B Cells and Diseases Flashcards
Define PIDs.
Primary Immunodeficiencies are disorders characterised by an impaired ability to produce a normal immune response that typically result in recurrent or severe infections. It is not caused by other diseases, treatments, or exposure to toxins.
Recall some examples of PID.
How do we usually treat PIDs?
- Usually by replacing the whole immune system if it’s completely missing (e.g. most severe forms, SCID, X-linked agmmaglobulinaemia)
- Gene therapy
- Haematopoietic stem cell transplantation (HSCT)
- Usually not the best option for patients with some form of immunity but increasing considered
- Replace antibodies through intravenous Ig (IVIg) or sub-cutaneous Ig (SCIg)
All lymphocytes descend from a ______________. Mention where the lymphocytes develop.
B cells develop in the ___________. Then, they exit through the ____________ to the periphery where thwy differentiate.
Bone marrow, sinusoids
B cells are precursors to ____________.
Plasma cells (antibody-secreting cell)
To make antibodies, a B cell must:
- Recognize antigen via immunoglobulin on the surface
- Surface membrane Immunoglobulin = B cell receptor
- Break down Ag and re-present to T-cells as peptides on surface MHC class II
- T cells then provide activation signals or ‘help’ (CD40L, ICOS, cytokines)
- Allow B-cell to differentiate to antibody-secreting cells (ASCs) and memory B cell
Recall what B cells do.
What is an antibody?
It is produced by plasma cells. They are globular proteins found in serum, interstitial fluids, and mucosal secretions. Their function is to identify and neutralise foreign pathogens (mark them for degradation). Each antibody binds to a specific antigen.
Describe the structure of antibodies.
They are made up of 4 chains: 2 identical Heavy (H), 2 identical Light (L) chains. It has two regions: variable and constant regions.
L+ H chains are paired by disulphide bond in each dimer. H + H paired by disulphide bonds in each tetramer. Heavy chains give ‘Y’ structure of Ab.
Describe the constant and variable regions of the antibodies.
The variable region serves as the ‘antigen-binding site’. It is extremely variable.
The constant region dictates the class/isotype of antibody
Recall the schematic representations of the antibodies’ classes.
Recall B cell activation and differentiation in reverse.
Briefly explain how B cells are clonally selected.
- A specific BCR recognizes the specific antigen
- The corresponding B-cell is ‘selected’ by the antigen
- They are then stimulated (clonal expansion) and differentiated (Ab secretion).
Note:
- B cells are quiescent unless stimulated to respond
- B cells usually need instructions from CD4 T cells to proliferate and differentiate
- B cell response to antigen changes with time (affinity and effector functions)
Both B and T cells undergo _________________ to remove cells that bind with high self affinity.
Negative Selection
Describe the mechanism of production of a diverse range of antibodies.
The immunoglobulin gene locus encodes for multiple genes for the variable and constant regions of both chains. There are three separate genes on 3 loci.
The variable region of the H chains is encoded by 3 separate segments (V, D, J), while for the L chain it is encoded by 2 segments (V, J). Each variable domain is a result of the recombination of the option from each segment.
Ig diversity comes partly from having:
- Random selection and rearrangement of VDJ (heavy) and VJ (light) genes at each locus;
- Independent rearrangement at Heavy and at Light chain loci; the pairing of various combinations
- 2 light chain genes (κ and λ) to choose from
- Imprecision of junctions (N region diversification, random insertion of nucleotides at junction regions, mediated by TdT)
- Somatic Hypermutation and Affinity Maturation: single point mutations in hypervariable regions and selection of highest binding receptors.
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Recall the Ig Gene rearrangement and B cell maturation sequence.
What is the RSS?
Recombination Signal Sequence is a unique nucleotide sequence that is the RAG recognition sequence, is adjacent to each mini-gene segment
What is RAG1/2?
Recombination Activating Genes are essential to Ig gene recombinati0on, they provide the recognition and DNA cleavage activity.
What is HMG1?
High Mobility Group 1 protein is a chromatin-binding, structural protein also required for rearrangement but not uniques to Ig gene rearrangement.
What is the Artemis/DNA-Protein Kinase catalytic subunit (DNA-PKcs)/Ku70,80 complex function?
It is involved in the recognition and synapsis of DNA ends.
Autophosphorylation of DNA-PKs activates Artemis, which is a nucelase that opens the coding end of the haripins.
What is the DNA Ligase IV/XRCC4?
The complex is responsible for joining the DNA ends.
What is the TdT?
Terminal deoxynucleotidyl transferase is unique to B cells and adds extra random nucleotides to the broken ends at V-D-J junctions (doesn’t work at L chain loci).
Mention the three steps of VDJ recombination.
Describe the structure of the RSS.
RSS has 3 elements:
- heptamer of 7 conserved nucleotides,
- spacer of 12/23 basepairs, and
- nonamer of 9 conserved nucleotides
Describe the 1st step in VDJ recombination.
Step 1 involves the selection of V and J regions to recombine.
RSS are recognised by RAG1 and RAG2, which are proteins that cut DNA at randomly selected RSS, making double-stranded DNA breaks. RAG1 and RAG2 forms a complex with HGM1.
The cut ends are then ligated to form:
- Coding joint (hairpins)
- V-J, then V-DJ for H chains
- V-J for L chains
- Signal end joints: loop of DNA deleting al intervening DNA
Describe the 2nd step of VDJ recombination.
It involves the non-homologous end joining (NHEJ).
DNA-protein kinases (DNA-PKcs) bind to each broken DNA ends and recruits Artemis, Ku70 & Ku80 to forms a complex. This complex then recognise and closes the DNA ends.
Autophosphorylation of DNA-PKcs activates Artemis, which opens the coding-end hairpins (essential for NHEJ DNA repair).
Describe the 3rd step of VDJ recombination.
It involves the DNA ends ligating together.
DNA-PKcs & XRCC4 (DNA Ligase) align DNA ends and recruit the TdT enzyme. TdT is a DNA polymerase that adds random nucleotide to coding-end in 5’ to 3’ direction. Exonucleases remove bases from coding ends and DNA polymerases fill in nucleotides compatible for joining. Processed coding ends ligated together by Ligase IV.
Describe the features of SCID.
Describe how can a newborn be screened for PID.
Signal Joint is a circular DNA segment that is a byproduct of a successful VDJ rearrangement in a B cell/T cell. It is called KRECS in B cells, TRECS in T cells. These circular DNA persist in cells and dilute with cell division as markers of new B cell output. Quantification of TRECs and KRECs by simple quantitative PCR would allow early detection of suspected PID. Absence of TRECS/KRECS indicate possible absence of T/B cells/
Explain how does the cell know when the Ig VH gene rearrangement is successful.
After the VDJ recombination and the formation of the H chain, the H chain pairs with two Surrogate Light Chains (SLC). SLC are them same in every B cell - not generated by rearrangement, which acts as a generic scaffolding as the real L chain are not yet arranged.
The IgH-SLC complex pairs with the signalling chains Igα and Igß to form the pre-BCR, which then sens the ‘success’ signal into the cell.
What marks the completion of the first stage of B cell development?
After the VJ recombination of the L chain, they paired with the existing H chain to form the complete Ig on the surface. IgHL complex pairs with Igα and Igß to form the BCR which sends another ‘success’ signal into the cell. This marks the end of the Ig gene rearrangement.
Mutation in BTK gene causes __________________. Describe the molecular cause of it.
X-linked agammaglobulinaemia (XLA):
- a kinase that is involved in the signalling pathway that signals rearrangement success
- mutation occur along the entire gene with no single mutation occurring >3%
- some result in residual Btk expression and signalling (variable phenotype)
- Mutation maintained in the population by de novo events
- BTK gene encoded on X-chromosome; thus almost exclusively affects males
Describe the primary immune response to a protein antigen.
Initial exposure to antigen mostly induces IgM secretion (low affinity, high avidity) followed by some IgG or IgA (higher affinity). Change in isotype or class of Ig typically occurs (class switch recombination). Antigen specificity remains unchanged. CSR from IgM to IgG1, or IgA1, or IgE is not random, but directed by signals from T cells and/or antigen.
Describe the memory response to a protein antigen.
- Secondary response
- faster kinetics
- mount a first IgM response, but rapidly produces IgG
- greater magnitude (more antibody) than the primary response
- not usually associated with further CSR, but maybe
- higher affinity than primary Ab (tighter binding of the antibody to antigen; through Somatic Hypermutation)
- faster kinetics
Describe the early stages of B and T cell activation.
Class switching mostly occur before SMH in the ____________ of the lymph node.
follicle
Recall the following regarding variable and constant Ig region.
Define somatic hypermutation.
It refers to the random introduction of point mutations into the V gene segments of the H and L chains to diversify binding to the antigen. This happens during B cell proliferation and occurs in the dark zone of GC.
Describe the process of affinity maturation in the GC.
It refers to activated B cells producing antibodies with increased affinity for antigen during the course of an immune response. A secondary response can elicit antibodies with several-fold greater affinity than in a primary response.
Affinity maturation primarily occurs on surface immunoglobulin of the germinal centre of B cells and as a direct result of somatic hypermutation (introduced by the AID) and selection by Tfh cells (higher affinity => drive proliferation).
Describe the process of CSR.
Naive B cells express IgM and IgD on their surface, with the transcription of the H chain genes starting from the promotor upstream of the VH segment. Thus, exons encoding the constant regions can’t be expressed until the variable segment is placed upstream of them.
Switch (S) regions are upstream of each C region. Only on activated B cell, AID enzyme is expressed, making double-stranded breaks in two S regions (after they make a switch loop). The ends are joined, DNA is repaired by nonhomologous end-joining (NHEJ), deleting an intervening chromosomal region. One of the constant isotype region is then expressed by the promoter.
Recall the order of the C regions and mention the cytokines that help direct Ig isotype switching.
Recall the features and functions of the Ig isotype.
Recall the 4 mechanisms to generate BCR diversity.
Describe the steps on how B cells solicit help from T cells.
- Activates B cells gather, process and present antigen to the T cells via the MHC class II
- After seeing Ag, activated T cells are co-stimulated (e.g. ICOSL)
- Co-stimulated T cells provide ‘help’
- CD40/CD40L
- activates NFkB signalling => induces proliferation
- induces AID-inducing signal
- Differentiation signal (e.g. IL-4, IL-21)
- With no help - B cells do nothing
- CD40/CD40L
Recall the defects in help signalling pathways that lead to PID.
Mention the susceptibilities of HIGM Syndrome.
Describe the management and therapy of HIGH syndromes.
Describe CVID.
Recall the effector functions of the Human Ig.
Recall the overview of the VDJ recombination process
Recall the major genetic causes of SCID.
Recall the final checkpoints of immature B cells before release from bone marrow.
