lymphocyte + antibodies

where are antibodies found (3 places)

serum, intestinal fluids and mucosal secretions

what cells produce antibodies

differentiated plasma cells

antibody structure: how many chains and regions?

4 chains, 2 light chains and 2 heavy chains and 2 regions, variable and constant

How are heavy and light chains bound?

How are the two heavy chains bound?

by disulphide bonds in each dimer

by disulphide bonds in each tetramer

why is constant region so important?

is the class/isotype of every Ab

ironically, the constant region is the one that is always different

where does B cell development occur?

bone marrow, where it matures from progenitive b cells

3 steps in b cell life

development, activation, differentiation

where does activation and differentiation of b cells occur?

periphery and secondary lymphoid tissues such as the spleen and lymph nodes

after a b-cell has developed in the bone marrow, how does it get to the periphery and 2ndary lymphoid tissues?

periphery circulation

LONG QS: to make an antibody, what 4 steps do B cells need to do?

1. recognise antigen via immunoglobulins on surface (SMIg) surface membrane Ig

2. break down the antigen and re-present it to T-cells as peptides MHCII on the surface

3. T cells then provide activation signals to cognate b cells by engagement of CD40/ligand complexes. Cytokines important → direct what type of anitbody b cell will produce

4. differentiate into an antibody secreting cell/memory cells

why are not all igG's the same?

• due to variable regions in the heavy and light chains which bind to specific epitopes of foreign antigens

2 things B and T cells both have in common

1. BCR, TCR = somatically assembled by VDJ mini segment rearrangement

2. both undergo negative selection to remove cells that bind with high self affinity to prevent autoimmune responses

purpose of VDJ rearrangement

• permits random generation of Antigen receptors for adaptive (specific) immune system

• unique BCR/TCR = each cell have specificity → ability to bind to single antigen

• population of cells with then be able to recognise a wide variety of specificity

where does the VDJ recomb attack for each BCR and TCR

BCR: heavy and light chain

TCR: alpha beta and y delta chain

First chain to undergo VDJ recomb

Heavy chain

Basic steps for heavy chain VDJ recomb

1 random D segment joins to 1 J

1 of 46 random V segments will recombine with DJ

Basic steps for light chain VDJ recomb

ONLY VJ involved,

ONLY ONE light chain will be rearranged to be presented as a BCR

LONG EXPLANATION: VDJ steps for heavy chain

1. both the random D and J is attached to a RSS specific DNA motif

2. RSS directs RAG proteins to the right stop

3. RAG1/2 + HMG1 form a complex that binds to the 2 random RSS segments

4. that complex brings strands from D and J together forming a loop between the RSS sequences

5. introduces NIX at the 5' to create a hairpin loop and signal ends

6. JOINING: DNAPK binds each broken end and recruits 3 other proteins: ( artemis ( scissors ), Ku70/80 ) to form a repair complex

7. artemis acts as a nuclease and cuts/opens coding ends ( hairpins ) and then the Ku70/80 attach to the cut pieces of the DJ strains and the 2 RSS on the signal ends

8. LIGATION: DNAPKcs and new XRCC4 are dna ligases that align the DNA ends and recruit the TdT enzyme

9. TdT: randomly inserts nucleotides (N) into the coding ends (5’ to 3’ direction) until it gets a complementary sequences and opposing ends can pair up

10. exonucleases remove mistmatched/unpaired nucleotides once DNA strand are lined up bases from coding ends

11. DNA polymerases fill in nucleotides compatible for joining

12. DNA ligase 4 comes at coding ends to form a joint

13. then you are left with a coding joint of just -DJ- and a circle signal joint with the two RSS segments

14. the signal joints can be used to track the normal development of b/t cells in new borns

LONG EXPLANATION: VJ steps for light chain

1. after heavy chain, it pairs with surrogate light chains (SLC) and Igalpha and beta → form a pre-BCR

2. pre-BCR sends the success signal to the B cell once attachment occurs

3. RAG1/2 are turned on again and initiate the VJ recomb

4. Light and Heavy chains pair forming the complete BCR

5. overall, receptor will consist of

   • Ig heavy and light chains,

   • Igalpha and beta for support and signalling

6. another success BCR signal is sent

7. both steps require signalling proteins downstream of BCR, BTK and BLNK

8. BCR now exist the bone marrow => periphery => spleen as a mature b cell

Tolerance in BCR

random generation of BCR = possibility of self specificity

• immature B cells screened for auto reactivity

• receptor editing initiated + rearranges alternate light chain

receptor editing

self reactive light chain is discarded , VJ rearrangement of remaining light chain (alternate light chain) to modify variable region to form a new BCR and is tested until non self-reactive

isotype switching or class switch recomb ( CSR )

B cells change the type of antibody they produce, switching from one isotype (like IgM) to another (like IgG, IgA, or IgE) while maintaining the same antigen specificity

LONG EXPLANATION: how isotype switching works / CSR

1. Naive B cells initially expresses IgM / D as their surface receptor

2. During an immune response, B cells encounter antigens + undergo differentiation.

3. DNA recomb → constant region of HC replaced, occurs in germinal centre (in light zone, core )

4. process guided by ‘switch regions’ in DNA, located upstream of each HC constant region gene

5. AID enzyme initiates DNA lesions in these switch regions, deaminating cytosine bases → uracil within switch regions

6. causes DNA breaks → results in deletion of intervening constant region genes + joining of the VDJ segment to the new constant region gene

7. repaired by NHEJ → joining the two s regions

8. DNA loop between the two s regions that are rejoined = deleted resulting in a new isotype

somatic hypermutation

in activated B cells,, high rate of point mutations in variable regions of Ig genes → increased antibody diversity + affinity maturation.

LONG EXPLANATION: how somatic hypermutation works

1. occurs in the dark zone of germinal centre, (in secondary lymphoid tissues) during B cell proliferation

2. initiated by AID (activation-induced cytidine) which deaminates cytosine (=uracil) in DNA of the Ig genes

3. uracils are then repaired by error-prone DNA repair mechanisms, leading to mutations in the V regions

4. B cells with antibodies that bind the antigen with higher affinity = selected for survival and proliferation = evolution of B cells that express BCRs with increased affinity for the antigen.

main difference in CSR and SHM

CSR occurs to the constant region and SHM occurs to the variable region

Lymphocyte development

1. Long term haematopoteic stem cells ⇒

2. haematopoteic stem cells ⇒

3. common lymphoid progenitors ⇒

4. B/T/NK cells

   • B cells: bone marrow

   • T cells: thymus

   • NK cells: fetal liver

polyclonal antibodies

collection of antibodies from different B cells that collectively recognise multiple epitopes on the same antigen → each individual antibody recognises a unique epitope

polyclonal antibody uses

COVID testing and pregnancy tests

polyclonal antibodies limitations

• batch to batch variation
• difficult to quanitify
• lower specificity
• therapeutic risks

polyclonal antibodies benefits

• broad reactivity
• rapid production
• recognise multiple epitopes

how to make polyclonal antibodies

1. inject an antigen into a rabbit

2. antigen activates B cells

3. plasma B cells produce polyclonal antibodies

4. we obtain the antiserum from rabbit containing the polyclonal antibodies

monoclonal antibodies

lab-produced proteins designed to mimic the body's natural antibodies, targeting specific antigens

monoclonal antibody uses

• theraputics like cancer and transplantation
• diagnostics
• imaging
• protein purification
• vaccine design

how to make monoclonal antibodies

1. immunisation of mice with cancer specific antigens to stimulate antibody production

2. isolation of plasma cells from the polyclonal resp.

3. fusion and generation of hybridomas ( plasma cells + myeloma )

4. selection fused cells, sorted into single cells and screened for desired antibody specificity in a HAT media

5. expansion of selected hybridoma to prodcue monoclonal antibodies

how can we change regular mAbs ( monoclonal antibodies )?

change the specificity
e.g bispecific, multi specific , multimeric, multifunctional

pros and cons of bi/multi specific mAbs vs normal mAbs

Pros:

• potential for large scale production

• more effiecient binding to target

• stability

Cons:

• need for external epitope

• may enhance toxicity if combined with classical immuno agents

Phage display

molecular biology technique → peptides/proteins displayed on surface of bacteriophages → can study protein interactions + identify ligands or antibodies with specific binding affinities

IgG

momomeric, 4 subclasses, major role in secondary immune response, longest half-life in circulation (20-24 days)

IgM

no hinge region, extra constant region + longer amino acid tail → allows it to multimerise, can exist as hexamer or in monomeric form on surface of naive B cells

IgA

detectable in blood but mainly found in mucous secretions, exists as monomer or dimer

IgD

basic structure, extended hinge region, easily digested, mostly found on surface of naive B cells within IgM

IgE

rare, present in really low levels, found on surface of basophils and mast cells, extra constant region, very flexible, cross link in large numbers, usually associated with its pathogenic role

B cells soliciting help from T cells

1. activated B cells gather, process and present antigen to T cells via MHC class II

2. after seeing Ag, activated T cells are co-stimulated

3. Co-stimualted T cells provide ‘help via: CD40L/CD40 = activates NFkappaB signalling, induces proliferation, induces AID-inducing signals, differentiation signals, if no CD4 help here, B cells do nothing

4 mechanisms to generate BCR diversity

1. somatic recombination

   • VDJ recombination of H chain (Pro-B to Pre-B cell stages) then VJ recombination of light chain (Pre-B cell to immature stage)

2. pairing of various heavy and light chains

   • Pre-BCR formed in Pre-B cell with surrogate light chain, final BCR formed in immature B cell — if successful, signal to B cells via BtK, BLNK, Syk (different signals for successful TCR rearrangement)

3. junctional diversity

   • random nucleotide addition during VDJ rearrangement of H chain by TdT

4. SMH and affinity maturation

   • single point maturations in hyper variable hotspots via AID and selection of high-affinity binding Ig