Humoral Immune Response. Dr. Iman Hussein Shehata Professor of Medical Microbiology and Immunology

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1 Humoral Immune Response Dr. Iman Hussein Shehata Professor of Medical Microbiology and Immunology

2 Intended Learning Outcomes By the end of this lesson the student is expected to: 1-Decribe the sequence of B cell activation 2-Describe the structure of immunoglobulin molecule. 3-List different isotypes of immunoglobulins 4-Compare between different isotypes of immunoglobulins as regards structure, serum concentration and function

3 Memory B cells Some activated B cells form memory cells, which can remain quiescent for long periods but are capable of being activated rapidly upon re- exposure to antigen Most memory B cells have surface IgG that serves as the antigen receptor. The presence of these cells explains the rapid appearance of antibody on second exposure to the same antigen

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5 Immunoglobulins The simplest antibody molecule is Y shaped, formed of four polypeptide chains: two heavy chains (H) and two light chains (L) chains. The four chains are linked by disulfide bonds. The light chain consists of one variable (VL) and one constant (CL) domain. The heavy chains consist of one variable (VH) and three constant (CH) domains.

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9 Change in amino acid sequence of the constant region of heavy chain gives 5 types of heavy chains; classifying immunoglobulins into 5 isotypes: The immunoglobulin isotype is named according to the heavy-chain isotype present IgG with γ heavy chain IgM with µ heavy chain IgE with heavy chain IgA with α heavy chain IgD with δ heavy chain

10 Heavy chain Isotype (Class Switching) Helper T cells stimulate activated B lymphocytes to produce antibodies of different heavy chain isotypes ( classes) Different antibody isotypes perform different functions so broadens the functional capabilities of humoral immune response. This is induced by a combination of CD40L mediated signals and cytokines

11 Role of cytokines in Class switching Cytokines produced by T helper and T Tfollicular helper T cells determine which heavy chain isotype is produced. Opsonizing IgG antibodies is stimulated by IL-10 and others (IFNγ and IL-4) Switching to IgE is stimulated by IL-4 Switching to IgA is stimulated by IL-5 anf TGF-β

12 The antibody isotype produced is also influenced by the site of immune responses IgA isotype is the major antibody in mucosal lymphoid tissues.

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15 The variable regions of both the light and heavy chain (antigen- binding fragment 2 Fab ends) are responsible for antigen-binding

16 Hyper variable regions In the variable region of both L and H chains there are 3 extremely variable regions that form the antigen binding site. The hypervariable regions form the region complementary in structure to the antigenic determinant of the epitope. These regions are involved in the formation of the paratope.

17 The paratope is the area of the immunoglobulin molecule that interacts specifically with the epitope. It is created by folding of the polypeptide chains which bring the hypervariable regions of the VH and VL domains into close proximity,resulting in a three dimensional structure that is complementary to the epitope

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21 whereas the constant region of the heavy chain (crystallizable fragment Fc portion) is responsible for various biologic functions of immunoglobulin molecule e.g., CH2 domain contains the complement binding site, CH3 domain is the site of attachment of IgG to receptors on neutrophils and macrophages.

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23 Idiotypes Antigen binding sites classify different immunoglobulins into different idiotypes. Each idiotype is unique for one antigen and produced by a specific clone of B cell.

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25 Immunoglobulins isotypes Immunoglobulin G (IgG) IgG is the most abundant class in serum, constitutes about 80% of the total serum immunoglobulin. There are 4 subclasses of IgG (IgG1- IgG2- IgG3- IgG4) due to the presence of 4 subtype of γ chain

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27 Biological activities of Ig G IgG is the most important class of immunoglobulin in secondary immune responses. It diffuses into extracellular spaces and neutralizes bacterial toxins as well as viruses fixes complement (IgG3 is the most potent complement activator). IgG1, 3, 4 cross the placenta and play an important role in protecting the fetus IgG is an opsonin with high affinity to Fc receptors on phagocytes

28 Immunoglobulin M (IgM) IgM is expressed in two forms Monomeric IgM : membrane bound antibody on B cells (B cell receptor). Pentameric IgM accounts for 5% 10% of the total serum immunoglobulin

29 Pentameric IgM contains the J chain five IgM monomers linked together via disulfide bonds. The J chain is a glycoprotein that links two of the 5 IgM Fc regions together

30 IgM is the first immunoglobulin class produced in a primary response to an antigen. IgM does not cross the placenta it is also the first immunoglobulin to be synthesized by the neonate.

31 raised IgM level in neonate indicates intrauterine infection

32 Because of its pentameric structure with 10 antigen-binding sites, serum IgM has a higher valency (higher number of binding sites) than the other isotypes. So, it is the most efficient immunoglobulin in agglutination, complement fixation (activation), Because of its large size, IgM does not diffuse well and therefore is found in very low concentrations in the intercellular tissue fluids.

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35 Immunoglobulin D (IgD) IgD constitutes about 0.2% of the total immunoglobulin in serum. IgD, together with IgM, is the major membrane bound immunoglobulin expressed by mature B cells, it acts as antigen receptor (B cell receptor)

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37 Immunoglobulin E (IgE) IgE concentration in serum is very low. IgE binds to Fc receptors on the membranes of eosinophils, basophils and mast cells. Cross-linkage of receptor bound IgE molecules by antigen (allergen) induces these cells to release their granular contents into the extracellular environment, a process known as degranulation giving rise to allergic manifestations.

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39 IgE is the main host defense against helminthes. IgE specific for worm proteins binds to receptors on eosinophils, causing eosinophils to release content of granules to kill the worm (antibody dependent cell mediated cytotoxicity). IgE does not fix complement or cross the placenta.

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41 Immunoglobulin A (IgA) Although IgA constitutes only 10% 15% of the total immunoglobulins in serum. it is the predominant immunoglobulin class in external secretions such as breast milk, saliva, tears, and mucus of the bronchial, genitourinary, and digestive tracts.

42 Secretory Ig A Secretory IgA is a dimer, which consists of a single short polypeptide J chain synthesized by submucosal plasma cells joined to two IgA monomeric subunits and a secretory piece. This secretory piece facilitates transport of secretory IgA across mucosal epithelium protects it from digestion by proteolytic enzymes

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45 The main functions of secretory IgA is to provide local immunity at mucous surfaces; by coating microorganisms, viruses or bacteria preventing their adherence to mucosal cells, thus preventing their entry into body tissues;

46 IgA in colostrum and milk protects the new born from infections.

47 IgA does not fix the complement and does not cross the placenta. There are 2 subclasses of IgA due to the presence of 2 types of α chain: IgA1 can be inactivated by an IgA protease produced by Neisseria, Pneumococci and H. influenza IgA2 more important in mucosal immunity against such pathogen

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50 Antibody mediated effector functions 1- Neutralization of toxins and viruses.

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54 Antibody mediated effector functions 2- Opsonization of microorganisms: antibodies coat microorganisms and render them more easily ingested by phagocytic cells; The Fc portion of IgG interacts with its receptors on the phagocyte surface to facilitate ingestion

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56 3-Complement activation through the classical pathway (IgM & IgG only).

57 Complement activation and ADCC

58 4-Antibody dependent Cellular Cytotoxicity (ADCC) a mechanism whereby an effector cell of the immune system actively lyses a target cell that has been bound by specific antibodies. Classical ADCC is mediated by NK cells, neutrophils, macrophages and eosinophils (perform ADCC against helminthes). These cells have specific receptors for the constant region of IgG (Fc γ)

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61 Primary and secondary immune responses The first contact of an exogenous antigen with an individual generates a primary humoral response. Second contact with the same antigen generates secondary immune response.

62 Main differences between 1ry and 2ry immune response Primary Immune Response Secondary Immune Response Responding B Cell Naive Memory cell Lag period following exposure to antigen Antigen Antibody Main Isotype produced Level of antibody 4-7 days 1-3 days 1-Thymus dependent and independent 2- High concentration is needed IgM Low 1-Thymus dependent only 2-Relatively low concentration IgG High

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64 These are highly specific antibodies produced against a single epitope by cells derived from a single clone of immortal cells. They are obtained by fusion of myeloma cell (malignant plasma cell) with a B cell producing antibody against a single epitope(derived from the spleen of mice immunized with this epitope)

65 The resulting cell called hybridoma acquires the property of being immortal from the myeloma cell and the single antibody specificity from the B cell. It can produce unlimited quantities of a highly specific monoclonal antibody. Monoclonal antibodies have many diagnostic and therapeutic applications

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67 Monoclonal antibodies have many diagnostic and therapeutic applications

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