Immunology Fall 2024: Lecture 11 Generation of Antibody Diversity (VDJ Recombination) Part 1

so today I'm going to talk about generation of antibody diversity and the set of slides that I've posted about this um are for both today and for Friday um so I'm not going to like repost another set of slides for Friday I'm just going to use the the rest of these um and um I want to remind you of a few things that we've already talked about in terms of setting up the problem that we are then going to be solving for the rest of today and on Friday um so I set you up with

this one uh problem last time um which was this issue that um if we think about the specificity of antibodies it is estimated that we can make about 10 to the 16th different antibodies um and um this is a bit of a problem compared to the number of genes in the genome um which is only sort of on the order of 20,000 um when scientists were trying to figure out the answer for this um there were a couple of other observations that they were sort of taking into account when they were trying to figure out

how this worked and so I just want to remind you of some of those observations um I used the complicated term on this slide you don't have to actually care what alic exclusion means yet we'll we'll actually see the definition of alic exclusion next week um but this is officially um something that's known as a l exclusion um which is that if you remember when we talked about those B cells um that are making antibodies we talked about the fact that each B cell makes homogeneous receptors so each B cell might make multiple receptors but

those receptors are going to be like identical so um for that 10 to the 16th different number of antibodies that we could make that requires 10 to the 16th different p that doesn't you can't have one B cell that's making more than one kind of antibody so that's one thing to um just make sure that we are aware of um we also have known for a very long time that our adaptive immune responses which are shown here in dark blue um change over time um so um you can see that the first time through perhaps

we have an antibody response we've got a little delay we make this nice response and then the second time that we are exposed to that same antigen we're going to have a larger response and a faster response and so somehow we have to be able to sort of retain the information that we previously seen this microbe and do something better and so there has to be some kind of longterm memory that is involved with all of this um this is involved with um our B cell and remember that our B cell is going to be

able to make a B cell receptor and an antibody that are basically going to be identical except for whether or not there's a transmembrane domain at the end um so the B cell receptor and the antibody are basically the same um so this is one of those observations that um scientists had made before they figured out how this process worked um they also knew that over time antibodies could change um particularly the antibodies made made during a response to a specific epitope could change and so we can see changes in the Affinity um or The

Binding strength of our antibody for antigen the antibody can get more specific and we can also see changes in isotype so we can see class switch um and um they we sort of had this Al this idea of clonal expansion um and the clonal selection Theory where the idea is that we might make those 10 to the 16th different B cells Each of which makes its own unique antibod so we have 10 to the 16th different B cells here it's shown as two because that's easier to draw than 10 to the 16th here's three because

three is also easier to draw than 10 to the 16th um but they're meant to be showing you some of those 10 to the6th different B cells making 10 to the 16th different antibodies you can notice make one of them um and then that one if it ever sees its antigen will um proliferate will make many many copies of itself and sort of make a clone army um so now you have a lot of those specific cells that can deal with the microb um and so all of these observations were out there when people were

trying to figure figure out how to deal with this diversity problem and some of them as I go through some of the answers you'll be like oh I see how that fits and works with some of these observations like I see where they got how they got that um but what I want to point out is that specifically when we think about things like the clonal expansion piece of this and when we think about say the primary and secondary response and the memory kind of part of this um one of the things that became pretty

evident right away was there had to be some sort of change or something going on that could be transmitted from generation of cells to generation of cells not necessarily from like generation of human to generation of human but from generation of cells cl to generation of cells like this cell had to be able to have some kind of change to make a particular receptor that it could then also make sure its progyny had and that had to be you know something that could happen over a long period of time if we were actually going to

have memory over a long period of time and so if we think about things that can um go across generations of cells what does that make us start realizing or thinking about the fact that we're going to have to imagine what can one cell give to its progyny cells what's one cell definitely going to give to its progyny cells H the genes specifically the DNA and so um some early scientists I used to go through some of the really there were some actually like alternate hypotheses and I used to go through them all and make

fun of the people who um came up with them every one of them who I would make fun of actually won a Nobel Prize for something else so like it's not like we should feel bad for them when I make fun of them but um so there are all of these alternate ideas about how this could work and one of the big things that people realized was we've got to involve the DNA because the DNA is the thing that's actually going to allow whatever this receptor thing is to be transmitted to progyny cells and so

if it was just a protein change for example that wouldn't be something you could easily transmit to progyny cells and that wouldn't be something you could easily have for memory because if the cell died you'd be out of luck whereas here if you have changes to genes changes to the DNA then we're going to be able to actually transfer that between generations of cells um so in general we've got a couple of different ways that we solve this problem this slide has a bunch of the technical terms for how we solve this problem when you

are going back to study for the exam this will be a helpful little outline of the answers to how we solve the diversity problem I haven't told you what all these words mean yet so if you look at this slide and are like I don't know what any of this means that's okay because I haven't told you yet but this is a little bit of an outline of what we're going to see and so if we're actually thinking about this antibody diversity problem we think about we've got you know 10 to the 16 different antibody

proteins and our genome only has um about 20,000 genes we've got two different sort of themes or ways of of solving this one is something known as combinatorial diversity which I'm going to spend most of the time today talking about um and there's also something called junctional diversity um and for both combinatorial diversity and junctional diversity there are two examples um and so we will see what all of these things mean um going forward and so the first thing that I want to um explain is combinatorial diversity and have us start thinking about combinatorial diversity

it turns out you kind of already know some stuff about combinatorial diversity you just don't know it as such but in order to make combinatorial diversity make some sense um I'm actually going to tell you uh Side Story So diversion time um so what we can imagine in our little diversion is that I decided that I was tired of this whole science thing and decided I was going to have another job and so and my other job was I was going to have a restaurant but the problem with me having a restaurant is I don't

know how to cook very many things and so my restaurant probably is going to have a very limited menu um and so we can imagine um the the fun joke about all of this is the not being able to cook part is not actually that much of an overstatement um it when I'm putting together this restaurant in this example but let's imagine that I know how to cook beef and I know how to cook broccoli even if I might struggle to spell broccoli okay so I know how to cook two dishes and if you look

at my menu you're going to find one thing on my menu beef and broccoli right now let's imagine I also learn how to cook chicken now I know how to cook three things chicken beef and broccoli I only got two things on my menu beef and broccoli chicken and broccoli right because we're thinking about my menu as a comb of a protein and of a vegetable but let's imagine I now really spend some time learning how to cook and let's imagine that I also learned how to cook these things so now I have five proteins

can cook beef chicken pork turkey tofu and we'll imagine I also learned how to cook carrots spinach corn and tomatoes I only know 10 recipes okay not like I'm not fancy I got 10 recipes it's all but if we start counting these not as 10 individual recipes but as combinations of things then my menu actually has 25 things on it so I got 10 recipes but because I count I'm going to count them as combos of dishes I'm going to have 25 dishes because I'm because my menu is going to have beef and broccoli beef

and carrots beef and spinach beef and corn beef and tomatoes chicken and broccoli chicken and carrots chicken and spinach chicken and corn chicken and tomatoes pork and broccoli pork and carrots and all the way through and so I only got 10 10 dishes but now I get way more than 10 things on my menu and you can see that if I start to add a even a relatively small number of more recipes but we don't count just the individual recipes as what I got but combos um I can get a really big list of dishes

on my restaurant menu from a really small number of recipes does that make sense that's combinatorial diversity um and we've got two different ways that combinatorial diversity is something that we can see in antibodies so one part of this is just like my menu that you see here we actually have instead of having a beef and broccoli Jean and a beef and carrots Jee and a beef and spinach Gene instead of needing to have a whole Gene for that we actually use combinations of genes and so each antibody is a combination of a heavy chain

and a light chain and so if we have five heavy chains just like I have five proteins and I have five light chains just like I have five vegetables that's only 10 genes and yet I can make 25 antibody proteins and so I'm using a small number of genes to get me a bigger number of proteins because I'm actually combining two different genes in this case the heavy chain and the light chain genes and by combining those together that lets me get to a bigger number of individual proteins from a smaller number of genes so

one way that we start to solve this problem is by combining heavy chains and light chains so if you remember when I told you about the antibody structure I told you how important it was that it's not just the heavy chain binds to the antigen or just the light chain it's the combination of the two and that's sort of one of the ways that we're getting um additional um additional diversity here one of the other things that people sort of noticed um is some of this information I had mentioned to you before about well the

B cell can sometimes have a transmembrane domain on this receptor and sometimes not um and we can even like class switch from IGM to IGG so people said well what if it's not just a combination of a heavy chain Gene and a light chain Gene but what if those genes had individual parts and so we were actually making combinations within that and so this is like if there I don't know how I would make like a combination within the beef um just really not that good at cooking um but the idea is well what if

we it wasn't just heavy chain was one gene but what if heavy chain was Little Jeans combined and so you could get a lot of heavy chains by doing combos again of the little genes and one thing people realize right away they're like wait this could work if the variable region and the constant region were encoded by different genes because then you could put a different variable region with different constant regions so if you had say a variable region you could put it with the MU constant region and make an IGM version of the antibody

or you could put it with a a gamma constant region and make it an IGG so maybe if the heavy chain Gene isn't really one gene but it has these little parts to it maybe we could explain pretty easily how we just sub out the part with a transmembrane domain or not or with different constant regions or not um and so we can sort of think about the variable region and the constant region as kind of being separate areas genetically um and so this whole thing sort of eventually led to this idea of the Min

Gene hypothesis and so what people realized was that in sort of this large section of the chromosome there might be a region that encodes the constant region so this you can see see the C for constant region and there might be some small Gene segments that we can put together in different combos to make the variable so if we want to make a b a billion variable regions we can have a small number of these two segments and then just put them together in different combos just like I was doing with my meals and so

we're going to do this and make combos so we can use a small number of genes to make a lot of heavy chains and then we're also going to have Min genes that come together to make light chains and so in the end we're going to have a list of a kind of small number of genes but get a massive number of proteins um and so the hypothesis basically was that there were these small mini genes shown here as things like V and J that are put together you can see there's a whole bunch of

choices in the DNA those genes are put together along with another Gene segment that encodes the constant region and that is how that sort of combination of these different mini genes would be how we can get to um genetic information encoding so many different proteins is by shuffling together all of these Min genes um and so you can see this from an older version of your textbook here um so this is only showing it it's only showing it with four Min genes and if they I wish they had used more than four Min genes because

it makes it so much easier with more than four um but you can see they've got two v's V1 V2 two J's J1 J2 and the idea is that during the development of the B cell when the B cell is growing up the B cell chooses a V and chooses a j and puts them together and when I say chooses a g b and chooses A J it's not really active Choice it's actually a random process um and when I say puts them together I mean it actually Cuts its DNA it cuts its genomic DNA

and pastes it back together so that those two Gene segments are next to each other and so you can see here that this B cell picked V1 and J1 this one picked V1 and J2 this one picked V2 and J1 this one picked V2 and J2 and so we got four different receptors out of this and four different genes that's why I wish they had used you know they use my example of 10 they would get 25 different receptors by pairing together the different v's and the different J's um and you can also see that

this hypothesis um meant that in fact the um DNA was cut in the B cell so the B cell actually Cuts this part of its DNA throws out this intervening DNA as trash and pastes together the V and the J that it has chosen um one of the um ways that scientists um um figured this out was that they looked at B cells that had undergone development and they also looked at other cells in the body of the same organism um and they often like to look at they they did different things with non-immune cells

but so we'll just say a non-immune cell they compared the DNA in a non-immune cell and the DNA in a B cell and they saw the B cell's DNA had been changed relative to the non-immune cell the B cell's DNA looked like this where some V and some J had been pasted together and some DNA had been thrown out and every other cell in the body's DNA at this region looked like this all separate um so um and each B cell had a different combo that it had made yeah Jonah we'll get there great great

question we'll get there um and um there was a scientist who actually one of the people who got the Nobel Prize for describing this process um was a guy named tonawa one of the predictions from this hypothesis um was that you could find the trash DNA in B cells early in their development and he found it found this the developing B cells all have these little Circles of DNA that they've thrown away um because they have done this process um and so we often refer to this process and you'll see this later as vdj Rec

combination um I will tell you what the d stands for in a bit um you can see that we' we're talking about these segments like V and J so that's where you can imagine that's where the V and the J come from in VJ or combination and the word Rec combination is a cutting is cutting and pasting DNA and making new DNA combinations um and so these are all kind of examples of what that how we're getting that combinatorial diversity in the way that we make B cell receptors or antibodies so we're combining heavy chains

and light chains we're actually combining variable region genes with constant region and in order to make the variable region we're actually combining um different Min genes together so you can see we sort of got we're doing combinations on multiple levels in order to get us big numbers of receptors from a small number of genes yes we'll get there that's coming too all of this are these are good predicting question um and so um in order to do this process and to make these genes we actually do vdj recombination twice in every V Cel once to

make a heavy chain and once to make a light chain when we make the heavy chain we combine three different Gene segments in order to make the variable region those three different Gene segments in the heavy chain are called v d and j there's your D Jonah um and so you can see that we're looking at just making the variable region of the heavy chain here we we get to pick a v we get to pick a d we get to pick a j whichever V we pick is going to encode amino acids about 1

to 101 these are only approximate numbers of that anal body protein whichever D we pick is going to encode amino acids 102 to 106 whichever J we pick is going to be approximately 107 to 123 and so you can see that we would put that v d and j together we throw out the intervening DNA and we have the constant region um in the case of um the light chain we just have a v segment and a j segment it's a different area of the chromosome you can see that the um V segment in the

light chain inodes um amino acids approximately 1 to 97 while J is encoding approximately 98 to 110 um I'm going to draw this out on the board as well um and this is Will partially get to your question David um note that in the problem set I'm going to ask you about some drawings to draw some things um I'm giving you most of the drawing right now but there's one part that I'm leaving out to right now that I'm going to add in on Friday because I don't want to complicate everything but you're going to

see kind of approximately what that drawing would look like or how I might draw this yeah Brooke the light chain doesn't have a d segment ad just a V and a d yes Jonah no there two different chromosomes two different sites Yep this is for the variable region yes um other questions let me go to the next slide before I just do my drawing because this Jonah your question was about chromosomes so the heavy chain is on chromosome 14 in humans um you can see we actually have two two light chain regions because Evolution um

one is on chromosome to one is on chromosome 22 um and so um I might draw I was going to draw this I might imagine a heavy chain Locus that has three V's three d's and three JS because I get a little lazy about drawing so if I did that I might say I have V1 V2 E3 I might have D1 I might have D2 I might have D3 I might have J1 I might have J2 I might have j3 and then as far as you're concerned we can just sort of have the constant regions

in reality there for a heavy chain there are five different sections of constant regions those five constant regions are mu for IGM Delta for IGG IG gamma Epsilon Alpha MDS give everyone apples so it's that order and so we basically have the genetic information for each of the constant regions at the end of this um setup for the variable region um as I mentioned um there's um if I was thinking about um this drawing compared to what I asked you for in the problem set there's one element that I have not drawn here that we

will get to later on if I were going to draw a light chain I might have this I might have two v's I might have two JS and then I would have my constant region it could either be Lambda or it could be Kappa depending on which chromosome we're on um sometimes you also will see um people will like do things like have an an H here see how there's a little H right there V little H that tells you it's a heavy chain if you got confused um and sometimes it'll be like a k

if it's Kappa that's telling you Kappa light chain or land delay chain so sometimes um we'll notice the the little eaches or things like that so that hopefully an answers your question Jonah about the chromosomes so these are in fact different loai on different chromosomes um and David I hope that that addresses your question at least to the level we're going to address it now about constant regions yep Brena and then were that's light yeah so those are light chains and we know they're light chains both because there's not a d and because the constant

region that's there is either Lambda or Kappa instead of the um heavy chain constant regions okay okay yeah for for heavy chain it's the five yeah for for for us yeah this is heavy chain so remember we talked about the five isotypes also remember they technically were like multiple G's and things I'm not actually drawing there's there's really multiple G's there but I'm not drawing them we're I'm just drawing that that that's complicated enough um so those allow you to make make the five different isotypes so you can make whatever variable region and then you

can pair it with whatever constant region um you might want to pair it with um so I'm going to switch the order of um a couple slides because I think this might make more sense um so here you can actually basically I'm just showing you again the same business but with the heavy chain now um and so now you can see this is what the heavy chain looked like originally in the DNA we had some V's we had some D's we had some J's we had some constant region stuff yes here it's showing M as

multiple exons I don't care if you know how many exons it is just know there's M constant region stuff you can see that the DNA is going to get changed so we're going to make some changes in the DNA we're then going to get an RNA transcript and that RNA is then going to be spliced and we're going to then get our protein and so you can see the VD and J region which is in the red green and yellow made up the variable region of that heavy chain and all this constant region business made

up the constant region and so this is sort of the process that we're going to see um we can also see this process happening um in the light chain so here you can see we've got the light chain DNA starts out with all of these Gene segments as being separate we combine V and J um in the DNA then we um have the RNA we'll do some splicing and then we'll make our light chain you can see the variable region is made from the VJ segments and the constant region is made from this constant region

segment um so I want us again um I'm going to think just about my heavy chain here that I've drawn you um and again if you took exactly what I showed you here on the problem set there's one thing that's still missing that we're still not at yet but I I want to to show you one other piece to this which is that this is what the DNA looks like before the B cell starts doing development when the B cell then starts doing development it picks a b picks a d picks a j and uses

those okay so this is sort of like the before DNA I can also draw you the after DNA let's imagine that this B cell used V2 D3 and J2 those just happen to be the ones I picked okay if those happen to be the ones that the B cell picked then this is what the after DNA would look like I said V2 right okay this is my after DNA so what the first thing that you should notice is that my three Gene segments that the B cell picked have actually been joined together and are next

to each other so we've got B2 D3 J2 all right next to each other nothing in between them all of the DNA in between like say the part that had V3 and the part that had D1 and the part that had D2 it got deleted we have removed that DNA we threw it in the DNA trash cane similarly this area with uh between D3 and J2 got thrown away in the DNA trash can but the stuff on the outside like j3 stayed there so we only do those recombination and rearrangements in between the segments we've

chosen the outside DNA does stays untouched so this is sort of the after um again with one set of elements not added um so there is a good thing about all of this and there is a tricky thing about well there are few tricky things about all of this um but the good thing I want to show you is if we think back to this slide here we can see a whole bunch of V's a whole bunch J's right and eventually we want to make an RNA with v and J so let's imagine we're in

a Cell that's not an immune cell it did not do this change to its DNA it does not have this after DNA here okay so we're going to start here with v and we're going to be RNA poas so we're going to make an RNA transcript from this okay so here I am am I'm making my RNA transcript I'm RNA polymerase I'm going to make this RNA then I'm going to make this RNA I'm make this RNA and what's going to happen now yes I am doing this on purpose what's gonna happen now no why

why is it going to stop it's not stop laughing it's just going to keep on going right is there any point to this how would you feel if you had to keep on going for 23 kilobases and you were RNA polymerase for in a kind of pointless way huh you might not cut it but you might do something else or skip it or fall off be like I'm tired I'm not doing this anymore and so RNA polymerase actually can't make it that far and RNA polymerase gets tired and so we never actually get good transcripts

from this but when we put these together and we throw out all that intervening space we've actually put the promoter that was by V really close to an enhancer that's at the end of this whole business and now the enhancer is really close to the promoter they can work work together RNA polymerase is like yeah I can do this and so we finally get a transcript before that we cut out all of that random all that stupid DNA that no one cares about and we aren't using RNA polymerase was never going to actually transcribe this

and so one of the nice things about making the change to the DNA and making and doing this recombination is that we're putting our promoter and enhancer really close together together we're getting rid of all the excess DNA we didn't need and now we could actually do transcription it's like possible where it wasn't possible before um so one other piece of this to um think about um I've mentioned to um and I'm going to a little bit later today but also very much on Friday go into like the enzymatic details I'm going to be like

drawing SE DNA sequence on the board there's going to be like nucleophilic attacks and stuff we're going to see some of that kind of stuff coming up on Friday and it's really going to focus on what's going on at either VD andj or v&j we're going to be kind of thinking about making those variable regions and what's goes on with the DNA for right now I'm just going to tell you you kind of leave the constant region Alone um and so you can see constant region just kind of keeps carrying down here okay let's imagine

that my B cell that I have over here on the board that shows V2 D3 and J2 let's imagine that my bell decides it doesn't look like those doesn't like V2 D3 J2 yes self yes little voice in my brain I am leaving something out when I say at this but it's okay for right now um can that B cell make a different Choice can that beell try something else what do you think okay you think no and the correct answer is no why not the DNA is gone so one of the things to remember

with this this recombination process is that when we change the DNA there's no going back we can't fix the DNA and like go back to the earlier thing once you've made that DNA change you're living with it for for the rest of your B cell life so it and in particular with the heavy chain it's pretty important because there's no other D's like you already threw away the rest of the D's you literally cannot choose any other ones um and so one of the kind of pieces of this is that when we think about some

of the steps in this process um we can think about are they steps that are happening to the DNA or are they steps that are happening to the RNA because the DNA steps once you make that DNA change you made that DNA change for the rest of your life but the an RNA change you can actually go back and play with a little bit you can make further changes to rnas and so for example when the B cell is making the decision about whether it wants to make secreted antibody or B cell receptor the transmembrane

protein it does that at the level of RNA so it can do both cuz the RNA it can just have the same old DNA and it can just do transcription in different ways and make different rnas but if you but any change that we see throughout this process that is a change at the level of the DNA you're stuck with forever and there's no way to further change it yeah Rena change uh yes there are instances where we're going to see the B cell wanting to change we're going to see them next week and the

bell is not going to is well we'll see reasons times when it times when there are situations where changes happen and situations where they don't but if the B cell wants to change and can't like wants to and it's a change that it is not possible to make with the DNA the B cell dies because it it's got no other it's got something sucky and it can't make any change yeah where's it going to get the the DNA to add back um that will I will be able to explain that better with the molecular stuff

that comes up more on Friday because we're GNA I'm gonna have to talk about some of the molecular details of that so um hold that till Friday um I will come back to you and ask you that exact same question after I have gone over a different part of this on Friday um um but what I want you to be be sort of aware of as you're thinking about some of the the things that are leading to these B cell receptor and antibody genes is that you want to think about are we changing the DNA

or are we changing the RNA um is it a thing we can go back on or not um so I originally had um My Little Restaurant I had 10 recipes and I made 25 dishes right how the combos work um um and I drew some B's D's and J's but I was lazy and didn't draw that many so how does how what does this actually look like numbers wise um so for we keep we're sort of still discovering some of this um but for the heavy chain we think there are about 45 V's 23 D's

and six JS and so if you actually multiply those together and get how many combos you can make to make how many different heavy chains you get 6,210 heavy chains and that's 6 210 heavy chains [Music] from 74 genes so you can see that this whole math of using combinations is buying us a lot of pro we get a lot of proteins for a small number of genes um with the light chain we either have Kappa or Lambda so we for kappas we have 41 v's and five J's for lambdas we've got 33 v's and

five J's so we can make 205 um kappas and 165 lambdas um and so that is what is it 370 light chains out of less than a 100 jeans so that's yeah that's so that's pretty good and then if we take all the different light chains and all the different heavy chains and try them in different pairs we're at we're at less than 200 genes if we add up 45 and 41 and 33 23 less than 200 genes 2 million antibodies 2.3 * 10 to the six antibodies so what I hope that you notice here

is that the combinatorial diversity is a big way that we are getting lots of protein combos out of a small number of genes um but there is sort of one other thing you might notice from these numbers 2.3 * 10 to the 6 um is a big number 2 million it's not 10 to the 16th which was the number I was trying to get us to so the combinatorial diversity doesn't get us all the way there we need something else that's going to ramp this up and get us the rest of the numbers um what

I also haven't specifically mentioned in talking about this is that um I haven't actually told specifically mentioned those Loops the cdrs which if you remember the cdrs were the little loops on the end of the imunoglobulin domain of the heavy chain and the light chain that are actually contacting the antien and so I haven't told you mention those Loops at all in all of this and so you might say what about those complimentarity determining regions those hyperv variable regions the regions that are the most variable the little Loops you can see these are my little

Loops here that are the three fingers on the heavy chain I know this is heavy chain because it has H and the light chain I know that's light chain because it has L they're that are actually making the contacts how do they fit into all of this um and for CDR and also what you can notice if you look at this is that in both heavy chain and light chain if we look at the amount of variability cdr3 is way more variable than the other ones like cdr3 is the super crazy hyper a lot mostest

variable region it turns out that cdr1 and two are pretty lame or relatively lame they're just encoded by whatever V segment you picked so there's like a 100 V segments and each of them encode Loops that would be cdr1 and C cdr2 but cdr3 the really really really variable one is interesting here because cdr3 is actually encoded by the DNA right where we did The Cutting and pasting right where we did the joining and so here this little box is showing you cdr3 and you can see in the heavy chain it's like encoded by the

area the end of the V the D and the J this really unique area that's special to this one B cell that picked this one combo for the light chain the V the the cdr1 and cdr2 are encoded by the uh V segment and the cdr3 is encoded by the junction of the V and the J so we're getting this we have this one Loop that's binding antigen that is super unique and variable and it's actually coming from how we joined together the DNA which is known as The Junction yes so it's actually for the

heavy chain it's go all goes all the way from v through D to J so it's actually the junction of all three of them for heavy um but you're exactly right for light yeah David it's totally random combinations um so at these sites in cdr3 I've mentioned the word that it's the junction it's the place where we pasted together the DNA right we pasted together the V that we've chosen the D that we've Chosen and the J that we've chosen or the V and the J that we've chosen if this is a light chain the

other way that we get diversity is that when we do that pasting which again is when we're really thinking about the cdr3 region we don't paste precisely sometimes we so here we're trying to paast together this V sequence that's pink and this J sequence that's yellow since it's a V and a j with no D in between we know it must be a light chain and sometimes we end up with this where we like deleted some sometimes we can actually add some extra ones and so our pasting at that Junction or the liation of the

two pieces of DNA together or as you can see here the joining is not precise and so sometimes we get a little bit of addition or subtraction of some base pairs there and that's how we get extra diversity here so even if we had two B cells that chose the same V the same D and the same J they could do their cut and paste a little differently in terms of how many base pairs they added or subtracted and as a result they would have different cdr3s and they would bind to slightly different antigens um

and so this is that's really all junctional diversity means is that we have this very imprecise joining of those two pieces of DNA um and so when we actually can uh when we actually add on all of the changes in how many base pair get added or subtracted with junctional diversity that's how we get to our final number of antibodies this textbook is showing you 10^ the 13 instead of 10 to the 16 it does not actually matter to us whether it's 10 to 13 or 10 to the 16 just know that the junctional diversity

on top of the combinatorial diversity is how we get a big number I see some puzzled looks and questions sounds good so when we're tting not precisely is it because we wasn't precise or just we that is part of the mechanism detail when I'm drawing sequences and like three prime hydroxy on when on Friday that I'm specifically going to address so that is exactly coming up on Friday great question yes um maybe yeah and you can imagine that well and so you you say the flaw in Precision in how this works um but realize that

in the organism so if you imagine um an organism that did not have this that organism would make fewer antibodies right and its baby might die of some infectious disease that it couldn't make an antibody to oh oh no so what what I hope that what I hope you see here and a million times throughout the semester is sometimes when I look at Immunology I look at and like yeah this is evolution because evolution is a mess and if I if I was gonna try to put this together myself I would not do some of

these weird things um and also you know I've mentioned this before but realize that you know the choosing of the v d and the J I say choosing that's a bad verb here it's completely random this is all a numbers game each B cell is basically getting a VD and a j that it uses and you're just hoping based on the number of B cells that you make that you get one of every combo do that you have another question there no okay um and so this is sort of another way of thinking about um

what I just said um so this process that I have just described to you of vdj re combination um this is one of the most important things to know about this this is one of those things that so many people have misconceptions about Immunology with this happens during development of your B cells when the B cells are developing in a primary lymphoid organ like the bone marrow you right now are making new B cells who are doing development and you've been doing it ever since you were a fetus and those B cells are just randomly

picking A V A D and A J all in the primary lymphoid organ this is happening before you see antigen before you get infected you right now have made one be sell of every possible combo already you got one of every single one the the some crazy pandemic that's going to happen in 20 years you already have one V cell that has made that vdj combo you already got it right now the reason but the issue is you only have one of it and one of it is not going to be enough to really protect

you the only thing that's going to really help you is if when you get a whole Army of it when it Finds Its antigen this pandemic pathogen in 20 years finds a antigen it gets a signal it basically gets a signal that says you are useful you are good we need more of you and it makes many copies of itself right now I don't think any of you have ever had Ebola I hope that none of you have ever had Ebola right now you all have B cells that recognize Ebola epitopes and what I sincerely

hope for you is that those B cells go around your body to different lymph nodes for the rest of your life and as one of my colleagues once described it those B cells die of unrequited love when you die because they never found their antigen and they never got to make a clone of themselves and do something so ideally you have one of every of every B cell making every antibody but one isn't enough to do much for you you need the whole Clone Army version of it and that only happens after it expands in

a secondary lymphoid organ after seeing antigen so every so you know we can talk about any antigen you want you already have one B cell right now that makes that antibody it's just waiting to maybe multiply and make more of itself yep Brook and gets a signal yeah but does it it randomly find an so it's basically it spends all of its time going around the body being like are you my antigen are you my antigen are you my antigen are it just circulates your body for your whole life no it just goes from secondary

from lymph node to lymph node around your body for your whole life being like can I find my antigen in this ly in this lymph node if I do then I know that region is where I should like you know if if go to and start doing surveillance but it spends its whole life looking for its one antigen okay and again in many cases you hope it never finds it yes does you every possible Bell you have you have yeah you already got it yes we can actually yeah yeah and there's another process that we're

going to talk about that happens late in the life of B cells that actually even ramps us up and makes it better but we're not we're we're still in the primary lymo organ we're not there yet yes so so so we so that's a great question um I say that this is adaptive because one of the things that we use when we when we are defining adaptive sometimes is ability to do bdj Rec combination to make that really specific receptor where the innate receptors did not involve BJ Rec combination okay shall be you have a

question okay um so yeah here you can see just another view of vdj combination happening in the absence of antigen so vdj recombination happens here to make this B cell before you ever got infected so vdj recombination is like here before all of the rest of this business so whenever you're sitting around doing nothing and your mom yells at you you can be like I'm doing vdj recombination um it's very complicated you need to give me some space um and the specific um details of exactly how that works in terms of molecular mechanisms are what

we're going to talk about on Friday as well as one other kind of caution or issue related to vdj um so I will see you guys in lab tomorrow um yeah that's it