CHK1 dosage in germinal center B cells controls humoral immunity

Abstract

Germinal center (GC) B cells are among the fastest replicating cells in our body, dividing every 4–8 h. DNA replication errors are intrinsically toxic to cells. How GC B cells exert control over the DNA damage response while introducing mutations in their antibody genes is poorly understood. Here, we show that the DNA damage response regulator Checkpoint kinase 1 (CHK1) is essential for GC B cell survival. Remarkably, effective antibody-mediated immunity relies on optimal CHK1 dosage. Chemical CHK1 inhibition or loss of one Chk1 allele impairs the survival of class-switched cells and curbs the amplitude of antibody production. Mechanistically, active B cell receptor signaling wires the outcome of CHK1-inhibition towards BIM-dependent apoptosis, whereas T cell help favors temporary cell cycle arrest. Our results predict that therapeutic CHK1 inhibition in cancer patients may prove potent in killing B cell lymphoma and leukemia cells addicted to B cell receptor signaling, but will most likely dampen humoral immunity.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. 1.

    Mesin L, Ersching J, Victora GD. Germinal center B cell dynamics. Immunity. 2016;45:471–82.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    De Silva NS, Klein U. Dynamics of B cells in germinal centres. Nat Rev Immunol. 2015;15:137–48.

    PubMed  PubMed Central  Google Scholar 

  3. 3.

    Shlomchik MJ, Weisel F. Germinal center selection and the development of memory B and plasma cells. Immunol Rev. 2012;247:52–63.

    PubMed  Google Scholar 

  4. 4.

    Victora GD, Schwickert TA, Fooksman DR, Kamphorst AO, Meyer-Hermann M, Dustin ML, et al. Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell. 2010;143:592–605.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Gitlin AD, Shulman Z, Nussenzweig MC. Clonal selection in the germinal centre by regulated proliferation and hypermutation. Nature. 2014;509:637–40.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Allen CDC, Okada T, Tang HL, Cyster JG. Imaging of germinal center selection events during affinity maturation. Science. 2007;315:528–31.

    CAS  PubMed  Google Scholar 

  7. 7.

    Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell. 2000;102:553–63.

    CAS  PubMed  Google Scholar 

  8. 8.

    Di Noia JM, Neuberger MS. Molecular mechanisms of antibody somatic hypermutation. Annu Rev Biochem. 2007;76:1–22.

    PubMed  Google Scholar 

  9. 9.

    Fazilleau N, Mark L, McHeyzer-Williams LJ, McHeyzer-Williams MG. Follicular helper T cells: lineage and location. Immunity. 2009;30:324–35.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Gitlin AD, Mayer CT, Oliveira TY, Shulman Z, Jones MJK, Koren A, et al. Humoral immunity. T cell help controls the speed of the cell cycle in germinal center B cells. Science. 2015;349:643–46.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Wortis HH, Teutsch M, Higer M, Zheng J, Parker DC. B-cell activation by crosslinking of surface IgM or ligation of CD40 involves alternative signal pathways and results in different B-cell phenotypes. Proc Natl Acad Sci USA. 1995;92:3348–52.

    CAS  PubMed  Google Scholar 

  12. 12.

    Otipoby KL, Waisman A, Derudder E, Srinivasan L, Franklin A, Rajewsky K. The B-cell antigen receptor integrates adaptive and innate immune signals. Proc Natl Acad Sci USA. 2015;112:12145–50.

    CAS  PubMed  Google Scholar 

  13. 13.

    Hauser AE, Junt T, Mempel TR, Sneddon MW, Kleinstein SH, Henrickson SE, et al. Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns. Immunity. 2007;26:655–67.

    CAS  PubMed  Google Scholar 

  14. 14.

    Kotsantis P, Petermann E, Boulton SJ. Mechanisms of oncogene-induced replication stress: jigsaw falling into place. Cancer Discov. 2018;8:537–55.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Daniel JA, Nussenzweig A. The AID-induced DNA damage response in chromatin. Mol Cell. 2013;50:309–21.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Robbiani DF, Nussenzweig MC. Chromosome translocation, B cell lymphoma, and activation-induced cytidine deaminase. Annu Rev Pathol. 2013;8:79–103.

    CAS  PubMed  Google Scholar 

  17. 17.

    Saldivar JC, Cortez D, Cimprich KA. The essential kinase ATR: ensuring faithful duplication of a challenging genome. Nat Rev Mol Cell Biol. 2017;18:622–36.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Ranuncolo SM, Polo JM, Dierov J, Singer M, Kuo T, Greally J, et al. Bcl-6 mediates the germinal center B cell phenotype and lymphomagenesis through transcriptional repression of the DNA-damage sensor ATR. Nat Immunol. 2007;8:705–14.

    CAS  PubMed  Google Scholar 

  19. 19.

    Ranuncolo SM, Polo JM, Melnick A. BCL6 represses CHEK1 and suppresses DNA damage pathways in normal and malignant B-cells. Blood Cells Mol Dis. 2008;41:95–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Phan RT, Dalla-Favera R. The BCL6 proto-oncogene suppresses p53 expression in germinal-centre B cells. Nature. 2004;432:635–39.

    CAS  PubMed  Google Scholar 

  21. 21.

    Phan RT, Saito M, Basso K, Niu H, Dalla-Favera R. BCL6 interacts with the transcription factor Miz-1 to suppress the cyclin-dependent kinase inhibitor p21 and cell cycle arrest in germinal center B cells. Nat Immunol. 2005;6:1054–60.

    CAS  PubMed  Google Scholar 

  22. 22.

    Lecona E, Fernandez-Capetillo O. Targeting ATR in cancer. Nat Rev Cancer. 2018;18:586–95.

    CAS  PubMed  Google Scholar 

  23. 23.

    Enders A, Bouillet P, Puthalakath H, Xu Y, Tarlinton DM, Strasser A. Loss of the pro-apoptotic BH3-only Bcl-2 family member Bim inhibits BCR stimulation-induced apoptosis and deletion of autoreactive B cells. J Exp Med. 2003;198:1119–26.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Labi V, Woess C, Tuzlak S, Erlacher M, Bouillet P, Strasser A, et al. Deregulated cell death and lymphocyte homeostasis cause premature lethality in mice lacking the BH3-only proteins Bim and Bmf. Blood. 2014;123:2652–62.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Woess C, Tuzlak S, Labi V, Drach M, Bertele D, Schneider P, et al. Combined loss of the BH3-only proteins Bim and Bmf restores B-cell development and function in TACI-Ig transgenic mice. Cell Death Differ. 2015;22:1477–88.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Delbridge ARD, Pang SHM, Vandenberg CJ, Grabow S, Aubrey BJ, Tai L, et al. RAG-induced DNA lesions activate proapoptotic BIM to suppress lymphomagenesis in p53-deficient mice. J Exp Med. 2016;213:2039–48.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Erlacher M, Michalak EM, Kelly PN, Labi V, Niederegger H, Coultas L, et al. BH3-only proteins Puma and Bim are rate-limiting for gamma-radiation- and glucocorticoid-induced apoptosis of lymphoid cells in vivo. Blood. 2005;106:4131–38.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Erlacher M, Labi V, Manzl C, Böck G, Tzankov A, Häcker G, et al. Puma cooperates with Bim, the rate-limiting BH3-only protein in cell death during lymphocyte development, in apoptosis induction. J Exp Med. 2006;203:2939–51.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Villunger A, Labi V, Bouillet P, Adams J, Strasser A. Can the analysis of BH3-only protein knockout mice clarify the issue of ‘direct versus indirect’ activation of Bax and Bak? Cell Death Differ. 2011;18:1545–46.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Gutierrez-Martinez P, Hogdal L, Nagai M, Kruta M, Singh R, Sarosiek K, et al. Diminished apoptotic priming and ATM signalling confer a survival advantage onto aged haematopoietic stem cells in response to DNA damage. Nat Cell Biol. 2018;20:413–21.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Hodgkin PD, Lee JH, Lyons AB. B cell differentiation and isotype switching is related to division cycle number. J Exp Med. 1996;184:277–81.

    CAS  PubMed  Google Scholar 

  32. 32.

    Wiedemann E-M, Peycheva M, Pavri R. DNA replication origins in immunoglobulin switch regions regulate class switch recombination in an R-loop-dependent manner. Cell Rep. 2016;17:2927–42.

    CAS  PubMed  Google Scholar 

  33. 33.

    Duffy KR, Wellard CJ, Markham JF, Zhou JHS, Holmberg R, Hawkins ED, et al. Activation-induced B cell fates are selected by intracellular stochastic competition. Science. 2012;335:338–41.

    CAS  PubMed  Google Scholar 

  34. 34.

    Zhang Y, Hunter T. Roles of Chk1 in cell biology and cancer therapy. Int J Cancer. 2014;134:1013–23.

    CAS  PubMed  Google Scholar 

  35. 35.

    Casola S, Cattoretti G, Uyttersprot N, Koralov SB, Seagal J, Segal J, et al. Tracking germinal center B cells expressing germ-line immunoglobulin gamma1 transcripts by conditional gene targeting. Proc Natl Acad Sci USA. 2006;103:7396–401.

    CAS  PubMed  Google Scholar 

  36. 36.

    Schuler F, Weiss JG, Lindner SE, Lohmüller M, Herzog S, Spiegl SF, et al. Checkpoint kinase 1 is essential for normal B cell development and lymphomagenesis. Nat Commun. 2017;8:1697.

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Sutherland DB, Suzuki K, Fagarasan S. Fostering of advanced mutualism with gut microbiota by Immunoglobulin A. Immunol Rev. 2016;270:20–31.

    CAS  PubMed  Google Scholar 

  38. 38.

    Klein F, Diskin R, Scheid JF, Gaebler C, Mouquet H, Georgiev IS, et al. Somatic mutations of the immunoglobulin framework are generally required for broad and potent HIV-1 neutralization. Cell. 2013;153:126–38.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Pappas L, Foglierini M, Piccoli L, Kallewaard NL, Turrini F, Silacci C, et al. Rapid development of broadly influenza neutralizing antibodies through redundant mutations. Nature. 2014;516:418–22.

    CAS  PubMed  Google Scholar 

  40. 40.

    Dogan I, Bertocci B, Vilmont V, Delbos F, Mégret J, Storck S, et al. Multiple layers of B cell memory with different effector functions. Nat Immunol. 2009;10:1292–99.

    CAS  PubMed  Google Scholar 

  41. 41.

    McHeyzer-Williams LJ, Milpied PJ, Okitsu SL, McHeyzer-Williams MG. Class-switched memory B cells remodel BCRs within secondary germinal centers. Nat Immunol. 2015;16:296–305.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Frankenberger S, Davari K, Fischer-Burkart S, Böttcher K, Tomi N-S, Zimber-Strobl U, et al. Checkpoint kinase 1 negatively regulates somatic hypermutation. Nucleic Acids Res. 2014;42:3666–74.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Wang Q, Kieffer-Kwon K-R, Oliveira TY, Mayer CT, Yao K, Pai J, et al. The cell cycle restricts activation-induced cytidine deaminase activity to early G1. J Exp Med. 2017;214:49–58.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Khalil AM, Cambier JC, Shlomchik MJ. B cell receptor signal transduction in the GC is short-circuited by high phosphatase activity. Science. 2012;336:1178–81.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Enomoto M, Goto H, Tomono Y, Kasahara K, Tsujimura K, Kiyono T, et al. Novel positive feedback loop between Cdk1 and Chk1 in the nucleus during G2/M transition. J Biol Chem. 2009;284:34223–30.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Mouhamad S, Besnault L, Auffredou MT, Leprince C, Bourgeade MF, Leca G, et al. B cell receptor-mediated apoptosis of human lymphocytes is associated with a new regulatory pathway of Bim isoform expression. J Immunol. 2004;172:2084–91.

    CAS  PubMed  Google Scholar 

  47. 47.

    Takada E, Furuhata M, Nakae S, Ichijo H, Sudo K, Mizuguchi J. Requirement of apoptosis-inducing kinase 1 for the induction of bronchial asthma following stimulation with ovalbumin. Int Arch Allergy Immunol. 2013;162:104–14.

    CAS  PubMed  Google Scholar 

  48. 48.

    Fischer SF, Bouillet P, O’Donnell K, Light A, Tarlinton DM, Strasser A. Proapoptotic BH3-only protein Bim is essential for developmentally programmed death of germinal center-derived memory B cells and antibody-forming cells. Blood. 2007;110:3978–84.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Greenow KR, Clarke AR, Jones RH. Chk1 deficiency in the mouse small intestine results in p53-independent crypt death and subsequent intestinal compensation. Oncogene. 2009;28:1443–53.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Boles NC, Peddibhotla S, Chen AJ, Goodell MA, Rosen JM. Chk1 haploinsufficiency results in anemia and defective erythropoiesis. PLoS ONE. 2010;5:e8581.

    PubMed  PubMed Central  Google Scholar 

  51. 51.

    Höglund A, Nilsson LM, Muralidharan SV, Hasvold LA, Merta P, Rudelius M, et al. Therapeutic implications for the induced levels of Chk1 in Myc-expressing cancer cells. Clin Cancer Res. 2011;17:7067–79.

    PubMed  Google Scholar 

  52. 52.

    Murga M, Campaner S, Lopez-Contreras AJ, Toledo LI, Soria R, Montaña MF, et al. Exploiting oncogene-induced replicative stress for the selective killing of Myc-driven tumors. Nat Struct Mol Biol. 2011;18:1331–35.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Ferrao PT, Bukczynska EP, Johnstone RW, McArthur GA. Efficacy of CHK inhibitors as single agents in MYC-driven lymphoma cells. Oncogene. 2012;31:1661–72.

    CAS  PubMed  Google Scholar 

  54. 54.

    Myklebust JH, Brody J, Kohrt HE, Kolstad A, Czerwinski DK, Wälchli S, et al. Distinct patterns of B-cell receptor signaling in non-Hodgkin lymphomas identified by single-cell profiling. Blood. 2017;129:759–70.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Lam MH, Liu Q, Elledge SJ, Rosen JM. Chk1 is haploinsufficient for multiple functions critical to tumor suppression. Cancer Cell. 2004;6:45–59.

    CAS  PubMed  Google Scholar 

  56. 56.

    Bouillet P, Metcalf D, Huang DC, Tarlinton DM, Kay TW, Köntgen F, et al. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science. 1999;286:1735–38.

    CAS  PubMed  Google Scholar 

  57. 57.

    Lang MJ, Brennan MS, O’Reilly LA, Ottina E, Czabotar PE, Whitlock E, et al. Characterisation of a novel A1-specific monoclonal antibody. Cell Death Dis. 2014;5:e1553–e1553.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Vikstrom I, Carotta S, Lüthje K, Peperzak V, Jost PJ, Glaser S, et al. Mcl-1 is essential for germinal center formation and B cell memory. Science. 2010;330:1095–99.

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82.

    CAS  Google Scholar 

Download references

Acknowledgements

We thank I. Gaggl and K. Rossi for expert technical assistance; M. Saurwein, N. Heinrich, and N. Schöpf for animal care; I. Lengenfelder and C. Rahm for administrative assistance; the present and former V.L. and A.V. lab members and E. Derudder for critical comments and suggestions; K. Rajewsky for Cg1-cre mice; A. Strasser for Bim mice, T.W. Mak for Chk1F mice, M. Herold for providing an antibody against BCL2a1 (clone 6D6), and B. Weinberger for technical help with ELISPOT analysis. This work was supported by grants from the Austrian Science Fund (FWF) to AV (P 26856; I1298) and to NH-K (P 28694-B30), the Tyrolean Science Fund to VL (UNI-0404/1696) and the Austrian Cancer Aid to VL (KH15017). KS was supported by the FWF-funded Doctoral College “Molecular Cell Biology and Oncology” (W1101), and received a DOC PhD fellowship from the Austrian Academy of Sciences (ÖAW).

Author information

Affiliations

Authors

Contributions

Conceptualization, methodology, project administration, writing—original draft, and writing—review & editing: VL; investigation: KS, BJ, JH, CS, AA and VL; resources and funding acquisition: VL, AV, NH-K; visualization: VL, KS and BJ.

Corresponding author

Correspondence to Verena Labi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Edited by C. Borner

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Schoeler, K., Jakic, B., Heppke, J. et al. CHK1 dosage in germinal center B cells controls humoral immunity. Cell Death Differ 26, 2551–2567 (2019). https://doi.org/10.1038/s41418-019-0318-5

Download citation

Further reading

Search