1. DEFINITION AND PROPERTIES OF ANTIGEN

2. Antigen (Ag) - any substance, which is recognized by the mature immune system of a given organism –antigenicity - specific reactivity with cells or molecules of the immune system –immunogenicity - capability to elicit an immune response –tolerogenicity - capability to induce immunological tolerance

3. part of the antigen which is recognized by a defined immunoglobulin (BCR / antibody) or by T cell receptor ANTIGENIC DETERMINANT (=EPITOPE)

4. B cell epitope T cell epitope recognized by B cells proteins polysaccharides lipids DNA steroids etc. (many artificial molecules) cell or matrix associated or soluble recognized by T cells proteins mainly (8-23 amino acids) requires processing by APC ! !

5. IMMUNOGLOBULIN STRUCTURE 2x Heavy chain (light blue) 2x light chain (dark blue) Variable regions  antigen binding Constant regions hinge region carbohydrate disulfide bond C H1 VLVL CLCL VHVH C H2 C H3 ! !

6. ANTIBODY DOMAINS AND THEIR FUNCTIONS !! Constant domain Effector functions Antigen recognition Variable domain

7. Antibody BCR (B cell receptor) ! ! MEMBRANE BOUND! Associated chains for signaling Transmembrane domain Cytoplasmic domain Antigen recognition and B cell activation SOLUBLE (freely circulating) Antigen recognition and effector functions. Produced by plasma cells

8. ANTIGEN BINDING Antigen Binding Fragment (Fab) Complement binding site Placental transfer Constant fragment (Fc) Binding to Fc receptors on phagocytic cells

9. DIFFERENT VARIABLE REGIONS  DIFFERENT ANTIGEN-BINDING SITES  DIFFERENT SPECIFICITIES

10. EFFECTOR FUNCTIONS OF ANTIBODIES Antibody-mediated immune responses NEUTRALIZATION OPSONIZATION ADCC (NK cell-mediated killing) MAST CELL DEGRANULATION COMPLEMENT FIXATION !!

11. Sequence variability of H/L- chain constant regions VARIABILITY IN DIFFERENT REGIONS OF THE Ig DETERMINES Ig SPECIFICITY OR CLASS Sequence variability of H/L- chain constant regions Isotype

13. OPSONIZATION ! ! Main opsonins: antibodies Complement fragments Acute-phase proteins Opsonization facilitate and accelerate the recognition of the pathogen by phaogocytes, opsonins form a bridge between pathogen and a phagocyte connecting them.

14. OPSONIZATION Flagging a pathogen Antigen binding portion (Fab) binds the pathogen, the Fc region binds phagocytic cells Fc-receptors speeding up the process of phagocytosis

15. FcRAffinity for ImmunoglobulinCell DistributionFunction FcγRI (CD64)High (K d < 10 -9 M); binds IgG1 and IgG3, can bind monomeric IgG Macrophages, neutrophils; also eosinophils Phagocytosis; activation of phagocytes FcγRIIA (CD32)Low (K d > 10 -7 M)Macrophages, neutrophils; eosinophils, platelets Phagocytosis; cell activation (inefficient) FcγRIIB (CD32)Low (K d > 10 -7 M)B lymphocytesFeedback inhibition of B cells FcγRIIC (CD32)Low (K d > 10 -7 M)Macrophages, neutrophils, NK cells Phagocytosis, cell activation FcγRIIIA (CD16)Low (K d > 10 -6 M)NK cellsAntibody-dependent cell-mediated cytotoxicity FcγRIIIB (CD16)Low (K d > 10 -6 M); GPI-linked protein NeutrophilsPhagocytosis (inefficient) FcΕRIHigh (K d > 10 -10 M); binds monomeric IgE Mast cells, basophils, eosinophils Cell activation (degranulation) FcΕRII (CD23)Low (K d > 10 -7 M)B lymphocytes, eosinophils, Langerhans cells Unknown FcαR (CD89)Low (K d > 10 -6 M)Neutrophils, eosinophils, monocytes Cell activation?

16. EFFECTOR FUNCTIONS OF ANTIBODIES Antibody-mediated immune responses NEUTRALIZATION OPSONIZATION ADCC MAST CELL DEGRANULATION COMPLEMENT FIXATION !!

17. NEUTRALIZATION Covering of the pathogen’s surface prevents replication and growth

18. Kb. 10 – 1000 million (10 7 - 9 ) different antigen receptors, unique specificity of B cells Kb. 10 – 1000 million (10 7 - 9 ) different antigen receptors, unique specificity of T cells ADAPTIVE IMMUNE SYSTEM Diversity of receptor strucure How can the antigen receptors of lymphocytes recognize extremly diverse antigens

19. Random hands, millions of variations

20. Random selection of gene segments ensures millions of different receptor (variable domains)

21. During B-cell development VH2 JH 65 VH 6 JH VH1VH3 DJH 27 D DDD JH DD SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS DD VH1VH2VH3 VH1VH2

22. HOW MANY IMMUNOGLOBULIN GENE SEGMENTS Variable (V) 40 30 65 Diversity (D)0027 Joining (J)546 Gene segmentsLight chainHeavy chain kappalambda Chromosome 2 kappa light chain gene segments Chromosome 22 lambda light chain gene segments Chromosome 14 heavy chain gene segments IMMUNOGLOBULIN CHAINS ARE ENCODED BY MULTIPLE GENE SEGMENTS ORGANIZATION OF IMMUNOGLOBULIN GENE SEGMENTS

23. VH D JH VLJL V-Domains C-Domains VH-D-JH VL-JL VARIABILITY OF B-CELL ANTIGEN RECEPTORS AND ANTIBODIES B cells of one individual 1 2 34

24. Estimates of combinatorial diversity Taking account of functional V D and J genes: 65 VH x 27 DH x 6JH = 10,530 combinations 40 V  x 5 J  = 200combinations 30 V  x 4 J = 120 combinations = 320 different light chains If H and L chains pair randomly as H 2 L 2 i.e. 10,530x 320 = 3,369,600 possibilities Due only to COMBINATORIAL diversity In practice, some H + L combinations do not occur as they are unstable Certain V and J genes are also used more frequently than others. There are other mechanisms that add diversity at the junctions between genes - JUNCTIONAL diversity GENERATES A POTENTIAL B-CELL REPERTOIRE

25. Clonal expansion of B cells

26. Forms of antibodies -memrane bound (BCR) -soluble (Plasma cells do not express antibody, but secrete it!!) Membrane bound and soulble antibodies recognize the same antigen Differenciation Plazma cell Secreted antibodies !

27. Several antibodies are expressed on B cells, (arround 100.000) but all of them with the same specificity ! !

28. Activation Clonal expansion B cell Antigen receptor, BCR Ag Clonal antigen receptors are expressed exclusively on T- and B lymphfocyties. ! ! Antigen recognition by specific BCR induces clonal expansion of the sepcific B cells.

29. Activation Clonal expansion Differencaition Plasma cells Antibody production Memory B cells Circulation Restricted lifespan (few days) Apoptosis ! Specific B cellsNon-specific B cells Antigen recognition by specific BCR induces clonal expansion and differentiation of the sepcific B cells.

30. Activation of specific B cells 1. Clonal expansion 2.Differen tiation Plasma cells, antibody production MEMORY B CELLS ! Antigen recognition by specific BCR induces clonal expansion and differentiation of the sepcific B cells.

31. During the B cell development in the bone marrow, self reactive B cells are deleted !

32. Several epitops of one microbes can be recognized by different B cells

33. POLICLONAL Response Ag B cell repertoar Specific, activated B cells Plasma cells Antigen specific antibodies

34. Activation 1. Clonal expansion 2.Differen tiation Plasma cells MEMORY B cells Antigen recognition by specific BCR induces clonal expansion and differentiation of the sepcific B cells. !

35. B – CELL ACTIVATION Where and how do all these things take place?

36. B-cell recycling in the absence of antigen (lymph node) B cells in blood Efferent lymph T cell area B cell area

37. Antigen enters node in afferent lymphatic Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y B cells leave blood & enter lymph node via high endothelial venules B cells proliferate rapidly GERMINAL CENTRE Transient structure of Intense proliferation Germinal centre releases B cells that differentiate into plasma cells Recirculating B cells are trapped by foreign antigens in lymphoid organs

38. Complement proteins Immune complex (1)antigen-(2) recognising antibody (3) complement components

39. The structure of the germinal centre FDC DZ LZ LZ: light zone DZ: dark zone FDC: follicular dendritic cell

40. Antigen is bound on the surface of follicular dendritic cells (FDC) FDC  FDC-s bind immune complexes ( Ag-Ab )  Ag detectable for 12 months following immunization  A single cell binds various antigens B cells recognize Ag on the surface of FDC

41. 1. B cells compete for the antigen High affinity B cells can grab the antigen !

42. B cell antigen presentation !

44. Antigen prezentation polysaccharides are not presented +++

45. 2. B cells compete for T cell help’ High affinit B cell can present the antigen to T cells !

49. INNATE IMMUNITY II Effector functions, elimination of pathogens 1.Phagocytosis 2.Killing with soluble mediators 3.Complement system 4.NK cell activation !!

50. Monocita/ makrofág DCHízó Sejt Granu locita NK sejtB-sejtT-sejtKomp lement recogni tion comm unicati on Effector function neutralizáció

51. EFFECTOR FUNCTIONS OF ANTIBODIES PLASMA CELL NEUTRALIZATION OPSONIZATION Binding of antibody increases phagocytosis Fc  R Fc  R CR1 Complement C3b COMPLEMENT ACTIVATION Opsonization by C3b PHAGOCYTES ENGULFMENT, DEGRADATION

52. cell surface antigen receptor on B cells allows B cells to sense their antigenic environment connects extracellular space with intracellular signalling machinery secreted antibody neutralization opsonization complement fixation NK cell –mediated killing Immunoglobulin Structure-Function Relationship