REVIEW: IMPORTANT INFORMATION TO DATEREVIEW: IMPORTANT INFORMATION TO DATEABOUT ===> B cells ABOUT ===> B cells & ANTIBODY ANTIBODY--------------------------------------------------------------------------------B cells ----> Express surface Antibody

= BCR (B-cell receptor)

B cells produce ----> Antibody

B cells when ---> Plasma cells properly stimulated

Plasma cells are ----> Antibody “factories”

Note: each B cell has a single type of antigen-binding site*---------------------------------------------------------------------------------* B cells are clonal (only the antigen-binding site type per cell)

LECTURE 10 GOALS:----------------------------------------------------------------------------1) Production of a pro-B cell

- IgH gene events: rearrangements

2) Production of a pre-B cell- IgH chain & surrogate light chain expression- IgL gene events: rearrangement

3) Production of a B cell- membrane versus secreted antibody

4) Generation of antibody diversity----------------------------------------------------------------------------

STAGES IN THE DEVELOPMENT OF STAGES IN THE DEVELOPMENT OF NAÏVE B CELLS* B CELLS*---------------------------------------------------------------------------------------------

Receptor Cell expression Receptor

pro-B cell None - - - - - -

pre-B cell pre-BCR pre B-cell receptor

B cell BCR surface Antibody

---------------------------------------------------------------------------------------------* naïve = development independent of foreign antigen

STAGES inSTAGES in naïve B CELL DEVELOPMENTB CELL DEVELOPMENT---------------------------------------------------------------------------------------------Cell Protein expressionpro-B cell BCR ( - )

pro-B cell, earlypro-B cell, late

pre-B cell pre-BCR (pre B-cell receptor)

B cell Surface Antibody---------------------------------------------------------------------------------------------

STAGES inSTAGES in naïve B CELL DEVELOPMENTB CELL DEVELOPMENT---------------------------------------------------------------------------------------------Cell Protein expressionpro-B cell BCR ( - )

pre-B cellpre-B cell, large pre-BCR (pre B-cell receptor)

surrogate IgL (+) IgH chain

pre-B cell, small cytoplasmic IgH chain

B cell Surface Antibody---------------------------------------------------------------------------------------------

STAGES inSTAGES in naïve B CELL DEVELOPMENTB CELL DEVELOPMENT---------------------------------------------------------------------------------------------Cell Protein expressionpro-B cell BCR ( - )

pre-B cell pre-BCR (pre B-cell receptor)

B cellnaïve immature Surface IgM (BCR expressed)

naïve mature Surface IgM & IgD (BCR expressed) ---------------------------------------------------------------------------------------------

Events that must occur in producing a viable B cell:Events that must occur in producing a viable B cell:---------------------------------------------------------------------------------Sequence of proteins expressed in the formation ofnaïve mature B cells (IgM+,IgD+ B cell)

Chain Cellular expressionIgH chain cytoplasm

Surrogate cell surface: pre-BCRlight chain* surrogate light chain + IgH

IgL BCR (surface antibody): surface IgM( or )

& chains IgM & IgD-----------------------------------------------------------------------------------* Two associated proteins: VpreB and 5

Events that occur in producing a functional Antibody---------------------------------------------------------------------------------MolecularEvent Consequence Cell stageDH --> JH DHJH Early pro-B cell

VH --> DHJH VHDHJH Late pro-B cell----------------------------------------------------------------------------------IgH chain & pre-BCR Large pre-B cellsurrogate IgLexpression

VL --> JL VLJL Small pre-B cell----------------------------------------------------------------------------------IgH & IgL BCR (surface Naïve immatureexpression IgM expression) B cell-----------------------------------------------------------------------------------

Events that occur in producing a functional Antibody---------------------------------------------------------------------------------MolecularEvent Consequence Cell stageDH --> JH DHJH Early pro-B cell

VH --> DHJH VHDHJH Late pro-B cell----------------------------------------------------------------------------------IgH chain & pre-BCR Large pre-B cellsurrogate IgL surfaceexpression expression

VL --> JL VLJL Small pre-B cell----------------------------------------------------------------------------------IgH & IgL BCR (surface Naïve immatureexpression IgM expression) B cell-----------------------------------------------------------------------------------

Events that occur in producing a functional Antibody---------------------------------------------------------------------------------Event Consequence Cell stageDH --> JH DHJH Early pro-B cell

VH --> DHJH VHDHJH Late pro-B cell---------------------------------------------------------------------------------- & surrogate IgL pre-BCR Large pre-B cell

VL --> JL VLJL Small pre-B cell----------------------------------------------------------------------------------IgH & IgL BCR, IgM Naïve immatureexpression B cell

& chains BCR: IgM & IgD Naïve immatureB cell

-----------------------------------------------------------------------------------

FORMATION OF A FUNCTIONAL HEAVY CHAIN FORMATION OF A FUNCTIONAL HEAVY CHAIN (IgH (IgH chain) chain): pro-B cell events-----------------------------------------------------------------------------------DNAEvents Consequence Cell stageDH --> JH DHJH Early pro-B cell

VH --> DHJH VHDHJH Late pro-B cell

- Review: organization of the IgH gene (chromosome 14)------------------------------------------------------------------------------------

U5-06-2

VH DH J H3 1 2 42 11 2

ORGANIZATION OF IgH GENE: Chr 14ORGANIZATION OF IgH GENE: Chr 14

ONE SELECTED TO MAKE UP CHUSED TO MAKE: V REGION

H

V, D, J

SEGMENTs

VARIABLEGENE

SEGMENTs

DIVERSITYGENE

SEGMENTs

JOININGGENE

SEGMENTS

CONSTANTGENE

2

= PSEUDOGENE

VH DH J H3 1 2 42 11 2

EACH SEGMENT IS AN EXON

U5-07-1

D H

SEGMENTS

DIVERSITY

N = 27

GENE

JH

SEGMENTS

JOINING

N = 6

GENE

IgH GENE ORGANIZATIONIgH GENE ORGANIZATION

V H

SEGMENTS

VARIABLE

N = 65

GENE

EACH V GENE SEGMENTIS MADE UP OF ALEADER EXON AND A V EXONH

L V HLEADER

EXONSEQUENCE

"EXON"

C1

EXON

C2

EXON

C3

EXON

C4 /SC

EXON

M

EXON

U5-08-1

MADE UP OF SEVERAL EXONSEACH CH GENE SEGMENT IS

V H DH J H3 1 2 42 11 2

SEGMENTs SEGMENTs SEGMENTs

VARIABLE DIVERSITY JOINING

GENE GENE GENE

M = TRANSMEMBRANE (aka TM)C = CYTOPLASMIC TAIL

SC = SECRETED FORMCARBOXY TERMINUS

SEGMENTSSEGMENTS

CONSTANTCONSTANTGENEGENE

EXON

C

EXONS MATCH w/ IgHEXONS MATCH w/ IgHconstant region domainsconstant region domains

IgH GENE CONSTANT REGION ORGANIZATIONIgH GENE CONSTANT REGION ORGANIZATION

D Segment ---> J Segment REARRANGEMENT

NOTE: ALL CONSTANTSEGMENTS ARE

NOT SHOWN

DH J H 3VH1...VH 51

FIRST STEP IN FORMATION OF A FUNCTIONAL IgH GENE:FIRST STEP IN FORMATION OF A FUNCTIONAL IgH GENE:D ---> J REARRANGEMENT D ---> J REARRANGEMENT (IRREVERSIBLE DNA EVENT)

U5-10-C

ACTION OF RECOMBINASE

(INCLUDES RAG 1 AND RAG 2)(RAG = Recombination Activating Genes)

(RANDOM)

DNA deletional event

Early pro-B cell: D ---> J : RESULT: DJ

3J H5

J H6

D H

VH1...V H511

V 2 -D 1 -J 5H H H

FORMATION OF FUNCTIONAL IgH HEAVY CHAIN:IRREVERSIBLE DNA REARRANGEMENT: V ----> DJ

U5-10-E

V ---> DJ REARRANGEMENT

VH31

VH 2

3

ACTION OF RECOMBINASE

(INCLUDES RAG 1 AND RAG 2)

Late pro-B cell: V ----> DJ : RESULT: VDJ

DNA deletional event

ARE UNSUCCESSFULARE UNSUCCESSFUL((SUCCESSFUL = "PRODUCTIVE")SUCCESSFUL = "PRODUCTIVE")

VDJ on IgH ALLELE "A"

PRODUCTIVE

PRE-B CELLDEVELOPS

NONPRODUCTIVE

IgH ALLELE "B"REARRANGES

VDJ on IgH ALLELE "B"

PRODUCTIVE

PRE-B CELLDEVELOPS

NO FURTHER IgHREARRANGEMENT"ALLELIC EXCLUSION"

NONPRODUCTIVE

CELLDIES U5-12-1

OUTCOME IF THE IgH VDJ REARRANGEMENTSOUTCOME IF THE IgH VDJ REARRANGEMENTS

FORMATION OF pre-B CELL: FORMATION OF pre-B CELL: RequirementsRequirements----------------------------------------------------------------------------- Expression of IgH chain- Expression of surrogate light chain- Surface expression of pre-BCR

VDJ

TRANSCRIPTION

3

IgH GENE

nascent mRNA

U5-11-A

U5-11-B

VDJSPLICED OUT

nascent mRNA

V D J

mature mRNA AAAAAA

Splicing of IgH Heavy Chain mRNA

NOTE: DO NOT CONFUSE THE RNA SPLICING EVENT

WITH THE DNA RECOMBINATION EVENTS OF VDJ JOINING

SPLICING

contantgene exons

nascent mRNA

mature mRNA for surface chain expression

DETAILED VIEW:Splicing of IgH Heavy Chain mRNA for SURFACE EXPRESSION

C1 C1 C1 C4/SC TM Cyto.

AAAAAA

Secretory-C-terminal (SC) portion of C4/SC exon not included in mRNA of chain of surface IgM

AAAAAAMaturemRNA

Translation

Variableregion/domain

C1 C2 C3 C4

TM

Cyto

IgL

RELATIONSHIP OF EXONS, mRNA splicing & DOMAINSRELATIONSHIP OF EXONS, mRNA splicing & DOMAINS

DOMAINS

Protein

Splicing of IgH Heavy Chain mRNA for SECRETION

nascent mRNA C1 C1 C1 C4/SC M Cyto.

mature mRNA for secretion

AAAAAA

M and Cytoplasmic exons not not includedin mRNA of chain of secreted IgM

V D J

C 1

IgH PROTEIN CHAIN

CDR1 CDR2 CDR3

FR = FRAMEWORK REGIONCDR = COMPLEMENTARITY DETERMINING REGION = HVRHVR = HYPERVARIABLE REGION

REVIEW: WU-KABAT PLOT for variability vs

amino acid position

U5-11-D

VH CH

V D JC1 C2 C3 C4

DOMAINS ILLUSTRATED

<=== Regions

FR1 FR2 FR3 FR4

V REGIONH

JUNCTIONS BETWEEN D & J and BETWEEN V & DJ:JUNCTIONS BETWEEN D & J and BETWEEN V & DJ:Site of diversification in 1Site of diversification in 100 amino acid sequence amino acid sequence

V

CDR1 CDR2FR1 FR2 FR3

J

C 1

FR4

V ---> DJ D ---> J

D

CDR3

Sequence variations in CDR3 affect epitope binding!

IMPRECISE RECOMBINATIONIMPRECISE RECOMBINATION

T C AC A TV SEGMENT D SEGMENT

C A T T C A ---HIS-SER---

T C A --SER---

C A T T C A

A --GLN---C AU5-19-2

C A T T C A

N REGION DIVERSITY: BASES ADDED at JUNCTIONSN REGION DIVERSITY: BASES ADDED at JUNCTIONS

N = A, T , C, or G

N N N IN FRAME

N NNN

ADDED BY TdT(TERMINAL

DEOXYNUCLEOTIDYLTRANSFERASE)

V

V OUT OFFRAME

D SEGMENT

D SEGMENT

V-DJJUNCTION

D-JJUNCTION

J

J

NOTE: DIVERSITY SEGMENT CAN BE READ IN MANY READING FRAMES!

15:1 - 15:1 - ARE THESE N-REGION ADDITIONS IN FRAMEARE THESE N-REGION ADDITIONS IN FRAMEOR OUT-OF-FRAME?OR OUT-OF-FRAME?

N = A, T , C, or G

NN N IN FRAME

NN N IN FRAME

ADDED BY TdT(TERMINAL

DEOXYNUCLEOTIDYLTRANSFERASE)

V

V

D SEGMENT

D SEGMENT

V-DJJUNCTION

D-JJUNCTION

J

J

U5-20-1

VH CHREGION REGION

CDR1 CDR2 CDR3

CDR1

CDR2

CDR3

EPITOPE

U5-11-E

SIMPLIFIED VIEW AS 6 CDRs (3 FROM IgH AND 3 FROM IgL)

MAKE UP THE SITE ON THE ANTIBODY WHERE ANTIGENIC

DETERMINANTS (EPITOPES) ARE BOUND

CHAIN, REGIONS, DOMAINS, CDR’s, EPITOPE BINDINGCHAIN, REGIONS, DOMAINS, CDR’s, EPITOPE BINDING

IgHchainSurrogate

light chain

pre-B cell Receptorpre-B cell Receptor(pre-BCR)(pre-BCR)

LARGE pre-B CELL: pre-BCR EXPRESSION

Expression ofpre-BCR is atrigger to initationof IgL generearrangement:

Transition to:Small pre-B cell

SMALL pre-B CELL: IgL rearrangement

IgL GENE ORGANIZATIONIgL GENE ORGANIZATION- separate genes for - separate genes for & &

- - gene: chr. 2; gene: chr. 2; gene: chr. 22 gene: chr. 22

Study

V K JK CKappa

KAPPA GENE ORGANIZATION:

KAPPA GENE REARRANGES before LAMBDA GENE

EXONs Single EXON(single domain)

N = 40 N = 5

V K J K C Kappa

J K C K

KAPPA GENE ORGANIZATIONKAPPA GENE ORGANIZATION

U5-13-2

VK

L V EXONsK

K

CK

VK JK C

V ---> J REARRANGEMENT

Kappa GENE

Kappa GENE

VK JK CK

Kappa GENE

ACTION OF RECOMBINASE

KAPPA LIGHT CHAIN V to J REARRANGEMENTKAPPA LIGHT CHAIN V to J REARRANGEMENTIRREVERSIBLE DNA EVENT: IRREVERSIBLE DNA EVENT: Small pre-B cell

U5-14-1

V 1K J 3K

Deletional event

TRANSLATION

mature mRNA AAAAAAAAA

U5-14-2

nascent mRNA

LIGHT CHAIN mRNA TRANSCRIPTION AND TRANSLATIONLIGHT CHAIN mRNA TRANSCRIPTION AND TRANSLATION

V JK K CK

Kappa GENE

TRANSCRIPTION

mRNA splicing

VJCKAPPA IgL (LIGHT) CHAINPROTEIN:

U5-15-1

VJ REARRANGEMENTSVJ REARRANGEMENTS

VJ, ALLELE "A"

PRODUCTIVE NONPRODUCTIVE

NAIVE IMMATURE B CELLDEVELOPS

KAPPA ALLELE "B"REARRANGES

VJ, ALLELE "B"

PRODUCTIVE NONPRODUCTIVE

NAIVE IMMATURE B CELLDEVELOPS

NO FURTHER IgLREARRANGEMENT"LIGHT CHAIN ISOTYPE EXCLUSION"

LAMBDA GENEREARRANGES

KAPPA GENE

ETC.

NEED PRODUCTIVE IgL REARRANGEMENT FOR FORMATION OFIMMATURE "NAIVE" B CELL

"IN FRAME" "NOT IN FRAME"

CONSEQUENCES OF "NONPRODUCTIVE KAPPA IgLCONSEQUENCES OF "NONPRODUCTIVE KAPPA IgL

(Expresses Kappa) SIMILAR TO KAPPA

IgHchain

IgL expression

BCR expressionBCR expression(IgM)(IgM)

Naïve immature B CELL: BCR (IgM) EXPRESSION

TRANSITION FROM NAÏVE TRANSITION FROM NAÏVE IMMATUREIMMATURE B CELL B CELL to to NAÏVE NAÏVE MATUREMATURE B CELL: B CELL: --------------------------------------------------------------------------------Simultaneous expression of IgM & IgD

- mRNA of B cell includes constant region in addition to constant region

- mRNA: termed a “long nuclear transcript”- mRNA alternative splicing results in either constant

region or constant region with the same VDJ-----------------------------------------------------------------------------------

3

VDJ

IgH GENE

VDJnascent mRNA

LONG NUCLEARTRANSCRIPT

U5-16-1

TRANSCRIPTIONTHRU MU and DELTA

TRANSLATION TRANSLATION

VDJ VDJ

VDJAAAAAA

VDJMU CHAIN

VDJDELTA CHAIN

ALTERNATIVE SPLICING OF IgH mRNA produces NAÏVE MATURE B CELLALTERNATIVE SPLICING OF IgH mRNA produces NAÏVE MATURE B CELL

U5-17-1IgM

plus L CHAIN

IgD

plus L CHAIN

PROTEIN

VDJAAAAAA

SPLICINGRNA

IgHchain

IgL expression

IgM expressionIgM expression

Naïve mature B CELL: IgM & IgD EXPRESSION

IgH chain

IgD expressionIgD expression

Antigen-binding sites of IgM & IgD are identical: B cell is still “clonal”

Naïve AntibodyRepertoire

Mature (naïve)B cell**

IgM

IgD

Naïve AntibodyRepertoire

Adaptive AntibodyRepertoire

“ Total” Antibody Repertoire = Naïve + Adaptive

Antigen

QUESTION: How can the immune system deal with an almost endless number of antigens that it has never been exposed to previously?

IMMUNE SYSTEM: Creates a “pre-existing” Antibody Repertoire* prior to any antigen exposure

REPERTOIRE = Sum total of the body’s different antibodies ~=> 10 billion different antibodies

CREATING THE NAÏVE ANTIBODY REPERTOIRE:CREATING THE NAÏVE ANTIBODY REPERTOIRE:CREATING DIVERSITY IN BCRCREATING DIVERSITY IN BCR----------------------------------------------------------------------------------Sources of diversity:

V D JDifferent VH’s, DH’s, JH’s IgH 65 27 6

VL’s, JL’s 40 --- 5 30 --- 4

Variations in junctions* Chain Junctions Incr. diversity- Imprecise recombination IgH 2 100- N-region diversity IgL 1 10

Various combinations of IgH and IgL---------------------------------------------------------------------------------* 10 fold incr. diversity with each junction

CREATING THE NAÏVE ANTIBODY REPERTOIRE:CREATING THE NAÏVE ANTIBODY REPERTOIRE:CREATING DIVERSITY IN BCRCREATING DIVERSITY IN BCR----------------------------------------------------------------------------------Sources of diversity: Combinatorial diversity

V D J CombinationsIgH 65 27 6 10,530 40 --- 5 200 30 --- 4 120

JunctionalCombinations (x) diversity Product

IgH ~10,000 100 1,000,000 200 10 2,000 120 10 1,200----------------------------------------------------------------------------------

CREATING THE NAÏVE ANTIBODY REPERTOIRE:CREATING THE NAÏVE ANTIBODY REPERTOIRE:CREATING DIVERSITY IN BCRCREATING DIVERSITY IN BCR---------------------------------------------------------------------------------

Combinatorial & Junctionaldiversity

IgH 1,000,000 2,000 1,200

[IgH (x) ] + [ IgH (x) ]

[ 106 x 2 x 103 ] + [ 106 x 1.2 x 103 ]

2x 109 + 1.2 x 109 = 3.2 x 103.2 x 1099

----------------------------------------------------------------------------------

V D J

mature mRNA

TRANSLATION TRANSLATION

AAAAAA

V D J

IgH CHAIN PROTEIN

DRAWING THE REGIONS IN ~CORRECT PROPORTIONS

VH

CH

REGION REGION

IgH CHAIN

U5-11-C