Skip to main content

TGF-βR inhibitor SB431542 restores immune suppression induced by regulatory B–T cell axis and decreases tumour burden in murine fibrosarcoma

Cancer Immunology, Immunotherapy volume 70, pages 153–168 (2021)Cite this article

Abstract

The contribution of immune cells in soft tissue sarcomas (STS) is not completely known and understanding their role is very essential for employing immunotherapy strategies. Here, we show that murine fibrosarcoma-conditioned medium promoted total spleen cell proliferation but inhibited T cell responses to mitogenic and allo-antigen-mediated stimulation. This increased proliferation was found to be in B cells resulting in generation of Breg further leading to Treg population. This was found to be the same in vitro and in vivo. The phenotype of these B cells was CD19+CD81+CD27+CD25+PD-L1hi and they secreted both IL-10 and TGF-β. These tumor evoked Bregs (tBreg), when co-cultured with B depleted T cells, suppressed their proliferation in response to anti-CD3/CD28 stimulation. tBreg-induced suppression of T cell responses was not abrogated by the inhibition or neutralization of IL-10 but by the small molecule inhibitor of TGFβ Receptor type I, SB431542. While SB531542 per se was not cytotoxic to tumor cells, administration of SB431542 in tumor-bearing mice (TBM) significantly reduced the tumor burden. In addition, the treatment significantly reduced Treg cells and rescued proliferation of T cells in response to mitogen and allo-antigen. Collectively, our results identify that tumor evoked Breg cells mediate T cell immune suppression through TGFβ-mediated pathway and that targeting the Breg–Treg axis can be potentially used as an immunotherapy agent.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

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

Abbreviations

FASL:

Fas ligand

GATA3:

GATA-binding protein 3

NT:

Non-tumor

RORγ:

RAR-related orphan receptor gamma

STS:

Soft tissue sarcoma

Tbet:

T-box transcription factor

TBM:

Tumor-bearing mouse

TCM:

Tumor-conditioned medium

TDLN:

Tumor draining lymph node

UT:

Untreated

References

  1. Fletcher CD (2014) The evolving classification of soft tissue tumours—an update based on the new 2013 WHO classification. Histopathology 64:2–11

    PubMed  Google Scholar 

  2. Lawrence W, Donegan WL, Natarajan N, Mettlin C, Beart R, Winchester D (1987) Adult soft tissue sarcomas. A pattern of care survey of the American College of Surgeons. Ann Surg 205:349–359

    PubMed  PubMed Central  Google Scholar 

  3. Christie-Large M, James SL, Tiessen L, Davies AM, Grimer RJ (2008) Imaging strategy for detecting lung metastases at presentation in patients with soft tissue sarcomas. Eur J Cancer 44:1841–1845

    CAS  PubMed  Google Scholar 

  4. Lindberg RD, Martin RG, Romsdahl MM, Barkley HT (1981) Conservative surgery and postoperative radiotherapy in 300 adults with soft-tissue sarcomas. Cancer 47:2391–2397

    CAS  PubMed  Google Scholar 

  5. Jacobs AJ, Michels R, Stein J, Levin AS (2015) Improvement in Overall Survival from Extremity Soft Tissue Sarcoma over Twenty Years. Sarcoma 2015:279601

    PubMed  PubMed Central  Google Scholar 

  6. Tanaka A, Sakaguchi S (2017) Regulatory T cells in cancer immunotherapy. Cell Res 27:109–118

    CAS  PubMed  Google Scholar 

  7. Crunkhorn S (2017) Cancer immunotherapy: targeting regulatory T cells. Nat Rev Drug Discov 16:754

    PubMed  Google Scholar 

  8. Pahl J, Cerwenka A (2017) Tricking the balance: NK cells in anti-cancer immunity. Immunobiology 222:11–20

    CAS  PubMed  Google Scholar 

  9. Noy R, Pollard JW (2014) Tumor-associated macrophages: from mechanisms to therapy. Immunity 41:49–61

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Brodt P, Gordon J (1978) Anti-tumor immunity in B lymphocyte-deprived mice. I. Immunity to a chemically induced tumor. J Immunol 121:359–362

    CAS  PubMed  Google Scholar 

  11. Nelson BH (2010) CD20 + B cells: the other tumor-infiltrating lymphocytes. J Immunol 185:4977–4982

    CAS  PubMed  Google Scholar 

  12. Gass JD (1985) Comparison of uveal melanoma growth rates with mitotic index and mortality. Arch Ophthalmol 103:924–931

    CAS  PubMed  Google Scholar 

  13. Zhou X, Su YX, Lao XM, Liang YJ, Liao GQ (2016) CD19(+)IL-10(+) regulatory B cells affect survival of tongue squamous cell carcinoma patients and induce resting CD4(+) T cells to CD4(+)Foxp3(+) regulatory T cells. Oral Oncol 53:27–35

    CAS  PubMed  Google Scholar 

  14. Zhou M, Wen Z, Cheng F, Ma J, Li W, Ren H et al (2016) Tumor-released autophagosomes induce IL-10-producing B cells with suppressive activity on T lymphocytes via TLR2-MyD88-NF-κB signal pathway. Oncoimmunology 5:e1180485

    PubMed  PubMed Central  Google Scholar 

  15. Kessel A, Haj T, Peri R, Snir A, Melamed D, Sabo E et al (2012) Human CD19(+)CD25(high) B regulatory cells suppress proliferation of CD4(+) T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells. Autoimmun Rev 11:670–677

    CAS  PubMed  Google Scholar 

  16. Olkhanud PB, Damdinsuren B, Bodogai M, Gress RE, Sen R, Wejksza K et al (2011) Tumor-evoked regulatory B cells promote breast cancer metastasis by converting resting CD4+ T cells to T-regulatory cells. Cancer Res 71:3505–3515

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Gray D, Gray M (2010) What are regulatory B cells? Eur J Immunol 40:2677–2679

    CAS  PubMed  Google Scholar 

  18. DiLillo DJ, Matsushita T, Tedder TF (2010) B10 cells and regulatory B cells balance immune responses during inflammation, autoimmunity, and cancer. Ann N Y Acad Sci 1183:38–57

    CAS  PubMed  Google Scholar 

  19. Jung J, Choe J, Li L, Choi YS (2000) Regulation of CD27 expression in the course of germinal center B cell differentiation: the pivotal role of IL-10. Eur J Immunol 30:2437–2443

    CAS  PubMed  Google Scholar 

  20. Murray PJ (2007) The JAK-STAT signaling pathway: input and output integration. J Immunol 178:2623–2629

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Wong SC, Puaux AL, Chittezhath M, Shalova I, Kajiji TS, Wang X et al (2010) Macrophage polarization to a unique phenotype driven by B cells. Eur J Immunol 40:2296–2307

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  24. van der Vlugt LE, Zinsou JF, Ozir-Fazalalikhan A, Kremsner PG, Yazdanbakhsh M, Adegnika AA et al (2014) Interleukin 10 (IL-10)-producing CD1dhi regulatory B cells from Schistosoma haematobium-infected individuals induce IL-10-positive T cells and suppress effector T-cell cytokines. J Infect Dis 210:1207–1216

    PubMed  Google Scholar 

  25. Mizoguchi A, Mizoguchi E, Takedatsu H, Blumberg RS, Bhan AK (2002) Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation. Immunity 16:219–230

    CAS  PubMed  Google Scholar 

  26. Mauri C, Bosma A (2012) Immune regulatory function of B cells. Annu Rev Immunol 30:221–241

    CAS  Google Scholar 

  27. Biragyn A, Lee-Chang C, Bodogai M (2014) Generation and identification of tumor-evoked regulatory B cells. Methods Mol Biol 1190:271–289

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Bodogai M, Lee Chang C, Wejksza K, Lai J, Merino M, Wersto RP et al (2013) Anti-CD20 antibody promotes cancer escape via enrichment of tumor-evoked regulatory B cells expressing low levels of CD20 and CD137L. Cancer Res 73:2127–2138

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Wejksza K, Lee-Chang C, Bodogai M, Bonzo J, Gonzalez FJ, Lehrmann E et al (2013) Cancer-produced metabolites of 5-lipoxygenase induce tumor-evoked regulatory B cells via peroxisome proliferator-activated receptor α. J Immunol 190:2575–2584

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Bailey SR, Nelson MH, Himes RA, Li Z, Mehrotra S, Paulos CM (2014) Th17 cells in cancer: the ultimate identity crisis. Front Immunol 5:276

    PubMed  PubMed Central  Google Scholar 

  31. Zhu X, Mulcahy LA, Mohammed RA, Lee AH, Franks HA, Kilpatrick L et al (2008) IL-17 expression by breast-cancer-associated macrophages: IL-17 promotes invasiveness of breast cancer cell lines. Breast Cancer Res 10:R95

    PubMed  PubMed Central  Google Scholar 

  32. Guo B, Li L, Guo J, Liu A, Wu J, Wang H et al (2017) M2 tumor-associated macrophages produce interleukin-17 to suppress oxaliplatin-induced apoptosis in hepatocellular carcinoma. Oncotarget 8:44465–44476

    PubMed  PubMed Central  Google Scholar 

  33. Vykhovanets EV, Maclennan GT, Vykhovanets OV, Gupta S (2011) IL-17 Expression by macrophages is associated with proliferative inflammatory atrophy lesions in prostate cancer patients. Int J Clin Exp Pathol 4:552–565

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Schlegel PM, Steiert I, Kötter I, Müller CA (2013) B cells contribute to heterogeneity of IL-17 producing cells in rheumatoid arthritis and healthy controls. PLoS ONE 8:e82580

    PubMed  PubMed Central  Google Scholar 

  35. Ma S, Cheng Q, Cai Y, Gong H, Wu Y, Yu X et al (2014) IL-17A produced by γδ T cells promotes tumor growth in hepatocellular carcinoma. Cancer Res 74:1969–1982

    CAS  PubMed  Google Scholar 

  36. Jung MK, Kwak JE, Shin EC (2017) IL-17A-Producing Foxp3. Immune Netw 17:276–286

    PubMed  PubMed Central  Google Scholar 

  37. Punt S, Fleuren GJ, Kritikou E, Lubberts E, Trimbos JB, Jordanova ES et al (2015) Angels and demons: Th17 cells represent a beneficial response, while neutrophil IL-17 is associated with poor prognosis in squamous cervical cancer. Oncoimmunology 4:e984539

    PubMed  PubMed Central  Google Scholar 

  38. Rashid RM, Achille NJ, Lee JM, Lathers DM, Young MR (2005) Decreased T-cell proliferation and skewed immune responses in LLC-bearing mice. J Environ Pathol Toxicol Oncol 24:175–192

    CAS  PubMed  Google Scholar 

  39. Chen ML, Yan BS, Bando Y, Kuchroo VK, Weiner HL (2008) Latency-associated peptide identifies a novel CD4 + CD25 + regulatory T cell subset with TGFbeta-mediated function and enhanced suppression of experimental autoimmune encephalomyelitis. J Immunol 180:7327–7337

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Rosser EC, Oleinika K, Tonon S, Doyle R, Bosma A, Carter NA et al (2014) Regulatory B cells are induced by gut microbiota-driven interleukin-1β and interleukin-6 production. Nat Med 20:1334–1339

    CAS  PubMed  Google Scholar 

  41. Menon M, Blair PA, Isenberg DA, Mauri C (2016) A regulatory feedback between plasmacytoid dendritic cells and regulatory B cells is aberrant in systemic lupus erythematosus. Immunity 44:683–697

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Schioppa T, Moore R, Thompson RG, Rosser EC, Kulbe H, Nedospasov S et al (2011) B regulatory cells and the tumor-promoting actions of TNF-α during squamous carcinogenesis. Proc Natl Acad Sci USA 108:10662–10667

    CAS  PubMed  Google Scholar 

  43. Horikawa M, Minard-Colin V, Matsushita T, Tedder TF (2011) Regulatory B cell production of IL-10 inhibits lymphoma depletion during CD20 immunotherapy in mice. J Clin Invest 121:4268–4280

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Inman GJ, Nicolás FJ, Callahan JF, Harling JD, Gaster LM, Reith AD et al (2002) SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 62:65–74

    CAS  PubMed  Google Scholar 

  45. Komai T, Inoue M, Okamura T, Morita K, Iwasaki Y, Sumitomo S et al (2018) Transforming growth factor-β and interleukin-10 synergistically regulate humoral immunity. Front Immunol 9:1364

    PubMed  PubMed Central  Google Scholar 

  46. Palomares O, Martín-Fontecha M, Lauener R, Traidl-Hoffmann C, Cavkaytar O, Akdis M et al (2014) Regulatory T cells and immune regulation of allergic diseases: roles of IL-10 and TGF-β. Genes Immun 15:511–520

    CAS  PubMed  Google Scholar 

  47. Chou WC, Levy DE, Lee CK (2006) STAT3 positively regulates an early step in B-cell development. Blood 108:3005–3011

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Ding C, Chen X, Dascani P, Hu X, Bolli R, Zhang HG et al (2016) STAT3 signaling in B cells is critical for germinal center maintenance and contributes to the pathogenesis of murine models of lupus. J Immunol 196:4477–4486

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Yang X, Yang J, Chu Y, Wang J, Guan M, Zhu X et al (2013) T follicular helper cells mediate expansion of regulatory B cells via IL-21 in Lupus-prone MRL/lpr mice. PLoS ONE 8:e62855

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Schmetterer KG, Pickl WF (2017) The IL-10/STAT3 axis: contributions to immune tolerance by thymus and peripherally derived regulatory T-cells. Eur J Immunol 47:1256–1265

    CAS  PubMed  Google Scholar 

  51. Schmitt N, Liu Y, Bentebibel SE, Munagala I, Bourdery L, Venuprasad K et al (2014) The cytokine TGF-β co-opts signaling via STAT3-STAT4 to promote the differentiation of human TFH cells. Nat Immunol 15:856–865

    PubMed  PubMed Central  Google Scholar 

  52. Mielle J, Audo R, Hahne M, Macia L, Combe B, Morel J et al (2018) IL-10 producing B cells ability to induce regulatory T cells is maintained in rheumatoid arthritis. Front Immunol 9:961

    PubMed  PubMed Central  Google Scholar 

  53. Lee KM, Stott RT, Zhao G, SooHoo J, Xiong W, Lian MM et al (2014) TGF-β-producing regulatory B cells induce regulatory T cells and promote transplantation tolerance. Eur J Immunol 44:1728–1736

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Shi J, Feng J, Xie J, Mei Z, Shi T, Wang S et al (2017) Targeted blockade of TGF-β and IL-6/JAK2/STAT3 pathways inhibits lung cancer growth promoted by bone marrow-derived myofibroblasts. Sci Rep 7:8660

    PubMed  PubMed Central  Google Scholar 

  55. Hjelmeland MD, Hjelmeland AB, Sathornsumetee S, Reese ED, Herbstreith MH, Laping NJ et al (2004) SB-431542, a small molecule transforming growth factor-beta-receptor antagonist, inhibits human glioma cell line proliferation and motility. Mol Cancer Ther 3:737–745

    CAS  PubMed  Google Scholar 

  56. Matsuyama S, Iwadate M, Kondo M, Saitoh M, Hanyu A, Shimizu K et al (2003) SB-431542 and Gleevec inhibit transforming growth factor-beta-induced proliferation of human osteosarcoma cells. Cancer Res 63:7791–7798

    CAS  PubMed  Google Scholar 

  57. Halder SK, Beauchamp RD, Datta PK (2005) A specific inhibitor of TGF-beta receptor kinase, SB-431542, as a potent antitumor agent for human cancers. Neoplasia 7:509–521

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Zhou HQ, Liu MS, Deng TB, Xie PB, Wang W, Shao T et al (2019) The TGF-β/Smad pathway inhibitor SB431542 enhances the antitumor effect of radiofrequency ablation on bladder cancer cells. Onco Targets Ther 12:7809–7821

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Sato M, Matsubara T, Adachi J, Hashimoto Y, Fukamizu K, Kishida M et al (2015) Differential proteome analysis identifies TGF-β-related pro-metastatic proteins in a 4T1 murine breast cancer model. PLoS ONE 10:e0126483

    PubMed  PubMed Central  Google Scholar 

  60. DiLillo DJ, Yanaba K, Tedder TF (2010) B cells are required for optimal CD4 + and CD8 + T cell tumor immunity: therapeutic B cell depletion enhances B16 melanoma growth in mice. J Immunol 184:4006–4016

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Kim S, Fridlender ZG, Dunn R, Kehry MR, Kapoor V, Blouin A et al (2008) B-cell depletion using an anti-CD20 antibody augments antitumor immune responses and immunotherapy in nonhematopoetic murine tumor models. J Immunother 31:446–457

    CAS  PubMed  Google Scholar 

  62. Maglioco A, Machuca DG, Badano MN, Nannini P, Camerano GV, Costa H et al (2017) B cells inhibit the antitumor immunity against an established murine fibrosarcoma. Oncol Lett 13:3225–3232

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Ribas A, Wolchok JD (2018) Cancer immunotherapy using checkpoint blockade. Science 359:1350–1355

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Mariathasan S, Turley SJ, Nickles D, Castiglioni A, Yuen K, Wang Y et al (2018) TGFβ attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells. Nature 554:544–548

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Tauriello DVF, Palomo-Ponce S, Stork D, Berenguer-Llergo A, Badia-Ramentol J, Iglesias M et al (2018) TGFβ drives immune evasion in genetically reconstituted colon cancer metastasis. Nature 554:538–543

    CAS  PubMed  Google Scholar 

  66. Bodogai M, Moritoh K, Lee-Chang C, Hollander CM, Sherman-Baust CA, Wersto RP et al (2015) Immunosuppressive and prometastatic functions of myeloid-derived suppressive cells rely upon education from tumor-associated B cells. Cancer Res 75:3456–3465

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Biragyn A, Lee-Chang C (2012) A new paradigm for an old story: the role of regulatory B cells in cancer. Front Immunol 3:206

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors acknowledge Mr Narendra Sidnalkar for his technical assistance.

Funding

The study was funded by Bhabha Atomic Research Centre, Government of India.

Author information

Authors and Affiliations

  1. Immunology Section, Radiation Biology & Health Sciences Division, Bio-Science Group, Bhabha Atomic Research Centre, Mumbai, 400 085, India

    Kavitha Premkumar & Bhavani S. Shankar

  2. Homi Bhabha National Institute, Anushaktinagar, Trombay, Mumbai, 400 094, India

    Kavitha Premkumar & Bhavani S. Shankar

Authors
  1. Kavitha Premkumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bhavani S. Shankar
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Ms. Kavitha Premkumar designed and performed the experiments, acquired the samples and analysed the data and wrote the manuscript. Dr Bhavani Shankar conceptualized and designed the study, analysed and interpreted the data, wrote and revised the manuscript. Both the co-authors approved the final version to be submitted.

Corresponding author

Correspondence to Bhavani S. Shankar.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval and ethical standards

All animal studies were approved and licenced by the Institutional Animal Ethics Committee (BARC/animalhouse/106/RBi/S/99/CPSEA), Bhabha Atomic Research Centre, Government of India, under the project no. BAEC/06/17 (dt 03.04.2017) and carried out in strict accordance with the guidelines issued by the institutional animal ethics committee regarding the maintenance and dissection of small animals.

Cell line authentication

The authenticated mouse fibrosarcoma cell line WEHI-164 was purchased from European Collection of Authenticated Cell Cultures (ECACC).

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 420 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Premkumar, K., Shankar, B.S. TGF-βR inhibitor SB431542 restores immune suppression induced by regulatory B–T cell axis and decreases tumour burden in murine fibrosarcoma. Cancer Immunol Immunother 70, 153–168 (2021). https://doi.org/10.1007/s00262-020-02666-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00262-020-02666-w

Keywords

  • Regulatory B cell
  • Breg–Treg axis
  • Immunosuppression
  • TGF-β signalling
  • SB431542
  • Fibrosarcoma