Humoral immune response

1. Immune Response I Dr. sufi h. z. Rahman Mbbs , md (immunology) Lecturer, medical faculty, AUCMS

2. Objectives Mechanism of non-specific immunity Process of phagocytosis Concept of specificity, immunological memory and non-self Induction process of B lymphocytes in forming antibodies Characteristics of different classes of antibodies and functions of each class Primary and secondary antibody response to antigens

3. Immune System Organs, cells and molecules the protect the host from infections and cancers Two different but coordinated immune systems are functioning to protect the host

4. Immune System Innate (inborn or non-specific) Immune System First line of defense Prevent microbial attachment, colonization and entry Disposes pathogens rapidly and nonspecifically if they can enter Adaptive (acquired or specific) Immune System Second line of defense when the innate system fails Devotes specialized sets of cells for each pathogen to identify, mark for disposal and retaining memory for future

5. Comparison between Innate and Adaptive Immunity Attribute Innate Immunity Adaptive Immunity Activation Active prior to exposure to any microbe or antigen Activated by exposure to microbes or antigens Lag phase Absent Response is immediate Present Response takes few days Specificity Limited Targets all pathogens High Targets specific pathogen Memory Absent Same response in 1 st and subsequent exposure Present Amplified response in subsequent exposure

6. Organization of the Immune System

7. Innate Immunity Skin Epidermis acts as mechanical barrier and retards entry of microorganisms Acidic e nvironment (pH 3- 5) retards growth of microorganisms Mucous membrane Normal microbial flora compete with pathogenic microorganisms for attachment and nutrients Mucus entraps foreign microorganisms Cilia of surface epithelium propel microbes out of the body Anatomical Barrier

8. Innate Immunity Fever response Inhibit growth of microorganisms Acidic environment Stomach: Kills microorganisms Vagina: Retards growth of microorganisms Chemical mediators Lysozyme of tear: Cleaves bacterial cell wall Interferon: Induces antiviral state in uninfected cells Complement: Lyses microbes or facilitate their phagocytosis Antibacterial peptides: Kill pathogenic bacteria Physiological Barrier

9. Innate Immunity Phagocytic cells Neutrophils Monocytes and Macrophages Dendritic cells Eosinophils (?) Detect Pathogen associated molecular pattern (PAMP) on the surface of microbes by their Pattern recognition receptors (PRR), internalize ( phagocytose ) and kill them Non-phagocytic killer cells Natural killer (NK) cells Induce apoptosis of virus infected and tumour cells Cellular Barrier

10. Innate Immunity Inflammatory response upon tissue damage or microbial infection induces leakage of vascular fluid containing antibacterial peptides to the site of damage or site of entry of microbes Inflammatory response also causes influx of phagocytic cells (neutrophils, monocytes and macrophages) to the affected site Antibacterial peptides and phagocytes then destroy the microbial agent Inflammatory Barrier

11. Innate Immunity Inflammation

12. Innate Immunity Phagocytic cells recognize PAMPs on microbial surface by their PRRs Extend pseudopodia and internalize the microbes in phagosome Kill microbes by Oxygen dependent mechanism Respiratory burst in the phagosomes produce reactive oxygen and reactive nitrogen intermediates that are extremely toxic to microbes Oxygen independent mechanism Phagosomes fuse with lysosomes and lysosomal enzymes kill microbes independent of ox ygen Phagocytosis

13. Innate Immunity Phagocytosis

14. Killing by NK cells Innate Immunity Bind to cells (normal, infected or tumour cells) by Activation receptor (AR) that provide activation signal to kill the cell Also bind to Class I MHC molecules on normal cells by Killer cell inhibitory receptors (KIR) that provide inhibitory signal Inhibitory signal is stronger than activation signal and normal cells are not killed As virus infected cells and tumour cells reduce Class I MHC molecule expression, they cannot provide inhibitory signal and are killed by NK cells NK cells induce apoptosis of virus infected cells and tumour cells

15. Innate Immune Response Role Eliminate microbes immediately Initiate Adaptive Immune Response

16. Adaptive Immunity Develops after exposure to pathogen or antigen Adaptive immunity has the following attributes Antigenic specificity Diversity Immunologic memory Self/ non-self recognition

17. Adaptive Immunity Antigenic specificity Capable of recognizing and selectively eliminating specific foreign microorganisms and molecules (i.e. foreign antigens) Can distinguish subtle differences among antigens Diversity Can generate tremendous diversity in recognition of molecules Can recognize billions of unique structures on foreign antigens This ability is generated by recombination of Immunoglobulin and T cell receptor (TCR) gene segments The immune system is capable of generating more than 10 9 clones of B cells and 10 10 clones of T cells, only a small fraction of which is displayed at any time Each clone is specific for one epitope (antigen)

18. Adaptive Immunity Immunologic memory Exhibits memory of an antigen to which it is exposed A second exposure to the same antigen induces a heightened state of immunological reactivity Because of this attribute, adaptive immune system can confer life-long immunity to many infectious agents after an initial encounter Self/ non-self recognition Capable of distinguishing self from non-self Normally responds only to foreign antigens This is due to development of self tolerance during maturation of B and T lymphocytes

19. Adaptive Immunity Adaptive immune system has two arms Provided by B lymphocytes Can recognize protein, polysaccharide, phospholipid and nucleic acid antigens Can act against soluble or free antigens Provides immunity to extracellular bacteria, viruses and toxins Causes Type I, II & III hypersensitivity Provided by T lymphocytes Can recognize only protein antigens Recognizes antigens presented by APCs with Class I or Class II MHC molecule Provides immunity to intracellular bacteria, viruses, fungi and protozoa Causes Type IV hypersensitivity Causes acute graft rejection

20. V= Variable, D= Diversity, J= Joining segment of Ig gene, H= Heavy chain, L= Light chain B cells Humoral Immune Response Maturation/ Ontogeny

21. Humoral Immune Response Mature B cells express membrane bound immunoglobulin ( mIg ) Naïve B cells express mIgM and mIgD on their surface mIgM acts as antigen receptor called B cell receptor (BCR) Each B cell contains 10 5 BCRs, all specific for a single epitope Each clone of B cell is specific for a single epitope 10 9 clones of B cells can recognize 10 9 epitopes B cells

22. Humoral Immune Response Mature B cells circulate in the blood and lymph and are carried to the secondary lymphoid organs e.g. lymph nodes and spleen Antigens are also carried by blood or lymph to the secondary lymphoid organs B cells usually meet their specific antigens in these organs An antigen binds to a B cell (or a clone of B cells) which carries mIgM specific for that antigen and activates it (clonal selection) B cell activated by an antigen starts to proliferate and increase number of cells (clonal expansion) After expansion, the cells differentiate to plasma cells and memory B cells B cell activation

23. Humoral Immune Response Binding of a TI antigen with mIgM on B cell surface activates the B cell specific for it B cell activation by TD and TI B cell antigens Binding of a TD antigen with mIgM on B cell surface provides stimulatory signal (1) Antigen is internalized & presented to T H cell CD40 on B cell also bind to CD40L on T H cell that provides costimulatory signal (2) These two signals and cytokines from T H cell activates the B cell TD= T H cell dependent, TI= T H cell independent

24. Humoral Immune Response B cell activation by TD and TI antigens

25. Humoral Immune Response Plasma cells secrete antibodies Secreted antibodies have the same specificity as the mIgM on the surface of B cell from which the plasma cell is derived Antibodies are the effector molecules of humoral immune response that bind with the antigen and eliminate the microbe Initially plasma cells secrete IgM antibody Within a few days antibody class switching occurs and they secrete IgG (or IgA or IgE ) Memory B cells have a long life span and provide heightened immune response if the person encounters the same antigen in future Antibody production

26. Humoral Immune Response Antigen Elimination Antibodies eliminate antigens by Neutralization of toxins and viruses Complement activation followed by Cytolysis Opsonization and phagocytosis Immune complex clearance Antibody dependent cell mediated cytotoxicity (ADCC)

27. Humoral Immune Response Antigen Elimination Antibody Dependent Cell Mediated Cytotoxicity (ADCC) Cells that have receptor for Fc portion of IgG ( Neutrohils , Macrophages and NK cells) or IgE ( Eosinophils ) induce apoptosis of the infected cells with bound IgG or IgE by: Neutrophils: Lytic enzymes Macrophages: Lytic enzymes, TNF Eosinophils : Lytic enzymes, perforins NK cells: Perforins , granzymes , TNF

28. Humoral Immune Response Antibody Antibodies are antigen binding proteins present on B cell membrane and secreted ( immunoglobulins ) by plasma cells Membrane-bound antibody confers antigenic specificity to B cells; antigen-specific proliferation of B-cell clone is elicited by the interaction of membrane-bound antibody with its specific antigen Secreted antibodies circulate in the blood, where they serve as the effectors of humoral immunity by searching and neutralizing antigens or marking them for elimination All antibodies share structural features, bind to antigen, and participate in a limited number of effector functions

29. Humoral Immune Response Antibody Molecule Consists of 4 peptide chains 2 identical heavy (H) chains (MW 50 kD ) 2 identical light (L) chains ( MW 25 kD ) Each L chain is bound to a H chain by disulfide bond and form 2 H-L combinations H-L combinations are held together by disulfide bond between H chains Amino acid sequence in amino terminal is variable ( V region) and form the antigen binding site ( hypervariable region or CDR) Amino acid sequence in carboxyl terminal is relatively constant (C region or Fc) and is responsible for biological function e.g. complement fixation, placental transfer, opsonization etc. MW= Molecular weight, kD = kilo Dalton, CDR= Complementarity determining region Fc= Fragment crystallizable

30. Humoral Immune Response There are 5 types of H chains: g, d, a, m and e and 2 types of L chains: k and l Both H and L chains have domain structure, 4- 5 domains in H and 2 domains in L chains An antibody molecule contains only one type of H chain and one type of L chain Depending on the type of H chain, antibodies are classified in to IgG , IgD , IgA, IgM and IgE Antibody

31. Humoral Immune Response IgG contains g H chain and is a monomer IgG has 4 subtypes IgG1, IgG2, IgG3 and IgG4 IgD contains d H chain and is a monomer IgE contains e H chain and is a monomer IgA contains a H chain and is a dimer where each monomer is joined together by a J chain and may contain a secretory component IgA has two subtypes: IgA1 and IgA2 IgM contains m H chain and is a pentamer where monomers are joined together by disulphide bonds and J chain Antibody Types and Subtypes These types are called isotypes because they are same in all members of the same species (cf. allotype and idiotype )

32. eosinophils

33. Humoral Immune Response Primary immune response Immune response that occurs after 1 st exposure to an antigen Secondary immune response Immune response that occurs after 2 nd or subsequent exposure to the same antigen Primary and Secondary Immune Response

34. Humoral Immune Response Primary and Secondary Immune Response Attribute Primary response Secondary response Antigen type Both T dependent and T independent Only T dependent Responding cells Naïve B or T cells Memory B or T cells Lag period Longer (4- 7 days) Shorter (1- 3 days) Peak response Occurs in 7- 10 days Occurs in 3- 5 days Magnitude Low High (100- 1000x) Antibody isotype IgM predominates IgG predominates Antibody affinity Lower Higher

35. Humoral Immune Response Primary and Secondary Immune Response Primary immune response IgM is produced first then class switch to IgG Secondary immune response IgM and IgG are produced simultaneously from the beginning with predominant IgG

36. Induction of Adaptive Immunity

37. Induction of Adaptive Immunity

38. Cooperation between Innate and Adaptive Immunity Adaptive immunity is not independent of innate immunity The phagocytic cells crucial to nonspecific immune responses are intimately involved in activating the specific immune response Various soluble factors produced by a specific immune response have been shown to augment the activity of these phagocytic cells Through the carefully regulated interplay of adaptive and innate immunity, the two systems work together to eliminate a foreign invader

39. Cooperation between Innate and Adaptive Immunity Component Innate Immune System Adaptive Immune System Macrophages Phagocytosis and killing of microorganisms Presentation of antigens of phagocytosed organisms to T lymphocytes, ADCC Dendritic cells Phagocytosis and killing of microorganisms Presentation of antigens of phagocytosed microorganisms to T lymphocytes Complements Activation by Alternative or Lectin pathway Activation by Classical pathway Neutrophils Phagocytosis of microbes Killing of microbes by ADCC Eosinophils Phagocytosis of microbes (?) Killing of parasites by ADCC NK cells Killing virus infected & cancer cells Killing of microbes by ADCC Basophils Inflammation Type I Hypersensitivity

40. Further Review Levinson W. Review of Medical Microbiology and Immunology . 11 th edition. McGraw Hill, 2008. Kindt TJ, Goldsby RA, Osborne BA. Kuby Immunology . 6 th ed. WH Freeman, 2006. Abbas AK, Lichman AH. Basic Immunology . 3 rd edition. Elsevier, 2011.