november 2, 2004. immunity adaptiveinnate cell mediatedhumoral antibodies effector systems fc...
Post on 29-Jan-2016
217 Views
Preview:
TRANSCRIPT
November 2, 2004
IMMUNITY
ADAPTIVE INNATE
CELL MEDIATED HUMORALANTIBODIES
EFFECTOR SYSTEMSFc ReceptorsComplement
RECEPTORSEFFECTORS
CellsMolecules
ANTIGENS
TCR
BCRSoluble Ab
MHC
B2 Cells
B1 Cells
B1 (Ly1)
B2 or conventional
Ly 1 (CD5) or B 1 B cellsAre very long-lived and self-replenishingDevelop early in ontogeny from fetal omentum and liver
After birth the stems cells for B-1 are not present in the bone marrow
In the adult are found predominately in the peritoneal and pleural cavitiesRare in lymph node, peripheral blood and spleen
Location
Ly 1 (CD5) or B 1 B cellsDisplay a limited V region repertoireLots of self-reactivity and recognition of bacterialantigensReactivity is broad specificity, low affinity
Produce large amounts of antibody; although <5%of B cells make >50% of all antibodies in serum
Ly 1 (CD5) or B 1 B cellsDo not require T cell help
Play a role in the innate immune response to infection; Abs mostly germline encoded
Make antibody response to polysaccharide Agsfound on bacteria (TI-2)
Rapid response - make Abs within 48 hours of antigen exposure
CD5 B cell binds capsular polysaccharide
CD 5 cell secretes IgM anti-polysaccharide antibody
IgM
IgM effective in activating complement thereby helping to remove bacteria
Innate response
Ly 1 (CD5) or B 1 B cells
Hypothesized to be primitive B cells
Abundant in autoimmune disease - remember specificity is often self-reactivity
In mice at birth most B cells are B-1
Most B cell leukemias are B-1 cells
Conventional B cell or B-2 B cellsAre the principal B cells in the secondary lymphoid organs
Stem cells continually generateArise from fetal liver and bone marrow in adult
Are the principal B cells in the adaptive immune responseHave extensive variable region repertoire that is generated somaticallyRequire T cell help and hence show memory
Ontogeny of Conventional B cellsIn the fetus, before there is a BM, hematopoiesis takes place in the liver
Although the fetal liver shuts off around birth, it is often referred to as the source of stem cells
In the adult, Ig gene rearrangement and the initialevents in B cell differentiation occur in the bonemarrow (BM)
Stem cells from the fetal liver persist for life around the periphery of the hollow bone space
Stem cells divide and migrate toward the large vein in the center of the bone
The spongy matrix of reticular cellsmacrophages and other supportingcells produce growth and differentiationfactors that guide development
Bone-marrowstromal cell
Pro-B cells
VLA-4
VCAM-1
SCFc-kit
IL-7receptor
IL-7
mIgM
Pre-B cells
Immature B cells
The adhesion molecule VLA-4 on the pro-B cells interacts with VCAM-1 on stromal cells
Bone-marrowstromal cell
Pro-B cells
VLA-4
VCAM-1
SCFc-kit
IL-7receptor
IL-7
mIgM
Pre-B cells
Immature B cells
c-Kit on the pro-B cells then interacts with stem-cell factor (SCF) on the stromal cell activating c-Kit and causing the pro-B to divide and differentiate into a pre-B cell
Bone-marrowstromal cell
Pro-B cells
VLA-4
VCAM-1
SCFc-kit
IL-7receptor
IL-7
mIgM
Pre-B cells
Immature B cells
Pre-B cells express the IL-7 receptor. IL-7 produced by the stromal cells drives the maturation process
B220
Pro-B Pre-B Immature Mature ActivatedPlasma cell
DH->JH
surrogateL chain IgM IgM IgD
IgM,IgD,IgGIgA or IgE
SecretedIgG, IgA,IgE or IgM
H chaingenes
DHJH VHDHJH VHDHJH VHDHJH VHDHJH VHDHJH
L-chaingenes
germ-line κ andλ
VLJ L- germ line
κ andλVLJ L VLJ L VLJ L
1/RAG2RAG
_+ + + _ _
TdT
L chain_ Surrogate
L chainκ orλ κ orλ κ orλ κ orλ
Secreted Ig _ _ _ _ Low levels μ, γ, α orε
Class switching _ _ _ _ _+Somaticmutation
_ _ _ _ _+
Membrane H chain
μ μ+δμ μ+δα, ε orγ
μ
B cell development
__+ + _ _ _ _
B220
Pro-B Pre-B Immature Mature ActivatedPlasma cell
DH->JH
surrogateL chain IgM IgM IgD
IgM,IgD,IgGIgA or IgE
SecretedIgG, IgA,IgE or IgM
H chaingenes
DHJH VHDHJH VHDHJH VHDHJH VHDHJH VHDHJH
B cell development
Note there is an ordered sequence of events for H chains
H chaingenesDHJHL-chaingenesgerm-line κ andλ
1/RAG2RAG
+TdT L chain_ Secreted Ig_ Classswitching_Somaticmutation_Membrane H chain_+
B220 is a molecule present on B cells and marks all cells of theB lineage
H chaingenesL-chaingenesgerm-line κ andλ
1/RAG2RAG
+TdT L chain Secreted Ig_ Classswitching_Somaticmutation_Membrane H chain
+VHDHJ HSurrogate L chain
μ
Surrogate L chain is comprised of Vpre-B and λ5 and is necessary to get expression of the pre-B cell receptor on the surface
H chaingenesL-chaingenesRAG1/RAG2+TdTL chainSecreted Ig_Class switching_Somaticmutation_MembraneH chainVHDHJHμVLJ Lκ orλ
The immature B cell expresses IgM on its surface
H chaingenesL-chaingenesRAG1/RAG2TdTL chainSecreted Ig_Class switching_Somaticmutation_MembraneH chainVHDHJHVLJLκ orλμ+δ
Mature B cells leave the bone marrow and populate the secondary lymphoid organs (spleen and lymph node)
The mature B cell expresses both IgM and IgD on its surface
H chaingenesL-chaingenesRAG1/RAG2TdTL chainSecreted Ig_Class switching_Somaticmutation_MembraneH chainVHDHJHVLJLκ orλμ+δ
Up to this point events occur in the absence of antigen stimulationThis development takes place in the bone marrow
H chaingenesL-chaingenesRAG1/RAG2TdTL chainSecreted IgClass switchingSomaticmutationMembraneH chainVHDHJHVLJLκ orλμ+δ Low levels++α, ε orγμ
Activation requires stimulation by antigen and occurs in the peripheral lymphoid tissue
H chaingenesL-chaingenesRAG1/RAG2TdTL chainSecreted IgClass switchingSomaticmutationMembraneH chainVHDHJHVLJLκ orλHigh levelsμ, γ, α orε
The plasma cell is the terminally differentiated B cell and is committed to the production of large quantities of a specific antibody
B220
Pro-B Pre-B Immature Mature ActivatedPlasma cell
DH->JH
surrogateL chain IgM IgM IgD
IgM,IgD,IgGIgA or IgE
SecretedIgG, IgA,IgE or IgM
H chaingenes
DHJH VHDHJH VHDHJH VHDHJH VHDHJH VHDHJH
L-chaingenes
germ-line κ andλ
VLJ L- germ line
κ andλVLJ L VLJ L VLJ L
1/RAG2RAG
_+ + + _ _
TdT
L chain_ Surrogate
L chainκ orλ κ orλ κ orλ κ orλ
Secreted Ig _ _ _ _ Low levels μ, γ, α orε
Class switching _ _ _ _ _+Somaticmutation
_ _ _ _ _+
Membrane H chain
μ μ+δμ μ+δα, ε orγ
μ
B cell development
__+ + _ _ _ _
T r a n s f e c t E S c e l l s a n d s e l e c t c e l l s w h i c h e x p r e s s t h e n e o g e n e
T a r g e t G e n e
T a r g e t G e n e
T a r g e t G e n e
H o m o l o g o u s
R e c o m b i n a t i o n
V e c t o r
n e o
n e o
n e o
V e c t o r
C h r o m o s o m e
C h r o m o s o m e
Vector for making a knock-out mouse
from recipient blasctocyst
cells, black are from ES cells.
Note that these are heterozygous (AB).
They are black because black coat
color is dominant. Mating of these mice
will give you AA, AB and BB mice.
A
are from
mouse stain B with black
coat.
Blastocoel cells are from
strain A with white coat.
Note that some mice will also be white if they are not from the ES cells
CD8
If you knock-out RAG1 or RAG2 the BCR and TCR remain unrearranged
Both B cell and T cell development is arrested
Surface Ig is the receptor for the B cell
Ig-α and Ig- associate with membrane bound heavy chain
Ig-α and Ig- have immunoreceptor tyrosine-based activation motifs (ITAM)
Activation through the BCR leads to phosphorylation of the ITAMs by Src family kinases and the recruitment and activation of Syk kinase with the signal transduced along several intracellular pathways
Pro-BB220CD43+
Pre-BB220CD43+
ImmatureBB220CD43-
Mature BB220CD43-
W T R A G- / -
I g - / -
S p l e e n
I g μ
B 2 2 0
T h y m u s
C D 8
C D 4
B o n e
m a r r o w
C D 4 3
B 2 2 0
Cells lacking Ig-α or Ig- do not have surface Ig and arrest in B cell developmentThere is no affect on T cells
Before L chain rearrangement a surrogate L chain comprised of Vpre-B and λ5 associates with the BCRDisruption of expression of the surrogate L chain blocks most B cell differentiation at the pre-B stage
Signaling through the BCR is important for establishing allelic exclusion and initiating L chain rearrangement
Both require expression of the membrane form of IgM
Tra1
2
3
4
1 2 3 4
m i c r o i n j e c t i o n
r e t r o v i r a l i n f e c t i o n
o v i d u c t t r a n s f e r
S o u t h e r n b l o t
a n a l y s i s
o f o f f s p r i n g t a i l
D N A
Transgenic mice have been used to show that expression of mIgM stops DNA rearrangement
Expression of a functional heavy chain on the membrane inhibits further heavy chain rearrangement
Murine IgM Murine IgM
EVH
CH
1 CH
2 CH
3 CH
4
s e c r e t e d μ
m e m b r a n e μ
V e c t o r N o . C l o n e s C l o n e s w i t h κ r e a r r a n g e m e n t
- - 1 1 0
μ s e c r e t e d1 9 0
μ m e m b r a n e 3 9 9
Expression of a functional heavy chain on the surface of a B cell also initiates L chain rearrangement
This was demonstrated by transfecting B cells and assaying for L chain rearrangement
Allelic exclusion guarantees that a B cell makes only one functional antibodySynthesis of a functional H chain turns off H chain rearrangement and turns on L chain rearrangementSynthesis of a functional Ab turns off L chain rearrangement
P r o g e n i t o r B C e l l
DH
JH P r o d u c t i v e
a l l e l e # 1
μ h e a v y c h a i n i n h i b i t s
r e a r r a n g e m e n t o f μ a l l e l e # 2
a n d i n d u c e s κ
r e a r r a n g e m e n t
P r o d u c t i v e
a l l e l e # 2
P r o d u c t i v e
a l l e l e # 1
μ + κ c h a i n s i n h i b i t
r e a r r a n g e m e n t o f κ a l l e l e
# 2 a n d λ r e a r r a n g e m e n t
N o n p r o d u c t i v e
a l l e l e # 1
N o n p r o d u c t i v e
a l l e l e # 2
P r o d u c t i v e
a l l e l e # 2
μ + κ c h a i n s i n h i b i t λ
r e a r r a n g e m e n t
μ + λ c h a i n s i n h i b i t
r e a r r a n g e m n t o f λ
a l l e l e # 2
N o n p r o d u c t i v e
a l l e l e # 2
C e l l d e a t h
P r o d u c t i v e
a l l e l e # 2
N o n p r o d u c t i v e
a l l e l e # 1
VH
DH
JH
VH
DH
JH
VH
DH
JH
N o n p r o d u c t i v e
a l l e l e # 1V
HD
HJ
H
N o n p r o d u c t i v e
a l l e l e # 2
C e l l d e a t h
Vκ
Jκ
Vκ
Jκ
Vλ
Jλ P r o d u c t i v e
a l l e l e # 1
Vλ
Jλ
μμ
μ
μμ
μ μ + κ
μ + κ
μ + λ
μ + λ
P r o g e n i t o r B C e l l
DH
JH P r o d u c t i v e
a l l e l e # 1
μ h e a v y c h a i n i n h i b i t s
r e a r r a n g e m e n t o f μ a l l e l e # 2
a n d i n d u c e s κ
r e a r r a n g e m e n t
P r o d u c t i v e
a l l e l e # 2
P r o d u c t i v e
a l l e l e # 1
μ + κ c h a i n s i n h i b i t
r e a r r a n g e m e n t o f κ a l l e l e
# 2 a n d λ r e a r r a n g e m e n t
N o n p r o d u c t i v e
a l l e l e # 1
N o n p r o d u c t i v e
a l l e l e # 2
P r o d u c t i v e
a l l e l e # 2
μ + κ c h a i n s i n h i b i t λ
r e a r r a n g e m e n t
μ + λ c h a i n s i n h i b i t
r e a r r a n g e m n t o f λ
a l l e l e # 2
N o n p r o d u c t i v e
a l l e l e # 2
C e l l d e a t h
P r o d u c t i v e
a l l e l e # 2
N o n p r o d u c t i v e
a l l e l e # 1
VH
DH
JH
VH
DH
JH
VH
DH
JH
N o n p r o d u c t i v e
a l l e l e # 1V
HD
HJ
H
N o n p r o d u c t i v e
a l l e l e # 2
C e l l d e a t h
Vκ
Jκ
Vκ
Jκ
Vλ
Jλ P r o d u c t i v e
a l l e l e # 1
Vλ
Jλ
μμ
μ
μμ
μ μ + κ
μ + κ
μ + λ
μ + λ
In most cases there is an ordered rearrangement of L chains with rearrangement first taking place on both alleles of the κ light chain locus
- If both of these are non productive then rearrangement begins at theλ locus
Light chain rearrangement
P r o g e n i t o r B C e l l
DH
JH P r o d u c t i v e
a l l e l e # 1
μ h e a v y c h a i n i n h i b i t s
r e a r r a n g e m e n t o f μ a l l e l e # 2
a n d i n d u c e s κ
r e a r r a n g e m e n t
P r o d u c t i v e
a l l e l e # 2
P r o d u c t i v e
a l l e l e # 1
μ + κ c h a i n s i n h i b i t
r e a r r a n g e m e n t o f κ a l l e l e
# 2 a n d λ r e a r r a n g e m e n t
N o n p r o d u c t i v e
a l l e l e # 1
N o n p r o d u c t i v e
a l l e l e # 2
P r o d u c t i v e
a l l e l e # 2
μ + κ c h a i n s i n h i b i t λ
r e a r r a n g e m e n t
μ + λ c h a i n s i n h i b i t
r e a r r a n g e m n t o f λ
a l l e l e # 2
N o n p r o d u c t i v e
a l l e l e # 2
C e l l d e a t h
P r o d u c t i v e
a l l e l e # 2
N o n p r o d u c t i v e
a l l e l e # 1
VH
DH
JH
VH
DH
JH
VH
DH
JH
N o n p r o d u c t i v e
a l l e l e # 1V
HD
HJ
H
N o n p r o d u c t i v e
a l l e l e # 2
C e l l d e a t h
Vκ
Jκ
Vκ
Jκ
Vλ
Jλ P r o d u c t i v e
a l l e l e # 1
Vλ
Jλ
μμ
μ
μμ
μ μ + κ
μ + κ
μ + λ
μ + λ
Cells which sustain non-productive Ig gene rearrangements and thus fail to express either functional Ig H-or L- chains are eliminated by apoptosis.
Immature bone marrow B cells expressing surface IgM that react with self-antigens are eliminated.
Mature B cells which co-express IgM and IgD exit the bone marrow and circulate to peripheral lymphoid tissues where the subsequent steps in B cell differentiation occur.
Only a small fraction of the total B lineage cells produced in the bone marrow survive to exit as mature peripheral B cells.
When immature B cell encounter self-antigen within the bone marrowthey die, presumably by apoptosis
These mice are transgenic for an Ab specific for Kk
Only a small fraction of the B cell produced in the bone marrow survive to exit and go to the peripheral lymphoid organs
Cells which sustain non-productive Ig rearrangements andfail to express functional surface Ig are eliminated byapoptosis
Immature bone marrow B cells expressing surface IgMthat reacts with self-antigens are eliminated
Mature B cells expressing IgM + IgD exit the bone marrow andpopulate the peripheral lymphoid organs
Further development is antigen dependent
Production of cells which will grow forever and produce a singlehomogeneous (monoclonal) antibody
These cells are called hybridomas
Antigen
Myeloma cells(HGPRT-)
Spleen cells
Clones each positiveculture
1. Culture in HAT medium
2. Test each supernatant for antibodies
Test each supernatantfor antibodies
Expand positive clones
Propagate
Monoclonal AbsMonoclonal Abs
It is now possible to immortalize a B cell producing a monoclonalantibody
The resulting cell lines are called hybridomas
Spleen cells makeantibody
Myeloma cells grow forever
De novo pathway
Phosphoribosyl pyrophosphage + Uridylate
Salvage pathway
Hypoxanthine + Thymidine
Nucleotides DNA
blocked by aminopterin
Catalyzed by HGPRT
and TK enzymes
HAT medium =
Hypoxanthine +
Aminopterin +Thymidine
In the presence of aminopterin cells must use the salvage pathway for DNA synthesis
For these experiments myeloma cells which can grow indefinitely but are deficient in either HGPRT or TK are used
De novo pathway
Phosphoribosyl pyrophosphate + Uridylate
Salvage pathway
Hypoxanthine + Thymidine
Nucleotides DNA
blocked by aminopterin
Catalyzed by HGPRT and TK enzymes
HAT medium =Hypoxanthine +Aminopterin +Thymidine
In the presence of aminopterin cells must use the salvage pathway for DNA synthesis
For these experiments myeloma cells which can grow indefinitely but are deficient in either HGPRT or TK are used
-if cells lack enzymes of salvage pathway they die
Myeloma
HGPRT-
Immortal
Spleen Cells
HGPRT+
Limited
Lifespan
Non-
selective
medium
fuse
inefficient so must
select few hybrids
Myeloma
HGPRT-
Immortal
Dies
Hybrid
HGPRT+
Immortal
Viable
Spleen Cells
HGPRT+
Limited
Lifespan
Dies
HAT
medium
Myeloma
HGPRT-
Immortal
Spleen Cells
HGPRT+
Limited
Lifespan
Non-
selective
medium
fuse
inefficient so must
select few hybrids
Myeloma
HGPRT-
Immortal
Dies
Hybrid
HGPRT+
Immortal
Viable
Spleen Cells
HGPRT+
Limited
Lifespan
Dies
HAT
medium
top related