1. B CELL DEVELOPMENT AND ACTIVATION In healthy people, there are mature B cells with the capacity to make antibodies to virtually any antigen. Bone marrow is the primary lymphoid organ in which B cell development occurs.

2. Following initial development in bone marrow, mature B cells migrate to various secondary lymphoid tissues, including lymph nodes, spleen, gut- associated lymphoid tissue and blood. There, mature B cells can interact with antigen, become activated, and further differentiate into antibody-secreting cells

3. B and T cells undergo distinct differentiation pathways. B cells are generated in the bone marrow, with mature B cells, which are ready to respond to antigen, then exiting and migrating to lymph nodes and spleen. T cells are generated in the thymus.

4. The development of B cells, starting from hematopoietic stem cells and ending with cells that produce antibodies, can be divided into four phases:

5. Phase 1 – development of B cells in bone marrow This first phase of B cell development is the generation of B cells in bone marrow. There, stem cells develop into pro-B cells, then pre-B cells, and finally mature B cells, which exit the bone marrow and migrate to secondary lymphoid organs. This phase of B cell development is not driven by contact with antigen: antigen independent. The DNA rearrangements that result in a functional cell-surface immunoglobulin molecule occur during this phase.

6. Phase 1 – development of B cells in bone marrow

7. Stem cells have both their H-chain and L-chain genes in germ-line, un-rearranged configuration The earliest cell that has made the commitment to the B cell lineage is the pro-B cell: pro-B cells have begun to rearrange their H-chain gene.

8. Once a B lineage cell expresses cell surface H-chain (  ) it is defined as a pre-B cell. However, the early pre-B cell receptor is not the final form of surface immunoglobulin: H-chain + surrogate light chain (molecule that mimics L-chain)

9. Further DNA rearrangements result in the formation of a functional L-chain, then IgM (H-chain + L-chain) is expressed on the cell surface. When a cell has productive rearrangements of both H- and L-chains) it becomes an immature B cell:

10. This first phase of B cell development in bone marrow is dependent on association with stromal cells. Stromal cells are non-lymphoid cells that provide an appropriate microenvironment for B cell development. Bone marrow stromal cells produce both cell surface- stimulatory molecules, as well as growth factors and cytokines, which help drive B cell development.

11. For a B cell to survive this phase of development, it must have productive rearrangements of both H-chain and L- chain. Failure to do this results in cell death - cells that have unproductive rearrangements (such as rearrangements that are not in a correct reading frame) are eliminated. A given B cell can undergo repeated rearrangements.

12. The rearrangements that result in functional H- and L-chains occur in a specific order:

13. Expression of a functional B cell receptor protein on the cell surface stops further rearrangement of the gene encoding that product:

14. Phase 2 – elimination of self-reactive cells Once B cells express a functional cell surface receptor for antigen (immature/mature B cell stage) they have the potential to be stimulated by contact with antigen, and to become antibody-secreting cells. Since the DNA rearrangements that result in functional H-chain and L-chain are not antigen-driven, a fraction of immature B cells will have a BCR that, by chance, reacts with some component of self - self antigen reactive cells These immature B cells are removed by clonal deletion, either in the bone marrow, or shortly after leaving the bone marrow.

15. Encounter with self-antigen results in apoptosis (death), or anergy (unresponsiveness) B cells that survive this step express surface IgD as well as IgM: mature B cells Goodnow experiments

16. After elimination of self-reactive B cells, mature cells, which express both cell surface IgM and IgD, are ready to leave the bone marrow, can interact with antigen in secondary lymphoid organs.

17. Phase 2 – elimination of self-reactive B cells, generation of mature IgM+, IgD+ cells:

18. Phase 3 – activation of B cells on contact with antigen Following the generation of a functional B cell receptor for Ag, and the removal of self-reactive cells, mature Ag- responsive B cells (IgM+, IgD+) emigrate from bone marrow. These mature B cells go to secondary lymphoid organs.

19. In the lymph node, B cells gather in primary lymphoid follicles, where they receive viability-promoting signals, interacting with follicular dendritic cells, and wait for antigen B cells enter lymph nodes via high endothelial venules (HEV) to reach these primary follicles. B cells can recirculate out via the lymphatic circulation, and back into blood.

20. What is meant by ‘B cell activation’? Go -> G1 of cell cycle (increase in size) upregulate – MHC class II – costimulatory molecules (B7-2) – adhesion molecules (ICAM1) – cytokine receptors (IL-2R) migrate to outer T zone – altered response to chemokines become receptive to T cell help – protected from fas enter mitosis if provided with submitogenic doses of other stimuli (LPS, CD40L, IL-4)

21. Types of antigen T-independent (TI) antigens - Type I –induce division/differentiation independently of BCR (polyclonal mitogens) LPS, bacterial (CpG) DNA T-independent (TI) antigens - Type II –induce division/differentiation by BCR signaling alone bacterial polysaccharides, repeating surface molecules on viruses T-dependent (TD) antigens –activate via BCR but depend on additional signals from helper T cells to cause division/differentiation any antigen containing protein Most pathogens contain both T-I and T-D antigens Only TD antigens can induce Germinal Center responses

22. T cell mitogenic BcR signal 'activation' signal but not mitogenic mitogenesis differentiation present Ag T-independent (TI) T-cell dependent (TD) Types of B cell Antigens -> most pathogens contain both T-independent and T-dependent antigens

23. Innate features of pathogens act as B cell costimulators pathogen multivalency –provides a level of BCR crosslinking optimal for activation many pathogens activate TLRs –TLR signaling synergizes with BCR signal many pathogens activate the complement cascade and become C3d coated –complement receptor (CR) crosslinking synergizes with BCR signal

24. Antigen-C3d complexes cross-link BCR and CR2- CD19 complex - increase sensitivity to antigen

25. Current Paradigm : B cellplasma cellsB cell plasma cells DC Emerging Model: DC B cell T-Independent (type II) Responses Multivalent Antigen

26. B cellplasma cells T-Dependent Responses DC Antigen T cell Dendritic Cell (DC) internalizes antigen (Ag), processes into peptides, presents peptides together with MHC molecules to T cells B cell binds Ag via surface Ig, transmits BCR signals and presents peptides to T cells, receives T cell help (growth and differentiation factors) Secretes Antibody (Ab)

27. Antigen-specific B cells are detained in the T cell-areas, where they interact with antigen, and with antigen-specific activated helper T cells. Stimulated antigen-specific B cells then proliferate and differentiate, eventually forming plasma cells and germinal centers:

28. B cells are antigen-presenting cells BCR cross-linking induces antigen internalization to endosomes antigen is proteolysed to peptides peptides associate with MHC class II MHC class II-peptide complexes traffic to surface of B cell\ B cells present antigen recognized by their BCR ~10 5 x more efficiently than other antigens

29. B cell antigen presentation and the concept of linked help protein sugar T Sugar Specific B cell Protein Specific T cell Antigen internalization, proteolysis -> presentation of peptides

30. HEL-specific (Ig-tg) B cells HEL-specific (TCR7 tg) T cells Interactions between antigen-specific B and T cells 1 day after HEL antigen injection

31. Onset of B-T interaction HEL-specific B cells HEL-specific T cells

32. B cells can interact with multiple T cells HEL-specific B cells HEL-specific T cells

33. Cardinal features of B - T interaction

34. However, signaling via the BCR alone is not sufficient to activate the B cell - Second signals (co-stimulatory signals) are necessary for activation.

35.  -  or  -deficient mice and human do not undergo isotype switching They only have IgM: hyper IgM syndrom

36. Laâbi, Y. and A. Strasser. Science 289:883, 2000 activation signal

37. Phase 4 – differentiation to antibody-secreting cells Some of the progeny of these antigen-activated B cells differentiate into IgM-secreting plasma cells (antibody- secreting cells).

38. Plasma cells: –terminally-differentiated cells –derived from activated B cells or memory cells –loaded with endoplasmic reticulum –devoted to protein (antibody) synthesis –no longer express surface immunoglobulin or MHC class II –no longer responsive to antigen contact –live for several weeks –migrate away from the site of initial contact with helper T cells, either to the medullary cords of the lymph nodes or to the bone marrow

39. Plasma cells Surface Surface High rate Growth Somatic Isotype Ig MHC II Ig secretion hypermut’n switch B B Mature B cell Plasma cell HighYes No Yes Yes Yes LowNo Yes No No No

40. Other antigen activated B cells give rise to germinal centers (GC), zones of proliferating activated B cells:

41. These germinal centers (GC) contains: –proliferating (D - centroblasts) B cells express low levels of Ig, especially IgD –differentiating (L - centrocytes) B cells express high levels of Ig

42. B cells can interact with an antigen that is bound to the surface of follicular dendritic cells in the lymph nodes. These cells trap and concentrate antigen, maximizing the interaction of antigen with B cells

43. Follicular Dendritic cells (stained blue)in the Germinal Centre

44. Retention of Antigens on Follicular Dendritic Cells Radiolabelled antigen localises on the surface of Follicular Dendritic cells and persists there, without internalisation, for very long periods

45. Maturation of Follicular Dendritic cells Club-shaped tips of developing dendritesFiliform dendrites Bead formation on dendrites

46. The veils of antigen-bearing dendritic cell surround the beads and the layer of immune complexes is thickened by transfer from the dendritic cell. These beads are then released and are then called ICCOSOMES Iccosome formation and release DC veils Iccosomes (black coated particles) bind to and are taken up by B cell surface immunoglobulin

47. Y Y Y Iccosomes bearing different antigens B Uptake of Iccosomes/Antigen by B cells Anti- B cell Surface Ig captures antigen Cross-linking of antigen receptor activates B cell Activated B cell expresses CD40 CD40

48. B B 2. Binding and internalisation via cell surface Ig induces the expression of CD40 3. Antigen enters exogenous antigen processing pathway and is degraded 4. Peptide fragments of antigen are loaded onto MHC molecules intracellularly. MHC/peptide complexes are then expressed at the cell surface Fate of Antigens Internalised by B cells 1. Capture by antigen specific Ig maximises uptake of a single antigen

49. This selection process involves competition for both antigen and for helper T cells. Antigen is trapped on the surface of follicular dendritic cells in the form of immune complexes (antigen + antibody complexes). B cells that bind to Ag with high affinity live, others die by apoptosis.

50. Centrocytes interact with T cells by presenting processed antigen to them via their MHC class II molecules. Centrocytes that receive co-signaling (via CD40, MHC class II and cytokines), as well as signaling via their antigen receptor survive Centrocytes that do not bind antigen and T cells with sufficient affinity die by apoptosis.

52. Germinal centers are where isotype switching and somatic hypermutation occur. Somatic hypermutation: –rapid mutation (hypermutation) of immunoglobulin genes –results in antigen-binding affinity that is higher, or lower, than its original binding affinity –selection by antigen results in the generation of BCR with increased affinity for antigen Only those B cells that have enhanced their antigen receptor’s binding affinity survive.

53. Isotype switching - change in the type of H-chain that is used by a given B cell, also occurs in germinal centers:

54. Isotype switching involves DNA rearrangements - replacement of one H-chain class gene with another Isotype switching also occurs in germinal centers Isotype switching is guided by the pattern of cytokines that are produced by helper T cells:

55. These somatically mutated and isotype switched B cells can then continue to differentiate into memory cells or plasma cells, producing: –IgG or other switched isotypes –much higher affinity Ab, due to somatic hypermutation increasing the antigen binding affinity –migrate from the secondary lymphoid organs to the bone marrow, where cytokines (IL-6, IL-11) produced by bone marrow stromal cells keep these cells viable and producing Abs

56. Together, these processes result in a 1-2 log increase in the number of antigen-specific B cells (clonal selection and expansion), an increase in antibody binding affinity for antigen (somatic hypermutation), and the expression of new Ig subclasses (Ig isotype switching):

57. Other germinal center B cells develop into memory B cells: –quiescent, long-lived –increased in frequency following primary responses –found in secondary lymphoid organs (spleen, lymph nodes, Peyer’s patch) –posses high-affinity, isotype-switched (IgG, IgA, IgE) antigen receptors –form a pool of cells ready to mount a rapid secondary antibody response, on subsequent exposure to antigen:

58. The combined result of clonal selection (expansion of pool size of Ag-reactive cells), somatic hypermutation, isotype switching, and the generation of memory cells, is the creation of a pool of cells that can respond rapidly and vigorously to subsequent contact with antigen, with high- affinity, IgG and IgA antibodies.

61. B cell cancers mirror these different stages of B cell development

62. Immunoglobulins:Structure and Function Definition: Glycoprotein molecules that are produced by plasma cells in response to an immunogen and which function as antibodies Immune serum Ag adsorbed serum 11 22  + - albumin globulins Mobility Amount of protein 

63. General Functions of Immunoglobulins Effector functions –Fixation of complement –Binding to various cells (Usually require Ag binding) Ag binding –Can result in protection

64. Immunoglobulin Structure Heavy & Light Chains Disulfide bonds –Inter-chain –Intra-chain C H1 VLVL CLCL VHVH C H2 C H3 Hinge Region Carbohydrate Disulfide bond

65. Immunoglobulin Structure Variable & Constant Regions –V L & C L –V H & C H Hinge Region C H1 VLVL CLCL VHVH C H2 C H3 Hinge Region Carbohydrate Disulfide bond

66. Structure of the Variable Region Hypervariable (HVR) or complimentarity determining regions (CDR) HVR3 FR1 FR2 FR3 FR4 HVR1 HVR2 Variability Index 25 75 50 100 Amino acid residue 150 100 50 0 Framework regions

67. Immunoglobulin Fragments: Structure/Function Relationships Fab –Ag binding –Valence = 1 –Specificty determined by V H and V L Papain Fc Fab Fc –Effector functions

68. Immunoglobulin Fragments: Structure/Function Relationships Ag Binding Complement Binding Site Placental Transfer Binding to Fc Receptors

69. Immunoglobulin Fragments: Structure/Function Relationships Fab –Ag binding Fc –Effector functions F(ab’) 2 Pepsin Fc Peptides F(ab’) 2

70. Human Immunoglobulin Classes IgG - Gamma heavy chains IgM - Mu heavy chains IgA - Alpha heavy chains IgD - Delta heavy chains IgE - Epsilon heavy chains

71. Human Immunoglobulin Subclasses IgG Subclasses –IgG1 - Gamma 1 heavy chains –IgG2 - Gamma 2 heavy chains –IgG3 - Gamma 3 heavy chains –IgG4 - Gamma 4 heavy chains IgA subclasses –IgA1 - Alpha 1 heavy chains –IgA2 - Alpha 2 heavy chains

72. Human Immunoglobulin Light Chain Types Kappa (  ) Lambda (  )

73. Human Immunoglobulin Light Chain Subtypes Lambda light chains –Lambda 1 (  1) –Lambda 2 (  2) –Lambda 3 (  3) –Lambda 4 (  4)

74. Immunoglobulins Nomenclature –IgM (kappa) –IgA1(lambda 2) –IgG Heterogeneity

75. Monomeric IgM IgM only exists as a monomer on the surface of B cells C  4 contains the transmembrane and cytoplasmic regions. These are removed by RNA splicing to produce secreted IgM Monomeric IgM has a very low affinity for antigen C4C4 C3C3 C2C2 C1C1 N.B. Only constant heavy chain domains are shown

76. C  3 binds C1q to initiate activation of the classical complement pathway C  1 binds C3b to facilitate uptake of opsonised antigens by macrophages C  4 mediates multimerisation (C  3 may also be involved) C4C4 C3C3 C2C2 C1C1 N.B. Only constant heavy chain domains are shown Polymeric IgM IgM forms pentamers and hexamers

77. C C C C C C Multimerisation of IgM C4C4 C3C3 C2C2 C C C4C4 C3C3 C2C2 C C C4C4 C3C3 C2C2 C C C4C4 C3C3 C2C2 C C C4C4 C3C3 C2C2 C C ss ss ss C C ss 1. Two IgM monomers in the ER (Fc regions only shown) 2. Cysteines in the J chain form disulphide bonds with cysteines from each monomer to form a dimer 3. A J chain detaches leaving the dimer disulphide bonded. 4. A J chain captures another IgM monomer and joins it to the dimer. 5. The cycle is repeated twice more 6. The J chain remains attached to the IgM pentamer.

78. Antigen-induced conformational changes in IgM Planar or ‘Starfish’ conformation found in solution. Does not fix complement Staple or ‘crab’ conformation of IgM Conformation change induced by binding to antigen. Efficient at fixing complement

79. IgM facts and figures Heavy chain:  - Mu Half-life: 5 to 10 days % of Ig in serum:10 Serum level (mgml -1 ): 0.25 - 3.1 Complement activation:++++ by classical pathway Interactions with cells: Phagocytes via C3b receptors Epithelial cells via polymeric Ig receptor Transplacental transfer: No Affinity for antigen:Monomeric IgM - low affinity - valency of 2 Pentameric IgM - high avidity - valency of 10

80. IgD facts and figures IgD is co-expressed with IgM on B cells due to differential RNA splicing Level of expression exceeds IgM on naïve B cells IgD plasma cells are found in the nasal mucosa - however the function of IgD in host defence is unknown - knockout mice inconclusive Ligation of IgD with antigen can activate, delete or anergise B cells Extended hinge region confers susceptibility to proteolytic degradation Heavy chain:  - Delta Half-life: 2 to 8 days % of Ig in serum:0.2 Serum level (mgml -1 ): 0.03 - 0.4 Complement activation:No Interactions with cells: T cells via lectin like IgD receptor Transplacental transfer: No

81. IgA dimerisation and secretion IgA is the major isotype of antibody secreted at mucosal sufaces Exists in serum as a monomer, but more usually as a J chain- linked dimer, that is formed in a similar manner to IgM pentamers. J C C S S S S C C S S S S C C ss IgA exists in two subclasses IgA1 is mostly found in serum and made by bone marrow B cells IgA2 is mostly found in mucosal secretions, colostrum and milk and is made by B cells located in the mucosae

82. Epithelial cell J C C S S S S C C S S S S C C ss Secretory IgA and transcytosis B J C C S S S S C C S S S S C C ss J C C S S S S C C S S S S C C ss J C C S S S S C C S S S S C C ss pIgR & IgA are internalised ‘Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR - the secretory component J C C S S S S C C S S S S C C ss IgA and pIgR are transported to the apical surface in vesicles B cells located in the submucosa produce dimeric IgA Polymeric Ig receptors are expressed on the basolateral surface of epithelial cells to capture IgA produced in the mucosa

83. IgA facts and figures Heavy chains:  1  or  2 - Alpha 1 or 2 Half-life: IgA1 5 - 7 days IgA2 4 - 6 days Serum levels (mgml -1 ): IgA1 1.4 - 4.2 IgA2 0.2 - 0.5 % of Ig in serum:IgA1 11 - 14 IgA2 1 - 4 Complement activation:IgA1 - by alternative and lectin pathway IgA2 - No Interactions with cells: Epithelial cells by pIgR Phagocytes by IgA receptor Transplacental transfer: No To reduce vulnerability to microbial proteases the hinge region of IgA2 is truncated, and in IgA1 the hinge is heavily glycosylated. IgA is inefficient at causing inflammation and elicits protection by excluding, binding, cross-linking microorganisms and facilitating phagocytosis

84. IgE facts and figures IgE appears late in evolution in accordance with its role in protecting against parasite infections Most IgE is absorbed onto the high affinity IgE receptors of effector cells IgE is also closely linked with allergic diseases Heavy chain:  - Epsilon Half-life: 1 - 5 days Serum level (mgml -1 ): 0.0001 - 0.0002 % of Ig in serum:0.004 Complement activation:No Interactions with cells: Via high affinity IgE receptors expressed by mast cells, eosinophils, basophils and Langerhans cells Via low affinity IgE receptor on B cells and monocytes Transplacental transfer: No

85. The high affinity IgE receptor (Fc  RI)  chain  chain 22 SS SS SS C1C1 C1C1 C2C2 C2C2 C3C3 C3C3 C4C4 C4C4 C1C1 C1C1 C2C2 C2C2 C3C3 C3C3 C4C4 C4C4 The IgE - Fc  RI interaction is the highest affinity of any Fc receptor with an extremely low dissociation rate. Binding of IgE to Fc  RI increases the half life of IgE C  3 of IgE interacts with the  chain of Fc  RI causing a conformational change.

86. IgG facts and figures Heavy chains:  1  2  3  4 - Gamma 1 - 4 Half-life: IgG1 21 - 24 days IgG2 21 - 24 days IgG3 7 - 8 days IgG4 21 - 24 days Serum level (mgml -1 ): IgG15 - 12IgG2 2 - 6 IgG3 0.5 - 1IgG4 0.2 - 1 % of Ig in serum:IgG145 - 53IgG2 11 - 15 IgG3 3 - 6IgG4 1 - 4 Complement activation:IgG1+++ IgG2 + IgG3 ++++ IgG4 No Interactions with cells: All subclasses via IgG receptors on macrophages and phagocytes Transplacental transfer: IgG1++IgG2 + IgG3 ++IgG4 ++

87. Carbohydrate is essential for complement activation Subltly different hinge regions between subclasses accounts for differing abilities to activate complement C1q binding motif is located on the C  2 domain

88. Fc  receptors ReceptorCell typeEffect of ligation Fc  RI Macrophages Neutrophils, Eosinophils, Dendritic cells Uptake, Respiratory burst Fc  RIIA Macrophages Neutrophils, Eosinophils, Platelets Langerhans cells Uptake, Granule release Fc  RIIB1 B cells, Mast CellsNo Uptake, Inhibition of stimulation Fc  RIIB2 Macrophages Neutrophils, Eosinophils Uptake, Inhibition of stimulation Fc  RIII NK cells, Eosinophils, Macrophages, Neutrophils Mast cellsInduction of killing (NK cells) High affinity Fc  receptors from the Ig superfamily: