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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)
STRUCTURE OF THE SPLEEN
NO LYMPHOID CIRCULATION
Filtration of blood borne antigens
RBC, platelet, granulocyte
Marginal sinus
(phagocytes)
Afferent lymph
Secondary
follicle (Ag)
Primary follicle
(no Ag)
B CELLS
B CELLS
Germinal
center (GC)
medulla
High endothelial
venule (HEV)
Trabecula
Collagen capsule
mature,naive
Mature,naive
BB-sejt
cell
FDC
Paracortex
cortex
T CELLS
Efferent lymph
vein
arthery
B CELLS
T CELLS
Memory B cell
Plasma cell
STRUCTURE OF LYMPH NODES
Pathogenic
factors
Antigen
Secretory IgA
MUCOSAL SURFACES
LC
LC
M-cell
DC
LN
LC
200 times larger than skin
surface
IgA
Lymphocytes
Macrophage
400m2
CYTOKINES
IL-8
plasma cell
MCP-1
TNF
MALT antibody producing cells
=
Spleen + lymph nodes +
bone marrow
Follicle
T-cells
Peyer-plaque
B-cells
HEV
Villi
s u b m uc osa
ORGANIZATION OF THE IMMUNE SYSTEM
LYMPHOCYTES CONGREGATE IN SPECIALIZED TISSUES
•
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
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
MIGRATION OF LYMPHOCYTES
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
Naive lymphocyte
L-selectin
CD34
HEV
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
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/effector/memory
lymphocyte
LFA-1
ICAM-1
VLA-4
VCAM-1
Activated endothel
ALTERED EXPRESSION OF
CELL SURFACE ADHESION
MOLECULES
MIGRATION OF LYMPHOCYTES IN CENTRAL AND
PERIPHERAL LYMPHOID ORGANS
B
BONE MARROW
MALT
SALT
BALT
SPLEEN
T
THYMUS
BLOOD
HEV
TISSUES
LYMPH NODES
Lymphatics
Thoracic duct
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 (25x109)/lymph nodes/day
B – CELL ACTIVATION
RECEPTOR MEDIATED CELL ACTIVATION
Ligand
Ligand
Cross - linking
Conformational change
SIGNAL
SIGNAL
CROSS – LINKING OF THE RECEPTOR INITIATES A
SIGNALING CASCADE
ligand
kinase activation
phosphorylation
recruitment of adaptors
SIGNAL
Activation of
transcription factors
Gene transcription
THE IgM B-CELL RECEPTOR
antigen binding
V V
V V
L
L
mIg molecule
H
H
ab
Btk
Syk
Phosphatases
Adaptors +
substrates
SHP-1
Lyn
Kinases
Ig-a/Ig-b heterodimer
Vav
SLP-65/BLNK
Signal transduction
PLC
HS1
SIGNALING UNITS OF THE B-CELL RECEPTOR
Ig-a/CD79a
Ig-b/CD79b
a b
Y
ITAM
Y
Ig domain + CHO
Y
Y
ITAM
ITAM: YxxL x7 YxxI
ITAM: Immunoreceptor Tyrosine-based Activation Motif
RECENT MODEL OF B-CELL RECEPTOR MEDIATED
SIGNALING
Subsequent activation of 2 kinases
1. Cross-linking
Ag
2. Src-family kinase activation
and ITAM phosphorylation
3. Syk recruitment and activation
4. SLP phosphorylation + Ca release
P
P
= ITAM
P
P
Lyn
Lyn
P
Syk
Syk
P
P
SLP
Calcium release
KINETICS OF LYMPHOCYTE ACTIVATION
ANTIGEN
SIGNAL1.
Resting limfocita
lymphocyte
Nyugvó
GG
0 0
Effector cell
Memory cell
G1
Co-receptor
Adhesion molecule
Cytokines
SIGNAL2.
Transport
Membrane change
RNA and protein synthesis
M
proliferation
G2
S
DNA synthesis
Lymphoblast
Resting lymphocyte G0
PTK activation
RNA synthesis
Free Ca++
Protein synthesis
Protein phosphorylation
DNA synthesis
0 10sec 1min 5min
1hr
6 hrs
12 hrs
24 hrs
THE CO-STIMULATORY ROLE OF CR2 (CD21)
COMPLEMENT RECEPTOR IN B – LYMPHOCYTES
C3d
ANTIGEN
Antigenic
determinant
CD21
CD19
TAPA=CD81
B-CELL
Y
Y
Enhanced B-cell activation
THE NEURAMIC ACID RECEPTOR CD22 INHIBITS
ACTIVATION THROUGH THE A B-CELL RECEPTOR
Mannose
Tissue cells
Bacterium
Neuraminic
(sialic) acid
Antigen
B Cell
CD22
Inhibited B cell activation
ANTIBODY DIVERSITY
STRUCTURE OF
IMMUNOGLOBULINS/ANTIBODIES
Heavy chain (H)
VH
VL
CH
Light chain (L)
CL
Antigen
Antigen
binding
antigénkötés
s
VL
s
s
s
s
s
s
ss
ss
Constans
konstadomains
ns dom ének
CH2 ss
s
s
CH3 ss
s
s
BINDING TO CELLS
DEGRADATION
TRANSPORT
effektor funkc iók
Effector functions
s
s
s
CL
s
COMPLEMENT ACTIVATION
s
CH1
s
va riábilis
d om ének
Variable
domains
s
s
S
s
S
s
s
s
s
VH
AMINO ACID SEQUENCE OF IMMUNOGLOBULINS
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
Gel electrophoresis
L
H
Reduction
50 kDa
Heavy chain
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
25 kDa
Light chain
Variable
Constant
GENETIC BACKGROUND OF ANTIBODY DIVERSITY
VH
VH
VL
VL
S–S
S–S
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?
MOLECULAR GENETICS OF IMMUNOGLOUBLINS
How can the bifunctional nature of antibodies be explained genetically?
In 1965, Dreyer & Bennett proposed that for
a single isotype of antibody there may be:
• 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.
This was genetic heresy as it violated the then accepted
notion that DNA was identical in every cell of an
individual
Proof of the Dreyer - Bennett hypothesis
V
V
V
V
V
V
V
V
V
V
V
V C
V
V
A single C region gene is
encoded in the germline
and separated from the
multiple V region genes
C
A mechanism to rearrange V and C
genes in the genome exists so that
they can fuse to form a complete
Immunoglobulin gene
Find a way to show the existence of multiple V genes and
rearrangement to the C gene
Approach
V
V
V
V
V
V
V
V
V
V
V
V C
V
Germline DNA
C
Rearranged DNA
V
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)
DOGMA OF MOLECULAR BIOLOGY
CHARACTERISTICS OF IMMUNOGLOBULIN SEQUENCE
1 GEN = 1 PROTEIN
THEORIES
1 GEN
Gen
High rate of somatic mutations in the V-region
V
C
Many GENES (10 000 – 100 000)
V1 C
Protein
V2 C
Vn C
Experiment of Susumi Tonegawa 1975 Basel
DNA-extraction
Digestion by restriction enzyme
Gel electrophoresis
Southern blot
Liver cell
Kb
B-cell
V-probe
6,0
4,0
1,5
V
C-probe
C
VC
6.0 Kb
4.0 Kb
V
C
1.5. Kb
V
C
B-cell
CONCLUSION
V and C genes get close to each other in B-cells only
V
V
V
C
B-CELL
There are many variable genes but only one constant gene
V
V
V
V
C
GERM LINE
Ig gene sequencing complicated the model
The structures of germline VL genes were similar for Vk, and Vl,
However there was an anomaly between germline and rearranged DNA:
VL
CL
~ 95aa
~ 100aa
L
LV
L
CL
~ 208aa
Where do the extra 13
amino acids come from?
LV
L
~ 95aa
JL
CL
~ 100aa
Some of the extra amino
acids are provided by
one of a small set of J or
JOINING regions
Further diversity in the Ig heavy chain
L VH DH JH
CH
The heavy chain was found to have further amino acids (0 – 8)
between the JH és CH genes
D (DIVERSITY) region
Each heavy chain requires 3 recombination events
JH to DH , VH to JHDH, and VHJHDH to CH
L VL
JL
CL
Each light chain requires 2 recombination events
VL to JL and VLJL to CL
IMMUNOGLOBULIN CHAINS ARE ENCODED BY MULTIPLE
GENE SEGMENTS
ORGANIZATION OF IMMUNOGLOBULIN GENE SEGMENTS
Chromosome 2
kappa light chain gene segments
Chromosome 22
lambda light chain gene segments
Chromosome 14
heavy chain gene segments
HOW MANY IMMUNOGLOBULIN GENE SEGMENTS
Gene segments
Light chain
Heavy chain
kappa
lambda
Variable (V)
132/40
105/30
123/65
Diversity (D)
0
0
27
Joining (J)
5
4
9
SOMATIC REARRANGEMENT OF KAPPA (κ) CHAIN GENE
SEGMENTS
Vκ Jκ
B-cell 2
Vκ
Vκ
80 Vκ
Vκ
Vκ
4 Jκ
Germ line
Jκ Jκ Jκ Jκ
During B-lymphocyte
development
Vκ
B-cell 1
DNA
Vκ
Vκ
Jk Jκ Jκ Jκ
EXPRESSION OF THE KAPPA CHAIN
Vκ
P
Vκ J
pA
J
E
Cκ
J
E
Cκ
Vκ-Jκ
Leader
Vκ J
Primary RNA transcript
Vκ J
Cκ
AAAA
mRNA
Translation
Vκ J
Cκ
Protein
Efficiency of somatic gene rearrangement?
SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE
SEGMENTS
120 VH
VH1
VH2
12 D
VH3
D
D
D
4 JH
D
JH JH JH JH
During B-cell development
VH1
VH2
VH3
VH1
D
D JH JH
VH2
D
D JH JH
VARIABILITY OF B-CELL ANTIGEN RECEPTORS AND
ANTIBODIES
B cells of one individual
2
3
1
4
V-Domains
C-Domains
VH
D
JH
VL
VH-D-JH
JL
VL-JL
ORDER OF REARRANGEMENTS OF IMMUNOGLOBULIN
GENE SEGMENTS
D – J recombination
V – DJ recombination
VDJ – δ transcription
Surrogate light chain
V – J recombination
VJ – k (or VJ - l) transcription
δ translation
k or l translation
B-sejt
mIgD
mIgM
Secreted IgM
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 Vk x 5 Jk = 145 combinations
30 Vl x 4 Jl = 120 combinations
= 265 different light chains
If H and L chains pair randomly as H2L2 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
THE RESULT OF SOMATIC GENE REARRANGEMENTS
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
INDEPENDENT OF ANTIGEN
OCCURS DURING B-CELL DEVELOPMENT IN THE
BONE MARROW
SYNTHESIS OF IMMUNOGLOBULINS
Secreted Ig
Membrane Ig
Golgi
ER
H and L chains are
synthesized on separated
ribosomes
CHAPERONES
Leader sequence
Ribosome
mRNA