Epi regulation of immunity

what are the 2 forms of cellular immunity?

acquired and inate

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what is acquired immunity?

Antibodies = results from clonal synthesis of antigen-specific immunogloblin from B lymphocytes and T cell receptors for T lymphocytes

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in acquired immunity what do B lymphocytes make?

immunogloblins (antibodies) that are antigen specific

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in acquired immunity what do T lymphocytes make?

T cell receptors

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what is innate immunity mediated by?

myeloid cells

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what are myeloid cells in innate immunity equipped with?

pattern recognition receptors (PRRs) that interact with pathogen-associated molecular pattern (PAMPs)

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what do both adaptive modulation of acquired and innate immunity specifically involve?

covalent histone modifications

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what could be a potential therapeutic strategy for chronic inflammatory disorders?

inhibition of epigenetic aspects of immune training- so you don't get as much inflammation

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what are the source of acquired immunity to specific antigens?

B and T lymphocytes

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Naive T and B cells undergo clonal expansion once they've become activated by infection what do theyresult in?

memory T and B lymphocytes have specific T cell recs or make specific antibodies to remember the pathogen

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every B lymphocyte (and terminally differentiated progeny- Plasma cells) produces what?

immunogloblins with a unique antigen specificity (Ig MADGE isotypes)

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what is the most terminally differentiated B cell you can get?

plasma cell that's secreting antibodies

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where do mature naive B cells circulate?

through lymphoid organs

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what are myeloid cells in innate immunity?

macs, monocytes, dendritic cells, microglia- don't have clonally specific recs for antigens but have PAMPS AND prrs

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Where does every B lymphocyte emerge from?

devs in bone marrow, emerges from spleen carries an immogloblin isoform on its surface

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what does a common lymphoid progenitor cell need to do to become a pro B cell?

common lymphoid progenitor cell has made a series of cell fate decisions and restrictions to become a pro b cells

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where do the immunogloblin gene arrangements that produce antigen specific B cell immunogloblins occur?

in the pro B cells (precursors)

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what do the pro B cells do once they've rearranged genes?

they synthesise proteins- heavy chain component is sent to the CS to pair with light chain

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when can a B cell precursor complete the process of maturation?

once it has a set of immunogloblins on its surface

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what is the process called in the pro B cell that generates the recombinant immunogloblin genes?

V(D)J recombination

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what works together with this recombination mechanism? why?

epigenetic and transcription machinery to ensure that any given pro B cell only rearranges one allele of any immunogloblin gene in any given cell so that the B cell produces a CS rec specific for one antigen

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what are immunogloblins?

multi-subunit proteins with variable and constant domains

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what is an immunogloblin made up of?

2 heavy chains and 2 light chains. Heavy and light chains are encoded by different genes, each polypeptide folds into globular domains held together by disulfide bridges

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what chain has variable domains in them?

both heavy and light chain

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when are the variable domains created?

by the process of PVD recombination (productive V(D)J)

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why have variable domains?

they are antigen specific

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what are the variable regions connected to?

constant regions

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how many DNAase I hypersensitive sites does the mouse Igh locus got?

2 DNA I hypersensitive sites

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where are the 2 DNAase I hypersensitive sites in the immunogloblin heavy chain locus?

one within Emu enhnacer and the other within the diversity segment Dq52

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what do the DNAase I hypersensitive sites mean?

that the DNA is open and accessible to TFs big enough for DNAase I to get in there

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what is Emu promoter and diversity segment in pro B cells open to?

TFs, epigenetic regulatory proteins and recombinases

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what is the gene that produces the heavy chain locus?

Igh2 (immunogloblin heavy chain)

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what does Igh have?

variable portion and constant region

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what does DNAase I hypersensitivity determine?

technique to determine whether chromatin is open or closed

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when do pro-B cells acquire this region of DNAase I hypersensitivity?

when pro-B cells become specificied then they open up these regions to TFs etc

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how did they find out it had DNAase I hypersensitivity (Igh locus)?

took nuceli from B cells, cultured treated with DNAase I- endonuclease nibbles away accesible regions

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how did they know which regions of the locus where DNAase I hypersensitive?

series of DNA probes

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what segments is there in the Igh locus?

segments that give rise to constant portions, diversity segments, variable diversity joing segments (VDJ recombination)

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what did they compare the Igh locus to in this experiment when treating with DNAase I?

genomic DNA from pro-B cells in which rearrangements are occurring and in T cells which the rearrangements of Igh locus weren't occuring (so control)

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what do you see in the proB cells but not the T cells with DNAase I hypersensitivity?

DNAase hypersensitive sites in B cells- get bands appearing where its cutting up the DNA

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what does this tell you?

as pro B cells get specified they reorganise their chromatin in this region in the Igh locus- diversity segment and Eu enhancer site

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when Eu enhancer within the Igh locus has been shown when its linked to reporter genes transfected into B cells to be transcribed has shown what?

that the Mu enhancer has B cell specific TF binding sites that activate the locus = transcriptional activity

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what kind of histone modification do Pro-B cells exihibit?

Pro-B cells exhibit high levels of Histone H3 and H4 acetylation

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where is there high levels of Histone H3 and H4 acetylation in the Igh locus

across the proximal Igh locus (D-J-C segments) peaking at the Jh-Eu enhancer region

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how did they study histone acetylation across the Igh locus?

chromatin immunoprecipitation- take an antibody that recognises acetylated lyisne on Histone H3 or H4, do a pull down then PCR the pull downs for each region across the locus then only proximal Igh locus + Jh-Eu enhancer had highly acetylated histon

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do you get this hyperacetylation in T cells?

no it's B cell specific

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what are the D-J-C segments where there's hyperacetylation actually called?

J = joining segments C segments = constant portions D segments = diversity segments

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What is the Jh-Eu enhancer regions specifically marked by?

high levels of histone H3K9 acetylation and H3K4 methylation in mouse Pro-B cells

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what established local chromatin accesibility in the Igh locus?

the Eu enhancer

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what does the variable segment give?

produces a variable heavy chain that has specificity for a particular antigen

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how many variable segments are there?

100's of them- the variable cluster extends for many hundreds of kbps is the biggest portion of the immunogloblin locus

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what was used to find lysine acetylation and methylation across the locus?

chIP seq analysis

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when they did the chIP seq analysis across the Igh locus what did they find?

high levels of H3K4 methylation and high levels of H3K9 acetylation in the diversity segment

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what does H3K4 methylation recruit? what's it made by?

trithorax- MSL2/trithorax makes the mark

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as you approach the Eu (mu) enhancer what do you get?

strong peak of H3K4 methylation

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where's the hypersensitive site?

in the mu enhancer

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why is there more than 1 constant segment?

because as B cells mature they go from early to late plasma cells their isotype switches from membrane bound Igm to secreted IgG

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what is recombined together?

a variable segment with a diversity segment then joined with a joining segment (which is then all added to a constant segment)

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what are the steps in recombination?

1) join the J segments to the D segments 2) take that D-J region and hook it up to a variable segment

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what are those 2 steps carried out by?

recombinase activating genes = rag1/2

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what do rag1/2 function as?

dimers- 1st of all join D + J together to form a D-J fusion and then they join that DJ fusion up a V segment

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why is the selection of which Variable segment joins random?

so in all the population of V cells all combinations are made but in any one B cell only one D-J segment is combined to one V segment

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what does it form when D-J-V are formed together?

it can form the heavy chain once its fused to a constant portion

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what is the connection by D-J and C made by?

splicing

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which recombination activating genes are essential for VDJ recombination?

both RAG1 + RAG2

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What do the rag recombinases bind to?

rag signal sequences- have same combo either side of the J segment and D segment

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what do RAG recombinases do when they bind to recombination signal sequences?

bind, cut and rejoin to give D-J fusions then again to give V-D-J fusions

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what are we trying to do by making a D-J fusion?

join a J segment to a D segment and trying to get rid of the stuff in the middle = RAG recombinases job

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what does Rag1 do?

portion of the recombianse that binds to the junction- it cuts and cleaves it but only if its bound to Rag2

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what does Rag2 do?

the key component that recruits the complex to the locus. Rag2 = has PHD finger

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what does the PHD finger that Rag2 has bind to?

the H3K4 methylation mark that's abundant in the J-D region

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how do they know Rag1+ 2 and H3K4 methylation are localised to the same DNA sequences in the mouse Igh recombination centre in the pro-B cells?

chromatin immunoprecipitation- map using antibodies against RAG across the Igh locus and antibodies against H3K4me3 = all H3k4me3 are colocalised with RAG1+2

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what does this tell us?

in order to recruit RAG to the Igh locus you need to methylate H3K4

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what does Rag2 bind to in vitro?

H3K4me3

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what else does Rag2 bind to?

pro-B cell chromatin from the mouse Igh locus (that's enriched in H3K4me3)

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evidence from mouse Igh locus that you need H3K4me3 to recruit RAG2?

immunoprecipitation taking chromatin from pro-b cells and precipitating it with an antibody for H3K4me3 and blotting with an antibody that picks up Rag2 = H3K4me3 also contains Rag2

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evidence in vitro you need H3K4me3 to recruit RAG2?

prepare columns covered in different forms of methylated histones and pour radiolabelled RAG2 on and it only binds to columns with H3K4 trimethyl lysine, then elute proteins find rag2 specifically associated with H3K4 trimethylation

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how does RAG1/2 recombinase interact with the D-J recombination signal sequences?

the D and J segments are methylated to recruit the Rag1/Rag2 complex- RAG2 binds with PHD finger to H3K4me3, get a turn complex, bend round to form U shaped structure RAG1 enzymes cleave DNA

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what does this cutting produce?

D-J fusion

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what does RAG1 cleaving the DNA do?

release this portion that is ligated to form a circular recombination product (That's discarded)

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what does RAG2 do?

recruiting- by binding to H3K4me3

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what does Rag1 do?

cutting and rejoining

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where does recombination first happen at the Igh locus in pro-B cells?

first make a D-J fusion then recombination between D and V segments to generate a D-J-V recombinat

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how does it select a variable portion to fuse to the D-J fusion?

needs to bring these variable portions of which there are hundreds of located many kilobases away need to bring them in close proximity

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how does it bring the variable segments in close proximity?

locus contraction

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what is the Igh locus in a pro-B cell?

a compact topologically associating domain in pro-B cells

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what did they use high C chromatin capture for?

to visualise domains of chromatin by virtue of intensity of signals

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what did they look at?

looked at the Igh locus in ES where there's no rearrangement (hypermeth or hyperacetylation) and then in proB cells where there is chromosomal rearrangements

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what did they see in the pro-B cells?

strong interacting triangle- mostly red indicates elements within it are close enough to be captured by the conformation capture technique

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what does the conformation capture technique involve?

chemical crosslinking of fragments that are in close proximity to each other and then a sequencing analysis to detect those close interactions

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what was this evidence for?

that the Igh locus is contracted into a compact domain (close enough for elements to interact)

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what did they do with fluorescence ISH?

took oligonucleotides that are specific for one of the variable domains labelled in red, labelled diversity segment in green and the 3rd oligonucleotide fluorescent for the joining segment and another for the constant portion

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what did they do with fluorescence ISH simplified?

got oligonucleotides against the V, D, J and constant portion all of different fluorescent colours, hybridises oligos to cells then microscopy image of Pro-B cell nucelus

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what did they find?

they found that the 3 signals constant, diversity segment and variable segment are all overlapping/colocalised in the pro-B cell but separate in the non B cell

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difference between pro-B cell and non-proB cell

regions are distinct- not condensed into a tight domain of contracted chromatin all spread out but int he pro-B cell are in a contracted tight locus

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the mu enhancer has high acetylation in what?

H3K9

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the mu enhancer has high methylation in what?

H3K4 (trithorax opens it open)

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what is Pax5?

a sequence specific DNA binding protein

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what does Pax5 do?

that promotes transcriptional activation of B-cell differentation genes and represses myeloid genes

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what drives the locus contraction?

Pax5

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what is Pax5 at the same time?

transactivator and transrepressor- actiavtes B cell genes and represses ones that are not so regulates B cell development

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where is Pax5 specifically expressed?

specifically in B cells and Pro B cells- dual function to repress non-b specific genes and promote B cell specific genes

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what experiment did they use to show that Pax5 is required for Igh locus contraction in a mouse ProB cell?

chromatin conformation capture- look at localsiation of probes that recognsie a variable portion and constant portion then look at where they are in Pax5 WT cells

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where did this show the constant and variable portions were?

close together (in WT)

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what happened to constant and variable portions when KO Pax5?

constant and variable region markers stay far apart

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what did this show?

pax5 is required to promote locus contraction to get signals (V and C to interact with each other)

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what is more separated in Pax5 KO cells?

Constant and variable genes

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what does Pax5 bind to?

15 specific DNA sequences within the distal Variable cluster

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what do these DNA sequences in the variable cluster also bind to?

CTCF (acetyltransferase?)

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what are the 15 DNA sequences in the variable cluster that pax5 binds to rich in?

active histone mods- H3K9 acetylation, H3K4me2 and H3k4me3

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what cluster does Pax5 bind to?

the Variable cluster

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what is each Pax5 binding site next to?

a variable domain

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what does the CTCF thats at these DNA sequences that Pax5 binds to doing?

loop forming protein- sits at the base of chromatin loops and organises the 3D structure of chromatin at a large scale- loop formation that is involved in this contraction process

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if you remove Pax5 what active histone mod do you lose?

the methylation- so methylation is partly dependent on Pax5 function

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what does each Pax4 binding sequence overlap with?

the CTCF binding site in the Variable domain

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what sequences does Pax5 bind to? where is it close to?

Pax5 binds close to recombinase sequence signals between Variable segments called PAIR sequences

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what does Pax5 interact with to facilitate Locus contraction to enable V-D-J rearrnagement?

Pax5 interacts with CTCF for locus contraction so V-D-J recombination can take place

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why do we need locus contraction?

in order to bring the variale segments close enough to fuse with D-J so all the variable segments have an equal chance of recombing

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what does trained immunity do?

increases cellular response to infections, adapts the immune system so you have memory cells that have made antibodies or T cell recs so when antigen present again the cellular response is bigger and faster to fight the infection

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what is adaptation in the innate immune system?

allows animals to respond better to an immune challenge when they're presented with a pathogen after already encountering it beore

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what is trained immunity thought to be dependent on?

Understood to be fully dependent on an epigenetic reprogramming of the immune cells responsible for the innate response, macrophages for example

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what is part of the effect of trained immunity?

to produce more pattern recognition recs by inducing transc and translation of PRRs to insert them into mac M so signals by PRR are more strongly transduced

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what is innate immunity conditioned by?

exposure to infectious pathogens, vaccines or tissue damage

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what is it called when the innate immune system is conditioned by pathogens?

innate immune memory- allows us to potentiate responses on repeat exposures

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is this trained immunity in the innate system pathogen specific?

no so exposure to one pathogen could substantiate the immune response and trained response o another pathogen- seems to depend on epigenetic mechanisms

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examples of experimentally-induced immune training?

immunisation with a fungal pathogen component can protect against bac infections, immunisation with a bacterial peptidoglycan fragment can generate innate immunity against protozoa

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what is the BCG vaccination process?

uses a microbacterium that can generate protection against fungal+protozoal infections = used as a therapy against bladded cabcer

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what is BCG immunisation beneficial for?

getting rid of bladder cancer

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what is the BCG vaccine against?

myobacterium tuberculosis

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what does this myobacterium protect against?

fungal and protozoa infections

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what did they show immunization of SCID mice (no lymphocytes) with the BCG vaccine protected against?

lethal fungus candida albicans

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what did the BCG vaccine in these SCID mice induce?

the induction of proinflammatory cytokine TNF alpha

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what are SCID mice dependent on for survival? why?

innate immunity as they have no B and T cells

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if you take SCID mice immunise half with BCG and give the other half saline then expose them to C albicans fungus what happens?

if they received the BCG fungus they survive but if they didn't they all die

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what is this an example of?

immune training within the innate immune system

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how is BCG thought to potentiate the immune system?

through a strong induction of TNF and cytokines like TNF alpha in the presence of C albicans, as TNF lapha production was much higher in animals that received BCG immunisation

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what can this be used for in humans?

can train this in humans- BCG can protect against infectious diseases and cancer

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what do pattern recognition recs on macs, monocytes and other WBCs bind to?

pathogen-associated molecular patterns (PAMPs)

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what happens when PAMPs bind to the pattern recognition rec on macs?

activate epigenetically regulated transcriptional responses to train the innate immune system- turns on genes responsible for producing trained immunity

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how was this phenomenon discovered?

animals immunised with a bacterial cell wall component LPS then either a couple of hours after immunisation or 24 hrs after animals sacrificed and macs analysed for the expression of genes

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what was the other difference?

they were given 1 or 2 shots of LPS- one at time 0 and other just before collect macs

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what 4 different states were they comparing?

completely naive, unimmunised or injected but at 20 hrs after macs collected and analysed

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when do they look at the immediate response the macs are showing

when they give LPS and it's still circulating

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what was the idea behind immunizing them a whole day before?

immunise them a whole day before, let the response clear out then have a look at what's left once the initial response has subsided (look at gene expr in macs)

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why was a 2nd dose of LPS given?

to capture the trained immune response

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what did they measure?

cytokine changes (to make sure something was detected) and gene expression

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how many classes of genes did they find?

2 classes

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what was the N+ L single condition?

N+ L- immunised for short period give a quick boost of pathogen into the immune system to capture any immediate rapid responding genes

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what was the double T+L condtiion?

T+L = immunised at time 0 with LPS and then administered with LPS than a short period of time later tissues collected

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what did they hsow with the short initial immune challenge in N+L?

the genes rapid response then declining

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what did they show with the double injection of LPS in the T+L condition?

moderate initial response but on the second challenge showed a much stronger transcriptional response

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what are the 2 classes of genes?

rapid response genes that decay or genes expr to a moderate level in initial challenge but show a much stronger expression with a stronger expression

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ones that had a rapid response and declined were probably what

epigenetically silenced

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the genes that showed trained immunity with a modest increase then much higher expression on 2nd challenge had what?

a modest amount of histone modification

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what is tolerised?

class T genes- the ones that are induced by first exposure to LPS but supressed by 2nd exposure to LPS rapid response then decays

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what is non-tolerised?

induced by first exposure to LPS nad induction enhanced by second exposure to LPS- become more active after 2nd exposure, low initial response, boosted by 2nd exposure

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what one is rapid response then decays?

tolerized

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what genes show low initial response but are boosted by 2nd exposure?

non-tolerized = trained immunity

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how do you get tolerised immunity (increases then decays)?

stimulation become active then are epigenetically silenced

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how do you get trained immunity?

stimulation, active gene expression, then restimulation more active marks put on -> gene transcription potentiated -> trained immunity

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what transiently increases then is supressed at tolerised gene prmoters?

active marks H3K4me3 and Histone acetylation of H4

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when are histone active marks persistently increased?

at non-tolerised gene promoters exhibiting immune training

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what does LPS binding induce?

binding of histone acetyltransferase CBP to regulatory sequences near to LPS-inducible genes in mouse macs

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what do each of the response genes that show immune training require

a much greater decoration with CBP- is localised to genes associated with a TF specific for macs called PU.1

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when is Pu1 recruited to these genes in macs?

independently of exposure to the immune challenge

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what is PU 1 normally?

a fate determinant of macs- sets up locci for induction but isn't able to bring that aboust until another mechanism puts CBP close to the PU1 TF genes to boost histone acetylation

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overexpression of these sorts of genes that confer trained immune response in macs is part of what pathological phenotype?

people with autoimmune disorders- get too much immune response

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what did this led us to think may about autoimmune disorders?

autoimmune disorders overexpress their inflammatory programmes because their immune system is overtrained

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what could be useful for treating autoimmune disorders in that case?

histone acetylation inhibitors because these inflammatory genes become too active

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what did it lead to the development of?

I-BET-

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what does IBET do?

a small molecule that mimics acetylated lysines that blocks the function of CBP and HAT enzymes

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what happens to the macs when IBET is administered to them?

level of histone acetylation of inflammatory genes like TNF nad IL-6 is reduced and the ability of proteins that bind acetylated lyisnes with bromodomains is also blocked

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Epi regulation of immunity

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