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