1. Immunoglobulins, immune response Martin Liška

2. 1. The structure of immunoglobulins

4. 2. Isotypes (in principle) classes of antibodies distinguished on the basis of H chain structure differences 5 types:  (IgM),  (IgD),  (IgG),  (IgA) and  (IgE) in addition, we can distinguish subtypes of antibodies within some classes (IgG, IgA) based on their H chain differences

5. 3. Domains and their biological function in principle: domains of V regions form a recognizing unit and domains of C regions determine secondary biological functions of antibody (i.e. biological half life, distribution in the body, binding complement, binding to cells through Fc- receptor)

6. 4. Variable region of Ig molecule hypervariable loops are concentrated at the spikes of variable regions where antigen binding sites are localized the binding site specifity is determined by amino acid sequences and both by morphology and shape of the loop

7. 5. The biological features of distinct Ig classes IgG the most abundant serum Ig the most important Ig of secondary immune response the only Ig which passes through the placenta the main opsonizing Ig activates complement via classical pathway biological half life 21 day

8. IgA found both in serum and seromucinous secretions defense of mucosa opsonization does not activate complement

9. IgM in pentamer form is found in serum; in monomer form is bound on membrane of B cells prevailing antibody of primary immune response high-effective agglutinant and cytolytic agent usually isohaemagglutinins and natural antibodies

10. the best classical way complement activator does not bind phagocytes Fc receptor, but substantially enhances phagocytosis through complement activation biological half life 6 days

11. IgD free form in serum, bound on B cells membrane antigen receptor on B cells

12. IgE in normal conditions low amounts in serum mainly bound on mast cells (binds through Fc  R) anti-helminth defense immediate type allergic reactions

13. Ig subclasses differences in H chain structure and biological properties IgG 1 a IgG 3 participate in defence against viral and bacterial protein antigens IgG 2 ensures defence against antigens which does not require help from T lymphocytes

14. 6. Allotypic and idiotypic variations allotypes = allelic variants of isotypes idiotypes = structural determinants localized in variable region connected with the ability of antigen binding idiotopes = unique set of antigenic determinants (epitopes) of variable portion of an antibody (idiotype is the sum of idiotopes) anti-idiotypic antibodies = directed against idiotypes, in principle reflect an antigen

15. 7. Genetic basis of Ig production a/ L chains genes  chain – genes located on chromosome 2 - V, J and C segments chain – encoded in similar complex of genes on chromosome 22

16. b/ genes encoding H chain more complicated localized on chromosome 14 V, D, J, C segments (genes encoding individual segments contain more regions compared with L chains) during completion of V/D/J exon, gene rearrangement occurs

18. Development of B lymphocytes Lymphoid progenitor → pro-B cells During maturation from pro-B cells into pre-B cells: Ig genes of the heavy chain recombine; pre-B cells express pre-BCR During maturation from pre-B cells into B cells: Ig genes of the light chain recombine Immature B cells express membrane IgM Mature B cells express membrane IgM and IgD = BCR and are able to respond to antigen in peripheral lymphoid tissues

19. Mechanisms contributing to antibody diversity: chance recombinations imprecise joining of V, D, J genes N-region additions extensive mutations involving variable- region genes after antigen exposure

20. Isotype switching during the immune response, plasma cells switch from producing IgM to IgG or to another Ig class (IgA, IgE) the switch involves a change in the H-chain constant domains (C H ) no changes in antigen-binding specifity ! (no alteration in the L chain or in the variable portion of H chain)

21. Allelic exclusion once the process of rearrangement on one of chromosomes is successful, then all attempts on second chromosome are stopped the same rule governs both for H- and L-chains every single B cell produces only one type of H- and one type of L-chain

22. Clonal restriction each B cell expresses identical copies of an antibody that is specific for single epitope when a B cell divides, the chromosomes in its progeny cells bear the selected allelic genes, and these genes do not undergo any further V/J or V/D/J rearrangements immunoglobulins produced by given B cell and its progeny are identical in epitope specifity and in  or  chain isotype

23. Clonal expansion proliferation of lymphocytes activated by reaction with an antigen all lymphocytes of generated clone have the identical antigenic specifity

24. Monoclonal antibodies immunoglobulins arising from a single clone of B cells, or more precisely cells artificially created by hybridisation of B lymphocytes of specific antigenic specifity (= produced Ig have the same antigenic specifity) with tumor cell (= cells are „immortal“)

25. The utilization of monoclonal antibodies: Diagnostics (flow cytometry, ELISA, autoantibodies etc.) Treatment (anti-IgE, anti-TNF- , anti-CD3)

26. Humoral immune response The recognition of antigen by specific Ig on the surface of naive B lymphocyte The binding of antigen cross-links Ig receptors of specific B cells and then activation signals are delivered inside the B cell; the necessary second signal is provided by a breakdown product of the complement protein C3 Clonal expansion of B cell and secretion of low levels of IgM

27. Humoral immune response Protein antigens activate antigen-specific T helper cells which stimulate B cell; antigen presentation of these antigens to T helper cells is required T helper cells exprime CD40L on their surface and secrete cytokines → proliferation and differentiation of antigen- specific B cells, isotype switching Affinity maturation = affinity of antibodies for protein antigens increases with prolonged or repeated exposure to the antigens (B cells migrate into follicles and form germinal centers → proliferate rapidly and their Ig V genes undergo extensive somatic mutations; at the same time, the antigen complexed with secreted antibody is displayed by FDC → B cells that recognize the antigen with high affinity are selected to survive)

28. Phases of humoral immune responses

29. Primary immune response First antigen exposure The amounts of antibody produced is smaller 2 types of antigens: T-dependent – help from antigen-specific T helper cells is required; protein antigens T-independent – antibody production is induced directly, without the involvement of T helper cells; typically polysaccharides, lipids

30. Secondary immune response Subsequent antigen exposure Higher amount of antibodies is produced With protein antigens, secondary responses show increased isotype switching and affinity maturation (= production of antibodies with increased affinity to antigen) Memory cells involvement

32. Affinity and avidity of antibodies affinity = the strength of the binding between a single binding site of a molecule (e.g.antibody) and a ligand avidity = expresses the strength of interaction of polyvalent antibody with a polyvalent antigen

33. Effector functions of antibodies Neutralization of microbes and their toxins Opsonization of microbes (binding to phagocytes through Fc-receptors, stimulation of their microbicidal activity) ADCC (Antibody-dependent cell-mediated cytotoxicity) – microbe, which is opsonized by IgG, is killed by NK-cell after binding of immunocomplex to Fc-receptor Complement system activation (classical pathway)

34. Ontogenesis of immune response a/ prenatal

35. Hematopoiesis Mesoblast – from 2nd (3rd) week of gestation Liver – from 6th (8th) week of gestation, in liver hematopoiesis persists whole prenatal period Bone marrow – from 10th (12th) week of g., from 20th week the main organ of hematopoiesis

36. T lymphocytes Precursors from week 7, from week 8-9 lymphocytes move into thyme, where they differentiate TCR gene segments rearrangement, expression of TCR on the surface of T lymphocytes Selection

37. B lymphocytes Precursors from day 8 Fetal B lymphocytes express IgM on their surface Synthesis of specific antibodies start at week 20-24, but IgA+M levels are in fact undetectable, IgG production starts after birth

38. Monocytes-macrophages Macrophages can be detected by week 3-4 Mature monocytes appear by month 5 in fetal circulation

39. Neutrophils Mature cells are detectable from week 12- 14

40. Postnatal B lymphocytes relative counts decrease after the birth respond to immunization presumably by IgM production, switching to other isotypes is slower slow increase of child’s own IgG connected with decrease of maternal IgG levels (by month 3-6) IgM reaches levels common i adults at the age of 1-3 yr., IgG+A between the age of 10-15 yr. Humoral response to polysaccharide antigen arises by the age of 2 yr.

41. T lymphocytes More than 90% are naive, but their numbers decrease in adult age Proliferation under mitogen stimulation similar to adults X response to specific antigens only after contact with them Lower cytotoxic activity of T lymphocytes

42. Innate imunity Newborns´phagocytes have generally decreased functional ability, activity of NK- cells is decreased Decreased total complement activity (concentration of its compounds is of 35- 70% of adults)

43. c/ Old age decreased cytotoxicity of NK-cells and macrophages decreased resistance against viral infections, decreased anti-tumour immunity switching from Th1 to Th2 weaker humoral response under new stimuli increased production of autoantibodies