1. PRODUCTION OF IMMUNOGLOBULINS

3. secondary response against antigen A primary response against antigen A level of antibodies napok primary response against antigen B Antigen A days Antigen A and B

4. Polyclonal antibody response Ag Immunserum Polyclonal antibody Ag Set of B-cells Activated B-cells Antibody- producing plasma-cells Antigen-specific antibodies

6. STRUCTURE OF IMMUNOGLOBULINS/ANTIBODIES COMPLEMENT ACTIVATION BINDING TO CELLS DEGRADATION TRANSPORT Light chain (L) Heavy chain (H) VL CL VH CH Antigen binding Variable domains Antigen Constans domains Effector functions

7. Antibodies with different isotypes differ in their Binding affinity, effector functions and their Transport. Carbohydrate antigens are usually recognized By IgM type antibodies. Differences in transport makes all the differece: Antibodies spec. to blood group antigens

8. Structures of the ABO blood group antigens Defined by specific enzymes inherited co-dominant genes (Mendelian rules)

9. Donors and recipients for blood transfusion - - - - - + - - - - ++ + + + +

10. Rhesus (Rh) blood group antigen (D) IgG type antibody- incomplete no direct agglutination but human immunglobulin-reactive 2. antibody can cause agglutination indirect agglutination POLYPEPTIDE TYPE ANTIGEN cytoplasm membrane extracellular space intracellular space

11. Pathological consequences of placental transport of IgG (hemolytic disease of the newborn)

12. Effects of agglutination in vivo ABO incompatibilityintravascular haemolysis ( complement mediated haemolysis) Rh incompatibilityhaemolytic disease of the newborn (erythroblastosis fetalis) ( opsonisation of red blood cells, which are then phagocytosed by macrophages and granulocytes) Rh profilaxis

13. ANTIBODY DIVERSITY

14. Multiple myeloma (MM) Plasma cell tumors – tumor cells reside in the bone marrow Produce immunoglobulins of monoclonal origin, serum concentration 50-100mg/ml Rodney Porter & Gerald Edelman 1959 – 1960 myeloma protein purification AMINO ACID SEQUENCE OF IMMUNOGLOBULINS 50 kDa Heavy chain 25 kDa Light chain Gel electrophoresis V ariable C onstant 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Reduction L H

15. GENETIC BACKGROUND OF ANTIBODY DIVERSITY VL VH Mechanism of the generation of variability? Different rules for encoding the variable and constant regions? Symmetric molecule  two identical VH and VL  both chromosomes encode the same sequence? S – S

16. Many GENES (10 000 – 100 000) V2V2V2V2 C2C2C2C2 VnVnVnVn CnCnCnCn V1V1V1V1 C1C1C1C1 1 GEN High rate of somatic mutations in the V-region VC GenGenGenGen Protein 1 GEN = 1 PROTEIN DOGMA OF MOLECULAR BIOLOGY CHARACTERISTICS OF IMMUNOGLOBULIN SEQUENCE THEORIES

17. MOLECULAR GENETICS OF IMMUNOGLOUBLINS A single C region gene encoded in the GERMLINE and separate from the V region genes Multiple choices of V region genes available A mechanism to rearrange V and C genes in the genome so that they can fuse to form a complete Immunoglobulin gene. In 1965, Dreyer & Bennett proposed that for a single isotype of antibody there may be: How can the bifunctional nature of antibodies be explained genetically? This was genetic heresy as it violated the then accepted notion that DNA was identical in every cell of an individual

18. The Dreyer - Bennett hypothesis V V V V V V V V V V V V V A mechanism to rearrange V and C genes in the genome exists so that they can fuse to form a complete Immunoglobulin gene C V C A single C region gene is encoded in the germline and separated from the multiple V region genes Find a way to show the existence of multiple V genes and rearrangement to the C gene

19. Approach Tools: A set of cDNA probes to specifically distinguish V regions from C regions DNA restriction enzymes to fragment DNA Examples of germline (e.g. placenta) and mature B cell DNA (e.g. a plasmacytoma/myeloma) C V V V V V V V V V Germline DNA C V V V V V Rearranged DNA

20. Liver cell B-cell 1.5. Kb B-cell V C 6.0 Kb V C 4.0 Kb DNA-extraction Digestion by restriction enzyme Gel electrophoresis Southern blot Experiment of Susumi Tonegawa 1975 Basel CKb6,0 1,5 V-probe 4,0 C V C-probe V

21. There are many variable genes but only one constant gene VCVVV GERM LINE V and C genes get close to each other in B-cells only C V VV B-CELL CONCLUSION

22. Ig gene sequencing complicated the model The structures of germline V L genes were similar for V , and V, However there was an anomaly between germline and rearranged DNA: Where do the extra 13 amino acids come from? CLCL VLVL ~ 95  ~ 100  L CLCL VLVL ~ 95  ~ 100  JLJL Some of the extra amino acids are provided by one of a small set of J or JOINING regions L CLCL VLVL ~ 208  L

23. Further diversity in the Ig heavy chain VLVL JLJL CLCL L CHCH VHVH JHJH DHDH L The heavy chain was found to have further amino acids (0 – 8) between the JH és CH genes D (DIVERSITY) region Each light chain requires 1 recombination events V L to J L Each heavy chain requires 2 recombination events J H to D H, V H to J H D H,

24. 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

25. During B-lymphocyte development JkJκJκJκJκJκJκVκVκVκVκVκVκ B-cell 1 JκJκVκVκ B-cell 2 40 Vκ 5 Jκ5 Jκ5 Jκ5 Jκ VκVκVκVκVκVκVκVκ JκJκ JκJκ JκJκ JκJκ Germ line SOMATIC REARRANGEMENT OF KAPPA (κ) CHAIN GENE SEGMENTS DNA

26. pA CCκCCκ E JJ Vκ-JκVκ-Jκ VκVκVκVκ P CCκCCκ JVκVκProtein mRNA CCκCCκ JVκVκ AAAA Translation EXPRESSION OF THE KAPPA CHAIN Primary RNA transcript CCκCCκ E JJVκVκ Leader Efficiency of somatic gene rearrangement?

27. 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

28. 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

29. D – J recombination V – DJ recombination VDJ –  δ transcription  δ translation Surrogate light chain V – J recombination VJ –  (or VJ - ) transcription  or translation B-sejt mIgD mIgM ORDER OF REARRANGEMENTS OF IMMUNOGLOBULIN GENE SEGMENTS Secreted IgM

30. Estimates of combinatorial diversity Taking account of functional V D and J genes: 65 VH x 27 DH x 6JH = 6,480 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. 6,480 x 320 = 2,073,600 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

31. 1.Combination of gene segments results in a huge number of various variable regions of the heavy and light chains expressed by different B-cells SOMATIC GENE REARRANGEMENT 2. Successful somatic rearrangement in one chromosome inhibits gene rearrangement in the other chromosome ALLELIC EXCLUSION 3. One B-cell produces only one type of heavy and one type of light chain COMMITMENT TO ONE TYPE OF ANTIGEN BINDING SITE 4. The B-cell pool consist of B-cells with differently rearranged immunoglobulin genes THE RESULT OF SOMATIC GENE REARRANGEMENTS INDEPENDENT OF ANTIGEN OCCURS DURING B-CELL DEVELOPMENT IN THE BONE MARROW

32. Evidence for allelic exclusion Allotypes can be identified by staining B cell surface Ig with antibodies a/a b/b a/b Y B b Y B a Y B b Y Y B ab Y B a AND ALLOTYPE- a polymorphism in the Heavy chain C region of Ig Suppression of H chain rearrangement by pre-B cell receptor prevents expression of two specificities of antibody per cell

33. Allelic exclusion is needed for efficient clonal selection All daughter cells must express the same Ig specificity otherwise the efficiency of the response would be compromised Suppression of H chain gene rearrangement helps to prevent the emergence of new daughter specificities during proliferation after clonal selection S. typhi Antibody S. typhi

34. Y Y YY Suppression of H chain gene rearrangement ensures only one specificty of Ab expressed per cell. Allelic exclusion prevents unwanted responses B Self antigen expressed by e.g. brain cells S. aureus Y Y Y Y Y B Y Y Y Y Y Y Y Anti S. aureus Antibodies Y Y Y Y Y Y Anti brain Abs One Ag receptor per cellIF there were two Ag receptors per cell Y Y Y Y Y Y Y Anti S. aureus Antibodies Prevents induction of unwanted responses by pathogens

35. Allelic exclusion is needed to prevent holes in the repertoire Exclusion of anti-brain B cells i.e. self tolerance Y Y B B One specificity of Ag receptor per cell S. aureus Anti-brain Ig AND anti-S. Aureus Ig Y Y Y B B IF there were two specificities of Ag receptor per cell Anti-brain Ig B B Deletion Anergy OR anti S.Aureus B cells will be excluded leaving a “hole in the repertoire” BUT Y Y Y B B

36. SYNTHESIS OF IMMUNOGLOBULINS ER Golgi mRNA Ribosome Leader sequence Membrane Ig Secreted Ig H and L chains are synthesized on separated ribosomes CHAPERONES

37. B – CELL ACTIVATION

38. Ligand SIGNAL Cross - linking Ligand Conformational change SIGNAL RECEPTOR MEDIATED CELL ACTIVATION

39. ligand kinase activation phosphorylation recruitment of adaptors CROSS – LINKING OF THE RECEPTOR INITIATES A SIGNALING CASCADESIGNAL Gene transcription Activation of transcription factors

40. (review) BCR signaling

41. Ig-  /CD79a Ig-  /CD79b ITAM: Immunoreceptor Tyrosine-based Activation Motif   Y Y Y Y ITAM ITAM Ig domain + CHO SIGNALING UNITS OF THE B-CELL RECEPTOR ITAM: YxxL x7 YxxI

42. KINETICS OF LYMPHOCYTE ACTIVATION ANTIGEN SIGNAL1. Co-receptor Adhesion molecule Cytokines SIGNAL2. Resting lymphocyte G 0 PTK activation RNA synthesis Free Ca++ Protein synthesis Protein phosphorylation DNA synthesis Lymphoblast 0 10sec 1min 5min 1hr 6 hrs 12 hrs 24 hrs Nyugvó limfocita G 0 proliferation DNA synthesis Effector cell Memory cell Transport Membrane change RNA and protein synthesis Resting lymphocyte G 0

43. Antigenic determinant C3d THE CO-STIMULATORY ROLE OF CR2 (CD21) COMPLEMENT RECEPTOR IN B – LYMPHOCYTES ANTIGEN CD21 CD19 YYYY TAPA=CD81 Enhanced B-cell activation B-CELL

44. THE NEURAMIC ACID RECEPTOR CD22 INHIBITS ACTIVATION THROUGH THE A B-CELL RECEPTOR B Cell Antigen Tissue cells Bacterium Mannose CD22 Neuraminic (sialic) acid Inhibited B cell activation

45. PLAZMA SEJT ANTIGÉN CITOKINEK B SEJT B cell differentiation is „helped” by T-cells IZOTÍPUS VÁLTÁS ÉS AFFINITÁS ÉRÉS CSAK T SEJT SEGÍTSÉGGEL MEGY VÉGBE HOGYAN LÁTJÁK A T SEJTEK AZ ANTIGÉNT? T SEJT (CD4+ helper)

46. 1)I read that monocytes have a phagocytic role. When is it? Don't they need to be activated and become macrophages to be able to phagocyte? 2) When do monocytes differentiate into macrophages? And when do they differentiate into dentritic cells? 3) I saw that sometimes macrophages are considered as innate immunity, and sometimes as acquired immunity. What is the difference within the macrophages? (when are they considered as innate, and when as 4) About the phagoctose process: what are ROI and NO? Aren't lysosomes enough to "digest" the antigen? 5) About the structure of the thymus: why is there macrophages and dentritic cells into it? What is their role?

47. 6) The recognition by toll receptors in unavoidable. But these toll receptors are part of the innate immunity. So are they used to attach the antigen to phagocytose it. And then, the antigen presentation enable the acquired immunity to make a receptor/antibody more precise for this specific antigen? 7) About the cytokines. I read that the IFN gamma are produced by T helper, and they activate the macrophages. Aren't the macrophages activated before? 8) Finally, I didn't understand few slides: Lecture 3-4: slides 31, 43, 44, 45