بسم اهلل الرحمن الرحيم سبحان اهلل والحمدهلل I OVERVIEW OF Immunology & SEROLOGY الحمد هلل Presented By DR. MAHMOUD KAMAL Mansour Assistant professor of Medical Microbiology & Immunology(SCU 2 Immune system •Thissystem is composed of primary lymphoid organs and secondary lymphoid organs •Primary lymphoid organs are contain thymus and adult bone marrow. • Secondary lymphoid organs are contain spleen, lymph node, tonsils and MALT. Cont….. All these cells of this system arise from polypotent stem cells of bone marrow through two main lines of differentiation, lymphoid lineage producing lymphocytes and Myeloid lineage producing phagocytes. The immune response of this system are mediated by a variety of cells and by the soluble mediators which they secrete. The function of this system to eliminate infectious agent or to minimize the damage they cause. 5 Cont….. Immune System It is formed of lymphoid organs, tissues and cells. It is divided into primary lymphoid organs(Thymus, Bone marrow). Secondary lymphoid organs (Bone marrow, lymph node, Spleen, Liver and MALT [BALT and GALT]). Composed of specific (B & T) & non-specific (NK, monocytes, neutrophils, Eosinophils, Basophils & Mast immunocompent cells). 3 4 7 8 IMPORTANT DEFINITIONS IMMUNOLOGY • This the science that deal with the Immunity , Serology, Autoimmune disease, Hypersensitivity, Allergies, Organ transplantation, Rejection of foreign tissue , Vaccination & Serology . The Immune system are responsible for all these mention Immunological functions & Immunological disorders. Immunity ☺ Immunity means protection. ☺There are two types of Immunity. • Innate Immunity ( non specific, non adaptive, natural ). • Acquired Immunity (specific, adaptive, acquired ). ☺ It is in vivo reaction deals with non specific immune defenses & specific humoral immunity (antigens, antibodies) & cell-mediated 10 Serology • It in vitro reaction between antigen & antibody in the laboratory IMMUNOGEN • It is a full antigen • It must be foreign substance that has the ability to provoke a specific IR. • It is usually proteins that more immunogenic than polysaccharides or lipids. 12 Cont… ☺ Immunogen It is a foreign substance or molecule that, when introduced into the body, it is capable of inducing an Immune response (IR). 10 Antigen • It refers to any foreign molecule that can be recognized by specific antibody, BCR, or a TCR and become the target of Immune response (IR). ☺All immunogens are antigens. اهلل أكبر 12 Cont… Antigen Allergen • Any foreign agent e.g. pollen, dust, animal dander that causes IgE- mediated reactions. Hapten It is a partial antigen (Incomplete Ag). It is a low molecular weight foreign substance. Alone cannot stimulate the immune system. carried on large protein to be full antigen(immunogenic), اهلل أكبر 14 Super antigen It is unusual antigen that can not be processed by APCs. It stimulates polyclonal lymphocytes. E.g. Toxic shock syndrome-1, pyrogenic toxin of Strep. 15 Epitope •It is the antigenic determinant of the Immunogenic molecule. • It is the smallest unit of a complex Immunogenic molecule. • An Immunogen can have one or more epitope. • Roughly the epitope has five amino acid or monosaccharide in size. Cont… Epitopes or antigenic determinants ☺Not all the antigen molecule is antigenic, only small parts of themolecule can bind antigen receptors and induce IR. These parts are called epitopes or antigenic determinants. ☺One antigen molecule may contain several epitope, each can combine with specific antigen receptor. 17 Factors Affecting IMMUNOGENCITY Must be foreign body (Non-Self Antigen). Large molecular weight (more than 10000 D). Chemical structure (tertiary/secondary/primary). Root of administration (ID/ SC/IV/Oral). Immunological status of host. presence of adjuvant. 17 اهلل أكبر 81 اهلل أكبر 91 اهلل أكبر اهلل أكبر 02 اهلل أكبر 12 اهلل أكبر 22 اهلل أكبر 32 Humoral Immune Response (Antibody-mediated IR) ☺Humoral IR depends on production of large amounts of specific antibodies by activated B cells. ☺Like T cells, there are millions clones of B cells, each one can specifically combine one foreign antigen through BCR, which consists of monomeric form of IgM. ☺The enormous diversity of the B cell repertoire depends on the ability of developing B cells to rearrange and modify their Ag receptors during the process of maturation in bone marrow. 24 ☺Maturation of B Cells consists of 3 Phases:- 1- proliferation. 2- expression of Ag R genes. 3- selection of cells expressing useful antigen receptors. 25 Steps of B cell maturation in BM (ontogeny):- ☺proliferation. • Proliferation of the earliest lymphocyte precursors is stimulated by IL-7 secreted by stromal cells in bone marrow. ☺Mature resting (virgin) B cells. • express, in addition to IgM, an IgD molecule, both with the same antigenic specificity. At this stage, B cells migrate to the peripheral lymphoid tissue. سبحان اهلل 26 Cont. (ontogeny) Steps of B cell maturation in Bone marrow:- ☺ Mature active B cells. • when mature B cells is activated by the specific antigen, it looses IgD and differentiate in several steps to plasma cells which secrete antibodies specific to the activating antigen. 27 Structure of B Cell antigen Receptor (BCR):- • B cell (together with IgD in case of virgin mature B cells (membrane Igs, monomeric IgM). • Ig-α/Ig-β heterodimers, disulfide-linked polypeptides containing cytoplasmic domains with ITAMs (Immunoreceptor Tyrosine-based Activation Motifs) are associated with the MIg subunits (two heterodimer molecules per BCR). 28 • The MIg is formed of 2 heavy and 2 light chains. • The light chain contains one variable and one constant domain. • Heavy chain contain one variable and 3 constant domains. • Variable regions of both heavy and light chains form the antigen binding site. • Variable domains containing three hyper variable regions (complementarity determining regions, CDRs). • CDRs bind antigenic determinants of the antigen or the epitope. 29 سبحان اهلل 13 Antibody • It is immunoglobulin molecule produced by animal in response to immunogen. It has the ability to combine specifically to antigenic part of this immunogen IMMUNOGLOBULIN • It is a glycoprotein composed of Heavy (H) an light (L) chains that functions as antibody. All antibodies are immunoglobulins but not all immunoglobulin has antibody function. Structure of Immunoglobulins • Immunoglobulins make up about 20% of plasma proteins. • Blood contains 3 types of globulins; alpha, beta and gamma. • When a serum sample is fractionated by electrophoresis, most immunoglobulins migrate as a broad band in gamma globulin fraction. + - albumin globulins 2 1 Immune serum Ag adsorbed serum Mobility 34 Immunoglobulins, Structure & Functions •Immunoglobulins are a big family of related but non identical glycoprotein. • Igs are the critical ingredients of the humoral IR, which is the function of lymphocytes. • Igs on the surface of B cells act as antigen receptors that can detect and bind a large number of antigens present in the environment. • On binding their cognate antigens, surface Igs initiate activation, proliferation and maturation of B cells with generation of plasma cells. • The Igs secreted by plasma cells are the Antibodies which will bind specifically to the antigens that trigger their production. Immunoglobulin variable regions • Variable regions of both the L and H chains mediate antigen binding and are the most heterogeneous portion of Ig molecules. • Variable regions consist of relatively invariant stretches called framework regions of 15-30 amino acids, separated by shorter regions (9-12 a. a.) of extreme variability called hypervariable regions (HVR). Cont….. Immunoglobulin variable regions • Hypervariable regions of both H and L chains together form the antigen binding site and determine the antigen specificity. Hypervariable regions are also called complementary-determining regions; CDR1-CDR3. They are complementary in structure to the antigen determinant or epitope on the antigen molecule. Cont…. Immunoglobulin variable regions • Each VH domain is positioned directly beside VL domain and this pair of domains together form the antigen binding site. Each basic unite contains 2 antigen binding sites (divalent) pratope. • The antigen specificity of a given Ig molecule is determined by the amino acid sequence of its VH and VL domains together. • The region between CH1 and CHs is called the hinge region and it has loose secondary structure making the Ig molecule flexible at this point. 37 cont …. Immunoglobulins ☺The 2 main characteristics of Immunoglobulins as antigen binding proteins are:- 1- Specificity: antibodies secreted by a clone of B cells will bind specifically to the antigen which trigger their secretion. 2- Diversity: different antibodies molecules secreted by different clones of B cells will bind different types of antigens. cont Immunoglobulins ☺Antibodies are bifunctional molecules; in addition to antigen binding, antibodies possess secondary biologic activities that are critical for host defense as complement fixation, acting as opsonins and crossing the placental barrier. Disulfide bond Carbohydrate CL VL CH2 CH3 CH1 Hinge Region 39 Structure of Ig Cont……… Structure of Immunoglobulins The four chain basic unite:- • Every immunoglobulin molecule is formed of four polypeptide chains; two identical heavy (H) chains and two identical light (L) chains. Cont……… Structure of Immunoglobulins • Some Igs are formed of only one basic unite, other formed of more than one. • All the H and L chains in a single Ig molecule are identical and can be presented by the general formula (H2L2)n. • The H & L chains are composed of folded globular domains, each of which is 100- 110 amino acid long and contain one intrachain disulphide bond. L chain always contain 2 domains, whereas H chain contains 4 or 5. اهلل أكبر 24 اهلل أكبر 34 اهلل أكبر 44 Enzymatic digestion products of Immunoglobulins Enzymatic digestion products of Immunoglobulins Effect of papain:- 1-Cleaves the molecule at the hinge region on the N-terminal side of the inter-heave chain disulphide bonding leading to formation of 3 fragments. 2- identical Fab fragments each consists of an entire light chain and VH and CH1 domains of the heavy chain. Each of these fragments contains one Ag binding site. 3-One Fc fragment consists of the carboxyl terminal of the 2 heavy chains held together by disulphide bond. Most of the secondary biologic functions of Igs are determined by the Fc fragment. 45 Papain الحمدهلل Fc Fab 46 Pepsin Effect of papsin Effect of pepsin Pepsin cleaves on the carboxyl terminal of the interheavy chain disulfide bond yielding one large fragment, F(ab)2, which corresponds to 2 disulfide linked Fab fragments and has divalent antigen binding capacity. The Fc region is extensively degraded Fc Peptides by pepsin and does not survive an intact fragment. F(ab’)2 47 Functions of Immunoglobulins 1- Agglutination of particulate antigens as bacterial cells (agglutinins). 2- Antitoxin antibodies: neutralize microbial toxins. 3- Neutralizing antibodies that attachment of microbes to mucosal surfaces (secretory IgA). Cont…. Functions of Immunoglobulins 4- Complement fixing antibodies. 5- Opsonization: Opsonins. 6- Antibody-dependent cellular cytotoxicity: important in immune response to helminthes by IgE سبحان اهلل 94 PARATOPE •This is the part of an antibody which is attach with epitope. • Also known as antigen binding site. Immunoglobulin Fragments: Structure/Function Relationships Ag Binding سبحان اهلل اهلل أكبر Complement Binding Site Binding to Fc Receptors Placental Transfer 51 IMMUNOGLOBULIN CLASS • A subdivision of immunoglobulin molecules based on unique antigenic determinants in the FC region of the H chain. • In human, there are five immunoglobulin classes ( IgG, IgA, IgM, IgD and IgE) سبحان اهلل اهلل أكبر Cont…. Immunoglobulin Classes IgG • Formed of one basic unite, antigen divalent. • Divided into 4 subclasses, Ig1-Ig4 . Cont…. IgG • the most abundant Ig in serum (75%). • the main Ig in the secondary immune response. • the only Ig that cross the placenta, and it is responsible for the protection of the newborn during the first months of life. سبحان اهلل Cont… IgG • Can fix and activate the complement. • can opsonizes, i.e., enhances phagocytosis of organism by phagocytic cell which have receptors for Fc region of IgG. سبحان اهلل Cont… IgG IgG1 IgG2 IgG3 IgG4 % of total IgG 70 20 6 4 Half life 23 23 7 23 Placental passage +++ + +++ +++ Complement fixation + + +++ - Binding to FcR +++ + +++ - IgA • Two types of IgA; serum and secretory. • Serum IgA accounts for only 10-15 % of serum Igs. • It is the predominant Ig formed by B cells in Peyer patches, tonsils and other sub mucosal lymphoid tissues. So, it is the most abundant Ig in saliva, tears, bronchial secretions, milk, intestinal mucosa, prostatic fluid and other secretions. الحمدهلل Cont…. IgA • It prevents attachment of pathogens as to mucous membranes. • Secretors IgA is usually formed of 2 polymerized basic units plus J polypeptide chain, which initiate polymerization, and a secretory component. • The secretory component is synthesized by epithelial cells and help in passage of IgA to the mucosal surface. • Serum IgA may be found in a monomeric or a dimeric form. Secretory IgA structure الحمدهلل اهلل أكبر Secretory Piece J Chain 55 IgM •Pentamer, containing J component. • H chain contains 4 domains. • 10% 0f serum Igs. Cont… IgM • The main Ig in the primary IR. • Expressed at the surface of B cells in the form of monomer as the Ag receptor. • It is the most efficient C fixing antibody. • Fc R for IgM exist but not well characterized. J Chain الحمدهلل اهلل أكبر 4C 75 IgD • It Has no known antibody function, but may act as antigen receptor on the surface of B cells. • It is a monomer and represent only o.2% of serum Igs. • It presents on the surface of some B cells together with IgM monomer in the stage of immature B cells. IgE • It is a monomer and H chains contain 4 domains. • Least concentration in serum; 0.004%. • Medically important for 2 reasons:- • mediates immediate type I hypersensitivity. • participates in host defense against helminthes (worms). الحمدهلل اهلل أكبر Allele • intra species variance at a particular gene locus. ALLogenic • Refers to intraspecies genetic variations. Isotypes • Normal individuals express the all 9 classes and subclasses of the H chains of Igs chains & the 2 types and subtypes of the L chains because each is encoded by a separate gene and is inherited independently; all together are called isotypes. Allotypes • Some of the genes coding for the different constant regions of H or L chains exist in more than one form within the population, the alternative forms (alleles) differ from one another in one or very few amino acid sequence. These minor alternative forms of a given Ig locus are called Allotypes. الحمدهلل اهلل أكبر Cont… Allotypes • In humans, allotypes have been found for y, α and ξ H chains and for ĸ L chain • This different allotypes can be immunogenic, for example, mother may be immunized against a different allotypes of the father during pregnancy. Idiotypes (self-type) • Refers to the unique Hypervariable region amino acid sequences of the homogenous Ig molecules produced by a single B cell clone. Thus, there are many idiotypes as there are many B cell clones (about 109 clones in an adult). AFFINITY • A measure of the binding strength between the epitope & paratope. This binding take place by the formation of multiple non-covalent bonds between the antigen & the amino acids of the binding site Avidity • A measure of the summation of binding strength between the multiple epitopes and paratopes. This binding take place by the formation of multiple non-covalent bonds between the antigens and the amino acids of the binding sites. These bonds namely hydrogen bonds. electrostatic. Van der Waals. hydropholic forces. 65 66 ANTIGEN PRESENTING CELLS • variety of cell types which process the antigen in a form that stimulate lymphocytes. • They included Macrophages, Langerhans cells, interdigitating cells, follicular dentritic cell and B cells. B cells act as APC with direct cell-to-cell contact •Ag presentation by B cell to TH and interaction is intensified through co -stimulatory molecules; CD40 and CD40 legend, B7 & CD28. • Activated T cells secrete IL4, 5, 6, 10, which activates proliferation and differentiation of B cells to plasma cells and memory cells. 96 الحمدهلل الحمدهلل الحمدهلل 07 Monoclonal Antibodies • Highly specific antibodies produced against a single epitope of an antigen by cells derived from a single clone of immortal cells. • These immortal cells are called hybridoma cells that are formed by fusion (hybridization) of a B cell producing antibodies against a single epitope and an immortal, continuously replicating myeloma cells. • The resulting hybridoma acquires the property of being immortal & the ability to produce monoclonal antibodies specific to a single epitope. Hybridoma • It is formed from fusion of B cell already immunized with the epitope of interest & myeloma cell, the fusion of hybridomas is encouraged by adding certain chemicals e.g. polyethylene glycol Technique of monoclonal antibody production • Spleen cells from suckling mice immunized with the antigenic epitope of interest are grown with myeloma cells in cell culture containing polyethylene glycol (PEG). • polyethylene glycol promotes fusion of cells resulting in formation of the hybridoma. • Then HAT culture medium is used which contain hypoxanthine, aminopterin and thymidine. In presence of HAT, myeloma cells will die and the spleenocytes will die naturally. • Resulting hybridoma are screened for antibody production, positive clones producing the antibody of interest are propagated in cell culture or freeze-dried at -70°C till used. 74 Uses of Monoclonal Antibodies Diagnostic uses 1- Identification of differentiation Ags on cells:- A- CD markers of lymphocytes subsets. B- Tissue typing (HLA). C- Tumor associated markers: CEA, alpha feto-protein. 2- Identification and typing of bacteria and viruses. 3- Hormonal assay. سبحان اهلل 75 Therapeutic uses 1- Antitumor therapy: by using MCA to tumor specific Ags alone or after coupling it with cytotoxin (e.g., Diphtheria toxin), or with radioactive substance (magic bullet therapy). 2- Immunosuppressive therapy: MCAs to T cells subsets (anti-CD3) to prevent graft rejection. 3- Treatment of drug toxicity, e.g. to neutralize digitalis toxicity. 4- Anti-RH D to prevent RH incompatibility. 5- Passive immunotherapy for protection from viral infections as CMV and VZV. POLYCLONAL ANTIBODIES • Antibodies that arise in animal in response to poly epitopes antigen. • They are formed by several different clones of B cells. سبحان اهلل Genetic origin of B cell antigenic diversity Light chain genes • There are 2 types of light chains; kappa (ĸ) and lambda (λ). • The light chains in a single molecule of Ig are of the same type, never both together. • Genes coding for ĸ chain exist on chromosome 2 and consist of 30-35 Vĸ segments and 5 Jĸ segments coding for the variable region and one Cĸ for the constant region. • Genes coding for λ chain exist on chromosome 22 and consist of 100 Vλ, 6 Jλ for variable domain and 6 Cλ for constant domain. سبحان اهلل 78 79 80 Rearrangement of Heavy chain genes • سبحان اهلل 81 82 83 Humeral IR Activation of B cells:- • Each clone will be activated by its specific Ag (clonal selection theory). • cross-linking of 2 or more BCRs by a bivalent or multivalent antigen cause a signal of activation which is transmitted to Ig-α/Ig-β heterodimer. This initiates ITAM phosphorylation by cytoplasmic protein kinases. • ITAMs phosphorylation generates a signal that is transmitted to the B cell nucleus via an intracellular signal transduction pathway. 84 B cell is activated, proliferate, differentiate to 1- plasma cells:- • which actively secrete antibody. • End-stage cells that do not proliferate and have no markers on its surface. 2- B memory cells :- • represent an expanded population of B cells able to respond to the inducing antigen. • They reside mainly within lymphoid follicles and survive for many years. 85 Activation and Clonal selection of B cells سبحان اهلل 86 Two types of antigens can bind BCR A. T-cell-independent antigens (TI-Ag):- • Can activate B cells without the help of T cells, No memory cells are produced. • These antigens Has the property that at high concentration can activate large number of B cells both specifically and non- specifically (polyclonal B cell activators)e.g. polysaccharides. 87 B- T-cell dependent Antigens:- • Most protein antigens need T cell help to induce Ab response. • T cell help can be provided in 2 forms: سبحان اهلل 88 B cells bind the Ag through BCRs • TH cells are stimulated by the presented antigen on APcs and secrete lymphokines which activates proliferation and differentiation of B cells to plasma cells and memory cells.. سبحان اهلل T-cell independent antigens سبحان اهلل 89 MHC MOLECULES • There are three major classes of molecules coded within the MHC (Class I, II, III). • The genes of MHC are located on the short arm of the chromosome six. • MHC class I molecules comprises a glycosylated heavy chain associated with B2- microglobulin سبحان اهلل MHC class I •Expressed on the surface of all nucleated body cells but not on mature RBCs. Cont… MHC class I Structure:- • A heavy chain molecule non-covalently associated with a small molecule, β2 microglobulin. • The class I molecule consists of 3 extracellular domains (α1, α2, α3), a transmembrane region and a cytoplasmic tail. • The groove between α1 and α2 form The peptide binding site. سبحان اهلل Cont… MHC class I Structure:- • There are 3 types of class I molecules; A, B, C coded by 3 genetic loci A, B, C. • β2 microglobulin is no polymorphic, coded by a gene on chromosome 15 and is essential for expression and stabilization of class I molecules. سبحان اهلل Human MHC genes • All of the MHC gene loci reside together as complex on the short arm of chromosome 6, class I loci A, B, C on one side and class II loci DP, DQ, DR on the other side. • The combination of these 6 loci on a single chromosome are inherited together and is called haplotype. • Because human inherit one chromosome from father and one from mother, so he has 6 loci for each class. • MHC genes are dominant, so all MHC proteins are expressed on the surface of body cells. So 2 proteins of each type are expressed. • Other genes related to MHC include:- 94 Major Histocompatibility complex MHC (HLA) • MHC antigens are group of proteins expressed on the surface of all body cells and are the main target of immunological reaction that lead to rejection of organ or tissue transplanted between unrelated individuals. • These proteins are also called HLA because they were first discovered on human leucocytes. HLA proteins behave as immunogenic markers that distinguish each person' cells from most others in the population and are the major barrier to histocompatibility. • MHC genes are the genes coding for the MHC proteins and are present on chromosome 6. • MHC is classified into 2 classes which differ in structure and tissue distribution. Class I represent antigens to Tc cells, class II represent to TH cells. MHC class II • Expressed only on immune cells, T, B, APCs. Structure:- • Two polypeptide chains, α and β, the extracellular region of each chain folds to form 2 domains; α1, α2 and β1, β2. • Peptide binding site is formed by α1 and β1. • Three types of class II molecules; DP, DQ, DR coded by 3 genetic loci, each of which include genes for one α and one β. 96 MHC Polymorphism • The most polymorphic gene complex. • At each locus of both class I and II there are multiple different alleles distributed in the human population. • For example, there are about 100 HLA-A alleles designate A1, A2 etc, more than 150 HLA-B alleles, more than 200 HLA-DR alleles and so on. • Polymorphism among MHC alleles involve amino acid sequence at the antigen binding site. So, each allelic form has its antigen binding properties. • Human inherits 2 different alleles at each locus, the advantage of this is the increase the range of different antigen peptides that can be presented to T cells. سبحان اهلل 97 Cont… MHC If the HLA proteins on the donor’s cells differ from those on the recipients cells an immune response occurs in the recipient. These are many alleles of the class I and class II genes. There are at least 40 HLA –A genes, 80 HLA – B genes and 10 HLA – C genes. Class I proteins are detected in laboratory by reacting lymphocytes with a battery of specific antibodies plus complement. سبحان اهلل Significance of MHC • Role in Ag processing and presentation. • Role in induction of immunological tolerance. • Role in alloreactivity and transplant rejection. • Relation of MHC to some diseases. • Paternity testing and forensic investigations. 98 Antigen processing and presentation • Depending on the source of antigen, processing can occur of either of 2 ways:- 1- Endocytic (class II, exogenous) pathway:- • Antigen sources are endocytosed extracellular proteins (host or foreign) and membrane proteins (host and foreign). • This pathway delivers peptides to MHC class II molecules which are expressed by macrophages and other professional antigen presenting cells (APCs) that present antigens to T4 (TH) cells. سبحان اهلل Cont… Antigen processing and presentation • Depending on the source of antigen, processing can occur of either of 2 ways:- 2- The cytosolic (class I, endogenous) pathway:- • Antigen source: endogenous Ags; cytosolic proteins of host or intracellular pathogen. • This pathway delivers peptide to MHC class I expressed on all nucleated body cells and present it to T8 (Tc) cells. Antigen processing pathways Endocytic pathway Cytosolic pathway Major Ag source endocytosed extracellular cytosolic proteins of host proteins (host or foreign) or intracellular pathogen, and membrane proteins viral, bacterial or (host and foreign). parasitic. Processing machinery Lysosomal enzymes in the Proteasomes, including lysosomes. LMPs Cell type where active Professional APCs All nucleated cells Site of Ag-MHC binding Endocytic vesicles, RER prelysosomes MHC utilized Class II Class I Present to T helper cells T cytotoxic cells. 100 Control of Ag processing and presentation • IFNy increases expression of class I molecules, TAP transporters and LMPs. • IFNy induce expression of class II molecules on some cell types as fibroblasts, endothelial cells, epithelial cells which normally do not express these molecules, enabling these cells to present Ag to TH cells. • On the other hand, some pathogens suppress these processes, for example herpes simplex virus produce protein that inactivate TAP transporters thus blocking endogenous pathway and so evade detection by the immune system. 101 Cont… Control of Ag processing and presentation ● in region of class II, 2 genes coding for TAPs, 2 genes coding for LMPS proteins and one gene coding for a heterodimer called DM. ● non classic HLA genes; E, F and G in the region of Class I. • HLA-G is expressed on the extra villous trophoblasts, the fetal cells directly in contact with maternal tissues. This protein provides inhibitory signals to T cells and NK cells and may serve in maintaining materno-fetal tolerance. • Other genes not related to histocompatibility or antigen presentation include genes coding for: C2, C4, BF factor, 2 heat shock proteins and 21 steroidogenic hydroxylase. 102 ADESETOPE ● is the part of MHC molecule which inks to the processed antigen. AGRETOPE ● It is the antigen fragment which interact with MHC Molecules Class II protein are first demonstrated by mixed lymphocytes reaction (MLR). MINOR HISTOCOMPATIBILITY ANTIGENS These are an unknown number of minor antigens encoded by genes at sites other than HLA locus. These minor antigens can induce a weak immune response that can result in slow reaction of a graft. 104 105 106 107 108 109 MINOR HISTOCOMPATIBILITY ANTIGENS •These are an unknown number of minor antigens encoded by genes at sites other than HLA locus. •These minor antigens can induce a weak immune response that can result in slow reaction of a graft. IMMUNE RESPONSE •Occurrence of this response need firstly recognition of the antigen. •Secondly mounting a reaction against this antigen ;These are achieved by innate or adaptive immunity. • there are two types of immune response primary & secondary IR Primary and secondary humeral IR Primary IR :- • Occurs the first time an individual encounters antigen. • Ag presentation is mainly by non B cells. • The lag period to reach maximum Ab concentration is long (up to 10 days). • The peak concentration is low. • The Abs formed are predominantly IgM & have low Ag affinity. 111 Secondary IR: by memory B cells • Ag presentation mostly by B cells. • Short lag period (2-5 days). • Peak concentration is high. • Main Abs are IgG in Lymph nodes and IgA in sub mucosal tissues. • Antigen affinity is high. 113 SECONDARY IMMUNE RESPONSE •This is occurred only for T-cell dependent antigen. • This occurred after a second encounter with the same antigen or a closely related antigens; One month or years after the primary response. •The lag period is typically 3-5 days. important events During the secondary IR A. Heavy chain (isotypes) class switch:- •plasma cells secrete Igs classes that differ from that of the parent cells. • It results from specialized type of DNA arrangement in the heavy chain genes, so that a new CH region is moved to become adjacent to the existing V/D/J Exon by deleting all intervening CH regions on the chromosome. 115 B- Somatic Hypermutation:- • During secondary IR, point mutation in the V/D/J and V/J exons occurs at high rates. • This may result in either increasing or decreasing the affinity of the resulting Igs to their target Ags. • Individual cells that express higher affinity mutants will be selected by their Ags. • This phenomenon is called affinity maturation and is responsible for the high antigen affinity of Igs produced in the secondary IR. 116 117 Heavy chain (isotype) class switch . INNATE IMMUNITY • It is non adaptive natural immunity. • It is not specific for certain pathogen. •It includes skin, mucous membranes, phagocyte and NK cells. ADAPTIVE IMMUNITY •It is highly specific for a particular pathogen. • The immunity improves with successive encounter with the same pathogen (memory cells). • It may be passive or acquired. • It may be humoral immunity, cell mediated or both. Acquired immunity is characterized by 1- High specificity to antigens which are recognized by specific immune cells. 2- Mainly targets peptide antigen derived from foreign protein. 3- Require prior exposure to antigen. 4- Development of immunological memory, that is why primary IR is weak while secondary IR is rapid and strong. 5- Wide Ag diversity. Cont…. Acquired immunity is characterized by Main components of acquired immunity:- 1- lymphocytes: T and B lymphocytes. 2- complement. 3- Macrophages. Lymphocytes:- • B-cells: responsible for the antibody- mediated (humoral) IR. • T-cells: responsible for cell-mediated immunity. • B and T cells combine with antigens in highly specific manner through cell surface antigen receptor (BCR and TCR respectively). Each lymphocyte or a clone of lymphocyte has a unique restricted specificity for an antigen (clonal restriction). • The human body contains About 108 clones of lymphocytes, each has one antigenic specificity. This range of possible specificity is known as primary lymphocyte repertoire. • TCRs only recognize peptide antigen bound to major histocompatibility complex (MHC) molecule on the surface of host cells. • T-cells are divided into 2 types:- 1- T helper (TH, T4) cells, represent 65% of total T cells. Recognize antigen on the surface of antigen presenting cells in association with MHC class II. 2- T cytotoxic (Tc, T8), 35% of T cells, recognize Ag in association with MHC class I. 122 These regions are clustered at the distal portion of the variable domain and contribute to the antigen-binding site (paratope) DOMAIN: A region of a peptide chain having a coherent tertiary structure. Immunoglobulins, TCR, MHC class I and MHC class II molecules have domains. The cumulative effect of several minor antigens can lead to a more rapid rejection response. These are no laboratory tests for detection of minor antigens. HYPER VARIABLE REGIONS:- There are three regions which contain the most variable areas of the variable domains of immunoglobulin and T-cell receptor chains. LYMPHOID CELLS •Lymphocytes are produced in the primary or central lymphoid organs (thymus and bone marrow) •The production rate is high (109 per day). •The size of lymphocytes, usually small (6-10 um). •Some of these cells migrate via the circulation into the secondary lymphoid tissues. lymphoid cells represent about 20% of the total leucocytes present in the adult circulation. The lymphoid tissue as a whole represents about 2% of the total weight. B CELLS represents about 5-15% of the circulating lymphoid pool. They are defined by the presence of surface immunoglobulin marker. They are developed in the bone marrow of the adult. The majority of B cells carry MHC class II antigens. B cells carry also complement receptors for C3b and C3d. It carry Fc receptor for exogenous IgG. CD19, CD20 and CD22 are the main markers currently used to identify human B cells. B cell markers • IgM (BCR), heterodimer Igα Igβ. • CD19, CD73, CD22, CD23. • Class I and II molecules. • CR1, CR2 (C3d R), CD40. • adhesion molecules (LFA-1, ICAM-1). They are the source of antibody development & humoral immunity. B cells do not require the thymus for its maturation. The life span of B lymphocytes are short (day or weeks). CMI Response •CMI is the function of T cells. •T cells recognize Ags by the TCR in the form of peptide fragments bound to class I or II MHC molecules. •The enormous diversity of the T cell repertoire depends on the ability of developing T cells to rearrange and modify their Ag receptors during the process of maturation. Structure of T cell Antigen Receptor (TCR):- •There are 2 types of TCR: 1- TCR-2 formed of 2 peptide chains designate α and β. TCR2- T cells constitute up to 95% of blood T cells. 2- TCR-1, the 2 peptide chains named ү and δ. TCR-1-T cells constitute <5% of blood cells. T CELLS •The ratio of the T cells to B cells is approximately 3:1 • T cells constitute 65-80% of the reticulating pool of lymphocytes. • They are defined by the presence of T cell antigen receptor (TCR) and CD2 molecules. Structure of TCR-2 • The amino terminal domains of α and β (α1 and β2) are pleomorphic and form the antigen binding site. Each clone of T cells has its own TCR that can recognize a particular combination of an Ag and MHC molecule (antigenic diversity). • The carboxyl terminal domains (α2 and β2) are constant. • α/β dimer is associated with a complex of proteins,CD3, formed of 6 peptide chains ү, δ, 2ε and 2 zeta. • CD3 is essential for stability of TCR and for transduction of signal of activation to inside the cell. cont. Structure of TCR-2 • Zeta and ε both have cytoplasmic domains with ITAMs (immune receptor tyrosine-based activation motifs) . • TCR interaction with antigen peptide bound to MHC molecules on APCs initiates ITAM phosphorylation by cytoplasmic protein kinases in T cells. • ITAMs phosphorylation generates a molecular "signal" that is transmitted to the T cell nucleus via an intracellular signal transduction pathway TCR α and β genes and TCR diversity •Genetic locus of α chain is located on chromosome 7 and consists of 100 V segments and 50 J segments for variable domain. There is only one gene for the constant domain. •Genetic locus of β chain is located on chromosome 15 and consists of 30 V segments, 2 D segments and 13 J segments for the variable domain and 2 constant genes. •Possible antigenic diversity of α chain=100х50=5000. •Possible antigenic diversity of β chain=30х2х13=780. •As antigen binding is formed by variable domains of α & β chains together, so the possible Ag diversity=5000х780=3900000. TCR α and β genes and TCR diversity Types of T cells • TCR-2 T cells are divided into 2 subsets:- 1- a subset carries CD4 marker. 2- a subset carries CD8 marker. T helper, T4, cells •Essential for activation of all other immune cells. •Functionally and according to cytokine secretion, TH is divided into types:- 1- TH1, secrete mainly IL2 and IFNy which stimulate phagocytic cells to kill intracellular pathogens and activate TC cells. So, TH1 are essential in immune response against intracellular pathogens as viruses, fungi, some bacteria as MT, Chlamydia, Rickettsia. 2- TH2, secrete IL4, IL5, IL6 and IL10, which stimulate activation, proliferation and differentiation of B cells and production of antibodies. So, TH2 are essential in immune response against extracellular pathogens. cytotoxic, T8, cells • toxic effects on virally infected cells and tumor cells. T cell ontogeny (maturation inside the thymus) •T cells originate from the stem cell in the bone marrow and then travel to thymus gland where they undergo several steps of maturation. Maturation consists of 3 processes; proliferation, expression of Ag R genes, selection of cells expressing useful Ag Rs. • Proliferation of the earliest lymphocyte precursors is stimulated by IL-7 secreted by stromal cells in thymus cortex. • Proliferation of developing lymphocytes maximize the number of cells expressing useful Ag R. • In the cortex of thymus, immature cells acquire CD2 marker, then CD3 together with TCR. At this stage, T cells lack both CD4 and CD8 (double negative thymocytes). • Then T cells express both CD4 and CD8 (double positive cells). • As T cells undergo maturation in medulla, the expression of TCRs increases and the cells lose either CD4 or CD8 and become single positive. Positive & negative selection of T cells & central tolerance. • Inside thymus, T cells learn to recognize Ag in combination with MHC class I or II molecules and to recognize self components as self (tolerance). Cortical epithelia in cortex and macrophages, dendrites cells in medulla are rich in HLA class I & II molecules loaded with self peptides. • Positive selection promotes survival of T cells whose TCRs can recognize peptide bound to self MHC molecules. This occurs in thymus cortex. Here, T cells expressing useful TCRs are selected. • Negative selection leads to deletion of T cells which are auto reactive to self components. This occurs in the medulla. • Mature immunocompetent T cells pass to circulation. T cell response to Antigen & activation •Ag presentation by APCs (macrophages, dendritic cells, Langerhan cells, interdigitating cells, B cells). •Interaction of TCR with its specific Ag (clonal selection). •Role of CD3 in transmitting signal of activation. •Interaction of CD4 or CD8 on TH and TC cells with constant regions of class II or class I MHC on APCs also send signal of activation. •IL1 secreted by macrophages also send a signal of activation. •Co stimulation by interaction of CD28 with B7 and CD40 ligand and CD40. •Role of adhesion molecules: LFA-1 and CD2 molecules on T cells bind to their ligands on APC (LFA-1 interacts with ICAM-1 and -2, CD-2 with LFA-3), promoting cell to cell contact and allowing T cells to survey APC for the appropriate Ag/MHC complex and also send signal of activation. •Role of CD45 ,a tyrosine phosphatase expressed on all leucocytes and is required for TCR signaling. •The final result is the expansion of the T cell clone specific to the activating Ag. T cell response to antigen CMI response and Effectors Functions of T cells 1- Ag presentation by APCs (macrophages, dendritic cells, Langerhan cells, interdigitating cells, B cells). 2- Activation of TH1 cells:- • Activated TH1 secrete IFNy and IL2. IL2 stimulate TH cells, through interaction with IL2 receptors, to proliferate into a clone of antigen specific cells (auto- activation). •A proportion of these cells become effectors cells releasing lymphokines, and the remainder become memory cells. •IL2 & IFNy activate macrophages to kill the intracellular pathogens. •IL2 & IFNy also activate TC and NK cells. 3- Activation of TH2 cells:- •Activated TH2 secrete IL4,5,6,10 and 13. these lymphokines chemoattractant and promote proliferation, differentiation of B cells, mast cells, eosinophils and basophils. •Activated B cells differentiate to plasma cells which secrete Abs to attack extracellular pathogens. •In addition, IL4 promotes B cell class switch to IgE, which bind Fcε receptors on mast cells, eosinophils and promote them to respond to Ags. This is important in IR against large multicellular pathogens as helminthes which can not be engulfed by macrophages. These cells will pour their enzymes to kill these Ags (exocytosis). • TH1 & TH2 cells derive from common precursor T cells, TH0 cell which can secrete both IFNy and IL4. • IL12 secreted by macrophage promote differentiation into TH1 • IL4, secreted by a minor T cells subset, promote differentiation into TH2. 4- Activation of Tc cells (CTLs):- • Respond to Ag in combination with class I MHC which is expressed on all nucleated cells. • Important in defense against virus infection by directly killing virally infected cells. Involved in IR to some intracellular bacteria as MT and Listeria. Play role in IR to tumor by killing malignant cells. Mechanisms of killing target cells 1- Perforin and granzyme effect. 2- Fas-Fas ligand. Regulation of CMI response 1- natural dissociation of antigen. 2- TH1/TH2 cross regulation. TH1 derived IFNy inhibits development of TH2 and TH2 derived IL10 inhibits IFNy production by TH1. 3- activated T cells express an important negative regulator called CTLA4 (common T lymphocyte antigen 4), which is similar to CD28, but unlike CD28, interaction of CTLA4 with B7 on APC inhibits T cell response. 4- Role of T suppressor (TS) cells, which suppress other T cells and also B cells. 5- feed back effect of IL2. TCR1-,y/δ, T cells • The majority of these cells are part of intraepithelial lymphocytes and they express CD8 marker. So, they may have toxic function and may play role in protection of mucosal surfaces. NATURAL KILLER CELLS (NK CELL) •Form about 5-10% of peripheral lymphocytes. • It is large granular lymphocytes. Cont…. Natural Killer Cells • Large granular lymphocytes that originate from BM. • Constitute about 15% of peripheral blood lymphocytes. • Markers:- CD16+ (low affinity Fc receptor), CD56+, but CD3-ve • Express IL2 receptors and proliferate in response to IL2. • Part of innate immunity, naturally kill tumor cells and virally infected cells without prior exposure. Like CTL, use perforins & granzymes to kill target cells. • Normal cells are not killed because inhibitory signals from MHC class (HLA E) inhibit NK activation. In tumor cells and virally infected cells, reduced expression or alteration of HLA E molecules interrupt inhibitory signals, allowing activation of NK cell and lysis. • Antibody-dependent cell mediated cytotoxicity: can kill antibody coated cells. Cytokines secreted by NK cells • IFNy, TNFα, and GMCSF., so have immunoregulatory abilities • Augment CMI response, enhance differentiation of TH toward TH1 and away from TH2. • Enhance production of granulocytes and monocytes. Cont.. NK- cells •They are called natural killer cells because they are active without prior exposure to its target (tumor cells, virus infected cells). • They are not specific for any virus. • They can kill without antibody but antibody enhances their effectiveness by a process called ADCC. • They do not have to pass through the thymus for maturation. Cont. NK- cells They have no immunologic memory or TCR. IL – 2 activated NK cells (LAK cells) are being used for the treatment of certain cancers. The presence of CD56 or CD16 is currently in use as a definitive marker. T cell precursors differentiate into immuno competent T cells within the thymus and acquire TCR, CD4 (helper) or CD8 (cytotoxic). The ratio between helper / cytotoxic 2:1. Helper T cells are divided into Th-1 and Th-2 cells. Th-2 cells help B cells but Th-1 cell help cytotoxic T cells and cell mediated immunity. Th- 1 cells produce IL – 2 and gamma interferon. Activated cytotoxic T cell (CD8) kill virus infected cells, tumor cells and allograft cells through its release of perforins or by induction of apoptosis. General objective: • To understand the role of innate immunity in protection against microbial infections. Specific educational objectives:- 1- understand the characters of innate immunity. 2- describe how does innate immunity recognize microbes. 3- know the components of innate immunity. 4- understand how microbes can evade innate immunity. 5- understand the role of innate immunity in stimulating adaptive immune response. سبحان اهلل Innate immunity is a natural inborn, first barrier to infections Features of the Innate Immunity • Inborn resistance that is present the first time a pathogen is encountered. • It does not require prior exposure. • It provides defense against wide range of microorganisms. • No acquired memory after first exposure. سبحان اهلل Recognition of microbes by the Innate Immune System 1- The components of innate immunity recognize structures that are specific for microbial pathogens and are not found on mammalian cells. Ex:- • LPS of gm-ve bacteria, Teichoic acid of Gm +ve bacteria, mannose-rich oligosaccharides found in microbial but not mammalian glycoprotein and N- formylmethionine which is typical for microbial proteins . • Phagocytes receptors recognize and respond to ds RNA which is found in many viruses but not in mammalian cells . ● molecular pattern and pattern recognition receptors. 2- Innate Immunity can recognize microbial components that are essential for survival and infectivity of the microbes, as LPS and Teichoic acid. So, a microbe cannot evade innate immunity by mutating or not expressing the target of recognition. 3- receptors of Innate Immunity are encoded in the germ line and are not produced by somatic recombination of genes as the receptors of acquired immunity. Therefore, the specificity of adaptive IR is much more diverse than that of innate immunity. 4- innate immunity respond in the same way to repeat exposure to the same microbe (no memory). Components of the innate immunity A- First line of defense:- consists of simple barriers to the entry of the organism inside the body: 1- Skin:- • Intact skin physically prevents microorganism and other potentially injurious agents from entering the tissue beneath. • Lactic acid and fatty acids in the sweat are toxic to many organisms. • Defensins, cysteine-rich peptides present in the skin of mammals and have broad- spectrum antibiotic effect that kill a wide range bacteria and fungi. 2- The epithelial surface (mucus membrane) • Mm lining respiratory and digestive tracts are bathed in a protective layer of mucus which can trap, dissolve and sweep away foreign substances • Epithelial cells produce peptide antibodies that kill bacteria. • Hair lining the mm of anterior nares help in trapping foreign substances which can be expelled by sneezing. • Cilia lining the trachea have a lashing movement to outside which help cleaning the mm. • Mucus membrane of urinary tract is cleansed by flushing of urine. • Intraepithelial T cells and B-1 cells can recognize and respond to microbes. 3- Lysozyme in tears and saliva is bactericidal. 4- acidic pH in stomach and vagina helps in destroying many bacteria. 5- Commensals (normal flora) present on the skin, conjunctiva, at the portal of entry as upper part of respiratory tract, mouth, GIT, genitourinary tract. They suppress the growth of pathogenic bacteria and fungi by:- • Competition for adherence sites and food. • Production of inhibitory substances as acids and colicin. B- Second line of defense: internal resistance factors:- If the organism succeeds to get through the first line of defense and enter blood or tissue, other non-specific defenses will operate:- I- Soluble proteins of innate immunity:- • recognize organisms through CHO or lipid molecules (in acquired immunity, immune cells recognize organisms through specific protein they express). 1- Lysozyme (mucopeptidase): present in all body secretion except CSF and urine. • It can destroy peptidoglycan mainly of gram bacteria. 2- Mannose-binding protein:- binds to sugar mannose in CHO on the surfaces of bacteria, viruses and parasites. Binding results in opsonization and alternative complement pathway activation. 3- C-reactive protein:- binds CHO or lipid determinants on many bacteria leading to opsonization and complement fixation. 4- LPS binding protein:- binds LPS of gm-ve bacteria and also binds to CD14 receptor on macrophages leading to opsonization. COMPLEMENT SYSTEM • The complement system consists of approximately 20 proteins that are present inactive in normal serum of human and other animal. • The term complement refers to the ability of these proteins to complement i.e. augment the effects of other components of the immune system. Cont….. Complement a naturally-occurring, self-regulating system consisting of about 30 proteins (e.g., components C1-C9; factors B, D, H, I) that can be nonspecifically activated by polysaccharides or lipopolysaccharides present on pathogens, including bacteria, fungi, etc. activation results in phagocytosis or lysis of the organism. Cont…. Complement is an important component of innate immunity. C1 is synthesized by intestine but C2, C3, C4 and C5 are synthesized by Macrophages. But C3, C4, C5, C6, C7, C8, C9 are synthesized by liver as the main source of complement. These are 3 main effects of complement:- • lysis of cells such as bacteria, allograft and tumor cells. • Generation of mediators that participate in inflammation and attract Macrophages. •Opsonization – I.e. enhancement of phagocytosis. • Complement is heat labile i.e. it is inactivated by heating serum at 56 0C for 30 minutes ( Antibodies are not inactivated at this temperature). There are two pathway, antigen-antibody complex activate C1 to form a protease which cleaves C2 and C4 to form a C4b2b complex (this is C3 convertase which cleaves C3 molecules into 2 fragments C3a and C3b. سبحان اهلل ACUTE PHASE PROTEIN • Serum proteins whose levels increase during infection or inflammatory reaction. • It includes C-reactive protein, mannose binding protein &fibrinogen. • C-reactive protein so called like that because of its ability to bind to the C-portion of pneumococcai and this binding promotes attachment of complement. Cont….. The acute phase proteins:- • The concentration of the soluble proteins increase 1000 folds during serious infection as a part of protective reaction called hepatic acute phase response. • In this response the liver increases synthesis of these proteins. • This response occurs when hepatocytes are exposed to certain cytokines as IL6, IL1 and TNFα which are produced by macrophages. • One of the most potent inducers of these cytokines is the bacterial LPS. II- Cellular barriers 1- Phagocytes:- Are leucocytes specialized for capture, ingestion and destruction of invading pathogens and foreign particles. There are 2 main types; ☺ polymorph nuclear leucocytes (PNL):- • Represent 60% of blood leucocytes. • Short-lived, die by apoptosis 12 hrs after being released from bone marrow. • Have abundant granules which store bactericidal agents as: Lysozyme, proteinases, collagenases, elastasis, lactoferrin, cathepsin G, defensins. PHAGOCYTES • They are composed from two types macrophages and microphages. • Macrophages are the most important group of long- lived phagocytic cells belong to the mono nuclear phagocyte lineage. • Their function is to engulf particles, including infectious agents. b- oxygen-independent: defensins, digestive enzymes 4- ingestion: of undigested materials by a process that is reverse of ingestion Other function is processing the antigen into a form in which it can be recognized by lymphocytes. Microphages are small phagocytic cells and are called polymorph nuclear neutrophils (PMN). These constitute the majority of the blood leucocytes about 60-70% and are 10-20 m in diameter. 2- ingestion (engulfment) • pseudopodia surround the pathogen, then fuse to form a vacuole called a phagosome • Formation of phagolysosomes by fusion with lysosomes (vacuoles containing a broad spectrum of digestive enzymes). 3- digestion/killing: occurs inside phagolysosomes Killing:→ a- oxygen-dependent • reactive oxygen intermediates - superoxide, hydrogen peroxide and hydroxyl radical - kill microbes directly by oxidizing their nucleic acid and proteins. • myeloperoxidase is activated by hydrogen peroxide to generate hypochlorous acid. POLYMORPHONUCLEAR GRANULOCYTES (PNG) • PNG are produced in the bone marrow at a rate of 80 million per minute and are short- lived (2-3 days), relative to monocytes /macrophages which may live for months or years. • Polymorphs like monocytes can adhere to endothelial cells lining the blood vessels and extravasate by process named diapedesis Recognition of microbes by neutrophils and macrophages Through receptors that recognize microbes: 1- Mannose receptors: macrophage lectins that bind terminal mannose residues of glycoproteins and glycolipids in microbial cell wall. 2- Scavenger receptors: bind a wide variety of polyanions on bacterial surfaces resulting in phagocytosis of bacteria . 3- Receptors for opsonins: Opsonins are various proteins that coat microbes and promote their phagocytosis. Macrophages have receptors for these opsonins: • CD14 which bind LPS binding protein, mannose binding protein receptors, C reactive protein binding receptor. • FcyRI which binds IgG • CR1 which binds C3b 4- Toll-like receptors (TLRs) (Pattern Recognition Receptors or PRRs) : • A number of receptors (about 10) that can recognize different components specific to microbes as LPS, peptidoglycan, bacterial lipoproteins, flagellin, double-stranded RNA of viruses, zymosan in fungi. • Binding TLRs to their ligands leads to phagocytosis and the release of inflammatory cytokines (IL-1, TNF-alpha and IL-6) by the phagocytes. 2- Natural killer (NK) cells:- •NK cells are the first line of defense to virus infection and cancer cells. NK has direct cytotoxic effect on these cells PMG are classified into Neutrophils, Eosinophils and Basophils on basis of the staining reactions of their granules with histological dyes NEUTROPHILS They are constitute over 90% of the circulating polymorphs. C5a, IL-8 and the products of certain bacteria are chemotactic agents for neutrophils. Neutrophils possess two main types of granules. The primary (azurophilic) granules are lysosomes containing acid hydrolases, myeloperoxidase and Lysozyme. The secondary granules contain lactoferrin and Lysozyme. Ingested organisms are present inside vacuoles termed phagosomes which fuse with lysosomes to form phagolysosomes. EOSINOPHILS Eosinophils comprise 2-5% of blood leucocytes. It has ability of phagocytosing and killing. It has only a bilobd nucleus and many cytoplasmic granules. ECF – A from T cell, mast cells and Basophil are attract Eosinophils. They bind worm larvae coated with IgG or IgE degranulation and release a toxic protein (major basic protein). Eosinophils also release histaminase and aryl sulphatase which inactivate histamine and SRS – A. BASOPHILS They are found in very small number in the circulation. They are characterized by deep violet blue granules. MAST CELLS They are not found in the circulation. They are found in the body tissues. There are two types of mast cell mucous member are mast cells and connective tissue mast cells. Mast cells and Basophils are contain granules filled with heparin, SRS –A and ECF – A. The stimulus for degranulation of these cells is usually an allergen. basophil and mast cells are carrying high affinity FC receptor for IgE (FCE RI) CHEMOTAXIS Increase directional migration of cells particularly in response to certain chemotactic factors e.g. C5a ANAPHYLATOXINS Complement peptides (C3a and C5a) which cause mast cell degranulation and smooth muscle contraction. The alternative pathway through many unrelated cell surface substances e.g.endotoxin, fungal cell wall and viral envelop, all these can initiate the process by binding C3 (H2O) and factor B. • This complex is cleaved by a protease factor D to produce C3b Bb. • This acts as a C3 convertase to generate more C3b OPSONIZATION The coating of an antigen or particle e.g. infectious agent by antibody, complement components or fibronectin. This coating facilitate uptake of the foreign particle into a phagocytic cell. Antibody Mediated Immunity Antibody synthesis typically involves the cooperation of 3 cells; macrophages, helper T cell and B cells. After processing by a macrophage, fragments of antigen appear on the surface of the macrophage in associated with class II MHC protein. These molecules bind to specific receptors on the surface of helper T cells which then produce lymphokines . These lymphokines as IL-2 (T cell growth factor), IL-4 (B cell growth factor) and IL-5 (B cell differentiation factor). These factors activate the antigen-specific B cell. The activated B cell proliferates and differentiate to form plasma cells that secrete large amount of immunoglobulins. T CELL INDEPENDENT ANTIGENS Antibody formation usually involves helper T cells. Certain antigens e.g. bacterial polysaccharides can activate B cells directly without the help of T cells. CELL MEDIATED IMMUNITY Cytotoxic T lymphocytes are also specific effectors of the cellular immune response particularly against virus infected cells. Viral envelop glycoprotein appear on the surface of the infected cell in associated with class I MHC Cytotoxic T cell binds via its antigen specific receptors to the viral antigen- class I MHC protein complex. This binding stimulate T cytotoxic cell and IL-2 produced by helper T cells, also stimulate T cytotoxic. These cytotoxic T cells kill specifically this virus infected cells not other virus infected cells. IV- Inflammatory response: • "early-warning" system whose signs include erythema (redness); edema (swelling); fever; pain. It is elicited by injury of tissue and by infection. • Histamine released from mast cells at site of tissue damage, and other inflammatory mediators released by macrophages as IL1, TNFα, leukotrienes, cause dilatation of local arterioles and capillaries with pouring of plasma in the inflamed area (edema). • The edema fluid carries proteins of innate immunity and fibrin. • Inflammatory mediators also induce the expression of adhesion molecules (integrins and selectin) on leucocytes and endothelial cells. These molecules cause leucocytes to adhere to endothelial cells of blood vessels, then migrate out of the capillaries (extravasation) towards the irritant. • Migration of leucocytes in the tissue is stimulated by certain substances present in the inflammatory exudate called chemokines (chemotaxis) as IL 8 secreted by macrophages and endothelial cells. • PNLs phagocytose microorganisms and digest them then undergo apoptosis. • Macrophages engulf leukocyte debris and microorganisms and pave the way for resolution of the local inflammatory process. • The beneficial effect of fever may be that antibody production and T- cell proliferation increase at higher body temperature. • Among the factors capable of inducing fever (pyogenes) are the endotoxin of gm-ve bacteria and IL1 released by macrophages.. Role of Innate Immunity in stimulating Adaptive Immune Response • Innate immune responses generate molecules that function as second signal, together with the antigen (first signal) to activate T & B lymphocytes. A. Macrophages activated by phagocytosis of antigen, express surface molecules, B7. • On presentation of Ag to TH cells by macrophages, B7 molecules interact with TH cell surface molecule CD28 resulting in a signal of activation to TH cells. •Activated macrophages secrete cytokines which can activate T cells:- 1- IL-12 which stimulates the differentiation of naïve T cells to TH1. 2- IL-1 which causes proliferation of TH cells B- Natural activation of complement by microbes leads to formation of some components as C3d. • C3d combine with a complement receptor on B cells (CR2) and this sends an activation signal to B cells. Evasion of Innate Immunity by Microbes Mechanism of immune evasion Ex. Of organism Mechanism Resistant to phagocytosis - Pneumococcai Capsular polysaccharide - Meningococci resists phagocytosis - H. influenza Resistant to reactive O2 radicals in Staphylococci Produce catalase→ breaks phagocytes down H2O2 Resistance to C activation - N. meningitidis - Sialic acid expression inhibit (alternative pathway) C3b binding to organism. - Streptococcus - M ptn blocks C3 binding to organism & C3b binding to C receptor Resistance to killing inside Mycobacterium Lipid in cell wall inhibits phagocytes fusion of phagosome and lysosomes CYTOKINES • These includes both lymphokines from lymphocytes and Monokines from macrophages. • there is no universal accepted definition for it. But for a molecule to be considered a cytokine must show the following features:- • It must be a protein or polypeptide usually glycosylated. • It must be a mediator of a component of inflammation. • It must have no intrinsic chemical or enzymatic activity. • It must bind to specific protein receptors on target cells. • Cytokines may be regarded as true hormones but differ from it as cytokines are biologically active in very small concentration (Picogram). • The target cell for cytokine maybe the cell of origin (autocrine action). • It may act on adjacent cell (Paracrine action). • It may act on distant cell (endocrine action). III- Interferons: - • Proteins of small molecular weight. • 3 types; α, β and y IFNs. • α and β are important in non-specific defense against virus infection. • They are released by any virally infected cell and when taken by other cells, protect them from the same virus and other viruses. • The protection through inducing formation of a translation inhibition protein which inhibits translation of viral genes. Cont… INTERFERONS • Interferon are host-coded proteins that inhibit viral replication. • They are produced by intact animals or cultured cells in response to viral infection or other inducer. • There are three group of interferon IFN-, , . • IFN - is synthesized predominantly by leukocytes. • IFN - is synthesized mainly by fibroblast. • IFN - is synthesized only by lymphocytes. • RNA viruses are stronger inducer of interferon than DNA viruses. • Interferon also can be induced by double- stranded RNA, bacterial endotoxin and small molecules like Tilorone . • IFN - is not produced in response to most viruses but is induced by mitogen stimulation. • Cloned interferon genes are being expressed in large amounts in bacteria and in yeast. • Interferon are produced soon < 48 hours after viral infection in intact animals. • Interferon plays a primary role in the defense of the host against viral infection. • IFN - increase expression of histocompatibility antigens and enhancement of natural killer cell activity. • The cell regulatory activity of IFN - is much greater than IFN - or IFN - . • Interferon are always host species-specific in functions. • Interferon activity is not specific for a given virus. • Interferon are potent so that very small amounts are required for function. • Interferon is not the antiviral agent, rather interferon induces an antiviral state by enhancing the synthesis Of other proteins that inhibit viral replication. • Interferon have some side effects like C.N.S, GIT and bone marrow suppressions. ANTIGEN –Antibody Reaction in the Laboratory • Characters of the reaction between antigen & antibody. It is specific that antibody combine only with the antigen which induced its formation. • This specificity in reaction of antigen an antibodies • An unknown bacterial culture can be identified by reacting it with known antisera (antibody)and vise versa unknown antibody in the serum of patient can be tested by using known bacterial cultures. 2- The observable result of the reaction is largely determined by the physical state of the antigen. ● If the antigen is in the form of particles. The resulting reaction is agglutination. ● If the antigen is in solution the resulting reaction is precipitation. • If the union of antigen and antibody occurs in the presence of a third normal component of serum called complement. The reaction is called complement fixation. • The presence of electrolytes is essential for the reaction to occur physiological saline and thus decreases the repulsion forces between particles, and facilitates their approximation by antibody molecules to form aggregates. • Zone phenomenon:- Observable union between antigen & antibody occurs best when both reactants are present in optimal proportions. No visible reaction occurs in the presence of excess antigen or excess antibody. AGGLUTINATION • There are several types of the agglutination reaction:- Cont….. agglutination Direct agglutination • this direct agglutination of antigen by its specific antibody can be performed in two ways. AGGLUTINATION Cont….. agglutination 1. Slide Agglutination – A drop of known serum is added to one of the saline suspensions of unknown colony and mixed well clumping occurs if the serum is specific to the organisms. Cont….. Agglutination 2-Tube Agglutination:- this is a quantitative test, which is used to determine the amount of Antibodies in the serum of patients. The classical application of the tube method is the widal test for enteric fever, brucellin test for diagnosis of brucellosis and weil felix reaction for typhus fever. Examples: • Identification and typing of Salmonella • Typing of streptococci • Typing of blood grouping • Important Note:– In the diagnosis of such infectious diseases two serum samples separated by 7- 10 days interval should be tested. Arising antibody titer of two or more is diagnostic. B- Ant globulin agglutination test (Coomb's test) • This test is used to determine the presence of Rh incompatibility which causes erythroblastosis foetalis. There are direct Coomb's test for detection incomplete Rh antibodies coating the RBc’s of ne born and indirect Coomb’s test to detect incomplete anti-Rh antibody. C- Passive Agglutination: – ● It is an agglutination reaction in which inert particles e.g. latex or RBC’s are coated with various antigen or antibodies. These particles are aggregated in the presence of specific antibody or antigen respectively. ● Examples of passive agglutination include the following immunologic tests pregnancy test, Rheumatoid factor, C-Reactive protein, anti- streptolysin O (ASO) and TPHA for syphilis. D- Co agglutination (CoA):- • CoA is based on the principle that killed staph aureus rich in protein A on their surface can bind IgG non specifically through the Fc region leaving specific Fab site free able to detect unknown bacterial culture e.g. gonococci or detection of bacterial antigens in body fluids e.g. serum, urine or C.S.F. E-Virus Haemagglutinating:- ● Some viruses e.g. influenza, Para influenza, mumps, adeno and yellow fever viruses can cause agglutination of RBC’s of man, chicken, guinea pig, rat and other animals. ● This reaction is used for the detection and titration of haemagglutinating viruses in culture materials. ● On the other hand inhibition of the haemagglutinating reaction can be used to detect antibodies in serum samples. This is known as the virus haemagglutination inhibition (HAI) test. PRECIPITATION ●This is an antigen antibody reaction in which the antigen is in solution. ● There are many ways of doing the precipitation reaction. ● Tube precipitation for detecting and identifying antigen as in typing of pneumococcai. Agar gel diffusion in this technique diffusion of antigen and antibody is allowed to occur in agar gel. Examples of this are: A. Double diffusion for grouping of streptococci e.g - Elek’s test showing a toxigenic strain of diphtheria. B. Single radial immuno diffusion for determination of IgG concentration. NOTE: Estimation of immunoglobulins is important in immuno deficiency syndromes e.g. agammaglobulinaemic children with very low or no IgG such children are treated by repeated injection of IgG which help to increase their resistance to pyogenic infection. TOXIN ANTITOXIN NEUTRALIZATION When a toxin is mix with the corresponding antitoxin, the toxic effect of toxin is neutralized. in vivo toxin antitoxin neutralization examples are: a. Schick’s test used to determine susceptibility to diphtheria b. in vivo virulence test for diphtheria (guinea pig). In vitro toxin antitoxin neutralization (antistreptolysin o titration) VIRUS NEUTRALIZATION When viruses are added to appropriate cells growing in Tissue culture, they will cause cell destruction called (CPE). Such virus infectivity can be inhibited by virus specific antibodies. IMMUNO FLUORESCENCE These are antigen – antibody reaction in which we use fluorescein labeled antibodies. Fluorescein is a dye which emits greenish fluorescence under .v. Fluorescein can be tagged to immunoglobulin molecules. There are two ways for doing the test ,direct and indirect immuno fluorescence. Direct immunofluorescence to determine if the dog has rabies. Indirect immunofluorescence is used in serologic diagnosis of syphilis to detect antitreponemal antibodies.(FTA). ENZYME linked Immunosorbant Assay (ELISA) The technique is very sensitive and avoid the hazards of radio activity. The method depend on conjugation of an enzyme to either antigen or antibody. Then the enzyme activity on a substrate is used as a quantitative measure. Solid-phase ELISA is widely used to measure antigen or antibody. The usual enzyme used is horse -radish perioxidase. To measure antibody the indirect method is used. To measure an antigen, the double antibody technique is used. Assessment Of Immune Competence Assessment of T and B cells competence, as well as phagocytic cell functions and complement are important in determining the immune status of individuals especially in the following :- Chronic infection Immunodeficiency syndromes Patient on immunotherapy e.g. Cancer patients. Assessment Of T Cell Competence This is through:- Enumerating the number of T cell using monoclonal antibodies or E rosette. Evaluation of T cell function using delayed hypersensitivity skin tests. Assessment Of B Cell Competence This is through:- Enumerating the number of B cell using polyclonal antibodies and monoclonal antibodies or by EA or EAC rosettes Evaluation of B cell functions using lymphocyte transformation, determination of the level of immunoglobulins by protein electrophoresis or quantitative of the different types of immunoglobulins by radial immuno diffusion, and lastly by active immunization and test the result in vivo. Assessment Of Phagocytic Functions This is through:- Assessment of chemo taxis. Assessment of ingestion or intracellular killing using nitro blue tetrazolium (NBT) reduction test. Assessment Of Complement This is through:- Measuring the quantity of the different components of the complement in the serum e.g. C3, C4 using radial immuno diffusion. Measuring the total hemolytic activity of the complement. Thank You The serum antibody concentration continues to rise for several weeks then declines. The first antibodies to appear are IgM followed by IgG or IgA. IgM levels decline earlier than IgG levels.