b cell develop,emt

1. B Cells and B Cell Development
© Dr. Colin R.A. Hewitt
crah1@le.ac.uk

2. The discovery of B cell immunity
1954 - Bruce Glick, Ohio State University
Studies on the function of the bursa of Fabricius, a lymphoid organ in
the cloacal region of the chicken
Bursectomy – no apparent effect
Bursectomised chickens
were later used in
experiments to raise
antibodies to Salmonella
antigens
None of the
bursectomised
chickens made
anti-Salmonella
antibodies
Bursa was later found to be the organ in which antibody producing cells
developed – antibody producing cells were thereafter called B cells
Mammals do not have a bursa of Fabricius

3. Transfer marked foetal
liver cells
Origin of B cells and organ of B cell
maturation
No Mature
B cells
After birth, development continues in the bone marrow
Normal bone marrow
Defective bone marrow
Mature marked
B cells
in periphery
B cell development starts in the foetal liver

4. B cell development in the bone marrow
B Regulates construction of an antigen receptor
Bone Marrow provides a
MATURATION & DIFFERENTIATION MICROENVIRONMENT
for B cell development
Ensures each cell has only one specificity
B
Checks and disposes of self-reactive B cells
B
Exports useful cells to the periphery
B
Provides a site for antibody production
B

5. Bone Marrow
E
M
M
S

7. Immature &
mature B
Central
Sinus
Progenitors Pre-B
Stromal cells
X
X
X
E
n
d
o
o
s
t
e
u
m
Macrophage
Scheme of B Cell Development in the Bone Marrow

8. Secreted
Factors - CYTOKINES
2. Secretion of cytokines by stromal cells
B
Bone marrow stromal cells nurture
developing B cells
Types of cytokines and cell-cell contacts needed at
each stage of differentiation are different
Stromal cell
1. Specific cell-cell contacts between stromal cells and developing B cells
Cell-cell contact

9. Bone marrow stromal cell
Maturing B cells

10. B
B
Stromal cell

11. Peripheral
Stages of B cell development
Stem Cell Early pro-B cell Late pro-B cell Large pre-B cell
Small pre-B cell Immature B cell Mature B cell
Each stage of development is defined by rearrangements of IgH
chain genes, IgL chain genes, expression of surface Ig,
expression of adhesion molecules and cytokine receptors

12. Early
pro-B
Kit
Receptor
Tyrosine
kinase
Stem cell
factor
Cell-bound
growth
factor
VLA-4
(Integrin)
Stem
Cytokines and cell-cell contacts at each
stage of differentiation are different
Stromal cell
Cell adhesion
molecules
VCAM-1
(Ig superfamily)

13. Early
pro-B
Interleukin-7
receptor
Stromal cell
Late
pro-B Pre-B
Interleukin-7
Growth factor
Cytokines and cell-cell contacts at each
stage of differentiation are different

14. Stages of differentiation in the bone marrow are
defined by Ig gene rearrangement
B CELL STAGE
IgH GENE
CONFIGURATION
Stem cell Early pro-B Late pro-B Large pre-B
Germline DH to JH VH to DHJH VHDHJH
Pre-B cell
receptor
expressed
Ig light chain gene has not yet rearranged

15. B cell receptor
Transiently expressed when VHDHJH CHm is productively rearranged
VpreB/l5 - the surrogate light chain, is required for surface expression
Iga & Igb signal
transduction
molecules
CHm
Heavy chain
VHDHJH
Light chain
VLJLCL
VpreB
l5
Ligand for the pre-B cell receptor may be galectin 1, heparan sulphate, other
pre-BCR or something as yet unknown
Pre-

16. Ligation of the pre-B cell receptor
1. Ensures only one specificty of
Ab expressed per cell
Large
Pre-B
Stromal cell
Unconfirmed ligand
of pre-B cell receptor
2. Triggers entry into cell cycle
ALLELIC EXCLUSION
Expression of a gene on one chromosome prevents expression of the
allele on the second chromosome
1. Suppresses further H chain rearrangement
2. Expands only the pre-B
cells with in frame VHDHJH joins

17. 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 a
b
Y
B
a AND
ALLOTYPE- polymorphism in the C region of Ig – one
allotype inherited from each parent
Suppression of H chain rearrangement by
pre-B cell receptor prevents expression of two
specificities of antibody per cell
(Refer back to Dreyer & Bennet hypothesis in Molecular Genetics
of Immunoglobulins lecture topic)

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

19. 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 prevent the emergence of
new daughter specificities during proliferation after clonal selection
S. typhi
Antibody
S. typhi

20. Allelic exclusion is needed to prevent holes in the repertoire
Exclusion of anti-brain B cells
i.e. self tolerance
B
B
One specificity of Ag
receptor per cell
S. aureus
Anti-brain Ig
AND
anti-S. aureus Ig
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
B
B

21. 1. Suppresses further H chain rearrangement
Ligation of the pre-B cell receptor
1. Ensures only one specificity of
Ab expressed per cell
Large
Pre-B
Stromal cell
Unconfirmed ligand
of pre-B cell receptor
2. Triggers entry into cell cycle
2. Expands only the pre-B
cells with in frame VHDHJH joins

22. Translation in frame 1
MKXLWFFLLLVAAPRWVLSQV
HLQESGPGLGKPPELKTPLGD
TTHTCPRCPEPKSCDTPPPCP
RCPEPKSCDTPPPCPRCPEPK
SCDTPPPCXXCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDXXVQFKWYVDG
VEVHNAKTKLREEQYNSTFRV
VSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPEE
MTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYNTTPPMLD
SDGSFFLYSKLTVDKSRWQQG
NIFSCSVMHEALHNRYTQKSLS
LSPGK*
Human IgG3 Heavy Chain
nucleotide sequence
ATGAAACANCTGTGGTTCTTCCTTCTCCTGG
TGGCAGCTCCCAGATGGGTCCTGTCCCAGG
TGCACCTGCAGGAGTCGGGCCCAGGACTGG
GGAAGCCTCCAGAGCTCAAAACCCCACTTGG
TGACACAACTCACACATGCCCACGGTGCCCA
GAGCCCAAATCTTGTGACACACCTCCCCCGT
GCCCACGGTGCCCAGAGCCCAAATCTTGTG
ACACACCTCCCCCATGCCCACGGTGCCCAG
AGCCCAAATCTTGTGACACACCTCCCCCGTG
CCCNNNGTGCCCAGCACCTGAACTCTTGGG
AGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGATACCCTTATGATTTCCCGGACCC
CTGAGGTCACGTGCGTGGTGGTGGACGTGA
GCCACGAAGACCCNNNNGTCCAGTTCAAGT
GGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCTGCGGGAGGAGCAGTACA
ACAGCACGTTCCGTGTGGTCAGCGTCCTCAC
CGTCCTGCACCAGGACTGGCTGAACGGCAA
GGAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGACAGCCCGAGGAGATGACCA
AGAACCAAGTCAGCCTGACCTGCCTGGTCAA
AGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAACACCACGCCTCCCATGCTGGACTCC
GACGGCTCCTTCTTCCTCTACAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACATCTTCTCATGCTCCGTGATGCATGAGGC
TCTGCACAACCGCTACACGCAGAAGAGCCTC
TCCCTGTCTCCGGGTAAATGA
Large pre-B cells need in frame VHDHJH joins to mature
Translation in frame 2
(no protein)
*
Translation in frame 3
ETXVVLPSPGGSSQMGPVPGA
PAGVGPRTGEASRAQNPTW*
Large
pre-B
Development
continues
Pre-B cell
receptor can
be activated
Development
arrests
Development
arrests

23. Large
Pre-B
Large
Pre-B
Large
Pre-B
Large
Pre-B Large
Pre-B
Large
Pre-B Large
Pre-B
Large
Pre-B Large
Pre-B
Large
Pre-B
Proliferation
Y
Immature
B cell
Light chain expressed
IgM displayed on surface
IgM
Ligation of the pre-B cell receptor triggers
entry into the cell cycle
Large
pre-B
Many large pre-B
cells with identical
pre-B receptors
Large
pre-B
Intracellular VDJCH chain
VL-JL rearranges
Proliferation
stops
Pre-receptor
not displayed
Small
pre-B

24. V D J C
D J
V C
D J
V C
D J
V
Germline
DH-JH joining
VH-DHJH joining
V J C
V C
J
V
Germline
VL-JL joining
Heavy and light chain rearrangement is potentially wasteful
Large
pre-B
Small
pre-B
With two “random” joins to generate a heavy chain
there is a 1:9 chance of a rearrangement of being in frame
With one “random” join to generate a light chain
there is a 1:3 chance of a rearrangement being of frame
There is, therefore, only a 1:27 chance of an in frame rearrangement
Out of frame rearrangements arrest further B cell maturation

25. B cells have several chances to successfully
rearrange Ig genes
Early Pro B Late Pro B Pre B
VH-DJH
On first
chromosome
VH-DJH
On second
chromosome
Immature B
NO
YES
NO
B
k on first
chromosome
k on second
chromosome
l on first
chromosome
l on second
chromosome
NO
NO
NO
NO
YES
YES
NO
YES
YES
YES
YES
Y
IgMk
B
Y
IgMl
B
DH-JH
On first
chromosome
DH-JH
On second
chromosome
YES
NO

26. B Y
Y
Y
Y
B
Small pre-B cell
No antigen receptor at cell surface
Unable to sense Ag environment
!!May be self-reactive!!
Immature B cell
Cell surface Ig expressed
Able to sense Ag environment
Can now be checked for self-reactivity
Acquisition of antigen specificity creates a
need
to check for recognition of self antigens
1. Physical removal from the repertoire DELETION
2. Paralysis of function ANERGY
3. Alteration of specificity RECEPTOR EDITING

27. B cell self tolerance: clonal deletion
Immature
B cell recognises
MULTIVALENT
self Ag
B
Clonal deletion by
apoptosis
Y
YB
Immature
B
B
Small
pre-B
Small pre-B cell
assembles Ig

28. B cell self tolerance: anergy
B
B
Anergic B cell
IgD normal IgM low
Immature
B cell recognises
soluble self Ag
No cross-linking
Y
Y
B
Immature
B
B
Small
pre-B
Small pre-B cell
assembles Ig
IgM
IgD
IgD
IgD

29. Receptor editing
A rearrangement encoding a self specific receptor can be replaced
V C
D J
V
V V
B
B
!!Receptor
recognises
self antigen!!
B Apoptosis
or anergy
B
B
Edited receptor now recognises
a different antigen and can be
rechecked for specificity
C
D J
V
V V
V
Arrest development
And reactivate
RAG-1 and RAG-2

30. Mature B cell
exported to the
periphery
B cell self tolerance: export of self tolerant B cells
IgD and IgM normal
IgM
IgD
IgD
IgD
IgD
IgM
IgM
IgM
Immature
B cell doesn’t
recognise any
self Ag
Y
Y
B
Immature
B
B
Small
pre-B
Small pre-B cell
assembles Ig
B

31. How can B cells express
IgM and IgD simultaneously?
Ca2
Ce
Cg4
Cg2
Ca1
Cg1
Cg3
Cd
Cm
Cm
Cd
Cg3
VDJ
Sg3
Cm
Cd
Cg3
VDJ
Cg1
Sg1
Ca1
Cg3
VDJ Ca1
Cg3
VDJ
IgG3 produced.
Switch from IgM
VDJ Ca1
IgA1 produced.
Switch from IgG3
VDJ Ca1
IgA1 produced.
Switch from IgM
N.B. Remember Molecular Genetics of Immunoglobulins lecture – No Cd switch region
Consider similarities with mechanism allowing secreted and membrane Ig by the same cell

32. Splicing of IgM and IgD RNA
Cm1 Cm2 Cm3 Cm4 Cd1 Cd2 Cd3
pA1
V D J
Cm1 Cm2 Cm3 Cm4 Cd1 Cd2 Cd3
V D J
AAA
RNA cleaved and
polyadenylated at pA2
V D J Cm IgM mRNA
V D J Cd IgD mRNA
Cg1
Cg3
Cd
Cm
V D J
Cm1 Cm2 Cm3 Cm4 Cd1 Cd2 Cd3 DNA
pA1 pA2
V D J
Two types of mRNA can be made simultaneously in the cell by differential usage of
alternative polyadenylation sites and splicing of the RNA
RNA cleaved and
polyadenylated at pA1
AAA

33. • B cells develop in the foetal liver and adult bone marrow
• Stages of B cell differentiation are defined by Ig gene rearrangement
• Pre-B cell receptor ligation is essential for B cell development
• Allelic exclusion is essential to the clonal nature of immunity
• B cells have several opportunities to rearrange their antigen
receptors
• IgM and IgD can be expressed simultaneously due to differential
RNA splicing
• So far, mostly about B cells in the bone marrow - what about mature
peripheral B cells?
Summary

34. Although Fab fragments bind to
membrane Ig (mIg), no signal is
transduced through the B cell
membrane
mIg
Fab anti-mIg
(Fab)2 anti-mIg Bridging (or ‘Cross-linking’) of
different mIg allows the B cell
receptor to transduce a weak signal
through the B cell membrane
Anti-(Fab)2
Extensive cross linking of (Fab)2
bound to mIg using an anti-(Fab)2
antibody enhances the signal
through the B cell membrane
What are the external signals that activate B cells?

35. Ring staining
Ig is evenly distributed
around the cell surface
Patching
Ig is aggregated in
uneven ‘clumps’ as
a result of mild
cross-linking of Ig
Capping
Ig is collected at a pole of the cell
in a ‘cap’ as a result of extensive
cross-linking of Ig

36. Transduction of signals by the B cell receptor
Iga
Igb
Intracytoplasmic
signalling domains
Extracellular antigen
recognition domains
The cytoplasmic domains of the Iga and
Igb contain Immunoreceptor Tyrosine -
based Activation Motifs (ITAMS) - 2
tyrosine residues separated by 9-12
amino acids - YXX[L/V]X6-9YXX[L/V]

37. Kinase domain Unique region
SH3 domain
SH2 domain
Enzyme domain
phosphorylates tyrosines
(to give phosphotyrosine)
Phosphotyrosine
receptor domain
Adaptor
protein
recruitment
domain
ITAM
binding
domain
• Phosphorylation is rapid, requires no protein synthesis or degradation to change
the biochemical activity of a target protein
• It is reversible via the action of phosphatases that remove phosphate
• Phosphorylation changes the properties of a protein by changing its conformation
• Changes in conformation may activate or inhibit a biochemical activity or create a
binding site for other proteins
Phosphorylation by Src kinases

38. Regulation of Src kinases
Kinase domain Unique region
SH3 domain
SH2 domain
Activating tyrosine residue
Inhibitory tyrosine residue
Phosphorylation of ‘Activating Tyrosine’ stimulates kinase activity
Kinase domain Unique region
SH3 domain
SH2 domain
Phosphorylation of ‘Inhibitory Tyrosine’ inhibits kinase activity
by blocking access to the Activating Tyrosine Residue

39. Regulation of Src kinases by Csk and CD45
Kinase domain Unique region
SH3 domain
SH2 domain
Kinase domain
Resting cells: Src
kinase is inactivated
by a constitutively
expressed C -
terminal Src kinase -
(Csk)
Kinase domain Unique region
SH3 domain
SH2 domain
C terminus
Activated cells: a
phosphatase associated
with the Leukocyte
Common Antigen - CD45,
removes the C terminus
phosphate allowing the
activating tyrosine to be
phosphorylated
The balance between Csk and CD45 phosphatase activity sets the threshold for
initiating receptor signalling

40. 3. Src kinases bind to
phosphorylated ITAMS
and are activated to
phosphorylate adjacent
ITAMS
P
ITAM
ITAM P P
ITAM
ITAM
2. Antigen clusters B cell receptors with
CD45 phosphatases.
Src kinases are phosphorylated
Phosphorylation of ITAMs by Src kinases
1. Csk inactived
Src interacts
with low affinity
with the ITAMs
of ‘resting’
receptors
P
ITAM P
and are activated to phosphorylate
ITAMS

41. One Syk binds to Iga, one to Igb - each Syk
transphosphorylates the other
Syk - 2 x SH2
domains spaced to
bind to two
phosphotyrosines on
an ITAM
Syk protein Tyrosine kinases
• CD45 phosphatase allows activation of Src family kinases Blk, Fyn & Lyn
• Receptor cross-linking activates Src kinases that phosphorylate ITAMs in
the Iga and Igb
ITAM
P
P
ITAM
P
P
ITAM
P
P
P P

42. CD21
(C3d receptor)
CD19
CD81
(TAPA-1)
Iga
Igb
CD45
The B cell co-receptor
The B cell co-receptor

43. Src family kinases
then bind the
phosphorylated
CD19
Antigen
recognition
C3d opsonised bacterium
• mIg and CD21 are cross-linked by antigen that has activated complement
C3d binds to
CD21, the
complement
receptor 2
(CR2)
P P
• CD21 is phosphorylated and receptor-associated kinases phosphorylate CD19
P P
Co-receptor phosphorylation
• Phosphorylated CD19 activates more Src family kinases
• Ligation of the co-receptor increases B cell receptor signalling 1000 -10,000 fold

44. BLNK
Cell membrane-associated B
cell Linker protein - BLNK -
contains many Tyrosine
residues
Activated Syk phosphorylates BLNK
P P P P
BLNK binds Tec kinases
Tec Tec Tec Tec
Tec kinases activate
phospholipase C- g (PLC-g)
PLC-g cleaves
phosphotidylinositol
bisphosphate (PIP2) to yield
diacylglycerol (DAG) and
inositol trisphosphate (IP3)
ITAM
P
P
ITAM
P
P
P P
Activated Syk phosphorylates
Guanine-nucleotide exchange factors
(GEFS) that in turn activate small GTP
binding proteins Ras and Rac
Ras and Rac activate the MAP
kinase cascade
Activation of signals that affect gene transcription

45. Transmission of signals from the cell
surface to the nucleus
• B cell-specific parts of the signalling cascade are associated with receptors
unique to B cells - mIg, CD19 etc.
• Subsequent signals that transmit signals to the nucleus are common to
many different types of cell.
• The ultimate goal is to activate the transcription of genes, the products of
which mediate host defence, proliferation, differentiation etc.
Once the B cell-specific parts of the cascade are complete, signalling to
the nucleus continues via three common signalling pathways via:
1.The mitogen-activated protein kinase (MAP kinase) pathway
2. Increased in intracellular Ca2+ mediated by IP3
3.The activation of Protein Kinase C mediated by DAG

46. • MAP Kinase cascade
Small G-protein-activated MAP kinases found in all multicellular animals -
activation of MAP kinases ultimately leads to phosphorylation of transcription
factors from the AP-1 family such as Fos and Jun.
• Increases in intracellular calcium via IP3
IP3, produced by PLC-g, binds to calcium channels in the ER and releases
intracellular stores of Ca++ into the cytosol. Increased intracellular [Ca++]
activate a phospatase, calcineurin, which in turn activates the transcription
factor NFAT.
• Activation of Protein Kinase C family members via DAG
DAG stays associated with the membrane and recruits protein kinase C
family members. The PKC, serine/threonine protein kinases, ultimately
activate the transcription factor NFkB
The activated transcription factors AP-1, NFAT and NFkB induce B cell
proliferation, differentiation and effector mechanisms
Simplified scheme linking antigen recognition
with transcription of B cell-specific genes

47. B cell recognises
non-self antigen
in periphery
Ig-secreting plasma cell
Differentiation in the periphery
Y
Y
Y
Y
Y
Y
B
Y Y
B
Y Y
B
Mature peripheral
B cell

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

49. You should know:
•Where B cells come from
•What happens to B cells in the bone marrow
•How B cell differentiation is linked with Ig gene rearrangement
•The B cell developmental ‘check points’ that ensure each cell
produces a single specificity of antibody that does not react with
self
•How B cells transmit information from the shape and charge of an
antigen through the cell membrane to allow the expression of
genes in the nucleus
What do mature B cells do once activated by an
antigen in the periphery?
Summary

50. Recirculating B cells normally pass through
lymphoid organs
B cells in
blood
Efferent
lymph
T cell area
B cell
area

51. Antigen enters
node in afferent
lymphatic
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
B cells leave blood &
enter lymph node via
high endothelial venules
B cells
proliferate
rapidly
GERMINAL CENTRE
Transient structure of
Intense proliferation
Germinal centre
releases B cells
that differentiate
into plasma cells
Recirculating B cells are trapped by foreign
antigens in lymphoid organs

52. B cells (stained brown) in the Germinal Centre
P = Paracortex, Mn = Mantle zone SC = Subcapsular zone

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

54. 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

55. Maturation of Follicular Dendritic cells
Club-shaped tips of developing dendrites Filiform dendrites
Bead formation on dendrites Bead formation on dendrites

56. Association of antigen with FDC
in the form of an immune complex with
C3b and antibodies attached
Antigen enters the germinal centre
Complement receptor 3
Ig Fc receptor
FDC surface
The filiform dendrites of FDC develop
beads coated with a thin layer of immune
complexes
The Immune complexes bind to
Fc and complement receptors
on the FDC dendrites

57. 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

58. 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

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

60. Y
T cell help to B cells
B
Signal 1
antigen & antigen
receptor
Th
1. T cell antigen receptor
2. Co-receptor (CD4)
3.CD40 Ligand
Th
Signal 2 - T cell help

61. T cell help - Signal 2
Y
B Th
Signal 2
Signal 1
B cells are inherently prone to die by apoptosis
Signal 1 & 2 upregulate Bcl-XL in the B cell and
Bcl-XL prevents apoptosis
Signal 1 & 2 thus allow the B cell to survive
T cells regulate the survival of B cells and thus
control the clonal selection of B cells
Cytokines
Cytokines
IL-4
IL-5
IL-6
IFN-g
TGF-b

62. T cell help - Signal 2 activates hypermutation
Y
B Th
Signal 2
Signal 1
Receipt of signal 2
by the B cell also
activates
hypermutation in the
CDR - encoding
parts of the Ig genes
Signal 2, and thus T cells,
regulate which B cells are
clonally selected.
Low affinity Ig takes up and
presents Ag to T cells
inefficiently.
Inefficient presentation to T
cells does not induce CD40.
With no signal 2 delivered
by CD40, low affinity B cells
die.
Only B cells with high affinity
Ig survive - This is affinity
maturation
Clone 1
Clone 2
Clone 3
Clone 4
Clone 5
Clone 6
Clone 7
Clone 8
Clone 9
Clone 10
CDR1
CDR2
CDR3
Day 6
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
Day 8 Day 12 Day 18
Deleterious mutation
Beneficial mutation
Neutral mutation
Lower affinity - Not clonally selected
Higher affinity - Clonally selected
Identical affinity - No influence on clonal selection

63. Control of Affinity & Affinity Maturation
Five B cell antigen
receptors -
all specific
for , but with
different affinities
due to somatic
hypermutation
of Ig genes in
the germinal centre
B B B B B
Only this cell, that has a high affinity for antigen can express CD40.
Only this cell can receive signal 2
Only this cell is rescued from apoptosis i.e. clonally selected
The cells with lower affinity receptors die of apoptosis by neglect

64. GC = Germinal Centre, TBM = Tingible Body Macrophages
Germinal Centre Macrophages (stained brown)
Clean Up Apoptotic Cells

65. Role of T cell cytokines in T cell help
Th
Signal 2
Y
B
Signal 1
Cytokines
IL-4
IL-5
IL-6
IFN-g
TGF-b
IgM IgG3 IgG1 IgG2b IgG2a IgE IgA
IL4 inhibits inhibits induces inhibits induces
IL-5 augments
IFN-g inhibits induces inhibits induces inhibits
TGF-b inhibits inhibits induces induces
PC
B
B
B B
B
B
B
B
B
B
B
B
B
B
Proliferation & Differentiation

66. Regulation of specificity - Cognate recognition
1. T cells can only help the B cells that present
antigen to them
2. B cells are best at presenting antigens that they
take up most efficiently
3. B cells are most efficient at taking up antigens that
their B cell antigen receptors bind to
4. T and B cells help each other to amplify immunity
specific for the same antigen
i.e. Regulates the Characteristics of Adaptive Immunity
Sharply focuses specificity - Pathogen specificity
Improves specificity & affinity - Better on 2nd exposure
Is specific antigen dependent - Learnt by experience
Seeds memory in T and B cell pools

67. Synaptic tethering of a B cell (red)
to a T cell (green)

68. T cell (in centre) surrounded by B cells with
cytoskeleton stained green

69. Antigen enters
node in afferent
lymphatic
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
B cells leave blood &
enter lymph node via
high endothelial venules
B cells
Rapidly
proliferate
in follicles
GERMINAL CENTRE
Transient structure of
Intense proliferation
Germinal centre
releases B cells
that differentiate
into plasma cells
Recirculating B cells are trapped by foreign
antigens in lymphoid organs

70. HEV
1. Antigen loaded dendritic cells
migrate from subcapsular
sinus to paracortical area
of the lymph node
DC
B
B
B
B
B
B
T
T
T
T
2. T cells migrate
through HEV and
are trapped by
antigen on DC
B
4. B cells migrate through
HEV - most pass through the
paracortex and primary follicle.
T
T
T
T
3. T cells
proliferate
Some interact with T cells and
proliferate to form a primary focus
B cells (90%) and T cells
(10%) migrate to form
a primary follicle
B
B
B
B
B
B
T
Primary follicle
formation

71. T cell motility in the lymph node

72. Primary Follicles become
secondary follicles
when germinal centres develop
Dark zone
Light zone B
T
Follicular dendritic
cells select useful
B cells
Germinal Centre Microanatomy
1. B cells (centroblasts) downregulate
surface Ig, proliferate, somatically
hypermutate their Ig genes.
AFFINITY MATURATION
2. B cells (centrocytes) upregulate
surface Ig, stop dividing and
receive costimulatory signals
from T cells and FDC
3. Apoptosis of
self-reactive &
unselected cells
4. Selected cells leave lymph
node as memory
cells or plasma cells

73. CD5
Two B cell lineages
B
B cell precursor
B
Mature B cell
B2 B cells
Plasma cell
Y
Y
Y
Y
Y
Y
PC
IgG
B
Y
Y
Y
Y
IgM - no other isotypes
B
Distinct B cell
precursor
?
?
B1 B cells
‘Primitive’ B cells found in
pleura and peritoneum

74. CD5
B
Y
Y
Y
Y
IgM
B-1 B Cells
IgM uses a distinctive & restricted range of V regions
Recognises repeating epitope Ag such as
phospholipid phosphotidyl choline & polysaccharides
NOT part of adaptive immune response:
No memory induced
Not more efficient on 2nd challenge
Present from birth
Few non-template encoded (N) regions in the IgM
NATURAL ANTIBODY
Can make Ig without T cell help

75. Comparison of B-1 and B-2 B cell properties
Property B-1 cells B-2 cells
N regions Few Extensive
V region repertoire Restricted Diverse
Location Peritoneum/pleura Everywhere
Renewal Self renewal in situ Bone marrow
Spontaneous Ig production High Low
Isotypes IgM IgM/G/A/D/E
Carbohydrate specificity Yes Rarely
Protein specificity Rarely Yes
Need T cell help No Yes
Somatic hypermutation of Ig No High
Memory development No Yes
Yes Rarely
Rarely Yes
No Yes
Carbohydrate specificity
Protein specificity
Need T cell help
Specificity & requirement for T cell help suggests strikingly different types
of antigens are seen by B-1 and B-2 B cells

76. T Independent Antigens (TI-2)
Immature B cells that bind to
multivalent self Ag undergo
apoptosis
B-2 cell repertoire is purged
of cells recognising
multivalent antigens during
development in the bone
marrow
Y
Y
Y
Y
IgM
Mature
B-1
Y
Y
Immature
B-2 Cell
Non-bone marrow derived B-1 cells
are directly stimulated by antigens
containing multivalent epitopes.
No T cells are necessary
Induces the expression of natural
antibodies specific for TI-2 antigens
TI-2 Antigen

77. LPS
LPS binding
protein
CD14
B Cell
Bacterial Lipopolysaccharides, (TI-1
antigens), bind to host LPS binding
protein in plasma
LPS/LPSBP is captured by CD14
on the B cell surface
TLR 4
Toll - like receptor 4 (TLR4) interacts
with the CD14/LPS/LPSBP complex
Activation of B cell
T Independent Antigens (TI-1)

78. Y Y Y Y Y Y
B B B B B B
Y Y Y Y Y Y
Y Y Y Y Y Y
Y Y Y Y Y Y
Y Y Y Y Y Y
Y Y Y Y Y Y
Y Y Y Y Y Y
T Independent Antigens (TI-1 e.g. LPS)
Six different B cells will require 6 different antigens to activate them
At high dose TI-1 antigens (like LPS) will POLYCLONALLY ACTIVATE all of
the B cells irrespective fo their specificity.
TI-1 antigens are called MITOGENS
LPS complexes with CD14, LPSBP & TLR4

79. T
Dependent
Antigens
TI-1
Antigens
TI-2
Antigens
Induce responses in babies Yes Yes
Induce responses in athymics No Yes Yes
Prime T cells Yes No No
Polyclonally activate B cells No Yes No
Require repeating epitopes No No Yes
T Dependent & Independent Antigens
No

80. Adult immature B cells that
bind to multivalent self Ag
undergo apoptosis
Y
Y
Y
Y
IgM
Mature
B-1
Y
Y
Immature
B-2 Cell
Adult non-bone marrow derived B-1
cells are directly stimulated by
antigens containing multivalent
epitopes produce IgM WITHOUT T
cell help.
TI-2 Antigen
Why are babies unresponsive to TI-2 antigens?
In adults:

81. Y
Y
Immature
B-1 Cell
TI-2 Antigen
Why are babies unresponsive to TI-2 antigens?
Immature B cells that bind to
multivalent self Ag undergo
apoptosis
As with adult B cells, immature B cells that bind multivalent self Ag
undergo apoptosis
Hence babies do not respond to TI-2 antigens.
Babies are, therefore susceptible to pathogens with multivalent
antigens such as those on pneumococcus
In babies:
All B cells, B-1 & B-2, are immature

82. T
Dependent
Antigens
TI-1
Antigens
TI-2
Antigens
Induces response in babies Yes Yes No
Induces response in athymia No Yes Yes
Primes T cells Yes No No
Polyclonally activates B cells No Yes No
Requires repeating epitopes No No Yes
T Dependent & Independent Antigens
Examples
TD: Diptheria toxin, influenza heamagglutinin, Mycobacterium tuberculosis
TI-1: Bacterial lipopolysaccharides, Brucella abortis
TI-2: Pneumococcal polysaccharides, Salmonella polymerised flagellin
TD: Activate B-1 and B-2 B cells
TI-1: Activate B-1 and B-2 B cells
TI-2: Activate only B-1 B cells

83. Immune effector mechanisms against
extracellular pathogens & toxins
NEUTRALISATION
Y
` `
Toxin release
blocked
Prevents
toxicity
NEUTRALISING ANTIBODIES
Adhesion to
host cells blocked
Prevents
invasion
Bacterium
Toxin

84. Fc receptor
binding
Effector mechanisms against
extracellular pathogens
OPSONISATION
OPSONISATION Phagocytosis
Bacteria in extracellular space
Ab
+

85. Effector mechanisms against
extracellular pathogens
COMPLEMENT Activation
Bacteria in plasma
Ab &
COMPLEMENT
+
Phagocytosis
binding
Complement &
Fc receptor
Lysis
Opsonisation

86. Summary
• B cell tolerance of self is by clonal deletion or anergy of self-reactive cells
• Receptor editing increases the efficiency of B cell development
• Follicular dendritic cells acquire antigen and transfer it to B cells
• T cell help to B cells is via CD40L and cytokines
• CD40 expression indirectly leads to Ig affinity maturation
• Germinal centre microanatomy & function
• There are two lineages of B cells - B1 and B2 B cells
• The dependency of B cells upon T cells varies