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Chronic lymphocytic leukemia

IL-10 production by CLL cells is enhanced in the anergic IGHV mutated subset and associates with reduced DNA methylation of the IL10 locus

Leukemia volume 31, pages 1686–1694 (2017)Cite this article

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Abstract

Chronic lymphocytic leukemias (CLLs) with unmutated (U-CLL) or mutated (M-CLL) IGHV have variable features of immunosuppression, possibly influenced by those CLL cells activated to produce interleukin 10 (IL-10). The two subsets differ in their levels of anergy, defined by low surface immunoglobulin M levels/signaling capacity, and in their DNA methylation profile, particularly variable in M-CLL. We have now found that levels of IL-10 produced by activated CLL cells were highly variable. Levels were higher in M-CLL than in U-CLL and correlated with anergy. DNA methylation analysis of IL10 locus revealed two previously uncharacterized ‘variably methylated regions’ (CLL-VMRs1/2) in the gene body, but similarly low methylation in the promoter of both U-CLL and M-CLL. CLL-VMR1/2 methylation was lower in M-CLL than in U-CLL and inversely correlated with IL-10 induction. A functional signal transducer and activator of transcription 3 (STAT3) binding site in CLL-VMR2 was confirmed by proximity ligation and luciferase assays, whereas inhibition of SYK-mediated STAT3 activation resulted in suppression of IL10. The data suggest epigenetic control of IL-10 production. Higher tumor load may compensate the reduced IL-10 production in U-CLL, accounting for clinical immunosuppression in both subsets. The observation that SYK inhibition also suppresses IL-10 provides a potential new rationale for therapeutic targeting and immunological rescue by SYK inhibitors in CLL.

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References

  1. Seifert M, Sellmann L, Bloehdorn J, Wein F, Stilgenbauer S, Durig J et al. Cellular origin and pathophysiology of chronic lymphocytic leukemia. J Exp Med 2012; 209: 2183–2198.

    Article  CAS  Google Scholar 

  2. Forconi F, Potter KN, Wheatley I, Darzentas N, Sozzi E, Stamatopoulos K et al. The normal IGHV1-69-derived B-cell repertoire contains stereotypic patterns characteristic of unmutated CLL. Blood 2010; 115: 71–77.

    Article  CAS  Google Scholar 

  3. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK . Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999; 94: 1848–1854.

    CAS  PubMed  Google Scholar 

  4. Moreira J, Rabe KG, Cerhan JR, Kay NE, Wilson JW, Call TG et al. Infectious complications among individuals with clinical monoclonal B-cell lymphocytosis (MBL): a cohort study of newly diagnosed cases compared to controls. Leukemia 2013; 27: 136–141.

    Article  CAS  Google Scholar 

  5. Forconi F, Moss P . Perturbation of the normal immune system in patients with CLL. Blood 2015; 126: 573–581.

    Article  CAS  Google Scholar 

  6. Solomon BM, Rabe KG, Slager SL, Brewer JD, Cerhan JR, Shanafelt TD . Overall and cancer-specific survival of patients with breast, colon, kidney, and lung cancers with and without chronic lymphocytic leukemia: a SEER population-based study. J Clin Oncol 2013; 31: 930–937.

    Article  Google Scholar 

  7. Morrison VA . Infectious complications of chronic lymphocytic leukaemia: pathogenesis, spectrum of infection, preventive approaches. Best Pract Res Clin Haematol 2010; 23: 145–153.

    Article  CAS  Google Scholar 

  8. Candando KM, Lykken JM, Tedder TF . B10 cell regulation of health and disease. Immunol Rev 2014; 259: 259–272.

    Article  CAS  Google Scholar 

  9. Yanaba K, Bouaziz JD, Matsushita T, Tsubata T, Tedder TF . The development and function of regulatory B cells expressing IL-10 (B10 cells) requires antigen receptor diversity and TLR signals. J Immunol 2009; 182: 7459–7472.

    Article  CAS  Google Scholar 

  10. Iwata Y, Matsushita T, Horikawa M, Dilillo DJ, Yanaba K, Venturi GM et al. Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells. Blood 2011; 117: 530–541.

    Article  CAS  Google Scholar 

  11. Matsushita T, Horikawa M, Iwata Y, Tedder TF . Regulatory B cells (B10 cells) and regulatory T cells have independent roles in controlling experimental autoimmune encephalomyelitis initiation and late-phase immunopathogenesis. J Immunol 2010; 185: 2240–2252.

    Article  CAS  Google Scholar 

  12. Yarkoni Y, Getahun A, Cambier JC . Molecular underpinning of B-cell anergy. Immunol Rev 2010; 237: 249–263.

    Article  CAS  Google Scholar 

  13. DiLillo DJ, Weinberg JB, Yoshizaki A, Horikawa M, Bryant JM, Iwata Y et al. Chronic lymphocytic leukemia and regulatory B cells share IL-10 competence and immunosuppressive function. Leukemia 2013; 27: 170–182.

    Article  CAS  Google Scholar 

  14. Rossi M, Gentile M, Toscano R, Recchia AG, Bossio S, Caruso N et al. Enumeration of interleukin-10-positive B cells from peripheral blood of patients with chronic lymphocytic leukemia. Leuk Lymphoma 2014; 55: 1394–1396.

    Article  Google Scholar 

  15. Herishanu Y, Perez-Galan P, Liu D, Biancotto A, Pittaluga S, Vire B et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 2011; 117: 563–574.

    Article  CAS  Google Scholar 

  16. Liu BS, Cao Y, Huizinga TW, Hafler DA, Toes RE . TLR-mediated STAT3 and ERK activation controls IL-10 secretion by human B cells. Eur J Immunol 2014; 44: 2121–2129.

    Article  CAS  Google Scholar 

  17. Levidou G, Sachanas S, Pangalis GA, Kalpadakis C, Yiakoumis X, Moschogiannis M et al. Immunohistochemical analysis of IL-6, IL-8/CXCR2 axis, Tyr p-STAT-3, and SOCS-3 in lymph nodes from patients with chronic lymphocytic leukemia: correlation between microvascular characteristics and prognostic significance. Biomed Res Int 2014; 2014: 251479.

    Article  Google Scholar 

  18. Jabara HH, McDonald DR, Janssen E, Massaad MJ, Ramesh N, Borzutzky A et al. DOCK8 functions as an adaptor that links TLR-MyD88 signaling to B cell activation. Nat Immunol 2012; 13: 612–620.

    Article  CAS  Google Scholar 

  19. Stevenson FK, Forconi F, Packham G . The meaning and relevance of B-cell receptor structure and function in chronic lymphocytic leukemia. Semin Hematol 2014; 51: 158–167.

    Article  CAS  Google Scholar 

  20. Packham G, Krysov S, Allen A, Savelyeva N, Steele AJ, Forconi F et al. The outcome of B-cell receptor signaling in chronic lymphocytic leukemia: proliferation or anergy. Haematologica 2014; 99: 1138–1148.

    Article  Google Scholar 

  21. Damle RN, Ghiotto F, Valetto A, Albesiano E, Fais F, Yan XJ et al. B-cell chronic lymphocytic leukemia cells express a surface membrane phenotype of activated, antigen-experienced B lymphocytes. Blood 2002; 99: 4087–4093.

    Article  CAS  Google Scholar 

  22. Mockridge CI, Potter KN, Wheatley I, Neville LA, Packham G, Stevenson FK . Reversible anergy of sIgM-mediated signaling in the two subsets of CLL defined by VH-gene mutational status. Blood 2007; 109: 4424–4431.

    Article  CAS  Google Scholar 

  23. Coelho V, Krysov S, Steele A, Sanchez Hidalgo M, Johnson PW, Chana PS et al. Identification in CLL of circulating intraclonal subgroups with varying B-cell receptor expression and function. Blood 2013; 122: 2664–2672.

    Article  CAS  Google Scholar 

  24. Apollonio B, Scielzo C, Bertilaccio MT, Ten Hacken E, Scarfo L, Ranghetti P et al. Targeting B-cell anergy in chronic lymphocytic leukemia. Blood 2013; 121: 3879–3888, S1–8.

    Article  Google Scholar 

  25. D'Avola A, Drennan S, Tracy I, Henderson I, Chiecchio L, Larrayoz M et al. Surface IgM expression and function associate with clinical behavior, genetic abnormalities and DNA methylation in CLL. Blood 2016; 128: 816–826.

    Article  CAS  Google Scholar 

  26. Queiros AC, Villamor N, Clot G, Martinez-Trillos A, Kulis M, Navarro A et al. A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact. Leukemia 2015; 29: 598–605.

    Article  CAS  Google Scholar 

  27. Broske AM, Vockentanz L, Kharazi S, Huska MR, Mancini E, Scheller M et al. DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction. Nat Genet 2009; 41: 1207–1215.

    Article  Google Scholar 

  28. Trowbridge JJ, Snow JW, Kim J, Orkin SH . DNA methyltransferase 1 is essential for and uniquely regulates hematopoietic stem and progenitor cells. Cell Stem Cell 2009; 5: 442–449.

    Article  CAS  Google Scholar 

  29. Kulis M, Heath S, Bibikova M, Queiros AC, Navarro A, Clot G et al. Epigenomic analysis detects widespread gene-body DNA hypomethylation in chronic lymphocytic leukemia. Nat Genet 2012; 44: 1236–1242.

    Article  CAS  Google Scholar 

  30. Oakes CC, Claus R, Gu L, Assenov Y, Hullein J, Zucknick M et al. Evolution of DNA methylation is linked to genetic aberrations in chronic lymphocytic leukemia. Cancer Discov 2014; 4: 348–361.

    Article  CAS  Google Scholar 

  31. Oakes CC, Seifert M, Assenov Y, Gu L, Przekopowitz M, Ruppert AS et al. DNA methylation dynamics during B cell maturation underlie a continuum of disease phenotypes in chronic lymphocytic leukemia. Nat Genet 2016; 48: 253–264.

    Article  CAS  Google Scholar 

  32. Cahill N, Bergh AC, Kanduri M, Goransson-Kultima H, Mansouri L, Isaksson A et al. 450K-array analysis of chronic lymphocytic leukemia cells reveals global DNA methylation to be relatively stable over time and similar in resting and proliferative compartments. Leukemia 2013; 27: 150–158.

    Article  CAS  Google Scholar 

  33. Tiffon C, Adams J, van der Fits L, Wen S, Townsend P, Ganesan A et al. The histone deacetylase inhibitors vorinostat and romidepsin downmodulate IL-10 expression in cutaneous T-cell lymphoma cells. Br J Pharmacol 2011; 162: 1590–1602.

    Article  CAS  Google Scholar 

  34. Teschendorff AE, Marabita F, Lechner M, Bartlett T, Tegner J, Gomez-Cabrero D et al. A beta-mixture quantile normalization method for correcting probe design bias in Illumina Infinium 450 k DNA methylation data. Bioinformatics 2013; 29: 189–196.

    Article  CAS  Google Scholar 

  35. Assenov Y, Muller F, Lutsik P, Walter J, Lengauer T, Bock C . Comprehensive analysis of DNA methylation data with RnBeads. Nat Methods 2014; 11: 1138–1140.

    Article  CAS  Google Scholar 

  36. Baer C, Oakes CC, Ruppert AS, Claus R, Kim-Wanner SZ, Mertens D et al. Epigenetic silencing of miR-708 enhances NF-kappaB signaling in chronic lymphocytic leukemia. Int J Cancer 2015; 137: 1352–1361.

    Article  CAS  Google Scholar 

  37. Gustafsdottir SM, Schlingemann J, Rada-Iglesias A, Schallmeiner E, Kamali-Moghaddam M, Wadelius C et al. In vitro analysis of DNA-protein interactions by proximity ligation. Proc Natl Acad Sci USA 2007; 104: 3067–3072.

    Article  CAS  Google Scholar 

  38. Mathelier A, Zhao X, Zhang AW, Parcy F, Worsley-Hunt R, Arenillas DJ et al. JASPAR 2014: an extensively expanded and updated open-access database of transcription factor binding profiles. Nucleic Acids Res 2014; 42 (Database issue): D142–D147.

    Article  CAS  Google Scholar 

  39. Blanchette M, Kent WJ, Riemer C, Elnitski L, Smit AF, Roskin KM et al. Aligning multiple genomic sequences with the threaded blockset aligner. Genome Res 2004; 14: 708–715.

    Article  CAS  Google Scholar 

  40. Andersson R, Gebhard C, Miguel-Escalada I, Hoof I, Bornholdt J, Boyd M et al. An atlas of active enhancers across human cell types and tissues. Nature 2014; 507: 455–461.

    Article  CAS  Google Scholar 

  41. Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A . Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001; 19: 683–765.

    Article  CAS  Google Scholar 

  42. Riches JC, Gribben JG . Immunomodulation and immune reconstitution in chronic lymphocytic leukemia. Semin Hematol 2014; 51: 228–234.

    Article  CAS  Google Scholar 

  43. Khoder A, Sarvaria A, Alsuliman A, Chew C, Sekine T, Cooper N et al. Regulatory B cells are enriched within the IgM memory and transitional subsets in healthy donors but are deficient in chronic GVHD. Blood 2014; 124: 2034–2045.

    Article  CAS  Google Scholar 

  44. Flores-Borja F, Bosma A, Ng D, Reddy V, Ehrenstein MR, Isenberg DA et al. CD19+CD24hiCD38hi B cells maintain regulatory T cells while limiting TH1 and TH17 differentiation. Sci Transl Med 2013; 5: 173ra23.

    Article  Google Scholar 

  45. Bouaziz JD, Calbo S, Maho-Vaillant M, Saussine A, Bagot M, Bensussan A et al. IL-10 produced by activated human B cells regulates CD4(+) T-cell activation in vitro. Eur J Immunol 2010; 40: 2686–2691.

    Article  CAS  Google Scholar 

  46. Fayad L, Keating MJ, Reuben JM, O'Brien S, Lee BN, Lerner S et al. Interleukin-6 and interleukin-10 levels in chronic lymphocytic leukemia: correlation with phenotypic characteristics and outcome. Blood 2001; 97: 256–263.

    Article  CAS  Google Scholar 

  47. Lech-Maranda E, Grzybowska-Izydorczyk O, Wyka K, Mlynarski W, Borowiec M, Antosik K et al. Serum tumor necrosis factor-alpha and interleukin-10 levels as markers to predict outcome of patients with chronic lymphocytic leukemia in different risk groups defined by the IGHV mutation status. Arch Immunol Ther Exp (Warsz) 2012; 60: 477–486.

    Article  CAS  Google Scholar 

  48. Kamper EF, Papaphilis AD, Angelopoulou MK, Kopeikina LT, Siakantaris MP, Pangalis GA et al. Serum levels of tetranectin, intercellular adhesion molecule-1 and interleukin-10 in B-chronic lymphocytic leukemia. Clin Biochem 1999; 32: 639–645.

    Article  CAS  Google Scholar 

  49. Yan XJ, Dozmorov I, Li W, Yancopoulos S, Sison C, Centola M et al. Identification of outcome-correlated cytokine clusters in chronic lymphocytic leukemia. Blood 2011; 118: 5201–5210.

    Article  CAS  Google Scholar 

  50. Cortes JE, Talpaz M, Cabanillas F, Seymour JF, Kurzrock R . Serum levels of interleukin-10 in patients with diffuse large cell lymphoma: lack of correlation with prognosis. Blood 1995; 85: 2516–2520.

    CAS  PubMed  Google Scholar 

  51. Saulep-Easton D, Vincent FB, Quah PS, Wei A, Ting SB, Croce CM et al. The BAFF receptor TACI controls IL-10 production by regulatory B cells and CLL B cells. Leukemia 2016; 30: 163–172.

    Article  CAS  Google Scholar 

  52. Schweighoffer E, Vanes L, Nys J, Cantrell D, McCleary S, Smithers N et al. The BAFF receptor transduces survival signals by co-opting the B cell receptor signaling pathway. Immunity 2013; 38: 475–488.

    Article  CAS  Google Scholar 

  53. Sharman J, Hawkins M, Kolibaba K, Boxer M, Klein L, Wu M et al. An open-label phase 2 trial of entospletinib (GS-9973), a selective spleen tyrosine kinase inhibitor, in chronic lymphocytic leukemia. Blood 2015; 125: 2336–2343.

    Article  CAS  Google Scholar 

  54. Alhakeem SS, Sindhava VJ, McKenna MK, Gachuki BW, Byrd JC, Muthusamy N et al. Role of B cell receptor signaling in IL-10 production by normal and malignant B-1 cells. Ann NY Acad Sci 2015; 1362: 239–249.

    Article  CAS  Google Scholar 

  55. Barr PM, Saylors GB, Spurgeon SE, Cheson BD, Greenwald DR, O'Brien SM et al. Phase 2 study of idelalisib and entospletinib: pneumonitis limits combination therapy in relapsed refractory CLL and NHL. Blood 2016; 127: 2411–2415.

    Article  CAS  Google Scholar 

  56. Hallek M, Fischer K, Fingerle-Rowson G, Fink AM, Busch R, Mayer J et al. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet 2010; 376: 1164–1174.

    Article  CAS  Google Scholar 

  57. Duty JA, Szodoray P, Zheng NY, Koelsch KA, Zhang Q, Swiatkowski M et al. Functional anergy in a subpopulation of naive B cells from healthy humans that express autoreactive immunoglobulin receptors. J Exp Med 2009; 206: 139–151.

    Article  Google Scholar 

  58. Garaud S, Morva A, Lemoine S, Hillion S, Bordron A, Pers JO et al. CD5 promotes IL-10 production in chronic lymphocytic leukemia B cells through STAT3 and NFAT2 activation. J Immunol 2011; 186: 4835–4844.

    Article  CAS  Google Scholar 

  59. Hedrich CM, Rauen T, Apostolidis SA, Grammatikos AP, Rodriguez Rodriguez N, Ioannidis C et al. Stat3 promotes IL-10 expression in lupus T cells through trans-activation and chromatin remodeling. Proc Natl Acad Sci USA 2014; 111: 13457–13462.

    Article  CAS  Google Scholar 

  60. Tsuji-Takayama K, Suzuki M, Yamamoto M, Harashima A, Okochi A, Otani T et al. The production of IL-10 by human regulatory T cells is enhanced by IL-2 through a STAT5-responsive intronic enhancer in the IL-10 locus. J Immunol 2008; 181: 3897–3905.

    Article  CAS  Google Scholar 

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Acknowledgements

This study was supported by Cancer Research UK (CRUK Centre Grant C34999/A18087), Southampton Cancer Research UK and NIHR Experimental Cancer Medicine Centres, University of Southampton, Bloodwise (Grants 16003, 14037 and 12021), Keanu Eyles Haematology Fellowship for the Cancer Immunology Centre, Istituto Toscano Tumori (Florence, Italy), Hairy Cell Leukemia Research Foundation (IL, USA), Heidelberg Center for Personalized Oncology (DKFZ-HIPO) (HIPO project H021), National Center for Tumor Diseases Precision Oncology Program, German Federal Ministry of Education and Research CancerEpiSys network (BMBF 031 6049C) and the Virtual Helmholtz Institute (VH-VI-404). CW was supported by the Helmholtz International Graduate School for Cancer Research.

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Author notes

  1. C C Oakes and F Forconi: These authors contributed equally to this work.

Authors and Affiliations

  1. Haematology Oncology Group, Cancer Sciences Unit, Cancer Research UK and NIHR Experimental Cancer Medicine Centres, Faculty of Medicine, University of Southampton, Southampton, UK

    S Drennan, A D'Avola, E Chrysostomou & F Forconi

  2. Wessex Investigational Sciences Hub laboratory, Cancer Sciences Unit, Cancer Research UK and NIHR Experimental Cancer Medicine Centres, Faculty of Medicine, University of Southampton, Southampton, UK

    Y Gao & A P Williams

  3. Division of Epigenomics and Cancer Risk Factors, The German Cancer Research Center (DKFZ), Heidelberg, Germany

    C Weigel, C Plass & C C Oakes

  4. Molecular Oncology Group, Cancer Sciences Unit, Cancer Research UK and NIHR Experimental Cancer Medicine Centres, Faculty of Medicine, University of Southampton, Southampton, UK

    A J Steele & G Packham

  5. Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany

    T Zenz

  6. Department of Medicine V, University of Heidelberg, Heidelberg, Germany

    T Zenz

  7. Medical Oncology, University Hospital Southampton National Health Service Trust, Southampton, UK

    P W Johnson

  8. Molecular Immunology Group, Cancer Sciences Unit, Cancer Research UK and NIHR Experimental Cancer Medicine Centres, Faculty of Medicine, University of Southampton, Southampton, UK

    F K Stevenson

  9. Division of Hematology, The Ohio State University, Columbus, OH, USA

    C C Oakes

  10. Haematology Department, University Hospital Southampton National Health Service Trust, Southampton, UK

    F Forconi

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Drennan, S., D'Avola, A., Gao, Y. et al. IL-10 production by CLL cells is enhanced in the anergic IGHV mutated subset and associates with reduced DNA methylation of the IL10 locus. Leukemia 31, 1686–1694 (2017). https://doi.org/10.1038/leu.2016.356

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