Humoral Immunity: Generation of Antibody Diversity Flashcards
Humoral immunity is part of ………..
Humoral immunity is mediated by ……..
adaptive immune system
antibodies secreted by B-cells
Antibodies are aka ………
Immunoglobulins, Ig
Antibodies are produced by ………
plasma cells
Antibodies fight against ………..
foreign pathogens + cancerous cells
How do antibodies work?
- Block pathogen from entering
- Tag pathogen for removal by other immune cells (opsonisation)
Antigen-independent stage is ………. B-cell activation
Antigen-dependent stage is ………. B-cell activation
Antigen-independent stage is before B-cell activation
Antigen-dependent stage is after B-cell activation
Antibodies have 2 versions:
1 - Membrane-Bound antibodies (B-cell Receptor). Anchored on B-cell membrane for weapon development
2 - Secreted antibodies - final, fully functional form of the antibody secreted by mature plasma cells.
Functions of antibodies
- antigen binding
- effector function
Describe the structure of antibodies
Y-shaped molecule: 2 heavy + 2 light chains
Heavy chain:
- 5 classes - γ,𝜀,ẟ,µ,𝜶,
- Subtypes - γ1-4 and 𝜶1-2
- Total 9 different heavy chains possible
- 4 domains - VH, CH1, CH2, CH3
Light chain:
- 2 classes - κ (kappa) , λ (lambda)
- 2 domains - VL, CL
A Heavy chain has 4 domains. Name these
VH = variable heavy domain CH1 = constant heavy domain CH2 = constant heavy domain CH3 = constant heavy domain
A Light chain has 2 domains. Name these
VL = Light Variable Region CL = Light Constant Region
What is the variable region of antibodies formed of?
- Variable region = VL + VH
- Fv bind antigens specifically - binds 1 pathogen and not another
- Diff antibodies have diff variable regions
- 2 antibodies can recognise diff parts of the same pathogen
(-CH1 supports????)
Constant region
Constant region - same for all Ab of same class
- All IgM have μ (mu) heavy chain
- All IgG have γ (gamma) heavy chain
Effector functions (activating complement, binding phagocytes) CH1,2 & 3 are the constant heavy domains
Plays a part in the biological activity of the antibody
Is the same for all Abs of the same class
(Can be adapted to deal w diff infections e.g. small viruses, big parasites)
Which regions of the antibody make up the antigen-binding portion?
Fab = VL + CL + VH + CH1
Is diff b/w antibodies secreted from diff B-cells
All Ab chains are made of ……
amino acids
- upstream NH3+ group
- downstream COO- group
What holds together heavy and light chains in Ab?
disulphide bonds b/w cysteine aa residues in chains
at hinge region
What is the role of disulphide bonds in antibody structure?
Disulphide bonds:
- Hold together heavy and light chains
- Stabilise the domains (intramolecular)
What is the role of the antibody hinge region?
Hinge region = flexibility + movement
b/w CH1 & CH2
(Ab is not rigid)
CH2 domain has ………
carbohydrate glycosylations
= anchors for immune cell interactions
Fv fragment is made of ……..
Fab fragment is made of ………
Fc fragment is made of ……..
- Fv fragment = VH + VL
- Fab fragment = VL + CL + VH + CH1
- Fc fragment = CH2 + CH3 (domains of the heavy chain)
(A domain is a part of a single chain.
A fragment is composed of domains from each of the 2 chains - heavy and light chain - working together in concert)
What is the CDR?
Complementarity Determining Regions:
- On variable regions (VH + VL)
- Where antibody interacts with antigens(on pathogen/tumour cell surface)
- CDRs of heavy chain and light chain are different
-3 loops of Light chain: L1 , L2 , L3
-3 loops of Heavy chain: H1 , H2 , H3
like 3 fingers
What is the role of the CDR?
CDR binds to antigen (fingers and apple)
CDR3 = most variable
What are the 4 main functions of antibodies to combat pathogens?
- Opsonization
- Neutralisation
- Complement / MAC
- Apoptosis
What is opsonisation?
Opsonisation = Abs tag pathogens to make pathogens more visible to immune cells (macrophages & NK cells) = ↑ susceptible to phagocytosis(macrophage)/ADCC(NK cell)
Outline how antibodies cause opsonization
Opsonisation = Abs tag a pathogen to make it more visible to other immune cells (macrophages, NK cells) = ↑ susceptible to phagocytosis
→Fv (CDRs) binds pathogen antigen
→Fc binds to FcR on macrophage/NK cell
→Macrophage = antibody-dependent cellular phagocytosis (ADCP) = engulf smaller pathogens
→NK cells = antibody-dependent cellular cytotoxicity (ADCC) = lyses target cell (perforin, granzymes, granulysin)
How do antibodies aid neutralisation of toxins?
Fv binds competitively to viral docking sites on cells / toxin active sites + neutralises.
= Prevents pathogen entry into host cell.
How do antibody immune complexes fight pathogens?
Form immune complexes (composed of antibodies + pathogens) that agglutinate + are removed by other immune cells
How do complement proteins aid immune response?
The immune complex can involve complement molecules (C1q, C1s and C1r) that promote inflammation, phagocytosis and MAC formation (damages membrane, causing cell lysis)
MAC = Membrane Attack Complexes
How many antibody classes are there?
5 different Ab classes with diff functions:
IgG IgA IgM IgE IgD
What is the difference between each antibody class?
Fc - Each antibody class expresses a different heavy chain constant region (same Fc for all Abs in the same class)
But the light chain and heavy chain variable regions remain the same for antibodies produced in the same B cell
(1 B-cell produces the same variable region - VL + VH)
What is the IgG antibody structure?
IgG has the classical canonical structure with 4 domains in a gamma chain
Describe the IgD structure
IgD delta chain has a longer hinge region
What is IgE structure like?
IgE epsilon chain has 5 domains
What enables IgA and IgM to polymerise?
The alpha and mu chains in IgA and IgM are similar to IgG but they have tail pieces at the end of CH3 for J chains to join. to facilitate polymerisation
Describe the structure of IgA (secretory)
Secretory IgA = 2 monomeric IgA joined by a J chain. Secretory component wraps around it, enabling IgA to be secreted into the mucus → good for respiratory infections
Outline the structure of IgM
IgM is composed of 5 monomeric IgG structures joined together by a J chain
How is antibody class determined?
The heavy chain+light chain variable regions are fixed by VDJ recombination
Which is the heaviest Antibody?
IgM has highest mw = pentamer
And IgA is also larger = dimer
Which is the main Ig in serum?
IgG is the main antibody in serum, followed by IgA
Which antibodies are able to fix complements?
Only IgM and IgG fix complements
Which antibody is able to provide immunity to foetus?
Only IgG can cross the placenta to provide immunity to the foetus
What is the role of heavy chain class switching?
Provides different effector functions to deal with different types of pathogens
Only affects Heavy chain constant region
What are the 2 types of Heavy chain class switching?
Minor class switch: differential splicing (mRNA level)
- IgM and IgD
- Doesn’t affect B cell DNA
Major class switch: DNA recombination
- IgM to IgG, IgA , IgE
- IgG to IgA, IgE
What causes class switching to occur?
Class switching occurs due to B-cell detecting cytokines(chemical signalling) released by T helper cells
Cytokines indicate which pathogens are present
What is the main signal to initiate class switching?
CD40L on T cell interacts with CD40 on B cells and cytokine signalling
Name the mechanism for major class switching (DNA)
CSR - class switch recombination
Outline how class switch recombination occurs
1) Cytokine signal
2) Switch regions
3) AID and DSB repair proteins
AID + other enzymes aid recombination b/w switch regions
What is the rule of Class Switching Recombination?
Switching only proceeds downstream
- IgM to IgG, IgA, IgE
- IgG to IgA, IgE
Once switched to IgG, cannot revert back to IgM bc IgM is upstream of IgG
How do heavy chain segments recombine?
Heavy chain gene loci undergo VDJ recombination and affinity maturation
Why can’t antibodies revert back to previous IgM class after recombination?
Switching only proceeds downstream.
Once switched to IgG, cannot revert back to IgM bc IgM is upstream of IgG.
Once switched to IgG, all the segments before will be removed ∴can’t revert back to IgM
What happens to the spliced antibody regions?
The segments cleaved out form a switch DNA circle once spliced
What are the 2 types of antibodies?
2 types of antibodies:
- membrane bound (BCR, B-Cell Receptor)
- Secreted form
What is the secreted form of antibody?
The secreted form is the final, fully functional form of the antibody secreted by mature plasma cells
Why are antibodies anchored to B cells before secretion?
Before it’s secreted, the antibody is anchored on B cells for weapon development
How do secreted and membrane bound antibody structure differ?
Secreted IgG has a tail piece, while the Membrane bound antibody has a transmembrane region+cytoplasmic tail(anchor)
Outline the components of the constant mu region before differential splicing occurs
Constant region of Cμ composed of μ1-4 + tail piece + stop codon + polyA tail followed by M1 and M2
There is also a second PolyA tail and stop codon
What do M1 and M2 gene segments code for?
M1 and M2 code for transmembrane region and the cytoplasmic tail
How are membrane-bound antibodies converted to secreted form?
Whole Cμ gene region is transcribed into mRNA and introns are spliced out to form mRNA for secreted antibodies
-Transcribe Cμ gene region to mRNA
-Splice out introns
= mRNA for secreted Ab
How are membrane-bound antibodies produced?
Whole Cμ region (upto second polyA tail) is transcribed and 8 regions including genes encoding tailpiece and stop signal will be spliced out
- Transcribe Cμ gene region
- Splice out genes encoding tailpiece + stop signal (keep genes encoding hydrophobic transmembrane region + cytoplasmic tail)
What is somatic recombination?
Somatic recombination = Alterating genetic information at the DNA level.
Involves processes: • V(D)J recombination • Tdt nucleotide addition • Somatic hypermutation • Class switching
Once these processes have occurred, they are irreversible - coded into B-cell DNA
Outline the different methods of somatic recombination
- V(D)J recombination
- Tdt nucleotide addition
- Somatic hypermutation
- Class switching
What is differential splicing?
Differential/alternative splicing: Alteration of genetic information at mRNA level
2 identical mRNA copies in a B cell will be altered differently to produce 2 different protein products
-e.g. IgM and IgD
Changes made to mRNA do not affect the original DNA in the B-cell
What are the products of differential splicing?
- IgM and IgD
- Membrane bound Ig
- Secreted Ig
What are the 2 stages of B cell development?
There are 2 stages to B cell development:
- Antigen-independent
- Antigen-dependent
Where do B cells originate?
B cells originate in bone marrow as stem cells that slowly differentiate into pro-B cells
BM:
Lymphoid progenitor stem cell -> Pro-B cell -> Pre-B cell -> Immature B cell
How is the heavy chain variable region coded for?
The Pro-B cell DNA undergoes D→J and V→DJ recombination to permanently code in the heavy chain variable region
What is the default Heavy chain constant region expressed by the B-cell?
The variable region will be expressed with the μ heavy chain (VH + μ chain) = the default first heavy chain expressed by B cells
What is a Pre-B cell?
Pre-B cell = B cell expressing a heavy chain (default μ heavy chain)
How is the light chain variable region coded for?
Pre-B cells undergo another V→J recombination to permanently code in the light chain variable + constant regions to become immature B cells(that express IgM and will mature overtime)
What are immature B cells?
Immature B cells express IgM and mature overtime
When do B cells become mature?
Once the B cells can express IgM and IgD (through differential splicing of mRNA) they become mature B cells and circulate between bloodstream, spleen and lymph nodes.
They then start their patrol, resting until they encounter a pathogen.
What is affinity maturation?
Affinity maturation = Fine-tuning antibody affinity to antigen:
- Body encounters a foreign pathogen = activates B-cells.
- B cells hone their ability to bind to a pathogen by affinity maturation in the germinal centre (GC) - only best survive.
- B-cells then receive information about what type of pathogen they are dealing with, and undergo class switching to the appropriate effector regions = B-cell R becomes IgG/IgA
What do majority B cells develop into?
Majority B cells further develop into Plasma cells that secrete the antibodies they encode
What is B cell first line defence?
As first line defence, some B cells expressing IgM differentiate into Plasma cells and enter circulation.
Secrete IgM as first defence.
What happens to B-cells after infection?
Some B-cells remain as memory B-cells = body remembers + is ready to fight off the same infection again
Describe the diversity of antibodies in the body
Body makes 1bn resting B cells that patrol our body
Each contains a unique ‘random’ BCR
(resting B-cell = mature naive B-cell)
How do B cells differentiate to provide so much diversity?
To generate 1bn resting B cells, lymphoid progenitor stem cells differentiate into Pro-B cells
(resting B-cell = mature naive B-cell)
What is the first recombination Pro-B cells undergo?
- Pro-B cell undergoes D → J recombination in DNA
- V segment then recombines with DJ segment (V → DJ) = hard-codes in the heavy chain variable region
This is then expressed with the μ (default) constant region.
Why can B cells not class switch during development?
VDJ is expressed with μ (default) constant region.
B cell can’t change class at this stage as pathogen type is unknown.
When does a Pro-B cell become a Pre-B cell?
Pro-B Cell becomes Pre-B cell when it can express a full heavy chain with a unique variable region
Describe the second recombination for the light chain region?
- Pre-B cell expresses a light chain placeholder that forms a pseudo-antibody with the heavy chain
- Pre-B cell undergoes another V→J recombination on its DNA to determine light chain (variable + constant regions)
How is additional diversity created among antibodies?
Additional diversity is generated via Junctional Flexibility and P + N nucleotide addition. During V->DJ recombination (heavy chain) and V->J recombination (light chain).
Random mechanisms.
Describe the structure of immature B cells
Immature B cell expresses full IgM(with the mu heavy chain) and either kappa / lambda light chain
When does an immature B cell become mature?
Becomes mature B cell (Naive B cells / resting B cells) when it has the capacity to produce both IgM and IgD through differential splicing of mRNA - quality control
Resting B-cell = Mature Naive B-cell
VDJ recombination generates diversity in the ………..
VJ recombination generates diversity in the ………..
VDJ recombination generates diversity in the heavy chain
VJ recombination generates diversity in the light chain
What is the role of V(D)J recombination?
No complete genes for Abs are inherited, only gene segments.
Arranging these gene segments in different combinations generates many Ig sequences
What gene segments are the light / heavy chains composed of?
The light and heavy chain gene loci are made up of different gene segments;
- Light chain gene segments: V, J and C
- Heavy chain gene segments: V, D, J and Cμ
The gene segments undergo recombination
Outline how the light chain segments recombine
Light chain: 1 V gene segment is randomly chosen to recombine with 1 J segment
Outline how heavy chain gene segments recombine
Heavy chain: Random VDJ segments recombine
VDJ segment encodes ……….
C segment encodes ………..
VDJ segment encodes the light/heavy chain variable fragment (Fv)
C segment encodes the constant domain (Fc)
What do C segments encode?
C segments encode constant domains (Fc)
What do J or D/J segments encode?
J or D/J segments code for CDR3
= 3rd finger protrusion that interacts w the antigen
What are the 3 genetic loci encoding for Ig?
2 for light chain:
- Kappa (κ) chr.2
- Lambda (λ) chr.22
1 for heavy chain chr.14
Located on different chromosomes
The VJ (light chain) and VDJ (heavy chain) genes are located in diff. parts of the human chr.
What is the simplest recombination?
V/J recombination on kappa light chain genes (chr 2)
What are the different possibilities of VJ recombination?
V segments far from J segments, but J are relatively close to C segments
- 40 Variable (V) segments
- 5 Joining (J) segments
- Constant region (C) segment
What is the role of the leader sequences?
Leader sequence in front of each V segment - directs protein to target
How are VJ segments recombined?
V + J segments are randomly chosen to form L, VJ, J, C segment ⇒ transcribed into mRNA
What happens to the extra J segment from VJ recombination?
- Extra J segment spliced out → mature mRNA (that contains L, V, J, C)
- mRNA is then translated into a.a sequences of light chain
L = Leader segment
How is the final kappa light chain formed?
A.a. is then folded and leader sequence is cleaved off once the protein reaches target ⇒ unique kappa light chain
How many different VDJ recombination segments are there?
- 51 Variable (V) segments
- 27 Diversity (D) segments
- 6 Joining (J) segments
- Constant region (C) segments
Outline the VDJ recombination that occurs on the heavy chain
First C regions are Cμ(codes for IgM heavy chain) and Cẟ(codes for IgD heavy chain)
∴ first recombination is D → J joining (randomly selected)
Random V segment is then joined
What happens to the VDJ recombined gene segments?
Recombined hard coded DNA is transcribed into mRNA transcripts
Outline the result of differential splicing of heavy chain gene segments
Section containing the extra J segment + Cμ/Cẟ is spliced out.
Differential splicing of mRNA occurs resulting in expression of either:
- LVDJCμ = IgM (extra J segment + Cẟ)
- LVDJCẟ = IgD (spliced out extra J segment + Cμ)
What signals initiate recombination?
Recombination signal sequences (RSS) required – conserved sequences upstream/downstream of VDJ gene segments
What are RSS made up of?
RSS are made of ‘Turns’ containing a heptamer and nonamer with a 12/23 bp spacer
Heptamer = 7bp DNA sequence, the same sequence in all turns Nonamer = 9bp DNA sequence
What are the diff types of turns?
1-turn = contains a 12bp spacer 2-turn = contains a 23bp spacer
What is the 1 turn/2 turn rule of recombination ~(aka 12/23 rule)?
Why do we have 2 types of turns?
Recombination only occurs between a segment with a 12bp spacer and a 23bp spacer
One-turn can recombine with two-turn (not one-turn and one-turn or two-turn and two-turn)
Prevents accidental recombination
Where are two-turns located?
Two-turns located:
- downstream of V segments of heavy and lambda light chains
- upstream of heavy chain and kappa chain J segments
Where are one-turns located?
One-turns located:
- Both sides of heavy chain D segments
- Upstream of lambda light chain J segment
- Downstream of kappa V segment
What are the different mechanisms of antibody diversity generation?
- Multiple germline V, D and J gene segments
- Combination V-J and V-D-J joining
- Junctional flexibility
- P-nucleotide addition
- N-nucleotide addition
- Combinatorial association of heavy and light chains = 5 heavy chain classes (GAMED), 2 light chain classes (kappa, lambda). combine these in diff ways to give diff combinations e.g. gamma lambda, gamma kappa, alpha kappa, alpha lambda etc.
- Somatic hypermutation during affinity maturation
What are autoantibodies?
Abs that recognise host’s own cells and cause autoimmune diseases
Abs are selected out by negative selection
What is junctional diversity?
Removal of nucleotides between gene segments v(D)J recombination
Junctional diversity is produced by Junctional Flexibility during V(D)J recombination, P and N nucleotide additions
Evaluate junctional diversity
Good: Antibody diversity
Bad: Also Generates Non-productive rearrangements (incorrect reading frame) – wasteful process
Outline the process of junctional diversity
- RAG1 / RAG2 enzymes bind to V/J 1-turn/2-turns
- Pulls segments together to form (major) hairpin
- DNA is nicked to form (minor) hairpin at end of gene
segments - Enzymes repair and process segment ends
- Forms:
- Coding joint of V+J segment
- Signal joint of turns + excess DNA b/w the 2 joining gene segments
Describe the different hairpins formed from junctional diversity?
Minor hairpin: between 2 strands of DNA
Major hairpin: whole DNA folded in half
How do the hairpins open and join?
RAG1/2 processing forms hairpins
Hairpin opened via Artemis
Exonucleases, TdT mess around with free DNA ends (add nucleotides)
Ends joined by another series of enzymes
How does Artemis enzyme open the hairpin?
Artemis randomly nicks 1 end of dsDNA
Nicked ends linearise to form overhanging ends
The DNA ends are processed by repair enzymes (exonucleases, TdT) these enzymes +/- free nucleotides to heavy chain.
The ends are ligated/rejoined together by enzymes
When does P nucleotide addition occur?
Repair enzymes fill overhang gaps from Artemis enzyme → P nucleotide addition
When are N nucleotide additions made?
Terminal deoxynucleotidyl Transferase (Tdt) : adds N nucleotides
Mostly in heavy chain
What are the effects of P+N nucleotide additions?
P+N nucleotide addition causes frameshifts, leading to new antibody formation regardless of previous V(D)J recombination selections
even if 2 B-cells select the same VDJ segments, still have diff Ab due to P+N additions
What is junctional flexibility?
Removal of nucleotides between gene segments during V(D)J recombination
Involves exonucleases
How does junctional flexibility cause frameshfits?
Artemis creates overhanging ends, causing mismatch nucleotides that need removing via exonucleases before repair enzyme can work - exonucleases can overtrim the ends, causing additions/deletions (frameshifts)
The joining of the signal joints is always …….
precise
Deletions by exonucleases causes aa sequence deletion = frameshifts = generates diff. antibody variable regions by diff B-cell lines
How do antibody genes differ from normal?
Allelic exclusion:
- Abs genes - Only 1 heavy chain allele and 1 light chain allele is expressed
- Apoptosis of unsuccessful heavy/light chain recombinations
- Ensures each B cell produces only 1 type of antibody
What is the role of allelic exclusion?
Ensure each B cell produces only 1 type of antibody
Activated B-cell vs Plasma cell
Activated B-cell = Membrane-bound Ab
Plasma cell = Secreted Ab
