Paper No.: 10: Module : 03: cells, BCR, TCR, Co-receptors, properties of antigen recognized by Development Team Principal Investigator: Co-Principal Investigator: Paper Coordinator: Content Writer: Content Reviewer: Prof. Neeta Sehgal Head, Department of Zoology, University of Delhi Prof. D.K. Singh Department of Zoology, University of Delhi Prof. Shibnath Majumder Department of Zoology, University of Delhi Dr. Rajni Arora Swami Shraddhanand College, University of Delhi Prof. Sukhmahendra Singh Banaras Hindu University 1
Description of Module Subject Name Paper Name Module Name/Title Module ID Keywords Zool 010: Antigen recognition by immune cells M03 Recognition units of B and T cells, BCR, TCR, Co-receptors, properties of antigen recognized by Adaptive Immunity, ligand, receptor, coreceptor, antigen recognition, signal transduction, B cell receptor/bcr, T cell receptor/tcr, Major Histocompatibility complex I and II/MHCI, MHCII, MHC restriction, cell activation, antigen processing and presentation Contents 1. Learning Objectives 2. Introduction 3. Recognition Unit of Lymphocytes 4. Structure of BCR Complex; mig and αβ Heterodimer 5. Properties of Antigen Recognised by BCR 6. Structure of TCR Recognition Unit 7. Properties of Antigen Recognised by TCR Cell 8. Summary 2
1. Learning Objectives The study of the chapter will help in understanding the following: The facts and concept of receptors. The difference of a receptor from co-receptor Recognition unit of. Structure of B cell receptor or BCR Structure of T cell receptor or TCR Role of BCR, TCR and co-receptors in antigen recognition The properties of antigen recognized by The structure and role of CD4 + and CD8 + in T cell activation 2. Introduction Immune system of our body is important and specially designed to recognise and respond to the variety of antigens. Broadly there are two ways of tackling with the antigen, that is by innate and the adaptive mechanism. Adaptive immune mechanism is initiated after the effort of innate immune responses towards the pathogen is almost over. Adaptive response starts late but lasts for a longer period. The main reason for the late onset is its very precise and species specific recognition of antigen unlike that of innate system where it responds to the general molecular patterns on the bacteria, virus, or other pathogens when they enter the body. The specificity and diversity in adaptive response is attributed to the presence of receptors on as BCR or B cell receptor and TCR or T cell receptors. BCR recognises the exogenous antigen but TCR recognises the endogenous antigen complexed with MHC (Major Histocompatibility Complex) present on the membrane of all the nucleated cells. The antigen properties thus differ for each of the receptor and accordingly there are structural variations in their recognition units which consist of BCR, TCR, coreceptors and other membrane associated accessory molecules. The recognition unit is a complex of receptors and co-receptors acting as transducer and enhancer to improve the binding ability with the antigen finally resulting in lymphocyte activation. The different structure of the two types of receptors on lymphocytes and the antigens they recognise is dealt with in this module. 3. Recognition Unit of Lymphocytes The are the two specialized cells of the adaptive immunity deputed to recognize and eliminate the intruders of the body in different ways. They vary in the nature of antigen they encounter during surveillance before they mount the immune response (Figure 1) as the antigen can be endogenous or exogenous. The specific recognition of the antigen is the most important function of the adaptive immunity which is attributed to the membrane receptors on the two lymphocytes. The receptors on the B cells are called B cell receptors or BCR and those located on the T cells are called T cell receptors or TCR. The BCR recognises exogenous antigen and forms antibody as the effector molecule after signal from Th/T helper cell through the release of cytokines. The Tc/Tcytotoxic lymphocyte cell when recognizes the antigen complexed with MHCI causes cytotoxicity by converting to CTL but the Th cell complexed with MHCII present on the membranes of APC/ antigen presenting cells, release the cytokines for activation of lymphocytes, macrophage and inflammatory 3
responses (Figure 1,2) The recognition is followed by response of lymphocytes to the antigen by producing effector molecules (antibody) or effector cells (CTL).The information of the receptors is transduced with the help of membrane co- receptors and the co -stimulatory molecules in the cell interior by protein kinases resulting in cell activation. Figure 1: Recognition and Response of different lymphocytes. Before we understand the receptors of B and T cells, let us know the basic structure of a receptor and a co-receptor. A receptor is any membrane molecule which receives signals from another molecule with which it binds specifically called the ligand (Figure 2). Structurally it has following three components or domains. 1. An extracellular domain that recognizes and binds with the ligand. 2. The transmembrane component that passes through the plasma membrane. 3. A cytoplasmic tail for transducing the signal from outside the cell to its interior. 4
Figure 2: Structure of a receptor: E: Extra cellulardomain, P: plasma membrane, I: intra cellular domain. Source: https://en.wikipedia.org The signal can be carried to the inside of cell by bringing a conformational change in the receptor on its cytoplasmic domain or by taking the help of certain accessory molecules if the receptor is handicapped in signal transduction due to its small cytoplasmic tail. The signal may be by opening of certain ionic channels (Figure 3) or activation of certain enzymes like Tyrosine Kinase which may trigger important changes in the cell s interior. A co-receptor is also a cell surface receptor and has the ability to bind the signal molecule in addition to a primary receptor for recognition of ligand. This helps in initiating various biological processes like opening of sodium channels but in the immune cells it is for activation of lymphocytes. (Figure 3, 4). The immune cells have number of CD (Cluster of Differentiation) co-receptors which may be monomers or dimers. They are all extracellular receptors. Figure 3: Opening of membrane channels by attachment of ligands. Source: www.en.wikipedia.org 5
Figure 4: Receptor, ligand interaction and biological response. In order to recognize the variety of antigens which may be either extracellular (exogenous) or (intracellular); the two types of receptors identify the antigen in a fundamentally different ways. The BCR is located on B cells and TCR is located on helper T cell (T H ) and cytotoxic T lymphocytes or killer cell (T CTL ) (Figure 5). The BCR or the membrane antibodies mig, recognize the antigen in its native form compared to most of TCR molecules which recognize the processed antigen located on the antigen presenting cells along with the MHC molecules. T helper cells recognize the antigen in association with MHC-II but the Cytotoxic T lymphocytes, CTL recognize it when it is presented with MHC- I membrane molecule. The TCR/T cell receptor also requires the co-receptors CD8+ and CD4+ on the cells. It is thus important for the BCR and TCR to exhibit molecular diversity to specifically identify the evolving pathogens and variety of newly transformed different cancer cells. Figure 5: BCR and TCR on. Source: www.wikibooks.org 6
4. Structure of BCR Complex; mig and αβ Heterodimer The BCR complex has two structurally and functionally different components working as a unit. They are BCR or mig (membrane antibody) and heterodimer of α (alpha) and β (beta) peptide chains (Figure 6). mig is for the recognition of antigen and αβ heterodimer is for signal transduction. The two functions when performed separately give the advantage of using the same cellular machinery for transduction on recognizing the variety of antigens. 4.1. BCR or mig Figure 6: Structure of BCR and TCR complex/unit. Membrane antibody has a monomeric Y shaped structure composed of four polypeptide chains and an anchoring unit so as to to fix it on the lipid rafts of the plasma membrane along with the other accessory molecules required for signal transduction. The IgM and IgD are the membrane receptors where the CH 4 Domain of IgM is hydrophobic to insert into the B cell membrane. The Fab component is oriented outside the cell and Fc is noncovalently involved in signal transduction by interacting with co-receptors and accessory molecules. The cytoplasmic tail of membrane antibody/ Fc is insufficient to transduce the signal on antigen recognition (Figure 6).Lipid rafts of the plasma membrane provide a platform for selective interaction of the mig with the signal molecules which carry the information to the cytoplasm for activation of B lymphocytes. The recognition of antigen requires the antibody crosslinking or clustering which initiates the process of B cell activation. The Y shaped membrane antibody structure has variable light, and heavy chains (V L and V H ) as discussed earlier, so variety of antigens are specifically identified and B cell is activated. 7
4.2. αβ Heterodimer It consists of two polypeptide chains alpha and beta which have a long cytoplasmic tail. The cytoplasmic tail of Igα is made of 61 amino acids but that of Igβ has 48 amino acids. If we compare the tail length of migm with other secretory antibodies, IgA has 14 aminoacids, IgG and IgE contain 28 amino acids, much shorter than the cytoplasmic tail length of αβ heterodimer. The cytoplasmic tail of the heterodimer has important component ITAM (Immunoreceptor Tyrosine Based Activation motif) which activates enzyme Tyrosine kinase (Figure 8). When an antigen binds, the Tyrosine in these ITAMs becomes phosphorylated by Tyrosine kinases which are important for transmitting the signal. The efficiency of B cell signal is also increased by the complement activation. The clustering of B cells also brings the complement binding receptors closer which further accentuates the signal strength as shown in Figure 7, 8. Figure 7: Signal transduction by BCR Recognition unit. The sequence of events for recognition and the activation of B cell is as follows: 8
Figure 8: Flow diagram to show the recognition of antigen and B cell activation. 5. Properties of Antigen Recognized by the BCR The BCR recognizes almost every macromolecule that is proteins, carbohydrates, lipids etc that can bring the clustering of the membrane antibody. The BCR identifies only a particular area of the macromolecule of protein antigen called antigenic determinant or epitope. When the macromolecule exhibits many antigenic determinants then it is called multivalent or polyvalent antigen. The B cells may recognize the antigen with epitopes which may be 1. Identical or nonidentical; Identical in chemical structure. 2. Repeating or non-repeating: Regular spaced identical epitopes or different. 3. Overlapping or non overlapping: Overlapping if epitopes are closely placed. 4. Conformational or non-conformational: Linear or three dimensional epitopes. 9
Since the pathogens display variety of multivalent antigens either proteins or carbohydrates, they induce the fast signal transduction across the B cell membrane due to clustering of BCR. The overlapping epitopes can result in antibody clustering better than the non-overlapping. The protein antigens may be linear or they assume the three dimensional structure. The denaturation of protein may result in disappearance of certain antigenic determinants which are called conformational epitopes. There are four types of noncovalent interactions involved in antigen and antibody interactions which are ionic, Hydrogen, Hydrophobic, and Vander Waal s forces. It should be very clear that not all the antigens which are recognized and bind with the receptors on lymphocytes are capable of triggering their activation. Those molecules which are activating the lymphocytes are called immunogens. There are small chemicals which may be successful in binding to the membrane antibody but do not activate the lymphocytes. These chemicals like Dinitrophenol are called haptens. They need the help of some carrier protein making a protein-carrier conjugate which converts them to immunogen (Figure 9). 6. Structure of TCR Recognition Unit Figure 9: Hapten and immunogen. T cells recognize the antigens by the T cell receptors or TCR. Each T cell has approximately50, 0000 to 100,000 identical receptors for antigen. They are clonally distributed which means each clone has one type of TCR. TCR is membrane bound only and is not secreted like BCR. It also has a three domain structures comprising of extra cytoplasmic part, transmembrane region and cytoplasmic tail as discussed earlier for BCR (Figure 10). 10
Figure 10: Structure of TCR. Source: www.wikipedia.org A complete recognition unit of T cells or TCR complex consists of the following important components: 1. Two α alpha and β beta or γ gamma and δ delta peptide chains expressed on surface of the T lymphocytes as TCR. 2. CD3complex.or invariant chain consisting of γ, δ, δ, ε, ε 3. Co- receptor molecule which is either CD4+ or CD8+ There are two distinct sub-types of T lymphocytes based on TCR composition that is the TCR with α and β or γ and δ peptide chains. There is difference in recognition of antigen as the alpha, beta T lymphocyte recognizes the antigen presented on MHC-Ia molecule which is a polymorphic form unlike that of MHC-Ib which is nonpolymorphic and the antigen complexed with MHCIb is recognized by gamma delta TCR. Gamma delta T cells have affinity for the nonprotein antigens which may not be complexed with MHC molecules. It recognizes the conserved structures on the invading microorganism. TCR is a highly variable heterodimer and is structurally homologous to Ig Superfamily. The heterodimer has two functionally different domains that is variable and constant (Figure 10). The Variable domain is responsible for the antigen recognition and binding but the constant domain is transmembrane with a small cytoplasmic tail. The transmembrane domain has a stretch of positively charged aminoacids which allow them to interact with the CD3 molecule.cd3 complex chains are called invariant as they have no variability in the amino acid sequence. It has three heterodimers of γδ, γε, δε and a homodimer of δδ chains or heterodimer δε. Homodimer of zeta is more prevalent on the T cell surface than the heterodimer peptide. The Gamma, delta and Eta chains belong to Immunoglobin superfamily but zeta chain is different and has a very long cytoplasmic tail consisting of about 113 amino acids. The cytoplasmic tail of CD3 is associated with the special peptide motifs called ITAM, or Immunoreceptor which are Tyrosine based Activation Motifs required for the activation signal of the T cell. There are three ITAM on δ and one each on delta, Eta and gamma chains CD3 molecule (Figure 6) The TCR trigerring also undergoes receptor clustering and confirmation changes in CD3 molecule along with receptors and MHC molecule. 11
Figure 11: Antigen and the recognition unit of T cells. Source: svg.common.wikimedia.org Figure 12: Antigen receptors and T lymphocytes. 12
The CD4 and CD8 co -receptors act as enhancers which increase the affinity of TCR with MHC-I or MHC-II molecules.cd4 is the co-receptor for Th cells which binds with the MHC-II of APC whereas CD8 bind with MHC-I exhibited on all the nucleated cells. T lymphocytes with CD8 surface glycoprotein are called CD8+ T cells and those cells with CD4 are called CD4+T cells. CD4 and CD8 have Ig fold domain like structure where CD4 is a 55KDa glycoprotein expressed on approximately 5-6 percent of T cells in human. It is a single peptide alpha monomer which is linked to the lck type of Tyrosine kinase in the cell membrane. It consists of four extracellular domains in peptide chain. It is the important receptor for the entry of HIV (human immunodeficiency virus.), which causes deadly disease AIDS (Aquired immunodeficiency syndrome). The cells with CD4 glycoprotein are more prone to attack by HIV. CD4 count which is a lab test done on AIDS victims indicate the health of patient. It is possible to perform a CD4 co-receptor blockade, using antibodies, in order to lower T cell activation and counteract various disorders leading to immunodeficiency and autoimmunity. CD8 is a 34KDa alpha/beta heterodimer both with extracellular, transmembrane and cytoplasmic domains (Figure 13). It is present on 30 % of T cells in blood of human. It consists of only one extracellular domain in the peptide and is associated with the lck Tyrosine Kinase in the cytoplasm. The extracellular domain of CD8-α interacts with the α 3 domain of class I MHC molecules which help in increasing its sensitivity and stabilizing the TCR and MHC interface (Figure 11, 1) required for the cell activation. Figure 13: Structure of Coreceptor, CD4 and CD8. The recognition of antigen by T lymphocyte results in signal transduction via reversible phosphorylation of Tyrosine by protein kinases and phosphatases. T lymphocytes use fyn, Src family of kinases in signalling to phosphorylate ITAM located on CD3 and Zeta chains. The signal step and 13
TCR triggering involves different kinases like Lck, fyn, ZAP70 and Tyrosine phosphatase CD45. It is membrane protein which has Tyrosine phosphatase to activate Src family kinases. The regulatory mechanism for weakening or attenuation of the immune receptor signaling is important to prevent reactions for self antigens. It is by the inhibitory receptors located on T and B cells which have ITIM (Immune Tyrosine Inhibitory Motifs) to activate phosphatases. 7. Properties of Antigen Recognized by T Cell The antigens recognized by T cells are small linear peptides loaded on MHC-I or MHC-II. They identify protein antigens which are processed and thus broken into small peptides. The reason only proteins are recognized is due to the peptide compatible structure of MHC. MHC molecules show polymorphism and have the ability to bind with the variety of peptides. Out of the large repertoire of MHC molecule, T cells recognize peptide which is complexed with one of the MHC molecule and this is called MHC restriction. T-helper cells with CD4+ are Class II MHC restricted whereas T- cytotoxic cells withcd8+ are MHC-I restricted. The polymorphic domain of aminoacid residues of MHC are near the peptide binding cleft whereas the nonpolymorphic Ig like domains of MHC molecule has the binding site for either of the two subpopulations of T cells that is Th or Tc. The class II restricted T helper cells/th cells are activated by the extracellular antigens which pass through the endosomal vesicles for antigen processing unlike that of class I restricted cytotoxic T cells which bind with the intracellular antigens produced by viral infections or cancer proteins after passing through cytosolic processing. As it is difficult for the T helper and T cytotoxic cells to distinguish the endogenous or exogenous antigens, they are directed by the two types of MHC molecules that is MHCI and MHCII..The T cells recognize not all the peptides which are processed but a few of them which are more compatible with the MHC and this is called immune-dominance. Gamma delta T cells are not MHC restricted as they recognize the antigen without MHC molecule also. There is a small population of Natural Killer T cells which have the characteristic markers of NK and T cells and they recognize non-protein antigens which are not MHC restricted. The recognition of antigen by the two different lymphocytes is by different mechanism. B cell recognition of epitopes of antigen has following features: 1) The interaction of B cells with the epitopes form binary complex that is Ag-Ab. 2) B cells bind with soluble antigens. 3) B cells do not require MHC molecules for epitope recognition. 4) B cells recognize antigens which may be proteins, polysaccharide and lipids in nature. 5) B cells recognize the epitopes which are accessible, hydrophilic, mobile peptides with sequential or non -sequential amino acids. T cell recognition of epitopes of antigen show following features; 1) The interaction with antigen and Tcell receptor forms a ternary complex that is Ag- MHC-Ab. 2) The T cells do not bind the soluble antigen. 3) The T cells require MHC molecule for presentation of the processed antigens. 4) The T cells recognize the antigens which are mostly protein in nature. 5) The epitopes recognized by the T cells are linear peptides due to processing of the antigens and are bound to the MHC molecule. 14
8. Summary The chapter helps to understand The receptor is a membrane glycoprotein and consists of extracytoplasmic, transmembrane and cytoplasmic tail Adaptive immune response is specific because of receptors on called BCR and TCR. They work in coordination with co- receptors and accessory molecules thus forming BCR and TCR recognition units or complexes. The BCR unit or complex consists of membrane antibody, mig and alpha, beta heterodimer as co receptors. The alpha beta heterodimers have ITAM for signal transduction. Membrane antibody clustering is important for antigen recognition by BCR and cell activation. ITAM triggers the signal to the cell interior by the activation of protein kinases. The T lymphocytes recognize the antigen complexed with MHC I or MHCII molecules associated with co-receptors CD4 and CD8 and invariant chains of CD3. T helper cells recognize antigen complexed with MHCII (onapc) and co-receptor CD4. T cytotoxic cells recognize the antigen complexed with MHCI and co- receptor CD8. CD4is a monomeric glycoprotein but CD8 is heterdimeric. CD8 interacts with alpha three domain of MHCI. There are three ITAM on δ and one each on delta, Eta and gamma chains of CD3 molecule. Tc and Th cells are MHC restricted. The epitope properties of antigen recognised by B cells are different from T cells. 15