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Production of interferon-gamma by tumor-sensitized T cells is essential for interleukin-12–induced complete tumor eradication
Production of interferon-gamma by tumor-sensitized T cells is essential for interleukin-12–induced complete tumor eradication Natalie C. Lee, MD, Kangla Tsung, PhD, and Jeffrey A. Norton, MD, San Francisco, Calif
Background. Interferon-γ (IFN-γ) is essential for eradication of established large tumors by interleukin12 (IL-12), but the critical source of IFN-γ has not been defined. Adoptive transfer of T cells into T cell–deficient mice allows for evaluation of the role of T cells and T cell production of IFN-γ in the antitumor immune response. Methods. Wild-type C57BL/6, IL-12 receptor-β1 knockout (IL-12Rβ1 KO), IFN-γ knockout (IFN-γ KO), and IFN-γ receptor-α knockout (IFN-γRα KO) mice were immunized and used as donors for adoptive transfer. Transfer of either splenocytes or CD90+ T cells was performed into recipient T cell receptor-β knockout (TCRβ KO) and IFN-γ/TCRβ double knockout mice bearing 14-day subcutaneous MCA207 tumors. Half of the mice were treated with IL-12, and cure rates were compared. Results. Transfer of either 1/4 immunized spleen equivalent or 107 immunized T cells into both TCRβ KO and IFN-γ/TCRβ KO mice resulted in 80% to 100% cure when given with IL-12. However, transfer of 107 immunized T cells from IFN-γ KO mice into TCRβ KO mice was ineffective with or without IL-12. T cell response to IL-12, but not IFN-γ, was required for tumor regression. Conclusions. Production of IFN-γ by IL-12–responsive tumor-sensitized T cells is both necessary and sufficient for complete tumor eradication induced by IL-12. T cells are the source, but not the target, of IFN-γ during tumor regression. (Surgery 2002;132:365-8.) From the Department of Surgery, University of California, San Francisco, and the Laboratory of Biological Therapy, Veterans Administration Medical Center, San Francisco, Calif
INTERLEUKIN-12 (IL-12) is a cytokine that induces a T cell–dependent antitumor response mediated by a type 1 T-helper (Th1) cell–directed process, stimulating production of interferon-γ (IFN-γ) and leading to eradication of established subcutaneous tumors.1 IFN-γ is a homodimeric protein produced by activated T cells and natural killer (NK) cells, as well as macrophages and dendritic cells, after IL12 stimulation.2-4 IFN-γ has numerous effects, including activation of effector cells such as macrophages, neutrophils, and NK cells5; up-regulation of costimulators and major histocompatibility complex molecules on antigen-presenting cells6; and stimulation of T cell proliferation and Supported by Veterans Administration Medical Research Services Merit Review Grants (K.T. and J.N.). Presented at the 63rd Annual Meeting of the Society of University Surgeons, Honolulu, Hawaii, February 14-16, 2002. Reprint requests: Jeffrey A. Norton, MD, 4150 Clement St, San Francisco, CA 94121. 11/6/125352 doi:10.1067/msy.2002.125352
differentiation to the Th1 subset of T helper cells.7,8 IFN-γ is essential for the antitumor response, because tumor regression is lost in IFN-γ knockout mice and in normal mice with antibody depletion of IFN-γ,9-11 but the critical source of IFN-γ has not been defined. In our previously reported model of adoptive transfer, tumor-sensitized T cells were shown to be essential for complete tumor regression induced by IL-12 and cyclophosphamide plus IL12.11,12 To determine whether the requirement for IFN-γ during the antitumor immune response is due to T cell production of IFN-γ, we performed adoptive transfer of splenocytes and T cells for reconstitution of the T cell population in T cell–deficient T cell receptor-β knockout (TCRβ KO) and IFN-γ/TCRβ double knockout mice. Furthermore, we transferred IL-12 receptor-β1 knockout (IL-12Rβ1 KO) T cells and IFN-γ receptor-α knockout (IFN-γRα KO) T cells into TCRβ KO mice to determine whether T cells are the target of IL-12, IFN-γ, or both during tumor regression. SURGERY 365
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Table I. Cure rates after adoptive transfer in TCRβ KO mice (requirement for IL-12–responsive tumorsensitized T cells) Donor No adoptive transfer 1/4 Naïve spleen 1/4 Immunized spleen 1/4 Immunized IL-12Rb1 KO spleen 107 Naïve T cells 107 Immunized T cells 107 Immunized IL-12Rb1 KO T cells
Recipient
Cure rate without IL-12
Cure rate with IL-12
TCRb KO TCRb KO TCRb KO TCRb KO TCRb KO TCRb KO TCRb KO
0/5 (0%) 0/5 (0%) 0/8 (0%) 0/5 (0%) 0/5 (0%) 2/10 (20%) 0/5 (0%)
0/5 (0%) 0/5 (0%) 9/9 (100%) 0/5 (0%) 0/5 (0%) 9/10 (90%) 0/5 (0%)
Table II. Cure rates after adoptive transfer in TCRβ KO and IFN-γ/TCRβ KO mice (requirement for T cell production of, but not response to, IFN-γ) Donor 1/4 Immunized spleen* 1/4 Immunized spleen 107 Immunized T cells* 107 Immunized T cells 107 Immunized IFN-γ KO T cells 107 Immunized IFN-γRα KO T cells
Recipient TCRβ KO IFN-γ/TCRβ KO TCRβ KO IFN-γ/TCRβ KO TCRβ KO TCRβ KO
Cure rate without IL-12
Cure rate with IL-12
0/8 (0%) 0/5 (0%) 2/10 (20%) 3/10 (30%) 0/5 (0%) 0/5 (0%)
9/9 (100%) 4/5 (80%) 9/10 (90%) 9/10 (90%) 0/5 (0%) 5/5 (100%)
*Same group as in Table I, listed for comparison.
METHODS MCA207, a methylcholanthrene-induced sarcoma in the C57BL/6 strain of mice, was obtained from the Surgery Branch of the National Cancer Institute (Dr J. Yang). Tumor cells were maintained in cell culture in RPMI 1640 medium with 10% heat-inactivated fetal calf serum, 2 mmol/L glutamine, 100 µg/mL streptomycin, 100 IU/mL penicillin, and 5 105 mol/L 2-mercaptoethanol. C57BL/6 wild-type mice were obtained from the Biological Testing Branch, NCI, NIH (Frederick, Md). TCRβ KO, IFN-γ KO, IFN-γ/TCRβ KO, IFNγRα KO, and IL-12Rβ1 KO mice in the C57BL/6 background were obtained from Jackson Laboratory (Bar Harbor, Maine). All mice used were 8- to 12-week-old females. C57BL/6 wild-type, IL-12Rβ1 KO, IFN-γ KO, and IFN-γRα KO donor mice were immunized subcutaneously on the flank with 1 106 irradiated (5000 rad) MCA207 cells in 0.2 mL saline. The immunization was repeated 1 week later, and the mice were challenged after 2 weeks with 5 105 live MCA207 cells in 0.2 mL saline subcutaneously on the opposite flank. Two to 4 weeks later the mice were used as donors for adoptive transfer. Spleens were harvested from donor mice on the day of transfer and processed into single cell suspension by using the blunt end of a 10-mL
syringe, followed by osmolysis to remove red blood cells and washing twice in phosphatebuffered saline. T cells were purified from a single cell suspension of splenocytes by positive selection with CD90 (Thy1.2) magnetic antibody beads (Miltenyi Biotec, Auburn, Calif). Purified T cells were greater than 93% CD3 positive and negative for NK1.1 by fluorescence-activated cell sorter analysis (not shown), indicating that the purified cells were comprised of T cells and not NK or NKT cells. TCRβ KO mice were inoculated with 5 105 MCA207 cells in 0.2 mL saline subcutaneously. Fourteen days later, when the tumors were 7 to 10 mm in diameter, adoptive transfer was performed via intravenous tail vein injection of either 1/4 spleen equivalent (approximately 2 107 splenocytes) or 107 T cells in 0.5 mL saline. Half of the mice received recombinant murine IL-12 (Genetics Institute, Cambridge, Mass). Treatment began 2 days after adoptive transfer, with 200 ng IL-12 in 0.5 mL of 1% mouse serum in saline intraperitoneally every other day for 3 doses and weekly doses thereafter. Tumor size was assessed by using calipers, and cure was defined as complete tumor regression persisting at least 60 days. Proportions of mice cured were compared statistically with Fisher exact test.
Lee et al 367
Surgery Volume 132, Number 2 RESULTS Presence of tumor-sensitized T cells responsive to IL-12 required for tumor rejection. Unlike normal C57BL/6 mice, TCRβ KO mice do not reject tumors with IL-12 treatment alone (Table I). Adoptive transfer of either 1/4 immunized spleen equivalent (approximately 2 107 splenocytes) or 107 immunized T cells is required for tumor regression. These T cells must be both tumor-sensitized and responsive to IL-12, because naïve T cells and immunized IL-12 receptor knockout T cells fail to cause tumor regression in response to IL-12 (Table I). Production of IFN-γ by non-T cells is not essential for tumor eradication. There was no difference in cure rate between TCRβ KO and IFN-γ/TCRβ KO mice, indicating that non-T cell production of IFN-γ is not essential for tumor regression (Table II). Transfer of 1/4 immunized spleen equivalent (approximately 2 107 splenocytes) into TCRβ KO and IFN-γ/TCRβ KO mice resulted in 80% to 100% cure when treated with IL-12. Likewise, adoptive transfer of 107 tumor-specific immunized T cells into both TCRβ KO and IFN-γ/TCRβ KO mice led to a 90% cure rate (Table II). Without IL12 treatment, cure was significantly reduced to 0% to 30% in these groups (P ≤ .02). T cell production of IFN-γ is necessary for tumor regression. On the other hand, T cell production of IFN-γ is required for effective tumor eradication, because transfer of 107 immunized T cells from IFN-γ KO mice into TCRβ KO mice was ineffective with or without IL-12 (Table II) (P = .002). T cell response to IFN-γ is not required for tumor rejection. T cell response to IFN-γ was not essential for tumor eradication, because IFN-γRα KO T cells were still able to effect cure with IL-12 when transferred into a T cell–deficient host (Table II), indicating that the T cells are not the target of IFN-γ. DISCUSSION IL-12 has been shown to possess the most broad and significant antitumor activity among all known cytokines and biologic response modifiers. Because IL-12 targets both NK and T cells,13 the antitumor activities of IL-12 have been demonstrated in 2 different types of experimental tumor models. One type, not addressed here, is in nonestablished tumor models in which the antitumor activity of IL12 is mainly mediated by NK or NKT cells. This antitumor activity is broad and nonspecific, is measured by degree of inhibition of tumor development but not tumor regression and cure, and is ineffective against large established tumors.14-16 In comparison, the IL-12–induced antitumor activity
examined here is mediated by T cells in the absence of NK cells and is dependent on IFN-γ.17,18 Unlike the nonestablished tumor models, the mechanism of IL-12–induced rejection of large established tumors has not been elucidated and is being studied here. It is known that both T cells and IFN-γ are critical elements.10-12 Because IFN-γ has multiple sources and pleiotropic effects among the various immune cells, it is difficult to pinpoint the mechanism of its involvement in the antitumor immune response. Previously we have demonstrated that the dramatic tumor rejection induced by IL-12 and cyclophosphamide plus IL-12 acts via a Th1-directed immune process.1 This process requires the presence of the essential Th1 cytokine IFN-γ, because we have demonstrated that IFN-γ knockout mice lose the ability to eradicate tumors.11 Because we have shown that IL-12–mediated tumor regression is also dependent on tumor-sensitized T cells,12 we were interested in whether these tumor-sensitized T cells or other non-T cells such as NK cells are the source of IFN-γ during tumor rejection. With this adoptive transfer model in T cell–deficient mice, we were able to manipulate both the T cell and host cell populations, allowing for precise characterization of the mechanism of the antitumor immune response. In our current study, we have shown that tumor-sensitized T cells are the critical source of IFN-γ production. Transfer of immunized T cells with the ability to produce IFNγ led to tumor regression even in IFN-γ–deficient hosts, indicating that T cell production of IFN-γ is sufficient for tumor eradication. Conversely, transfer of IFN-γ KO T cells into IFN-γ–competent hosts failed to cause tumor eradication, also indicating that T cell production of IFN-γ is essential. Finally, T cells lacking IFN-γ receptor were still able to effect tumor rejection. Therefore, T cell production of IFN-γ is both necessary and sufficient for tumor regression, but T cell response to IFN-γ is not required. T cells are the source, but not the target, of IFN-γ during tumor rejection. The results suggest a mechanism for the IL-12–induced antitumor immune response, in which T cells primed by specific tumor antigens respond to IL-12 by secreting IFN-γ, which in turn activates other effector cells, possibly macrophages,19 that lead to tumor eradication.
REFERENCES 1. Tsung K, Meko JB, Peplinski GR, Tsung YL, Norton JA. IL12 induces T helper 1-directed antitumor response. J Immunol 1997;158:3359-65.
368 Lee et al
2. Gately MK, Warrier RR, Honasoge S, Carvajal DM, Faherty DA, Connaughton SE, et al. Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-gamma in vivo. Int Immunol 1994;6:157-67. 3. Gessani S, Belardelli F. IFN-gamma expression in macrophages and its possible biological significance. Cytokine Growth Factor Rev 1998;9:117-23. 4. Ohteki T, Fukao T, Suzue K, Maki C, Ito M, Nakamura M, et al. Interleukin 12-dependent interferon gamma production by CD8alpha+ lymphoid dendritic cells. J Exp Med 1999;189:1981-6. 5. Boehm U, Klamp T, Groot M, Howard JC. Cellular responses to interferon-gamma. Annu Rev Immunol 1997;15:749-95. 6. Gerrard TL, Dyer DR, Zoon KC, zur Nedden D, Siegel JP. Modulation of class I and class II histocompatibility antigens on human T cell lines by IFN-gamma. J Immunol 1988;140:3450-5. 7. Bertagnolli MM, Lin BY, Young D, Herrmann SH. IL-12 augments antigen-dependent proliferation of activated T lymphocytes. J Immunol 1992;149:3778-83. 8. Bradley LM, Dalton DK, Croft M. A direct role for IFNgamma in regulation of Th1 cell development. J Immunol 1996;157:1350-8. 9. Brunda MJ, Luistro L, Hendrzak JA, Fountoulakis M, Garotta G, Gately MK. Role of interferon-gamma in mediating the antitumor efficacy of interleukin-12. J Immunother Emphasis Tumor Immunol 1995;17:71-7. 10. Nastala CL, Edington HD, McKinney TG, Tahara H, Nalesnik MA, Brunda MJ, et al. Recombinant IL-12 administration induces tumor regression in association with IFNgamma production. J Immunol 1994;153:1697-706. 11. Tsung K, Meko JB, Tsung YL, Peplinski GR, Norton JA. Immune response against large tumors eradicated by treat-
Surgery August 2002
12.
13.
14.
15.
16.
17.
18.
19.
ment with cyclophosphamide and IL-12. J Immunol 1998;160:1369-77. Le HN, Lee NC, Tsung K, Norton JA. Pre-existing tumorsensitized T cells are essential for eradication of established tumors by IL-12 and cyclophosphamide plus IL-12. J Immunol 2001;167:6765-72. Wolf SF, Temple PA, Kobayashi M, Young D, Dicig M, Lowe L, et al. Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells. J Immunol 1991; 146:3074-81. Takeda K, Seki S, Ogasawara K, Anzai R, Hashimoto W, Sugiura K, et al. Liver NK1.1+ CD4+ alpha beta T cells activated by IL-12 as a major effector in inhibition of experimental tumor metastasis. J Immunol 1996;156:3366-73. Cui J, Shin T, Kawano T, Sato H, Kondo E, Toura I, et al. Requirement for Valpha14 NKT cells in IL-12-mediated rejection of tumors. Science 1997;278:1623-6. Shin T, Nakayama T, Akutsu Y, Motohashi S, Shibata Y, Harada M, et al. Inhibition of tumor metastasis by adoptive transfer of IL-12-activated Valpha14 NKT cells. Int J Cancer 2001;91:523-8. Iwasaki M, Yu WG, Uekusa Y, Nakajima C, Yang YF, Gao P, et al. Differential IL-12 responsiveness of T cells but not of NK cells from tumor-bearing mice in IL-12-responsive versus unresponsive tumor models. Int Immunol 2000;12:701-9. Yu WG, Yamamoto N, Takenaka H, Mu J, Tai XG, Zou JP, et al. Molecular mechanisms underlying IFN-gamma-mediated tumor growth inhibition induced during tumor immunotherapy with rIL-12. Int Immunol 1996;8:855-65. Pace JL, Russell SW, Torres BA, Johnson HM, Gray PW. Recombinant mouse gamma interferon induces the priming step in macrophage activation for tumor cell killing. J Immunol 1983;130:2011-3.
Background. Interferon-γ (IFN-γ) is essential for eradication of established large tumors by interleukin12 (IL-12), but the critical source of IFN-γ has not been defined. Adoptive transfer of T cells into T cell–deficient mice allows for evaluation of the role of T cells and T cell production of IFN-γ in the antitumor immune response. Methods. Wild-type C57BL/6, IL-12 receptor-β1 knockout (IL-12Rβ1 KO), IFN-γ knockout (IFN-γ KO), and IFN-γ receptor-α knockout (IFN-γRα KO) mice were immunized and used as donors for adoptive transfer. Transfer of either splenocytes or CD90+ T cells was performed into recipient T cell receptor-β knockout (TCRβ KO) and IFN-γ/TCRβ double knockout mice bearing 14-day subcutaneous MCA207 tumors. Half of the mice were treated with IL-12, and cure rates were compared. Results. Transfer of either 1/4 immunized spleen equivalent or 107 immunized T cells into both TCRβ KO and IFN-γ/TCRβ KO mice resulted in 80% to 100% cure when given with IL-12. However, transfer of 107 immunized T cells from IFN-γ KO mice into TCRβ KO mice was ineffective with or without IL-12. T cell response to IL-12, but not IFN-γ, was required for tumor regression. Conclusions. Production of IFN-γ by IL-12–responsive tumor-sensitized T cells is both necessary and sufficient for complete tumor eradication induced by IL-12. T cells are the source, but not the target, of IFN-γ during tumor regression. (Surgery 2002;132:365-8.) From the Department of Surgery, University of California, San Francisco, and the Laboratory of Biological Therapy, Veterans Administration Medical Center, San Francisco, Calif
INTERLEUKIN-12 (IL-12) is a cytokine that induces a T cell–dependent antitumor response mediated by a type 1 T-helper (Th1) cell–directed process, stimulating production of interferon-γ (IFN-γ) and leading to eradication of established subcutaneous tumors.1 IFN-γ is a homodimeric protein produced by activated T cells and natural killer (NK) cells, as well as macrophages and dendritic cells, after IL12 stimulation.2-4 IFN-γ has numerous effects, including activation of effector cells such as macrophages, neutrophils, and NK cells5; up-regulation of costimulators and major histocompatibility complex molecules on antigen-presenting cells6; and stimulation of T cell proliferation and Supported by Veterans Administration Medical Research Services Merit Review Grants (K.T. and J.N.). Presented at the 63rd Annual Meeting of the Society of University Surgeons, Honolulu, Hawaii, February 14-16, 2002. Reprint requests: Jeffrey A. Norton, MD, 4150 Clement St, San Francisco, CA 94121. 11/6/125352 doi:10.1067/msy.2002.125352
differentiation to the Th1 subset of T helper cells.7,8 IFN-γ is essential for the antitumor response, because tumor regression is lost in IFN-γ knockout mice and in normal mice with antibody depletion of IFN-γ,9-11 but the critical source of IFN-γ has not been defined. In our previously reported model of adoptive transfer, tumor-sensitized T cells were shown to be essential for complete tumor regression induced by IL-12 and cyclophosphamide plus IL12.11,12 To determine whether the requirement for IFN-γ during the antitumor immune response is due to T cell production of IFN-γ, we performed adoptive transfer of splenocytes and T cells for reconstitution of the T cell population in T cell–deficient T cell receptor-β knockout (TCRβ KO) and IFN-γ/TCRβ double knockout mice. Furthermore, we transferred IL-12 receptor-β1 knockout (IL-12Rβ1 KO) T cells and IFN-γ receptor-α knockout (IFN-γRα KO) T cells into TCRβ KO mice to determine whether T cells are the target of IL-12, IFN-γ, or both during tumor regression. SURGERY 365
366 Lee et al
Surgery August 2002
Table I. Cure rates after adoptive transfer in TCRβ KO mice (requirement for IL-12–responsive tumorsensitized T cells) Donor No adoptive transfer 1/4 Naïve spleen 1/4 Immunized spleen 1/4 Immunized IL-12Rb1 KO spleen 107 Naïve T cells 107 Immunized T cells 107 Immunized IL-12Rb1 KO T cells
Recipient
Cure rate without IL-12
Cure rate with IL-12
TCRb KO TCRb KO TCRb KO TCRb KO TCRb KO TCRb KO TCRb KO
0/5 (0%) 0/5 (0%) 0/8 (0%) 0/5 (0%) 0/5 (0%) 2/10 (20%) 0/5 (0%)
0/5 (0%) 0/5 (0%) 9/9 (100%) 0/5 (0%) 0/5 (0%) 9/10 (90%) 0/5 (0%)
Table II. Cure rates after adoptive transfer in TCRβ KO and IFN-γ/TCRβ KO mice (requirement for T cell production of, but not response to, IFN-γ) Donor 1/4 Immunized spleen* 1/4 Immunized spleen 107 Immunized T cells* 107 Immunized T cells 107 Immunized IFN-γ KO T cells 107 Immunized IFN-γRα KO T cells
Recipient TCRβ KO IFN-γ/TCRβ KO TCRβ KO IFN-γ/TCRβ KO TCRβ KO TCRβ KO
Cure rate without IL-12
Cure rate with IL-12
0/8 (0%) 0/5 (0%) 2/10 (20%) 3/10 (30%) 0/5 (0%) 0/5 (0%)
9/9 (100%) 4/5 (80%) 9/10 (90%) 9/10 (90%) 0/5 (0%) 5/5 (100%)
*Same group as in Table I, listed for comparison.
METHODS MCA207, a methylcholanthrene-induced sarcoma in the C57BL/6 strain of mice, was obtained from the Surgery Branch of the National Cancer Institute (Dr J. Yang). Tumor cells were maintained in cell culture in RPMI 1640 medium with 10% heat-inactivated fetal calf serum, 2 mmol/L glutamine, 100 µg/mL streptomycin, 100 IU/mL penicillin, and 5 105 mol/L 2-mercaptoethanol. C57BL/6 wild-type mice were obtained from the Biological Testing Branch, NCI, NIH (Frederick, Md). TCRβ KO, IFN-γ KO, IFN-γ/TCRβ KO, IFNγRα KO, and IL-12Rβ1 KO mice in the C57BL/6 background were obtained from Jackson Laboratory (Bar Harbor, Maine). All mice used were 8- to 12-week-old females. C57BL/6 wild-type, IL-12Rβ1 KO, IFN-γ KO, and IFN-γRα KO donor mice were immunized subcutaneously on the flank with 1 106 irradiated (5000 rad) MCA207 cells in 0.2 mL saline. The immunization was repeated 1 week later, and the mice were challenged after 2 weeks with 5 105 live MCA207 cells in 0.2 mL saline subcutaneously on the opposite flank. Two to 4 weeks later the mice were used as donors for adoptive transfer. Spleens were harvested from donor mice on the day of transfer and processed into single cell suspension by using the blunt end of a 10-mL
syringe, followed by osmolysis to remove red blood cells and washing twice in phosphatebuffered saline. T cells were purified from a single cell suspension of splenocytes by positive selection with CD90 (Thy1.2) magnetic antibody beads (Miltenyi Biotec, Auburn, Calif). Purified T cells were greater than 93% CD3 positive and negative for NK1.1 by fluorescence-activated cell sorter analysis (not shown), indicating that the purified cells were comprised of T cells and not NK or NKT cells. TCRβ KO mice were inoculated with 5 105 MCA207 cells in 0.2 mL saline subcutaneously. Fourteen days later, when the tumors were 7 to 10 mm in diameter, adoptive transfer was performed via intravenous tail vein injection of either 1/4 spleen equivalent (approximately 2 107 splenocytes) or 107 T cells in 0.5 mL saline. Half of the mice received recombinant murine IL-12 (Genetics Institute, Cambridge, Mass). Treatment began 2 days after adoptive transfer, with 200 ng IL-12 in 0.5 mL of 1% mouse serum in saline intraperitoneally every other day for 3 doses and weekly doses thereafter. Tumor size was assessed by using calipers, and cure was defined as complete tumor regression persisting at least 60 days. Proportions of mice cured were compared statistically with Fisher exact test.
Lee et al 367
Surgery Volume 132, Number 2 RESULTS Presence of tumor-sensitized T cells responsive to IL-12 required for tumor rejection. Unlike normal C57BL/6 mice, TCRβ KO mice do not reject tumors with IL-12 treatment alone (Table I). Adoptive transfer of either 1/4 immunized spleen equivalent (approximately 2 107 splenocytes) or 107 immunized T cells is required for tumor regression. These T cells must be both tumor-sensitized and responsive to IL-12, because naïve T cells and immunized IL-12 receptor knockout T cells fail to cause tumor regression in response to IL-12 (Table I). Production of IFN-γ by non-T cells is not essential for tumor eradication. There was no difference in cure rate between TCRβ KO and IFN-γ/TCRβ KO mice, indicating that non-T cell production of IFN-γ is not essential for tumor regression (Table II). Transfer of 1/4 immunized spleen equivalent (approximately 2 107 splenocytes) into TCRβ KO and IFN-γ/TCRβ KO mice resulted in 80% to 100% cure when treated with IL-12. Likewise, adoptive transfer of 107 tumor-specific immunized T cells into both TCRβ KO and IFN-γ/TCRβ KO mice led to a 90% cure rate (Table II). Without IL12 treatment, cure was significantly reduced to 0% to 30% in these groups (P ≤ .02). T cell production of IFN-γ is necessary for tumor regression. On the other hand, T cell production of IFN-γ is required for effective tumor eradication, because transfer of 107 immunized T cells from IFN-γ KO mice into TCRβ KO mice was ineffective with or without IL-12 (Table II) (P = .002). T cell response to IFN-γ is not required for tumor rejection. T cell response to IFN-γ was not essential for tumor eradication, because IFN-γRα KO T cells were still able to effect cure with IL-12 when transferred into a T cell–deficient host (Table II), indicating that the T cells are not the target of IFN-γ. DISCUSSION IL-12 has been shown to possess the most broad and significant antitumor activity among all known cytokines and biologic response modifiers. Because IL-12 targets both NK and T cells,13 the antitumor activities of IL-12 have been demonstrated in 2 different types of experimental tumor models. One type, not addressed here, is in nonestablished tumor models in which the antitumor activity of IL12 is mainly mediated by NK or NKT cells. This antitumor activity is broad and nonspecific, is measured by degree of inhibition of tumor development but not tumor regression and cure, and is ineffective against large established tumors.14-16 In comparison, the IL-12–induced antitumor activity
examined here is mediated by T cells in the absence of NK cells and is dependent on IFN-γ.17,18 Unlike the nonestablished tumor models, the mechanism of IL-12–induced rejection of large established tumors has not been elucidated and is being studied here. It is known that both T cells and IFN-γ are critical elements.10-12 Because IFN-γ has multiple sources and pleiotropic effects among the various immune cells, it is difficult to pinpoint the mechanism of its involvement in the antitumor immune response. Previously we have demonstrated that the dramatic tumor rejection induced by IL-12 and cyclophosphamide plus IL-12 acts via a Th1-directed immune process.1 This process requires the presence of the essential Th1 cytokine IFN-γ, because we have demonstrated that IFN-γ knockout mice lose the ability to eradicate tumors.11 Because we have shown that IL-12–mediated tumor regression is also dependent on tumor-sensitized T cells,12 we were interested in whether these tumor-sensitized T cells or other non-T cells such as NK cells are the source of IFN-γ during tumor rejection. With this adoptive transfer model in T cell–deficient mice, we were able to manipulate both the T cell and host cell populations, allowing for precise characterization of the mechanism of the antitumor immune response. In our current study, we have shown that tumor-sensitized T cells are the critical source of IFN-γ production. Transfer of immunized T cells with the ability to produce IFNγ led to tumor regression even in IFN-γ–deficient hosts, indicating that T cell production of IFN-γ is sufficient for tumor eradication. Conversely, transfer of IFN-γ KO T cells into IFN-γ–competent hosts failed to cause tumor eradication, also indicating that T cell production of IFN-γ is essential. Finally, T cells lacking IFN-γ receptor were still able to effect tumor rejection. Therefore, T cell production of IFN-γ is both necessary and sufficient for tumor regression, but T cell response to IFN-γ is not required. T cells are the source, but not the target, of IFN-γ during tumor rejection. The results suggest a mechanism for the IL-12–induced antitumor immune response, in which T cells primed by specific tumor antigens respond to IL-12 by secreting IFN-γ, which in turn activates other effector cells, possibly macrophages,19 that lead to tumor eradication.
REFERENCES 1. Tsung K, Meko JB, Peplinski GR, Tsung YL, Norton JA. IL12 induces T helper 1-directed antitumor response. J Immunol 1997;158:3359-65.
368 Lee et al
2. Gately MK, Warrier RR, Honasoge S, Carvajal DM, Faherty DA, Connaughton SE, et al. Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-gamma in vivo. Int Immunol 1994;6:157-67. 3. Gessani S, Belardelli F. IFN-gamma expression in macrophages and its possible biological significance. Cytokine Growth Factor Rev 1998;9:117-23. 4. Ohteki T, Fukao T, Suzue K, Maki C, Ito M, Nakamura M, et al. Interleukin 12-dependent interferon gamma production by CD8alpha+ lymphoid dendritic cells. J Exp Med 1999;189:1981-6. 5. Boehm U, Klamp T, Groot M, Howard JC. Cellular responses to interferon-gamma. Annu Rev Immunol 1997;15:749-95. 6. Gerrard TL, Dyer DR, Zoon KC, zur Nedden D, Siegel JP. Modulation of class I and class II histocompatibility antigens on human T cell lines by IFN-gamma. J Immunol 1988;140:3450-5. 7. Bertagnolli MM, Lin BY, Young D, Herrmann SH. IL-12 augments antigen-dependent proliferation of activated T lymphocytes. J Immunol 1992;149:3778-83. 8. Bradley LM, Dalton DK, Croft M. A direct role for IFNgamma in regulation of Th1 cell development. J Immunol 1996;157:1350-8. 9. Brunda MJ, Luistro L, Hendrzak JA, Fountoulakis M, Garotta G, Gately MK. Role of interferon-gamma in mediating the antitumor efficacy of interleukin-12. J Immunother Emphasis Tumor Immunol 1995;17:71-7. 10. Nastala CL, Edington HD, McKinney TG, Tahara H, Nalesnik MA, Brunda MJ, et al. Recombinant IL-12 administration induces tumor regression in association with IFNgamma production. J Immunol 1994;153:1697-706. 11. Tsung K, Meko JB, Tsung YL, Peplinski GR, Norton JA. Immune response against large tumors eradicated by treat-
Surgery August 2002
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