1
Q
Two arms of adaptive immunity
A
humoral and cell mediaed
2
Q
Membrane bound vs secreted IgG
A
- Membrane Bound: B cell receptor (BCR)
- Secreted: Antibody
3
Q
Prototypical antibody structure
A
- Variable region at the top - rest is the constant region
- Framework region is a subdivision of the variable region that acts as a scaffold for the CDRs
- It is made up of 2 identical heavy chains and 2 identical light chains
- Heavy Chain has 4-5 domains
- Light Chain has 2 domains
- Each domain is made up of 110 aas
- Heavy and light chains are joined by disulfide bonds
4
Q
Know how to draw prototypical antibody structure (Slide 6) - know where the disulfide bonds are
A
5
Q
Where does the diversity of antigen recognition by antibodies come from (structurally)
A
- In the variable region there are hypervariable loops of aas - CDRs (6 of them)(complementarity determining regions)
- They are supported by framework regions (conserved beta sheets)
6
Q
CDRs recognize based on …
A
- Complementary shape and charge (noncovalent interaction)
7
Q
Conformational vs Linear Determinants
A
- Conformational determinant - determinant is lost by denaturation (antibody cannot bind)
- Linear determinant - antibody binds only to denatured protein or to both denatured and native protein
8
Q
VDJ recombination
A
- somatic recombination of gene fragments of the Ig heavy and light chain loci
9
Q
What are the main players of VDJ recombination
A
RAG1 and RAG1 - they work together to shuffle and rejoin V, D, and J segments
10
Q
Can B and T cells complete their development without VDJ recombination?
A
No, this is why RAG knockout mice have no B or T cells
11
Q
What determines the specificity of an antibody
A
the 6 CDR loops
12
Q
Recombination Signal Sequences
A
V D, and J fragments are flanks by RSS containing a heptamer and a nonamer separated by 1 (12 bp) or 2 (23) helical turns
13
Q
12/23 rule
A
- ensures precise and ordered V(D)J recombination (V binds to J etc.)
- A gene segment flanked by an RSS with a 12 bp spacer can only be joined to one flanked by a 23 bp spacer RSS
14
Q
Generation of Junctional Diversity
A
- lot of slippage at the junction regions
Random nucleotides added at the joints between gene segments as a result of the addition and subtraction of nucleotides during the recombination process:
- You add some random nucleotides (not in groups of 3 so you create alternative reading frames)
- You add random N (non-template) and P (palindromic) nucleotides
15
Q
4 things that lead to Ab Diversity
A
- VDJ recombination between germline and encoded exonal variants
- Junctional Diversity
- Random pairing of Heavy and Light Chains
- Somatic Hyper mutation
16
Q
Life of a B Cell
A
Bone Marrow:
1. Rearrangement: B cell precursor rearranges its immunoglobulin genes (generation of BCRs)
- Tolerance: Negative Selection of B cells
Periphery:
3. Activation: Mature B cell bound to cognate antigen is activated and migrates to the peripheral lymphoid organs
- Maturation: Activated B cells gives rise to plasma (antibody secretion in bone marrow and lymphoid tissue) and memory cells (only in lymphoid tissue)
17
Q
Negative selection of B cells in the bone marrow is somewhat rudimentary compared to the testing of T cells in the Thymus because…
A
- There are ways to control B cells if they see something that is self in the periphery
18
Q
Rearrangement and Expression of Ig Genes during B Cell Development - Know how to draw this
A
- Stem Cell: Nothing is expressed yet and no rearrangement has yet begun - Ig gene segments are still in germline configuration
- Early Pro B Cell: D-J rearrangement of heavy chain ON BOTH CHROMOSOMES (rest is only 1)
- Late Pro B Cell: V-DJ rearrangement of heavy chain
- Large Pre-B Cell: VDJ of heavy chain rearranged. mu chain transiently expressed at the surface as part of pre BCR, most of it is still in the cell, the light chain at this moment is just a surrogate (dummy)
- Small Pre-B Cell: V-J rearranging of light chain (Pre BCR not expressed on surface anymore - only the mu heavy chain is expressed in the cytoplasm)
- Immature B Cell: VJ of light chain has been rearranged. IgM is expressed on the cell surface
- Mature B Cell: IgD and IgM made from alternatively spliced transcripts
19
Q
Outcomes for B cells during Negative selection for the following cases:
1. Multivalent self-molecule
2. Soluble self-molecule
3. Low affinity non-cross-linking self-molecules
4. No self-reaction
A
- Apoptosis or receptor editing (if new receptor is still self-reactive cell dies)
- Migrates to the periphery: Anergy
- Migrates to the periphery -> matures but is clonally ignorant - in this case it depends on the avidity of the binding
- Migrates to periphery -> matures
20
Q
B Cell Activation and Signaling
A
- Clustering of BCRs through antigen binding permits signaling through: Ig- alpha/beta a.k.a. CD79 A/B - these initiate signaling
- Leads to the activation of the same 4 transcription factors as T cell signaling
21
Q
Thymus dependent antigens
A
- 1st signal through the BCR
- 2nd signal through Help T cells: MHC-2, CD40:CD40L, cytokines
- Leads to affinity maturation
22
Q
Affinity maturation
A
- activated B cells produce antibodies with increased affinity for antigen during the course of an immune response - occurs through somatic hypermutation + class switching
- Antibodies of greater affinity are produced by somatic hypermutation of V region genes
- B cells whose V regions have accumulated deleterious mutations and can no longer bind antigen die.
- B cells whose V regions have acquired mutations that improve the affinity of the B-cell receptor for antigen are able to compete more effectively for antigen and receive signals that drive their proliferation and expansion.
23
Q
Thymus independent antigen
A
- Ti antigens can deliver both signals to B cells
- But no affinity maturation and no memory B cells
- 2nd Signal is through a PRR (i.e. TLR)
- no class switching (only IgM)
24
Q
T-cell help for B cells requires what?
A
Priming by professional APC
25
B Cell maturation and germinal center formation
1. B cells that encounter antigen at the T cell- B cell border becomes activated.
2. They then form primary foci in the medullary cords.
3. Some cells then migrate to the primary follicle, forming a germinal center
4. Plasma cells migrate to the medullary cords or leave via the efferent lymphatics and migrate to the bone marrow.
26
Germinal Center
- transiently formed structures within B cell zone (follicles) in secondary lymphoid organs (lymph nodes, ileal Peyer's patches, and the spleen)
- where mature B cells are activated, proliferate, differentiate, and mutate their antibody genes
- Through somatic hypermutation aimed at achieving higher affinity, during a normal immune response
27
AID enzyme role and where is it expressed
- Catalyzes Isotype (class) switching and somatic hypermutation
- Expressed only in germinal center B cells
28
Somatic hypermutation
- Catalyzed by AID
- occurs in mature activated B cells
- Introduces mutations into the rearranged Ig V regions
29
Class Switching
- All B cell progeny have the exact same Vh region except for some mutations from somatic hyper mutation
- In contrast to, that B cell’s progeny may express several different C-region isotypes
as the cells mature and proliferate during the immune response.
- The first antigen receptors expressed by B cells are IgM and IgD.
- Later in the immune response, the same assembled V region may be expressed in IgG, IgA, or IgE antibodies.
- Achieved through irreversible DNA recombination - class switch recombination
- only occurs in activated B cells
30
IgG
- Highest affinity and greatest specificity
- Several subclasses with different Fc regions
- All subclasses neutralize
- IgG1 is the most adept at opsonization
- IgG1 and IgG3 mediate ADCC (in humans)
- Defends against microorganisms and toxins
- OK complement fixer
- Good opsonizer
- Helps NK cell kill (ADCC)
- Can cross the placenta and provide the fetus with adaptive protection
31
IgE
- Mediates immunity to eukaryotic pathogens as well as allergy
- Cross linking of IgE receptor on the surface of mast cells causes degranulation
- Histamine release
- Increased vascular permeability
- Chemoattraction of innate inflammatory cells
32
IgA
- Dimeric
- Forms in response to tolerogenic TGF-β and IL-10 secretion
- Secreted Ig at the mucosal surfaces (produced a lot in the gut)
- Participates in both neutralization of mucosal pathogens and tolerance to commensal organisms
33
IgM
- First line of defense (first antibody made)
- Great complement fixer
- Good opsonizer
34
How does heavy antibiotic use cause allergy
- by killing bacteria and thus allowing fungus to grow
- and to produce an allergic reaction against a given antigen, an individual must
first be exposed to the antigen under conditions that result in the production
of IgE antibodies (i.e. fungus)
35
Antibodies fit into the complement cascade through which pathway?
Classical
36
Difference between complement fixation with IgM and IgG
- Both bind antigens on bacterial surface
- But C1 binds to a single IgM
- C1q binds to 2 or more IgGs
37
Functions of antibodies
- Neutralize pathogen toxins
- Prevent pathogen adherence
- Opsonize pathogen for
- Destruction by the complement cascade
- Phagocytosis via Fc/FcRγ interaction
- Neutrophils
- Macrophages
- Dendritic cells
- ADCC via Fc/FcRγ interaction
- All of the above plus NK cells
38
Name of the surrogate light chain during BCR development
lambda5+VpreB
39
Most B cells are lost during rearrangement of which chain? Why?
- Heavy Chain
- During rearrangement of the heavy chain, the B cell tries for a successful rearrangement on two chromosomes (sequentially) if it fails on both the cell dies
- During the process for the light chain, it also goes through two chromosomes because it also goes through a kappa and a lambda locus on each chromosome
- It will attempt kappa on each gene then lambda
40
Most antibodies in humans are lambda or kappa? Why?
Kappa because it is the first locus to be rearranged during development of the light chain of the BCR
41
What fraction of joining during VDJ recombination will be successful? Why?
1/3 due to DNA reading frame (2/3 of rearranged genes are out of frame)
42
How many successful joining are required for heavy and light chain respectively during VDJ recombination in BCR development? Why?
- Heavy requires 2 (D-J rearrangement and V-DJ rearrangement)
- Light chain only 1 (just V-J rearrangement)
43
Ig-alpha and beta are expressed on the cell surface right beside the BCRs
44
allelic exclusion
- a state in which on 1 of the two alleles on a gene is expressed in a diploid cell
- A B cell enforces allelic exclusion to prevent successful rearrangement at both heavy chain alleles (one on each chromosome), which would result in producing two receptors with different antigen specificities
- occurs at the light chain as well, pre BCR signals to downregulate RAG
45
Name 2 reasons for why a B cell may not want to express BCR with differing specificities
- Diluted Response: It would be less efficient at binding to a specific pathogen. Since signaling is initiated by a clustering of receptors
- Autoimmunity Risk: One receptor might target a virus, while the other targets your thyroid. If that cell gets activated, it’s a double-edged sword you don't want.
46
Opsonization vs Complement System
- Opsonization phagocyte binds to Fc portion of bound antibody and eats the pathogen
- Complement open holes in the pathogen
47
Affinity maturation and class switching require
T cell help
48
How do mismatch repair pathways contribute to somatic hypermutation?
- They are initiated by changes to the DNA by AID, they then change the DNA even more because this process in B cells is error prone.
49
Class Switch Recombination
- Cytokines from Th cells activate transcription from cryptic promoters exposing S regions to homologous recombination (probably).
50
S (switch regions)
- present in the heavy chain locus, upstream of C (constant) regions
- stretches of repetitive DNA that guide class switch recombination
51
ADCC - Antibody Dependent Cellular Cytotoxicity
- Cell recognizes Fc portion of bound antibody and initiates killing enzymes etc.
52
True or False: Class switching contributes to antibody variable region diversity?
No since it only deals with the constant region, but it does contribute to overall antibody diversity
53
What is minimally required for the formation of a germinal center?
- immunogen
- B cells
- T Cells
- Follicular DCs
- an area of active cell division that forms within a surrounding region of resting B cells in the primary follicle.
- Proliferating germinal center B cells displace the resting B cells toward the periphery of the follicle, forming a mantle zone of resting cells around the two distinguishable areas of activated B cells, called the light zone and
the dark zone.
GS-IY-6200
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