1. Humoral Responses to Infection 2015 MICROBIOLOGY & INFECTIOUS DISEASES Lecture 10 Ikuo Tsunoda, MD, PhD Associate Professor Department of Microbiology and Immunology itsunoda@hotmail.comitsunoda@hotmail.com, http://tsunodalaboratory.w eb.fc2.com/ http://tsunodalaboratory.w eb.fc2.com/ itsunoda@hotmail.com http://tsunodalaboratory.w eb.fc2.com/ July 22, 2015 https://www.facebook.com/pages/Tsunoda- laboratory/406138889435131?fref=nf

2. Objectives Structures of immunoglobulins and their proteolytic digestion Structures of immunoglobulins and their proteolytic digestion Difference in biologic properties among antibody classes Difference in biologic properties among antibody classes VDJ recombination and class switching of immunoglobulin VDJ recombination and class switching of immunoglobulin Transfer of IgG from mother to fetus (via FcRn) Transfer of IgG from mother to fetus (via FcRn) Mucosal secretion of IgA (via poly-Ig receptor) Mucosal secretion of IgA (via poly-Ig receptor) Primary and secondary immunoglobulin responses Primary and secondary immunoglobulin responses Effector functions of antibody Effector functions of antibody To understand;

3. Humoral immune responses “Antibody” = immunoglobulin is the primary component of the humoral immunity “Antibody” = immunoglobulin is the primary component of the humoral immunity “Antigen” = a molecule recognized by antibody or T cell “Antigen” = a molecule recognized by antibody or T cell “Epitope” is the molecular structure in an antigen that interacts with antibody or T cell “Epitope” is the molecular structure in an antigen that interacts with antibody or T cell Proteins (best antigen) >> carbohydrates (weak) >> lipids, nucleic acids (poor antigen) Proteins (best antigen) >> carbohydrates (weak) >> lipids, nucleic acids (poor antigen)

4. Antibodies are composed of polypeptides with both variable and constant regions The immunoglobulin G (IgG) molecule Hinge Note: 2 identical heavy chains (green) 2 identical light chains (yellow) Note: Location of the variable (red) and constant (blue) regions in the antibody molecule.

5. The immunoglobulin constant (C) regions    Any of two identical light chains can pair with any two identical heavy chains to make a complete immunoglobulin molecule. The constant (C) regions have little variability in amino acid sequence. Two different kinds of C regions for light chains : kappa (κ) lambda (λ)

6. Five antibody classes (isotypes), determined by the constant region of the heavy chain The Structural Organization of the Human Immunoglobulin Classes Five different kinds of C regions for H chains : gamma (γ) chains, IgG mu (µ) chains, IgM delta (δ) chains, IgD alpha (α) chains, IgA epsilon (ε) chains, IgE

7. Each chain is folded into multiple ‘Ig’ domains The heavy chain constant domains (CH) : The “biological activity” region Responsible for interactions with other proteins (eg, complement), cells (eg, mast cells), and tissues that result in the effector functions of immunoglobulins Both H and L chains contain multiple loops of around 110 amino acids each.

8. Each chain is folded into multiple ‘Ig’ domains The variable domains (VH, VL) : The “antigen-binding” region Together, these domains compose the sole antigen-binding region of the immunoglobulin molecule. Both H and L chains contain multiple loops of around 110 amino acids each. The V regions are the first 110 amino acids at the N-terminus of both H and L chains. This is the region that gives antibody specificity for a given antigen.

9. Proteolytic digestion of IgG Papain treatment produces Fab fragments and Fc fragment Fab, antigen-binding; Fc, crystalizable or constant region Fc portion interacts with host systems Complement activation Binding to Fc receptor (FcR) on macrophages and other cells

10. Immunoglobulins Basic structure Basic structure 2 identical heavy chains 2 identical light chains Heavy and Light chains Heavy and Light chains variable (V) and constant (C) regions disulfide bonds link the chains together light chains have 2 domains, 1V and 1C heavy chains have 4-5 domains, 1V and 3-4 C heavy chains: 5 classes, μ, γ, α, δ, ε light chains: 2 classes, κ and λ Membrane bound (mIg) or secreted (sIg) Membrane bound (mIg) or secreted (sIg)

11. Antibody recognizes only a small region (epitope) on the surface of a large moleculeAntibody recognizes only a small region (epitope) on the surface of a large molecule Antibody binds an epitope formed by a specific sequence (linear epitope) or a three-dimensional structure (conformational epitope)Antibody binds an epitope formed by a specific sequence (linear epitope) or a three-dimensional structure (conformational epitope)

12. Antigens can bind in pockets, grooves, extended surface, or protruding surfaces in the binding sites of antibodies

13. The process of rearrangement of the Ig locus – called VDJ recombination. During B cell development, V, D, and J gene segments rearrange, creating functional genes that encode immunoglobulin transcripts. Genetic recombination at the DNA level and processing at the RNA level to produce functional mRNA

14. 51276 variable genesconstant gene For example, a rearranged heavy chain locus is composed of : one V gene segment (from 51) one D gene segment (from 27) one J gene segment (from 6) one C gene segment During B cell maturation, V, D, and J gene segments rearrange, creating functional genes that encode immunoglobulin transcripts. *After maturation, the variable gene segment rearrangment (and therefore the epitope specificity) does not change. V 23 D 14 J3J3

15. Thus, 2.64 x 10 6 DIFFERENT specificities are possible from the process of VDJ recombination alone 8262 x (200 + 120) = 2643840 = 2.64 x 10 6 8262 x (200 + 120) = 2.64 x 10 6 A given immunoglobulin has either κ chains or λ chains, never one of each

16. No two B cells are likely to secrete the same Ig (unless they are clonal progeny), due to the number of different rearrangements that are possible within the variable region of the immunoglobulin locus Note – There are several other forms of antibody diversification (eg, somatic hypermutation) that greatly increase the number of distinct antibodies that the immune system can generate. Thus, 2.64 x 10 6 DIFFERENT specificities are possible from the process of VDJ recombination alone (more than 10 11 different specificities in total)

17. Murray textbook, p75 Number of gene segment, wrong information: κ chain: 300 V, 5J H chain: 300- 1000 V, 12D, 9J

18. Naïve B cells die if they do not see antigen (Ag) Naïve B cells that encounter antigen (plus T cell help) : 1. become activated 2. undergo class switching 3. differentiate into either : plasma cells [antibody (Ab) secreting] or memory cells (non-secreting) The Life of a B cell

19. When membrane- bound Ig (mIg) on the B cell surface binds to it’s specific antigen, the B cell becomes activated.

20. Proper activation of the B cell typically requires contact with CD4 helper T (TH) cells.

21. Activated B cells differentiate into : plasma cells (Ab secreting) plasma cells (Ab secreting)or memory cells (non-secreting) memory cells (non-secreting)

22. Class switching (IgM to IgG, IgE, or IgA) occurs in response to different cytokines produced by CD4 helper T cells Class switching does not change the variable region

23. The usage of heavy chain constant gene segments changes with B cell activation in a process termed class switching 2 345 1 but...... epitope specificity does not change !

24. Structures of the five major classes of secreted antibody Important because antibody classes differ with regard to biological properties

25. IgG and IgA are divided into subclasses Subclasses differ in the number and arrangement of the interchain disulfide bonds.

26. 13.5 mg/ml 0.0003 mg/ml vs. The principal immunoglobulins in plasma are IgG >> IgA > IgM

27. FunctionIgGIgMIgAIgDIgE Complement Activation ++++--- Opsonization+---- ADCC+---- Placental transport+---- B cell antigen receptor Naïve-+-+- Memory+-+-+ Mucosal secretions--+-- Mast cell degranulation----+ Antigen binding+++++ Biological properties of antibody classes differ

28. IgG is the only isotype to cross the placenta The transfer of IgG from mother to fetus (placental transfer) is mediated by an IgG transport protein in the placenta, FcRn, which binds to the Fc portion of IgG Infant produces its own IgG at 6 months IgG levels are low from the age of 3 months to 1 year

29. to the fetal circulation Maternal blood Syncytio- trophoblast Endothelial cells of fetal capillary Villi

30. FcRn: neonatal Fc receptor for IgG

31. Syncytiotrophoblasts are bathed in maternal blood and internalize serum containing maternal IgG. FcRn is expressed in the internal vesicles of the syncytiotrophoblast. On acidification in the endosome, FcRn binds to maternal IgG and transcytoses it to the fetal circulation where it is released at physiological pH.

32. Dimeric IgA is the predominant in mucosal secretions The delivery of antibody to mucosal surfaces requires their movement across epithelial cell layers Transport is mediated by the polymeric immunoglobulin receptor (pIgR) IgA in colostrum, intestinal and respiratory tracts, saliva, tears Some pathogens produce proteolytic enzymes that can cleave IgA Haemophilus influenzae (respiratory), Neisseria gonorrhoeae (genital mucosa) Secretory component: part of the cleaved pIgR associated with the IgA

33. IgE is found associated with mast cells beneath epithelial surface, including respiratroy tract, gastrointesitnal tract, and skin IgD exists as membrane IgD, which serves with IgM as an antigen receptor on B cells, and activate B cell growth In healthy individuals the Ig concentration in the central nervous system (CNS) is low. Immunoglobulin isotypes are selectively distributed

34. The humoral immune response to infectious pathogens

35. The primary response to an initial antigen exposure long lag phase long lag phase IgM is the first reponder IgM is the first reponder gradual switch to IgG gradual switch to IgG low affinity antibodies low affinity antibodies

36. The secondary response to antigen exposure reflects immunological memory. short lag phase short lag phase very rapid rise in titer very rapid rise in titer IgG predominates IgG predominates high affinity antibodies high affinity antibodies

37. EFFECTOR FUNCTIONS OF ANTIBODIES Opsonization – the promotion of phagocytosis of antigens by macrophages and neutrophils. Protein molecules called Fc receptors (FcR), which bind the constant region of antibody, are present on the surfaces of phagocytes. Activation of complement – IgM (most effective) and most subclasses of IgG can activate the classical complement pathway Antibody-dependent cell-mediated cytotoxicity (ADCC) – The linking of antibody bound to target cells (virus infected cells, or some tumor cells) with FcR of natural killer cells (NK cells), neutrophils, macrophages, or eosinophils can result in killing of the target cell. Neutralization of viruses and bacteria – Prevent attachment to cell receptors. Neutralization of toxins

38. Antibody-dependent opsonization of bacteria facilitates phagocytosis of the pathogen opsonization (op´sŏn-ī-zā´shŭn) The process by which bacteria and other cells are altered in such a manner that they are more readily and more efficiently engulfed by phagocytes. opsonin (op´sŏ-nin) Any blood serum protein that binds to antigens, enhancing phagocytosis (e.g., C3b of the complement system, specific antibodies). [G. opson, boiled meat, opsonin: to prepare for a meal] IgG FcγR

39. Antibody-dependent activation of the complement pathway Potential outcomes : - lysis of bacteria via MAC - induction of inflammatory response - phagocytosis of bacteria IgM is the most efficient immunoglobulin for fixing (binding) complement (Murray p 102) “Complement fixation” = “Complement activation”

40. Antibody-dependent cellular (cell-mediated) cytotoxicity (ADCC) kills the infected target cell =FcγR

41. Infection of cells can be blocked by neutralizing antibodies

43. Antibody can prevent the attachment of bacteria to cell surface

44. Many common diseases are caused by bacterial toxins

45. Neutralization by IgG antibodies can protect cells from toxins

47. FunctionIgGIgMIgAIgDIgE Complement Activation ++++--- Opsonization+---- ADCC+----+-+ Placental transport+---- B cell antigen receptor Naïve-+-+- Memory+-+-+ Mucosal secretions--+-- Mast cell degranulation----+ Antigen binding+++++ Biological properties of antibody classes differ Murray p73, Table 9-4 is inaccurate helminth killing

48. Effector functions of antibodies http://www.studentconsult.com/content/9780323054706/abbas_sped-up_animations/index.html https://www.facebook.com/406138889435131/videos/vb.406138889435131/866119870103695/?type=2&theater

49. Questions (Murray p78) What is wrong with each of the following statements, and why? The laboratory tested a baby for IgM maternal antibodies. The laboratory tested a baby for IgM maternal antibodies. An investigator attempted to use fluorescent-labeled F(ab') 2 fragments to locate class II major histocompatibility complex molecules on the cell surface of antigen-presenting cells without cross-linking (binding two molecules together) these cell surface molecules. An investigator attempted to use fluorescent-labeled F(ab') 2 fragments to locate class II major histocompatibility complex molecules on the cell surface of antigen-presenting cells without cross-linking (binding two molecules together) these cell surface molecules. A patient is diagnosed as having been infected with a specific strain of influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of anti- influenza IgG in serum taken from the patient at the initial visit (within 2 days of symptoms). A patient is diagnosed as having been infected with a specific strain of influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of anti- influenza IgG in serum taken from the patient at the initial visit (within 2 days of symptoms). A patient was considered unable to use the complement systems because of a T-cell deficiency, which precluded the ability to promote class switching of B cells. A patient was considered unable to use the complement systems because of a T-cell deficiency, which precluded the ability to promote class switching of B cells. Analysis of immunoglobulin genes from B cells taken from the patient described in statement 4 did not contain recombined VDJ variable- region gene sequences. Analysis of immunoglobulin genes from B cells taken from the patient described in statement 4 did not contain recombined VDJ variable- region gene sequences. A patient was considered to have a B-cell deficiency because serum levels of IgE and IgD were undetectable despite proper concentrations of IgG and IgM. A patient was considered to have a B-cell deficiency because serum levels of IgE and IgD were undetectable despite proper concentrations of IgG and IgM.

50. Anti-HLA (MHC, major histocompatibility complex) antibody cross-link two HLA molecules, activating PI3K and mTOR Example of cross-linking by antibody

51. Proteolytic digestion of IgG DigestionIgG Antigen binding/ Immunostaining Cross-linking /binding two molecules together Fc mediated functions ADCC/ opsonization no treatment IgG+++ pepsinF(ab’)2++- papainFab+--

52. When IgG molecules are incubated with papain, producing three fragments of similar size: two Fab fragment and one Fc fragment. Papain digestion: pa-pa-in, three syllables = 3 components Pepsin digestion produces 1) one F(ab')2 fragment and 2) numerous small peptides of the Fc portion. http://www.piercenet.com/browse.cfm?fldID=4E03B016-5056-8A76-4ECA-982DA6CAAC8A Pepsin digestion: pep-sin, two syllables = 2 components How to remember papain versus pepsin digestion of IgG? Papain =PAPA(YA) + IN By Mr. Tameem Islam Medical Student, LSUHSC

53. http://tsunodalaboratory.blog.fc2.com/blog-entry-120.html = =

54. Questions (Murray p78) What is wrong with each of the following statements, and why? The laboratory tested a baby for IgM maternal antibodies. The laboratory tested a baby for IgM maternal antibodies. An investigator attempted to use fluorescent-labeled F(ab') 2 fragments to locate class II major histocompatibility complex molecules on the cell surface of antigen-presenting cells without cross-linking (binding two molecules together) these cell surface molecules. An investigator attempted to use fluorescent-labeled F(ab') 2 fragments to locate class II major histocompatibility complex molecules on the cell surface of antigen-presenting cells without cross-linking (binding two molecules together) these cell surface molecules. A patient is diagnosed as having been infected with a specific strain of influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of anti- influenza IgG in serum taken from the patient at the initial visit (within 2 days of symptoms). A patient is diagnosed as having been infected with a specific strain of influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of anti- influenza IgG in serum taken from the patient at the initial visit (within 2 days of symptoms). A patient was considered unable to use the complement systems because of a T-cell deficiency, which precluded the ability to promote class switching of B cells. A patient was considered unable to use the complement systems because of a T-cell deficiency, which precluded the ability to promote class switching of B cells. Analysis of immunoglobulin genes from B cells taken from the patient described in statement 4 did not contain recombined VDJ variable- region gene sequences. Analysis of immunoglobulin genes from B cells taken from the patient described in statement 4 did not contain recombined VDJ variable- region gene sequences. A patient was considered to have a B-cell deficiency because serum levels of IgE and IgD were undetectable despite proper concentrations of IgG and IgM. A patient was considered to have a B-cell deficiency because serum levels of IgE and IgD were undetectable despite proper concentrations of IgG and IgM.

55. Time course of immune responses. The primary response occurs after a lag period. The immunoglobulin (Ig) M response is the earliest response. The secondary immune response (anamnestic response) reaches a higher titer, lasts longer, and consists predominantly of IgG. Figure 9-15

56. Questions (Murray p78) What is wrong with each of the following statements, and why? The laboratory tested a baby for IgM maternal antibodies. The laboratory tested a baby for IgM maternal antibodies. An investigator attempted to use fluorescent-labeled F(ab') 2 fragments to locate class II major histocompatibility complex molecules on the cell surface of antigen-presenting cells without cross-linking (binding two molecules together) these cell surface molecules. An investigator attempted to use fluorescent-labeled F(ab') 2 fragments to locate class II major histocompatibility complex molecules on the cell surface of antigen-presenting cells without cross-linking (binding two molecules together) these cell surface molecules. A patient is diagnosed as having been infected with a specific strain of influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of anti- influenza IgG in serum taken from the patient at the initial visit (within 2 days of symptoms). A patient is diagnosed as having been infected with a specific strain of influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of anti- influenza IgG in serum taken from the patient at the initial visit (within 2 days of symptoms). A patient was considered unable to use the complement systems because of a T-cell deficiency, which precluded the ability to promote class switching of B cells. A patient was considered unable to use the complement systems because of a T-cell deficiency, which precluded the ability to promote class switching of B cells. Analysis of immunoglobulin genes from B cells taken from the patient described in statement 4 did not contain recombined VDJ variable- region gene sequences. Analysis of immunoglobulin genes from B cells taken from the patient described in statement 4 did not contain recombined VDJ variable- region gene sequences. A patient was considered to have a B-cell deficiency because serum levels of IgE and IgD were undetectable despite proper concentrations of IgG and IgM. A patient was considered to have a B-cell deficiency because serum levels of IgE and IgD were undetectable despite proper concentrations of IgG and IgM.

57. Naïve B cells die if they do not see antigen (Ag) Naïve B cells that encounter antigen (plus T cell help) : 1. become activated 2. undergo class switching 3. differentiate into either : plasma cells [antibody (Ab) secreting] or memory cells (non-secreting) The Life of a B cell IgM, IgD

58. Questions (Murray p78) What is wrong with each of the following statements, and why? The laboratory tested a baby for IgM maternal antibodies. The laboratory tested a baby for IgM maternal antibodies. An investigator attempted to use fluorescent-labeled F(ab') 2 fragments to locate class II major histocompatibility complex molecules on the cell surface of antigen-presenting cells without cross-linking (binding two molecules together) these cell surface molecules. An investigator attempted to use fluorescent-labeled F(ab') 2 fragments to locate class II major histocompatibility complex molecules on the cell surface of antigen-presenting cells without cross-linking (binding two molecules together) these cell surface molecules. A patient is diagnosed as having been infected with a specific strain of influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of anti- influenza IgG in serum taken from the patient at the initial visit (within 2 days of symptoms). A patient is diagnosed as having been infected with a specific strain of influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of anti- influenza IgG in serum taken from the patient at the initial visit (within 2 days of symptoms). A patient was considered unable to use the complement systems because of a T-cell deficiency, which precluded the ability to promote class switching of B cells. A patient was considered unable to use the complement systems because of a T-cell deficiency, which precluded the ability to promote class switching of B cells. Analysis of immunoglobulin genes from B cells taken from the patient described in statement 4 did not contain recombined VDJ variable- region gene sequences. Analysis of immunoglobulin genes from B cells taken from the patient described in statement 4 did not contain recombined VDJ variable- region gene sequences. A patient was considered to have a B-cell deficiency because serum levels of IgE and IgD were undetectable despite proper concentrations of IgG and IgM. A patient was considered to have a B-cell deficiency because serum levels of IgE and IgD were undetectable despite proper concentrations of IgG and IgM.

59. The usage of heavy chain constant gene segments changes with B cell activation in a process termed class switching 2 345 1 but...... epitope specificity does not change !

60. Answers (Murray p78) What is wrong with each of the following statements, and why? The laboratory tested a baby for IgG maternal antibodies. The laboratory tested a baby for IgG maternal antibodies. An investigator attempted to use fluorescent-labeled Fab fragments to locate class II major histocompatibility complex molecules on the cell surface of antigen-presenting cells without cross-linking (binding two molecules together) these cell surface molecules. An investigator attempted to use fluorescent-labeled Fab fragments to locate class II major histocompatibility complex molecules on the cell surface of antigen-presenting cells without cross-linking (binding two molecules together) these cell surface molecules. A patient is diagnosed as having been infected with a specific strain of influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of anti- influenza IgM in serum taken from the patient at the initial visit (within 2 days of symptoms). A patient is diagnosed as having been infected with a specific strain of influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of anti- influenza IgM in serum taken from the patient at the initial visit (within 2 days of symptoms). A patient was still considered able to use the complement system, despite a T-cell deficiency, which precluded the ability to promote class switching of B cells. A patient was still considered able to use the complement system, despite a T-cell deficiency, which precluded the ability to promote class switching of B cells. Analysis of immunoglobulin genes from B cells taken from the patient described in statement 4 contained recombined VDJ variable-region gene sequences. Analysis of immunoglobulin genes from B cells taken from the patient described in statement 4 contained recombined VDJ variable-region gene sequences. A patient was not considered to have a B-cell deficiency despite undetectable serum levels of IgE and IgD, because of proper concentrations of IgG and IgM. A patient was not considered to have a B-cell deficiency despite undetectable serum levels of IgE and IgD, because of proper concentrations of IgG and IgM.

61. studentconsult.com

62. Every B cell expresses a single type of immunoglobulin. The specificity of that immunoglobulin is determined by the process of VDJ recombination – rearrangement of the DNA that encodes the variable region.