Microarray analysis indicates that different subsets of B cells express specific “gene signatures.”

Microarray analysis indicates that differentsubsets of B cells expressspecific “gene signatures.”

Adaptive ImmunityAdaptive Immunity

• Central objective:

• Protect against foreign invaders• Create memorymemory of invasion to prevent

recurrent infection• Response must be highly specificspecific to

the invader

Ig Isotypes: Classes and SubclassesIg Isotypes: Classes and Subclasses

Ig isotypes are defined by the C-terminal portion of

the molecule There are 5 classes of Ig and there are 4 sub-classes of IgG.

Isotype class determines effector function.

Ig IsotypesIg Isotypes

Ig Isotypes Localize to Different Ig Isotypes Localize to Different Anatomical Sites in the Body Anatomical Sites in the Body

Ig Isotypes Localize to Different Ig Isotypes Localize to Different Anatomical Sites in the Body Anatomical Sites in the Body

Ig Isotypes Have Different Effector Ig Isotypes Have Different Effector FunctionsFunctions

When Do Isotypes When Do Isotypes Matter?Matter?

The primary -> secondary immune response:

•Increase Ig titer

•Switch from IgM to IgG

•Increase Ag binding affinity

When Do Isotypes Matter?When Do Isotypes Matter?

Slide 2

Primary ->Secondary Immune Response TransitionPrimary ->Secondary Immune Response Transition

Primary/Secondary Immune Primary/Secondary Immune ResponseResponse

Slide 2

Events associated with Events associated with an immune response an immune response over timeover time:

Increase of IgM titer

Increase of IgM titer is specific to the immunizing Ag

Isotypes Switch: IgM->IgXIsotypes Switch: IgM->IgX

Three Types of Switch:

RNA levelRNA level•IgM->IgD• Membrane->Secreted

DNA level•Class Switch Recombination

IgM and IgDIgM and IgD Are Coexpressed in Are Coexpressed in Mature B CellsMature B Cells

Differentiation to the Differentiation to the mature B cell stage mature B cell stage is marked by the co-is marked by the co-expression of IgM expression of IgM and IgD on the and IgD on the plasma membraneplasma membrane

A Shift of IgM to IgM/IgD A Shift of IgM to IgM/IgD Expression in Mature B cellsExpression in Mature B cells

Mechanism:Mechanism:Alternative RNA Alternative RNA polyadenylation and polyadenylation and splicingsplicing

In Mature B Cells Ig is Located on In Mature B Cells Ig is Located on the Mb and Functions as the BCR the Mb and Functions as the BCR

Location of Ig on the cell surface is transmb and requires the presence of the mb exon

In Plasma Cells Ig is SecretedIn Plasma Cells Ig is Secreted

Secretion of Ig from the cell requires the presence of the secretion exon

In mature B In mature B cells Ig is cells Ig is located on the located on the plasma mb.plasma mb.Ig is secreted Ig is secreted from terminally from terminally differentiated differentiated plasma cellsplasma cells

Secreted Ig Promotes Critical Functions Secreted Ig Promotes Critical Functions

In mature B In mature B cells Ig is cells Ig is located on the located on the plasma mb.plasma mb.Ig is secreted Ig is secreted from terminally from terminally differentiated differentiated plasma cellsplasma cells

Secreted IgM and IgA are Multimerized Secreted IgM and IgA are Multimerized by J Chainby J Chain

Isotypes Switch: IgM->IgXIsotypes Switch: IgM->IgX

Three Types:

RNA levelRNA level•IgM->IgD• Membrane->Secreted

DNA levelDNA level•Class Switch Recombination

C Regions Are Arrayed in the C Regions Are Arrayed in the IgH LocusIgH Locus

VDJ S S3 S1 S2bC C3 C1E C2b

Looping Out and Deletion Model of SRLooping Out and Deletion Model of SR

VDJ C3S3CCSE S1 C1

mRNA

mRNA3 germline transcr ipt

A.

C

C

S S3

VDJ C3 S1 C1

B.

S S3 C3VDJ E S1 C1

3 mRNA

C.

S

C

S3

C+

Germline Transcripts are Required for Germline Transcripts are Required for SRSR

VDJ S S3 S1 S2bC C3 C1E C2b

S3 C3 S2b C2b

LPS:

S1 C1LPS + IL4:

I3

I1

I2b

P

mRNA

mRNA

sterile transcript

mRNA

When Do Isotypes When Do Isotypes Matter?Matter?

The primary -> secondary immune response:

•Increase Ig titer

•Switch from IgM to IgG

•Increase Ag binding affinityIncrease Ag binding affinity

Affinity Maturation and Ig Affinity Maturation and Ig Isotype SwitchIsotype Switch

In a primary immune response, affinity of Ig for its Ag is usually not high enough to immediately clear the pathogen from the system. However, affinity is increased by somatic hypermutation (SHM).

SHM alters V genes at the DNA levelDNA level

Primary/Secondary Immune Primary/Secondary Immune ResponseResponse

Slide 2Acquisition of Acquisition of mutations in the mutations in the V region of the V region of the VH and VL VH and VL proteinsproteins: nonrandom nonrandom distributiondistribution

Protein levelProtein level

Primary/Secondary Immune ResponsePrimary/Secondary Immune Response

Slide 2

The position of The position of the newly the newly acquired acquired mutations is in mutations is in the antigen the antigen binding binding pocket. pocket.

Protein levelProtein level

Somatic Hypermutation and Affinity Somatic Hypermutation and Affinity MaturationMaturation

In SHM, individual nucleotides in VJ or VDJ units are mutated, thus adjusting the specificity of and potentially increasing the affinity for Ag.

Somatic Hypermutation and Affinity Somatic Hypermutation and Affinity Maturation cont...Maturation cont...

Mutations translate into a change in the Ag binding pocket of the Ab. Some mutations are deleterious while others are neutral or advantageous for Ab:Ag fit. Some deleterious mutations generate autoreactive Ig.

Question: Question: are mutations random or directed at are mutations random or directed at the level of the genes?the level of the genes?

Somatic Hypermutation and Affinity Somatic Hypermutation and Affinity MaturationMaturation

DNA levelDNA level

Somatic Hypermutation and Affinity Somatic Hypermutation and Affinity Maturation cont...Maturation cont...

Selection of high affinity B cell clones leads to affinity maturation of the immune response. B cells expressing high affinity BCR are favored by clonal selection. This occurs when Ab:Ag complexes are highly compatible and possess a “good fit”. The high affinity Ab:Ag interaction transduces a strong signal to the B cell to proliferate.

Clonal Clonal Selection: Type ISelection: Type I

Clonal selection occurs following VDJ in the BM to delete B cells expressing autoreactive BCR.

Mutations which increase affinity of Ab for Ag will create a BCR which promotes strong proliferation. Selection also occurs in the periphery following SHM on the GC to delete newly autoreactive BCR and to preclude the escape of self-reactive B cells.

Clonal Clonal Selection: Type IISelection: Type II

SHM Occurs in SHM Occurs in the Germinal the Germinal

Center B CellsCenter B Cells

The Surprising Link between CSR, The Surprising Link between CSR, SHM and Gene ConversionSHM and Gene Conversion

Activation induced deaminase (AIDAID) is responsible for CSR and SHM.

It is the only B cell specific gene product required for both these genetic alterations.

VDJ C3S3CCSE S1 C1

mRNA mRNA

Germline transcript

C2bS2b S2a C2a

A. Isotype class switching

Pseudogene

RAD54, RAD51 paralogues

+ AID

CHSS

VDJ

C

C

C3

C1 C2b

Switch Recombination

AID Induced DSBs

SS1

1 mRNA

B. Somatic hypermutation C. Gene conversion

SS

mRNA

AID Induced Mutations

VDJ CH

The Surprising Link Between The Surprising Link Between CSR, SHM and Gene ConversionCSR, SHM and Gene Conversion

How does AID work?How does AID work?AID has homology to the APOBEC family of proteins which are involved in RNA

editing AID is also a cytidine deaminase and converts dC ->uracilUracil is mutagenic and triggers the BER

pathway

AID Deaminates dC and Triggers the BER AID Deaminates dC and Triggers the BER PathwayPathway

G U

U G

AID

GC G

C

AID

DSBs

G

G

APE

G5’ 3’

G3’ 5’

G5’

3’

G3’

5’

UNG

G5’ 3’

G3’ 5’

Tpol

What happens when AID is What happens when AID is mutated?mutated?

What happens when UNG is What happens when UNG is mutated?mutated?

What happens when DSB repair What happens when DSB repair proteins are mutated?proteins are mutated?

Hyper-IgM Syndrome: No Hyper-IgM Syndrome: No Secondary IsotypesSecondary Isotypes

Four Types: Cause????Four Types: Cause????Mutations in: •CD40 (T cell); CD40L (B cell)•AID•UNG•Subgroup with unknown cause

CSR Requires DNA Repair CSR Requires DNA Repair ProteinsProteins

DNA Repair Proteins Involved in CSR:DNA Repair Proteins Involved in CSR:DNA-PKcs, Ku70, Ku80, H2AX, NBSl, ATMMMR (Msh2, Msh6,Pms2, Mlhl)

The EndThe End

End of part IIEnd of part II

U

G

ReplicationG->A; C->TTransitions

SHMUNG Replication

Transitions and Transversions

MMR Transitions and Transversions

Error prone repair

A.

G U

U G UNG APE DSBs

MMR

CSR andGene conversion

B.

U=T During ReplicationU=T During Replication

BCR and TCR Break the Rules of Mendelian Genetics

Monospecific Ag receptors:Monospecific Ag receptors:

Only one H chain and one L chain is expressed per B/T cellwhereas each cell has 2 alleles for H and L chains