Meeting 3

Meeting 3 - Effector Mechanisms of Humoral Immunity Look at...

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Unformatted text preview: Effector Mechanisms of Humoral Immunity Look at consequences of effectors and know overall concepts. On Tuesday we've talked about antibodies. We're now going to look at Fc receptors and complement which are the effectors of the immune response. They can be used in the adaptive AND the innate response. They are produced by cells but they perform their function at a site quite distant. They go into the blood stream and do its work. They are mediated through the heavy chain constant region. Ab with different constant regions have different funcitonal properties. IgM have 5 building bloccks held by J chain or 6. It activates the classical pathway of complement. The first ab made during immune response are low affinity but have many binding sites thus high avidity. IgG is in our blood. It coats ags to help uptake. It's very important in ADCC mediated by NK or Mphages. IgG is the only Ab that crosses placenta. IgA is the Ab on our mucosal surface. It is important since the vast ajority of Ags past through the mucosa. IgE is for allergy. IgD is a mystery antibody. It is on naiive B cells. It's a surface and secreted immunoglobulin. It is conserved throughout species. They thought it was important. Conan. Little to no phenotype in mouse. These effectors are mediated though the heavy chain. They are turned on through the binding of the variable region which is responsible for turning on effector mechanisms. Technique: Immmunofluorescence Direct: cells expressing the ag will fluoresce. Indirect: Make an antibody to the antibody and fluoresce that secondary antibody. They recognize the same thing. It's easier to lable one secondary antibody. Technique breakthrough: FACS It can measure the fluorescent intensity of a single cell. it mcan measure the level of fluorescence and do analysis or FLw. It can sort cells and it can look for cells that express a certain Ag. Data analysis is easiest in 1D. Cell number vs. fluorescence. There must always be a control shown here in white. The analysis is red expressing for ex. CD4. Another example shows a population that doesn't express an antigen very well and it's heterogenous. 2D FACS: Poeple want to look at the comaprison of 2 things now it's up to 11 dimensional. It's to see if they are labeling one or both or none of the antigen and give a 2D plot. Each odt is a single cell. A = CD4 and B = CD8 Importance is each dot represents an x and y coordinate of one cell. Flow cytometry: All these cells at top left express IFN-gamma. The ones to the right express CD4. 25% express IFNgamma and CD4. Antibodies are good at clearing pathogens because they bind to Fc receptors. It is responsible forthe effector function. Fab is for specificity. (Lost recording =() Most of the effectors will be given these CD numbers. It's bad because it's just a number and doesn't give you information. FcgammaR1 or CD64 is a high affinity. t makes gamma chain with an ITAM motif. They are present on a lot of molecules. There's a companion ITIM = immunoreceptor tyrosine based inhibitory motif. Ab binds to the antigen and cross links to the receptor. Remember that this FcgammaR1 can trigger AdCC and phagocytosis in response to cross-linking. It has its own cytoplasmic tail associated motifs. It comes in two flavors. For 2A is an activating motif. For 2B is an inhibitory signal. We'll see this several times. When we crosslink a ITIM, you actually get inhibitiory. 2A there's no counterpart in the mouse. 2B is an inhibitory. It can act as an inhbiitory receptor on many cells. On B cells, on it's surface is immunglobulin. When you crosslink those Abs it only proliferates when Fab'2. If you have 2B it will give an inhibitory signal. IMPORTANT: go back and review. FcgammaR3 or CD16 Needs the cahins like on R1 It's very important receptor for phagocytosis. Improtant: Through the Fc receptor, if they have ITAM or ITIM to turn on/off immune function. ADCC: Antibody dependent cell mediated cytotoxicity This is an exaple of thow this can be employed. It profvides protection against tumor growth. Left: given turmor with Her2/neu. Antibody was made to bind that. Used to treat Her2/neu expressing breast cancer. If you give Herceptin, the tumor growth is much slower. How does that provide protect? Through Fc Receptors because you can make a mouse without the gamma chain that means FcR2 and FcR3 won't work. when those mice don't express the gamma chain (right) the tumor now grows even when there is a herceptin. That shows that the Fcgamma is improtant for preventing tumor growth. It's a system that has to have to be kept in checks and balances. they treated with an Ab with a low dose. If you do WT mice, the tumor growth is not protected against with a low dose. But if you remove the inhibitory receptor, we now can see protection. there is a balance between activaitng and inhibotory receptors. However if you coligate, you'll get inhibition. (explain again please) These are Mphages and add ovaalbumin. Labeled antigen with fluorescence (FITC) and look at where the antigen is. We see that there's veryu little uptake. But if you mix OVA with antibody and make an immunecomplex, we see very effective uptake and fluorescence. Complement How was complement discovered? Rabbits were immunized with sheep redblood cels. Now you take antiserum and add to SRBC yuou would see lysis. However, if you heat the antiserum and now added to SRBC, you'd see no lysis. NOW if you add a guinea pig or any non immunized animal and take serum and add it to SRB, there's no lysis. But if you take heated serum and the serum from pig together, you get lysis. this shows that lysis was dependent on two factors, Ag specific agen that is heat stable and a separate agen that is heat labile that complements Ab and causes lysis. Hence the name complement. We now know it's a very important effector mechanism. it funciotns in the lysis of cells. This was used to demonstrate in that experiment but one of the least important functions since many bacteria have cell walls resistent to lysis. Opsonization is another function. The cells are importnat in phagocytosis and they have complement receptors and facilitates uptake by the cell. Inflammation very important in privoidng protection against infection but you don't want TOo much. There are peptides generated during complement such as C3a, C5a etc. They also act as chemoattractants and will now recruit the desired cells to the site of infection. Dr. zack will talk more about how cells are whizzing through the blood vessel and move up this chemical gradient of chemoattractants. 4 Immune clearnace: deposit these complexes in the liver to be degraded. 5 Enhanced immune response and B cells have receprs called Cd21 for complements. If you don't have complement in an animal, you don't have an effective immune response. 6 Virus neutralization. Remember these in a broad sense. How does complement work? we'll go over main parts.Conversion of C3 to C3b. There are 3 pathway for complement to be activated. It's an effector system for the adaptive pathway and called it classical pathway. however, it also is imprtant for innate mannose binding lectin pathway or the alternative pathway. C3: All are converted from C3? All the pathways focus on C3. It is cleaved and realease C3a and C3b. C3b? has available thioester and will bind to the cell membrane. Don't need to memorize products. C3 cleaves to C3b and further cleaves and binds to the membrane and act as opson and help phagocytic cells. KNOW THIS! Classical pathway gives this Ag-Ab complex and works with C1 that acts as a protease and cleave C4, C1 and eventually C3. Know that they are cleaved via C1 which is activated from Ag-Ab complex. Once C3 is activated, it will activate C5. C5b will interact with the rest of the membranes such as MAC which will form holes in membranes. C1q will be actiavted to C1. C1q has globular parts which will now with an Fc of an antibody.l Imp: C1q has low affinity for Fc. Will only be activated when more of its globular parts activated which means they have to be close together in order to be bound by Fc. Here's C1q has these heads and associated with these proteases. are activated by binding with Fc. DNK stalk. If C1q could be activated easily, you'd get activation from circulatin g Fc. These proteases now cleave C1 and ultmately cleave C3. Imp: activation by C1q bindint to Abs leads to the production of proeases etc. IgG has only two sites to combine complement and only one can be combined at a time so you need two IgG. That can only hapen when you have tehme bound to the surface of the antigen. What about IgM? In circulation, IgM is very rigid. When it is in circulation, the Fc that would bind to C1q is not available. However, when IgM is bound to an antigen, there's a conformation change, now the Fc can bind. Each single molecle of IgM bound to an Ag you can get C1q binding effective since there are many on one molecule. Lectin pathway is not actiavted by C1q. But it's mannose binding. But it looks a lot like C1q since it has proteases. But it's binding specific because of the lectin. Mannose binding lectin will bind to the carbohydrates on pathogens and activate complement pathway. it's in innate because it's always present and not generated through adaptive pathway. However, once it binds to the carbohydrate it uses the same pathway as C1q. It will activate to C2 and C4 and get C3 and then goes to complement. A third way of actvating complement is alternative pathway. There's always a little bit of spontaneous cleavage of C3 but usually they degrade and nothing happens. But when there's a pathogen pathway, C3bBb will be activated which can now ccleave C3. when the appropriate elicity agaen is present. B and Factor D protect C3 to make C3bBb only in an appropriate microenvironment. Cleavage products are very important effector moleucles and most important are C5a, C3a, and C4a. They're called anaphylatoxins. They are small peptides ... etc. When they bind to receptors, those pro-inflammatory efectors are released. Too much inflammation and many diseases reflect over activation of inflammation. They've ben trying to neutralize theses anaphylotoxins. Cleavage products of C3b are opsonins. MAC C5b will spontaneously associate with C7 and C8 to make C5b678 and then it iwill associate with C9 to make a hole in the membrane. These holes puncture the cell to cause lysis. Here's these nice bacteria and now they've been incubated with complement and ab and now their contents are oozing out. This is a slide. DNM all these factors. Complement activation is limited and directed to the appropriate targets. Many proteins can block c3. the imporatnt concept is that complement action is tightly controlledc. Complement rectors are broadly rxpressed and these outcomes are important. Immune complex clearance, opsonization, enhances immune response and mediator release. These anaphlytoxins will be direcctly inflammatory and lead to inflammatory mediators. Let's summarize the functions of complement. Cell lysis, forms complex on cell membrane. Opsonization: we coat the bacteria with cleavage products of C3b and recognized by phagocyte. Activation of inflammatory response by anaphlyotoxins: granuloctes get dengrulated and also form a gradient to get extravasation from the blood to help clear the infection. Complement activation is important in clearing immune complexes. There are receptors on RBC and those RBCs are brough to the spleen and they take off the immune compelexes. Complement and Fc receptors will synergize and promote phagocytosis. If you have a bacteria coated with IgG will activate complement and will now be coated by both Fc receptor and C3b and both of those will be recognized by the phagocyte and be taken up and cleared. consequnces: Cell lysis, inflammatory response, opsionization,k viral neutralization, and clearance of immune complexes. You can see where if you're lacking complement, all of these will be impaired. Remember that if you're missing complement, wihat will be the funcitonal consequences....
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