Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Adjuvant immunotherapy of C6 glioma in rats with pertussis toxin



In spite of the recent advances in surgery and antitumor drugs, the brain tumors, like glioblastoma, have shown a poor prognosis. The aim of this study was to determine the effect of pertussis toxin (PTx) as immunomodulatory molecule on glial tumors induced by C6 glioma cells.


Given the pleiotropic effect of PTx on the immune system, we analyzed the effect of PTx on CD4+/CD25+/FoxP3+ (Treg) cells like as immunotherapeutic adjuvant. Thirty rats with a glial tumor of 1.5 cm in diameter were separated in two groups: the first group was treated with PTx and the second group was non-treated (controls). Tumoral volume was measured weekly; tumor, blood and spleen were taken for analysis of subpopulations of T cells, apoptotic index and cytokine contents, in both groups.


We observed a significant decrease in tumor volume in the PTx group; this was associated with a decreased in the number of Treg cells, in both spleen and tumor. The percentage of apoptotic cells was increased as compared with that of controls. The production of proinflammatory cytokines was increased in mRNA for IL-6 as well as a small increase in the mRNA expression of perforin and granzime in tumors from rats treated with PTx. No changes were found in the mRNA expression of MCP-1 and MIP-1α.


These results suggest that PTx could be an immunotherapeutic adjuvant in the integral therapy against glial tumors.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8


  1. Agarwal RK, Sun SH, Su SB, Chan CC, Caspi RR (2002) Pertussis toxin alters the innate and the adaptive immune responses in a pertussis-dependent model of autoimmunity. J Neuroimmunol 129:133–140

  2. Alt C, Laschinger M, Engelhardt B (2002) Functional expression of the lymphoid chemokines CCL19 (ELC) and CCL 21 (SLC) at the blood-brain barrier suggests their involvement in G-protein-dependent lymphocyte recruitment into the central nervous system during experimental autoimmune encephalomyelitis. Eur J Immunol 32:2133–2144

  3. Arrieta O, Guevara P, Reyes S, Ortiz A, Rembao D, Sotelo J (1998) Protamine inhibits angiogenesis and growth of C6 rat glioma; a synergistic effect when combined with carmustine. Eur J Cancer 34:2101–2106

  4. Backlund PS Jr, Meade BD, Manclark CR, Cantoni GL, Aksamit RR (1985) Pertussis toxin inhibition of chemotaxis and the ADP-ribosylation of a membrane protein in a human-mouse hybrid cell line. Proc Natl Acad Sci USA 82:2637–2641

  5. Barnett B, Kryczek I, Cheng P, Zou W, Curiel TJ (2005) Regulatory T cells in ovarian cancer: biology and therapeutic potential. Am J Reprod Immunol 54:369–377

  6. Benda P, Lightbody J, Sato G, Levine L, Sweet W (1968) Differentiated rat glial cell strain in tissue culture. Science 161:370–371

  7. Bruckener KE, el Baya A, Galla HJ, Schmidt MA (2003) Permeabilization in a cerebral endothelial barrier model by pertussis toxin involves the PKC effector pathway and is abolished by elevated levels of cAMP. J Cell Sci 116:1837–1846

  8. Buckner JC (2003) Factors influencing survival in high-grade gliomas. Semin Oncol 30:10–14

  9. Cassan C, Piaggio E, Zappulla JP, Mars LT, Couturier N, Bucciarelli F, Desbois S, Bauer J, Gonzalez-Dunia D, Liblau RS (2006) Pertussis toxin reduces the number of splenic Foxp3+ regulatory T cells. J Immunol 177:1552–1560

  10. Chattopadhyay S, Chakraborty NG, Mukherji B (2005) Regulatory T cells and tumor immunity. Cancer Immunol Immunother 54:1153–1161

  11. Chen X, Winkler-Pickett RT, Carbonetti NH, Ortaldo JR, Oppenheim JJ, Howard OM (2006) Pertussis toxin as an adjuvant suppresses the number and function of CD4+CD25+ T regulatory cells. Eur J Immunol 36:671–680

  12. Chen X, Howard OM, Oppenheim JJ (2007) Pertussis toxin by inducing IL-6 promotes the generation of IL-17-producing CD4 cells. J Immunol 178:6123–6129

  13. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, Evdemon-Hogan M, Conejo-Garcia JR, Zhang L, Burow M, Zhu Y, Wei S, Kryczek I, Daniel B, Gordon A, Myers L, Lackner A, Disis ML, Knutson KL, Chen L, Zou W (2004) Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10:942–949

  14. Cyster JG, Goodnow CC (1995) Pertussis toxin inhibits migration of B and T lymphocytes into splenic white pulp cords. J Exp Med 182:581–586

  15. Darabi K, Karulin AY, Boehm BO, Hofstetter HH, Fabry Z, LaManna JC, Chavez JC, Tary-Lehmann M, Lehmann PV (2004) The third signal in T cell-mediated autoimmune disease? J Immunol 173:92–99

  16. Deorah S, Lynch CF, Sibenaller ZA, Ryken TC (2006) Trends in brain cancer incidence, survival in the United States: Surveillance, Epidemiology, End Results Program, 1973 to 2001. Neurosurg Focus 20:E1

  17. El Andaloussi A, Lesniak MS (2006) An increase in CD4+CD25+FOXP3+ regulatory T cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme. Neuro Oncol 8:234–243

  18. El Andaloussi A, Lesniak MS (2007) CD4+CD25+FoxP3+ T-cell infiltration and heme oxygenase-1 expression correlate with tumor grade in human gliomas. J Neurooncol 83:145–152

  19. El Andaloussi A, Han Y, Lesniak MS (2006) Prolongation of survival following depletion of CD4+CD25+ regulatory T cells in mice with experimental brain tumors. J Neurosurg 105:430–437

  20. Fecci PE, Mitchell DA, Whitesides JF, Xie W, Friedman AH, Archer GE, Herndon JE 2nd, Bigner DD, Dranoff G, Sampson JH (2006) Increased regulatory T-cell fraction amidst a diminished CD4 compartment explains cellular immune defects in patients with malignant glioma. Cancer Res 66:3294–3302

  21. Garcia-Lora A, Algarra I, Garrido F (2003) MHC class I antigens, immune surveillance, and tumor immune escape. J Cell Physiol 195:346–355

  22. Golgher D, Jones E, Powrie F, Elliott T, Gallimore A (2002) Depletion of CD25+ regulatory cells uncovers immune responses to shared murine tumor rejection antigens. Eur J Immunol 32:3267–3275

  23. Guevara P, Sotelo J (1999) C6 rat glioma grown into the peritoneal cavity, a large source of tumoral cells for subcutaneous transplant of glioma. J Neurooncol 44:91–92

  24. Hart MN, Linthicum DS, Waldschmidt MM, Tassell SK, Schelper RL, Robinson RA (1987) Experimental autoimmune inflammatory myopathy. J Neuropathol Exp Neurol 46:511–521

  25. He J, Gurunathan S, Iwasaki A, Ash-Shaheed B, Kelsall BL (2000) Primary role for Gi protein signaling in the regulation of interleukin 12 production and the induction of T helper cell type 1 responses. J Exp Med 191:1605–1610

  26. Hofstetter HH, Shive CL, Forsthuber TG (2002) Pertussis toxin modulates the immune response to neuroantigens injected in incomplete Freund’s adjuvant: induction of Th1 cells and experimental autoimmune encephalomyelitis in the presence of high frequencies of Th2 cells. J Immunol 169:117–125

  27. Hou W, Wu Y, Sun S, Shi M, Sun Y, Yang C, Pei G, Gu Y, Zhong C, Sun B (2003) Pertussis toxin enhances Th1 responses by stimulation of dendritic cells. J Immunol 170:1728–1736

  28. Ichihara F, Kono K, Takahashi A, Kawaida H, Sugai H, Fujii H (2003) Increased populations of regulatory T cells in peripheral blood and tumor-infiltrating lymphocytes in patients with gastric and esophageal cancers. Clin Cancer Res 9:4404–4408

  29. Jager D, Jager E, Knuth A (2001) Immune responses to tumour antigens: implications for antigen specific immunotherapy of cancer. J Clin Pathol 54:669–674

  30. Kohno S, Munoz JA, Williams TM, Teuscher C, Bernard CC, Tung KS (1983) Immunopathology of murine experimental allergic orchitis. J Immunol 130:2675–2682

  31. Li M, Liu J, Zhang SZ, Zhou Y, Guo YW, Chen Q, Ke YQ, Jiang XD, Cai YQ (2011) Cellular immunologic response to primary cryoablation of C6 gliomas in rats. Technol Cancer Res Treat 10:95–100

  32. Linehan DC, Goedegebuure PS (2005) CD25+CD4+ regulatory T-cells in cancer. Immunol Res 32:155–168

  33. Linthicum DS (1982) Development of acute autoimmune encephalomyelitis in mice: factors regulating the effector phase of the disease. Immunobiology 162:211–220

  34. Linthicum DS, Frelinger JA (1982) Acute autoimmune encephalomyelitis in mice. II. Susceptibility is controlled by the combination of H-2 and histamine sensitization genes. J Exp Med 156:31–40

  35. Liyanage UK, Moore TT, Joo HG, Tanaka Y, Herrmann V, Doherty G, Drebin JA, Strasberg SM, Eberlein TJ, Goedegebuure PS, Linehan DC (2002) Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol 169:2756–2761

  36. Lopez-Gonzalez MA, Sotelo J (2000) Brain tumors in Mexico: characteristics and prognosis of glioblastoma. Surg Neurol 53:157–162

  37. Mascart F, Verscheure V, Malfroot A, Hainaut M, Pierard D, Temerman S, Peltier A, Debrie AS, Levy J, Del Giudice G, Locht C (2003) Bordetella pertussis infection in 2-month-old infants promotes type 1 T cell responses. J Immunol 170:1504–1509

  38. Mielcarek N, Hornquist EH, Johansson BR, Locht C, Abraham SN, Holmgren J (2001) Interaction of Bordetella pertussis with mast cells, modulation of cytokine secretion by pertussis toxin. Cell Microbiol 3:181–188

  39. Munoz JJ, Bernard CC, Mackay IR (1984) Elicitation of experimental allergic encephalomyelitis (EAE) in mice with the aid of pertussigen. Cell Immunol 83:92–100

  40. Nencioni L, Pizza MG, Volpini G, De Magistris MT, Giovannoni F, Rappuoli R (1991) Properties of the B oligomer of pertussis toxin. Infect Immun 59:4732–4734

  41. Onizuka S, Tawara I, Shimizu J, Sakaguchi S, Fujita T, Nakayama E (1999) Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody. Cancer Res 59:3128–3133

  42. Papaspyridonos M, McNeill E, de Bono JP, Smith A, Burnand KG, Channon KM, Greaves DR (2008) Galectin-3 is an amplifier of inflammation in atherosclerotic plaque progression through macrophage activation and monocyte chemoattraction. Arterioscler Thromb Vasc Biol 28:433–440

  43. Pineda B, Estrada-Parra S, Pedraza-Medina B, Rodriguez-Ropon A, Perez R, Arrieta O (2005) Interstitial transfer factor as adjuvant immunotherapy for experimental glioma. J Exp Clin Cancer Res 24:575–583

  44. Ryan M, McCarthy L, Rappuoli R, Mahon BP, Mills KH (1998) Pertussis toxin potentiates Th1 and Th2 responses to co-injected antigen: adjuvant action is associated with enhanced regulatory cytokine production and expression of the co-stimulatory molecules B7–1, B7–2 and CD28. Int Immunol 10:651–662

  45. Shimizu J, Yamazaki S, Sakaguchi S (1999) Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J Immunol 163:5211–5218

  46. Shive CL, Hofstetter H, Arredondo L, Shaw C, Forsthuber TG (2000) The enhanced antigen-specific production of cytokines induced by pertussis toxin is due to clonal expansion of T cells and not to altered effector functions of long-term memory cells. Eur J Immunol 30:2422–2431

  47. Strojnik T, Kavalar R, Zajc I, Diamandis EP, Oikonomopoulou K, Lah TT (2009) Prognostic impact of CD68 and kallikrein 6 in human glioma. Anticancer Res 29:3269–3279

  48. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996

  49. Tomayko MM, Reynolds CP (1989) Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 24:148–154

  50. Tonon S, Goriely S, Aksoy E, Pradier O, Del Giudice G, Trannoy E, Willems F, Goldman M, De Wit D (2002) Bordetella pertussis toxin induces the release of inflammatory cytokines and dendritic cell activation in whole blood: impaired responses in human newborns. Eur J Immunol 32:3118–3125

  51. Velasquez-Perez L, Jimenez-Marcial ME (2003) Clinical-histopathologic concordance of tumors of the nervous system at the Manuel Velasco Suarez National Institute of Neurology and Neurosurgery in Mexico City. Arch Pathol Lab Med 127:187–192

  52. Woo EY, Chu CS, Goletz TJ, Schlienger K, Yeh H, Coukos G, Rubin SC, Kaiser LR, June CH (2001) Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer. Cancer Res 61:4766–4772

  53. Zhu X, Lu C, Xiao B, Qiao J, Sun Y (2005) An experimental study of dendritic cells mediated immunotherapy against intracranial gliomas in rats. J Neurooncol 74:9–17

Download references


This work was partially supported by CONACyT-México grants (90550) and ICyTDF (PICSA10-143).

Conflict of interest


Author information

Correspondence to Benjamín Pineda.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Orozco-Morales, M., Sánchez-García, F., Guevara-Salazar, P. et al. Adjuvant immunotherapy of C6 glioma in rats with pertussis toxin. J Cancer Res Clin Oncol 138, 23–33 (2012).

Download citation


  • Pertussis toxin
  • Glioblastoma multiforme
  • Immunotherapeutic
  • Regulatory T cells
  • C6 glioma