1. Humoral Immunity & Immunoglobulin Structure and Function Dr. Adel Almogren

2. 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.

7. B cell activation T-independent antibody response generally have 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

9. 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 Domain 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 

10. CH3

11. CH2

12. CH3 CH2 CH1

13. CH3 CH2 CH1 VH1

14. CH3 CH2 CH1 VH1 CL

15. CH3 CH2 CH1 VH1 CL VL

16. CH3 CH2 CH1 VH1 CL VL

17. Hinge CH3 CH2 CH1 VH CL VL Elbow

18. Fb Fv CH3CH3 CH2CH2 Fb Fv Hinge Elbow CH3CH3 CH2CH2 Fb Fv Flexibility and motion of immunoglobulins

19. 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)

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

21. 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

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

23. Structure and function of the Fc region CH3CH3 CH2CH2 IgA IgD IgG CH4CH4 CH3CH3 CH2CH2 IgE IgM The hinge region is replaced by an additional Ig domain 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

24. Monomeric IgM IgM only exists as a monomer on the surface of B cells Monomeric IgM has a very low affinity for antigen 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

25. C C C C C C Multimerisation of IgM 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

26. 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

27. IgD facts and figures ??IgD & IgM ?? 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

28. IgA dimerisation and secretion 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, 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

29. 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 Polymeric Ig receptors are expressed on the basolateral surface of epithelial cells to capture IgA produced in the mucosa

30. 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

31. 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

32. 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: IgG1++IgG2 + IgG3 ++IgG4 ++ The neonatal Fc  receptor may be responsible!

33. 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

34. 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)

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

36. Antibody Dependent Cell Mediated Cytotoxicity (ADCC) 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.