1. REVIEW: IMPORTANT INFORMATION TO DATE ABOUT ===> B cells ANTIBODY ABOUT ===> B cells & 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)

2. 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 ----------------------------------------------------------------------------

3. STAGES IN THE DEVELOPMENT OF B CELLS* STAGES IN THE DEVELOPMENT OF NAÏVE B CELLS* --------------------------------------------------------------------------------------------- Receptor CellexpressionReceptor pro-B cellNone- - - - - - pre-B cellpre-BCR pre B-cell receptor B cellBCRsurface Antibody --------------------------------------------------------------------------------------------- * naïve = development independent of foreign antigen

4. STAGES inB CELL DEVELOPMENT STAGES in naïve B CELL DEVELOPMENT --------------------------------------------------------------------------------------------- CellProtein expression pro-B cellBCR ( - ) pro-B cell, early pro-B cell, late pre-B cellpre-BCR (pre B-cell receptor) B cellSurface Antibody ---------------------------------------------------------------------------------------------

5. STAGES inB CELL DEVELOPMENT STAGES in naïve B CELL DEVELOPMENT --------------------------------------------------------------------------------------------- CellProtein expression pro-B cellBCR ( - ) pre-B cell pre-B cell, largepre-BCR (pre B-cell receptor) surrogate IgL (+) IgH  chain pre-B cell, small cytoplasmic IgH  chain B cellSurface Antibody ---------------------------------------------------------------------------------------------

6. STAGES inB CELL DEVELOPMENT STAGES in naïve B CELL DEVELOPMENT --------------------------------------------------------------------------------------------- CellProtein expression pro-B cellBCR ( - ) pre-B cellpre-BCR (pre B-cell receptor) B cell naïve immature Surface IgM (BCR expressed) naïve mature Surface IgM & IgD (BCR expressed) ---------------------------------------------------------------------------------------------

7. Events that must occur in producing a viable B cell: --------------------------------------------------------------------------------- Sequence of proteins expressed in the formation of naïve mature B cells (IgM+,IgD+ B cell) ChainCellular expression IgH  chaincytoplasm Surrogatecell surface: pre-BCR light chain*surrogate light chain + IgH IgLBCR (surface antibody): surface IgM (  or  )  &  chainsIgM & IgD ----------------------------------------------------------------------------------- * Two associated proteins: VpreB and  5

8. Events that occur in producing a functional Antibody --------------------------------------------------------------------------------- Molecular EventConsequenceCell stage D H --> J H D H J H Early pro-B cell V H --> D H J H V H D H J H Late pro-B cell ---------------------------------------------------------------------------------- IgH  chain &pre-BCRLarge pre-B cell surrogate IgL expression V L --> J L V L J L Small pre-B cell ---------------------------------------------------------------------------------- IgH  & IgLBCR (surfaceNaïve immature expressionIgM expression)B cell -----------------------------------------------------------------------------------

9. Events that occur in producing a functional Antibody --------------------------------------------------------------------------------- Molecular EventConsequenceCell stage D H --> J H D H J H Early pro-B cell V H --> D H J H V H D H J H Late pro-B cell ---------------------------------------------------------------------------------- IgH  chain &pre-BCRLarge pre-B cell surrogate IgLsurface expression V L --> J L V L J L Small pre-B cell ---------------------------------------------------------------------------------- IgH  & IgLBCR (surfaceNaïve immature expressionIgM expression)B cell -----------------------------------------------------------------------------------

10. Events that occur in producing a functional Antibody --------------------------------------------------------------------------------- EventConsequenceCell stage D H --> J H D H J H Early pro-B cell V H --> D H J H V H D H J H Late pro-B cell ----------------------------------------------------------------------------------  & surrogate IgL pre-BCRLarge pre-B cell V L --> J L V L J L Small pre-B cell ---------------------------------------------------------------------------------- IgH  & IgLBCR, IgMNaïve immature expressionB cell  &  chainsBCR: IgM & IgDNaïve immature B cell -----------------------------------------------------------------------------------

11. FORMATION OF A FUNCTIONAL HEAVY CHAIN (IgH  chain) (IgH  chain) : pro-B cell events ----------------------------------------------------------------------------------- DNA EventsConsequenceCell stage D H --> J H D H J H Early pro-B cell V H --> D H J H V H D H J H Late pro-B cell - Review:organization of the IgH gene (chromosome 14) ------------------------------------------------------------------------------------

12. U5-06-2 V H D H J H 3124 21 12 ORGANIZATION OF IgH GENE: Chr 14 ONE SELECTED TO MAKE UP C H USED TO MAKE: V REGION H V, D, J SEGMENTs VARIABLE GENE SEGMENTs DIVERSITY GENE SEGMENTs JOINING GENE SEGMENTS CONSTANT GENE 2 = PSEUDOGENE

13. V H D H J H 3124 21 12 EACH SEGMENT IS AN EXON U5-07-1 D H SEGMENTS DIVERSITY N = 27 GENE J H SEGMENTS JOINING N = 6 GENE IgH GENE ORGANIZATION V H SEGMENTS VARIABLE N = 65 GENE EACH V GENE SEGMENT IS MADE UP OF A LEADER EXON AND A V EXON H LV H LEADER EXON SEQUENCE "EXON"

14. C1C1 EXON C2C2 C3C3 C  4 /SC EXON M U5-08-1 MADE UP OF SEVERAL EXONS EACH C H GENE SEGMENT IS V H D H J H 3124 21 1 2 SEGMENTs VARIABLEDIVERSITYJOINING GENE M = TRANSMEMBRANE (aka TM) C = CYTOPLASMIC TAIL SC = SECRETED FORM CARBOXY TERMINUS SEGMENTSCONSTANTGENE EXON C EXONS MATCH w/ IgH constant region domains IgH GENE CONSTANT REGION ORGANIZATION

15. D Segment ---> J Segment REARRANGEMENT NOTE: ALL CONSTANT SEGMENTS ARE NOT SHOWN D H J H 3 V H 1...V H 51 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 3 J H 5 J H 6 D H V H 1...V H 51 1

16. V 2 -D 1 -J 5 HH H FORMATION OF FUNCTIONAL IgH HEAVY CHAIN: IRREVERSIBLE DNA REARRANGEMENT: V ----> DJ U5-10-E V ---> DJ REARRANGEMENT V H 3 1 V H 2 3 ACTION OF RECOMBINASE (INCLUDES RAG 1 AND RAG 2) Late pro-B cell: V ----> DJ : RESULT: VDJ DNA deletional event

17. ARE UNSUCCESSFUL (SUCCESSFUL = "PRODUCTIVE") VDJ on IgH ALLELE "A" PRODUCTIVE PRE-B CELL DEVELOPS NONPRODUCTIVE IgH ALLELE "B" REARRANGES VDJ on IgH ALLELE "B" PRODUCTIVE PRE-B CELL DEVELOPS NO FURTHER IgH REARRANGEMENT "ALLELIC EXCLUSION" NONPRODUCTIVE CELL DIES U5-12-1 OUTCOME IF THE IgH VDJ REARRANGEMENTS

18. FORMATION OF pre-B CELL: Requirements ---------------------------------------------------------------------------- - Expression of IgH  chain - Expression of surrogate light chain - Surface expression of pre-BCR VDJ TRANSCRIPTION 3 IgH GENE nascent mRNA U5-11-A

19. U5-11-B VDJ SPLICED 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  contant gene exons

20. nascent mRNA mature mRNA for surface  chain expression DETAILED VIEW: Splicing of IgH Heavy Chain mRNA for SURFACE EXPRESSION C1C1 C1C1 C1C1 C  4/SC TMCyto. AAAAAA Secretory-C-terminal (SC) portion of C  4/SC exon not included in mRNA of  chain of surface IgM

21. AAAAAA Mature mRNA Translation Variable region/domain C1C1C2C2C3C3C4C4 TM Cyto IgL RELATIONSHIP OF EXONS, mRNA splicing & DOMAINS DOMAINS Protein

22. Splicing of IgH Heavy Chain mRNA for SECRETION nascent mRNA C1C1 C1C1 C1C1 C  4/SC MCyto. mature mRNA for secretion AAAAAA not M and Cytoplasmic exons not included in mRNA of  chain of secreted IgM

23. VDJ C 1 IgH PROTEIN CHAIN CDR1CDR2CDR3 FR = FRAMEWORK REGION CDR = COMPLEMENTARITY DETERMINING REGION = HVR HVR = HYPERVARIABLE REGION REVIEW: WU-KABAT PLOT for variability vs amino acid position U5-11-D V H C H VDJ C1C1 C2C2 C3C3 C4C4 DOMAINS ILLUSTRATED <=== Regions FR1 FR2 FR3 FR4 V REGION H

24. JUNCTIONS BETWEEN D & J and BETWEEN V & DJ: Site of diversification in 1 0 amino acid sequence V CDR1CDR2 FR1 FR2 FR3 J C 1 FR4 V ---> DJ D ---> J D CDR3 Sequence variations in CDR3 affect epitope binding!

25. IMPRECISE RECOMBINATION TCACAT V SEGMENT D SEGMENT CAT TCA ---HIS-SER--- TCA --SER--- CATTCA A --GLN---CA U5-19-2 CATTCA

26. N REGION DIVERSITY: BASES ADDED at JUNCTIONS N = A, T, C, or G NNN IN FRAME NNNN ADDED BY TdT (TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE) V V OUT OF FRAME D SEGMENT V-DJ JUNCTION D-J JUNCTION JJ NOTE: DIVERSITY SEGMENT CAN BE READ IN MANY READING FRAMES!

27. 15:1 - ARE THESE N-REGION ADDITIONS IN FRAME OR OUT-OF-FRAME? N = A, T, C, or G NNN IN FRAME NNN ADDED BY TdT (TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE) V V D SEGMENT V-DJ JUNCTION D-J JUNCTION J J U5-20-1

28. V H C H REGION CDR1CDR2CDR3 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 BINDING

29. IgH  chain Surrogate light chain pre-B cell Receptor (pre-BCR) LARGE pre-B CELL: pre-BCR EXPRESSION Expression of pre-BCR is a trigger to initation of IgL gene rearrangement: Transition to: Small pre-B cell

30. SMALL pre-B CELL: IgL rearrangement IgL GENE ORGANIZATION - separate genes for  &  -  gene: chr. 2;  gene: chr. 22 Study V K J K C Kappa KAPPA GENE ORGANIZATION: KAPPA GENE REARRANGES before LAMBDA GENE

31. EXONs Single EXON (single domain) N = 40N = 5 V K J K C Kappa J K C K KAPPA GENE ORGANIZATION U5-13-2 V K LV EXONs K

32. K C K V K J K C V ---> J REARRANGEMENT Kappa GENE V K J K C K ACTION OF RECOMBINASE KAPPA LIGHT CHAIN V to J REARRANGEMENT IRREVERSIBLE DNA EVENT: IRREVERSIBLE DNA EVENT: Small pre-B cell U5-14-1 V 1 K J 3 K Deletional event

33. TRANSLATION mature mRNA AAAAAAAAA U5-14-2 nascent mRNA LIGHT CHAIN mRNA TRANSCRIPTION AND TRANSLATION V J K K C K Kappa GENE TRANSCRIPTION mRNA splicing VJC KAPPA IgL (LIGHT) CHAIN PROTEIN:

34. U5-15-1 VJ REARRANGEMENTS VJ, ALLELE "A" PRODUCTIVENONPRODUCTIVE NAIVE IMMATURE B CELL DEVELOPS KAPPA ALLELE "B" REARRANGES VJ, ALLELE "B" PRODUCTIVENONPRODUCTIVE NAIVE IMMATURE B CELL DEVELOPS NO FURTHER IgL REARRANGEMENT "LIGHT CHAIN ISOTYPE EXCLUSION" LAMBDA GENE REARRANGES KAPPA GENE ETC. NEED PRODUCTIVE IgL REARRANGEMENT FOR FORMATION OF IMMATURE "NAIVE" B CELL "IN FRAME""NOT IN FRAME" CONSEQUENCES OF "NONPRODUCTIVE KAPPA IgL (Expresses Kappa) SIMILAR TO KAPPA

35. IgH  chain IgL expression BCR expression (IgM) Naïve immature B CELL: BCR (IgM) EXPRESSION

36. TRANSITION FROM NAÏVE IMMATURE B CELL to NAÏVE MATURE 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 VDJ nascent mRNA LONG NUCLEAR TRANSCRIPT U5-16-1 TRANSCRIPTION THRU MU and DELTA

37. TRANSLATION VDJ AAAAAA VDJ MU CHAIN VDJ DELTA CHAIN ALTERNATIVE SPLICING OF IgH mRNA produces NAÏVE MATURE B CELL U5-17-1 IgM plus L CHAIN IgD plus L CHAIN PROTEIN VDJ AAAAAA SPLICING RNA

38. IgH  chain IgL expression IgM expression Naïve mature B CELL: IgM & IgD EXPRESSION IgH  chain IgD expression Antigen-binding sites of IgM & IgD are identical: B cell is still “clonal”

39. Naïve Antibody Repertoire Mature (naïve) B cell** IgM IgD

40. Naïve Antibody Repertoire Adaptive Antibody Repertoire “Total” Antibody Repertoire = Naïve + Adaptive Antigen

41. 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

42. CREATING THE NAÏVE ANTIBODY REPERTOIRE: CREATING DIVERSITY IN BCR ---------------------------------------------------------------------------------- Sources of diversity: VDJ Different V H ’s, D H ’s, J H ’sIgH65276 V L ’s, J L ’s  40---5  30---4 Variations in junctions*Chain JunctionsIncr. diversity - Imprecise recombination IgH2 100 - N-region diversity IgL1 10 Various combinations of IgH and IgL --------------------------------------------------------------------------------- * 10 fold incr. diversity with each junction

43. CREATING THE NAÏVE ANTIBODY REPERTOIRE: CREATING DIVERSITY IN BCR ---------------------------------------------------------------------------------- Sources of diversity:Combinatorial diversity VDJCombinations IgH6527610,530  40---5200  30---4120 Junctional Combinations (x) diversity Product IgH ~10,0001001,000,000  200102,000  120101,200 ----------------------------------------------------------------------------------

44. CREATING THE NAÏVE ANTIBODY REPERTOIRE: CREATING DIVERSITY IN BCR --------------------------------------------------------------------------------- Combinatorial & Junctional diversity IgH 1,000,000  2,000  1,200 [IgH (x)  ] + [ IgH (x)  ] [ 10 6 x 2 x 10 3 ] + [ 10 6 x 1.2 x 10 3 ] 3.2 x 10 9 2x 10 9 + 1.2 x 10 9 = 3.2 x 10 9 ----------------------------------------------------------------------------------

45. V D J mature mRNATRANSLATION AAAAAA V D J IgH CHAIN PROTEIN DRAWING THE REGIONS IN ~CORRECT PROPORTIONS V H C H REGION IgH CHAIN U5-11-C