Immunology. B lymphocytes & Antibodies , Ruhr-Universität Bochum Marcus Peters, marcus.peters@rub.de

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1 Immunology B lymphocytes & Antibodies , Ruhr-Universität Bochum Marcus Peters, marcus.peters@rub.de

2 What is an antibody? An antibody is a glycoprotein, which specifically binds to a substance, i.e. the antigen. Based on its unique structure each antibody molecule can bind specifically and selectively to its appropriate antigen. All antibodies have a similar structure, and they are summarized under the term immunoglobulins (Ig). Antibodies are produced by cells of the B lymphoid lineage, termed plasma cells. Antibodies are produced in response to stimulation with an immunogen. 2

3 Immunoglobulin function Either to link an antigen to its elimination mechanism (effector system) or to neutralize a potential harmful antigen directly. Antibodies induce complement activation and cellular elimination mechanisms that include phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC). All immunoglobulin classes can be expressed on B-cell surfaces where they act as antigen receptors. 3

4 The structure of a typical antibody molecule 2 heavy chains (C H ), 4-5 domains 2 light chains (C L ), 2 domains The chains are linked (C H -C H und C H -C L ) by disulfide bonds. Every chain has a variable (N-terminal) and a constant (C-terminal) domain. The variable domains develop the antigen binding side (2 per Ab). The constant domains mediate the antibody function (e.g. receptor binding) 4

5 The structure of a typical antibody molecule The single domains in particular the C H2 bear carbohydrate structures The single domains and chains are twisted around each other. 5

6 Constant domain or C domain, refers to the immunoglobulin C H and C L regions encoded by the corresponding constant exon. The domain has a globular compact structure that consists of two antiparallel twisted b sheets. There is only minor variability in the primary structure of the constant domains. 6

7 Fc fragment Fab F(ab ) 2 Fab Peptides IgG Fc pfc 7

8 Immunoglobulin superfamily Protein molecules sharing 15 % AA homology with Ig proteins and possess one or more Ig domain belong to this large protein family Several molecules participate in the immune response and show similarities in structure, e.g. TCR MHC I and MHC II CD2, CD3, CD4, CD8, CD28, LFA-3 IgG-receptor 8

9 Complementarity-determining region (CDR) refers to the hypervariable regions in an immunoglobulin molecule that build the threedimensional cavity where an epitope binds to the antibody molecule. Both the heavy and the light chains each contribute hypervariable regions to the antigen binding site of the antibody molecule. 9

10 The binding of an antigen The binding of an antigen by an antibody is never covalent. 10

11 The binding of an antigen Antibody binding always between epitope (antigen) and paratope (antibody) 11

12 Development of the antibody diversity Possibilities: 1) Germline theory: Each antibody has an own gene But: 1x10 11 different antibodies exist 2) Somatic diversification: Only a few existing gene sequences change themselves and accomplish thereby the needful variety 12

13 Somatic recombination Combinatorial diversity: Light chain : 40 V L -segments 5 J L -segments 40 x 5 =

14 Somatic recombination Combinatorial diversity: Heavy chain: 51 V H -segments 27 D H -segments 6 J H -segments 51 x 27 x 6 = heavy chains 14

15 Somatic recombination 200 light chains x heavy chains = 1,6 x

16 Junctional diversity During VDJ recombination the enzyme recombination activating gene (RAG1&2) induces palindromic nucleotides (P-nucleotides) At the junction of the gene segments nucleotides are introduced by the enzyme Terminal deoxynucleotidyl transferase (N-nucleotides). Due to this process there is a high chance for the induction of frame shift mutation 16

17 Somatic hypermutation Affinity maturing of the antigen binding sites takes place in the secondary lymphatic organs (only in activated B-cells). Mutations occur in the whole variable region of the antibody. Mutations appear in certain regions (hot spots). B cells become apoptotic when somatic hypermutation resulted in reduced binding affinity of the antibody (during affinity maturation) 17

18 Somatic Hypermutation Antigen binding site Complementary Diversity Regions (CDRs) CDR1 CDR3 CDR3 Antikörper 1 Antikörper 2 Antikörper 3 AS AS AS

19 Summary Antibodies can be raised against almost every molecule The antibody diversity is based on three different mechanisms: 1) Combinatorial diversity (Somatic recombination) 2) Junctional diversity (Inaccurate junction) 3) Somatic hypermutation (Affinity maturing in germinal centres) 19

20 B-cell development B-cells develop continuously in the bone marrow, they derive from lymphatic progenitor cells The environment (stromal cells of the marrow) delivers the necessary milieu (surface molecules and cytokines) for the development The immunoglobulin genes are rearranged 20

21 B-cell development The heavy chain is expressed with a surrogate L-chain to stabilize the pre-b-cellreceptor After successful expression of the heavy chain the pre-b-cell divides, before the rearrangement of the light chain starts 21

22 B-cell development Beginning of the V-J-rearrangement of the -locus of the light chain, in case of an unsuccessful rearrangement, the -Locus becomes rearranged 22

23 B-cell development - Summary 23

24 B-cell development In the bone marrow: Selection on self-tolerance Contact to antigens in the environment of the bone marrow is leading to apoptosis induction in the immature B-cell 24

25 B-cell development after leaving the bone marrow Self-tolerant, naive B-cells emigrate of the bone marrow. Naive B-cells circulate through the blood in the secondary lymphatic organs. If the B-cells in the secondary lymphatic organs encounter their specific antigen, they become activated. Without antigen contact they circulate back with the lymph in the bloodstream. Lifetime of a not-activated B-cell: approx. 3 days. 25

26 Antibody classes - Isotypes Serum concentration (mg/dl) Ig IgG IgM IgA IgD IgE <

27 Antibody classes - Isotypes High avidity due to pentamer formation! 27

28 Affinity & Avidity IgM compensates its low affinity with high avidity. Affinity = Binding strength between one antigen binding side and the antigen. Avidity = Binding strength between more antigen binding sides and one multivalent antigen. Association Dissociation Affinity Avidity 28

29 Antibody classes - Isotypes IgA forms dimers Secretion of the antibody (Garland Science 2005) 29

30 Antibody classes - Isotypes c = IgM, c = IgD, c = IgG, c = IgE, c = IgA = Pseudogene The expression of the genes of the constantan region changes during the maturation of the B-cell = Isotype-Switch 30

31 Antibody classes - Isotypes During the Isotype- Switch the gene segment located between the switching signal sequences is excised develoment just in one direction 31

32 Antibody classes - Isotypes Specific DNA-sequences (Switch regions) regulate the recombination The isotype-switch takes place in the lymph node (germinal centre) It requires a specific milieu of Cytokines, which induce transcription of enzymes involved in the switching process: transcription factors 32

33 Cytokines involved in isotype-switching Heavy chain J H 1-6 C µ C C 3 C 1 wc C 1 C 2 C 4 C C 2 IL-4 IL-5 IFN- TGF-b = induces = increases the synthesis = inhibits 33

34 concentration The serum-concentration of specific antibodies generated during infection to fight the infectious agens Contact Begin of disease Incubation time Acute phase Early antibodies IgM Convalescence Late- or protection-antibodies IgG IgA time 34

35 Antibody classes - function IgG can be transferred to the Embryo via the placenta IgG and IgM are the dominant Akclasses in the plasma IgA is secreted in all mucous membranes ( protective barrier) IgA can be transferred to the infant by lactation IgE can be find on mast cells in the skin and the mucous membranes 35

36 Antibody classes - function 1) Neutralisation (Viruses, bacteria, toxins): IgG and IgA 36

37 Antibody classes - function 2) Opsonisation (supports phagocytosis): IgG1 and IgG3 37

38 Cytolytic mechanisms of NK-cells ADCC (antibody-dependent cellular cytotoxicity): Lysis of cells opsonized by an antibody by induction of apoptosis via secretion of granzyme and perforin 38

39 Antibody receptors The antibody binding receptor determines also the mechanism 39

40 Antibody classes - function 3) Complement activation (classic way): IgG and IgM 40

41 Antibody classes - function The distribution of the antibody receptors on the different cells of the immune system/the tissue determines the site of activity of the antibody. Function IgM IgD IgG IgA IgE Neutralisation Opsonisation Mast cell sensitization Complement activation Transport via epithelia Transport via placenta Diffusion into the tissue Serum level [mg/ml] /

42 B-cell activation Naive B-cells need 2 signals for activation: First signal: signalling through the B-cell receptor (BCR) = Surface-Immunoglobulin The second signal can be submitted on two ways: 1) Thymus-dependent (via already activated T-helpercells) = TD-antigens (thymus dependent) 2) Thymus-independent (activtion without Tcell help) = TI- antigen (thymus independent) 42

43 Thymus-independent (TI)-B-cell activation TI-1-antigens activate a further receptor on the surface, e.g. TLR-4 against LPS or a complement receptor = co-stimulatory signal TI-2-antigens activate the B-cell by crosslinking the BCR via a large molecule containing antigens without costimulation 43

44 TI-B-cell activation TI (1+2)-activation : B-cell Plasma cell no germinal centre no memory cells no affinity maturation isotype switch to IgG3 and IgG2 via an unknown mechanism is described in the literature 44

45 Thymus-dependent-B-cell activation Binding of an antigen to its specific BCR Internalization of the Antigen/BCR complex Degradation of proteins and binding to MHCII 45

46 Thymus-dependent-B-cell activation Linked recognition = T-cell and B-cell recognise the same antigen (the B cell via the BCR; and the T-cell via the TCR) CD4 T-cell receptor MHC II Peptide 46

47 Thymus-dependent-B-cell activation CD4 T-cell receptor Peptide MHC II A T-cell does not express co-stimulatory signals for B cells until the cell is activated by a professional antigen-presenting cell (APC) (dendritic cell)! 47

48 Thymus-dependent-B-cell activation In particular IL-4! 48

49 Summary: TD-B-cell activation Signals for the B-cell activation: 1) Binding of an antigen to the BCR (B-cell receptor = membranous immunoglobulin) 2) Signals given by a T-cell, which recognised the MHC-IIpresented peptide via TCR and the co-receptor CD4: Stimulation of CD40 (B-cell) by CD40-ligand (T-cell) Cytokines 49

50 TD-B-cell activation Function of the effector cytokines: Proliferation of the B-cell Isotype-switch After activation B cells develop to: B-memory cells Antibody-secreting plasma cells 50

51 Primary humoral immune response After recognizing a specific antigen, T-cells are activated by dendritic cells in the lymph nodes Nature Immunology 7(2006), green = dendritic cells red = T-cells 51

52 Primary humoral immune response The primary activation of the naive B-cell by an activated Th-cell occurs also in the secondary lymph organs. B-cells immigrate via venules with high endothelium (HEV) into the lymph organs. 52

53 Primary humoral immune response After activation of the B cell by a specific T- cell, these cells generate a primary focus, in which both cell types proliferate. green = T-cells red = B-cells Nature Immunology 7(2006),

54 Primary humoral immune response Some of the activated B-cells migrate from the primary focus to the medulla strands Plasma cells (antibody production, Isotype-switch). 54

55 Secondary humoral immune response Some B- and T-cells leave the primary foci and move in a primary lymphfollicle, where they build germinal centres. The B-cells proliferate quickly (centroblasts) and pass through the somatic hyper mutation. 55

56 FDCs play an important role in affinity maturation 1) FDC = Follicular dendritic cells FDCs are found in the germinal centres and attract B-cells by the release of chemokines. FDCs present antigens not on MHC but by immune complexes bound to Fc- and complement receptors. 56

57 Tfh cells play an important role in affinity maturation In the light zone of the germinal centre B cells compete for binding to antigen complexes bound on FDCs and therefore for stimulation by Tfh cells needed for survival! Follicular T helper cells (Tfh) 57

58 Summary: Life cycle of B-lymphocytes Lymph follicle, Germinal centre 6 days Activation of the B-cell by T-cells Primary Foci 3 days Hypermutation, Affinity Affinitätsreifung maturation Rezirkulation recirculation Ab-release Low affinity Medulla strands Apoptosis Plasma - zellen cells Ab-release High affinity!! Gedächtnis Memory - zellen cells Sec. lymphatic organs long-lasting Langlebige Plasma plasma - zellen cells Bone marrow 58

59 Immunological memory Memory cells long-lasting Plasma cells B-memory cells (Memory B-cells) circulate. They need stimulation by an antigen for antibody production and divide continuously in low frequency. Long-lasting plasma cells occur after the second antigen contact They move into the bone marrow (important for their survival). They need no antigen for antibody production. They survive for years/decades. 59