nm0237: so can we start now please just past nine o'clock can i remind you today before we start the official lecture that you've got video discussion sessions er the third grou-, the third er period here okay same rooms but different members of staff we will be discussing the tests and so on i think you might have found this test perhaps more difficult than the previous one but er after all you are beginning to learn more of the subject so er please er make sure that you go through it okay so let's start with er the subject then can i have hush please at the back er we're dealing with if you remember the lymph nodes okay and especially we were talking about stimulation of B-cells which occur in the lymph node er if you take a typical lymph node whereabouts in the lymph node do you normally find B-cell activity cortex paracortex medulla ss: nm0237: sorry ss: nm0237: cortex okay mainly occurring in these follicles the secondary follicles here which we've discussed before but also there's some B-cell activity outside of the follicles er still in the cortex in the sort of non-follicular area if you like as well so there's activity in both zones but predominantly it's in the follicles so this picture don't bother to don't bother to try to copy it down but this tries to show an enlarged picture of the cortex area here and the paracortex area showing a follicle here with a germinal centre which is where you find the B- cells proliferating and also the sort of cortex outside of the follicle round here and you'll notice in both that it pictures er macrophages which are in effect antigen presenting cells and also these follicular dendritic cells which again are antigen presenting cells remember A-P-Cs antigen presenting cells and er there does appear to be B-cell stimulation in both areas but the B-cells in the follicles are different to those outside of the follicles in fact in the follicles you have cells which are called B-two cells and outside you have B- one cells and they do appear to respond to different types of antigen okay so there's two sorts of B-cells in the follicles B-two cells outside of the follicles B-one cells still in the cortex but they do appear to respond to er different sorts of antigen and if you look in these especially in the follicles then you will find B-cells proliferating you get mytosis and maturation and so on and the memory cells will stay there but remember the plasma cells once they're produced the plasma cells secreting the antibodies will go to the medulla area and secrete the antibody from there okay there we can to go the medulla area and also they may go off to other lymph nodes and so on okay so what we want to know now is to to try to answer the question er how does antigen stimulate a B-cell how do the B-cells become stimulated to produce antibodies so really today's lecture is about this topic about how B-cells become stimulated by antigen ultimately to produce er antibodies and er i would say right from the start that i don't think anyone knows the complete story er but er one can make pointers in that direction so okay you get lots of different forms of antigen there's no doubt Professor namex's told you er and some of them are able to stimulate some of these antigens are able to stimulate B-cells directly this picture outlines how this might be here's a B-cell remember it's got I-G on its surface immuno-globulin molecules on its surface and one way is for antigen to cross-link these I-G molecules directly cross-linking so you've in fact got a polymeric form of antigen an antigen which contains lots of repeater units so that all these groups on the outside remember will have to be the same to attach to the I-G on any one B-cell because all these receptors are the same okay so it's a polymeric molecule repeating molecule er which can stimulate B-cells directly and in fact what happens is in all these cases that if you've got a B-cell with its Y-chains on the surface like that i'm not going to draw them all but all round what happens is the antigen has to cross-link individual I-G molecules it has to link at least two together and obviously you see in that first case you've got cross-linking between a lot of these molecules and all the once this happens all the I-G is taken to one end of the cell a process called capping the I-G with the antigen on it goes to one end of the cell and then it's taken into the cell by endocytosis so you get capping and endocytosis and that's what appears to stimulate the B-cell okay material has to cross-link the I-G taken into the cell after it goes to one end like that so one method is to use polymeric molecules the second method is apparently you can get equivalent stimulation by antigen binding to a a a an I-G chain but also a mitogenic effect that also appears to be able to stimulate a B-cell you get some cross-linking of Y-chains but also linkage to a mitogenic receptor on the B-cell and the third sort of response is where again you get cross-linking but you get some complement binding as well remember B-cells have a complement receptor on you don't know what complement is yet except it's a polypeptide but that can attach to the antigen so you get multiple linking I-G linking there immuno-globulin linking there and complement linking there and that also will encourage this procedure to start to occur so there's at least three sorts of bonding polymeric mitogenic complement bonding so i'll give you some examples of this on a an overhead and you can er copy them down so antigen can directly stimulate B-cells you can have polymeric molecules long linear molecules usually relatively firm ones and examples of those are pneumococcal polysaccharides of er bacteria er diamino acid polymers and also artificial er compounds like polyvinyl pyrrolidone pyrrolidine sorry okay and they cross-link the antigen receptors to the B on the B-cell just as the first example i gave you an example of a mitogenic one of bacterial lipopolysaccharides they bind to the antigen receptor on the B-cell possibly more than one Y-chain but they also seem to offer a mitogenic stimulus and then thirdly you can activate via complement binding no specific example now these antigens they're probably the exception rather than the rule they're perhaps somewhat strange antigens in the sense that they can stimulate B-cells directly and it appears that predominantly the sort of B-cell that is stimulated are these ones that are outside of the follicles they seem to be the sort that can be stimulated directly without any other assistance probably macrophages present this stuff this antigen to the B-cells but they don't seem to need any further assistance to produce er a an antibody response but the vast majority of antigens are poor direct B-cell stimulators most of them are poor things like a lot of parasites for example like sheep red blood cells which are l-, used in a lot of immune experiments and so on these are relatively poor direct B-cell stimulators they don't do this sort of thing and also a lot of antigens are degradable and so are not present and able to activate the B-cell for fairly lengthy periods of time so the predominant B-cell's er stimulation is not by the sort of antigen which i've already mentioned so the majority of antigens the majority of antigens for instance sheep red blood cells as i say which is sort of thing that immunologists have tended to use do not have the above molecular characteristics they're not linear they're not mitogenic they might well not bind complements in any sort of way but you still get immune responses against these other antigens okay they're not of this character up here but you still get these responses and what you find is that the first three sorts these er er polymeric mitogenic or complement binding ones tend not to use any cells that are derived from the thymus they don't depend upon T-cells so no T-cells required okay but for the majority of antigens like sheep blood cells if you're going to get an immune response against them you need some T-helper cells you need T-helper cells to get a significant immune response and therefore the majority of antigens like sheep red blood cells are called thymus-dependent antigens so the first three groups are thymus-independent antigens don't need T- cells to help the B-cells the last group are thymus-dependent antigens needing T-cells to help the B-cell and these are predominantly the ones that occur in the follicles these B-two B-cell okay so what we'll spend most of today really talking about is the main re-, sort of response that you get with er as i say in the follicles and they'll be involving B-cells they'll be involving T-helper cells and also antigen presenting cells are important in this story okay so this is all happening then inside the lymph node follicle or the equivalent area in the spleen or wherever in these follicles in there and you've got these follicular dendritic cells which are at least one form of antigen presenting cell in there so how do we know that T-cells were involved well there's some evidence right from the start remember i showed you this last time er where we had chickens they'd taken out the thymus or the bursa at birth or had left them intact and remember that when you measure the antibody production taking out the bursa taking out the B-cells antibodies taken out completely if you take out the thymus you get a bit of antibody production but it's not as good as if you leave both systems in together so clearly the T-cells are playing a role even in that experiment there but you can go further than that you can use mice and do various nasty things to them and you can show that T-cells and B-cells are important okay so let's consider the situation of a lethally irradiated mouse this is wa-, a mouse a mouse of an inbred strain so you can have lots of different mice almost all the same and they've been irradiated and er they would die shortly afterwards but okay they'd probably die anyway of something or other but what happens to them is that the irradiation knocks out lots of er different cell types including the lymphocytes okay so irradiation knocks out the lymphocytes and then you can take these mice and you can repopulate them with lymphocytes from the same inbred strain of mice their their er brothers and sisters and so on and remember if it's an inbred strain these mice are all genetically identical so really transferring the cells from one mouse to another will not make any difference there shouldn't be any nasty responses as a result okay so you've got some lethally irradiated mice and you populate them with or repopulate them with lymphocytes of different sorts and give them an antigen sheep red blood cells S-R-B-C so you inoculate one mouse or possibly if it's experimental you'd have to have some replicates of course inoculate T-cells and sheep red blood cells you don't get any antigen any antibodies pretty obviously if you inoculate them with B-cells you take your mouse your lethally irradiated mouse put in some B-cells and then give it sheep red blood cells as the antigen you get a bit of I-G-M and no I-G-G little bit of I-G-M not very much and some I-G-G and you do the third experiment this lucky mouse gets both T and B-cells and you give it sheep red blood cells and it produces a lot of I-G-M and I-G-G which suggests that although the B-cells do produce antibodies you need some help from T-cells to produce adequate quantities of antibodies that is these T-helper cells are required T-helper cells are required and the same sort of thing can happen in mice which are genetically deprived of their thymus in the mouse world there are some mice known as nude mice jolly chilly but they they certainly haven't got any hair nude mice have no hair but that's not the important thing the important things about nude mice is that they don't have a thymus they're thymus-deficient they're athymic as it's called oops pen seems to be on the way out athymic genetically don't have a thymus so they've got B-cells but no T-cells so they're a bit like these mice here although they're not at death's door but they haven't got a thymus but if you get some nude mice and put T-cells into them from another inbred mouse which is normal very similar to them but normal then these athymic mice these nude mice instead of not producing antibodies will suddenly start to produce antibodies so you can give nude mice which haven't got their T-cells so you give them T-cells of the right sort and they'll start to produce antibodies whereas they didn't produce antibodies before so T-cells and B-cells then have to cooperate to produce an immune response okay so what about macrophages antigen presenting cells and similar sorts of er cells are possibly important as well and you can show this by various er culture experiments as is shown here you can separate out the spleen cells don't worry about the technique but you can separate spleen cells out and you can er isolate them and grow them in culture and so on quite happily in a petri dish as i say the metho-, the methodology doesn't matter you can divide them into two populations you can divide them into those that stick to glass glass adherent cells and you can also have non-glass adherent cells and the glass adherent cells are the macrophages they have the ability to stick to glass or plastic as long as it's clean and non-glass adherent cells will include the lymphocytes okay so you take your mouse you take out the spleen you separate the cells and you do the following experiment again using sheep red blood cells if you like as the antigen perfectly possible so you take your mouse spleen cells you divide them into three lots you have a complete set you have some glass adherent ones and you have glass non-adherent ones and if you have the complete cells you present these cells with sheep red blood cells as the antigen you get antibodies produced in due course you have the glass adherent ones you present antigen no antibody you have glass non-adherent ones you present antigen you get no antibody if you remix the sets the glass adherent ones and glass non-adherent ones you get antibody produced so clearly glass adherent cells er are necessary for the production of antibody but no one would say that macrophages actually produce it but they're clearly important for its production and so the glass non-adherent ones need the glass adherent ones to make the antibody macrophages are needed to produce antibody so these are cells from a line of mice from strain of mice and so they're all from one mouse basically you can take one spleen of one mouse and do this experiment and not sure this is going to work actually okay er move it up a bit sorry we'll take the top off here so these are all all the same all cells from one mouse and so because they're from one mouse they're all going to have the same M-H-C group let's say for the sake of argument they're all H-two-K they're all of the M-H-C same M-H-C type major histocompatibility complex remember all cells from one animal will have the sem-, same M-H-C type and similarly with an inbred strain of mice if you got a lot of siblings of one inbred strain they'll all be of the same M-H-C type so you could take the glass adherent cells from one mouse and the glass non-adherent cells from another mouse all of the same inbred line and you'd still get the same response when you mix them you've got antibodies produced but if we do a devious experiment where we take two lines of mice one of one M-H-C type and one of another M-H-C type say H-two-K and H-two-D and then we try and get the cells to cooperate as in below the line here okay so you've got glass adherent cells which are H-two-K from one mouse which is H-two- K glass non-adherent cells from a mouse which is H-two-D now and you put them together you don't get any antibodies produced you don't get antibodies produced when the macrophages and the lymphocytes come from mice of different M- H-C type and this is what is called M-H-C-two or sorry M-H-C restriction the response is M-H-C restricted okay because the macrophages and the lymphocytes have to be of the same M-H-C type and in fact it's M-H-C-two restricted because it's that molecule which is the important one it's the M-H-C-two molecules that are important for reasons which i won't explain in detail here but will become obvious a bit later on so it's M-H-C-two restriction that's applying here it the cells have to be of the same M-H-C ti-, type before you get the response okay so what goes on between these cells you've got three sorts of cells you got T-helper cells you got antigen presenting cells or macrophages and you got B-cells and somehow you've got to get them together to make antibodies well early on the theory was that you got a response something like this this is what you would find in many early textbooks you have an antigen here which consists of a complicated molecule it could for instance consist of a carrier protein such as er chicken gamma globulin can somebody stop talking at the back please chicken gamma globulin C-G-G and it's known that you can make artificial antigens by sticking a the small molecular group on called a hapten for instance dinitrophenol and i expect Professor namex has told you about that dinitrophenol and it's known that the antibody will recognize the dinitrophenol as long as it's attached to a carrier molecule if it's free on its own the B-cell won't recognize it the B-cell won't recognize the dinitrophenol on its own but it'll recognize it and make antibodies if it's atch-, attached to chicken gamma globulin and of course the hapten could be replaced in nature by a normal epitope antigenic determinant so the picture was something like this that you had T-helper cells recognizing one end of the molecule the B-cell recognizing the bit that one's interested in against which will make the antibody and the T-helper cell helps the B-cell whatever help might mean okay and that's described by the way in Weir and er Stewart thirty-nine and page thirty-nine and forty and Roitt Brostoff and Male on pages one-four-two and one-four-three if you're interested in that so that was the early picture you've got the T-helper cell and the B-cell cooperating but you've got a third sort of cell an antigen presenting cell and can anyone see what the problem is remembering that obviously this gap here is vastly magnified i mean this T- helper cell would be much closer to the B-cell than i've shown but can anyone see what the problem is if you got the third cell as well coming into it pretty naive problem really but you may not notice it you can't haven't got enough space for anything to be going on because this T-helper cell will be close to the B-cell how do you get a macrophage putting its paw in so to speak or its er er pseudopodium in to get this to get into contact with the antigen so there's something wrong with this story because there must be more to it than that because you've got a third cell and how does it make contact okay so let's try to explain what actually goes on let's start just by saying one or two things about antigen presenting cells you've seen this picture before but an antigen presenting cell can present antigen in a variety of ways it can present it on its surface just as a lump attached by say complement so you get the antigen presenting cell some complement and then the antigen stuck on the surface like that for example you could have er simple lumps of antigen presented in that way with complement but also you can have the antigen taken in and processed and remember during the processing it can be exposed or reprocessed and put on the surface in the cleft in the M-H-C-two molecule the M-H-C-two molecule is assembled inside er er er er little vacuoles inside the macrophage bits of antigen are stuck in the cleft it's exocytosed and exposed on the surface so presentation can be in the form of M-H-C-two there's a bit of antigen in that cleft there a little bit of processed antigen this is much larger this bit here this is a little bit of processed antigen okay a few amino acids for example so there are at least two ways in which antigen presenting cells can present antigen on their surface so if you could look at the diagram that i gave the early-comers unfortunately i haven't got enough copies of it to go round i thought it was photocopied more than that i'll get you copies of it er in time for the er video at eleven o'clock i'll get you some more copies of it but you've got a picture like this which summarizes what goes on and so just note could you that you've got a T-helper cell and the B- cell cooperating and two antigen presenting cells here and here okay and we'll go through this story and if you haven't got the picture you can certainly get most of this copied down from the pictures as i put them up in sequence okay so let's go through the stages first of all talking about this side then this side and then the interaction between the two this side that side then the interaction between the two so let's start with the T-helper cell somewhere in the lymph node probably in the paracortex there are T-helper cells which will be specific for the antigen there's specificity of fit between the T-cell receptor and the antigen and the M-H-C-two molecule here okay you get specificity of fit there's a few of these T-helper cells somewhere in the paracortex of a lymph node and an antigen presenting cell presents this antigen then in combination with M-H-C-two on its surface and the T-cell receptor recognizes the antigen and the M-H-C like as is shown there there is a fit between the M-H-C and the antigen and the T-cell receptor there's also a subsidiary molecule C-D-four which helps the connection don't worry too much if you don't understand that at this stage i'll ex-, have to explain that to you in a later lecture so the T-helper cell connects up to the antigen presenting cell because of this specificity of fit across here and messages are exchanged between the two cells that's what these squiggly arrows mean and the T-helper cell becomes stimulated will undergo mitosis will undergo maturation and will produce a clone of daughter cells okay so the T-helper cell receives some of this antigen after it's processed by an antigen presenting cell showing it in in the M-H-C-two context and mitosis and maturation result and you'll notice M- H-C-two is important and that's why it's M-H-C-two restricted because the T- cell receptor will only recognize its own M-H-C-two sort so an H-two-K T-helper cell will recognize an H-two-K antigen presenting cell but an H-two-K T-helper cell will not recognize an H-two-D type antigen presenting cell that's why there's M-H-C- two restriction okay so the T-helper cell produces a clone of daughter cells as a result of that stimulation and some of them will probably migrate to those follicles that we keep talking about but remember it takes some time for this to take place it isn't an instantaneous thing for instance mitosis will take some time for a start okay now let's go to the other side of the story this is in the follicle you've got an antigen presenting cell there like the dendritic follicular cell this time it presents antigen in a different way bound to the surface perhaps with the aid of complement or something or other but it presents a blob of antigen not in terms of M-H-C-two now but just on the surface so you've got a distinct blob of antigen presented to the B-cell you get cross-linking as i said early on the I-G cross-links capping endocytosis and the antigen molecule or er it's more than a molecule it's a lump is taken into the B-cell okay the antigen's taken into the B-cell and it's broken up it's broken up into small fragments that's what this is supposed to represent this is broken up into that and these small fragments are small enough to be expressed on the M-H-C-two molecule on the B-cell okay so the B- cell has M-H-C-two molecules just like that antigen presenting cell that we had over this side before and it expresses bits of antigen in the M-H-C-two cleft so the B-cell has started to be doing things with the antigen and you'll notice that this response down here is not M-H-C-two restricted because there's no M-H- C-two involved down here there's M-H-C-two up here but not down here so the B- cell has started to process the antigen for which it is specific and it's presented some of this antigen in its M-H-C-two and so when these T-helper cells come along which is a bit of a mind-boggling event because how does a rare cell meet another rare cell i don't know but this is what is said to occur you've got this B-cell now with the antigen in its cleft it recognizes a T- helper cell which has still got the same T-cell receptor specific for it it's the same one as recognized the antigen presenting cell way back down here the two come along they interconnect there's the antigen there's the M-H-C- two there's the T-helper cell and you find then that once the T-helper cell makes contact with the B-cell it then gives the T- helper s-, the T-helper cell then gives the B-cell some help okay so the antigen is broken down by the B-cell put into the M-H-C-two cleft T-helper cell recognizes it with its T-cell receptor for which it speci-, it's specific for this M-H-C antigen pattern there also C-D-four is involved and once that connection is made there are in fact a number of other surface molecules but as long as that connection is made you then get help going abro-, across from the T-helper cell to the B-cell and once that occurs the B-cell is then stimulated to produce a clone or memory cells plasma cells and to produce antibodies okay so you end up with a picture which is on the sheet that i've given at least some of you slightly drawn slightly differently but basically it's the same antigen presenting cell one T-helper cell B-cell and antigen presenting cell two so you've had that bit of the story then that bit of the story then that bit of the story and as a result the B-cells are stimulated to produce a clone of daughter cells memory cells and plasma cells and the plasma cells will produce antibody and if you find that there are difficulties with that i mean don't hesitate to discuss it in the discussion sessions after the video and you'll find that when i come to deal with T-cells i'll give you a similar picture er and show that the process is approximately the same with cell mediated immunity okay now the final thing i think we need to discuss this morning is what this help is these dotted these jagged arrows are in-, intended to indicate help help goes from the T-helper cell to the antigen presenting cell across here and back again i've shown it mainly going in that one way and also there's er cross-help from the T-helper cell to the B-cell there are two ways in which help can be received in which one cell can stimulate another cell you've got two cells here let's call them A and B help is sometimes received by direct molecular interaction you get receptors interacting between the two cells okay so they're in close proximity very close proximity and these receptors interact with each other they fit each other perhaps in some way or another and a message is sent across as a result that's one way that help can be transferred and in fact the help is often not just one way it's two way and the other way the this help occurs is through molecules being secreted and molecules are secreted by cell A and they act on receptors on cell B and so help can be transferred in that way and remember that in this picture these cells are far closer together than i've shown here because these receptor groups are very small and in fact a secretion to diffuse across need only diffuse a very very short distance and these secreted factors so far as immunology s-, is concerned are called cytokines cytokines okay and most of them not all of them are called by the er abbreviation I-L so you got I-L-one up to at least I-L-twenty and probably further okay and I-L stands for interleukin okay which simply means between leukocytes er though it's mainly between lymphocytes but certainly I-L-one for example is secreted by macrophages so it's not always just lymphocytes that secrete er cytokines or interleukin it can be other cells interleukin-one as i say being secreted by macrophages and if you look at the cytokines in bold in this response here they are many and quite complicated but just to try to keep it relatively simple they're as follows you've got your antigen presenting cell the one that reacts with the T-helper cell called a T-helper-two cell here okay and the B-cell which was stimulated by another antigen presenting cell you'll notice then that the antigen presenting cell secretes I-L-one as i've already said over there it can act on the B-cell directly can also act on the T-helper cell and that stimulates the both sets of cells but notice the T-helper cells remember they underwent mitosis they can self-stimulate they can stimulate themselves with the s-, er interleukin I-L-two so T-helper cells can stimulate themselves they're autostimulated by interleukin-two and then T-helper-two cells can stimulate B-cells by a number of cytokines some I-L-two but perhaps not very importantly but more importantly I-L-four and I-L-five I-L-four and I- L-five stimulate B-cells and I-L-six encourages the actual secretion of antibodies I-L-six encourages the secretion of antibodies so I-L-four and I-L-five are the prime helpers I-L-two perhaps a bit and I-L-six actually encourages the secretion of the antibodies and then finally remember that when you get an immune response you get a change of I-G production you start off with I-G-M and then later on you get I-G-G being produced there is a class switch remember and the class switch from I-G- M to I- G-G is controlled predominantly by an increase of secretion of I-L-four the I-L- four level converts the B-cells from being I-G-M secretors to being I-G-G secretors a predominance of I-G of I-L-four causes this class switch from secreting I-G-M to I-G-G now i just want to leave you with the thought about these different sorts of B-cells remember that i talked about early on that B- cells in the follicles of the lymph node are the normal ones that we've been discussing here using T-helper cells but outside of the follicles you get B- cells these B-one cells that respond to strange antigens things like these polymeric ones and so on but they don't use T-cells at all and what you find is that in the follicles the B- cells will produce I-G-M and then I-G-G just as we've described here outside of the B-cell outside of the follicle rather these B-one B-cells not using T-cells only produce I-G-M and the reason for this is that since these B-cells are being stimulated on their own they may get some I-L-one help from antigen presenting cells but they don't get any of this help because T-helper cells are not involved so these B-one lymphocytes outside the follicle don't have T-cells and they don't get any of this cytokine help down here they might get some I-L- one coming from an antigen presenting cell but they don't get any of this and therefore you find that this sort of T-cell will only produce I-G-M it will never undergo a class switch so you get a very deficient sort of response against things like polymeric antigens er like the mitogenic ones and the complement ones which are responded to by these B-one B-cells only if if the antigen is not responded to by a B-two B-cell it will only produce an I-G-M response because you've got no T T-helper cell help therefore you haven't got these cytokines so the B-cell stays at that position and can't switch to that position there okay so with that thought that the end just to put up a piece of paper about what is relevant in the books er Weir and Stewart in fact hasn't altered since last time because there's not a great deal in this area in that early part of chapter four but i've added chapter eleven now to what you should have a look at in Roitt Brostoff and Male some of it you won't understand but some of it is relevant to what i've said today okay thank you very much there is a handout which i'll give you