MODULE 3 - JO'S LECTURES Flashcards
where do B cells develop?
in the bone marrow
where do B cells get activated?
in the spleen or lymph nodes by naked antigen via BCR
what are some of the things B cells can do?
secrete antibody
present antigen on MHCII to CD4 T cells to activate them
can class switch (first isotype IgM)
can undergo somatic hypermutation (increasing ab affinity)
undergo clonal expansion
can differentiate in antibody producing plasma cells
how does BCR signal activate B cells with no signalling component?
has closely associated signalling components which are the invariant molecules Ig-alpha and Ig-beta
these closely associate with membrane bound Ig (BCR) to transduce signal into B cell
what are the clinical reasons it is important to understand B cell activation?
in order to:
develop new cures for cancer (B cell malignancies like lymphoma and leukaemia)
develop new treatments for AI diseases
make new vaccines
what are kinases?
enzymes that phosphorylate themselves and other proteins
phosphorylation can generate binding sites for other proteins to allow signal to transmit along chain
what are phosphotases?
take phosphate off things stopping signal transduction
what is the first messenger in BCR signalling?
naked antigen
what does the BCR consist of?
cell surface Ig (with two light and two heavy chains) + invariant signalling proteins Igalpha and Igbeta
Igalpha and Igbeta are connected by a disulphide-link to make them a heterodimer
what does the BCR co-receptor do and what does it consist of?
CD21 - complement receptor 2; binds complement-coupled antigen
CD19 - augments signalling through BCR and also recruits PI3 kinase
B cell equivalent of CD4/CD8; decreases BCR signalling threshold by amplifying BCR signals allowing B cells to respond to low levels of antigen
also includes CD81 which trafficks CD19 to cell surface
how and why can BCRs move around on the cell surface?
on lipid rafts
clustering together all the molecules is necessary to send a good signal down (I think this is called receptor clustering)
this clustering/aggregation is usually caused by highly repetitive structures e.g. flagellin
what is an example of T cell independent antigen?
highly repetitive structures like polysaccharide
how can antigen which is typically T-cell-dependent activate a BCR without T cell help?
monovalent soluble antigen can cause the Src family kinase Lyn to ‘disturb’ the actin cytoskeleton which obscures phosphorylation sites and open the BCR allowing signal transduction
how can antigen-bound complement fragments trigger the BCR?
antigen-bound complement fragments like C3d or C3b can bind to CD21; the antigen binds to the antibody (membrane bound?) resulting in cross-linking
outline the main intracellular signalling pathway for the BCR?
tyrosine phosphorylation is main mechanism by which transmembrane receptors transduce signals
phosphotyrosine residues are recognised by proteins containing the phospho-dependent binding domain SH2
SH2 domain proteins can act to recruit other molecules as enzymes or as transcription factors
what are the main tyrosines responsible for BCR signal transduction?
ITIMs (immunoreceptor tyrosine-based inhibitory motifs)
ITAMs (immunoreceptor tyrosine-based activation motifs)
these are found on Igalpha and Igbeta
how do ITAMs get phosphorylated BCR?
Src family kinases Fyn, Blk and Lyn associate with Igalpha and Igbeta
when ligand binds BCR and receptor clustering occurs the kinases phosphorylate tyrosines in the ITAMs on Igalpha and Igbeta
this allows Syk (another Src family kinase) to come in and bind the ITAMs on Igbeta too thus activating Syk
what does the Src family kinase Syk do?
Syk has two SH2 domains and binds ITAMs on Igbeta
by binding to phosphorylated site Syk becomes activated
Syk then phosphorylates the scaffold protein BLNK which induces formation of a membrane-associated signalling complex called the signalsome
what does BLNK do?
has multiple sites for tyrosine phosphorylation
it recruits other SH2 domain proteins to form the multi-protein signalling complex called the signalsome
what are the three pathways that can be activated by BCR signal transduction?
PLCgamma2 pathway (main pathway)
PI3K pathway
ERK pathway
outline the PLCgamma2 pathway of BCR signalling?
activated by Bruton’s tyrosine kinase (Btk) which gets recruited to the signalsome
this activates PLCgamma2 which then hydrolyses PIP2 into IP3 and DAG
this leads to increased intracellular calcium which cause PKC to get activated
PKC has multiple functions but the main one is activate MAPkinases which leads to TF activation
PKC also activates IKK which turns of inhibitory signal to NFkappB thus activating? it/turning it on
calcium flux also leads to activation of calcineurin which activates the TF NFAT
activation of these TFs leads to increased B cell activation and maturation
outline the PI3K pathway of BCR signalling?
phosphorylation of CD19 co-receptor by LYN activates PI3K
this leads to PIP3 production which recruits BCR signalling components to the plasma membrane and activates them
AKT is the main mediator of PI3K pathway and it leads to activation of genes which regulate cell cycle and apoptosis (leads to inactivation genes involved in apoptosis)
AKT activation induces pro-survival genes through IKK and mTOR (controls cell growth and proliferation)
overall the PI3K pathway promotes B cell growth and survival
outline the ERK pathway of BCR signalling?
RAS activation leads to ERK phosphorylation and dimer formation which allows it to translocate across nucleus to induce proliferation, survival and differentiation promoting genes
ERK can also be activated by PLCgamma2 pathway
how is BCR signalling negatively regulated?
ITIM phosphorylation turns shit off
it recruits phosphotases that dephosphorylate adaptor proteins like BLNK and also activates cellular calcium releases impairing calcium mediated signalling
how is BCR signalling negatively regulated?
ITIM phosphorylation turns shit off
it recruits phosphotases that dephosphorylate adaptor proteins like BLNK and also activates cellular calcium releases impairing calcium mediated signalling
what are two key defects of B cell development and differentiation?
Bruton’s tyrosine kinase (Btk) deficiency
Igalpha deficiency (agammaglobulinaemia)
what is Bruton’s tyrosine kinase deficiency?
Bruton’s disease or X-linked agammaglobulinemia means there is low or no Igs in blood (all isotypes)
Btk gene on X chromosome so only males get it
Btk is a crucial component of the signalsome as it phosphorylates and activates PLCgamma2. If no Btk this means no B cell proliferation, activation or maturation
symptoms include frequent infections and delayed growth
treatment involves regular intravenous gamma globulin and prompt treatment of any cuts/infections
what is Igalpha deficiency?
Igalpha crucial signalling component of the BCR
agammaglobulinaemia
symptoms include frequent infections and delayed growth and treatment involves regular intravenous gamma globulin and prompt treatment of any cuts/infections
what are the four key phases of B cell development?
BCRs form in bone marrow and B cell precursors rearrange Ig genes
negative selection occurs in bone marrow, immature B cell bound to self Ag is removed
B cells migrate to periphery, mature B cells bound to foreign antigen get activated
activated B cells give rise to plasma and memory cells leading to antibody secretion and memory cells in bone marrow and lymphoid tissue
how do autoreactive B cells get a second chance?
light chain encoded by kappa and lambda so if you are autoreactive you get a second chance to express other genes
but if those are still self-reactive you die
what occurs during antigen independent B cell development?
this is the first part of B cell development in the bone marrow where you get generation of lymphocyte diversity through Ig gene rearrangement
the B cell precursors here are stem cell, then pro B cell and then pre B cell
what occurs during antigen dependent B cell development?
the second half of B cell development which involves immature B cell, mature B cell, germinal centre B cell, memory and plasma B cells
important things which happen here include somatic hypermutation and antibody class switching (these occur once mature B cell binds foreign antigen)
what the fuck is the germinal centre?
mostly composed of proliferating B cells and 10% antigen-specific T cells
is very dynamic - grows as immune response increases and shrinks and disappears once infection is cleared
germinal centres generally found in lymph node or spleen
why is it beneficial that IgM gets secreted in pentameric form?
IgM gets secreted very early on in infection so tends to have very low binding affinity for antigen as hasn’t undergone somatic hypermutation
so instead its avidity gets increased by having lots of antigen binding points
what is antibody class switching?
genes for different isotypes ordered from IgM, IgD, IgG, IgE and then IgA
cytokines can induce RNA transcript production in “switch regions” near the heavy chain segment they preferentially induce (switch regions located just ahead of isotope genes)
so basically excises DNA for that isotope allowing the next to be expressed
once this DNA excised B cell cannot go back to making that isotype
how do switch regions allow class switching?
switch region sequences cause formation of secondary DNA structures that promote pausing of RNA polymerase II and binding of activation-induced deaminase (AID) which then introduces DNA breaks
AID can only bind ssDNA (like where gene transcription is occurring and RNA poly II has been paused)
AID also only expressed in activated B cells (it gets upregulated on activation)
how does AID drive somatic hypermutation?
AID works by deamination of cytosine to uridine which causes the mismatch repair system to try repair this resulting in point mutations (somatic hypermutation) and the good ones which increase affinity are selected for (affinity maturation)
this occurs in the IgV (variable) regions which encode protein that binds antigen
how does AID drive class switching and how is this different to the way it drives somatic hypermutation?
AID deaminates switch region instead of IgV region which results in double stranded breaks
repair mechanism for ds breaks just excises out the affected area and any genes (for any isotope) within that get lost
this means that once this DNA is looped out you can’t get those genes back and express that class of Ig and the next class on will be expressed
so the excising of these ds breaks allows for class switch recombination and the expression of new classes of antibody
how do cytokines regulate class switching?
cytokines regulate the gene transcription of switch regions
e.g. IL-4 enhances gene expression of switch region ahead of IgE so AID will deaminase this one
prob coming from an antigen-specific T cell in the germinal centre
what genes are expressed in a naive B cell and what happens when you add LPS and IL-4 to it?
recombined VDJ region encoding variable genes and also constant genes encoded by Cmew and Cdelta (making IgM and IgD)
if you add LPS these genes get upregulated but if you add IL-4 too you start to see gene expression in switch regions preceding IgG1 and IgE
outline somatic hypermutation?
occurs after antigen-driven B cell activation as part of germinal centre response and after receiving T cell signals
involves the introduction of point mutations to rearranged V region genes, ones that increase antigen affinity outcompete the others
50% chance that B cell will acquire a mutation in antibody it encodes at each division
requires activation of AID which deaminated DNA leading to base pair mismatches, lesion repair induces mutations
doesn’t have the same sequences that pause RNA polymerase II as switch regions do, no-one knows how it occurs in IgV gene for somatic hypermutation
how do 3’ enhancers ensure AID recruitment is specific to the discussed regions?
3’ enhancers after C regions enable chromosomal looping and this forms transcriptional domains that control transcriptional levels enabling AID to bind
without the 3’ enhancers there is no somatic hypermutation or class switching
what antibody isotype is best at opsonisation?
IgG1
what antibody isotype is best at sensitisation of mast cells?
IgE
what antibody isotype is best at complement activation?
IgM
what are the key functions of antibody isotypes?
neutralisation
opsonisation
sensitisation of mast cells
complement activation
each isotype is good at some of these to varying extents i.e. they all have different roles
how are antibody isotypes distributed?
distribution varied as they go different places to exert function
only ones that get through to placenta are IgG isotypes
all are found in serum
what are the two cells which exhibit B cell memory?
plasma cells
memory B cells
what are the differences between short-lived and long-lived plasma cells?
short-lived plasma cells reside in the lymph node or spleen
long-lived plasma cells reside in the bone marrow where they receive survival signals from stromal cells so that they can last months. These are a source of long lasting high affinity class switched antibody
both are antibody factories
what are memory B cells?
arise from germinal centre reaction (occurs in lymph node or spleen) and have inherited the genetic changes from this meaning they express high-affinity antibody and have undergone antibody class switching
they express higher levels of MHCII and co-stimulatory molecules than naive B cells so that they are ready to interact with T cells
thye populate the spleen and lymph nodes and circulate through the blood and divide slowly if at all and express surface Ig but do not secrete antibody and quickly generate antibody producing plasma cells if they re-encounter antigen
most current vaccines rely on B cell memory
what are memory T cells?
long-lived cells which survive after the contraction of effector phase resulting in a higher precursor frequency
have different activation requirements making them much easier to activate and respond upon reinfection and this also means different cell surface proteins from naive and effector cells
cytokines IL-7 and IL-15 important for memory T cell survival as well as periodic contact with MHC-peptide
divide more frequently than naive T cells
why are memory T cells better than naive?
proliferate more than naive T cells and in response to lower amounts of antigen
require less co-stimulation for activation than naive cells
produce cytokine faster and retain their polarised phenotype
may be strategically positioned in the tissue where the pathogen is most likely to be re-encountered
compare and contrast central and effector memory T cells?
central memory - express lymph-node homing molecules, slower than effector memory cells to produce cytokine, generally found in secondary lymphoid tissues e.g. spleen, lymph-node
effector memory - lack lymph-node homing molecule expression, rapidly produce cytokine upon antigenic stimulation, generally found circulating blood and tissues (might pass through lymph-node briefly)
