B cells and antibody production Flashcards
whats the basic structure of Ab?
- 2 identical light chains Constant and variable domains (VLCL). Encoded by rearranging variable, joining and constant elements (VLJLCL)
- 2 identical heavy chains Constant and variable domains (CHVH). Encoded by rearranging variable, joining, diversity and constant elements (VHDHJHCH)
what are antibodies?
Soluble plasma proteins
Monomers, dimers or pentamers
ISOTYPE - IgA IgD IgE IgG IgM
Effector functions: fix complement - neutralise - block - opsonise - interact with cells etc.
what are B cell antigen receptors?
- Cell surface-bound Ig of any isotype.
- Only found on B cells
- Always a monomer
- No effector functions
- Senses the antigenic environment of the B cell
- Connects the extracellular environment with signalling pathways
- Activates B cell effector function
- Only one antigen specificity per B cell
how are Immunoglobulins bifunctional?
- Conserved enough to interact with small number of specialised molecules.
- Allow changes to interactions during an immune response.
- Allow extensive variation between antibodies to match the infinite number of potential antigens but retain specificity throughout the immune response
- requires the protein to be both conserved and infinitely variable
what is the Immunoglobulin fold?
- a β barrel of 7 (CL) or 8 (VL) strands connected by loops and held together with a disulphide bond
The domains all look the same how can domain structure account for bifunctionality?
Examine genes encoding VH & VL domains
how does Organisation of Ig VH domain genes Maximise diverse specificity?
- COMBINATORIAL DIVERSITY - VH1 domains made from any combination of V gene with a D gene and a J gene
- JUNCTIONAL DIVERSITY - Imprecise breaking and joining of DNA when V, D and J genes recombine
how is the Ig heavy chain gene rearranged?
- RNA splicing joins the CH domains.
- Imprecise breaking and rejoining of DNA between the V and D genes and the D and J genes creates diversity of sequence and specificity
How does diversity arise from Ig light chain gene rearrangements (κ and λ)
- any V gene being able to join to any J gene
- deliberate errors in breaking and rejoining DNA
- insertion of new nucleotides between the join
what are Hypervariable sequences in Ig?
- COMPLEMENTARITY DETERMINING REGIONS (CDRs)
Distinct regions of high variability suggested region of antibody interacted with antigens. - More conserved parts acted as a FRAMEWORK (FR), on to which the hypervariable regions were suspended
how does Organisation of Ig CH chain genes Maximise effector function?
- the ability to switch to, one of several isotype-defining C genes
- Each CH chain domain is encoded by a separate exon
Summarise Bifunctionality of antibodies explained
by domain structure
- Fc- Structurally conserved end Effector functions
- Fab- Structurally diverse end Antigen recognition
- Light chain C domains κ or λ
- Heavy chain V domain
- Heavy chain C domains 3 x α, δ, γ, or 4 x μ, ε
- Papain cleavage sites- 2 x Fab 1 x Fc
- Domain structure allows diverse specificity with conserved Fc-dependent effector function
Why are they called ‘B’ cells?
1954 - Bruce Glick, Ohio State University
Functional studies of the bursa of Fabricius - a lymphoid organ in the cloacal region of birds
what was Bruce Glick’s experiment?
- Bursectomy – no apparent effect
- Bursectomised chickens re-used to raise anti-Salmonella antibodies
- Bursectomised chickens did not make anti-Salmonella antibodies
- The Bursa therefore the organ which antibody producing cells developed
- No bursa of Fabricius in mammals - bone marrow
how is Bone Marrow a maturation & differentiation microenvironment for B cells
- Regulates construction of antigen receptors
- Ensures each cell has one specificity
- Checks and disposes of self-reactive B cells
- Exports useful cells to the periphery
- Is a site of antibody production
how do Stromal cells nurture developing B cells?
- Contact between stromal cells and developing B cells
2. Secrete CYTOKINES at each stage of differentiation
how do Cytokines and cell-cell interactions regulate differentiation?
Different cytokines and cell-cell contacts are required at each stage of differentiation
how is The stage of differentiation is defined by Ig gene rearrangement
Stem cell- Germline unrearranged
Early pro-B- DH to JH
Late pro-B- VH to DHJH
Large pre-B- VHDHJH, PRE-B CELL RECEPTOR expressed
what are the Consequences of pre-B cell receptor ligation?
- Suppresses further H chain rearrangement
- Triggers entry into cell cycle
- Ensures only one specificty of Ab expressed per cell
- Expands only the pre-B cells with in frame VHDHJH joins
what is ALLELIC EXCLUSION
Expression of a gene on one chromosome prevents expression of the allele on the second chromosome
what is the Evidence for allelic exclusion of Ig genes?
- one allotype is inherited from each parent
- Allotypes identified by staining B cell surface Ig with antibodies
- Suppression of IgH gene rearrangement after pre-B cell receptor ligation prevents two specificities of antibody per B cell
why is Allelic exclusion important?
prevents autoimmunity
Suppression of IgH gene rearrangement ensures only one specificity of Ab per cell –may prevent pathogens provoking undesirable responses
why is allelic exclusion needed to prevent ‘holes in the repertoire?
- Two specificities of Ag receptor per cell – anti-brain Ig AND anti S. aureus
- Infection with S. aureus due to self tolerance
- ‘Hole’ in the repertoire of B cell specificities allows infection to proceed unchecked.
what are the Consequences of pre-B cell receptor ligation?
- Suppresses further H chain rearrangement
- Triggers entry into cell cycle
- Ensures only one specificty of Ab exparessed per cell
- Expands only the pre-B cells with in frame VHDHJH joins
why are Large pre-B cells need in frame VHDHJH joins to mature?
Only B cells with potential to make a B cell receptor can pass this stage
why is Heavy and light chain rearrangement wasteful?
- Two “random” joins – only a 1:9 chance of a rearrangement being in frame
- One “random” join only a 1:3 chance of a rearrangement being of frame
- Only a 1:27 chance of an in frame rearrangement per B cell
- Out of frame rearrangements arrest further B cell maturation
how do B cells have more than one chance to
rearrange heavy chains
- DH-JH or VH-DJH On first chromosome 14
2. DH-JH or VH-DJH On second chromosome 14
why is Heavy and light chain rearrangement wasteful?
- Only a 1:27 chance of an in frame rearrangement per B cell
- Out of frame rearrangements arrest further B cell maturation
what are Lymph nodes
A secondary lymphoid organ in which lymphocytes and antigens encounter each other
describe Lymphocyte recirculation through 2° lymphoid tissue
Non-lymphoid cells
Pass through the blood vessels in the lymph node and continue arterio-venous circulation
Lymphoid cells
Adhere to and squeeze between HIGH ENDOTHELIAL VENULES (HEV), then percolate through the lymph node and exit via the efferent lymphatic vessel
how do Cells with antigen receptors check for recognition of self antigens
EXPORT DELETION ANERGY STALLED MATURATION RECEPTOR EDITING
explain Germinal Centre formation
- Antigen drains in lymph and on cells into afferent lymphatic.
- Ag activated B cells increase CCR7 expression and move to CCL19/21 rich T/B zone border
- GERMINAL CENTRE forms – a transient structure of intense proliferation
- B cells released by germinal centre differentiate into plasma & memory cells
- Mature B cells leave blood via
HEV and home to the B cell follicles - CXCL13/CXCR5 dependent
whats the difference between Follicular dendritic cells
and DCs?
FDC- Probably develop from mesenchymal precursors
DC- Bone marrow derived
DC are the only antigen presenting cell that can present antigen to naïve (as opposed to memory) T cells.
DC are crucial to establishing the primary immune response
explain Germinal Centre formation
- Antigen drains in lymph and on cells into afferent lymphatic.
- Ag activated B cells increase CCR7 expression and move to CCL19/21 rich T/B zone border
- GERMINAL CENTRE forms
- B cells released by germinal centre differentiate into plasma & memory cells
- Mature B cells leave blood via HEV and home to the B cell follicles - CXCL13/CXCR5 dependent
describe FDC bead formation & release
- Veils on antigen-bearing DC surround the FDC beads.
- Immune complexes transferred from DC to FDC beads.
- B cells also involved in transport of immune complexes to FDC
- Immune complexes bind to and are taken up by B cell
surface immunoglobulin
explain Germinal Centre formation
- Antigen drains in lymph
- Ag activated B cells and move to T/B zone border
- GERMINAL CENTRE forms
- B cells released by germinal centre differentiate into plasma & memory cells
- Mature B cells leave blood via HEV and home to the B cell follicles
describe FDC bead formation & release
- antigen-bearing DC surround FDC beads.
- Immune complexes transferred from DC to FDC beads.
- B cells also involved in transport of immune complexes to FDC
- Immune complexes bind to and are taken up by B cell
surface immunoglobulin
how do T cells help B cells survive?
- Signal 1 antigen & antigen receptor
Signal 2 delivered via CD40 and cytokines in the germinal centre - B cells are inherently prone to die by apoptosis
- Signals 1 & 2 upregulate Bcl-XL, which rescues B cells from apoptosis
- Signal 2 from T cells allows the B cell to survive
- T cells control the CLONAL SELECTION of B cells
where do T cells control B cell clonal selection?
germinal centre
what is affinity maturation?
- Cells accumulate mutations in their CDR
- are selected or neglected on the basis of antigen binding affinity
- affinity ‘matures’ throughout the response
how do T helper cells control affinity maturation?
- if a cell has a higher affinity for antigen, it will
- bind more antigen (receive more signal 1)
- present more antigen to T cells (solicit and receive more signal 2)
- express more CD40 (receive more signal 2)
- express more cytokine receptors (receive more signal 2) - Receipt of more signal 1 and 2 gives the cell a selective advantage i.e. it is rescued from apoptosis and is clonally selected.
- Cells with lower affinity receptors are neglected and die by apoptosis
how do T cells help B cells survive?
T cells control CLONAL SELECTION of B cells
1. Signal 1 antigen & antigen receptor
Signal 2 delivered via CD40 and cytokines in the germinal centre
- B cells are inherently prone to die by apoptosis
- Signals 1 & 2 upregulate Bcl-XL, which rescues B cells from apoptosis
- Signal 2 from T cells allows the B cell to survive
how do T helper cells control antibody isotype switching in the germinal centre?
- Diverse effector function arises from ability to switch isotype-defining C genes
- Class Switch Recombination (CSR) is T cell dependent and takes place in the germinal centre
what are Switch regions?
Repetitive regions of DNA upstream of C regions
how do switch regions work?
- Switching similar to V(D)J recombination.
2. Occurs after B cell activation by antigen and help from T cells in germinal centres
what is Switch recombination?
At each recombination constant regions are deleted from the genome
An IgE - secreting B cell will never be able to switch to IgM, IgD, IgG1-4 or IgA1
what is an ALLOTyPE?
a polymorphism in a conserved region of Ig
what are High endothelial venules?
specialised properties to allow lymphocytes and no other types of cells into the lymph node
what is the GERMINAL CENTRE?
a transient structure of intense proliferation
