---start immuno 2.3.97--- questions from the last lecture? no. today we're going to go on from there and talk about how T cells function (we started that last time, discussed production of cytokines and cell/cell contact) also today we'll go through MAJOR Tcell functions and see where effects are contact mediated and where they're cytokine mediated. Remember we're talking about T cells that can perform 3 different functions: CYTOTOXIC T cells: kill target cell INFLAMMATORY T cells: produces cytokines, activates macros, which kill targets eg tumor cells, intracellular pathogens, parasites, etc. HELPER T cells: work via soluble mediators AND contact - eg, to help B cells need both... CONTACT So if you have a T cell and a B cell, there are some molecules on the surfaces of each that need to be triggered to effect the HELP: CD40 on the B cell must bind CD40 ligand on the T cell. B7 on the B cell interacts with CD28 on the T cell. MHC II on the B cell needs to interact with the TcR. there are other less important interactions as well. The CD40-CD40L interaction is most CRITICAL: without it, mice can't develop strong B cell response. CYTOKINES: IL-2 IL-4: mediates class switch to IgE IL-5 IL-6 these are the ones made by T cells which help trigger B cell to produce antibody, in addition to contact mechanism. also in fact after a primary immunization you have a certain amount of Ab response, but after a secondary, you get MORE Ab and you get class switching - because the T cells are helping. a primary response you get low levels of IgM, only B cells are responding. A secondary response get higher levels, get class switching, because T cells are helping. Now: Ag recognized by B/T must have epitopes on the same molecule to effect T cell help. this is called linked recognition. if you have a large molecule, say, a globular protein, you will have epitopes recognized by B cells, and epitopes recognized by T cells. not necessarily the same epitopes...in fact, usually NOT the same epitopes. but for T cells to help B cells, the epitopes MUST be on the same molecule..LINKED RECOGNITION...you need this in order to bring B and T cells into proximity....B cell processes antigen, expresses processed peptide Ag, and T cell recognizes it. SO the epitopes have to be on same molecule so B cell can act as APC. experiments were done... DNP; BSA: dinitrophenol - can't induce immune response on its own, is a hapten, no T cell epitope...can't be seen by T cells. BSA: bovine serum antigen: has T cell epitope so you bind them together, and immunize animal with DNP;BSA, and then challege and see what response you get...you get a good Ab response, because the hapten is linked to a globular protein. but if you take DNP and link it to a different globular protein, it doesn't matter what it is, just a different one... DNP;HEL--> immunize with this, then challenge with DNP;BSA and you get only a primary response to the challenge. primary Tcell secondary antiDNP response DNP;BSA BSA DNP;BSA +++ DNP;HEL HEL DNP;BSA +/- DNP;BSA & HEL BSA/HEL DNP;HEL +++ DNP;BSA & HEL BSA/HEL DNP;BGG +/- DNP;BSA & HEL BSA/HEL DNP;BGG & HEL +/- So, what's going on? well, the T cell is primed in the first case, to recognize BSA, and the B cells recognize DNP. you have a B cell specific for DNP, right? If the DNP is linked to BSA, those B cells can take the molecule up, into the class II pathway, and they'll express on their surface a BSA peptide. So now a T cell recognizing BSA will interact with the B cell, and its surface molecules. note: the memory cells still need T cell help, is important to realize. so. if you immunize with DNP-BSA & unlinked HEL, and challenge with DNP;HEL, you get a good response, because the first immunization created T cells which recognize HEL and BSA. So, this LINKED recognition is important in the secondary response where b cell acts as APC. one more.... In the final examples above, you don't get a good response, because your T cell may recognize HEL, but in the final example, HEL isn't linked to DNP, so it won't work. LINKED RECOGNITION, remember. this is important, for specificity of response, strength of response, when you develop a vaccine, it has to follow these rules to be effective. so your pathogen may be best controlled by Ab to a particular epitope. so you might think, ok, we'll make this vaccine using this epitope on a carrier- BUT you aren't doing a good job, because you aren't going to induce an appropriate T cell response. you need the T cell epitope on their somewhere as well. moving on to second kind of major way in which T cells function... CELL MEDIATED IMMUNITY (CMI) - this is a vague term. if you take lymphocytes out of an animal and stimulate with a pathogen...will they proliferate? this is one way of testing CMI response. another way, more classic, is "delayed type hypersensitivity" response. you inject Ag into skin, and 24-72 hrs later you see thickening, erythema, etc - due to infiltration of monocytes and lymphocytes into the area. so clinically you can check if an animal has been exposed and has good CMI response by injecting Ag and looking later - eg, TB test. so that test is a measure of your exposure to a particular pathogen or to related organisms (cross reactivity) another simiilar mechanism is contact sensitivity, eg poison ivy rxn, oils bind skin proteins and change them, provoking an immune response. so in vitro you can do that first test, measureing lymphocyte proliferation, but it is easier to do the skin test for delayed type hypersensitivity - this is going to measure animals ability to have CMI response to this pathogen. what cells are involved? MACROPHAGEs: infected macrophage has pathogen in it, and Ag on surface. Without being activated, macrophages can kill a range of bacteria and protozoa - which aren't really pathogens, because they can be killed by unactivated macrophages, so they don't cause disease, see. but other kinds of bacteria and protozoa are taken up by macrophages and multiply within them and LIVE in there, and aren't killed by the inactive mphage. but when cytokines activate the macrophage, killing activity is enhanced. T cells will activate the macrophage via cytokine release. in terms of macrophage activation, IFN-gamma is the major cytokine involved. When the macrophages are activated, there is fusion with lysosomes and destruction of mycobacteria in lysosomes in macrophages. TNF can be made by macros, and feeds back and enhances cell's ability to be activated. so a good way of getting rid of intracellular pathogens is via activation of macros. activated macros ALSO kill extracellular pathogens, etc. can bind to tumor cell, or worm, and release toxic substances, and kill them. there are two major - there are varieties of ways macrophages can killa fter beign activated: enzymes, low pH, toxic enzymes....two main pathways: respiratory burst: leads to production of toxic oxygen molecules, including H2O2, very important to kill some kinds of pathogens Nitric oxide production: other pathway. this is only produced upon activation, when inducible NO-synthetase is expressed, leading to production of NO. NO has variety of normal functions, and in addition can be made by macros and is very toxic and can kill bacteria and other targets. also has vasodilatory and other effects. unlike T/B cells, where specificity exists at level of Ig and/or TcR, activated macrophages do NOT have specificity. If you immunize against mycobacteria, and get activated mphages, the mphages don't know they're only supposed to kill mycobacteria...there's no specificity there at all. old expt: animals immunized against listeria. immunize IP challenge result listeria listeria listeria killed why? T cells recognize listeria, make gamma interferon, and activated macrophages kill the listeria. BUT immunize IP challenge result mycobact. listeria listeria not killed why? When you challenge with listeria, you don't activate the T cells. The T cells are specific for mycobacteria. BUT: immunize IP challenge Result mycobacteria listeria + mycobacteria both killed because your T cells recognize the mycobacteria when challenged, and start activating macrophages via gamma interferon...THEN, those macrophages will kill BOTH pathogens because they are not specific for anything. ---break--- note: there's some confusion re: the slide... point: once the macrophage is activated, it acts nonspecifically. BUT- to get the macrophage activated, all you need is gamma IFN production. now, the mphage can act as an APC, but that isn't REQUIRED for that mphage to become activated. it's just that APC function is another part of the macrophage's job. The T cell could be activated somewhere else and make IFN, and that will activate the mphage. you can add gamma IFN to isolated mphages in test tube, and that will activate them. TNF is also a cytokine which is made by the macrophage itself, and it feeds back onto TNF receptors on the macrophage, further activating itself. note: these activated macrophages aren't PERMANENTLY activated...activation decays, and mphage returns to resting state. this is important regulatory fact. activated mphages make toxic compounds, if they stayed activated all the time, it woudl be a pathologic condition....remember the superantigen...that activates too many mphages, leads to pathological condition. other function of T cells: not to activate other cells, but to specifically kill target cells. think of viral infections. if you have a T cell which recognizes a target cell, that t cell has a variety of molecules it can release: PERFORIN ENZYMES TNF all of which can act on the target cell and lead to lysis of that cell. how does T cell recognize target cell? MHC I. CD8+ T cell will see virally infected Class I expressing cell. the TcR will recognize foreign Ag w/MHC I on the surface of the infected cell. any cell in the body,just about, can be a target cell. Class I MHC is expressed on all nucleated cells. so, you have a virus hanging out in cytoplasm of infected cell. proteins are broken down by proteosomes, peptides go to ER, bind class I, and go to cell surface. a CYTOTOXIC T CELL recognizes it and releases perforin, enzymes, TNF...killing target cell. again, if you have a layer of infected cells, with class I and foreign peptides on surface, the T cell can kill these cells as above - and you can watch the target cells undergoing cell death, neat slides shown. macrophages eat the remains of the dying cells. PERFORIN very important. is present in granules in cytotoxic t cells. when the T cell contacts the target cell, the granules get released. you can see the space between the t cell and target cell being filled up by granules being released. the molecules then polymerize into a structure which opens a pore in the surface membrane of the target cell. this perforin is very similar to the 9th component of complement (MAC, C9) ok. so, questions? no. ok, well, we've gone through how T cells act as effector cells: activating mphages helping b cells make ab acting as cytotoxic t cells. when you have an immune response, do you get all these effector mechanisms generated? well, no. why not? well, hmmm. it makes some sense, because some pathogens are best cleared by some types of immune response. in viral infxn, cytotoxic t cells are VERY important and effective. Ab kind of helpful too. but, if you have a mycobacterium, Ab not helpful at all - it's all in macrophages. so you want to activate macrophages to kill this bug. so particular pathogens are best beaten by particular pathways. virus bacteria fungi protozoa worms major classes of pathogens. many different pathogens provoke different immune responses. different kind of Ab classes, and different T cell effects.some parts of response more important than others. so type of response generated is going to determine if you can control pathogen. some dz are more clear cut in showing us balance between immune responses... CMI vs Humoral immunity TB, leprosy, leishmaniasis, Johne's dz, HIV(?): best handled by CMI you can get patients presenting with these infxns and showing different immune responses. whether or not they control the organism determines if they are controlling organism and/or healing. with these dz, patients having CMI are better able to heal, have low numbers of organisms; patients having humoral response are NOT getting better, have high numbers of organisms so there are these two types of immune response. we know T cells are important in generating HELP for humoral response, and also for activating macrophages for CMI response...how come if you have good CMI, you don't always have good T cell help for B cells? looking at populations of CD4 cells, two categories are found: TH1: are able to activate macrophages TH2: are primarily involved in helping B cells make Ab. one of the main ways T cells activate macrophage is via IFN gamma production T cells use IL-4 and IL-5 to help B cells. it is differential production of cytokines which defines these subsets. T cells leave thymus as naive T. contact Ag, proliforate, become immature effector cell: TH0 then become either inflammatory TH1 or B cell helper TH2 cell again. major subsets: alpha-beta and gamma-delta alpha-beta has two subsets: CD4 and CD8. CD8 are cytotoxic T cells CD4 are CMI macrophage activating TH1 cells or B cell helping TH2 cells. TH1 cells make IFN-g and TH2 cells make IL-4, IL-5, IL-10. so, knowing that these subsets exist, what questions leap to mind? what is important to know to know more about immune system? well, what determines whether or not you get TH1 or TH2 cells developing from the TH0 population? what kind of cell surface markers do they have? these cells have no reliable surface markers, which is unusual compared to other populations of T cells. these cells are differentiated only by the cytokines they produce. how long do they produce the cytokines? just as mphages are activated and deactivated, same happens here. these cells will produce their effects and then go into a resting state, and maybe become a T memory cell or something. the follow up to this question is, if they are a TH2, and then they rest, when challenged, can they be TH1 cells in the future, or are they committed to being TH2? well, they appear to be committed to being one or the other, and seemingly do not switch between subtypes. what about regulation of these cells? if you have a dominant TH1 response...does it remain a dominant response? if you have many T cell clones responding, can you force a TH2 response to emerge when you are getting primarily a TH1 response? in autoimmune dz, you want to turn off TH1 response... So T cells come out of thymus and THEN differentiate into these populations. what determines which line they follow? can you regulate or modulate the populations once they exist? (not on an individual cell level...but within a cell population, can you "turn off" TH1 and "turn on" TH2?) what are implications of that? What determines why a particular T cell becomes a TH1 or TH2 cell? the answer is, really, given to us by looking at infxs dzs. it turns out that understanding how infxs dz induces immune response gives us the answer. after exposure to infxs dz, innate immune response occurs right away - no t cells recognizing Ag, and no Ab...just the innate immune response eg macrophages, NK cells, possibly gd T cells. All of these cells make particular cytokines. these have been measured and are quite important in determining if naive cells become TH1 or TH2. some pathogens induce IL-12 production by macrophages, and also partly due to IFNg from NK cells, and these influence naive cells, causing them to become TH1 cells. Other pathogens cause production of other cytokines, eg IL-4, and this causes naive T cells to become TH2 cells. this was discovered by looking at infxns, but can be experimentally checked out...eg, knock out IL-4 gene and animal can't make a TH2 response anymore.... so major cytokine for driving TH1 development is IL-12 TH2 IL-4 so, interferon gamma made by TH1 cells ALSO inhibits development of TH2 cells. similarly, if you have many TH2 cells, they make IL-10, which inhibit proliferation of TH1 cells. so if you start having one kind of response, it generally becomes the dominant response quickly. regulation can also occur at effector arm level. IFN-g inhibits ability of IL-4 to act on B cells, and similarly IL-4 inhibits ability of IFN-g to act on mphages. so there are two levels of regulation here. final question is...why is this information important and how is it useful? why is it important to know the answers to these questions? how does it help in a clinical setting? he's leaving these up to us... someone suggests that this information could be useful in treating/dxing autoimmune dz. eg, if you have patient w/autoimmune dz, how do you control it? you could globally immunosuppress the patient, but that's really dangerous. what's the alternative? immunotherapy involving regulation of these cellular responses, via treating with cytokines, eg IL-10, to inhibit TH1 response. if you have pt with infxs disease, who's making the wrong immune response, maybe you can regulate the response via cytokines, etc. ---end---