2. ESF workshop Leeds

3. Plasma cells Activators: Blimp-1, XBP1 Repressors: Pax5, Bcl6
BCR and CD40 signalling
Cytokines: IL-6, -10, -21
TLRs (TLR4, TLR9)
MicroRNAs : miR-155 BIC

4. B cell activation and formation of plasma cells
Shapiro-Shelef & Calame (2005) Nat. Rev. Immunol. 5:

5. Transcriptional regulation of plasma cell differentiation
Nera & Lassila (2006) Scand. J. Immunol. 64:

6. Pax5 Bcl6 Paired box protein Groucho, HDACs
Guardian of B cell identity and function
B cell commitment
-crucial for B cell fate
Represses plasma cell formation
Proto-oncogene
BTB/POZ Zn finger repressor (HDACs)
Required for GC formation
Tolerance for genotoxic stress (p53, ATR)
Represses plasma cell formation
Proto-oncogene

7. Blimp-1 / PRDI-BF1 (prdm1)
Zn finger repressor HDAC
Master regulator of plasma cell formation
B cells can initiate plasma cell differentiation without Blimp-1 upregulation
5-15 % of GC B cells are Blimp-1+ and Bcl6-
Long lived plasma cells need Blimp-1
Blimp-1 is suggested not to be required for memory B cell formation

8. AID (Aicda) Activation induced cytidine deaminase
Needed for somatic hypermutation, class switch recombination and gene conversion
transcriptional regulation (Pax5, E2A)
intracellular compartmentalization
posttranslational modification
posttranscriptional regulation (miR-155/Bic)
UNG Uracil-DNA glycosylase

9. Durandy et al. (2005). Immunol. Rev.203: 67-79.

10. Todd et al Nature Rev. Immunol 8, 663-674, 2008

11. Disruption of Pax5 in DT40 B cells
Nera et al. (2006) Immunity 24:

12. Slow growth and decreased sIgM expression
Log Fluorescence Intensity
Nera et al. (2006) Immunity 24:

13. Defective BCR signaling in the absence of Pax5
BCR cross-linking
H2O2
Nera et al. (2006) Immunity 24:

14. Loss of Pax5 promotes plasma cell differentiation
Nera et al. (2006) Immunity 24:

15. Conclusions Loss of Pax5 promotes plasma cell differentiation
Pax5 target genes include components of BCR signalling, Ig diversification (AID) and Ig secretion (XBP-1)
Pax5 positively regulates B cell identity as well as GC B cell program
Pax5 represses indirectly Blimp-1 via Bcl6

16. Nera & Lassila (2006) Scand. J. Immunol. 64: 190-199.

17. Nutt et al. (2007) Nat. Rev. Immunol. 7: 923-927

18. Interaction / repression
Bcl BTB/POZ (bric-a-brac, tramtrack, broad complex/poxvirus and Zn domain family)
Bcl6
Interaction / repression
DNA binding
F1 F2 F3 F4 F5 F6
BTB/POZ
Structure
N -
- C
IRF4
BCoR
Evi-9
PLZF
BAZF
SMRT
Sin3A
HDAC
Jun
Miz-1

19. Bcl6 Master regulator of GC transcriptional programme
Facilitates Ig affinity maturation (SHM, CSR)
DNA damage sensing by regulating p53,ATR
Repress plasma cell diffrentiation via Blimp1
Inhibition of memory B cell formation

20. Control of PC differentiation
Martins & Calame (2008) Ann Rev Immunol. 26:

21. Disruption of Bcl6 in DT40 B cellsA p1 p2
deleted
bsr-F
neo-F F1
Bcl6-bsr
Bcl6-neo
bsr
neo
Bcl6 gene R2 WT
Bcl6 +/-
Bcl6 -/-
bsr
neo
Bcl6
Relative number
of transcripts WT
Bcl6-/-
Bcl6-/- /Bcl6 B C

22. Slow growth and decreased sIgM expressionB
sIgM
CD45 WT
Bcl6-/-
Bcl6-/- /Bcl6
Log Fluorescence Intensity
101
103
Time (h)
Cell density (x106/ml)

23. Bcl6 downregulation leads to plasma cell differentiationWT
Bcl6-/-
0 min
0.5 h
1 h
2 h
4 h
Ig µ
Ig λ 23

25. Loss of Bcl6 downregulates EBF and Pax5 but upregulates E2A expression

26. AID and UNG require Bcl6 expression and Bcl6 represses Blimp-1

27. Conclusions
Bcl6 downregulation leads to exit from GC program and differentiation into plasma cells
Loss of Bcl6 supresses the plasma cell repression function of Pax5 and MITF
Bcl6 controls double stranded DNA breaks by allowing AID, UNG, RAD54, BRCA2 expression
and thus SHM, CSR and gene conversion.
4. Bcl6 target genes include components of BCR signalling, Ig diversification and secretion
Bcl6 is a direct repressor of Blimp-1

29. Todd et al Nature Rev. Immunol 8, 663-674, 2008

30. H. Singh Nat Immunol 8,119, 2006

31. Laslo et al Cell 2006