1. PERIPHERAL LYMPHOID ORGANS Spleen Lymph nodes Epithelial cell – associated lymphoid tissues Skin-associated lymphoid tissue (SALT) Mucosa-associated lymphoid tissue (MALT) Gut-associated lymphoid tissue (GALT) Bronchial tract-associated lymphoid tissue(BALT)

2. STRUCTURE OF THE SPLEEN NO LYMPHOID CIRCULATION Filtration of blood borne antigens RBC, platelet, granulocyte

3. Collagen capsule Marginal sinus (phagocytes) Paracortex cortex Trabecula High endothelial venule (HEV) Afferent lymph Efferent lymph Primary follicle (no Ag) B CELLS Secondary follicle (Ag) Germinal center (GC) B CELLS T CELLS STRUCTURE OF LYMPH NODES Plasma cell Memory B cell Mature,naive B cell T CELLS

4. Antigen Secretory IgA Pathogenic factors CYTOKINES IL-8 MCP-1 TNF IgA LN Follicle B-cells T-cells Villi MUCOSAL SURFACES 400m 2 200 times larger than skin surface MALT antibody producing cells = Spleen + lymph nodes + bone marrow

5. ORGANIZATION OF THE IMMUNE SYSTEM CENTRAL (PRIMARY) LYMPHOID ORGANS –Bone marrow –Thymus DEVELOPMENT TO THE STAGE OF ANTIGEN RECOGNITION (TCR, BCR, self) PERIPHERAL (SECONDARY) LYMPHOID ORGANS –Spleen –Lymph nodes –Skin-associated lymphoid tissue (SALT) –Mucosa-associated lymphoid tissue (MALT) –Gut-associated lymphoid tissue (GALT) –Bronchial tract-associated lymphoid tissue (BALT) ACTIVATION AND DIFFERENTIATION TO EFFECTOR CELLS (filtration, foreign, connection, collect the antigens) BLOOD AND LYMPH CIRCULATION (lymphocytes – sentinels) –Lymphatics – collect leaking plasma (interstitial fluid) in connective tissues –Lymph – cells and fluid –No pump– one way valves ensure direction – edema –Several liters (3 – 5) of lymph gets back to the blood daily – vena cava superior LYMPHOCYTES CONGREGATE IN SPECIALIZED TISSUES

6. LYMPHOCYTE RECIRCULATION 1. Homing – most lymphocytes reside in lymphoid organs, few in circulation 2. Recruitment - chemokines Few antigen-specific lymphocytes should migrate to the site of antigen ANTIGEN RECOGNITION (lymph node) The appropriate effector lymphocyte populations shoud migrate to the site of antigen EFFECTOR/MEMORY CELLS (tissue, lymphoid tissue) 3. Migration Among tissues, organs Lymph node - Lymph node, Lymph node - Tissues BLOOD CIRCULATION - LYMPHATICS 4. Adhesion molecules HOMING RECEPTORS Antigen independent appearance (dependent on activation state of lymphocyte)Selectins Integrins Ig supergene family molecules LIGANDS FOR VASCULAR ENDOTHELIAL CELL RECEPTORS Adressin ligands INTERACTION WITH THE EXTRAVASCULAR CONNECTIVE TISSUE Binding, detachement

7. NAIVE LYMPHOCYTES Homing to lymphoid tissues Homing receptor on naive lymphocyte L-selectin – carbohydrate binding Ligand on HEV- mucin-like adressin CD34+ and GlyCAM-1 HIGH ENDOTHELIAL VENULES HEV Lymphocytes slow down and bind to HEV LFA-1 integrin – ICAM-1/2 Ig family CCL21 chemokine and CCR7 chemokine receptor MIGRATION OF LYMPHOCYTES HEV CD34 L-selectin Naive lymphocyte

8. EFFECTOR/MEMORY LYMPHOCYTES Return to the site of stimulation (antigen) Mucosal surface- MADCAM-1 Retention in spleen, lymph node LFA-1 – ICAM-1/2 integrin – cell and extracellular matrix Migration through activated endothelial cells of inflammed tissues Lamina propria in gut Mucosal epithelium Dermis in skin Activated endothel LFA-1 VLA-4 VCAM-1ICAM-1 Activated/effector/memory lymphocyte ALTERED EXPRESSION OF CELL SURFACE ADHESION MOLECULES

9. 1.The central lymphoid organs are not connected to lymphatics – Isolated from the environment 2.The spleen has no lymph circulation – immune response to blood borne antigens 3.HEV – high endothelial venules – special entry sites of blood circulating lymphocytes to peripheral lymphoid organs 4.1 lymph node circle/hour, 25 billion lymphocytes (25x10 9 )/lymph nodes/day BONE MARROWTHYMUS MALT SALT BALT HEV BLOOD Thoracic duct Lymphatics TISSUES SPLEEN LYMPH NODES MIGRATION OF LYMPHOCYTES IN CENTRAL AND PERIPHERAL LYMPHOID ORGANS

11. B – CELL ACTIVATION

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

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

14. a a antigen binding mIg molecule HH LL V V V V   Ig-  /Ig-  heterodimer THE IgM B-CELL RECEPTOR Signal transduction Lyn Kinases Syk Btk SHP-1 Phosphatases SLP-65/BLNK PLC HS1 Vav Adaptors + substrates

15. 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

16. Ag RECENT MODEL OF B-CELL RECEPTOR MEDIATED SIGNALING Subsequent activation of 2 kinases = ITAM 1. Cross-linking Lyn 2. Src-family kinase activation 4. SLP phosphorylation + Ca release SLP P Calcium release P P P P and ITAM phosphorylation 3. Syk recruitment and activation Syk Syk PP

17. 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

18. 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

19. 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

20. ANTIBODY DIVERSITY

21. 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

22. 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

23. 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 for the same sequence? S – S

24. 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

25. Proof of 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

26. 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

27. Many GENES (10 000 – 100 000) V2V2V2V2C VnVnVnVnC V1V1V1V1C 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

28. 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 VCKb6,0 1,5 V-probe 4,0 C V C-probe Experiment of Susumi Tonegawa 1975 Basel

29. 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 Fehérje GénGénGénGén GÉN SZEGMENSEK SZOMATIKUS ÁTRENDEZŐDÉSE EGY GÉNNÉ

30. 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

31. 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 2 recombination events V L to J L and V L J L to C L Each heavy chain requires 3 recombination events J H to D H, V H to J H D H, and V H J H D H to C H

32. HOW MANY IMMUNOGLOBULIN GENE SEGMENTS Variable (V) 132/40 105/30 123/65 Diversity (D)0027 Joining (J)549 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

33. During B-lymphocyte development JkJκJκJκJκJκJκVκVκVκVκVκVκ B-cell 1 JκJκVκVκ B-cell 2 80 Vκ 4 Jκ VκVκVκVκVκVκVκVκ JκJκ JκJκ JκJκ JκJκ Germ line SOMATIC REARRANGEMENT OF KAPPA (κ) CHAIN GENE SEGMENTS DNA

34. 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?

35. During B-cell development VH2 JH 120 VH 4 JH VH1VH3 DJH 12 D DDD JH DD SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS DD VH1VH2VH3 VH1VH2

36. 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

37. 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

38. Estimates of combinatorial diversity Taking account of functional V D and J genes: 40 VH x 27 DH x 6JH = 6,480 combinations D can be read in 3 frames: 6,480 x 3 = 19,440 combinations 29 V  x 5 J  = 145 combinations 30 V  x 4 J = 120 combinations = 265 different light chains If H and L chains pair randomly as H 2 L 2 i.e. 19,440 x 265 = 5,151,600 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

39. 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

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