MODULE 1 - ALEX'S LECTURES Flashcards
what is a payoff of having an adaptive immune system and why?
autoimmunity
because it involves rearranging DNA in B and T cells
what are key components of an adaptive immune system?
MHC molecules, T and B lymphocytes
what are some selection pressures that have shaped our immune systems and how have we coped/responded to this?
selection pressures = unknown emerging diseases
responses = generation of diversity in B and T cells (adaptive immune system)
what is a key problem with somatic cell DNA rearrangements of gremlin encoded receptors in B and T cells?
autoreactivity
what was the Cambrian explosion and what might have lead to mass extinctions following this?
a great increase in multicellular life thus coinciding with an increase in innate and adaptive immunity
lack of adaptive immune system may have lead to extinctions after this due to emergence of things like viruses
also could’ve been due to decreasing oxygen/increasing sulphur
what are immunoglobulins?
similar shaped proteins which make up the backbone of TCR and BCR systems
what are variable lymphocyte receptors (VLRs)?
structures in agnathans (jawless fish) which have LRRs and similar functions to immunoglobulin
do agnathans have toll-like receptors?
yes
how do you know if a fish has an adaptive immune system?
if it has a jaw
i.e. NOT an agnathan
why did adaptive immunity evolve in jawed fish?
jaw allows increased prey range so more microbial exposure
skeletal meals increase risk of gut/tissue perforation
longer lifespan and extended reproductive age so greater chance of infection
how did agnathans and normal vertebrates benefit from whole genome duplication (when you get an extra copy of a chromosome)?
extra copy of chromosome allows some of the genes to mutate and take on new functions e.g. developing MHC
what are genome duplications often accompanied by?
morphological and functional innovations
this is because genetic changes in one copy of a gene allows diversification of function in a new ‘paralogue’
what is a paralogue and what aspect of the adaptive immune system do they form the basis of?
the genetically different copy of a gene following genome duplication
likely form the basis of the MHC system by allowing new functions of classical lipid-binding MHC allowing it to bind peptides
what is RAG?
recombinase activating gene
an enzyme which allows for rearrangement of DNA and catalyses immunoglobulin recombination which is necessary for TCR and BCR rearrangements
likely derived from ancestral transposon
RAGs are very specific to rearranging BCR and TCR
how did a transposon go on to form RAG?
original transposon encoded transposase enzyme which allowed it to move and integrate transposase recognised sequences
it jumped into our genomes and took on new function and also duplicated into RAG1 and RAG2 which both catalyses rearrangement of T and B cells
they are also separated from original integration sites
when are RAG genes most expressed/active?
during B and T cell development when they get their first receptors
it was thought agnathans such as hagfish have no adaptive immune response. what was recently discovered which disproved this?
it was found they didn’t miss out on an Ig-based adaptive immune systems, they just developed totally different receptors called VLRs
what are VLRs (variable lymphocyte receptors)?
VLRs are leucine rich repeat proteins (similar to TLR structure) which are in gremlin configuration in hagfish T and B-like cells prior to diversification
they undergo recombination with flanking sequences which involves homology as opposed to looping out like us
ultimately hagfish/lamprey lymphocytes (which are similar to our B and T cells but different) secrete soluble VLR in their blood (just like Ig in us!)
what are the two hagfish lymphocyte-like cells and what are their functions?
two distinct types of cell - VLRA and VLRB
VLRA seems to act like a T cell with cell surface receptors acting as a trigger
VLRB seems to act like a B cell by secreting VLRs (as if its Ig antibody)
TLRs and mannose receptors of the innate system are germline encoded, what does this mean?
they don’t change and are expressed all through your body in many cells
this works as they sense conserved microbial structures?
antibody and TCRs of the adaptive system are in germline state in most cells of your body and so are not expressed, so how are they expressed in B and T cells?
in B and T cells they have mechanisms for rearrangement of germline genes allowing them to be expressed as proteins
expression can only occur once rearrangement occurs
this gene rearrangment involves looping out variable genes (requires RAG enzyme) thus bringing together other genes which can be transcribed as a single transcriptional unit in B and T cells
hagfish VLRs (variable lymphocyte receptors) are not rearranged like human antibody and TCRs, so how does their expression occur?
they undergo gene conversion where germline genes fold together and some homology occurs between VLR subunits leading to gene conversion rather than looping
gene rearrangement and gene conversion are both examples of what?
somatic cell DNA alterations (also remember these can lead to autoimmunity, the big pitfall of adaptive systems of immunity)
what is DSCAM?
a gene in drosophila which is involved in neuronal wiring but also host defence by opsonising bacteria to facilitate uptake by phagocytes
splicing of the clusters of exons which are in DSCAM can produce thousands of isoforms, so why is this not an adaptive system?
because for it to be adaptive you need a lymphocyte to replicate that isoform (cloning) rather than just randomly produce them
i.e. if you immunise a drosophila it won’t have a stronger immune response next time
sea urchins have 222 TLRs well we have 10, why have they still not got an adaptive immune system?
for it to be adaptive the sea urchin phagocytes would need to divide keep select TLRs on their surface not just everyone i.e. expression is not clonally distributed
so if you immunise a sea urchin it will not change its immune response
what do the drosophila and sea urchin examples tell us about adaptive immune system requirements?
no clonal selection = no memory response = no adaptive system
in immature T or B cells, the TCR/BCR genes are in germline state. What does rearrangement of these genes do?
rearrangement generates diversity by looping out variable genes allowing others to be brought together and be expressed as a single unit
this is how mature (naive) T and B cells express unique antigen receptors
this also means in germline state Ig genes not expressed (BCR genes)
what is clonal selection?
selective expansion or lymphocytes that interact with antigen
one B or T cell has one type of receptor on its surface with thousands of copies of it. If that receptor is for the right antigen then that lymphocyte will undergo clonal expansion
what is a neutrophil?
produced in the bone marrow
the most abundant leukocyte but also short-lived
produce ROS and neutrophil extracellular traps (NETS)
what is the major component of pus?
dead and dying neutrophils
what does ROS production by neutrophils lead to?
breakdown of the neutrophil and thus the release of DNA and histones (neutrophil extracellular traps or NETS)
what do neutrophil extracellular traps (NETS) do?
bind bacteria
what are dendritic cells?
APCs which can activate naive T cells
express both MHCI and II (upregulate MHCII especially after activation) and also express costimulatory molecules
migratory function as they ferry antigen from the periphery to the lymph node
why are dendritic cells the best APC for activating naive (mature) T cells??
they are the most potent APC
when dendritic cells migrate to the lymph node what is the first section of it that they go to?
the T cell zone (makes sense cause they wanna present that antigen asap, get there via chemokinetic signals)
what is the main immune cell involved in transplant rejection?
dendritic cells
what are the dendritic cell subsets derived from?
common myeloid progenitor cells
what are the three main subsets of dendritic cells?
langerhans cells
dermal dendritic cells
thymic dendritic cells
all these have primary roles which they are best suited for
what are langerhans cells?
a subset of dendritic cells which are found in the epidermis
what are dermal dendritic cells?
dendritic cells specialised in cross-priming
what are thymic dendritic cells?
dendritic cells which mediate central tolerance
what is cross-priming?
allows peptides from exogenous antigen being processed in the endosome to leak into the cytoplasm of the dendritic cell and be presented very quickly onto MHCI to activate cytotoxic (CD8) T cells
what does MHCII-peptide activate?
helper (CD4) T cells
what are conventional dendritic cells?
the main subset of DCs involved in stimulating naive memory T cells
what cytokines do plasmacytoid dendritic cells release and why?
release large amounts of interferon type I (IFNa) which tells neighbouring cells to downregulate protein transcription and upregulate MHC expression
IFNa is very effective against viral infections
why are plasmacytoid DCs called that?
cause they look like plasma cells as they have a lot of golgi in them which allows for production of lots of cytokines
what does lack of co-stimulation lead to?
anergy (non-responsiveness) or cell death (apoptosis)
they would have received signal one (MHC-peptide presentation) but signal two (costimulation) is required for activation
signal 2 (costimulation) only occurs if the dendritic cell has been activated
this guards against autoimmunity and is a mechanism of peripheral tolerance
just signal 1 =
OFF
signal 1 + signal 2 =
ON
how are dendritic cells activated?
stimulation by signals from stressed cells or microbes (via TLR) causing the DC to express costimulatory molecules (CD80/86) on its surface as well as upregulate MHC expression
this activation can also be thought of as maturation, where the DC becomes competent to activate naive T cells
what are macrophages?
have many functions in the body, mainly antigen clearance
precursor is monocyte in blood
inappropriate differentiation of macrophages can lead to atherosclerosis (heart disease) as they can express coagulation factors
outline the interaction between a CD4 (helper) T cell and a macrophage?
presentation of intracellular antigens by macrophages leads to interactions with CD4 T cells which then release IFN-gamma which activates anti-bacteria activity in macrophage
so macrophage activation by CD4 T cells enhances antimicrobial immunity
what do natural killer cells do?
recognise inhibitory receptors (MHCI) on normal cells
positive signal builds up and unless inhibited by signal from MHC NK cell becomes activated and lyses target cell
are primarily an innate cell but are essential for development of tumour immunity
can infiltrate lymph nodes and contribute to T cell activation
why are natural killer cells considered innate cells?
they don’t undergo generation of diversity like T and B cells do
what is antibody-dependent cellular cytotoxicity (ADCC)?
the other way NK cells can kill infected or cancerous host cells
NK cells have CD16 Fc receptors which are good at recognising and binding the Fc domain on cell-bound antibodies (ABs which have bound antigen on surface of target cell)
this binding causes cross-linking of CD16 triggering degranulation into a lytic synapse killing the infected/tumour cell via apoptosis
this could be how giving someone therapeutic antibodies can help cancer patients
other than ADCC, what else are Fc receptors critical for?
complement
what is the name of the Fc receptor on NK cells involved in antibody-dependent cellular cytotoxicity (ADCC)?
CD16
when might we need to use engineered T cells (CAR T cells)?
if a cancer has gotten too big or the tumour isn’t very hot (doesn’t mutate as much so fewer peptides on surface so illicits a weaker immune response)
how do you make CAR T cells?
take a bunch of blood from patient, cycle it through aphaeresis machine so it spins out the plasma, grow up the T cells from this in vitro
put crippled lentivirus in which has CAR (chimeric antigen receptor) which is a spliced antibody component with the cytoplasmic domains CD3 (involved in signal 1) and CD28 (involved in signal 2)
how do CAR T cells work?
you can splice the antibody component (CAR) to be specific for whatever antigen you want to target e.g. tumour antigens
so the CAR T cells have their original TCRs but now also have a chimeric receptor which when triggered activates the T cell allowing it to target the cancer cell
this is proven to be very effective against B cell lymphomas and relapsed/refractory disease
where is complement made?
the liver
where are Toll-Like receptors (TLRs) most active?
the endosome
but we also have some in the cytoplasm looking for microbial nucleic acids which looks different to our mRNA which you might find in the cytoplasm
what is the convertase for most complement pathways?
C2b and C4b
what is the broad idea behind the complement pathways?
different pathways which lead to activation of C3 complement via cleavage to C3b which is deposited on microbial surface thus stimulating membrane attack complex which leads to lysis of cell
outline the classical complement pathway?
antibody (especially IgM) activates this pathway by triggering C1 molecules to bind to microbial cell surface which activates proteases that cleave C2 and C4 to form C2b-C4b convertase which then cleaves C3 to C3b (active complement) which is deposited on cell surface leading to formation of membrane attack complex
outline the lectin complement pathway?
carbohydrates (e.g. LPS) activate this pathway by triggering mannose-binding lectin molecules to bind to the microbial cell surface which activates proteases that cleave C2 and C4 into C2b-C4b convertase which cleaves C3 to active complement (C3b) which can then be deposited on microbial cell surface leading to formation of membrane attack complex
outline the alternative complement pathway?
occurs via spontaneous hydrolysis (of C3 to C3b which then becomes its own convertase by complexing with some molecules) so is always occurring unless inhibited by transmembrane molecules called complement inhibitors in our membranes which deflect any complement from our cell surface
microbes don’t have complement inhibitors which is why they get fucked up by complement and we don’t
so our way of switching off the alternative pathway is complement inhibitors
what happens to someone who has a deficiency in the enzyme that locks complement inhibitors into our membranes?
they piss red cause of all the dead lysed RBCs that end up in their urine
what is occurring on a molecular level when complement binds to cell surfaces?
C3 binds to microbial membrane once its thioester domain (TED) which sits in the C3 alpha domain is activated
TED activation is inhibited by the inhibitory beta domain which blocks it. Once C3 cleaves beta-chain this exposes highly reactive alpha chain TED creating the active complement molecule C3b. C3b is then immediately bound to adjacent microbes or inactivated by hydrolysis
what happens to any C3b that has nothing to bind to?
it gets inactivated quickly by water (via hydrolysis)
what is beneficial about the TED being so reactive?
it will react with and bind pretty much anything on the microbial cell surface
what happens with C3a and C5a (leftovers which float away after cleavage of C3)?
they act as inflammatory signals allowing other innate cells like neutrophils to come and help clean up the mess
what do complement inhibitors do?
prevent complement from binding and/or inactivate the complement
what happens from CD59 deficiency?
decreased complement inhibition causing paroxysmal nocturnal haemoglobinuria (PNH) which is where you piss red in the morning cause of your urine retention exposing all the lysed RBCs
what is CD59?
it is a complement inhibitor in the membrane of host cells which prevents the assembly of the membrane attack complex (i.e. inhibits the later components of complement which form the complex/pore)
what happens when bacteria stimulate the release of IL-6 from macrophages?
IL-6 goes and acts on the liver to induce release of acute phase reactants which are inflammation markers such as C-reactive protein (CRP)
what is C-reactive protein (CRP) and what does it do?
it is an acute phase reactant which binds phosphocholine which is common on bacterial cell surfaces
it is also an important biomarker in clinical scenarios for bacterial infections (not really for viral infections)
how did Toll-like receptors (TLRs) get their name?
they look very similar to drosophila toll receptors
what are toll-like receptors (TLRs)?
dimers with leucine-rich repeat (LRR) domains that bind PAMPs and DAMPs (pathogen and damage associated molecular patterns)
do TLRs only recognise and bind pathogen associated molecules?
no they also do so for commensals however we usually have firewalls up keeping the commensals from becoming invasive
however if body is compromised (e.g. immunosuppressants) then commensals may become more invasive like pathogens making them opportunistic pathogens
what is similar and different in the immune responses to gram-neg (pink) and gram-pos(purple)?
they display different molecules so are recognised by different TLRs in the innate system
gram-negative cell membranes have LPS so are detected by TLR-4
gram-positive bacteria cell membranes have lipoteichoic acid so are detected by TLR-2
both types of bacterial membrane are destroyed by lysozyme, they just have different recognition systems
so the innate system can distinguish between different classes of microbes (also like fungi and shit)
what is lysozyme?
an important enzyme of the innate system that can destroy both classes of bacterial membrane
why is the innate system distinguishing between types of microbe important?
so it can have an effective and specialised adaptive response (i.e. different T cells for different microbes)
how can we detect so many different molecules with only 10 different TLR’s?
TLRs are homodimers (TLR3, 4 etc.) but they can also form heterodimers which changes their secondary and tertiary structure allowing recognition of lots of molecules
why are TLRs that recognise nucleic acids most efficient in the endosome/lysosome?
because the low pH protonates the microbial nucleic acids making recognition by the TLRs easier and more efficient
what is the TIR domain of TLRs?
the TLRs recruit different adapter proteins to their TIR domain allowing them to activate different signalling pathways
what are some of the key signalling pathways activated by TLRs?
NF-kappaB transcription factor activation
MAP kinase pathway leading to downstream signalling to activate transcription factor AP-1
interferon regulating factor (IRF) pathways (ramps up the interferon response e.g. down regulate protein transcription and up regular MHC expression so more commonly activated by viral infections)
the first two pathways are more generalised activation pathways associated with inflammation
why might you find the IRF pathways to be more active for TLRs that detect nucleic acids?
cause it ramps up interferon responses which is important for viral infections
what is the general process of TLR activation and signalling?
LRRs detect and bind ligands
TIR domain recruits adaptor molecules
signal transduction mediated via phosphorylation leads to activation of transcription factors
transcription factors translocate across nuclear membrane into nucleus and initiate transcription of usually silent genes
are DCs and innate immune cell or an adaptive immune cell?
neither
they are more like a bridge between the innate and adaptive response
this is cause they have lots of molecules involved in adaptive immunity (e.g. MHC) but also lots involved in innate immunity (e.g. TLRs)
they are also very involved in kicking off inflammation via IRF pathways (especially plasmocytoid DCs)
which immune cells have TLRs?
pretty much all of them I think
what do nucleic acid sensors in the cytoplasm do?
detect nucleic acids that shouldn’t be there and stimulate type I interferon production which acts on neighbouring cells via IFNAR triggering the JAK-STAT pathway which prevents from viral infections
some TLRs are a type of nucleic acid sensor
what are RIG-I and MDA5?
nucleic acid sensors (not TLRs or TFs) which detect short (uncapped) dsRNA and long dsRNA
when activated RIG-I or MDA5 coalesce on mitochondria with MAVS (a protein) to activate IRF and NF-kappaB transcription factors
this induces interferon type I (IFN-a and IFN-b) transcription along with other anti-viral genes
why can RIGI and MDA5 usually detect viral RNA even if it isn’t double stranded?
viruses in a cell usually overproduce nucleic acids so the overabundance of viral RNA tangles up and forms detectable ds structures
how do we distinguish host RNA/DNA from microbial RNA/DNA?
it is in a strange location e.g. DNA in cytoplasm, RNA in endosome
OR
it has an unusual structure e.g. CpG-rich DNA, uncapped RNA
we have multiple systems to detect different kinds of nucleic acids in our cytoplasm
what does RIG-I do?
recognises uncapped RNA with 5’-triphosphate (therefor no methyl guanosine)
RIG-I activation results in type I interferon production as well as classic NF-kappaB induced genes
how many amino acids are in a peptide which binds MHC class I?
8-9 amino acids
how many amino acids are in a peptide which binds MHC class II?
13-25 amino acids
MHCI and II can be considered peptide receptors, true or false?
true!
which cells in your body express MHCI?
all of them except red blood cells
what makes a cell an antigen presenting cell?
it must express MHCII
which cells express MHCII?
B cells
macrophages
dendritic cells
epithelial cells of the thymus
so all of these are APCs
T cells can pick up some MHCII in mice but not in humans
why is MHCII so enriched in epithelial cells of the thymus?
these cells are often involved in deletion of autoreactive T cells
outline the structure of MHCI?
has a single alpha chain with a beta2-microglobulin
most polymorphisms found in peptide binding groove of alpha chain
has a single transmembrane domain with a short cytoplasmic tail
outline the structure of MHCII?
has two chains - an alpha chain and a beta chain
most polymorphisms found in peptide binding groove on the beta chain
has two transmembrane domain both with a short cytoplasmic tail
what is the purpose of the transmembrane domain/s in MHC?
both MHCI and II have to be transported to the cell surface in vesicles, during which the transmembrane domain is embedded in the membrane
if it is not in a membrane at any time MHC will get all fucked up
outline the structure of the peptide binding groove of MHCI and II (similarities and differences)?
both have a beta sheet on the bottom of the groove with alpha helices around the side
MHCII binding groove has slightly more open ends allowing longer peptides to bind
what is MHC-peptide binding mediated by and what does this mean for amino acid binding in general?
anchor residues
so not all amino acids residues in a peptide are needed for binding, there will be conserved amino acids (anchor residues) which are critical for binding
there will be a similar pattern of anchor residues between different peptides which bind the same type of MHC
how can peptides be released (eluted) from MHC and why is this useful?
using acidic buffers (pH2-3)
this allows us to then collect the peptides and figure out what peptides are bound by different types of MHC
why might some peptides bulge?
if they are long they often bulge
this is allg because as long as the anchor residues are hydrogen bonding with MHC molecule the other amino acids don’t have to bind
which MHC class possesses conserved amino acid residues at either end of the binding groove and why?
MHCI has conserved amino acid residues at either end of the groove which bind the two ends of the peptide constraining it to a length of about 9 amino acids
MHCII lacks these conserved amino acids and instead is in an open conformation allowing longer peptides to fit in the binding groove
what types of bonds do anchor residues form with conserved amino acids in MHC class I?
hydrogen bonds or weak electrostatic forces
NEVER covalent
what do the differences in the ends of the alpha helices at either end of the binding groove allow MHCII?
allow it to be in extended open conformation
how are MHC alleles expressed?
co-dominantly i.e. all at once
what are polymorphisms?
mutations that have been selected by evolution and distributed throughout the species
the majority of polymorphisms in MHC are in the regions which contact the peptide
what is a consensus sequence and how does this relate to MHC?
a consensus sequence is the most frequent sequence in a population
if you look at the MHC consensus sequence you will find very few mutations outside the peptide binding groove. This probably means that any mutation outside of the groove is probably harmful and so is negatively selected for. It also means that MHC has a lot of tolerance/flexibility for mutations in the binding groove
where do we find some of the most variable genes in our body?
the MHC class II beta chain
which end of the alpha chain in MHCI is the C terminus?
the cytoplasmic tail
why is the alpha chain relatively invariant (doesn’t have many mutations)?
probably because its structure/function gets fucked if there are too many mutations
what are the three types of MHCII we have in our body?
HLA-DP
HLA-DQ
HLA-DR
you will have two of each of these on different chromosomes, one set either parent and usually of differing sequences
they will all be co-dominantly expressed on every APC in your body
what are the three types of MHCI we have in our body?
HLA-A
HLA-B
HLA-C
you will have two of each of these on different chromosomes, one set either parent and usually of differing sequences
they will all be co-dominantly expressed on every cell in your body (apart from RBCs)
what is non-classical MHC?
can bind lipids and other non-peptide shit
have fewer mutations and are more conserved than normal MHC
what are the two MIC types of non-classical MHC and what do they do?
MICA
MICB
both are stress ligands that are very important for activating NK cells
can be unregulated during infection or in cancer cells which is effective as it make the NK cell angry
what is CD1 family?
another form of non-classical MHC which presents glycolipids to NK-T cells
what are NK-T cells?
basically just NK cells with a TCR (therefor not an innate cell)
the TCR on NK-T cells is quite invariant and recognises molecules presented by CD1
MHC-peptide interactions and MHC-peptide interactions with the TCR are…
low affinity
they are weak, non-covalent interactions
the TCR recognises MHC-peptide as an…
entire fucking complex
what are the three sources of peptide for MHCI loading?
classical pathway - proteins degraded to peptides within cytoplasm and destined for MHCI via ubiquitin tagging (often old proteins that need to be turned over)
cross priming (allows exogenous antigen to enter MHCI pathway)
early protein translation fragments (DRIPS)
what are DRIPS?
defective ribosomal products (DRiPs) aka primary translation products (PTPs)
the junk of translation e.g. improper RNA splicing, translations of frameshift, improperly folded proteins, stalling ribosomes
these preferentially enter the MHCI pathway and are a compelling explanation for how MHCI loading occurs so quickly (ubiquitin tagging and degradation can take a while)
often may be a result of a viral infection as this results in a burst of protein synthesis and thus a lot of waste peptides (RNA which didn’t turn into a 100% product of protein)
why are DRIPS so much faster than normally derived peptides?
peptides derived from mature proteins (e.g. viral and host-derived) reach the proteasome in a slow fashion
in contrast the junk of translation (DRiPs) are immediately degraded by the proteasome
both pathways end up providing MHCI with peptide in the ER
how can DRIPS be used to increase the efficiency of vaccines?
you could change the structure of mRNA in vaccines so as to encourage DRIP formation and subsequently encourage CD8 T cell formation
which MHCI loading pathway/s is the proteasome important for?
all of them
its like the final blender which trims down peptides to about 9 amino acids
the proteasome is present in the cytoplasm
what is ubiquitin?
a protein used to tag other proteins that need to be degraded
it then basically drags them kicking and screaming to the proteasome
what is the immunoproteasome?
induced by interferons (so upregulated during infection) and alters the processing of peptides
increased protein cleavage after hydrophobic residues and decreased cleavage after acidic residues resulting in the generation of peptides able to be better transported by TAP and with better terminal anchor residues for insertion into MHCI
basically just like the proteasome but better at blending the peptides so MHC loaded a bit more efficiently
why is it not good to have the immunoproteasome on all the time?
it works by generating peptides from your own self proteins meaning that it can predispose you to autoimmunity
so we just have it set up as an inducible system (by interferons) which can be upregulated during infection
what does the Transporter associated with Antigen Processing (TAP) do?
is a complex (Tap1 and Tap2) that imports peptides into the ER for MHCI loading once the proteasome has done its job
how does the TAP complex bind and transport proteasome-processed peptide?
Tap1 and 2 have a hydrophobic transmembrane domain with an ATP-binding cassette (ABC) domain in the cytoplasm
once this domain binds the processed peptide it is transported across the ER membrane into the lumen of the ER for MHCI loading
outline the process of MHCI maturation and loading?
immature (cause has a chaperone called calnexin bound) MHCI sits partly folded in ER waiting for beta2-microglobulin to bind
once b2m binds it releases calnexin and binds a complex of chaperone proteins (calreticulin and ERp57) and binds to TAP via a chaperone called tapasin
TAP delivers peptide to ER (maybe after a bit of trimming from ERAAP) and it binds MHCI well the chaperones fuck off, thus completing MHCI folding
vesicle starts to form (remember MHC transmembrane domains must always be in a membrane otherwise it’ll fall apart and won’t reach cell surface
what is Endoplasmic Reticulum Aminopeptidase associated with Antigen Processing (ERAAP)?
an enzyme which chills in the ER and ensures the peptides are the right length for MHC
if they aren’t ERAAP will give them a trim so that they are
unregulated by interferon-gamma (so more trimming when there’s interferon around such as during a viral infection which makes sense cause that’s when you’d really want to be looking after the peptides)
what is CIITA?
the master transcription factor which controls MHCII expression
is only active in APCs thus ensuring specific expression of MHCII by APCs
what is the most abundantly expressed MHC allele?
HLA-DR (MHC co-dominantly expressed but you find DR a bit more on most cells than the other MHC)
it has the highest number of polymorphisms
why are you absolutely fucked if you don’t have CIITA?
cause you won’t have any MHCII on your cell surfaces
what occurs during MHCII processing?
invariant chain (li) is the chaperone which holds MHCII together well it waits for peptide
once in endosome invariant chain cleaved by acidic environment leaving a short peptide fragment (CLIP) still bound to MHCII
endocytose antigens are degraded to peptides in endosome, but CLIP blocks them from binding MHCII
HLA-DM enzyme binds MHCII making it release CLIP thus allowing the peptides to bind
MHCII then travels to cell surface
which class of MHC undergoes exogenous processing?
MHCII
what does the enzyme HLA-DM do?
binds to and stabilises MHCII thus catalysing the release of CLIP
what does allogeneic mean?
relating to tissues or cells that are genetically dissimilar and thus immunologically incompatible despite being from individuals of the same species
what are the two main types of graft rejection?
direct (relating to MHC)
indirect (relating to different peptides within graft from enzymes with polymorphisms)
what are the two possible outcomes when T cells are presented with MHC from another individual?
they either ignore it or react very strongly (alloreaction)
alloreaction occurs in about 1-5% of T cells which is enough to cause graft rejection
how does the thymus screen T cells to ensure they behave normally?
it has lots of MHC which it displays to the T cells and if they react strongly they are deleted (negatively selected) and if they react too weakly or don’t recognise at all they aren’t selected for cause they useless and will never recognise MHC (death by neglect)
if they have the perfect intermediate affinity (moderate signal) then they are positively selected
what does it mean when Alex says “1-5% of T cells can be alloreactive”?
regardless of the peptide in the groove, 1-5% will respond vigorously to someone else’s MHC
what can alloreaction do in regards to the transplant?
cause transplant rejection
what are the three isotopes of MHCI?
HLA-A
HLA-B
HLA-C
what are the three isotypes of MHCII?
HLA-DR
HLA-DP
HLA-DQ
how the fuck can there be three isotypes of MHCI and MHCII?
there genes are lying next to each other on the chromosome but they make different proteins with similar functions
probably are recognised by different T cells too
and then you’ve got two of each one on different chromosomes cause paternal and maternal chromosome
so expressing six of each type of MHC codominantly at any given time so 12 polymorphic sets of MHC at once
what are the most important HLA alleles to match for transplants and why?
HLA-A
HLA-B
HLA-DR
because although they are codominantly expressed some genes are stronger expressed and have more enriched polymorphisms making them more important to match
how do you read HLA types e.g. read HLA-A*02:01?
HLA-A*02:01
HLA-A - gene
02 - allele group
01 - protein type
and I’m pretty sure protein type is always different, allele group is the one you have to try match
they see what type you are using PCR
what is the base cause for alloreaction/transplant rejection?
polymorphic MHC molecules between individuals and T cell restriction to host MHC
why are HLA alleles generally not lost from a population?
cause they are codominantly expressed so no recessive shit so very commonly passed on to offspring and if they are they will certainly be expressed by offspring
e.g. HLA-A24 common for aboriginals and isn’t lost from that population
why does graft survival decrease as the years go on?
indirect antigen presentation leading to chronic transplant rejection which is a progressive sort of rejection that can occur years after transplant
what is acute transplant rejection?
occurs within the first weeks following transplant and due to direct antigen presentation from DCs and MHC within the graft
outline the challenges with finding transplant donors in regards to the ethnic bias of some HLA allele groups?
HLA-A*24 needs to be matched and is very common in Māori and Aboriginals
not many Māori and Pasifika donors due to differing cultural perspectives on tissue donation/transplantation
so it ends up being quite hard to match this key allele
why is it less common for Māori and Pasifika to have a 6 out of 6 match for MHC in transplants?
much harder to find a correct match for certain alleles important in some ethnicities cause less donors
how do antibodies play a major role in transplant rejection?
people can have HLA antibodies which are present in normal individuals and may arise following pregnancy, transfusions, previous transplants or cross-reaction with pathogen/commensal structures
can also have ABO blood antigen antibodies which arise from stimulation from oligosaccharides on gut commensals
both antibody types can be present in host and cause hyperacute transplant rejection
donors that might cause a reaction are eliminated by pre-transplant screening
why does having a fucking baby inside you stimulate development of HLA antibodies?
cause you are getting exposed to the HLA of another person
what is hyperacute rejection and why does it usually occur when the recipient has HLA or ABO antibodies?
hyperacute rejection way worse than acute rejection
it occurs when antibody and complement bind and usually occurs during xenogeneic transplants
antibodies and graft rejection stimulate complement which attracts neutrophils which causes a bunch of cell destruction through release of lytic enzymes
it also activates platelet and coagulation pathways which causes further problems
occurs very rapidly
how does acute graft rejection occur via direct recognition of allogeneic MHC?
in the donated kidney there are lots of the donors dendritic cells which are loaded with MHC. Once put in the recipient the DCs behave relatively normal and migrate to the lymph node but when they do this they encounter host T cells which will scan the donor DCs which results in about 1-5% of T cells getting fucked off (direct allorecognition) and start homing back to the graft to wreak havoc
what are the two ways we can mitigate acute graft rejection?
matching recipient and donor MHC alleles
using immunosuppressants on recipient
why doesn’t it matter what peptide is being presented during direct allorecognition?
because it is the MHC that is important
this is what the T cell is recognising and turning alloreactive against
what the fuck is indirect allorecognition?
involves host T cell (like direct allorecognition) but this time the host APC is getting involved
host APCs may be taking up different proteins to what’s usually in their body from the graft (e.g. polymorphic enzymes). Often these are gender specific antigens donor MHC (most polymorphic structure in our body)
so the donor MHC gets broken down and its weird peptides are presented by host APCs over the years leading to eventual chronic rejection of graft
what is H-Y antigen?
a male specific histocompatibility antigen often responsible for indirect allorecognition
what are minor histocompatibility antigens?
the donor specific antigens presented on host APCs leading to indirect allorecognition
what is Graf versus Host Disease (GvHD)?
occurs when haematopoietic cells are transferred to recipient (e.g. for treatment of leukaemia)
T cells mediate GvHD which is a bad thing
what you are hoping for is for both T cells and NK cells to participate in graft versus leukaemia (GvL)
so when you transplant bone marrow you are hoping for an element of GvL and minimised GvHD
which class of T cells is involved in GvHD?
both CD4 and CD8
where does gene rearrangement take place for T cells?
thymus
they move from venues in the cortex and then enter the medulla prior to leaving the thymus
outline the process of T cell maturation?
CD4 and CD8 T cells go through the process of a double negative –> double positive –> single positive stages (CD4+ or CD8+)
beta chain is rearranged first and if expression successful alpha chain rearranged next
once both beta and alpha chain rearranged the cell becomes double positive meaning it can interact with MHCI and II (if autoreactive it gets deleted at this stage)
then undergoes positive selection, if interacts nicely with MHCI –> CD8 and if nice interaction with MHCII –> CD4
what occurs during the double negative stage?
beta chain rearrangement and expression
expression of pre-TCR which is just a beta chain with invariant holding it in place
following beta selection, what occurs next?
TCR alpha chain locus rearrangement
stimulates expression of CD4 and CD8 coreceptors
stimulates proliferation
stops additional TCR beta chain locus rearrangements via allelic exclusion
what the fuck is allelic exclusion?
successful rearrangement at cell surface not only keeps T cell alive and stimulates rearrangement of alpha chain but also stops beta chain rearrangement on the other chromosome by basically telling it to sit the fuck down and be quiet in germline
this is called allelic exclusion
once alpha chain rearrangement of the TCR and expression has finished, what happens?
T cell becomes double positive
undergoes negative selection (high affinity) or positive selection (intermediate affinity) making it single positive and thus it is released to carry out effector function (cytotoxic or helper)
why are antibodies able to recognise way more variety of shape than TCRs?
antibody has to be able to recognise almost an infinite variety of shapes, while TCRs are confined to recognise MHC/peptide
so following thymic selection TCRs are much less variable in shape than antibody
describe the TCR complex?
TCR involves the alpha and beta chain with two transmembrane domains and two small cytoplasmic tails
TCR complex involves other chains without which it cannot signal including zeta, epsilon-gamma and epsilon-delta
these chains have signalling parts called ITAMS which cause phosphorylation and calcium flux leading to T cell activation. So TCR signalling is done by these signalling molecules in the complex
TCR is positively charged so attracts all these other components and they coalesce as a cluster/complex
charge of transmembrane domains mediates association between different TCR molecules in the complex
what is the zeta chain?
the chain in the TCR complex with the most signalling parts (ITAMS)
it has three of them and there are two zeta chains so six ITAMS all together
these ITAMS are phosphorylated making proteins in the chain coalesce causing phosphorylation and calcium flux leading to T cell activation
zeta chain ITAMs are what is borrowed and put into CAR-T cell as it has three strong signalling domains
how many ITAMS does epsilon-gamma or epsilon delta chains have?
one
and there’s two chains in the complex so two
what mediates the coalescence of all the TCR complex chains?
the charge of their transmembrane domains
TCR transmembrane domains positive and other chains transmembrane domains negative
what are the complementarity determining regions (CDR domains) of the TCR?
these are the most variable parts of the TCR and what allow it to be unique between cells
CDR1 and CDR2 mainly contact MHC (germline encoded in V genes)
CDR3 contacts mainly peptide (non-germline encoded (hypervariable) by J junctions in alpha chain or DJ junction in beta chain, messily joined making this where the variation occurs due to nucleotide addition)
what is the beta chain of the TCR analogous to?
the heavy chain in antibody
and the alpha chain is analogous to light chain
during thymic selection, what parts of the TCR are interacting with MHC?
pretty much just CDR1 and CDR2
what does it mean that the NK-T cells TCR is relatively invariant?
all NK-T cells seem to have the same kind of TCR
this also means that if you come up with this kind of TCR during gene rearrangement you are more likely to become a NK-T cell
what do NK-T cells do?
they have some NK like features but are T cells
important in cytokine secretion and cancer recognition
they recognise a variety of ligands other than peptides; mainly glycolipids which are usually associated with the MHCI like molecule (non-classical MHC) CD1
what are gamma delta T cells?
rearrange an entirely different set of genes to alpha beta T cells (alpha and beta genes don’t even bother due to allelic exclusion)
recognise a variety of structures (some glycolipids, others unknown)
what are MAIT cells?
recognise vitamin B metabolite commonly produced by bacteria as a byproduct of bacterial metabolism
like NK-T cells has a semi-invariant alpha beta TCR
produce lots of cytokines and can have cytotoxic functions
what is the ligand for alpha beta T cells?
MHC + peptide
what is the ligand for gamma delta T cells?
CD1 (plus many other ligands) + glycolipids
what is the ligand for NK-T cells?
CD1 + glycolipids
what is the ligand for MAIT cells?
MR1 + vitamin B metabolite (which is released by actively growing bacteria)
what are Chimeric Antigen Receptor (CAR) T cells?
engineered T cells expressing a CAR comprised of tumour-binding antibody fragment (scFv)
scFv is fused to T cell signalling components in cytoplasm (e.g. CD28, CD3-zeta)
when CAR binds to surface tumour antigen this leads to T cell activation
what is scFv?
single chain fragment variable - just the variable part of the heavy and light chain of an antibody spliced together with a flexible linker between them
then put on a T cell to make it a CAR-T cell
give an example of CAR-T cell treatment?
Emily Whitehead CAR-T cell therapy to treat leukaemia
treated with a T cell based anti-CD19 CAR therapy
also got rid of all non-malignant (normal) B cells
this is because the leukaemia was in B cells which express CD19 so best way to target all tumour cells was make it target CD19
as a result Emily has no B cells (but also no leukaemia)
outline the structure of the chimeric antigen receptor of a CAR-T cell?
extracellular domain which just has the variable part of heavy and light chain of antibody spliced together with flexible linkage
transmembrane domain to embed in T cell
costimulatory domain (usually CD28)
signalling domain (CD3 zeta with all three ITAMS)
these last two domains are cytoplasmic
CAR can give you signal 1 and 2 without MHC or any molecules on T cell having to be stimulated
what happens when a CAR-T cell encounters a tumour?
the tumour antigen is recognised and bound by the heavy and light chain regions of CAR, CARS will coalesce together on cell surface and you get massive T cell activation from aggregation of cytoplasmic domains (CD28 and CD3-zeta)
the CAR-T cell (can be CD4 or CD8) then fucks up the tumour cell either with granzyme/perforin or via cytokines
CD4 and CD8 CAR-T cells generally used as 1:1 ration for most effective therapy
once you spliced together the heavy anbd light variable domains of anti-cancer antibody onto cytoplasmic domains of CD3-zeta and CD28, how to you transduce into patients T cells?
take T cells out of patient via aphaeresis
using a retrovirus which has CAR inserted within its genome so that it jumps into genome of T cell
inject those T cells back into patient as CAR-T cells
what is the effectivity of CAR-T cells?
good response rates in B cell lymphomas
effective against relapsed/refractory disease
outline the structure of antibody?
two heavy chains, two light chains divided into constant and variable regions
light chains are either kappa or lambda (two genes which contribute to formation of light chain, one of each on each chromosome so altogether four contributing to diversity)
five main isotypes: IgD, IgM, IgG, IgA and IgE
what is the ratio of kappa to lambda light chains in the body and what does this mean?
ratio of kappa:lambda is 2:1
this means that kappa gene is rearranged first as most of the time it works and so your body has more kappa (allelic exclusion) but if it doesn’t work then lambda is the next option
what are antibodies very similar in structure to?
TCR
light chain and heavy chain in antibodies are analogous to alpha chain and beta chain in TCRs
both have CDR
when do antibodies not have a transmembrane domain?
when secreted
what allows the domains of the antibody to fold into shape?
flexible bits joining them
what are complementarity determining regions (CDR)?
these are the main points of contact to antigen
they are hyper variable
both the light chain and heavy chain have three CDR
what are two key structural components of an Ig domain?
CDR and beta strands
why are antibodies so high affinity to the shit they bind i.e. why are they good at binding?
because the CDR are hypervariable
why is CDR3 so much more hypervariable than CDR1 and 2?
CDR1 and 2 are encoded by V genes in both heavy and light chain
CD3 encoded by V and J genes in light chain and V, D and J genes in heavy chain
this is the same for both B cell and T cell CDR
what is the order of rearrangement during the generation of diversity in B cells?
heavy chain rearranged first and if this is successful then light chain rearranged next
what is similar about the generation of diversity in both B and T cells?
both antibody heavy chain and TCR-beta chain undergo VDJ rearrangement before TCR-alpha chain and light chain
both TCR-alpha chain and antibody light chain undergo VJ rearrangement after TCR-beta chain and heavy chain rearrangement
how can we cleave antibody to give distinct fragments called Fc and Fab?
using papain protease
what is the Fab region of antibody involved in?
antigen binding
what is Fc region of antibody involved in?
binds to Fc receptors
FcR can trigger NK cells, macrophage, DCs and can also stimulate antigen uptake and complement activation
what part of the antibody does papain cut?
cuts close to the hinge region of the antibody
this separates it out into two Fab regions and an Fc region
what is the hinge region of antibody important for?
allows antibody to be flexible and bind both widely and closely spaced cell surface determinants
it has some rigidity which is why it is referred to as a hinge and not a linker
what are the antibody hinge and constant (Fc) domains useful for in the lab?
used as a scaffold for protein expression which is very useful for studying your receptor of interest
this is because usually a receptor is embedded in a membrane and if you isolate it it gets fucked cause hydrophobic and wants to bind a membrane
but if you truncate it and fuse it onto the Fc domain of some human IgG this makes it very soluble and thus easy to isolate
having an Fc domain doesn’t affect receptor function
what are the two kinds of B cell activation?
thymus dependent (mediated by CD4 T cells)
thymus independent (TLR and Ig cross linking)
what is thymus dependent (TD) B cell activation?
T helper cell mediated activation of B cells. Normally mediated by CD40L and IL-4
often results in high affinity, class switched IgG antibodies
what is thymus independent (TI) B cell activation?
occurs when BCR is cross linked and when strong TLR signalling occurs
rapid and useful response against bacteria, normally produces low affinity antibodies which recognise lipids and carbohydrates
what do naive B cells express?
IgM and IgD as membrane bound forms
B cells producing antibody specific for given antigen over 10,000 times more likely to bind and internalise specific antigen
how do activated B cells secrete soluble forms of Ig?
by splicing out transmembrane domains during RNA processing
why is it that conventional thymus dependent B cells need help to produce antibody?
by needing two antigen-specific lymphocytes to be present guards against unwanted B cell activation (autoimmunity)
are TI B cells at risk of causing autoimmunity since they don’t require other antigen-specific lymphocytes to be activated?
not really cause they produce IgM which is a low affinity antibody and so not associated with autoimmune disorder
plus if they are activated in the absence of microbial signalling this induces peripheral tolerance and deletion
what are some of the functions of effector T cells?
support antibody production
kill infected cells
make more cytokines
enhance innate immune system
why is there a need for different effector responses?
different types of organisms
different route of infection
different site of infection
different molecules etc
what are the main CD4+ T cell subsets?
Th1
Th2
Th17
Treg
also Th9 and T follicular helper
