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. 1998 Jan 15;101(2):429–441. doi: 10.1172/JCI1348

Importance of cyclophosphamide-induced bystander effect on T cells for a successful tumor eradication in response to adoptive immunotherapy in mice.

E Proietti 1, G Greco 1, B Garrone 1, S Baccarini 1, C Mauri 1, M Venditti 1, D Carlei 1, F Belardelli 1
PMCID: PMC508583  PMID: 9435316

Abstract

Cyclophosphamide (CTX) increases the antitumor effectiveness of adoptive immunotherapy in mice, and combined immunotherapy regimens are now used in some clinical trials. However, the mechanisms underlying the synergistic antitumor responses are still unclear. The purpose of this study was (a) to evaluate the antitumor response to CTX and adoptive immunotherapy in mice bearing four different syngeneic tumors (two responsive in vivo to CTX and two resistant); and (b) to define the mechanism(s) of the CTX-immunotherapy synergism. Tumor-bearing DBA/2 mice were treated with a single injection of CTX followed by an intravenous infusion of tumor-immune spleen cells. In all the four tumor models, a single CTX injection resulted in an impressive antitumor response to the subsequent injection of spleen cells from mice immunized with homologous tumor cells independently of the in vivo response to CTX alone. Detailed analysis of the antitumor mechanisms in mice transplanted with metastatic Friend leukemia cells revealed that (a) the effectiveness of this combined therapy was dependent neither on the CTX-induced reduction of tumor burden nor on CTX-induced inhibition of some putative tumor-induced suppressor cells; (b) the CTX/immune cells' regimen strongly protected the mice from subsequent injection of FLC, provided the animals were also preinoculated with inactivated homologous tumor together with the immune spleen cells; (c) CD4(+) T immune lymphocytes were the major cell type responsible for the antitumor activity; (d) the combined therapy was ineffective in mice treated with antiasialo-GM1 or anti-IFN-alpha/beta antibodies; (e) spleen and/ or bone marrow cells from CTX-treated mice produced soluble factors that assisted in proliferation of the spleen cells. Altogether, these results indicate that CTX acts via bystander effects, possibly through production of T cell growth factors occurring during the rebound events after drug administration, which may sustain the proliferation, survival, and activity of the transferred immune T lymphocytes. Thus, our findings indicate the need for reappraisal of the mechanisms underlying the synergistic effects of CTX and adoptive immunotherapy, and may provide new insights into the definition of new and more effective strategies with chemotherapy and adoptive immunotherapy for cancer patients.

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Selected References

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  1. Abrams J. S., Eiseman J. L., Melink T. J., Sridhara R., Hiponia D. J., Bell M. M., Belani C. P., Adler W. H., Aisner J. Immunomodulation of interleukin-2 by cyclophosphamide: a phase IB trial. J Immunother Emphasis Tumor Immunol. 1993 Jul;14(1):56–64. doi: 10.1097/00002371-199307000-00008. [DOI] [PubMed] [Google Scholar]
  2. Affabris E., Jemma C., Rossi G. B. Isolation of interferon-resistant variants of Friend erythroleukemia cells: effects of interferon and ouabain. Virology. 1982 Jul 30;120(2):441–452. doi: 10.1016/0042-6822(82)90044-7. [DOI] [PubMed] [Google Scholar]
  3. Awwad M., North R. J. Cyclophosphamide-induced immunologically mediated regression of a cyclophosphamide-resistant murine tumor: a consequence of eliminating precursor L3T4+ suppressor T-cells. Cancer Res. 1989 Apr 1;49(7):1649–1654. [PubMed] [Google Scholar]
  4. Belardelli F., Ciolli V., Testa U., Montesoro E., Bulgarini D., Proietti E., Borghi P., Sestili P., Locardi C., Peschle C. Anti-tumor effects of interleukin-2 and interleukin-1 in mice transplanted with different syngeneic tumors. Int J Cancer. 1989 Dec 15;44(6):1108–1116. doi: 10.1002/ijc.2910440629. [DOI] [PubMed] [Google Scholar]
  5. Belardelli F., Gabriele L., Proietti E., Sestili P., Peretti M., Rozera C., Gresser I. Synergistic anti-tumor effects of combined IL-1/IFN-alpha/beta therapy in mice injected with metastatic Friend erythroleukemia cells. Int J Cancer. 1991 Sep 9;49(2):274–278. doi: 10.1002/ijc.2910490222. [DOI] [PubMed] [Google Scholar]
  6. Belardelli F., Gresser I., Maury C., Maunoury M. T. Antitumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend leukemia cells. II. Role of host mechanisms. Int J Cancer. 1982 Dec 15;30(6):821–825. doi: 10.1002/ijc.2910300622. [DOI] [PubMed] [Google Scholar]
  7. Belardelli F., Proietti E., Ciolli V., Sestili P., Carpinelli G., Di Vito M., Ferretti A., Woodrow D., Boraschi D., Podo F. Interleukin-1 beta induces tumor necrosis and early morphologic and metabolic changes in transplantable mouse tumors. Similarities with the anti-tumor effects of tumor necrosis factor alpha or beta. Int J Cancer. 1989 Jul 15;44(1):116–123. doi: 10.1002/ijc.2910440121. [DOI] [PubMed] [Google Scholar]
  8. Ciolli V., Gabriele L., Sestili P., Varano F., Proietti E., Gresser I., Testa U., Montesoro E., Bulgarini D., Mariani G. Combined interleukin 1/interleukin 2 therapy of mice injected with highly metastatic Friend leukemia cells: host antitumor mechanisms and marked effects on established metastases. J Exp Med. 1991 Feb 1;173(2):313–322. doi: 10.1084/jem.173.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Contegiacomo A., Pizzi C., De Marchis L., Alimandi M., Delrio P., Di Palma E., Petrella G., Ottini L., French D., Frati L. High cell kinetics is associated with amplification of the int-2, bcl-1, myc and erbB-2 proto-oncogenes and loss of heterozygosity at the DF3 locus in primary breast cancers. Int J Cancer. 1995 Mar 29;61(1):1–6. doi: 10.1002/ijc.2910610102. [DOI] [PubMed] [Google Scholar]
  10. Evans R. Combination therapy by using cyclophosphamide and tumor-sensitized lymphocytes: a possible mechanism of action. J Immunol. 1983 Jun;130(6):2511–2513. [PubMed] [Google Scholar]
  11. Ferrantini M., Proietti E., Santodonato L., Gabriele L., Peretti M., Plavec I., Meyer F., Kaido T., Gresser I., Belardelli F. Alpha 1-interferon gene transfer into metastatic Friend leukemia cells abrogated tumorigenicity in immunocompetent mice: antitumor therapy by means of interferon-producing cells. Cancer Res. 1993 Mar 1;53(5):1107–1112. [PubMed] [Google Scholar]
  12. Gabriele L., Proietti E., Greco G., Venditti M., Gresser I., Schirrmacher V., Von Hoegen P., Testa U., Modesti A., Cianfriglia M. Isolation and characterization of a metastatic Eb-like tumor variant highly responsive to interleukin (IL)-2 and to combination cytokine therapy with IL-2/IL-1 beta and IL-1 beta/interferon-alpha/beta. Invasion Metastasis. 1993;13(3):147–162. [PubMed] [Google Scholar]
  13. Glaser M. Regulation of specific cell-mediated cytotoxic response against SV40-induced tumor associated antigens by depletion of suppressor T cells with cyclophosphamide in mice. J Exp Med. 1979 Mar 1;149(3):774–779. doi: 10.1084/jem.149.3.774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gold J. E., Malamud S. C., LaRosa F., Osband M. E. Adoptive chemoimmunotherapy using ex vivo activated memory T-cells and cyclophosphamide: tumor lysis syndrome of a metastatic soft tissue sarcoma. Am J Hematol. 1993 Sep;44(1):42–47. doi: 10.1002/ajh.2830440109. [DOI] [PubMed] [Google Scholar]
  15. Gold J. E., Osband M. E. Autolymphocyte therapy--I. In vivo tumour-specific adoptive cellular therapy of murine melanoma and carcinoma using ex vivo activated memory T-lymphocytes. Eur J Cancer. 1994;30A(12):1871–1882. doi: 10.1016/0959-8049(94)00339-7. [DOI] [PubMed] [Google Scholar]
  16. Gold J. E., Osband M. E. Autolymphocyte therapy. II. Dependence of in vivo anti-tumor specificity and long-term immunity against murine melanoma and carcinoma on ex vivo activated donor memory T-cells. Clin Immunol Immunopathol. 1994 Jun;71(3):325–332. doi: 10.1006/clin.1994.1093. [DOI] [PubMed] [Google Scholar]
  17. Gold J. E., Ross S. D., Krellenstein D. J., LaRosa F., Malamud S. C., Osband M. E. Adoptive transfer of ex vivo activated memory T-cells with or without cyclophosphamide for advanced metastatic melanoma: results in 36 patients. Eur J Cancer. 1995;31A(5):698–708. doi: 10.1016/0959-8049(94)00523-8. [DOI] [PubMed] [Google Scholar]
  18. Gold J. E., Zachary D. T., Osband M. E. Adoptive transfer of ex vivo-activated memory T-cell subsets with cyclophosphamide provides effective tumor-specific chemoimmunotherapy of advanced metastatic murine melanoma and carcinoma. Int J Cancer. 1995 May 16;61(4):580–586. doi: 10.1002/ijc.2910610424. [DOI] [PubMed] [Google Scholar]
  19. Gold J. E., Zachary D. T., Osband M. E. Adoptively transferred ex vivo activated memory T cells with cyclophosphamide: effective tumor-specific chemoimmunotherapy of advanced metastatic murine melanoma and carcinoma. Clin Immunol Immunopathol. 1994 Oct;73(1):115–122. doi: 10.1006/clin.1994.1177. [DOI] [PubMed] [Google Scholar]
  20. Greenberg P. D., Kern D. E., Cheever M. A. Therapy of disseminated murine leukemia with cyclophosphamide and immune Lyt-1+,2- T cells. Tumor eradication does not require participation of cytotoxic T cells. J Exp Med. 1985 May 1;161(5):1122–1134. doi: 10.1084/jem.161.5.1122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gresser I., Carnaud C., Maury C., Sala A., Eid P., Woodrow D., Maunoury M. T., Belardelli F. Host humoral and cellular immune mechanisms in the continued suppression of Friend erythroleukemia metastases after interferon alpha/beta treatment in mice. J Exp Med. 1991 May 1;173(5):1193–1203. doi: 10.1084/jem.173.5.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gresser I., Kaido T., Maury C., Woodrow D., Moss J., Belardelli F. Interaction of IFN alpha/beta with host cells essential to the early inhibition of Friend erythroleukemia visceral metastases in mice. Int J Cancer. 1994 May 15;57(4):604–611. doi: 10.1002/ijc.2910570427. [DOI] [PubMed] [Google Scholar]
  23. Gresser I., Maury C., Carnaud C., De Maeyer E., Maunoury M. T., Belardelli F. Anti-tumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend erythroleukemia cells. VIII. Role of the immune system in the inhibition of visceral metastases. Int J Cancer. 1990 Sep 15;46(3):468–474. doi: 10.1002/ijc.2910460324. [DOI] [PubMed] [Google Scholar]
  24. Gresser I., Maury C., Kaido T., Bandu M. T., Tovey M. G., Maunoury M. T., Fantuzzi L., Gessani S., Greco G., Belardelli F. The essential role of endogenous IFN alpha/beta in the anti-metastatic action of sensitized T lymphocytes in mice injected with Friend erythroleukemia cells. Int J Cancer. 1995 Nov 27;63(5):726–731. doi: 10.1002/ijc.2910630520. [DOI] [PubMed] [Google Scholar]
  25. Gresser I., Maury C., Woodrow D., Moss J., Grütter M. G., Vignaux F., Belardelli F., Maunoury M. T. Interferon treatment markedly inhibits the development of tumor metastases in the liver and spleen and increases survival time of mice after intravenous inoculation of Friend erythroleukemia cells. Int J Cancer. 1988 Jan 15;41(1):135–142. doi: 10.1002/ijc.2910410124. [DOI] [PubMed] [Google Scholar]
  26. Gresser I., Tovey M. G., Bandu M. E., Maury C., Brouty-Boyé D. Role of interferon in the pathogenesis of virus diseases in mice as demonstrated by the use of anti-interferon serum. I. Rapid evolution of encephalomyocarditis virus infection. J Exp Med. 1976 Nov 2;144(5):1305–1315. doi: 10.1084/jem.144.5.1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hengst J. C., Mokyr M. B., Dray S. Importance of timing in cyclophosphamide therapy of MOPC-315 tumor-bearing mice. Cancer Res. 1980 Jul;40(7):2135–2141. [PubMed] [Google Scholar]
  28. Hoover S. K., Barrett S. K., Turk T. M., Lee T. C., Bear H. D. Cyclophosphamide and abrogation of tumor-induced suppressor T cell activity. Cancer Immunol Immunother. 1990;31(2):121–127. doi: 10.1007/BF01742376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Katsanis E., Bausero M. A., Ochoa A. C., Loeffler C. M., Blazar B. R., Leonard A. S., Anderson P. M. Importance in timing of cyclophosphamide on the enhancement of interleukin-2-induced cytolysis. Cancer Immunol Immunother. 1991;34(2):74–78. doi: 10.1007/BF01741339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kedar E., Ben-Aziz R., Epstein E., Leshem B. Chemo-immunotherapy of murine tumors using interleukin-2 (IL-2) and cyclophosphamide. IL-2 can facilitate or inhibit tumor growth depending on the sequence of treatment and the tumor type. Cancer Immunol Immunother. 1989;29(1):74–78. doi: 10.1007/BF00199920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kedar E., Rutkowski Y., Leshem B. Chemo-immunotherapy of murine solid tumors: enhanced therapeutic effects by interleukin-2 combined with interferon alpha and the role of specific T cells. Cancer Immunol Immunother. 1992;35(1):63–68. doi: 10.1007/BF01741057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kolb J. P., Poupon M. F., Lespinats G. M., Sabolovic D., Loisillier F. Splenic modifications induced by cyclophosphamide in C3H/He, nude, and "B" mice. J Immunol. 1977 May;118(5):1595–1599. [PubMed] [Google Scholar]
  33. Laude M., Russo K. L., Mokyr M. B., Dray S. Cure of mice bearing a late-stage, highly metastatic, drug-resistant tumor by adoptive chemoimmunotherapy. Cancer Immunol Immunother. 1993;36(4):229–236. doi: 10.1007/BF01740904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mitchell M. S. Combining chemotherapy with biological response modifiers in treatment of cancer. J Natl Cancer Inst. 1988 Nov 16;80(18):1445–1450. doi: 10.1093/jnci/80.18.1445. [DOI] [PubMed] [Google Scholar]
  35. Mulé J. J., Shu S., Schwarz S. L., Rosenberg S. A. Adoptive immunotherapy of established pulmonary metastases with LAK cells and recombinant interleukin-2. Science. 1984 Sep 28;225(4669):1487–1489. doi: 10.1126/science.6332379. [DOI] [PubMed] [Google Scholar]
  36. North R. J., Awwad M. Elimination of cycling CD4+ suppressor T cells with an anti-mitotic drug releases non-cycling CD8+ T cells to cause regression of an advanced lymphoma. Immunology. 1990 Sep;71(1):90–95. [PMC free article] [PubMed] [Google Scholar]
  37. Ootsu K., Gotoh K., Houkan T. Therapeutic efficacy of human recombinant interleukin-2 (TGP-3) alone or in combination with cyclophosphamide and immunocompetent cells in allogeneic, semi-syngeneic, and syngeneic murine tumors. Cancer Immunol Immunother. 1989;30(2):71–80. doi: 10.1007/BF01665956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Osband M. E., Lavin P. T., Babayan R. K., Graham S., Lamm D. L., Parker B., Sawczuk I., Ross S., Krane R. J. Effect of autolymphocyte therapy on survival and quality of life in patients with metastatic renal-cell carcinoma. Lancet. 1990 Apr 28;335(8696):994–998. doi: 10.1016/0140-6736(90)91064-h. [DOI] [PubMed] [Google Scholar]
  39. Proietti E., Tritarelli E., Gabriele L., Testa U., Greco G., Pelosi E., Gabbianelli M., Belardelli F., Peschle C. Combined interleukin 1 beta/interleukin 2 treatment in mice: synergistic myelostimulatory activity and protection against cyclophosphamide-induced myelosuppression. Cancer Res. 1993 Feb 1;53(3):569–576. [PubMed] [Google Scholar]
  40. Puddu P., Locardi C., Sestili P., Varano F., Petrini C., Modesti A., Masuelli L., Gresser I., Belardelli F. Human immunodeficiency virus (HIV)-infected tumor xenografts as an in vivo model for antiviral therapy: role of alpha/beta interferon in restriction of tumor growth in nude mice injected with HIV-infected U937 tumor cells. J Virol. 1991 May;65(5):2245–2253. doi: 10.1128/jvi.65.5.2245-2253.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Radov L. A., Haskill J. S., Korn J. H. Host immune potentiation of drug responses to a murine mammary adenocarcinoma. Int J Cancer. 1976 Jun 15;17(6):773–779. doi: 10.1002/ijc.2910170613. [DOI] [PubMed] [Google Scholar]
  42. Rakhmilevich A. L., North R. J. Elimination of CD4+ T cells in mice bearing an advanced sarcoma augments the antitumor action of interleukin-2. Cancer Immunol Immunother. 1994 Feb;38(2):107–112. doi: 10.1007/BF01526205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Reissmann T., Voegeli R., Pohl J., Hilgard P. Augmentation of host antitumor immunity by low doses of cyclophosphamide and mafosfamide in two animal tumor models. Cancer Immunol Immunother. 1989;28(3):179–184. doi: 10.1007/BF00204986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rocha B., Dautigny N., Pereira P. Peripheral T lymphocytes: expansion potential and homeostatic regulation of pool sizes and CD4/CD8 ratios in vivo. Eur J Immunol. 1989 May;19(5):905–911. doi: 10.1002/eji.1830190518. [DOI] [PubMed] [Google Scholar]
  45. Schwartz A., Askenase P. W., Gershon R. K. Regulation of delayed-type hypersensitivity reactions by cyclophosphamide-sensitive T cells. J Immunol. 1978 Oct;121(4):1573–1577. [PubMed] [Google Scholar]
  46. Shu S., Chou T., Rosenberg S. A. In vitro sensitization and expansion with viable tumor cells and interleukin 2 in the generation of specific therapeutic effector cells. J Immunol. 1986 May 15;136(10):3891–3898. [PubMed] [Google Scholar]
  47. Testa U., Martucci R., Rutella S., Scambia G., Sica S., Benedetti Panici P., Pierelli L., Menichella G., Leone G., Mancuso S. Autologous stem cell transplantation: release of early and late acting growth factors relates with hematopoietic ablation and recovery. Blood. 1994 Nov 15;84(10):3532–3539. [PubMed] [Google Scholar]
  48. Tough D. F., Borrow P., Sprent J. Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science. 1996 Jun 28;272(5270):1947–1950. doi: 10.1126/science.272.5270.1947. [DOI] [PubMed] [Google Scholar]
  49. Tritarelli E., Greco G., Testa U., Belardelli F., Peschle C., Proietti E. Combined interleukin-1 beta/interleukin-6 treatment in mice: synergistic myelostimulatory activity and myelorestorative effect after cyclophosphamide-induced myelosuppression. Cancer Res. 1994 Dec 15;54(24):6469–6476. [PubMed] [Google Scholar]
  50. Turk J. L., Parker D. Effect of cyclophosphamide on immunological control mechanisms. Immunol Rev. 1982;65:99–113. doi: 10.1111/j.1600-065x.1982.tb00429.x. [DOI] [PubMed] [Google Scholar]
  51. Unutmaz D., Pileri P., Abrignani S. Antigen-independent activation of naive and memory resting T cells by a cytokine combination. J Exp Med. 1994 Sep 1;180(3):1159–1164. doi: 10.1084/jem.180.3.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vidović D., Marusić M., Culo F. Interference of anti-tumor and immunosuppressive effects of cyclophosphamide in tumor-bearing rats. Analysis of factors determining resistance or susceptibility to a subsequent tumor challenge. Cancer Immunol Immunother. 1982;14(1):36–40. doi: 10.1007/BF00199430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yoshizawa H., Sakai K., Chang A. E., Shu S. Y. Activation by anti-CD3 of tumor-draining lymph node cells for specific adoptive immunotherapy. Cell Immunol. 1991 May;134(2):473–479. doi: 10.1016/0008-8749(91)90318-6. [DOI] [PubMed] [Google Scholar]

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