At this point, we turn to the process of B cell activation. Here is a review slide to remind you where we've been. So, at the top here, we've looked at the problems and processes of synthesizing the immunoglobulin receptor in the membrane. That is, we went through gene rearrangement, negative selection, the maturation process that involve putting the D chain as well as the M chain into the surface of the cell, and then getting it ready to be released into the circulation. So, basically, we wound up with a mature but naive B cell, that is, we got this far here. Of course, now, it's heading out into the circulation and we have to look at the process of activation. So, here's a reminder. This is what the cell looks like. So, to survive, this cell is going to have to bind antigen, develop a signal to the interior, and then begin to differentiate, divide, and ultimately, secrete antibodies. As a reminder, this is the second half of that review slide, and we're starting here at the top with this cell here, the mature B cell, and really, in this clip, we're only going to get through activation. We're going to look at this process whereby we activate it, and then in the clip after that, we will turn to how the T cell promotes the rest of the processes shown here in this illustration. Okay. So, you've seen this one before, and we know that to survive, the cell must activate, that is, encounter antigen that will bind to its receptors. There we have it. When it binds to those receptors, that's going to set up a signal to the interior, and the cell will begin to activate, develop, and divide. Now, it turns out, there are a number of ways to set up the signal into the interior, and we're going to eventually call this signal one. That means that to begin the process of activation, the B cell has to bind to foreign antigen, but to keep going, it's going to need more than just that. That is, it's going to need a second signal. Usually, this comes from interaction with a TH cell, but first, we're going to look at two different other ways that you can activate a B cell without the intervention of a TH cell, and those are called thymus-independent reactions, the antigens are called thymus-independent antigens and that distinguishes it from the thymus-dependent version which involves the TH cells. Thymus-independent antigens T-1. One antigen, and we'll come back to this at various times in this course, but one antigen that really upsets your immune system is lipopolysaccharide found in the surfaces of gram-negative cells. So, if one of those things binds to a receptor recognition region that just happens to recognize it, that sets off a strong signal. The strong signal is not enough, however, and in this case, the second signal comes when the lipopolysaccharide binds to a toll-like receptor designed to activate when it sees it. So, these two signals together will produce the cell that begins to divide, and then will ultimately begin to secrete antibodies that will bind to the lipopolysaccharide and hopefully, that will make it easier for cells to phagocytize the pathogen and get rid of them. The second kind of thymus-independent antigens T-2 works in a very similar kind of way, but in this case, we're looking at things like flagellin, the thing that's in the bacterial flagella, capsule polysaccharides around the outside of bacteria, and other large repeating molecules. When these things bind to a cell that can recognize them, this often means that because of their length, they bind many, many 10,15,16, or more immunoglobulin receptors together. The mere fact that you're clustering all of these guys, and we'll see banging them together, will often activate the cell all by itself. So, we have a very strong first signal. On the other hand, it seems that there needs to be some sort of additional potentiation and that is not nearly as defined the second signal as we had in the previous slide, but includes anything like proinflammatory cytokines. Here, we have IL-2, IL-3, IFN gamma if it receives these, and for some reason, it also seems to require some kind of intervention from either macrophages and sentinel dendritic cells, and of course, these guys also secrete their own cytokines. This second signal is kind of nebulous in its requirement, but in any case, we have two different ways of producing an active B cell assuming its receptor is set up to receive these from what are essentially pattern recognition signals. Now, the downside of this is you will not make memory cells, you will not class switch, all of those things will require intervention of a T cell. We'll talk more about that later. But the first signal is set off in much the same way whether you have a T-1, T-2 or TH-dependent, that is thymus-dependent B-cell. So, we're going to go back and look at that activation signal, signal one, which is used by both kinds of cells. When an antigen cross-links these two immunoglobulin receptors, it's going to bind one and then eventually, the other will hook on to it, you can see that what happens is, the Alpha-Beta receptors at the interior will also be drawn together and will tend to bang into each other. That's going to make changes in them that are going to activate them and begin the process of setting off a signaling sequence to the interior. So here, I've got them spread out. So, we've got these activated ITAM. So, here are the Alpha and Beta immunoglobulin signaling molecules and their cytoplasmic portions have some Immunotyrosine Activation Motif or ITAM that's capable of picking up a phosphate. So, once they been activated, they will attract the enzymes that put the phosphate on here. These that I'm showing here are called src-like kinases, a kinase is something that puts a phosphate on things. That actually isn't the only thing attracted to these ITAMs. This is a sort of a very complex process, but in any event, we have these kinases coming in here, and they're going to add phosphates to the ITAMs extending into the cytoplasm. Once you do that, you're going to provide a docking site that's going to set off the next series of reactions. So here, we have this here. Once the src-like kinases have added their phosphates, they leave. Once the phosphates are there, they will attract a whole bunch of other things that dock to them, and in particular, we're looking at a class of kinases called syk. These kinases are actually very powerful and are capable of setting off a whole bunch of different reactions. We're going to look at three of them. Now, just as an aside, if you look up B cell activation in a number of different textbooks or on the web, you're going to see one very complicated diagram after the other, and they don't all going to look exactly the same. The problem with this stuff is that so many different things are happening. What I'm doing with this is basically looking at three overarching kinds of responses you get after you have activated the first stage in this series. One important pathway you get after you take one of the membrane phospholipids and hydrolyze it into diacylglyceride or DAG and PIP or phosphoinositol. These compounds again trigger a wide variety of responses. One branch results in calcium release in the ER that activates calmodulin, which in turn activates a number of other pathways. But one important one is the removal of a phosphate from NFAT which activates it and then NFAT becomes an active transcription factor, goes into the nucleus, and turns on the genes necessary for the cell to divide and then changes developmental pathway and produce antibodies. Similarly, a second version of this branch leads to the removal of an inhibitor, inhibitor kappa B, from an inactive NF kappa B molecule, and this then becomes also an active transcription factor, enters the nucleus, and up-regulates inflammatory genes and will again help the cell to develop into an active B cell. Notice that the transcription factors in this pathway or branch pathway are both in the ready, they're inhibited, and they're activated by removing an inhibitor, that's a very common developmental pathway in many cell biology systems as well as developmental and immunological. A second pathway that's used in this is the very classic common RAS-RAF-MEK-ERK pathway and this is a phosphorylation pathway, where the first one phosphorylates to number of the second, and then they phosphorylate the third, and they phosphorylate ERK, and then ERK goes on in and phosphorylates multiple transcription factors that will then bind to the DNA and promote B cell division and differentiation. So, the pathway is something that's used in a number of different developmental promotions. But in this case, it ends with ERK phosphorylating transcription factors that will then go in and up-regulate an immune response. So, in this case, we're not removing an inhibitor, we're adding a phosphate from the ERK, but it's the same deal the transcription factors are in the ready, and they're waiting to be activated. The last thing we look at is the phosphorylation of membrane, phosphoinositol. Okay. So, what you've got is a phosphoinositol in the membrane that has two phosphates. You add a third one to it and when you do that, this attracts various compounds proteins to the membrane and results in the down-regulation of apoptosis. So that also helps the cell to survive and that's going to be necessary as part of this procedure. So, I have been a bit sketchy about this pathway, but I think maybe what is really important is for you to see that a signal outside is transduced to the interior via phosphorylation and then that sets off multiple branching pathways that will then cooperate in a variety of ways to promote the development of the B cell. There is one final issue here, and then how do you turn it all off? Anyone who's seen Fantasia and poor Mickey Mouse fighting off all of those brooms understands that when you have a system where something multiplies, and multiplies, and multiplies, that you might want some way of turning it back off again. So, what you have at the surface of a plasma cell, that's what's being shown here secreting antibodies, is a number of Fc receptors that will judge how many antibodies are out there. Now, they're actually unusual Fc receptors because while I'm showing them here as being attached to antibodies, what they really are is receptors for antibody-antigen complexes. When all these antibody-antigen complexes build up, then a B cell essentially knows that help has been successful. In other words, it's made the right kind of antibodies, these antibodies are doing their job, the antigens are getting tied up, and it can sort of start to slack off a bit. So, in this case, the antigen-antibody complexes, when they bind to the Fc receptors, actually essentially shut down the plasma cell. So, we have down-regulation of antibody production in here and eventually a plasma cell will only last for a couple of weeks before it dies a natural death of apoptosis. So, that's it for the first stages of activation of B cells, and in the next clip, we're going to see how T cells will then also promote this process and lead to class switching and the production of an even more efficient recognition region.
