B cells structure, signalling and repertoire diversity Flashcards
LO
- B cell differentiation in context of location.
- General antibody structure.
- B-cell receptor (BCR) structure.
- Changes in antibody structure during B cell responses to foreign antigens.
- Central and peripheral mechanisms of tolerance to self-antigens.
- Common elements of BCR signalling pathways from membrane to nucleus.
- How knowledge of antibody structure is used to design more effective and safer monoclonal antibodies for immunotherapy.
Tell me about the development of hematopoietic cells in the bone marrow
- The myeloid and lymphoid cellular lineages develop from pluripotent stem cells due to the regulated expression of genes specific for each cell type
- B cells are the only ones which produce antibodies (plasma cells)
- Antibodies are inserted in the B cell plasma membrane from where they test their binding ability to molecules in the environment
- B cells communicate binding interactions with antigen to the cell nucleus and the B cell takes appropriate action
Tell me about the primary and secondary lymphoid organs
Both B and T cells begin development in bone marrow
In bone marrow, B cells undergo genetic rearrangement in the cell nucleus, whereas immature T cell go to thymus, where they undergo a similar process
Both B and T cells leave their respective primary lymphoid organ and traffic to secondary lymphoid organs such as spleen and lymph where they encounter antigens as mature cells
Tell me about peripheral blood CD5+ and CD5- B cells
When peripheral blood is taken from a vein and analysed for B cell composition based on presence of markers CD19 and CD5, two separate B cell lineages are found:
Tell me about antibody structures
Antibodies are Y shaped glycoproteins consisting of 2 identical heavy chains and 2 identical light chains (hence monomer has 4 chains).
They have
2 x Fab (fragment antigen binding)
2 x Fc (Fragment cystallisable)
Tell me about the Fab regions on the antibody
2x Fab (fragment antigen binding)-
Bind antigens
Both are identical in structure and bond the same antigen epitope
Both beta pleated sheets and loops
Loops are what contacts antigen
Sheet supports loop
Tell me about the Fc regions on antibodies
2x Fc (fragment crystallisable)
classes or isotypes: IgM, IgD, IgG, IgE, IgA- can bind to Fc receptors
Binds to Fc receptors on the surface such as B cells, NKC, follicular dendritic cells, monocytes, neutrophils, eosinophils, basophils, platelets, and mast cells
Antibody structure
Tell me about the heavy chain variable domain in amino acid sequences
- Molecular biologists determined the amino acid sequences of antibody H (heavy) and L (light) chains
- H chains were arranged into 7 families based on sequence similarity (as shown above)
- Within a family, similar framework regions (FR) sequences alternate with very different complementarity determining regions (CDR)
- The CDR3 sequence is different for each heavy chain
Tell me about the constant region isotypes (classes) and region binding reactivities in antibodies
Amino acid sequences beyond FR4 in both H and L chains are more constant. Hence, antibodies are divided into the variable and constant regions
Each region is composed of domains, and each domain has a name e.g. CH1, CH2, CH3 and CH4
The hinge region is flexible
The 5 antibody classes are distinguished based on the amino acid sequence of the H chain constant region. They differ in biological function.
There are 2 L chain classes: kappa (k) and lambda (symbol like an upside-down y). There are no known biological differences between them.
H and L chains are joined via disulphide bridges
Tell me about the functional role of carbohydrates in antibodies
The % carbohydrate in the H chain constant regions differs between the antibody isotypes. Glycosylation sites are also found in the variable regions
Here they are both germlines encoded and generated during somatic hypermutation
The CH2 domains of alpha, delta, gamma and CH3 domains of mu and epsilon are made accessible to the aqueous environment.
Carbohydrates…
- enhance solubility by masking a hydrophobic patch on CH2.
- influence local peptide conformation.
- Is required for complement fixation.
- Is required for binding to FcR by IgG and IgE.
- Necessary for secretion from cells (IgA, IgE).
- Increases rate of clearance of immune complexes from the serum by the liver.
Tell me about the beta barrel domain in the antibodies
An antibody domain consists of a sandwich of two layers of b-sheets held together by a disulphide bond between FR1 and FR3.
The 9 antiparallel b strands are linked by b turns and loops. The strands are composed of framework regions (FR).
The loops form the complementarity determining regions (CDR) which contact antigen.
Tell me about the isotypes expressed at different stages during B cell differentiation
Tell me the stages and what happens during B cell differentiation and recirculation
Properties of the major classes of antibodies
Tell me about some therapeutics with antibodies
All currently approved therapeutic antibodies are of the IgG class. They do not provide efficient surveillance of the tissue compartment.
The hypothesis is that IgE immunotherapy can recruit innate immune cells using FceRs.
When antigen specific IgE antibody is attached to a mast cell via the FceRIa, multivalent allergen cross-links multiple IgE antibodies which in turn causes FceRIa to become cross-linked on the surface of mast cells which in turn causes degranulation and possible anaphylaxis.
There is now evidence that the IGHV3 variable region of a therapeutic IgE antibody (e.g., Trastuzumab- anti-Her2) can be bound by Staphylococcus protein A (SpA) outside of the antigen combining site and trigger mast cell degranulation.
The conclusions based on the basic scientific investigation of antibody structure have widespread relevance to the biotechnology industry by influencing the design of IgE immunotherapeutics.
Tell me about the B cell receptor (BCR) structure
The BCR is attached to the cell surface and consists of 2 molecules not covalently bound to each other:
- Immunoglobulin/ Antibody- antigen recognition component.
- CD79a/b- signalling component
CD79a/b- Concensus Immunoreceptor Tyrosine-based Activation Motif (ITAM): YxxLxxxxxxxYxxL (2Y and 2L in precise spacing)
Tell me about the antibodies that B cell produce
What are they crucial for?
Each B cell produces many identical antibodies, each with the same specificity (monospecific). This is crucial for the cross-linking of surface antibodies to generate intracellular signals.
BCR expression and signalling (e.g., sub-threshold or tonic signal through the BCR) are essential for developing and mature B cells.
IgM-BCRs and IgD-BCRs are localized in different protein islands.
All light chains are the same isotype (either kappa OR lambda in a healthy cell) on a B cell.
Each B cell can only make one type of light chain and one type of heavy chain
Tell me the peripheral B cell response to antigens and the different phases involved in this process
What are the only cells that produce antibodies?
B cells
What are the two types of antibodies that B cells produce?
- Membrane bound antibodies (monomers)
- Soluble (secreted) antibodies
Tell me about Membrane bound antibodies: monomers
Membrane bound antibodies: monomers
B cells respond to the binding of foreign antigens via membrane bound antibodies by sending signals to the nucleus to induce B cell maintenance, activation, proliferation, differentiation and silencing.
Tell me about soluble secreted antibodies
Soluble (secreted) antibodies:
Bind to and mark pathogens for destruction.
Can passively bind via Fc receptors to the surface of non-B cells.
Membrane bound and soluble structure of the different antibody classes
Tell me about the primary and secondary protective antibody response and how they differ in kinetics, magnitude and isotype
The primary response is slower, peaking at 7-14 days compared to 3-5 days in the secondary response
Weaker (less antibody is produced) and is characterised by IgM antibodies compared to IgG antibodies, or other class-switched isotypes in the secondary response
What is required to generate a signal in antibodies?
BCR cross-linking is required to generate a signal
What does BCR aggregation rapdily activate?
The Src family kinases Lyn, Blk and Fyn as well as the Syk and Blk tyrosine kinases
Tell me the different types of outcomes od BCR signalling and what each may be due to
There are many factors that can influence BCR signalling outcomes, tell me what some of the outcomes of BCR signalling are determined by?
- The maturation state of the cell (e.g., immature, mature).
- The nature of the antigen (e.g., size, valence, solubility, foreign or self).
- The magnitude and duration of BCR signalling (influenced by the affinity of antigen binding).
- Signals from other receptors (e.g.CD40, TLR, IL-21 receptor, BAFF-R, CD45, CD19, CD22 and FcγRIIB1) are not parallel, separate pathways. They crosstalk.
What is self-tolerance?
A state of unresponsiveness to self-antigens
Tell me about the mechanism that tolerance refers to
Tolerance refers to the mechanisms that control self/non-self-discrimination which protect an individual from anti-self-immune attack by self-reactive lymphocytes.
What do Autoimmune diseases involve and how is it characterised?
Autoimmune diseases involve the failure of the elimination and inhibition mechanisms that maintain self-tolerance by the immune system.
Autoimmune disease is characterized by the destruction of host cells by autoantibodies and/or self-reactive T cells.
How is the balance between generating a protective antibody response and autoimmune disease controlled?
In both the primary and secondary lymphoid tissue
Where does central B cell tolerance occur?
In the bone marrow
What are the different ways in which negative selection of anti-self B cells in the bone marrow can occur by?
- clonal deletion
- receptor editing
- Anergy (non-responsiveness)
Tell me about clonal deletion and negative selection of anti-self B cells in the bone marrow
Clonal deletion of immature high-affinity self-reactive B cells by apoptosis if self-antigen is multivalent and immobilised (e.g., expressed on cell membrane)
Tell me about Receptor editing and negative selection of anti-self B cells in the bone marrow
Receptor editing: a different light chain pairs with the heavy chain in immature B cells to eliminate self-specificity when the self-antigen is multivalent and cross-links BCR. Predominant mechanism
Tell me about anergy and negative selection of anti-self B cells in the bone marrow
Anergy (non-responsive): self-reactive immature B cells exposed to large amounts of soluble, weakly cross linking, low valence self-antigen in the bone marrow migrate to the periphery where they are anergic
However, not all self-antigens are represented in bone marrow (e.g., post-transitionally modified proteins found only in the periphery or are tissue specific molecules)
B-cell self tolerance
Tell me the different mechanisms of inactivation of seld-reactive B cells in the secondary lymphoid tissue
- Clonal deletion by apoptosis if the antigen is strongly cross-linking.
- Anergy- cell survival but unresponsive to soluble antigen.
- Receptor editing- L chain and rare IGHV rearrangement.
- Physical separation of cells from antigen prevents cellular activation
- Generation of regulatory cells:
- B-cell suppression via Treg effects on T cells
- B-cell suppression via Breg cells (IL-10 and TGF-beta production)
What happens if mature peripheral B cells encounter multivalent self-antigens in the absence of T helper cells?
If mature peripheral B cells encounter multivalent self-antigen in the absence of T cell help, they do not receive co-stimulatory signals and fail to enter the primary follicle and become trapped in the T cell zone and die by apoptosis.
What happens in mature peripheral B cells recognise soluble self-antigens in the absence of T helper cells?
if mature peripheral B cells recognize soluble self-antigen in the absence of T cell help, they produce few surface IgM receptors and become anergic. They express Fas and are killed by Fas-L expressing CD4+ T cells. This is called activation-induced cell death (AICD).
Mechanisms of peripheral B cell self-tolerance
Autoreactive B cell subsets
- Healthy individuals have mature, recirculating self-reactive lymphocytes.
- 55% of bone marrow B cells were found to be polyreactive/autospecific.
- 24% of naïve B cell antibody specificities in the peripheral blood were found to be polyreactive/autoreactive.
- 7% of circulating naïve B cells are anergic, an indication that they are possibly self-reactive.
- >50% of IgD antibodies were found to be autoreactive.
- 31% of IgG memory cell antibodies were weakly autoreactive. Most autoreactivity in this subset is due to somatic hypermutations.
In what % of the population does autoimmunity occur in?
What is it a result of?
What are important determinants of the outcome?
Autoimmunity occurs in ~3-8% of populations in developed countries (e.g., cold agglutinin disease, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus and certain types of diabetes).
Autoimmunity is the result of the combination of genetic predisposition and environment.
The physical nature of the self-antigen (soluble vs membrane bound, size, valence, affinity) is an important determinant of outcome.
B cell receptor signalling pathways
What are the cellular locations of signalling events?
- Plasma membrane
- Cytoplasm
- Nucleus
Tell me about the signalling events that occur at the plasma membrane
Pre-existing inactive molecules are activated by phosphorylation.
There is a balance between phosphorylation (kinase activation) and dephosphorylation (phosphatase activation).
Tell me about the signalling events that occur at the cytoplasm
Cytoplasm
- Phosphorylation cascade
- Calcium release
- Inactive transcription factors are activated.
- Activated factors translocate into the nucleus.
Tell me about the signalling events which occur at the nucleus
Alteration of transcriptional programme.
How have signalling pathways been used as therapeutic targets?
Increased expression of BCR associated kinases is associated with a shorter treatment-free interval in some diseases.
Signaling therefore is a therapeutic target in some diseases.
Tell me some of the types of therapy that have been created?
Immunosuppressive drugs (small molecule compounds).
Immunotherapy (monoclonal antibodies).
Tell me about SYK inhibitors
SYK inhibitors
SYK-mediated B-cell receptor signalling inhibitors have shown positive results in the treatment of allergy, autoimmune diseases and B cell lineage malignancies.
Tell me about BTK inhibitors
BTK inhibitors
Ibrutinib- drug
Tell me about the Inhibitors of calcineurin-dependent activation of NFAT
Inhibitors of calcineurin-dependent activation of NFAT
Cyclosporin A: Inhibition of B cell proliferation following ligation of surface Ig
Tacrolimus: Induction of apoptosis following B-cell activation
Tell me about the inhibitors of the ERK pathway
ERK pathway inhibitors
- The ERK pathway is deregulated in approximately one-third of all human cancers.
- The ERK pathway has been a focus for drug discovery for almost 15 years with Ras, Raf and MEK as the main targets.
Tell me about the phospholipase C (PLC) pathway and how it leads to signal activation
Tell me about Bruton’s Tyrosine kinase (BTK)
- Btk is a cytoplasmic tyrosine kinase required for normal B cell differentiation.
- Btk is activated following signalling through the BCR and pre-BCR.
- Btk is located on the inner side of the plasma membrane where it is phosphorylated.
- Btk phosphorylates PLCg2 leading to calcium flux and activation of the NF-kB and NFAT pathways.
- Ibrutinib is a small molecule drug that binds and inactivates BTK. It is the most broadly used inhibitor in B cell lymphoma (CLL, MCL, MZL, ABC-DLBCL)
Tell me the steps to the cytoplasmic calcium flux process
A graph showing the calcium flux in normal B cells via IgM and IgD
The effects of extracellular calcium on Ca2+ flux profiles
FACS separation of B cells
Variable levels of Ca2+ are induced in cases of CLL by ligation of sIgM using polyclonal F(ab’)2 anti-IgM
Common calcium flux trace in CLL cells following IgD ligation
Tell me the stages to the Ras/Raf/MEK/ERK kinase pathway and in which part of the cell each stage occurs in
Cytoplasm:
- Ras is a monomeric G protein which is inactive when bound to GDP.
Ras-GDP (inactive).
- DAG recruits RasGRP (guanine nucleotide releasing protein) to membrane where it activates Ras.
Ras-GTP (active)
- Ras-GTP binds Raf (a MAP kinase kinase kinase).
MAP = Mitogen Activated Protein
- Activated Raf binds to MEK (a MAP kinase kinase).
- Activated MEK phosphorylates ERK (a MAP kinase).
Nucleus:
- Phosphorylated ERK enters the nucleus.
- The transcription factor AP-1 is induced leading to transcriptional activation of several genes
Tell me the stages to the NF-kappaB activation pathway
L1 summary
- Immunoglobulins are made only by B cells.
- Immunoglobulins are bivalent having a “Y” shaped structure.
- The BCR is made up of surface immunoglobulins which binds cognate antigen and the CD79a/b component which sends intracellular signals to the cell nucleus.
- BCR signalling pathways are therapeutic targets.
LO L2
- The genetic basis of antibody variable region diversity
- The genetic mechanisms involved in class (isotype) switching
- The mechanisms of variable region somatic hypermutation
How many chromsomes do humans have,
what three pairs of chromosomes are involved in antibody production and what loci are each of them in?
Humans have 23 pairs of chromosomes (46 chromosomes in total).
Three pairs of chromosomes are involved in antibody production:
- Chromosome 14- heavy chain locus
- Chromosome 2- kappa chain locus
- Chromosome 22- lambda chain locus
How many immunoglobulin genes are there?
There are approximately 20,000 genes in a human B cell.
Approximately 1012 different immunoglobulins can be generated by the B cell population at any one time.
How can so many different proteins be made from so few genes?
Because the DNA encoding antibody polypeptide chains is arranged as separated gene segments which recombine.
The IGHV region locus showing correspondence with antibody protein regions
What are the light chain chromosomal loci?
Tell me about the segments they contain
What are the stages to combinatorial diversity of gene segments?
Tell me about somatic recombination and where it occurs
In the bone marrow in the absence of antigen:
- Heavy chains rearrange first (one chromosome 14 then the other if the first rearrangement is not productive)
- Light chain rearrangement takes place next (usually kappa followed by lambda).
IGHD –> IGHJ, IGHV –> IGHD-IGHJ = heavy chain
IGKV –> IGKJ (kappa before lambda) = light chain
B cells are an accident waiting to happen because the DNA is cut into fragments and pasted together
What are the human gene segment repertoires?
What are the two types of combinatorial diversity?
Combinatorial diversity (1): gene segment selection
There is a reservoir of multiple V(D)J gene segments that are randomly assembled.
Combinatorial diversity (2): H and L chain pairs
The association of different variable heavy and light chains to form antibody recognition sites.
Tell me about the anatomy of a gene segment and what each of the recombination signal sequences (RSS) consist of?
Each recombination signal sequence (RSS) consists of:
1.Heptamer- 7 bp 5’CACAGTG3’ (well conserved).
The palindromic heptamer has at least two roles:
i) enhances binding of the recombination activating proteins (RAG) to the RSS
ii) specifies the site of DNA cleavage. The first three nucleotides of the heptamer (5′-CAC3’) are particularly important.
2.Spacer- 12±1nt or 23± nt.
The spacer is variable in sequence. Spacer lengths are important. The primary role of the spacer is to properly align the heptamer and nonamer.
3.Nonamer- 9 bp 5’ACAAAAACC3’ (conserved).
The A (or T) rich nonamer is a sequence-specific binding site for the nonamer-binding domain of RAG1 and functions to anchor the RAG proteins to the DNA.
Tell me about the recombination activating proteins RAG1/2
The RAG1 core contains a well-defined nonamer-binding domain and a heptamer-binding domain responsible for interactions with both the heptamer and RAG2. Zinc finger region B (ZnB) is thought to be important for RAG2 interactions.
The RAG2 core is crucial for DNA cleavage activity. It interacts with RAG1 and enhances the DNA-binding affinity and specificity of the RAG complex, although RAG2 has little or no DNA binding activity on its own.
What are the recombination signal sequences and spacers?
Tell me about the cleavage efficiency of RSS by RAG1/2
The dominant factor determining IGHV segment utilization is that RAG1/2 cleaves individual RSS with different efficiencies.
IGHV4-34 (VH4 is from that family, 34 is the 34th from the constant region?) has the most efficient RSS of all human IGHV genes and hence is over-represented in the developing and naïve B cells (6-8% in healthy people). (Yu K et al. JBC 277:5040, 2002.)
What is the 12/23 rule?
RSS with 12 bp spacers (1 turn of DNA helix) usually pair with RSS with 23 bp (2 turns of DNA helix) spacers. This is known as the 12:23 rule.
What does the 12:23 rule ensure?
- that IGHV–IGHV rearrangements do not occur.
- inclusion of the D gene segment.
- VDJ joining is restricted to biologically productive events.
- ensures that a pair of RSS control the activation of the catalytic activity of the recombination activating proteins (RAG) preventing RAG induced genomic instability.
VDJ recombination
Explain junctional diversity and enzymatic alteration in the coding joing
Nucleotides are gained and lost at the junctions (junctional diversity). Approximately 2 out of 3 rearrangements are non-productive due to frameshifts.
Palindromic (P) nucleotides are short inverted-repeat sequences resulting from uneven cleavage of dsDNA and filling in by DNA polymerase.
Nucleolytic activity removes nucleotides.
Terminal deoxynucleotidyl transferase (TdT) inserts non-templated nucleotides. Up to 20 nucleotides can be added. These sequences are not encoded in the germline.
Enzymatic activity is the major source of CDR3 diversity. The CDR3 nucleotide sequence is characteristic of each B cell
What are the two stages in which somatic V(D)J recombination occur in, in the bone marrow?
- DNA recognition and cleavage
- End processing and joining
Explain what happens in each of the stages of somatic V(D)J recombination
1. DNA recognition and cleavage:
- Lymphocyte specific recombination activating proteins (RAG-1 and RAG-2) protein complexes randomly bind gene segments and bring together the gene segments to
- be recombined and cut double-stranded breaks in
- the DNA exactly at the junction of the
- gene segment and its RSS.
2. End processing and joining
- Require in addition to RAG1/RAG2, general dsDNA repair enzymes of the non-homologous end joining (NHEJ) pathway (e.g. Artemis, TdT, DNA ligase IV).
B cells in the secondary lymphoid organ (lymph node)
Germinal centre in secondary follicle
What does the molecular mechanism of somatic hypermutation in IGHV and IGLV involve?
involves error-prone DNA repair
involves activation-induced cytidine deaminase (AID).
Generally, tell me about the point mutations in somatic hypermutation in IGHV and IGLV
deletions (4-7%), duplications (1%)
Frequency of mutation: 1 x10-3 per base pair per cell generation. (~106 more frequent than by spontaneous mutation).
What are the mutation hotspots and outcomes of somatic hypermutation in IGHV and IGLV?
Mutation hotspots:
RGYW (R=A or G; Y=C or T; W=A or T). bCDR contain more hotspots than FR in the germline sequence.
Outcome:
Affinity maturation usually resulting in increased affinity of antigen binding due to antigen selection.
When was Activation-induced cytidine deaminase (AID) first discovered and what is its main roles?
First discovered in 1999. Is specifically induced in activated B cells.
198 amino acid protein that initiates both somatic hypermutation (SHM) and class switch recombination (CSR) by producing U:G mismatches in DNA. Different domains of AID are involved in SHM and CSR.
AID catalyses the deamination (-NH2) of cytidine to uridine producing a U:G mismatch in DNA.
What is somatic hypermutation and class switch recombination supported by?
SHM and CSR are supported by a pathway involving uracil DNA glycosylase (ung) which removes uracil from DNA. The gapped DNA may be a substrate for error-prone repair.
How are uracils converted to UNG?
Uracils are converted by UNG and apyrimidinic endonuclease (APE1) into single stranded nicks in the DNA on both strands
What does class switching involve?
Tell me about this
What do cytokines determine?
What are the functional outcomes of SHM and CSR?
- Generation of stop codons in in-frame sequences stops protein production.
- Codon insertions/deletions (multiples of 3 nucleotides) which do not disrupt an in-frame sequence create diversity and increase affinity of Ag binding.
- Insertions or deletions that are not multiples of 3 result in an out of frame sequence which generates a nonsense peptide.
- Generating broadly neutralizing antibodies can be protective. (e.g., anti-HIV).
- Generation of auto specific antibodies which can cause disease.
- DNA damage during SHM and CSR may account for B cells being prone to malignant transformation (e.g., lymphoma).
What are the different mechanisms of generating antibody diversity?
