MMI 528 Exam #2 - UW-Madison

Antigen

any molecule or part of a molecule that can be specifically bound by antigen receptors:

-antibody (Ab)

-B cell receptors (BCRs)

-T cell receptors (TCRs)

do antigen receptors recognize the whole antigen?

No, only parts of it. Typically, multiple parts of antigens are recognized by B and T cells.

Epitopes

The specific part of an antigen that is recognized by an antibody, BCR, or TCR. The antigen receptor and epitope fit together like a lock and key.

What must happen for a TCR to bind an antigen?

Antigens must be broken down into smaller pieces, called peptides, bound by MHC molecules, and displayed on the cell surface.

Conformational Epitope

Formed by parts of a protein that far apart in sequence but come together when the proteins folds.

What are conformational epitopes recognized by?

-Only recognized by BCR/antibodies

-Only recognized when the protein is in its natural, folded shape

Linear Epitopes

Parts of a protein that form a single, continuous sequence

What are linear epitopes recognized by?

-TCRs ONLY recognize linear epitopes

-Can be recognized by BCR/antibodies

Immunogens (antigens)

Antigens that induce adaptive immune responses

Factors that determine immunogenicity

1. Foreigness

2. Molecular size

3. Structural Complexity

4. Route and dose of antigen exposure

Types of antigens (from least to most immunogenic)

1. Autoantigens

2. Tumor neoantigens

3. Alloantigens

4. Xenoantigens

5. Microbial antigens

Physical form of antigens

Organic

-proteins>polysaccharides, bacterial coat proteins, flagella, toxins, viral proteins, etc.

-pure lipids and nucleic acids are poor immunogens

Inorganic: least immunologic threat and uncommon

-nickel sensitivities

Structural complexity of antigens

The more complex (quaternary structure) antigens are the more immunogenic.

Molecular size in relation to immunogenicity

the bigger the better, lol

-Larger antigens typically have more surface area (epitopes) where antibodies can bind or total amino acids that can be processed for T cells

Haptens (Incomplete Antigens)

Small molecules that can bind to a T and B cell receptor/antibodies but are too small to trigger an immune response on their own.

Ex.: Antibiotics, medications, poison ivy, nickle

-To become immunogenic, a hapten must be attached to a larger carrier molecule

Ex.: proteins, red blood cells, skin cells

Can haptens be recognized even when not attached to a larger carrier molecule?

Yes. Haptens bound to larger moelcules can become immunogenic, and once an adaptive immune response is stimulated, they can be recognized even when not attached to the larger molecule.

Ex. Penecillin on RBCs

Tolerogens

antigens that induce tolerance (self-antigens).

Ex. Allergy shots attempt to reduce allergy symptoms by inducing tolerance tp the allergens

What are immunologists dirty little secret?

Foreign proteins alone are not necessarily immunogenic. In order to induce immune responses you need to include additional factors to induce immunity.

Adjuvants

Any substance administered with an anitgen to enhance immunogenicity

-PAMPs!

-Often added to vaccines to enhance immune repsonses

What cells express MHC class 1?

all nucleated cells

What cells express MHC class II?

antigen presenting cells

What are the three components of MHC class 1?

1. membrane anchored alpha chain

-highly polymorphic (multiple distinct gene variants within a population)

2. B2-microglobulin

-highly conserved, stabilizes interaction between the peptide and alpha chain

3. peptide

-the peptide binding groove is formed between the alpha 1 and alpha 2 domains

What are the components of MHC class II?

1. membrane bound alpha chain

2. membrane bound beta chain

3. peptide

-Both alpha and beta chains are polymorphic

-Peptide binding groove formed between alpha and beta chains

How many amino acids can MHC class I accomodate?

peptides that are 8 to 10 amino acids

-hot dog fits in the bun

How many amino acids can MHC class II accomodate?

peptides that are at least 13 amino acids long

-foot-long hotdog hangs out of bun

How does MHC class I bind peptides?

-Non-covalent bonds hold the peptide in the MHC-1 binding groove

-Anchor residues at either end of the peptide "anchor" it to the MHC class I.

-The middle of the peptide bulges out of the MHC-1 to interact with T cell receptors.

MHC 1 anchor residues

Anchor residues are specialized for specific amino acids. TCRs are also specific to bulge amino acids. This is why we need polymorphic MHC-1 genes.

MHC binding motif

distinctive amino acid sequence that allows peptides to bind specific MHC

MHC-1 binding grooves

Contains pockets at each end of the binding groove that holds peptides. Each MHC-1 has differently shaped pockets, which can bind distinct amino acids.

Anchor residue

The specific amino acids of the peptide that fit within the MHC-1 molecule, anchoring the peptide in place.

How does MHC class II molecules bind peptides?

-The peptides extend outside the peptide-binding groove

-Anchor residues are not localized at the ends of the peptides, rather they are distributed the length of the peptide

-MHC-II binding pockets accomodate a wider range of peptides than MHC-1

3 ways that MHC genetics diversifies antigen presentation

1. MHC molecules are polygenic

2. MHC genes are highly polymorphic and have many variants

3. MHC genes are expressed from both inherited alleles (co-dominant)

Polygenic

Multiple genes produce proteins with the same function

-Humans have 3 MHC class I loci: HLA-A, HLA-B, HLA-C

Polymorphism

Multiple gene variants (alleles) within a population

Haplotype

A group of alleles of different genes on a single chromosome that are closely enough linked to be inherited usually as a unit

co-dominant expression

Both parental alleles of each MHC gene are expressed

-Humans can express up to 6 different MHC-1 molecules on all nucleated cells

Way the adaptive immune system maximizes diversity of antigen recognition

1. MHC diversity within population and co-dominant expression

2. Peptide processing by immunoproteasome

3. combining two protein chains

4. random combination of gene segments to form TCR and BCR

How are extraceullular antigens processed?

The exogenous pathway by professional APC for MHC-II binding and presentation to CD4+ T cells.

How are cytosolic antigens processed?

The endogenous pathway for MHC-I binding and presentation to CD8+ T cells

4 stages of the endogenous pathway

1. Origin

-cytosolic (inside cell) antigens

2. Degredation

-breakdown of antigens into peptide occurs in the cytoplasm

3. MHC-peptide complex formation

-peptides loaded into MHC-I in the endoplasmic recticulum

4. Antigen presentation

-transport of MHC-peptide complexes to the cell surface

How does a protesome chop proteins into peptides?

1. Ubiquitin (Ub) targets proteins for proteasome degredation

2. 19S cap on proteasome regulates the entry of proteins w/ Ub tags into the proteasome

3. Protein bonds are broken in core of proteasome

Constitutive proteasome

the version of the proteasome present in normal uninfected cells

Immunoproteasome

a specialized form that is always present in professional APCs and can be induced in other cell types after exposure to IFN-gamma.

How do cytosolic peptides get into the endoplasmic reticulum (ER)?

TAP proteins

-transporter associated with antigen processing

-preferentially transports peptides 8-16 amino acids long

-expression of TAP is increased by IFNgamma

Describe the process of assembly and loading of MHC-1

1. Chaperones Calnexin and ERp57 stabalize MHC-I alpha chain and promote folding.

2. Beta2-microglobulin displaces calnexin.

3. Chaperones tapasin and calreticulin joins the MHC-I alpha chain and ERp57 to form the peptide loading complex.

4. Tapasin brings TAP into close contact with MHC-I and ERAP trims peptides entering ER to 8-10 amino acids to bind MHC-I

5. PLC promotes binding of peptides with high affinity for MHC-I.

6. Once bound to peptide, MHC-I is stabilized, dissociates from PLC, and travels to cell surface.

What is the half life of high affinity peptide-MHC-I interactions?

>24 hours

What is the half life of low affinity peptide-MHC-I interactions?

<6 hours

4 stages of exogenous antigen (MHC-II) processing

1. Origin

-extraceullar antigens are brought into cells for processing

2. Degradation

-breakdown of antigens into peptides occurs in endosomes

3. MHC-peptide complex formation

-peptides loaded into MHC-II in late endosomes

4. Antigen presentation

-transport of MHC-II/peptide complexes to the cell surface

How can exogenous antigens be uptook for intracellular processing?

Professional APCs

-phagocytosis

-pinocytosis

-Fc and complement-mediated endocytosis

B cells

-BCR-mediated uptake

What prevents premature loading of self proteins to MHC-II in ER?

Invariant chain: stabilizes immature MHC-II and prevents premature loading of the peptide-binding groove.

Invariant chain in guiding MHC-II to late endosomes

The invariant chain helps direct the MHC-II molecule to late endosomes, containing exogenous peptides.

Once in late endosomes, the invariant chain is degraded by pH-dependent proteases and leaves the CLIP peptide (class-II associated invariant chain peptide) in the binding groove.

How is CLIP displaced from MHC-II?

-CLIP blocks binding of exogenous peptides to MHC-II.

-HLA-DM associates with MHC-II and promotes the release of CLIP and the binding of high affinity peptides.

-HLA-DM does not have a peptide-binding groove.

-Once peptides are loaded into the MHC-II, they are transported to the cell surface.

professional APCs can use the endogenous pathway to process cytosolic pathogens and display MHC-I on their surface.

False. APCs use the exogenous pathway and display pathogenic peptides via MHC-II.

Can naive CD8 T cells be activated by an epithelial cell presenting an antigen via MHC-I?

Nope!! A naive CD8 T cell must first be activated by professional antigen presenting cells.

How do naive CD8+ T cells get activated when professional APCs pickup extracellular antigens?

Cross-presentation pathway

-Cross-presentation is the process by which professional APCs can present extracellular antigens on MHC-1 molecules.

-This process effectively "crosses" antigens from the exogenous pathway into the endogenous pathway enabling the presentation of external antigens on MHC-I.

-Activates CD8+ T cells against antigens not produced within the pAPCs itself.

How to antigens interact with receptors?

non-covalent bonds (electrostatic bonds) that are inherently reversible

affinity

strength of a single binding interaction between one receptor and one epitope

avidity

collective strength of binding between multiple antigen receptors and multiple epitopes

Cognate antigens

the specific antigen recognized by an individual antigen receptor

Antigenic valency

the number of identical epitopes on a single antigen

Ag-receptor valency

the number of antigen binding sites possessed by an antigen receptor

Antigen 1 has a valency of 5 while antigen 2 has a valency of 10. Which antigen is more effective at activating B cells?

Antigen 2.

Highly valent antigens are more effective at activating B cells than antigens with low valency.

Describe BCR and Ab antigen recognition mechanisms

-They recognize native antigens in their natural, fully-folded form

-They recognize antigens that are outside of cells, whether attached to cell surfaces or soluble

Describe antibody structure

Fab region - contains variable / hypervariable regions, Light and Heavy chains, recognizes the antigens.

Fc region - constant, Carboxyl terminal, Complement binding (IgG and IgM), Carbohydrate side chains, determines isotype of antibody (IgG, IgM...)

-can change through class switching

Heavy chain contribute to Fc and Fab region

Light chain contribute to Fab region only

Complementary determining regions (CDRs)

-Are highly variable portions of antigen receptors that directly contact antigens

-CDRs are found at the tips of antigen receptors

-CDRs determine what specific antigen a BCR or TCR can bind to

-Both chains have 3 CDRs, these 6 CDRs from the antigen binding site

Are antibodies flexible?

Yes, the accommodate variety of binding angles (far apart or close epitopes)

How to BCRs transform into Abs?

B cells modify their mRNA splicing to transform membrane bound BCRs into secretable antibodies. This process replaces hydrophobic amino acids in the BCR with hydrophilic ones in antibodies. The resulting changes in solubility allow antibodies to be secreted from B cells.

What transmembrane accessory molecules do BCRs require for effective signal transduction?

CD21 (complement receptor 2)

-co-receptor, enhances Ag binding

CD19

-co-receptor, assists with signal transduction

Iga and IgB

-co-stimulatory molecules

-contain Immunoreceptor Tyrosine Activation Motifs (ITAMs)

-critical for signaling that a BCR has a bound antigen

-lead to the activation of B cells and antibody secretion

How do TCRs recognize antigens?

TCRs recognize peptides presented by MHC-I or MHC-II molecules, not free antigens

TCRs must engage both MHC and peptide to activate T cells?

True

TCRs have high affinity interactions?

False. Low affinity because it must bind both MHC and peptide.

Why do BCRs have high affinity interactions?

Because they can mirror antigen in shape and electrostatic charge

What role does CD3 play in TCRs?

CD3 is a complex of six molecules that are associated with TCRs.

-co-stimulatory molecules

-similar role to Iga and IgB for BCRs

-all CD3s have ITAMS

-required for TCR signaling and activating T cells

What role do CD4 and CD8 play in TCR-MHC interactions?

CD4 and CD8 molecules are co-receptors that stabilize TCR-MHC interactions, thereby increasing avidity of the engagement.

How many BCRs can the immune system generate?

100 billion

Germ-line theories of antigen diversity

Each Ag-receptor is coded independently in the genome

Dreyer-Bennet hypothesis of antigen diversity

AG-receptor genes are coded by separate variable (V) and constant (C) regions

How to prove the Dreyer-Bennet hypothesis?

Use restriction enzymes to cut skin cell DNA to get a range of fragment sizes, then sort these fragments using gel electrophoresis and detect where V and C regions are with probes. Then cut B cell DNA and compare to the skin cell DNA.

What was found that in the skin cell DNS, some V regions were missing and in the B cell DNA, the V and C probes detected the same fragment.

They discovered an additional region that is incorporated into V and C called the joining region (J).

How does recombination occur for the heavy chain?

-two recombination events must occur

J to D --> JD to V --> VDJ

-Heavy chains have an extra segment that adds ~8 amino acids between the J and C segments called the DIVERSITY (D) region.

How does recombination occur for the light chain?

V to J --> VJ

-Only one recombination event

BCR light chain gene locus has multiple V, D, and J gene segments to promote diversity.

FALSE. BCR light chain loci only have V and J gene segments. No D segments.

The heavy chain has multiple V, D, and J gene segments.

combinatorial diversity

The random combination of different gene segments during antigen receptor formation.

How does recombination of these gene segments occur?

Each antigen receptor gene segment has a recombination signal sequence (RSS) upstream, downstream, or both. RSS elements guide enzymes involved in somatic recombination to the correct location in the DNA and ensure proper joining of gene segments during recombination.

What are the 3 parts of an RSS?

1. conserved heptamer (7 bps)

2. spacer region (either 12 or 23 base pairs)

3. conserved nonomer (9bps)

What is the 12/23 rule in regards to RSS elements?

Recombination only occurs between one 12-bp RSS and one 23-bp RSS containing gene segment.

This rule helps ensure correct gene segment pairing.

-for example, one variable gene segment cannot bind to another variable gene segment.

Describe how the 12/23 rule guides proper gene segment joining

RAG recombinases bring complementary 12-bp and 23-bp gene segments together, aligning the heptameters and nonomers in the RSS elements to form a DNA loop.

-the 12/23 RSS pairing creates a sharp turn in the DNA

-the 12/12 or 23/23 pairing cannot form a sharp turn in the DNA

RAG recombinases recognize the sharp turns in the DNA created by the 12/23 RSS pairings, allowing for their correct positioning to cut DNA

-RAG genes are only expressed in B and T cells during development

What are the two joints formed by RAG cutting

1. Coding joint: new joint b/w gene segments in the gneome

2. Signal joint: joint in excised DNA containing RSS elements

What are the steps in Ag-receptor gene recombination?

1. RAG and associated proteins bind to RSS elements

2. The two RAG complexes bind to each other and bring 12-RSS and 23-RSS gene segments together

3. RAG makes single-stranded nicks in the DNA and forms a hairpin bind b/w the two DNA strands

-hairpin bonds prevent the DNA from unwinding

4. Artemis exonuclease binds the ends of the DNA strands and cleaves the hairpins at the end of the joining segments leaving the ends open for palindromic nucleotides to be added to the coding joint, which are not in the original sequence, thereby adding diversity.

Where can Artemis cut the hairpin?

At different locations along the hairpin bond leaving either a:

-3' overhang

-5' overhang

-blunt end

What's another way junctional diversity is achieved in addition to adding P-nucleotides to the ends of the joining segments?

Terminal deoxynucleotidyl transferase (TdT) can add up to 20 random non-templated (N) nucleotides to the P nucleotide ends of the single stranded junction sites. However, exonucleases trim back nucleotides, they can remove a random number of N and P nucleotides (0-14bps) into the coding joint, creating an additional layer of diversity.

DNA polymerases fill the gaps with complementary nucleotides and DNA ligase

What accounts for most of the diversity observed in antigen receptors?

Junctional diversity

T cell receptor rearrangement

T cells undergo very similar gene rearrangements to B cell receptors.

-B chain undergoes VDJ recombination similar to BCR heavy chain

-a chain undergoes VJ recombination, similar to BCR light chain

5 mechanisms for generating antigen receptor diversity in naive cells

1. combinatorial diversity

2. pairing of two different receptor chains

3. addition of palindromic P-nucleotides by Artemis cutting

4. addition of non-templated N-nucleotides by TdT

5. exonuclease trimming

Give an overview of B cell development

Bone marrow is the primary lymphoid tissue for B cells, providing specialized microenvironment for B cell maturation.

B cell precursors interact with various stromal cells in the bone marrow, which regulate B cell receptor rearrangement and cell development.

Most immature B cells leave the bone marrow and complete their maturation in the spleen.

Mature naive B cells leave the spleen and circulate among secondary lymphoid tissues, searching for their specific (cognate) antigen.

What are the stages of B cell development?

1. Stem cell

2. Early pro-B cell

3. Late pro-B cell

4. pre-B cell

5. Immature B cell

What stage is a B cell in when D recombines to J?

Early pro-B cell

What stage is a B cell in when D-J recombines to V?

Late pro-B cell

What stage is the B cell when the heavy chain is completely recombined (V-D-J)?

Large pre-B cell

Does V-D-J need to be in-frame for maturation to continue?

Yes.

mRNA is translated in codons (triplets). The addition of nucleotides not in multiples of 3 (a codon) during VDJ rearrangement causes frameshift mutations, resulting in nonfunctional heavy chains.

What is the first checkpoint of B cell maturation?

Large pre-B cells must demonstrate that the rearranged heavy chain is functional. Only then will the cell proceed to rearranging light genes.