Humoral Immunity; Antibodies and the life cycle of B cells Flashcards
Define antibody
Antibodies – Class of proteins called Immunoglobulins that Directly Bind to Specific Antigen. They are produced by Plasma Cells (Activated B-Cells) in response to infection/immunisation.
Describe the structure of an antibody
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What is the nomenclature of antibodies?
• Fab = Antigen Binding Site
• FC = Constant Region
• VH = Variable Region of a Heavy Chain
• VL = Variable Region of a Light Chain
CH = Constant Region of a Heavy Chain
• CL = Constant Region of a Light Chain
• CDR = Complementarity Determining Region
What is the CDR?
- These are 3 finger like protrusions in the variable region and they are the ones interacting with the antigen
- They are located in the variable light and heavy regions
What are the 4 functions of antibodies?
They bind specifically to their corresponding antigens, leading to:
- Neutralisation of Pathogens/Toxins -> Phagocytosed.
- Opsinisation of Pathogen -> Marks them for destruction by Phagocytes.
- Activation of Complement -> Lysis of Extracellular Bacteria -> Phagocytosed.
- Ab-Dependent Cellular Cytoxicity (ADCC) -> Lysis of a Target Cell that has been bound by Specific Antibodies.
a. NK-Cells -> Lysis of a Pathogen-Infected Cell.
b. Eosinophils (Via IgE) -> Kills Parasites that are too big for phagocytosis
What heavy chain does IgM/D/G/A/E have?
What is the function of them
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What is heavy chain class switching?
- This only affects the heavy chain constant region
* It produces different effector functions to deal with different pathogens
What are the two types of heavy chain class switching?
- Minor (does not affect DNA of the B-cell): this occurs between IgM and IgD at the Mrna level
- Major: DNA recombination: IgM ->G/A/E and IgG ->A/E
How does the B-cell know what class to switch to?
They do this by sensing chemicals around released from the T-cells
What does Major class switching (class switching recombination) require?
- Cytokine signals
- Switch regions
- AID and DSB repair proteins
Compare and contrast membrane vs secreted Ig
Same constant and variable chains but secreted ig has tail piece, whilst membrane IgG has a transmembrane cytoplasmic tail.
How do we code for either a secreted or membrane Ig?
- The DNA has genes (for example for IgM), which are u1,2,3,4,5 and then a polyA tail and a stop codon.
- Then M1 and M2- transmembrane region and cytoplasmic tail.
- Then a stop codon and a polyA tail.
- For secretory Ig- this will be transcribed into mRNA (up to first stop codon) and splicing too.
- For membrane bound Ig, the whole region up to the second polyA tail will be transcribed into RNA.
- 8 regions, including genes encoding tail piece and stop signal, will be spliced out.
What is somatic recombination and give some examples?
• Somatic occurs at the DNA level
Examples: • V(D)J recombination • Tdt nucleotide addition • Somatic hypermutation • Class switching • Any changed at the DNA level during somatic recombination, it will affect mRNA and the protein
What is differential splicing and give some examples?
• Differential splicing occurs at the Mrna level
Examples:
• Is the formation of IgM and IgD
• Membrane bound and secreted Ig
Describe the life cycle of the B-cell (independent)
- Stem cell in bone marrow differentiates to pro-B cell.
- DNA in pro-b undergoes D-J and V-DJ recombination to permanently code in heavy chain variable region.
- Variable region is expressed with mu, default first heavy chain expressed by b cell. Called Pre-B cell- only heavy chain but placeholder light chain (psuedoantibody).
- V-J recombination to code in (k or l) light chain and constant to become immature B cell. This will express IgM and will mature over time. During the V(D)J recombination, can also give additional diversity through junctional flexibility and P/N nucleotide addition. Though random, can produce millions of unique B cells.
- Can also express IgD through differential splicing. Once both expressed- it is mature and can circulate in blood stream, spleen and lymph nodes. Naïve b cell.
Describe the life cycle of the B-cell (dependant)
- Activation- B cell will complete ability maturation in germinal centre (GC). Only most suited to pathogen will survive.
- B cell will receive information about the type of pathogen and undergo class switching to the appropriate effector regions. BCR becomes IgG or IgA.
- Majority of B cell will further develop onto plasma cells, secreting more Ig.
- Some B cells coding for IgM will differentiate into plasma cells secreting IgM as first defence.
- After infection, some B cells remain as memory B cells.
What is VDJ and VJ recombination for variable fragment diversity?
How is this done in the light and heavy chains?
What does the J gene code for?
Create diversity in the heavy chain and light chain variable regions.
• Complete Antibody genes are not inherited, only gene segments are.
• Arranging these gene segments in different combinations generate many Ig sequences
Light chain:
• Consists of VJC segments
• The gene segments under recombination (one V segment is selected along with one J segment)
• The DNA will now encode a complete antibody with the V/J AND VDJ segments encoding for the light and heavy chain variables
• THE C SEGMENTS CODE FOR THE CONSTANT DOMAINS
Heavy chain:
• Consists of VDJC segments
• The gene segments under recombination (one V segment is selected along with one J segment and d segment)
• The DNA will now encode a complete antibody with the V/J AND VDJ segments encoding for the light and heavy chain variables
• THE C SEGMENTS CODE FOR THE CONSTANT DOMAINS
THE J GENE CODES FOR CDR3 – INTERACTS WITH ANTIGEN
Describe VJ recombination of kappa light chain chromosome 2
Describe VDJ recombination of gamma heavy chain genes chromosome 14
Gene consists of:
• 40 V segments (30 in lambda).
• 5 J segments (4 in lambda).
• C segment.
V and J segments farther from each other than J and C.
- In front of each V segment is a leader sequence that tell cell where protein will end up.
- V and J segments are randomly chosen to form a leader and an extra j segment and C are added.
- Transcribed and spliced (extra J removed, as well as other parts) to form mature RNA, with only leader, V, J and C with poly-A tail.
- Translation into amino acid chain and then folded into protein.
- Leader cleaved off once protein reached its destination.
- Lambda chain recombination is similar but more complex.
Genes consist of: • 51 V segment. • 27 diversity (D) segments. • 6 J segments. • Constant region.
- First recombination is D to J joining.
- V segment is also be joined.
- Transcription and differential splicing.
- Begin with leading sequence, then VDJ and Cu.
- Eventually translated into IgM heavy chain.
- Or begin with leading sequence, then VDJ and Cd.
- Eventually translated into IgD heavy chain, signalling maturity
Describe the mechanism of VDJ recombination
- Recombination signal sequences (RSS) are conserved sequences up/downstream of gene segments.
- RSS made up of turns consisting of a heptamer (conserved 7 base pair DNA sequence- always the same) and then a 12 or 23 bp spacer and then a nonamer (conserved 9 base pair DNA sequence).
- 2 types of turns: two turn (23 spacer) and one turn (12 spacer).
- DNA recombination only occurs between segment with a 12bp spacer or a 23bp spacer. One-turn/two-turn or 12/23 rule.
- 2 turns are downstream of the V segment and upstream of J segment in both the heavy and light chain.
- The 1 turns are on both sides of the heavy chain D segments, upstream of the lambda light chain J segment and downstream of the kappa light chain V segment.
- Heavy chain- 12/23 or one-turn/two-turn join between D and J and then D and V, preventing other types of recombinations. Same principle for the light chains.
- RAG1 and RAG2 enzymes bind to the turns and pulls them together to form a major hairpin.
- DNA is then cleaved at both RSS turns and retains hairpin shape at ends of gene segment. This is the minor hair pin.
- Many enzymes repair and process joints- forming coding joint with the V and J next to each other in light chain or D and J next to each other in heavy chain.
- Signal joint produced with turns and other DNA between the gene segments.
What is Combinatorial diversity?
What other mechanisms produce diversity?
- How many possible combinations from heavy chain V,D,J and light chain V,J?
- Heavy chain= V(51), D (27) and J (6)= 51 x 27 x 6= 8262.
- Light chain kappa= V (40) and J (5)= 40 x 5= 200.
- Together= 8262 x 200 x 120= 198,288,000= 1.98 x 108 possible combinations.
- Need 1 billion but this is less than 200 million so therefore there are additional mechanisms to generate diversity.
- 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 Somatic hypermutation during affinity maturation (next lecture)
Why is junctional diversity good and bad?
Describe the mechanism for it?
- Through junctional flexibility during VDJ recombination, P and N nucleotide additions.
- Good= antibody diversity.
- Bad= non-productive rearrangements (incorrect reading frame due to loss of stop codon)- which makes this a wasteful process.
Mechanism:
• Minor hairpin- opened by enzyme Artemis. Artemis cleaves one strand of the double stranded DNA randomly. Nicked ends will linearize forming overhanging ends.
• Repair enzyme will fill in gaps leading to P-nucleotide addition.
• Terminal deoxynucleotidyl transferase (tDt) then adds N-nucleotides between the two ends before the ends are ligated together again.
• N-nucleotide addition occurs almost exclusively in heavy chain.
• Addition of P and N-nucleotides before joining the segments together causes addition of amino acids which may shift reading frame- this produces even more diversity.
What is junctional flexibility?
What enzyme does it require?
- Removal of nucleotides between gene segments during V(D)J recombination.
- Involves exonucleases.
- Once Artemis cleaves strand, there are overhanging ends.
- If there is enough complementarity, these ends can overlap (between the two DNA segments).
- There will be mismatches that will be removed by exonucleases before repair enzyme can work.
- Exonuclease can sometimes over-trim the end, removing a number of nucleotides of varying amounts. Even if same V and J segments are chosen for a kappa light chain, this process will still create diversity as different lengths will be removed, leading to changes in amino acids, in frameshift so brand-new unique proteins produced.
- Joining of signal joints (RSS/RSS) is always precise.
What is Allelic exclusion?
- Two copies of Ig gene (one from each parent).
- Only one heavy chain allele and one light chain kappa and light chain lambda are expressed. ½ of the possible ones.
- Order of loci rearrangement- heavy chain first allele (if successful move on), then kappa, then lambda. If 1st chain does not produce a functional chain, the 2nd allele is used.
- These ensures that each B cell only makes one type of antibody.
Describe what happens when the body encounters a pathogen
- When the body encounters a pathogen a subset of naïve B-cells will become activated.
- The B-cells will then make clones of itself through clonal expansion
- Some of these clones will secrete IgM to initially fight the pathogen
- The rest of the B-cells will migrate to the lymph nodes to wait for T-cell activation
- The b-cells will engulf the pathogen and present the antigen to the T helper via the MHC 2 receptor
- Dendritic cells will also engulf and present to the T-helper cells
- CD40/L allows the B-cell to recognise the T-cell, cytokines are also produced by the T-helper cell.
- The B-cells will now undergo affinity maturation and class switching, and differentiate into plasma cells where they secrete antibodies.
Describe Clonal expansion and affinity maturation
Naïve B-cells (not exposed to antigen) has a unique receptor, the b-cell which has the best binding affinity will be selected for, it will proliferate.
Affinity maturation will then occur:
• This aims to improve the affinity of the antibody for the antigen
• Mutations in the V segment allow this so the antibody binds better.
• This process occurs in the germinal centre of the lymph node of the GC
• T-follicular helper cells (T-cells that can only enter the GC) and follicular dendritic cells (FDC) assist in affinity maturation
