2.3 biol 453 Major histocompatibility Complex

Describe the concept of a haplotype as it relates to meiotic recombination and inheritance of MHC genes as groups that rarely are separated by recombination

- Allelic forms of MHC genes are inherited in linked groups called haplotypes; recombination is rare - Individuals inherit one haplotype from each parent - Mice can be selectively bred to have more standardized genotypes

What does Ii(CD74) & HLA-DM do and what's its role in the exogenous pathway?

- An invariant chain composed of Ii (CD74) guides the transport of MHC-II to endocytic vesicles - Ii prevents peptides from binding MHC-II groove too early - Ii uses sorting signals in cyto tail to direct MHC-II- containing vesicles to peptide-containing compartments - Ii initially degraded by proteolytic activity within endocytic compartment to class II-associated invariant chain (CLIP) - HLA-DM exchanges CLIP out of the grove for a peptide fragment - HLA-DO regulates HLA-DM;has role in placement of self peptides

Be able to describe how a we stern blot is performed after SDS/PAGE. Why is the advantage of working with a blot over working with a gel?

- Can identify a specific protein in a complex protein mixture (lysate) - IDs and quantifies specific protein - Separate proteins by electro- phoresis (SDS/PAGE) based on size - Transfer gel pattern to nylon membrane (blot) - Expose membrane w/ Ab to protein of interest - Identify where Ab is bound using enzyme-tagged assay - Followed by substrate exposure to identify protein band

Describe the following terms and their role within the endogenous pathway: Chaperones, ERp57, Calnexin, calreticulin, tapasin, ER aminopeptidase, and ERAP1.

- Chaperones aid peptide/MHC-I assembly -ERp57, Calnexin, calreticulin, and tapasin help fold MHC class I and put it in close proximity to TAP -ER aminopeptidase, ERAP1, trims long peptides to a suitable size for MHC class I groves

Describe the Cross Presentation of exogenous antigens.

- Dendritic cells appear to be the primary cross- presenting cell - Exogenous antigens are redirected to the endogenous presentation pathway - This allows for their presentation on MHC class I molecules, priming CD8+ T-cell responses - Dendritic cells are the only APCs (so far) known to do this

What is the role of the MHC proteins in tissue rejection?

- Details of MHC and tissue rejection worked out with inbred strains of mice - k haplotype = H2-Kk, H2-Sk, H2-Dk H2-Kk, H2-Sk, H2-Dk - b haplotype H2-Kb, H2-Sb, H2-Db H2-Kb, H2-Sb, H2-Db -Heterozygote H2-Kk, H2-Sk, H2-Dk H2-Kb, H2-Sb, H2-Db

Know the general organization of the HLA locus in humans. How many Class I, II, III genes there are in the complex? How is the HLA complex organized differently than the H-2 complex

- Humans have 3 MHC-I genes HLA-A, HLA-B, HLA-C - MHC-II are dimers with a and ß chains - Human have 3 pairs HLA-DPa, HLA-DPß; HLA-DQa, HLA-DQß HLA-DRa, HLA-DRß

Describe the process of MHC-I presenting intracellular antigen peptides.

- Includes self proteins - Provides a way for "checking" that cells are self AND are generally healthy - Often used to show which cells have been infected with viruses, endobacteria, or are otherwise abnormal

Describe what it means to be co-dominantly expressed in terms of the number of different MHC-I and MHC-II protein heterodimers that can be expressed on any one cell. Know why there can be up to six types of Class I proteins and 12 types of Class II proteins expressed.

- MHC alleles are co-dominantly expressed - Most humans are heterozygous for each gene - For MHC Class I = means a cell can express up to six different MHC I proteins - This gives good chance for person to have some capability of presenting all possible encountered antigen peptides -Also makes organ transplantation difficult -Non-matching MHC patterns will result in rejection

Describe Class I MHC-peptide interaction. Describe the bulge and what it results in.

- MHC-I present peptide antigens to CD8+ T cells - Peptides are derived from endogenous intracellular pros. or processed pros. from intracellular microbes. - some amino acids(2,3,9) anchor the peptide in the groove. - Other amino acids are available to interact with a TCR as bulge.

Describe Class II MHC-peptide interaction. Describe the bulge and what it results in.

- MHC-II present peptide antigens to CD4+ T cells - Peptides are derived from exogenous extracellular antigens or pathogens that are internalized and processed. (they eat, they digest, they poop, they present). - Peptide fragments are longer than the once that bind to the MHC-I. - Some amino acids from the alpha-1 and Beta-1 domain hold the peptide in the groove at the floor. - Longer peptides are not anchored, which means there is no bulging. The fact that there is no bulging results in the groove being open at both ends.

Sandwhich ELISA what is it? What it do?

- Measures Ag presence - Can quantify the amount of Ag in a sample - Useful for measurement of soluble cytokine concentrations

Competitive ELISA what is it? What it do?

- Measures amount of Ag in a sample - More Ag in original sample the lower the final signal - Works on same principle as the RIA

Know the general organization of the H-2 locus in mice. Know in general how many Class I, Class II, Class III genes there are in the complex.

- Mice have 2-3 MHC-I genes - Not all strains have H2-L - MHC-I locus split H2-K, H2-D, H2-L - MHC-II are dimers with a and ß chains - Mice have 2 pairs H2-IAa, H2-IAß; H2-IEa, H2-I Eß

Describe the process of MHC-II presenting excracellular antigen peptides.

- More restricted - Generally found on cells involved in immune responses - On professional antigen presenters - Also found on non- professional antigen presenters - Helps to direct responses against threats—things that should not be in our systems

Describe the Endogenous Pathway for antigen presentation.

- Peptides are generated by proteasome digestion - Ubiquitin "tags" intracellular proteins for degradation - Tagged proteins are fed into the proteasome - Subtle variant known as an immuno- proteasome cleaves proteins into fragments that pair better with MHC molecules

Describe how peptides are transported in the Endogenous pathway and what happens to them.

- Peptides are transported from the cytosol to the rough endoplasmic reticulum (RER) - Transporter-associated with Antigen Processing (TAP) molecules in the RER membrane move the fragments - MHC-I molecules lie in wait in the RER after their translation by ribosomes on the surface

Describe the process of presentation of non-peptide antigens.

- Some non-protein Ag can be recognized by T cells - Presentation is not via classic MHC molecules - Occurs by CD1 family of non-classical MHC class-I molecules; looks like MHC-I with ß2 microglobulin - Have deep pockets like "shoe" for hydrophobic Ags - Five human CD1 genes are known - Very little polymorphism - Most function similarly to MHC-II molecules: process thru exogenous pathway with endosomes, etc

Describe a generalized antigen-binding pocket for MHC-1, and how antigen presentation occurs.

- Structure of the antigen-binding cleft: i. Beta sheet forming the floor of the cleft. ii. Two alpha helices forming the walls.

Explain the redirection method in dendritic cells and the significance of a "license" during the cross presentation of exogenous antigens.

- The actual redirection method is unclear - To prevent redirection of self antigens into APC pathways, dendritic cells may need "license" - If DC can present foreign antigen to CD4+ helper T cell, it gets "license" to redirect exogenous Ag into the endogenous pathway - "License" might be back/forth cytokine signal between the APC/TH cell

Describe a generalized antigen-binding pocket for MHC-II, and how antigen presentation occurs.

Both chains pass through the membrane. - The peptide-binding cleft is formed by pairing alpha 1 and B1 domains. This binding pocket is able to bind larger processed peptides compared to the class 1 molecule.

Describe CD-1 pockets.

CD1 pockets are deep like foot in a shoe.

If I were to ask you what I mean when I say that Class I and Class II molecules exhibit polymorphism, how would you respond?

Class I and Class II molecules have different shapes and sizes. Any one MHC can bind to many different peptides, and some peptides bind to more than one MHC.

What does ELISA do?

ELISA (enzyme-linked immunosorbent assay) - Uses Abs or antigens covalently bound to enzymes - Types of ELISAs a. Indirect ELISA = detects presence and conc. of an Ab in a sample - This is the method of choice to detect presence of serum Ab against HIV - Uses a secondary Ab (IgE) conjugated to an assay system (enzyme) than can be quantified

Understand the general differences between Class I MHCs and Class II MHCs in terms of structure, cell-type expression, processing of peptides, interactions with TCRs, and what types of T cells they each present to

Everything you need to know is in the picture.

True or False. Whole antigens can be recognized by T cells.

FALSE. - Whole antigens cannot be recognized by T cells - Only processed antigen peptide fragments can be recognized

understand the general organization of the MHC Class I genes.

MHC Class I have exon/intron arrangements that reflect each domain being encoded by an exon -Domains a. Leader = signal sequence b. a1 = holds processed peptide c. a2 = holds processed peptide d. a3 = Ig domain e. Tm = transmembrane domain f. C + C = cytoplasmic domain

Understand the general organization of the MHC class II genes.

MHC Class II have exon/ intron arrangements that reflect each domain - Recall there are two chains (a, ß), each has same domains a. Leader = signal sequence b. a1 or ß1 = holds processed peptide c. a2 or ß2 = Ig domain d. Tm + C1 = transmembrane plus part of cytoplasmic e. C2+C3 = cytoplasmic - a chain has one fewer C

Be able to draw the domain structure of a MHC-I molecule and be able to l ist the 3 extracellular domains, and describe which parts are needed for antigen presentation. Also know how ß-2 microglobulin is associated with the larger chain.

MHC-1 is found on most cell; not on human RBCs. Has three domains: alpha 1, alpha 2, and alpha 3. The alpha 1 and alpha 2 domains form a cleft region (Hand like molecule) that binds a processed peptide fragment. The alpha 3 domain(nearest the membrane) has another immunoglobulin domain, and these MHC proteins are considered part of the ig superfamily. CD8 (represents the side of the MHC) and is a target for T(c) cells acting as a coreceptor. There is also a smaller subunit that's considered a part of the class one MHC that is ubiquitous meaning that it's not sequence specific. IMPORTANT: You need the large subunit MHC-1 and the B-2 microglobulin to form the fully functional class-1 molecule.

Where are most of the proteins required for endogenous antigen processing found?

Many of these proteins are found embedded in the MHC Class II complex.

Why do you expect the different alleles of MHC genes to have most of their differences in the nucleotides coding for the amino acids within the binding pockets and not in other parts of the proteins?

Most of the genetic differences are going to be noticed in the coding for the amino acids within the binding pockets because the binding pockets are responsible for binding to the different arrays of antigens. - The human population is outbred, so homozygotes for any one classical HLA gene are extremely rare - Plus the HLA-I locus is one of the most polymorphic for genes that encode proteins; > 1000 functional alleles for the genes involved in Ag presentation: HLA-A, HLA-B, HLA-C

Be able to draw a picture highlighting the general differences between the class-I and Class-II molecules

Note: it's very important to not only be able to write down the general structure of the two different molecules, but also understand what the structures look like visually.

Be able to describe how allelic variation contributes to the vast number of polymorphism exhibited by Class I (especially) and Class II MHC proteins.

There are 100s to 1000s of different allelic variants that exist in humans. The chances that any of the possible six class I molecules expressed match the person next you are next to nothing.

Be able to draw the domain structure of an MHC-II molecule and be able to list the two extracellular domains for each chain. Be able to describe how the two chains are associated with each other, and which parts are needed for antigen association

These are found on All professional APCs(Dendritic, Macrophages, b-cells), but some are found on some unprofessional APC's in special circumstances. Also a heterodimer, you have two important chains an alpha chain and a beta chain these come together. The alpha chain has two important extracellular domains an alpha 1 domain which is involved in binding antigens and an alpha 2 domain(closer to the membrane). The Beta-chain has a Beta 1 domain acts as another hand holding the antigen and the Beta 2 domain(nearest the membrane) will be another immunoglobulin containing domain.

Describe how MHC expression can change depending on the conditions.

a. Genetic regulatory components - Some promoters drive up transcription of MHCs during times of infection b. Viral interference - Viruses try to shut down MHC-I expression b/c it targets the cells they have infected c. Cytokine-mediated signaling - Some cytokines expressed during infection/disease can drive up/down MHC expression d. MHC-II alleles and antigen presentation - Different capability to present antigens may dictate overall strength of response from individual to individual

What are the available enzyme systems for ELISA assays and what do they do.

a. ß-galactosidase (ß-Gal) - Cleaves ONPG to the yellow product = ONP - Measure accumulation with spectophotometer; absorption at 420 nm b. Alkaline phosphatase (AP) - Cleaves pNPP to nitro-phenol = yellow product - Absorbs at 450 nm c. Horseradish peroxidase (HRP) - Converts DAP (carcinogenic) into brown product that absorbs at 600 nm