of MHC class I molecule have different binding groove characteristics that likely result in altered ability to bind to peptides. For instance, each allele may have specificity for peptides that have a different array of anchor residues.

Answer 37

If you were to sequence each of those proteins and map where the differences lie along the entire MHC class I alpha chain, you could determine where the differences are in the fully folded protein. This has been done and is shown in the lower panel. You can see some red bars that represent amino acid positions where lots of changes are observed between the different alleles. The two upper panels show that these amino acid differences map to the binding groove of the MHC molecule. This indicates that the different alleles of MHC class I molecule have different binding groove characteristics that likely result in altered ability to bind to peptides. For instance, each allele may have specificity for peptides that have a different array of anchor residues.

Question 38

T or F. there are only five alleles of HLA-DR alpha

Answer 38

F. there are only 3

In general, amino acid differences between the different alleles map to regions of the binding grove of the MHC class II molecule, resulting in distinct peptide binding capacity between the different alleles.

Question 39

Why is polygenic and polymorphic expression of MHC class I expression important?

Answer 39

Each person inherits one copy of chromosome 6 from their mother and one from their father. Each of these copies of chromosome 6 has a single gene that encodes HLA-A, a single gene that encodes HLA-B, and a single gene that encodes HLA-C. Each of these loci express an MHC class I protein that has distinct peptide binding characteristics. Also remember that the genes encoded on both chromosomes will be expressed.

If an individual inherits homozygously at each of these loci, they will express a total of three different isoforms of MHC class I. This is the minimum number of MHC class I isoforms that can be inherited.

MHC class II expression is more complicated because for each locus (HLA-DP, HLA-DQ, or HLA-DR) there are polymorphic genes that encode both the alpha and beta chains of MHC class II molecules. Therefore, minimal inheritance of MHC class II requires that all six of these genes are inherited homozygously. If this occurs, the individual will express three different isoforms of MHC class II.

Question 40

Additional note on inheritance of HLA-DR.

Answer 40

Each copy of chromosome 6 includes a gene that encodes the HLA-DR beta 1 gene. Some people inherit an additional gene that encodes either a beta 3, beta 4, or beta 5 HLA-DR gene (i.e. 2 HLA-DR beta genes). The product of each of these additional beta chain genes can pair with the HLA-DR alpha chain to form an additional isoform of MHC class II.

Question 41

In the case of MHC class I inheritance, it is pretty simple. If a person inherits heterozygously at HLA-A, HLA-B, and HLA-C, that person will express __ distinct isoforms of MHC class I molecules on all nucleated cells in his/her body. I hope you will think about the implications of this. Is it better to inherit minimally or maximally? Why?

Answer 41

6

Question 42

Maximal inheritance of MHC class II isoforms

Answer 42

Please note that the alpha chains expressed at each class II locus (HLA-DP, HLA-DQ, and HLA-DR) can only pair with beta chains expressed from that same locus.

As you can see, this person has inherited a different allele of each alpha and beta chain gene of HLA-DP, HLA-DQ, and HLA-DR. In addition, this person has inherited a second HLA-DR beta chain gene on both the paternal and maternal copies of chromosome 6, and they are also different alleles. Therefore, this person has inherited genes that code for 2 HLA-DP alpha chains and 2 HLA-DP beta chains.

Both of the alpha chains can pair with either of the beta chains (2 x 2 = 4 HLA-DP proteins). Same thing for the HLA- DQ genes: 2 HLA-DQ alpha chains and 2 HLA-DQ beta chains that can each pair with each other (2 x 2 = 4 HLA-DQ proteins).

HLA-DR is a bit different because this person inherited an additional beta chain coding region of each copy of chromosome 6, and they were different alleles. Maximum expression therefore is 4 different beta chain genes and 2 different alpha chain genes that can all pair to create a functional MHC class II protein (2 alpha x 4 beta = 8 HLA-DR molecules).

Is this a good thing, or a bad thing for this individual?

Question 43

Although we are not really talking about transplant immunology until the second half of this course, you might want to give some thought to the implications of the polygeny and polymorphism of MHC expression. Remember, any particular person will only be tolerant to the MHC class I or MHC class II isoforms (alleles) that they have inherited and express on their cells. Each person will have T cells in their repertoire that will recognize determinants derived from any MHC isoform that they do not express on their cells. This is the basis of transplant rejection.

Answer 43

Although we are not really talking about transplant immunology until the second half of this course, you might want to give some thought to the implications of the polygeny and polymorphism of MHC expression. Remember, any particular person will only be tolerant to the MHC class I or MHC class II isoforms (alleles) that they have inherited and express on their cells. Each person will have T cells in their repertoire that will recognize determinants derived from any MHC isoform that they do not express on their cells. This is the basis of transplant rejection.

Question 44

T or F. The more isoforms of MHC class I and MHC class II a person expresses, the higher the number of peptides that person will be able to present to naïve T cells.

Answer 44

T. If I had the choice, I would want to inherit heterozygously at each MHC locus so that my antigen-presenting cells can present the largest array of peptides possible to naïve T cells. This would give my immune system the ability to produce the largest possible array of effector T cell specificities.

Question 45

T or F. Any tissue bearing non-self MHC will be rejected

Answer 45

T. T-cell mediated immune response

MHC molecules serve as alloantigens that result in rejection of transplanted tissues. This is why tissue typing is so important for successful transplantation. Tissue typing is primarily meant to determine the degree of MHC haplotype match; the better the match, the better the chance for success.

Question 46

What is a syngeneic transplant (isograft)?

Answer 46

transplant between genetically identical individuals

Question 47

What is an allogeneic transplant (allograft)?

Answer 47

transplant between genetically different individuals

Question 48

What is a xenograft?

Answer 48

transplant of tissue from a different species