Exam 1 (Lectures 7-9) - Antigen Binding Site

Question 1

Describe the antigen-binding site of an Ig molecule (ie the structure and function).

Answer 1

1) VH and VL domains in hypervariable regions where the amino acids vary greatly from antibody produced by different clones.

2) Approximately 3 regions within each variable domain of the antibody are not next to each other in the linear structure but will be next to each other in the folded protein.

3) They vary in size and shape.

Question 2

Define antibody affinity.

Answer 2

Strength of binding that depends on shape and bonding ability.
- sum of attractive and repulsive forces between molecules

Question 3

Explain linear and conformational epitopes and explain how mishandling a protein antigen might impact antibody binding to the antigen.

Answer 3

1) Linear epitope = antibody binds to parts of antigen that are adjacent to each other in molecule
- not as detrimentally affected by desaturation, as the epitope will not be separated but new epitopes can be created

2) Conformational epitope = parts of epitope are together due to binding
- desaturation will destroy the conformation so antibody will not bind the antigen

Question 4

Antibody Isotype Characteristics and Functions (SECTION 2)

Question 5

List the 5 isotypes (classes) of antibody and their unique characteristics, location, and primary functions.

Answer 5

1) IgM
- Pentamer with 5 heavy chain domains (4 constant), held together with disulfide bonds and J chain, can bind 10 identical
epitopes, found intravascularly.
- Functions: Complement activation via classical pathway, agglutination, neutralization of toxins and viruses

2) IgG
- Monomer with 4 heavy chain domains (3 constant), has 2 binding sites, found in bloodstream and interstitial fluid
- Functions: complement activation via classical pathway, agglutination, neutralization of viruses and toxins, opsonization
of microbes for phagocytosis by neutrophils and macrophages, systemic immunity in newborns (passive),
antibody-dependent cell-mediated cytotoxicity

3) Secretory IgA
- Dimer with 4 heavy chain domains (2 monomers held together by J chain), secretory component added as transported to
mucosal surface so it will not be degraded while on mucosal surfaces (monomer located in blood; dimer located in
mucosal surfaces)
- Functions: neutralize toxins and viruses, block the entry of bacteria, found in mother’s milk and helps provide intestinal
immunity to suckling neonate

4) IgE
- Five domains in heavy chain (4 constant); 2 binding sites, high affinity for FcE receptors on mast cells so mainly found
attached to mast cells (does not need to bind antigen to bind to FcE receptor), bind FcR receptors on eosinophils after
binding antigen
- Function: defense against helminthic parasites, mediator of immediate hypersensitivity (allergies)

5) IgD
- Located on B cell receptor; marks B cells as mature

Question 6

List 7 ways antibody can function in protection from disease, which portion of the antibody molecule (Fc or antigen-binding site) is responsible for that function, and what isotypes are important for that function.

Answer 6

1)Agglutination = Antigen binding site; IgM and IgG

2) Neutralize toxins or viruses = Antigen binding site; IgM, IgG, and IgA

3) Block attachment = Antigen binding site; IgA

4) Complement fixation via classical pathway = Fc; IgM and IgG

5) Opsonization for phagocytosis by neutrophils and macrophages = Fc; IgG

6) Antibody dependent cell-mediated cytotoxicity = Fc; IgG

7) Sensitization of mast cells = Fc; IgE

Question 7

Cytokines: Regulators of the Immune Response (SECTION 3)

Question 8

Explain and define cytokines in general: where do they come from, what do they do, and where do they act (autocrine, paracrine, and endocrine). List several general properties of cytokines.

Answer 8

1) Large group (60 different) of secreted proteins that regulate intensity and duration of innate and adaptive immune response.

2) Activity receptor mediated

3) Autocrine = act on cell that produced it
Paracrine = action on another cell nearby
Endocrine = action on a distant location

4) General properties:
- secretion is brief and limited
- only produced when needed
- individual cytokines are produced by multiple diverse cell types
- cytokine actions may be local and/or systemic
- most cellular responses to cytokines require transcription and translation

Question 9

Explain what is meant by cytokines milieu.

Answer 9

1) Mixture of cytokines in the environment

2) The mixture regulates cell function and trafficking but it is complex and hard to replicate with therapies

Question 10

List and explain mechanisms for down-regulating/blocking cytokines and give two examples of how these are used clinically.

Answer 10

1) Receptor antagonist
- Something binds to the receptor so cytokines can’t; but it doesn’t stimulate signal

2) Release of soluble receptors to soak up or neutralize cytokines
- Something binds to cytokines so it cannot bind to cells

3) Cytokines of opposite effect to counteract response
- One cytokine binds and stimulates response by another cytokine; also binds and suppresses the response

4) Deceptor receptor
- Decoy receptor that will bind cytokine and create no signal

Clinical Examples:
- IRAP (interleukin 1 receptor antagonist) used for equine arthritis
- Apoquel inhibits pruritogenic cytokines and proinflammatory cytokines to help with allergies

Question 11

Major Histocompatibility Complex (SECTION 4)

Question 12

List the molecules that present antigen to the adaptive immune system and the molecules the adaptive immune system uses to bind antigens.

Answer 12

1) Molecules that present antigens:
- MHC1
- MHCII

2) Molecules used to bind antigens:
- TCR
- BCR

Question 13

Describe the immunoglobulin superfamily proteins, including examples of molecules in the family and the common structure required for membership.

Answer 13

1) Family of proteins that contain characteristic immunoglobulin domains that are 110 AAs homology regions

2) Examples: BCR, TCR, MHCII, MHCI, CD3, CD4, CD8

Question 14

Compare and contrast MHCI and MHCII, including structure, location, cells and molecules that recognize and bind to them and the key points about the antigens that end up on each.

Answer 14

1) MHCI
- Structure = 1 alpha chain with one constant and 2 variable domains; one transmembrane protein and beta-2
microglobulin chain (stabilizer protein)
- variable chains form peptide binding groove that is closed structure that is the binding site for TCR
- Function = present cytosolic peptide antigen in its binding groove to CD8 T Cell
- allows any infected cancerous uncleared cell to be detected
- binding of effector CTL results in cytotoxicity and killing of cell
- Location = on membrane of all nucleated cell
- Antigen = binds endogenous antigens of 9 AAs

2) MHCII
- Structure = alpha chain with 1 constant and 1 variable domain; beta chain with 1 constant and 1 variable; each chain contains transmembrane portion.
- Function = present exogenous peptide antigens to CD4 T cells
- Location = on membranes of specialized antigen-presenting cells
- Antigens = bind exogenous antigens of 9+ AAs

Question 15

Pathways of Antigen Processing and Presentation (SECTION 5)

Question 16

Label a diagram and describe the steps for antigen processing and presentation to T cells. (Steps for both the endogenous pathway of antigen presentation and the exogenous pathway of antigen presentation).

Answer 16

1) Endogenous Pathway
- Occurs when antigen is in cytosol of cell and antigen ends up on MHCI and recognized by CD8 T cells
- Ubiquitin binds to cytoplasmic protein and targets it to proteosome
- TAP binds to cytosolic peptides from proteosome and transports them to lumen of ER
- Peptide is trimmed to 9 AAs by ER resident aminopeptidase and then binds to empty MHCI in ER
- MHCI + peptide moves to Golgi and transported to exocytic vesicle to cell surface
- Antigen + MHCI available for CD8 T cell recognition on cell surface

2) Exogenous Pathway
- Occurs when antigen is outside cell and internalized by endocytosis/phagocytosis and ends up on MHCII to be recognized
by CD4
- APC internalizes antigen via phagocytosis/endocytosis endolysosome is formed
- Lysosomal enzymes digest antigen into short peptides
- MHCII molecule is synthesized in RER
- Alpha and beta chains come together and from groove
- Invariant chain fills peptide groove while MHCII is transported through Golgi
- Exocytic vesicle of RER fuses with endosome/phagolysosome containing antigen peptides
- Invariant chain is partially digested and removed from groove and antigen from the phagolysosome is loaded into groove
- MHCII with antigen in groove is transported to membrane where antigen + MHCII are available for CD4 T cell recognition

Question 17

Explain the added benefits with regard to the immune response of using ISCOMS (immune-stimulatory complexes) to deliver an inactivated antigen vaccine.

Answer 17

1) ISCOMS = adjuvant used to improve immune response stimulated by vaccine
- lipid micelles that fuse with cell membranes

2) They carry proteins (killed vaccines or purified proteins) into cytosol of cell so that it can be presented on MHCI and stimulate CD8 cytotoxicity T cell response via the endogenous pathway; normally these proteins would just be presented via exogenous pathway through MHCII

Question 18

MHC and Transplantation (SECTION 6)

Question 19

Explain MHC heterozygote advantage and include how many possible different MHC molecules an individual can have.

Answer 19

1) The heterozygote advantage allows an animal to be able to bind and present a greater variety of antigen/MHC combination than homozygous animals.

2) Fully heterozygote animal has 6 different MHCI molecules and 6-8 different MHCII alpha and beta chains to from more than 6 combinations of alpha and beta chains to make MHCII molecules.

Question 20

Explain the role of MHC in tissue transplantation and the cells of the immune system responsible for rejection; compare this to immune mechanism that mediates the rejection because of RBC antigens.

Answer 20

1) Host T cells are activated by foreign MHC molecules present in transplanted tissue resulting in CTL killing of transplanted cells and cytokine release by TH cells; cytotoxic T cells bind to the foreign MHCI and result in killing of the cell

2) Blood type matching will prevent acute rejection caused by IgM antibodies to blood group antigens
- IgM will agglutinate the RBC together and will also lyse them via activation of complement

Question 21

Generation of Antigen Receptors (SECTION 7)

Question 22

Explain how antibody diversity is generated.

Answer 22

1) Random gene rearrangement occurs from germ line heavy chain DNA

2) Specificity is determined by variable portion of heavy and light chains
- genes for variable domain of heavy chains has variable region (V), diversity region (D), and joining (J)
- variable portion of heavy chain results from the random selection of one of each region
- genes for variable domain of light chain has variable (V) either kappa or lambda, and joining (J) either kappa or lambda
- variable portion of light chain results from the random selection of one of each region
- all of the selected DNA segments are brought to one segment by looping and cutting portions of DNA and recombination
by recombinase

3) Heavy chain constant domains
- cell rearranges DNA and selects one segment from V, D, and J regions and all gene segments for constant regions
- RNA transcript is made by transcribing V, D, J region and Constant regions.
- mRNA transcript made of VDJ + C and is translated into IgM
- cell will undergo maturation and tagged with IgD so it expresses the BCRs of IgM and IgD on its membrane
- cell will maintain mRNA expression of constant portion of heavy chain with DNA still able to code for other constant
heavy chains for IgG, IgE, IgA

Question 23

On the stick figure identify the domains that are coded for by the B cell heavy chain DNA VDJ regions, the constant regions, and the light chain VJ region and kappa/lambda.

Question 24

Describe where and what is happening during the following stages of B cell development.

Answer 24

1) Formation of the antigen binding site
- occurs in the primary lymphoid tissue
- formation of heavy and light variable chains occurs based on information (in the question about how antibody diversity is generated); these will be the antigen binding sites

2) Negative selection
- occurs in primary lymphoid tissue
- any B cell that meets antigen in primary lymphoid tissue will undergo apoptosis

3) Class switching
- occurs in memory B cells at secondary lymphoid tissues
- signal released from mixture of cytokine and T helper cells that are triggered when mature naive B cell meets its antigen and clonally expands —> DNA of cell undergoes recombination and switch to IgG, IgE, or IgA depending on what portions are removed by recombinase along with IgM and IgD coding regions (change only occurs on constant heavy chain).

4) Affinity maturation
- occurs in secondary lymphoid tissue
- the first antibody produced has low affinity by which somatic mutations via hyper mutation in VDJ regions of DNA the antibodies with many mutations (and in turn low affinity) will undergo apoptosis leaving the antibody with high affinity.

Question 25

Describe with drawings how the DNA changes as a B cell develops and matures, include: the germ line B cell DNA for the heavy chain, mature naive B cell DNA for the heavy chain and memory B cell DNA for the heavy chain in a B cell that will make IgG when it becomes a plasma cell

Question 26

Explain what “isotypes switching while maintaining antigen specificity” means.

Answer 26

1) When isotype switching occurs when the only constant heavy chain changes resulting in a different isotype since the constant heavy chain determine isotype.

2) No changes are made to the variable domains of both heavy and light chains as well as the constant light chain

Question 27

Compare and contrast the BCR of a B cell making IgM and a clone of that cell that has switched to another isotype. (Don’t forget about the light chain).

Answer 27

1) The BCR of a B cell making IgM will have the same variable domains for both the heavy and light chains and constant light chain as the clone of the cell that switched to another isotype. The BCR of the B cell making IgM will have the constant heavy chain that specifies IgM while the clone of the cell will have the constant heavy chain that specifies the isotype that it has switched to.

Question 28

Compare and contrast the immunoglobulin made by a B cell and the plasma cells differentiated from its clones.

Answer 28

1) B cells = immunoglobulin has transmembrane portion coded by DNA and secured Ig in the B cell membrane as the BCR

2) Plasma cell Ig is made without the transmembrane portion so Ig is secreted and not maintained in the membrane

Question 29

Describe what antibody affinity maturation is and explain the mechanisms that result in the population of antibody increasing in affinity.

Answer 29

1) Antibody affinity maturation allows the antibody to be selected for the antibody with the highest affinity for its antigen.

2) Somatic mutation occurs via hyper mutation in the VDJ region of DNA; antibody will have many mutations in lower affinity which will undergo apoptosis while antibody with higher affinity will be selected for

3) Antibody that is selected will undergo mitosis and more clone creation and plasma cells from clones that secrete high affinity antibody