I took my first immunology class at UCSD in the spring of 2004. I've always been interested in signaling (how cells take information from the outside and translate that to the inside) but the subject matter of this class was set to disappoint - in terms of signaling, it more or less stopped at the outer membrane of cells. Even though looking back, I can see now that a subject as vast as immunology has to cut some corners in a 10 week course, early on that quarter I was a bit frustrated. But just before the first midterm, we started learning about one of the most bizarre behaviors of cells that I've ever learned about, the thing that would get me hooked on immunology and make it my passion. This is the molecular magic that can generate a functionally limitless number of different genes that allow B-cells to make antibodies that recognize almost any chemical structure that has ever existed or will ever exist. It's so important for the immune system that its acronym amongst immunologists is GoD - Generation of Diversity.
GoD is made possible by a complex series of steps that, for reasons that will soon be come apparent, we call V(D)J recombination. But in order to explain why V(D)J is so amazing, we have to take a few steps back and look at some of the underlying principals of biology: DNA, genes and proteins. For all living things that we know of (except a few types of virus), genes are encoded by DNA. The word gene gets thrown around a lot, but the simplest way to think about a gene is as a code that tells a cell to make a particular protein. Antibodies are proteins, so you would be correct to assume that there are genes that code for antibodies written on the DNA within your cell. The trouble is, humans have between 20,000 and 30,000 genes, but the average person is capable of making about 10 billion (with a b) different types of antibodies that all bind unique molecular shapes. There simply isn't enough room in your genome to fit a gene for every antibody you can produce. What in GoD's name is going on here?
It didn't take the early immunologists long to figure out that this was a problem, or to start looking for solutions. The first thing that became apparent is that all of those individual, unique antibodies are actually far more similar than they are different. As I mentioned a couple weeks ago, antibodies are shaped like a Y, and it's the end of the two arms of the Y that are actually the business end that stick to things. The bottom (or butt according to Abby) within particular classes of antibodies are are remarkably similar within individuals, and even across the population.
So, one solution to the not-enough-room-in-the-DNA problem is to have one copy of a code for all of the stuff that remains constant, and a bunch of different copies of the code for the part that's variable.
This is sort of how it works, but it's a bit more complicated. Even if you vary only a tiny part of the gene, getting to 10 billion would still take up more space than the entire rest of your genome. Instead, the gene that codes for the variable part of the antibody protein is broken up into a bunch of different segments. The end of the gene still codes for the constant region, but the variable region is split up into 3 segments, and there are multiple versions of each of these segments. In humans, there are about 100 versions of the V (variable) segment, 30 of the D (diversity) segment and 6 J (joining) segments1. During the development of the B-cell, one V, one D and one J are brought together to form a complete piece.
In order for B-cells to accomplish this, they actually break apart their DNA. An enzyme randomly picks a D and a J, cuts across both strands of DNA and then stitches them back together. Then, the same enzyme grabs a random V as well as the new DJ segment, cuts the DNA again and mashes V to DJ.
This is astonishing - double-strand breaks are incredibly dangerous, but B-cells in your body are doing it all the time. As Abbie mentioned with regards to class-switching:
This is an abomination. This should not happen (HELLO??? CANCER!!! We have a million safe-guards in our DNA to kill cells that start doing crazy stuff like CUT UP THEIR OWN DNA!!!), but in this case, it does, for a very good reason.
The reason for doing this at the DNA level is a topic for another post, but we're not through with GoD just yet. The algebra-obsessed among you might have noticed that 100 x 30 x 6 does not equal 10 billion. This is true, but I've left out a few things. First, an antibody isn't just a single protein, it's actually four proteins stuck together - two copies of a heavy chain, and two copies of a light chain.
The variable region of the light chain is also spliced together (though it only has V and J segments), and it's the combination of heavy and light chain variable regions that ends up sticking to the antibody's target. Plus, there are two different light chain genes, either of which can be combined with the heavy chain. Plus, you have two copies of each of these genes (one from mom and one from dad). You can't get a V from mom pairing with a DJ from dad, but you can get a light chain from one and a heavy chain from the other. Even still, if you do all the math, this combinatorial diversity will only get you to a few hundred thousand possible antibodies - a far cry from the true extent of the average person's antibody repertoire.
The final piece of GoD is what we call "junctional diversity." When the DNA is severed durring V(D)J recombination, it's not always a clean cut, and some extra nucleotides must be added to fill the gap. In addition, there's an enzyme whose only job is to add in random nucleotides to the junction. This randomness can be extreme and it's here that GoD mangages to reach the 10 billion mark. This is also the reason that every individual has their own set of antibodies. The inherent randomness, from the selection of V's, D's and J's, to the jagged cuts to the random inserted nucleotides means that not even identical twins will have the same repertoire, or even similar repertoires.every individual has their own set of antibodies. The inherant randomness, from the selection of V's, D's and J's, to the jagged cuts to the random inserted nucleotides, means that not even identical twins will have the same repertoire.
1Matsuda and Hanjo, "Organization of the Human Immunoglobulin Heavy-Chain Locus" Advances in Immunology. Volume 62, 1996, Pages 1-29
The problem is I can see the problem, but I can't understand it at a level I'd like. On the other hand, seeing GoD helps me to see Yaweh or is that Jehovah or is that Allah or is that nirvana...well, I'm not smellin' any teen spirit....okay, okay, this is fantastically cool shit. It really makes me resent my misspent youth...Kevin, and Kevin's cool science buddies...you are on the cutting edge of cool! Keep up the good work and make an old fool use his noggin'. Good post, thanx!
Loved this post. I work for a local biotech and occasionally teach some basic immunology to sales reps. This is so well written, and I wish I could have read it years ago! Would have been very helpful then, and still is now!
I share the same excitement about how GoD is a wonderful piece of evolutionary advantage. Just imagine if Darwin had know about this, he would have died the happier man on earth.
I would like to remember that TCR and BCR are generated by the same kind of recombination, antibody are "just BCR without it's tail". It turn out that B-cell keeps the specificity between it's BCR and antibody producing (it's still can edit it )
Thanks for this nice post, i hope that more of this came out.
Then there's 'Error-prone' DNA repair in 'Germinal Centers' (spleen and ? ) Invented by Sharks? - presumably there's a system that eliminates self-antigens and selects for stronger binding to Aliens...
@ Mike - feel free to ask any questions to clarify. Or is it that you're not even sure what questions to have?
@ Adam & Rafael - Thanks! Happy to be of service.
@ g bruno - Not quite. This isn't error prone copying like HIV or something like that. There are enzymes who's job it is to go and cause these mutations. And what I'm talking about here occurs during b cell development in the bone marrow. There IS a mechanism to edit in the germinal center (I'll have a post on that later) but it's also done by a specific enzyme. And you're also right, there are a bunch of systems put in place to avoid attacking self.
As for the evolutionary origins, we know that all vertebrae have the system, and nothing outside chordata does (the lawless fish have a convrrgemtly evolved system similar in concept), but the full e discussion of that is also a topic for a different post.
What about the B cells?!
I was reading this article on Nature and thought of your post about the B cells. This section threw me off:
Porter's group is one the first to report results from a generation of chimeric receptors that include both an antibody to target the cancer and part of a receptor that amplifies the T-cell response. This time, the doctored T cells proliferated more than 1,000-fold in the body, and were still present at high levels six months after the treatment.
June credits this expansion and persistence for the study's dramatic results: two patients in complete remission and a third showing a partial response. The treatment kills off normal antibody-producing B cells too, but patients can be given regular infusions of antibodies to compensate for this, Porter says.
I do not know very much about infusions of antibodies, but it seems the cells needed to produce them being destroyed by these engineered T cells should be a bigger concern. If those cells remain in the patients body for an extended period of time and have sleeper T cells left throughout the body then the patient would need IVIg, which I think I am correct in thinking that is what is meant by "infusions of antibodies" but please let me know if that is incorrect, for an undetermined amount of time. As long as the engineered T cells are in the body, it cannot produce B cells, right? From my understanding it takes a lot of plasma to produce this antibody replacing drug, it is already high in demand as well as cost, and it is already in danger of being in short supply. Like I said, I am not very knowledgeable in this area and was hoping you might be able to respond to some of my questions/thoughts.
Is there any other way for these patients to receive antibodies without the use of plasma?
If there is not, then it would seem that these T cells could cause a bit of trouble. I mean, can the B cells even be replaced after the T cells are no longer present? What if the T cell are always present or the patient's cancer comes back and they need more of the engineered T cells? They would have to continue receiving infusions.
Does it seems like it should be a bigger concern or am I totally off?
Okay, look, I've got to confess. Prior to reading this I'd drank several good beers: oatmeal milk stouts, mocha porters and IPAs. Don't get me wrong, I only drank one or two of each. While doing this I'd turned on some blues and spent time floating on my back in a pool, gazing up at the stars and the nearly full moon. I was very mellow and my mind felt very expansive. Reading this stuff was the sort of conversation I wish I'd been able to have 25-30 years ago...while having a few brews. Very deep stuff and the given variables and combinations create a sort of stunning realization or the chaos from which life springs. Now, having said all of that, it is well written and straight forward. But, it creates many questions simply about the nature of biological reality...and in a buzzed mellow sort of mind, to a laymen who can't have those conversations on a regular basis....it was more than a little mind blowing. Again, I appreciate your work. Please keep it up.