1. Humoral Immunity (immunity mediated by antibodies) & Immunoglobulin (antibody) Structure and Function Dr. Adel Almogren

2. Immune system : 1 – Innate 2 – Adaptive : A- Humoral (B-cell) B – Cell Mediated (T-cell) If the antibody level that is specific for a certain disease decreased, usually person will experience that disease again.

3. Humoral () Immunity Humoral (Antibody-Mediated) Immunity + Involves production of antibodies against foreign antigens. + Antibodies are produced by a subset of lymphocytes called B cells. + B cells that are stimulated will actively secrete antibodies and are called plasma cells. + Antibodies are found in extracellular fluids (blood plasma, lymph, mucus, etc.) and the surface of B cells. + Defense against bacteria, bacterial toxins, and viruses that circulate freely in body fluids, before they enter cells. + Also cause certain reactions against transplanted tissue.

4. How Do B Cells Produce Antibodies? u u B cells develop from stem cells in the bone marrow of adults (liver of fetuses). u u After maturation B cells migrate to lymphoid organs (lymph node or spleen). u u Clonal Selection : When a B cell encounters an antigen it recognizes, it is stimulated and divides into many clones called plasma cells, which actively secrete antibodies, and memory B cells u u Each B cell produces antibodies that will recognize only one antigenic determinant. only one antigenic determinant.. Maybe there are more than one antigen on a same surface of the foreign body, so for each one we produce specific antibody.

7. Both T cell, B cell have.. 1- effector, 2- memory Macrophages will ingest the antigen and break it and take the antigenic part of it. It will produce it through it’s surface to Th cell. The Th cell will be activated against this antigen and produce (secrete) chemicals that is going to stimulate B cell to produce antibody.

8. Antibody Production The activation of B cell to produce antibody is through: T T - - Dependent Antigens: Dependent Antigens (Th cell): u u Antibody production requires assistance from T helper cells. u u A macrophage cells ingest antigen and presents it to T H H cell. u u T T H H cell stimulates B cells specific for antigen to become plasma cells. u u Antigens are mainly proteins on viruses, bacteria, foreign red blood cells, and hapten - - carrier molecules. T T - - Independent Antigens: Independent Antigens (the antigen it self): u u Antibody production does not require assistance from T cells. u u Antigens are mainly polysaccharides or lipopolysaccharides with repeating subunits (bacterial capsules). u u Weaker immune response than for T - - dependent antigens.

9. B cell activation T-independent antibody response generally have )important) 1. no memory 2. no isotype switching 3. no somatic mutations Thymus-dependent because T cells are required Thymus-independent because T cells are not needed Some responses require T help whereas other do not

10. Antibody – Antigen interaction: agglutination (eg. Blood group test) precipitation of soluble antigen (like agglutination but for very small antigen)

11. CLCL VLVL S S S S S S S S CH3CH3 CH2CH2 CH1CH1 VHVH Fc Fab F(ab) 2 Domains are folded, compact, protease resistant structures 2 identical light chains (L) small 2 identical heavy chains (H) large Domain (quaternary structure) Structure of Immunoglobulins Pepsin cleavage sites - 1 x (Fab) 2 & 1 x Fc Papain cleavage sites - 2 x Fab 1 x Fc Light chain C domains  or Heavy chain C domains  or 

12. Antibodies are Y shaped (upper part of that Y are hypervariable area which binds to an antigen). The one molecule of antibody has 4 peptide chains : 2 heavy (large) chains + 2 light (small) chains. Each light chain binds to heavy chain by (disulfide bond) and by (uncovalent interaction), also disulfide bond connect the parts of antibody structure. The (Fab) will bind to an antigen, but the (Fc) will bind to effector cells like macrophages. The (V H ) or (the variable area) is part of antibody determines the specificity of antibody, so it differs among the types of antibodies.

13. The segments with highly variable sequence are called V regions (V L and V H ). The constant regions are called C regions (C L and C H ). The chemical content of antibody is glycoprotein.

14. CH3

15. CH2

16. CH3 CH2 CH1

17. CH3 CH2 CH1 VH1

18. CH3 CH2 CH1 VH1 CL

19. CH3 CH2 CH1 VH1 CL VL

20. CH3 CH2 CH1 VH1 CL VL

21. Hinge CH3 CH2 CH1 VH CL VL Elbow

22. Fb Fv CH3CH3 CH2CH2 Fb Fv Hinge Elbow CH3CH3 CH2CH2 Fb Fv Flexibility and motion of immunoglobulins There are 2 movements.. Small by elbow, and large by Hinge. Elbow : more back and forward. (between Fv and Fb). Hinge : more horizontal. (between Fb and CH 2 )

23. Hypervariable regions FR1FR2FR3FR4CDR2CDR3CDR1 Amino acid No. Variability 80 100 60 40 20 406080100120 Most hypervariable regions coincided with antigen contact points - the COMPLEMENTARITY DETERMINING REGIONS (CDRs) Y

24. Hypervariable CDRs are located on loops at the end of the Fv regions

25. The sequences of the hypervariable loops are highly variable amongst antibodies of different specificities Variable amino acid sequence in the hypervariable loops accounts for the diversity of antigens that can be recognised by a repertoire of antibodies Hypervariable loops and framework: Summary

26. Antibody + complement- mediated damage to E. coli Healthy E. coli Electron micrographs of the effect of antibodies and complement upon bacteria

27. Structure and function of the Fc region CH3CH3 CH2CH2 IgA IgD IgG … Nothing in hinge Doesn’t contain C H 4 CH4CH4 CH3CH3 CH2CH2 IgE IgM The hinge region is replaced by an additional Ig domain C H 4 … it contain C H 4 Fc structure is common to all specificities of antibody within an ISOTYPE (although there are allotypes) The structure acts as a receptor for complement proteins and a ligand for cellular binding sites IgM and IgD work As receptors on B cell surface

28. Monomeric IgM onlymonomer IgM only exists as a monomer on the surface of B cells pentamerichexameric Monomeric IgM has a very low affinity for antigen.. IgM is monomeric when it is on B cell surface, but when it is released in the circulation it is either pentameric or hexameric. C4C4 C3C3 C2C2 C1C1 N.B. Only constant heavy chain domains are shown IgM forms pentamers and hexamers

29. C C C C C C Multimerisation of IgM (pentameric) C4C4 C3C3 C2C2 C C C4C4 C3C3 C2C2 C C C4C4 C3C3 C2C2 C C C4C4 C3C3 C2C2 C C C4C4 C3C3 C2C2 C C ss ss ss C C ss IgM is the strongest and largest antibody. It has 10 light chains and 10 heavy chains. It has 5 subunit and 10 (Fab)s. J chain is a bond that connects two or more antibody together.

30. IgM facts and figures Heavy chain:  - Mu Half-life: 5 to 10 days % of Ig in serum:10 Serum level (mgml -1 ): 0.25 - 3.1 Complement activation:++++ by classical pathway Interactions with cells: Phagocytes via C3b receptors Epithelial cells via polymeric Ig receptor Transplacental transfer: No Affinity for antigen:Monomeric IgM - low affinity - valency of 2 Pentameric IgM - high avidity - valency of 10

31. IgD facts and figures ??IgD & IgM ?? Both are maturation markers of B cells Heavy chain:  - Delta Half-life: 2 to 8 days % of Ig in serum:0.2 Serum level (mgml -1 ): 0.03 - 0.4 Complement activation:No Interactions with cells: T cells via lectin like IgD receptor Transplacental transfer: No

32. IgA dimerisation and secretion mucus memberane IgA is present in mucus memberane. IgA is the major isotype of antibody secreted at mucosal surfaces Exists in serum as a monomer, but more usually as a J chain- linked dimer (2 antibodies connected together by J chain), that is formed in a similar manner to IgM pentamers. J C C S S S S C C S S S S C C ss IgA exists in two subclasses IgA1 is mostly found in serum and made by bone marrow B cells IgA2 is found in higher concentration in mucosal secretions, colostrum

33. Epithelial cell J C C S S S S C C S S S S C C ss Secretory IgA and transcytosis B J C C S S S S C C S S S S C C ss J C C S S S S C C S S S S C C ss J C C S S S S C C S S S S C C ss pIgR & IgA are internalised ‘Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR - the secretory component J C C S S S S C C S S S S C C ss IgA and pIgR are transported to the apical surface in vesicles B cells located in the submucosa produce dimeric IgA (because the [plag] receptors specific for dimer IgA). Polymeric Ig receptors are expressed on the basolateral surface of epithelial cells to capture IgA produced in the mucosa 1 2 3

34. IgA facts and figures Heavy chains:  1  or  2 - Alpha 1 or 2 Half-life: IgA1 5 - 7 days IgA2 4 - 6 days Serum levels (mgml -1 ): IgA1 1.4 - 4.2 IgA2 0.2 - 0.5 % of Ig in serum:IgA1 11 - 14 IgA2 1 - 4 Complement activation:IgA1 - by alternative and lectin pathway IgA2 - No Interactions with cells: Epithelial cells by pIgR Phagocytes by IgA receptor Transplacental transfer: No

35. IgE facts and figures its role in protecting against parasitic infections IgE is also closely linked with allergic diseases Heavy chain:  - Epsilon Half-life: 1 - 5 days Serum level (mgml -1 ): 0.0001 - 0.0002 % of Ig in serum:0.004 Complement activation:No Interactions with cells: Via high affinity IgE receptors expressed by mast cells, eosinophils, basophils and Langerhans cells Via low affinity IgE receptor on B cells and monocytes Transplacental transfer: No

36. IgG facts and figures Heavy chains:  1  2  3  4 - Gamma 1 - 4 Half-life: IgG1 21 - 24 days IgG2 21 - 24 days IgG3 7 - 8 days IgG4 21 - 24 days Serum level (mgml -1 ): IgG15 - 12IgG2 2 - 6 IgG3 0.5 - 1IgG4 0.2 - 1 % of Ig in serum:IgG145 - 53IgG2 11 - 15 IgG3 3 - 6IgG4 1 - 4 Complement activation:IgG1+++ IgG2 + IgG3 ++++ IgG4 No Interactions with cells: All subclasses via IgG receptors on macrophages and phagocytes Transplacental transfer Transplacental transfer: IgG1++IgG2 + IgG3 ++IgG4 ++ The neonatal Fc  receptor may be responsible! (in animals not in humans).

37. IgG : Has more capability to activate the complements. hydrops fetalis The only antibody that can cross the placenta and may cause hydrops fetalis

38. Carbohydrate is essential for complement activation Subltly different hinge regions between subclasses accounts for differing abilities to activate complement C1q binding motif is located on the C  2 domain

39. Fc  receptors ReceptorCell typeEffect of ligation Fc  RI Macrophages Neutrophils, Eosinophils, Dendritic cells Uptake, Respiratory burst Fc  RIIA Macrophages Neutrophils, Eosinophils, Platelets Langerhans cells Uptake, Granule release Fc  RIIB1 B cells, Mast CellsNo Uptake, Inhibition of stimulation Fc  RIIB2 Macrophages Neutrophils, Eosinophils Uptake, Inhibition of stimulation Fc  RIII NK cells, Eosinophils, Macrophages, Neutrophils Mast cellsInduction of killing (NK cells)

40. Hinge Fv Fb Fab CH3 CH2 CH1 VH1 CL VL Fc Elbow Carbohydrate

41. may cause Myasthenia gravis Antibody Dependent Cell Mediated Cytotoxicity (ADCC) – may cause Myasthenia gravis - Target cell is covered with antibodies, leaving Fc portion sticking outwards. Natural killer and other nonspecific cells that have receptors for Fc region are stimulated to kill targeted cells. Target organism is lysed by substances secreted by attacking cells. Used to destroy large organisms that cannot be phagocytosed.