The distinct role of CD4+ and CD8+ T-cells during the anti-tumour effects of targeted superantigens

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To target T-cells to the tumour area we created a recombinant protein of the bacterial superantigen (SAg) Staphylococcal enterotoxin A (SEA) and the Fab-fragment of a tumour-reactive antibody. This antibody-targeted SAg immunotherapy therapy has been shown to be highly efficient, eliminating > 95% of the pulmonary metastasis in mice carrying established melanoma micrometastases. Earlier studies demonstrated that elimination of the C215-expressing B16-melanoma lung metastasis was dependent on interferon (IFN)-γ release and expression of perforin. In the present study, therapeutic effector functions were analysed both locally at the tumour site and systemically in the spleen. In order to elucidate the role of each T-cell subset during Fab–SEA therapy, CD4 knock-out (KO) and CD8 KO mice were used. Tumour size reduction was statistically significant in Fab–SEA-based tumour therapy in both types of T-cell-deficient mice compared to wild-type mice. CD4 KO mice displayed a drastic reduction in the number of tumour-infiltrating macrophages and CD8+ T-cells. Therapy-induced accumulation of perforin-containing cells at the tumour site was significantly impaired in CD8 KO mice, and marginally in CD4 KO mice. Moreover, CD4 KO mice failed to produce substantial amounts of the tumour suppressive cytokine IFN-γ. This is in sharp contrast to normal mice where a massive local release was recorded. CD8 KO mice displayed a spontaneous production of interleukin (IL)-4 and IL-10 locally in the tumour. Neither normal nor CD4 KO mice produced detectable levels of these Th-2-associated cytokines. The high level of IL-10 was demonstrated to inhibit Fab–SEA tumour therapy, since the therapeutic efficacy was significantly higher in IL-10 KO mice. These results illustrate the importance of a finely tuned cellular collaboration to regulate the various phases of an efficient anti-tumour immune response.


  1. Andersson, J, Nagy, S, Bjork, L, Abrams, J, Holm, S & Andersson, U (1992). Bacterial toxin-induced cytokine production studied at the single-cell level. Immunol Rev 127,

  2. Aruga, A, Aruga, E, Tanigawa, K, Bishop, DK, Sondak, VK & Chang, AE (1997). Type 1 versus type 2 cytokine release by Vbeta T cell subpopulations determines in vivo antitumor reactivity: IL-10 mediates a suppressive role. J Immunol 159: 664–673.

  3. Battegay, M, Moskophidis, D, Rahemtulla, A, Hengartner, H, Mak, TW & Zinkernagel, RM (1994). Enhanced establishment of a virus carrier state in adult CD4+ T-cell-deficient mice. J Virol 68: 4700–4704.

  4. Coppola, MA & Blackman, MA (1997). Bacterial superantigens reactivate antigen-specific CD8+ memory T cells. Int. Immunol 9: 1393–1403.

  5. de Waal Malefyt, R, Abrams, J, Bennett, B, Figdor, CG & de Vries, JE (1991a). Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med 174: 1209–1220.

  6. de Waal Malefyt, R, Haanen, J, Spits, H, Roncarolo, MG, te Velde, A, Figdor, C, Johnson, K, Kastelein, R, Yssel, H & de Vries, JE (1991b). Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression. J Exp Med 174: 915–924.

  7. Dhein, J, Walczak, H, Baumler, C, Debatin, KM & Krammer, PH (1995). Autocrine T-cell suicide mediated by APO-1/(Fas/CD95). Nature 373: 438–441.

  8. Ding, L, Linsley, PS, Huang, LY, Germain, RN & Shevach, EM (1993). IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression. J Immunol 151: 1224–1234.

  9. Dohlsten, M, Lando, PA, Hedlund, G, Trowsdale, J & Kalland, T (1990). Targeting of human cytotoxic T lymphocytes to MHC class II-expressing cells by staphylococcal enterotoxins. Immunology 71: 96–100.

  10. Dohlsten, M, Sundstedt, A, Bjorklund, M, Hedlund, G & Kalland, T (1993). Superantigen-induced cytokines suppress growth of human colon-carcinoma cells. Int J Cancer 54: 482–488.

  11. Dohlsten, M, Abrahmsen, L, Bjork, P, Lando, PA, Hedlund, G, Forsberg, G, Brodin, T, Gascoigne, NR, Forberg, C & Lind, PETAL (1994). Monoclonal antibody-superantigen fusion proteins: tumor-specific agents for T-cell-based tumor therapy. Proc Natl Acad Sci USA 91: 8945–8949.

  12. Dohlsten, M, Hansson, J, Ohlsson, L, Litton, M & Kalland, T (1995a). Antibody-targeted superantigens are potent inducers of tumor-infiltrating T lymphocytes in vivo. Proc Natl Acad Sci USA 92: 9791–9795.

  13. Dohlsten, M, Lando, PA, Bjork, P, Abrahmsen, L, Ohlsson, L, Lind, P & Kalland, T (1995b). Immunotherapy of human colon cancer by antibody-targeted superantigens. Cancer Immunol Immunother 41: 162–168.

  14. Fiorentino, DF, Zlotnik, A, Vieira, P, Mosmann, TR, Howard, M, Moore, KW & O’Garra, A (1991). IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J Immunol 146: 3444–3451.

  15. Fitzpatrick, L, Makrigiannis, AP, Kaiser, M & Hoskin, D (1996). Anti-CD3-activated killer T cells: interferon-gamma and interleukin-10 cross-regulate granzyme B expression and the induction of major histocompatibity complex-unrestricted cytotoxicity. J Interferon Cytokine Res 16: 537–546.

  16. Fung Leung, WP, Schilham, MW, Rahemtulla, A, Kundig, TM, Vollenweider, M, Potter, J, van Ewijk, W & Mak, TW (1991). CD8 is needed for development of cytotoxic T cells but not helper T cells. Cell 65: 443–449.

  17. Giovarelli, M, Musiani, P, Modesti, A, Dellabona, P, Casorati, G, Allione, A, Consalvo, M, Cavallo, F, di Pierro, F & de Giovanni, CETAL (1995). Local release of IL-10 by transfected mouse mammary adenocarcinoma cells does not suppress but enhances antitumor reaction and elicits a strong cytotoxic lymphocyte and antibody-dependent immune memory. J Immunol 155: 3112–3123.

  18. Groux, H, Bigler, M, de Vries, JE & Roncarolo, MG (1996). Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells. J Exp Med 184: 19–29.

  19. Groux, H, Bigler, M, de Vries, JE & Roncarolo, MG (1998). Inhibitory and stimulatory effects of IL-10 on human CD8+ T cells. J Immunol 160: 3188–3193.

  20. Herrmann, T, Maryanski, JL, Romero, P, Fleischer, B & MacDonald, HR (1990). Activation of MHC class I-restricted CD8+ CTL by microbial T cell mitogens. Dependence upon MHC class II expression of the target cells and V beta usage of the responder T cells. J Immunol 144: 1181–1186.

  21. Hom, SS, Topalian, SL, Simonis, T, Mancini, M & Rosenberg, SA (1991). Common expression of melanoma tumor-associated antigens recognized by human tumor infiltrating lymphocytes: analysis by human lymphocyte antigen restriction. J Immunother 10: 153–164.

  22. Janeway, CA Jr, Yagi, J, Conrad, PJ, Katz, ME, Jones, B, Vroegop, S & Buxser, S (1989). T-cell responses to Mls and to bacterial proteins that mimic its behavior. Immunol Rev 107,

  23. Kagi, D, Ledermann, B, Burki, K, Hengartner, H & Zinkernagel, RM (1994). CD8+ T cell-mediated protection against an intracellular bacterium by perforin-dependent cytotoxicity. Eur J Immunol 24: 3068–3072.

  24. Kahn, M, Sugawara, H, Mcgowan, P, Okuno, K, Nagoya, S, Hellstrom, KE, Hellstrom, I & Greenberg, P (1991). CD4+ T cell clones specific for the human p97 melanoma-associated antigen can eradicate pulmonary metastases from a murine tumor expressing the p97 antigen. J Immunol 146: 3235–3241.

  25. Kern, DE, Klarnet, JP, Jensen, MC & Greenberg, PD (1986). Requirement for recognition of class II molecules and processed tumor antigen for optimal generation of syngeneic tumor-specific class I-restricted CTL. J Immunol 136: 4303–4310.

  26. Klarnet, JP, Kern, DE, Okuno, K, Holt, C, Lilly, F & Greenberg, PD (1989). FBL-reactive CD8+ cytotoxic and CD4+ helper T lymphocytes recognize distinct Friend murine leukemia virus-encoded antigens. J Exp Med 169: 457–467.

  27. Krieger, NR, Yin, DP & Garrison Fathman, C (1996). CD4+ but not CD8+ cells are essential for allorejection. J Exp Med 184: 2013–2018.

  28. Langford, MP, Stanton, GJ & Johnson, HM (1978). Biological effects of staphylococcal enterotoxin A on human peripheral lymphocytes. Infect Immun 22: 62–68.

  29. Lee, WT & Vitetta, ES (1992). Memory T cells are anergic to superantigen staphylococcal enterotoxin B. J Exp Med 176: 575–579.

  30. Litton, MJ, Sander, B, Murphy, E, O’Garra, A & Abrams, JS (1994). Early expression of cytokines in lymph nodes after treatment in vivo with Staphylococcus enterotoxin B. J Immunol Methods 175: 47–58.

  31. Litton, MJ, Dohlsten, M, Lando, PA, Kalland, T, Ohlsson, L, Andersson, J & Andersson, U (1996). Antibody-targeted superantigen therapy induces tumor-infiltrating lymphocytes, excessive cytokine production, and apoptosis in human colon carcinoma. Eur J Immunol 26: 1–9.

  32. Litton, MJ, Dohlsten, M, Hansson, J, Rosendahl, A, Ohlsson, L, Kalland, T, Andersson, J & Andersson, U (1997). Tumor therapy with an antibody-targeted superantigen generates a dichotomy between local and systemic immune responses. Am J Pathol 150: 1607–1618.

  33. Mosmann, TR & Coffman, RL (1989). TH-1 and TH-2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7: 1967–1990.

  34. Oswald, IP, Gazzinelli, RT, Sher, A & James, SL (1992). IL-10 synergizes with IL-4 and transforming growth factor-β to inhibit macrophage cytotoxic activity. J Immunol 148: 3578–3582.

  35. Pace, JL & Russell, SW (1981). Activation of mouse macrophages for tumor cell killing. I. Quantitative analysis of interactions between lymphokine and lipopolysaccharide. J Immunol 126: 1863–1867.

  36. Rahemtulla, A, Fung Leung, WP, Schilham, MW, Kundig, TM, Sambhara, SR, Narendran, A, Arabian, A, Wakeham, A, Paige, CJ & Zinkernagel, RM et al (1991). Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4. Nature 353: 180–184.

  37. Rosenberg, SA, Lotze, MT, Muul, LM, Leitman, S, Chang, AE, Ettinghausen, SE, Matory, YL, Skibber, JM, Shiloni, E & Vetto, JT et al (1985). Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 313: 1485–1492.

  38. Rosendahl, A, Hansson, J, Sundstedt, A, Kalland, T & Dohlsten, M (1996). Immune response during tumor therapy with antibody-superantigen fusion proteins. Int J Cancer 68: 109–113.

  39. Rosendahl, A, Kristensson, K, Hansson, J, Ohlsson, L, Kalland, T & Dohlsten, M (1998a). Repeated treatment with antibody-targeted superantigens strongly inhibits tumor growth. Int J Cancer 76: 274–283.

  40. Rosendahl, A, Kristensson, K, Hansson, J, Riesbeck, K, Kalland, T & Dohlsten, M (1998b). Perforin and IFN-gamma are involved in the antitumor effects of antibody-targeted superantigens. J Immunol 160: 5309–5313.

  41. Scherer, MT, Ignatowicz, L, Winslow, GM, Kappler, JW & Marrack, P (1993). Superantigens: bacterial and viral proteins that manipulate the immune system. Annu Rev Cell Biol 9,

  42. Schmidt Wolf, IG, Dejbakhsh Jones, S, Ginzton, N, Greenberg, P & Strober, S (1992). T-cell subsets and suppressor cells in human bone marrow. Blood 80: 3242–3250.

  43. Schreiber, RD (1984). Identification of gamma-interferon as a murine macrophage-activating factor for tumor cytotoxicity. Contemp Top Immunobiol 13,

  44. Sher, A, Gazzinelli, RT, Oswald, IP, Clerici, M, Kullberg, M, Pearce, EJ, Berzofsky, JA, Mosmann, TR, James, SL & Morse, HC, III (1992). Role of T-cell derived cytokines in the downregulation of immune responses in parasitic and retroviral infection. Immunol Rev 127: 183–204.

  45. Sundstedt, A, Hoiden, I, Rosendahl, A, Kalland, T, van Rooijen, N & Dohlsten, M (1997). Immunoregulatory role of IL-10 during superantigen-induced hyporesponsiveness in vivo. J Immunol 158: 180–186.

  46. White, J, Herman, A, Pullen, AM, Kubo, R, Kappler, JW & Marrack, P (1989). The V beta-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell 56: 27–35.

  47. Willems, F, Marchant, A, DelVille, JP, Gerard, C, Delvaux, A, Velu, T, de Boer, M & Goldman, M (1994). Interleukin-10 inhibits B7 and intercellular adhesion molecule-1 expression on human monocytes. Eur J Immunol 24: 1007–1009.

  48. Yoshimoto, T & Paul, WE (1994). CD4pos, NK1.1pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3. J Exp Med 179: 1285–1295.

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Correspondence to A Rosendahl.

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About this article


  • tumour therapy
  • superantigen
  • cytokines
  • perforin
  • knock out mice
  • T-cells

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