1. B Cell Activation and the Humoral Immune Response

2. Each B cell after stimulation with Ag, proliferate and produces 4000 daughter cells and these cells produced Ab molecules.

3. Humoral (Antibody-Mediated) Immunity
Involves production of antibodies against foreign antigens.
Antibodies are produced by a subset of lymphocytes called B cells.
B cells that are stimulated will actively secrete antibodies and are called plasma cells.
Antibodies are found in extracellular fluids (blood plasma, lymph, mucus, etc.) and the surface of B cells.
Defense against bacteria, bacterial toxins, and viruses that circulate freely in body fluids, before they enter cells.
Also cause certain reactions against transplanted tissue.

5. Antibody Protection of the Host

6. Humoral immune response properties
Humoral immune response against non-protein Ag dose not need to helper T cells.
Activation of B cells resulted in memory or plasma cells.
Isotypes switching and affinity maturation are helper T cell dependent.
The quantity and quality of primary and secondary humoral immune response is different.

7. Dynamics of Antibody Production
Initial antibody produced in IgM
Lasts days
Followed by production of IgG
Lasts 4-5 days
Without continued antigenic challenge antibody levels drop off, although IgG may continue to be produced.

9. Comparison of Primary and Secondary Responses
Time course
Antibody titer
Antibody class
Antibody affinity
Study Guide
Compare and contrast the primary and secondary immune responses.

10. Immunologic Memory Virgin lymphocyte pool PRIMARY RESPONSE
effector cells
memory cell pool
Study Guide
What is the significance of immunologic memory?
SECONDARY RESPONSE
effector cells
memory cell pool

12. ITAM, immuno receptor tyrosin- based activation motif

13. CR2,CD19,CD81= BCR Complex, in this model of activation using complement receptor CD19 is phosphorylated and enhance B cell activation. In this way antigen is 1000 times more immunogen

14. Types of B cell Antigens
T-independent (TI)
T-cell dependent (TD)
present
T cell Ag
T-independent antigens
bacterial polysaccharides
low dose LPS
repeating surface molecules on viruses
TLR signaling can synergize with this type of signal
T-dependent antigens
usually require a protein component, but B cell may recognize a non-protein part of the complex (e.g. a surface sugar or lipid of a virus)
most pathogens contain both T-independent and T-dependent antigens
mitogenic
'activation' signal
mitogenesis
BcR signal
but not mitogenic
differentiation

15. Types of antigen T-independent (TI) antigens T-dependent (TD) antigens
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

17. B cell activation Some responses require T help whereas other do not
Thymus-dependent because T cells are required
Thymus-independent because T cells are not needed
T-independent antibody response generally have 1. no memory 2. no isotype switching 3. no somatic mutations

18. T-Independent (type II) Responses
Old Paradigm:
Multivalent Antigen
B cell
B cell
plasma cells
B cell
plasma cells
DC/Mø
New Model:
(BAFF)

19. T-Dependent Responses
Antigen DC
T cell
T cell
B cell
plasma cells
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)

20. 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 ~105 x more efficiently than other antigens ( BCR needs lower dose)

21. B cells are antigen-presenting cells

22. B cell antigen presentation and the concept of linked help
protein
sugar T
Protein Specific
T cell
CD40L
Cytokines
BCR
endosome
MHC II
Polysaccharide Specific
B cell

23. Cardinal features of B - T interaction

24. Role of CD40 in B cell activation
TCR triggering up-regulates CD40L on T cell
CD40 signaling promotes B cell activation
CD40L-deficiency = 'hyper-IgM syndrome' (no isotype switching, no Germinal Centers)
CD40 signaling also important in DC, MØ function
Germinal center formation is CD40-CD40L interaction dependent
increased expression of cell cycle
molecules,
survival molecules, cytokines,
promotes isotype switching

25. B-T interaction summary
BCR signals induce costimulatory molecules (B7.2/CD86, ICAM1 etc), prosurvival molecules (e.g. Bclxl), entry to G1, antigen presentation
CD40L triggers CD40 -> synergizes with BCR signals to promote mitosis
T cell derived cytokines promote proliferation, differentiation, isotype switching:
IL2, IL6 promote differentiation
IL4 -> IgG1, IgE
IFNg -> IgG2a, IgG3
TGFb and IL5 -> IgA
Many other molecules involved in T-B interactions
ICOS/ICOSL (essential for GC response)
SLAMM/SAP (promote prolonged B-T interaction essential for GC response)
ICAM1/LFA1, CD30/CD30L, CD27L/CD27, OX40L/OX40, ...

26. 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
Some induce type I IFNs
IFNa/bR signaling synergizes with BCR
Many activate the complement cascade and become C3d coated
- complement receptor (CR) crosslinking synergizes with BCR signal

27. After appropriate activation the B cell differentiates into an antibody secreting cell, also known as a Plasma Cell
B cell
Plasma Cell
membrane Ig
secretory Ig
blimp1
XBP1
Electron microscopy Images from Molecular Biology of the Cell
Alberts et al., 3rd Edition., 1994

28. Production of membrane vs secreted Ig

29. B cell antibody response - clonal replication enters into a
higher order upon plasma cell differentation
3 days
12 divisions
1 day
differentiation
1 day
104 Ab/cell/sec
naive
B cell
activated B cells
plasma cells
antibodies
4,096
>1012 1
212 = 4,096
bacteria - dividing every ~60 min
5 days = 2120 divisions = 1.3x1036

30. Plasma Cells are antibody secreting cells
Two types:
1. Plasma cells generated early in the primary response
- short-lived (~ few days)
- typically low affinity
- form in T-independent and T-dependent responses
- home to red pulp of spleen, medullary cords of lymph nodes
- IgM but also IgG and other isotypes
2. Plasma cells generated later in the primary response and that predominate in secondary responses
- arise predominantly from germinal centers (in primary) or from memory B cells (in secondary)
- long-lived (possibly several months)
- often home to bone marrow, gut, lactating mammary gland
- predominantly isotype switched

31. BCR isotype switching
AID dependent

32. Ig Heavy chain class (isotype) switching
neutralization
neutralization

33. Affinity Maturation
Affinity maturation occurs in germinal centers and is the result of somatic hypermutation of Ig-genes in dividing B cells followed by selection of high affinity B cells by antigen displayed by FDCs
The high affinity B cells emerging in germinal centers give rise to long-lived plasma cells and memory B cells

34. Antibody Affinity Maturation
Germinal Center
Low affinity B
High affinity B
mutation
selection VH VL Ag VH VL VH VL Ag VH VL

41. Germinal Center Organization
FDC
High Affinity T T
memory B cell
Light Zone
FDC CC CC
FDC CC T
plasma cell
Selection, proliferation, differentiation CC
FDC
FDC CC CC MØ
FDC MØ CB CB CB CB CB
Dark Zone CB CB CB
Certainly a lot of work has been done on fixed tissue sections and with isolated cells and this has led to a general model that proliferation and somatic hypermutation occur predominantly in the dark zone while selection occurs in the light zone. Strong experimental evidence has been obtained to indicate that there is a movement of cells from dark to light zone. In our own studies, work that was presented in workshop here 2 yrs ago by Chris Allen, we showed that CB positioning in the DZ is CXCR4 dependent whereas movement to the LZ involves CXCR5. But, we have known very little about the dynamics… CB
T – T cell
FDC – follicular dendritic cell
MØ – macrophage
CC - centrocyte
CB - centroblast
Proliferation, somatic hypermutation CB CB CB CB CB CB
Low Affinity B
CC, doubling time 6-12 hr , within 5 days each B cell produces 5000 cells, FDC:CR-1,2,3+, MHCII+

43. Germinal Centers
Function: to generate B cells that produce antibodies with increased affinity for the inducing antigen
=> affinity maturation
Germinal Center Reaction:
Activated B cells give rise to Centroblasts
- localize in follicle, undergo rapid cell division and turn on machinery that causes somatic mutation in V-regions
Centroblasts give rise to Centrocytes
- migrate to the FDC-rich region of the Germinal Center
- survival is dependent on interaction with FDC-bound Ag and presentation of Ag to T cells
- centrocytes that successfully compete to bind antigen (e.g. by having higher affinity BCR) and to receive T cell help are selected and may differentiate into long-lived plasma cells or memory B cells

45. Germinal Center mantle zone GC light zone GC dark zone T zone
(CD23+ naive B cells)
GC light zone
(CD23++ FDC
and centrocytes)
GC dark zone
(Ki67 cell cycle antigen+ centroblasts)
T zone
from Liu et al., Immunology Today 13, (1992)

46. Affinity maturation is T-dependent
CD3e LZ
IgD DZ
T cells are enriched in the light zone
Represent ~10% of GC cells

47. Somatic hyper mutation occurred in V H and VL gene and the neucleotide sequences of mutated IgG is 50% more than germinal gene and resulted in change of 10 a.a. in variable area.

48. Selection Model
GC B cells that have improved affinity capture more antigen in a given amount of time, receiving a stronger BCR signal and presenting more MHC-peptide complexes to GC T cells, outcompeting surrounding B cells for T cell help

50. Memory B cells Generated during the primary response
best characterized for T-dependent responses involving GC but some evidence exists for memory to TI antigens
Small, recirculating cells
Typically isotype switched (e.g. IgG+ or IgA+)
Typically have higher affinity for the inducing Ag
Longer lived than naïve B cells
Persistence of memory B cells after an immune response ensures that we have increased numbers of B cells specific for the antigen and ready to respond on re-encounter
May have intrinsic differences that promote greater clonal expansion and more rapid differentiation to plasma cells
differences in cytoplasmic domains of IgG vs IgM/D
upregulation of TLRs

52. Down-regulation of B cell response by pre-formed IgG

55. Antibody Dependent Cell Mediated Cytotoxicity (ADCC)
Target cell is covered with antibodies, leaving Fc portion sticking outwards.
Natural killer and other nonspecific cells that have receptors for Fc region are stimulated to kill targeted cells.
Target organism is lysed by substances secreted by attacking cells.
Used to destroy large organisms that cannot be phagocytosed.

60. FcgRIIIA

65. Receptor Cell type Effect of ligation
FcgRI Macrophages Neutrophils,
Eosinophils, Dendritic cells Uptake, Respiratory burst
FcgRIIA Macrophages Neutrophils,
Eosinophils, Platelets Langerhans cells Uptake, Granule release
FcgRIIB1 B cells, Mast Cells No Uptake, Inhibition of stimulation
FcgRIIB2 Macrophages Neutrophils,
Eosinophils Uptake, Inhibition of stimulation
FcgRIII NK cells, Eosinophils, Macrophages, Neutrophils Mast cells Induction of killing (NK cells)

66. THANKS