Humoral Immunity; Antibodies and the life cycle of B cells Flashcards
State the types of antibodies?
- Membrane bound (B-cell receptor BC) OR secreted antibody
- Secreted antibody is released by plasma cells
- Membrane bound / BCR is found on activated B cells
Describe the difference between membrane and secreted Ig in terms of its structure?
- Both have same constant and variable region (if same clas)
- Difference between secreted + membrane Ig structure
- Secreted Ig -> Tail piece
- Membrane Ig -> Hydrophobic transmembrane region + Cytoplasmic tail
Describe the complete structure of an antibody in terms of general bonding?
- General bonding:
- String of AA ( NH3+ + COO- ends) -> Chains joined via disulphide bonds
Describe the complete structure of an antibody in terms of regions
- Regions/fragments:
- Variable region: Different -> Contains complementarity determining regions/ antigen binding site (Fab) (VH + VL)
- Constant region -> biological activity + Same for all Ab of same class
• Hinge -> flexibility
• Glycosylation (CHO bond) on CH2 domain -> interaction to other immune cells
Describe the complete structure of an antibody in terms of chains?
- Chains:
- Light (2 chains/4 domains): k or \ chain
- Heavy (2 chains 4 domains):
- u,s,v, a or & chain (9 different HCs) (VD)
- y1, y2, y3, y4
- a1, a2
State the major antibody effector functions once antigen binded? (3) (PART 1)
- Virus & toxin neutralisation: Prevents pathogen-host binding
- Opsonization (tagging of pathogen -> increased visibility for immunity)
a. ADCP (antibody-dependent cellular phagocytosis)
I. Recruits Macrophages via FC binding on M
II. Phagocytosis -> smaller pathogens
State the major antibody effector functions once antigen binded? (3) (PART 2)
b. ADCC (antibody-dependent cellular cytotoxicity)
I. Recruits NK cells
II. NK-induced apoptosis -> infected or cancerous cells
3. Complement fixing/ MAC formation (CDC)
a. MAC = Membrane attack complex -> complement-dependent cytotoxicity (CDC)
b. Binds to complements (c1q, c1s, c1r) -> Causes phagocytosis or lysis
State the 5 different classes of antibodies describing its differences
- IgG: Main AB of secondary response
- IgD: Indicates mature B cells: only AB not secreted
- IgE: Allergy/ anti-parasites
- IgA: Secreted into mucous, tears, saliva
- IgM: Main AB of primary response
- All differ in HC constant region + function
- IgA and IgM both have J chains, but IgA has secretory components
- BCR = B-cell receptor
Describe heavy chain class switching, its purpose and the 2 types?
- Only affects heavy chain CONSTANT region
- Allows for Different effector functions - deal with different pathogens
- Minor: Differential splicing (mRNA level) -> Doesn’t affect DNA: IgM and IgD (last lecture)
- Major: DNA recombination: IgM to IgG, IgA, IgE. IgG to IgA, IgE
Describe the two factors required for class switching to occur?
- CD40L on T cell interacts with CD40 on B cells + cytokine signalling -> signals what Ig to convert to depending on what cytokines are signalled
- VD
Describe the mechanism behind major recombination of class switching
- Known as class switch recombination
- Factors required: 1. Cytokine signal 2. Switch regions 3. AID and DSB (double strand break) repair proteins
- Recombination occurs between switch regions
- Switching only proceeds downstream: IgM to IgG, IgA, IgE. IgG to IgA, Ig
Provide a summary for this section
- Antibodies - secreted by B cells to neutralize pathogens; B-cell receptor
- Structure - 2 HC, 2LC: domain vs fragment. membrane-bound vs secreted
- VH and VL CDRs bind to antigen
- 5 classes of antibodies - different effector functions to deal with different pathogens
- Class switching: Heavy chain constant region change, the rest stays the same
Life cycle of B cells
VD
Generation of B cells (life cycle)
What’s the Difference between somatic recombination vs differential splicing?
- Somatic recombination: Alteration of DNA level -> irreversible once coded -> e.g. V(D)J recombination, Tdt nucleotide addition, Somatic hypermutation + Class switching
- Differential splicing -> Changes at mRNA level -> E.g. IgM and IgD, Membrane bound + secreted Ig
Describe key principle behind how unique antibodies are formed?
Body -> formation of many resting B cells -> each with unique, random BCR
Antigen-independent phase of B cell life cycle
Describe the antigen-independent phase within the life cycle of B cells?
Bone marrow:
- Stem cell -> Pro-B cell -> Pre- B cell -> immature B cell - mature/resting/naive recirculating B cell (IgM OR IgD)
- Pro-B to Pre-B -> 1) D-J recom. 2) V-DJ recom. -> codes HCVR + u constant R
- Pre-B to immature -> placeholder LC expressed + V-J recom. -> LCV + constant
- Immature-mature -> Additional diversity mechanism -> junctional flexibility + P & N nucleotide addition ->random + generates unique B cells
- Mature -> Quality control -> IgM or ID production via differential splicing
V(D)J recombination
What is inherited in terms of antibody genes and what does it generate?
- Only Gene segments -> arranged in different combinations -> different lg generated
Describe what happens in V(D)J recombination?
- LC (VJC) + HC (VDJC(u)) -> specific segments chosen
- LC - VJ recombination -> variable region
- HC - VDJ recombination -> VR
- J or D/J -> codes CDR3 (most variable region)
State the 3 genetic loci + chromosome that codes for lg
- LC -> kappa (к/C2) + lambda (\/C22) -> VJ
- HC -> C14 -> VDJ
Describe VJ recom. of kappa light chain gene + number of segments?
- 40 Variable (V), 5 Joining (J) , Constant region (C)
- Germline k-chain DNA -> random segment (VJ) chosen -> Rearranged K-chain -> transcription -> Splicing -> translation
Describe VDJ recom. of heavy chain gene + number of segments? VD
51 Variable (V), 27 Diversity (D), 6 Joining (J), Constant region (C)
What are recombination signal sequences and state the 2 types?
- RSS: Conserved sequences upstream or downstream of gene segments
- Types -> Turn -> heptamer + nonamer + spacer
- One turn -> 12 op S
- Two turn -> 23 bp S
Describe the one-turn/two-turn rule in V(D)J recombination?
Recombination: ONLY between one turn + two turn -> Prevents D/V/J recombination
Describe the complete mechanism behind V (D) J recombination? VD
- Diversity generated after minor hairpin formation
- Enzymes bind turn + pull together -> Major hairpin formed (whole DNA folded in half) -> cleavage at hairpin site (random) via artemis -> Minor hairpin formed (between 2 strands) with overhanging ends -> exonuclease + TdT repair & joining via add or removing nucleotides -> P nucleotides addition to overhang -> N/TDT nucleotide addition via terminal deoxynucleotidiyl transferase (Tdt)
(mostly HC) -> coding + signal joint formed
State 7 mechanisms involved in the generation of antibody 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 HC + LC, Somatic -
- Hypermutation during affinity maturation
Describe the mechanisms involved within junctional diversity stating good + bad points?
- Junctional flexibility during V(D)J recombination, P + N nucleotide additions
- Good: Antibody diversity
- Bad: Non-productive rearrangements (incorrect reading frame) - wasteful process
Describe junctional diversity?
- Removal of mismatched nucleotides via exonuclease -> between gene segments during V(D)J recombination -> Precise mechanisms unknown -> Signal joint is precise (no removal)
- Happens at the end of VDJ after P and N nucleotide addition
Describe allelic exclusion?
Two copies of Ig (mum + dad) -> In cells, both genes expressed - AB gene is different: Only 1 HC + LC allele expressed
- Antibody exclusion is where B cell make one type of Antibody
- Order of rearrangement -> Heavy > kappa> lambda -> Each one - 1st allele then 2nd -> if recombination is not successful -> cell becomes apoptosed
Summary
- Life cycle of B cell - antigen-independent phase (bone marrow)
- B cell receptor diversity at this stage generated by:
- Multiple germline genes
- Heavy and light chain combinations
- V (D)J recombination (one-turn/two-turn rule)
- Additional diversity: P and N nucleotide addition; Junctional flexibility
- Mature, naive B cells expresses IgM and IgD
- Somatic recombination vs differential splicing
- Allelic exclusion
Mature B cell -> spleen/lymph node -> activated B cell (via binding pathogen + Th cell) -> Affinity maturation (GC) : 1. clonal expansion 2. Somatic hypermutation 3. Selection -> cycle occurs several times -> class switching (app. effector functions) -> plasma cells (some move into blood)
State the processes involved within B cell activation?
1) T-cell independent -> Pathogen encountered -> diff. + clonal expansion of activated B cells - Same time T cell activated
2) T-cell dependent -> 3 signals required -> Antigen binding to BCRs, Co-stimulation by activated Th cell specific to same antigen, Th cell-derived cytokines production
- signal transduction pathway -> BC binding -> Tyrosine kinase activation
- cell pro., diff. + survival
Describe the purpose of clonal expansion
Pathogen attacks B cell -> combines + activates -> B cell cloned -> affinity maturation
What is affinity maturation and state key features?
- Improves affinity of AB to antigen -> somatic mutations in lg V genes -> selection of high-affinity B cells (AB) for specific antigen
- Site of AM -> Germinal centre in Lymph node -> Aided via T follicular helper cells (only T cells that can enter GC) + Follicular dendritic cells
Describe how affinity maturation occurs?
- GC -> Dark zone + light zone
- Dark zone -> clonal expansion -> AID (Activation-induced cvtidine deaminase) causes somatic hypermutation: point mutations in DNA of B cells -> Light zone -> Selection -> Increased affinity or decreased affinity pathway
- Increased Affinity -> FDC presents antigen -> AB compete for limited antigens -> Successful AB present antigen to TfH cell -> survival sign to AB
- Decreased affinity -> cells apoptose
- B cells survive whole cycle -> enter dark zone -> Repeat cycle until AB affinity is increased
State the relationship between AB affinity and cycles of affinity maturation?
Increased cycles -> Increased AB (present) affinity to antigen
