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Antigen Recognition and Antibodies

Hadeel Al-Kofahi Ziad Al-Nasser Tuesday, 5/7/2011

Lec # 3 Tuesday 5-7-2011 Hadeel al-kofahi

Good morning:

Well continue with the important concepts regarding the immune system. Last time we gave you an overview about the differences between the innate and adaptive immune systems and from those how we can pass through many different principles, Also we talked about the primary Immune response, secondary immune response, memory cells, the nature of the products of the primary and secondary immune sys., vaccination (well keep crossing on that continuously), and we said that the innate as well as the adaptive sys. Work together, help each other, & communicate through the cytokines, or the lymphokines & whatever soluble mediators, So they act on each other to suppress or activate depending on the needed case.
Then we ended up in the concept of antigen presenting cells and its role in the establishment

of an immune response, and we introduced some principles about antigen presenting cells. And those cells including [1macrophages, 2dedentric cells, 3B cells which produce antibodies with specificity, interdegetating cells] have to take the antigen, processing it inside, and then hook it into the major histocompatibility antigen and then present the antigen with the MHC into the surface So the T helper cells must recognize the antigen plus the MHC as one unit, then the helper T cells appear to cooperate and start to produce cytokines that are needed for the immune system. Another important concept in the immune sys. is about the active and the passive immune systems; The active immune response; when theres a direct involvement of the immune cells that will lead to the interaction and production of the products either immunoglobulin or activated cells plus memory cells. And this active immune response could be natural or artificial, Examples: Natural: if we are infected with microbes, bacteria, viruses and so on, then our body is going to interact and present these bacteria to the cells and then immunoglobulin will develop against those viruses, bacteria etc. Artificial: through vaccination, we can get organisms then inactivate them or their toxins and then inject them to our bodies.

The Passive immune response; when the products of the immune sys. that are already present in our bodies can be transferred into another person. It could be natural or artificial, Examples: Natural: when the mother thats immunized has immune products, so the immunoglobulin in the mother will pass into her baby and thiss a natural passive immunity, cuz the baby doesnt do any effort to produce those immunoglobulins, they pass directly from the mother. Artificial: when we make immunoglobulins, and as we will see we can make monoclonal antibodies, raise them in animals and then give them to the people, like anti-serum that we use to protect against hepatitis B , also anti rabies antibodies when someone is bitten by rabid dog ,so we give anti-rabies immunoglobulins, they inject them into the abdomen in order to neutralize any rabies viruses that could have access into our bodies. Also we can transfer cells from one person to another, if the cells are compatible with the MHC of the other person we can transfer them, or we can take the cells activate them then return them back to the same patient. The passive or the active immune systems can be cellular or humeral, mainly we are dealing in the passive immunity with immunoglobulins, or Cells as I told u but not frequently as Igs. Phases of an immune response: When theres an active immune response, the immune response will pass into three stages: I. II. Cognitive phase ( :) here the antigen has to attach to receptors, and then antigen presentation, cells activation & cytokine production have to take place. Activation phase: when the recognition has taken place, the activation will take place, cells proliferation, clonal expansion and selection will occur, plasma cells also will be produced. III. Effector phase: when changes start to occur with certain outcomes like immunoglobulins with specificity, activated T cytotoxic cells or other cells, and this will lead to eventual elimination for the antigen.

Antigen recognition molecules in the adaptive immune sys.: three main areas I want u to know: 1. MHC: and we talked about the polymorphism and how important to be diverse so we can be more effective in the immune response.

2. The T cells receptors [T helper cells, T cytotoxic cells]: the more the types and shapes the better the immune response I could have. 3. Immunoglobulins: the same thing here; how many different shapes I could have. Antigen recognition molecules -all of them- have similar molecular structure, & amino acids polypeptide chains which we call them immunoglobulin domains or immunoglobulin supergenes family, they are very similar but there are differences for sure. The outcome is antigen recognition, the main purpose of all these variations that we are going to talk about is to recognize the antigen and to have an induction for the immune response. So well keep talking about the innate and adaptive and how these recognition molecules are part of adaptive immune response. But do we have antigen recognition molecules for the innate immune sys. ? Yes, [the patter-recognition molecules], which is not specific compared to MHC , T cell receptors or Igs. What about the complement and its function?? The complement sys. is NOT specific although its activation sometimes could have a specific immune response y3ne when antigen antibody reaction takes place ,complement will be fixed and then activated and the outcome is the activation of the lymphoid cells as we are going to see.

Mannan binding lectins; Mannan is a sugar that covers the surfaces of certain micro-

organisms and this will lead to the activation of the complement. The complement means {complemented antigen antibody reaction}, so the essence is to have antigen antibody Rx and the complement continues or adds supplementary to the immune sys. , its a very effective way of defense. So when we talk about Mannan binding lectins we are talking about non-specific immune response thats going to activate the complement sys. as well. Even the complement has a mechanism to recognize self-versus non-self, when the complement is Dock [means go from the bottom to the surface of the cell], so if that cell is self, death reaction will occur, if the cells are foreign so the process will continue, the complement will be activated and the outcome is an immune defense as we are going to see. The adaptive immune response is related to specificity. So we are going to have so many genes thats going to involve, and those genes are the genes that we are born with, and the more different genes from parents I have, the more or the higher chance of diversity and many different shapes I could have.

Again the antigen recognition molecules are: 1) B cells receptor: The B cell receptors when activated will give me plasma cells and antibodies with the same morphology and shapes of the Igs that are present on its surface. And remember that B cell receptors are immunoglobulin of the IgM type, a monomer of IgM thats present on the surface of B cells. This Ig is supposed to have so many different shapes, with one shape for every cell, And what determines the particular shape are the genes. Also u will see how these genes are present on different chromosomes so well have gene rearrangement to give a probability of 1011 different specificities that each one of us has it. 2) T cells receptors: the same thing here, genes are present on different chromosomes so well have gene rearrangement To give a probability on T cells much more which could reach to 1018 or 1019 different specificities that we born with . Again the more our genes are diversed, the more the probability to have different shapes. 3) Human leukocyte antigen: The same thing here about the HLA antigens; the major histocompatibility complex as well, here we have the co-dominant type of inheritance, means half of those sets of genes are from the mother and half from the father. We have a maximum of 12 genes, and a minimum of 6 genes (3 for class one and 3 for class two). The variation of genes we call it allele, to give different allelic forms, so we get one allele from the father and one from the mother, and each of us has two alleles for every HLA (e.g.: A1A2, A2A3, if the mother and father with the same alleles then well get for example A1A1, and A1 has a different shape from A2, so the probability to have these different shapes will determine the diversity).

The T & B cell receptors:

They apply to the process of clonal selection, y3ne already we have those shapes on the surface, they will bind to antigens and clones will be activated by proliferation and then they go to the effector stage. One type or shape of a specific receptor will be present on the surface of a cell; we dont see more than one shape on the surface of a cell. Diversity (having so many shapes) is generated by gene rearrangement and joining; and well see how those genes are going to be rearranged, what are the chromosomes where genes are located in and how those genes are going to be cut , sliced and joined together , that will produce proteins to make the B cell receptors, T cell receptors or immunoglobulins. So the immunoglobulin (as a polypeptide chain or a protein) comes from many genes and those will be rearranged as well talk about that.

Another term is the somatic hyper mutation; when a primary immune response develops,

immunoglobulins will be produced and then memory cells, Later when u activate the memory cells the nature of the Igs that are going to develop is going to have a better fit to that certain antigen. We call this [fit]: an [affinity] or [the strength of binding]. But why they are going to have a better fit? Cuz they are going into a process called somatic hyper mutation, which means they make changes at the area where its going to bind to the antigenic determinant to make it a perfect fit. Its not just like Lock and key , the idea of lock and key is in the perfect situation, but sometimes you might not have a perfect fit, and we will tell its importance in the cross reactions. Cross Reaction: when an antibody whichs produced against certain antigen could react with another antigen, not perfectly but to a level that could induce a reaction and hypersensitivity. This none-perfect fit occurs after the primary Rx but after the secondary Rx the fit is going to be perfect, and the perfection of the fit is mediated by the somatic hypermutation and the changes that occur in that particular area. The immunoglobulin supergene family [includes the immunoglobulins, the B cell receptor, the T cell receptor, and the MHC] have immunoglobulin folds, that means they are formed from polypeptide chains that are folded to form domains, and those domains are important for the function of that particular protein on the surface of the cell or even when its free as the immunoglobulins.

Major Histocompatibility Complex (MHC):

Their inheritance; there are sets of genes that we inherit from our parents and we call this

type of inheritance a co-dominant type of inheritance. Those genes are coded or present on the short arm of the chromosome number 6. The area on the chromosome where the genes are located we call it Loci, singular Locus, so we have (locus A, Locus B or Locus C) or (Area A, B or C) which represent class 1 MHC. We have many different allelic forms that are present on each locus: For locus B we have over 500 different allelic forms. Locus A one hundred and something. Locus C maybe 80-90.

MHC play a role in graft rejection or compatibility; Each one of us has two allelic forms, so each one of us has a haplotype like blood groups and finger prints. And this haplotype antigen is present on every cell in my body (thiss so interesting!), so if I want to transplant a tissue from me to u, then if mine the same as yours so nothing is going to happen but if we are different then your immune system is going to react with those HLA antigens, And thats why its important to do tissue matching. We have 6 allelic types for class 1 in locus A, B, and C, and the same, 6 forms for class 2. Class 2 is involved in antigen presentation to T helper cells, so MHC class 2 are present on antigen presenting cells [B cells, macrophages, dendritic cells , interdegetating cells] So B cells have MHC class 2 for example , But do B cells have class 1 as well ? Yes, Cuz class 1 is present in ALL nucleated cells of our body so its not present in red blood cells cuz RBCs are anucleated while class 2 is only in antigen presenting cells.
All the classes of MHC look like immunoglobulin in away, Cuz they belong to immunoglobulin

supergene family. T cell receptors recognize Ig if only presented with class 2 MHC molecules. Y3ne when the antigen presenting cells present the antigen to T helper cells, the T helper cell will accept this only if the antigen is presented with class 2 MHC molecule. But B cells bind directly with the antigen without presentation. T helper cells are the orchestrator cells that are going to help even the B cells. So thiss the difference btw the B cells & the helper T cell receptors binding.

Pic A: Thiss what we mean by the diversity, each one of us has a different haplotype and shapes as we see, and we will react against these different shapes. MHC is polygenic and polymorphic; each individual has multiple different MHC genes. But every cell in each individual expresses the same set of MHC molecules. Pic B: kol wa7ad 3leeh 5atem exactly like a finger print, and thiss going to be reflected on the lymphocyte as u can see here , Every B or T lymphocyte expresses a different antigen-recognition molecule created from multiple gene segments that undergo somatic rearrangement.

The function of MHC:

The function of the MHC is to Capture and display antigens from cells associated with microbes like viruses, so when we are infected with a virus, the virus goes to tissue cells, then its going to be processed and presented. Now the presentation of the virus to T cytotoxic cells that are going to kill the virally infected cells is through class 1 MHC, so class 1 presents the antigen to the killer cells, while class 2 presents the antigen to the T helper cells . The genes of MHC are the most variable, and in order to have so diverse genes, remember >>> ( .) There are 6 different allelic forms on the surface of every lymphoid cell. All allelic forms are the same in every individual.

Antigen antibody Structure, Ch 4

Now well start talking about the second antigen recognition molecules which are the immunoglobulins. Igs come from B cells that have B cell receptors, B cell receptors bind the antigen, then the antigen is taken inside, processed and bind to T helper cell, T helper cell produces cytokines which will act on B cell, So B cell will differentiate to produce a clone of plasma cells and Igs of the same specificity. The B cell receptors are IgM monomers, so the IgM is the first antibody thats going to be produced. All the Igs are similar morphologically but differ antigenically. They are polypeptide chains represented in four chains model by having two light chains and two heavy chains. Also Igs are polymeric sometimes for example when we have two of the monomers attached to each other we call them dimers, five units are pentamers (IgM is a pentamer). So although the antigens have diverse structures, the Igs have extensive repertoire of different binding sites, and when exposed to an antigen, these immunoglobulins are going to be produced with different biological roles for each Ig class. The variation of Igs that they will develop depends on their biological function, So we get any specific Ig according to the function I want the Ig to do, for example ; IgM is a monomer on the surface of B cells ,and when the B cell is activated it becomes a plasma cell and it goes into the pentameric stage [pentameric means five units], each one of the units has two arms for attachment , so we will get Ten different attachment sites (or Valence) for IgM ,Ten sites the IgM is going to bind to antigens, bacteria, viruses or whatever.

Some features of IgM: IgM is the first Ab thats going to be produced in uterus. It has a valence of ten. It fixes the complement as well. Its a huge molecule. It doesnt cross the placenta, and the significance of that is to know that the baby is going to make its own IgM not having it from the mother, so if u detect an IgM antibodies, this means that the baby has been infected in uterus, & thats why in congenital infections we rely on IgM antibodies. The IgG antibodies are present on the surface of B cells, they complement the function of the receptors, so when we talk about the receptors which are the IgM well see the IgG is attached near it. So the function of IgG is to help the stability of IgM, and its a sign of maturity [Maturity means the B cells are mature & ready to act]. IgG antibodies are the most common antibodies in our bodies, & it is the most common Ab produced as a secondary immune response. IgG antibodies are of four types [1,2,3 & 4]. IgG4 cant cross the placenta while IgG 1,2 & 3 can cross the placenta, So when they cross the placenta, they provide the baby with passive immunity. IgG fix the complement. Also it has the longest have life. Two of the IgG act as anti-toxins ,so when u are infected with bacteria that produces toxins, they give u anti-toxins which are IgG. IgE antibody: Its the Ab of inflammation, this Ig we see it in parasitic infection or in allergy, thiss the one that makes mast cells and basophils to be triggered and produce the vasoactive amines and other mediators, so when we say allergy and hypersensitivity type 1 Abs we mean IgE Abs. It doesnt mean that IgE Ab is a bad Ab, No! Its an Ab of inflammation, but when we have mast cells and basophils activated for wrong reasons then we could have anaphylactic shock and the patient could die as a sequel of that. IgE Abs have the lowest concentration in the serum, while IgG Abs have the highest concentration in the serum. IgA antibodies: Mainly they are present as dimers (two monomers adhere to each other), and those are present on the surface of mucus membranes, where ever mucus membranes are located. You could see them in the serum, but then they pass and adhere to the surface of mucus membranes then pass through to the outside. So IgA Abs are important as a first line of defense, for example when you are infected with viruses, or bacteria like Neisseria gonorrhea on the mucus membranes , if u have IgA specific on the surface so they are going to neutralize the bacteria or the virus.

As u can see those Igs belong to Igs supergene family, they have two parts; the first one is the binding part to the antigen which we call it Fab [the fragment of antigen binding], and this area that binds determine the specificity and the shape that has to fit as a lock and key. The other part we call It the Fc portion [the crystallizable fragment or the fraction crystallizable], the Fc function is for the biological function as we will see, this one fixes the complement , and can pass through the placenta to bind to certain receptors on the cells.

Antigens: -Antigens and immunogens: Antigens are substances required to produce antibodies, they react

with the product of the immune system. Immunogens are also substances that have the ability to induce immune response and we use those two terms sometimes interchangeably to refer to the same thing. -Those are proteins of high molecular weight [not less than 6000 Daltons]. The higher the molecular weight, the higher the antigenicity. They have to be complex and foreign. -Now, Can carbohydrates be antigenic or immunogenic? Yes. Also u could have glycoproteins together, proteins or carbohydrates or both. - What about the nucleic acids and fatty acids?? Both are none or poor antigenic. Sometimes, Cuz they are not present in our body as pure molecules, so they are present with protein components. For example; the nucleic acids are bound into proteins that we call them [Distomons], so when we see anti-DNA anti bodies, we means Abs against nucleic acids that are bound to those proteins. Also lipids dont induce an immune response by their own, but if they are bound to proteins as [lipoproteins], then they can induce an immune response. -We have a substance we call it a Hapten, and Its a substance on its own cant induce an immune response, but if u hook it into [a protein carrier] then it can produce Abs against the hapten and the hapten carrier together. Its so important to understand the nature of haptens. For Example; penicillin act as a hapten, this means when u give it to a patient, on its own cant induce an immune response, but if u hook it into a protein carrier, the structure will be changed, and it

could induce an immune response, if the patient is reactive and produces hypersensitivity to penicillin. -The term epitope or antigenic determinant; its the most specific part of the antigen that fix into the receptor, its counterpart we call it paratope [The paratope is the part of an antibody which recognizes an antigen, the antigen-binding site of an antibody Wiki]. - the types of the epitopes could be linear or discontinuous; the linear epitope can fit into the T cell receptors, while the discontinuous can fit into the Igs or B cell receptors.

-We have a term we call it adjuvants ( ;)they are substances if u mix antigen with them, they will enhance its antigenicity. For example [the Freunds adjuvant] where we mix it with a substance of mycobacterium tuberculosis in oil, the oil will make it slowly to be released in your body , that means it increases the time thats going the antigens to be exposed to the lymphoid cells, thats why they are going to enhance the immune response, so simply if u mix an antigen with oil and water as such we call it [incomplete adjuvant], but if u mix it with mycobacterium tuberculosis antigen that brings macrophages into the area to act as antigen presenting cell, then we get [complete Freunds adjuvant], so if I want to enhance antigenicity I use adjuvant.

Freund's adjuvant: is a solution of antigen emulsified in mineral oil and used as an immunopotentiator (booster). The complete form, Freund's Complete Adjuvantis composed of inactivated and dried mycobacteria (usually M. tuberculosis), whereas the incomplete form lacks the mycobacterial components (hence just the water in oil emulsion).-Wiki

So thiss what we mean by hapten.

Here, thiss the linear epitope which fits into the Yshaped immunoglobulin, and you can see the fab region whichs the antigen binding site, that can bind to antigens. You can also see the discontinuous epitope, created from amino acid residues located in different parts of the polypeptide chain, y3ne the part goes in and out in a discontinuous way.

Antibodies: -Antibodies react with Ags that stimulate their production, But can we have antibodies that

react with antigens which are not responsible for their production? Yes! And thiss what we call it [cross-reacting Abs]. -Igs are seen in the serum, and about 20% of the proteins in our serum are immunoglobulins, whichs a huge percentage! Those Igs are present in the gamma portion of the serum when we do protein electrophoresis, where we separate the albumin, alpha 1, alpha 2, beta, and the gamma proteins from each other. The gamma part where the Igs mainly are present during electrophoresis. -We have Polyclonal versus monoclonal antibodies; each B cell produces a different type of Ab, The polyclonal Abs come out from different B cells, but if all the Igs come originally only from one cell we call it monoclonal, can we do that? Yes of course, by the industry of [Hypridoma] or [monoclonal Abs production].

You can see in the picture that, the albumin is the most negative charge so thiss the first wave youll see, and then the alpha1, alpha2, beta and finally the gamma wave. And as u can see, most of the Igs are under the gamma part. From the figure, see {IgG, IgM, IgD, IgA,} where they are located.

Now, how can I localize those Igs, & how can we test them??

By immune electrophoresis. Here we inject Abs to an animal, so they will produce antibodies against the primary Abs. So if I inject IgG or a serum from a patient into a rabbit and then we take the serum, as a result the serum of the rabbit will have Igs against the human Igs, after that if you put them in a gel and react the human serum proteins with the ones we get from the animal, you will have antibodies against antibodies that will make precipitation lines, So if u put anti IgG it will show u that line only, if anti IgA so it will give you that line, so by default u can realize where each immunoglobulin is going to be located, when u do the electrophoresis. Its so simple to do it in the lab, where we can determine how much Igs we have, if we have over production or under production.

Thiss the protein electrophoresis; you can see the albumin, the alpha, beta and the gamma regions. In (a), u can see the polyclonal band of Igs in the gamma portion compared to the monoclonal band in (c) which represent one type of Igs that comes out from one of the cells. In (c), we have hypogammaglobulinemia or what we call it [monoclonal gammopathy] which means one type of the Igs is predominant because one type of the cells is producing large number of one type of Ig.

Theres a disease which is called multiple myeloma or plasmacytoma, where plasma cells go into a malignant transform stage, and produce antibodies continuously, so we call it monoclonal gammopathies, and according to this we can see large amount of one type of Igs. Sometimes we could have polyclonal malignancies where all types of Igs increase in number. If u have immunesuppression then u wont see immunoglobulin in the gamma area.

Production of monoclonal antibodies (Hybridoma)

This method has been discovered by two German scientists who won the Nobel Prize in 1983 for the ability to make Igs in the lab. But how can we do that? The main idea is to make a B cell that proliferate indefinitely & forever, and then Ill harvest the outcomes. For example, if I want to make Abs against the hepatitis B surface antigen that binds to the liver cells (passive vaccine by production of Abs that will neutralize the HBsA), we do the following: 1) We bring the hepatitis B surface antigens then we vaccinate or immunize a mouse by those antigens, so well have then memory cells that will start to give me more proliferation of B cells. 2) Then Ill go to the spleen of the mouse [Cuz the spleen contains lots of B cells and T cells at the same time], so we expect to see B cells that are specific for hepatitis B surface antigen, plus other B cells at the same time. 3) We take those B cells and mix them with myeloma cells. As we said, we want B cells to multiply indefinitely and forever, But whats the cell that can survive indefinitely & forever? Its the cancerous cell. So I get cancerous cells for the B cell [called the myeloma cell, & the myeloma is a plasmacytoma], and we put them with the B cells, after that we fuse them together by using a substance called polyethylene glycol, or we can use an electric current or even viruses, and the cells will be fused. After that, what Im going to get? A hybridoma; a mixture of B cells and myeloma cells, so Ill have the benefit of both. 4) The cells are cultured in a selective medium. Only the fused cells survive after several days. Then we start to make cloning, by bringing a plate and putting those fused cells in the plate and then we disperse the tissue, then we get one hybridoma in each well.

5) Those hybridoma are going to give me Igs against the hepatitis B surface antigen and other antigens that the mouse has been exposed we can put the HB surface antigen in those Igs, And then we see which one is going to react. 6) when we know which the hybridoma that we want is, we take it out and put it In another plate or u can put it in the peritoneal cavity of the rabbit, its a plasma cell thats malignant in a way so when we put it in the peritoneal cavity, continuously they will give me antibodies against HB surface antigens, so every few days I aspirate the liquid from the peritoneal cavity of the rabbit that contains very large amounts of Igs.

So thiss what we call hybridoma or monoclonal antibodies production, and by this technique u can make Abs against any antigenic determinant that u want, for diagnostic purposes in the lab, treatment purposes, or as passive vaccines.

The Enddddddd
Done by: Hadeel Al-kofahi.