~
Cancer Immunol Immunother(1990) 31: 243 - 249
ancer mmunolggy mmunotherapy
© Springer-Verlag 1990
Effect of recombinant human tumor necrosis factor 0{ on the induction of antibody-dependent cellular cytotoxicity in the treatment of established B16 melanoma liver nodules A. Eisenthal and J. K. Melntosh SurgeryBranch, National CancerInstitute, National Institutes of Health, Bethesda, MD 20892, USA Received 3 August 1989/Accepted10 January 1990
Summary. Incubation of C3H/Hen thymocytes in the presence of recombinant human tumor necrosis factor o~ (TNF) and interleukin-2 (IL-2) augmented the generation of antibody-dependent cellular cytotoxicity (ADCC) when compared to cells cultured in TNF or IL-2 alone. This effect was optimal when 100-200 units/ml IL-2 was used together with 103-104 units/ml TNF. TNF alone at any concentration could not mediate the induction of ADCC. Similar to the results obtained in vitro, TNF, when given alone, had no effect on the generation of ADCC in vivo. The addition, however, of TNF to IL-2, given at 10 000 and 20000 but not 40000 units, enhanced the IL-2-induced ADCC on a per-cell basis. Furthermore, TNF enhanced the total ADCC activity in various organs including the liver, spleen and thymus as a result of an increase in the number of mononuclear cells isolated from these organs. The increase in total ADCC activity was optimal when 110000220000 units ( 5 - 1 0 gg) TNF were employed together with IL-2. The combined treatment with TNF and IL-2 also increased the intracellular benzyloxycarbonyl-l-L-lysinethiobenzyl-ester esterase content in cells isolated from the livers of mice treated with these cytokines. On the basis of these results we treated mice bearing a single B 16 melanoma nodule with TNF and TNF + IL-2 given with or without anti-B 16 monoclonal antibody. We found that TNF administration augmented the anti-tumor effect of specific antiB 16 antibodies, and the addition of IL-2 further increased this anti-tumor effect.
Introduetion
lating factors [6] can induce antibody-dependent cellular cytotoxicity (ADCC) in different cell populations such as natural killer (NK)/lymphokine-activated killer (LAK) cells [20, 27, 34] monocytes/macrophages and polymorphonuclear cells [8, 10, 11]. We have previously shown that IL-2 induced in vitro asialo GM1 ÷, Fc-receptor ÷ cells, which mediate ADCC against antibody-coated fresh tumor targets [7]. This effect in vitro correlated with the ability of IL-2 both to induce ADCC in vivo (A. Eisenthal, unpublished results) and to mediate an anti-tumor effect against established B 16 liver metastases when combined with specific anti-B16 melanoma monoclonal antibody [6]. We have shown that the induction of ADCC in vitro could be further augmented by the addition of IL- 1 and tumor necrosis factor 0{ (TNF) to cells cultured in IL-2 [5]. The possible role of TNF in the induction of ADCC was also suggested by its ability to up-regulate the density of Fc receptors, which are essential for ADCC, on murine peritoneal macrophages [2]. TNF was also shown to cause the rapid necrosis of murine tumors growing as a subcutaneous as weil as a single liver tumor [1, 3, 4, 17]. In the present study we tested the rote of TNF in the generation of ADCC in vitro and in vivo and in mediating, when combined with IL-2 and anti-B-16 mAb, an anti-tumor effect against established B16 liver metastases. We found that TNF increased, both in vitro and in vivo, the induction of ADCC mediated by IL-2 and enhanced, when combined with antiB16 mAb, the anti-tumor effect on a single B16 liver nodule.
Materials and methods Animals. C57BL/6 and C3H/Hen female mice were obtained from the
Several cytokines including interleukin-2 (IL-2) [14, 19, 24, 29], interferon 0{, [3, 7 (IFNc~, -13, -7) [15, 24, 32], and macrophage and granulocyte-macrophage colony-stimu-
Offprint requests to: S. A. Rosenberg, SurgeryBranch, National Cancer Institute, Building 10, Room 2B42, Bethesda,MD 20892 USA
Small Animal Section, Veterinary Resources Branch, NIH, Bethesda, Md, and were used when they were 8 - 16 weeks old. Tumors. Weakly immunogenicB 16 melanoma tumor cells syngeneicto C57BL/6 mice were obtained from Dr. Ovejera of Frederick Cancer Center, Frederick, Md, and were serially transplanted subcutaneouslyin syngeneicmice [6]. The EL4 lymphomasyngeneicto C57BL/6mice was passaged in vivo as ascites tumors [7].
244
Antibodies. Anti-B16 melanoma mAb of the IgG2b isotype was produced and purified as previously described [6]. (B 10.AxA/J) F1 anti-B 10 (anti-H2b) was produced as described elsewhere [26] and kindly supplied by Dr. David Sachs (Immunology Branch, National Cancer Institute). A fluorescein-isothiocyanate-conjugated rat anti-(Fc receptor) monoclonal antibody (2.4G2) [31] was a gift from Dr. David Segal (National Cancer Institute).
Cytokines. Human IL-2 was kindly supplied by Hoffman-LaRoche Inc., (Nutley, NJ). Purified material had a specific activity of 1.5 x 107 units/mg, and had no detectable endotoxin by standard Limulus amoebocyte lysate assay. Recombinant human TNFc~ was kindly provided by the Cetus Corp. (Emeryville, Calif) and had a specific activity of 2.2 x 107 units/mg as determined in vitro by the murine L929 assay [25]. The endotoxin level in the purified preparation was less than 0.2 ng/2.2 x 107 units TNFc~ measured in a standard Limulus assay.
Preparation of single-cell suspensions from solid tumors. Single-cell suspensions of B 16 melanoma were prepared as previously described [6]. Briefly, a subcutaneous tumor was excised, minced with scissors into fragments and stirred in a triple-enzyme mixture of hyaluronidase, deoxyribonuclease, and collagenase (Sigma Chemical Co., St. Louis, Mo) for 3 0 - 60 min. The suspension was then collected, passed through 100-gauge nylon mesh (Nitex), washed three times with Hanks' balanced salts solution (HBSS) and resuspended at the appropriate cell concentration for injection. EL4 lymphoma cells were isolated from C57BL/6 mice by washing the peritoneal cavity with HBSS. The cells were then washed twice in HBSS and resuspended in complete medium.
Isolation of cellsfrom various organs of cytokine-primed mice. Liver and lungs were excised, minced into 1 - 3 - m m fragments and stirred in the triple-enzymë mixture described above for 2 - 3 h. The cells were then transferred to a lymphocyte M gradient and centrifuged at room temperature for 20 min; the interphase was collected and the erythrocytes lysed by resuspending the cell pellet in 10% buffered amonium chloride solution (NIH Media Section). The cells were then washed three times in HBSS and resuspended in complete medium. Spleen and thymus were excised, crushed with the hub of a syringe, passed through 100-gauge nylon mesh (Nitex), and washed three times after lysing the erythrocytes as described above.
51Cr-release assay of cytotoxicity. Fresh EL4 tumor cells were labeled with 5ICr (New England Nuclear, Boston, Mass) for 1 h at 37 °, washed three times with complete medium and recounted before dilution and incubation in triplicate with effector cell at varying ratios in 96-weil round-bottom plates (Costar), as previously described [7]. To test for ADCC 10 gl]well alloantisera at the appropriate dilution was incubated with the target cells for 2 0 - 3 0 min in 37°C before the addition of effector cells. The optimal concentrations of alloantisera to achieve maximal target lysis were determined in titration experiments (between 1 : 30 and 1 : 80 [7]. Spontaneous release of 51Cr was measured after incubation of cells with complete medium only and total release was measured after incubation of cells in 0.1 M HC1. Plates were harvested as previously described [7] and radioactivity was counted. Spontaneous release was
Cancer Immunol Immunother(1990) 31: 243 - 249
ancer mmunolggy mmunotherapy
© Springer-Verlag 1990
Effect of recombinant human tumor necrosis factor 0{ on the induction of antibody-dependent cellular cytotoxicity in the treatment of established B16 melanoma liver nodules A. Eisenthal and J. K. Melntosh SurgeryBranch, National CancerInstitute, National Institutes of Health, Bethesda, MD 20892, USA Received 3 August 1989/Accepted10 January 1990
Summary. Incubation of C3H/Hen thymocytes in the presence of recombinant human tumor necrosis factor o~ (TNF) and interleukin-2 (IL-2) augmented the generation of antibody-dependent cellular cytotoxicity (ADCC) when compared to cells cultured in TNF or IL-2 alone. This effect was optimal when 100-200 units/ml IL-2 was used together with 103-104 units/ml TNF. TNF alone at any concentration could not mediate the induction of ADCC. Similar to the results obtained in vitro, TNF, when given alone, had no effect on the generation of ADCC in vivo. The addition, however, of TNF to IL-2, given at 10 000 and 20000 but not 40000 units, enhanced the IL-2-induced ADCC on a per-cell basis. Furthermore, TNF enhanced the total ADCC activity in various organs including the liver, spleen and thymus as a result of an increase in the number of mononuclear cells isolated from these organs. The increase in total ADCC activity was optimal when 110000220000 units ( 5 - 1 0 gg) TNF were employed together with IL-2. The combined treatment with TNF and IL-2 also increased the intracellular benzyloxycarbonyl-l-L-lysinethiobenzyl-ester esterase content in cells isolated from the livers of mice treated with these cytokines. On the basis of these results we treated mice bearing a single B 16 melanoma nodule with TNF and TNF + IL-2 given with or without anti-B 16 monoclonal antibody. We found that TNF administration augmented the anti-tumor effect of specific antiB 16 antibodies, and the addition of IL-2 further increased this anti-tumor effect.
Introduetion
lating factors [6] can induce antibody-dependent cellular cytotoxicity (ADCC) in different cell populations such as natural killer (NK)/lymphokine-activated killer (LAK) cells [20, 27, 34] monocytes/macrophages and polymorphonuclear cells [8, 10, 11]. We have previously shown that IL-2 induced in vitro asialo GM1 ÷, Fc-receptor ÷ cells, which mediate ADCC against antibody-coated fresh tumor targets [7]. This effect in vitro correlated with the ability of IL-2 both to induce ADCC in vivo (A. Eisenthal, unpublished results) and to mediate an anti-tumor effect against established B 16 liver metastases when combined with specific anti-B16 melanoma monoclonal antibody [6]. We have shown that the induction of ADCC in vitro could be further augmented by the addition of IL- 1 and tumor necrosis factor 0{ (TNF) to cells cultured in IL-2 [5]. The possible role of TNF in the induction of ADCC was also suggested by its ability to up-regulate the density of Fc receptors, which are essential for ADCC, on murine peritoneal macrophages [2]. TNF was also shown to cause the rapid necrosis of murine tumors growing as a subcutaneous as weil as a single liver tumor [1, 3, 4, 17]. In the present study we tested the rote of TNF in the generation of ADCC in vitro and in vivo and in mediating, when combined with IL-2 and anti-B-16 mAb, an anti-tumor effect against established B16 liver metastases. We found that TNF increased, both in vitro and in vivo, the induction of ADCC mediated by IL-2 and enhanced, when combined with antiB16 mAb, the anti-tumor effect on a single B16 liver nodule.
Materials and methods Animals. C57BL/6 and C3H/Hen female mice were obtained from the
Several cytokines including interleukin-2 (IL-2) [14, 19, 24, 29], interferon 0{, [3, 7 (IFNc~, -13, -7) [15, 24, 32], and macrophage and granulocyte-macrophage colony-stimu-
Offprint requests to: S. A. Rosenberg, SurgeryBranch, National Cancer Institute, Building 10, Room 2B42, Bethesda,MD 20892 USA
Small Animal Section, Veterinary Resources Branch, NIH, Bethesda, Md, and were used when they were 8 - 16 weeks old. Tumors. Weakly immunogenicB 16 melanoma tumor cells syngeneicto C57BL/6 mice were obtained from Dr. Ovejera of Frederick Cancer Center, Frederick, Md, and were serially transplanted subcutaneouslyin syngeneicmice [6]. The EL4 lymphomasyngeneicto C57BL/6mice was passaged in vivo as ascites tumors [7].
244
Antibodies. Anti-B16 melanoma mAb of the IgG2b isotype was produced and purified as previously described [6]. (B 10.AxA/J) F1 anti-B 10 (anti-H2b) was produced as described elsewhere [26] and kindly supplied by Dr. David Sachs (Immunology Branch, National Cancer Institute). A fluorescein-isothiocyanate-conjugated rat anti-(Fc receptor) monoclonal antibody (2.4G2) [31] was a gift from Dr. David Segal (National Cancer Institute).
Cytokines. Human IL-2 was kindly supplied by Hoffman-LaRoche Inc., (Nutley, NJ). Purified material had a specific activity of 1.5 x 107 units/mg, and had no detectable endotoxin by standard Limulus amoebocyte lysate assay. Recombinant human TNFc~ was kindly provided by the Cetus Corp. (Emeryville, Calif) and had a specific activity of 2.2 x 107 units/mg as determined in vitro by the murine L929 assay [25]. The endotoxin level in the purified preparation was less than 0.2 ng/2.2 x 107 units TNFc~ measured in a standard Limulus assay.
Preparation of single-cell suspensions from solid tumors. Single-cell suspensions of B 16 melanoma were prepared as previously described [6]. Briefly, a subcutaneous tumor was excised, minced with scissors into fragments and stirred in a triple-enzyme mixture of hyaluronidase, deoxyribonuclease, and collagenase (Sigma Chemical Co., St. Louis, Mo) for 3 0 - 60 min. The suspension was then collected, passed through 100-gauge nylon mesh (Nitex), washed three times with Hanks' balanced salts solution (HBSS) and resuspended at the appropriate cell concentration for injection. EL4 lymphoma cells were isolated from C57BL/6 mice by washing the peritoneal cavity with HBSS. The cells were then washed twice in HBSS and resuspended in complete medium.
Isolation of cellsfrom various organs of cytokine-primed mice. Liver and lungs were excised, minced into 1 - 3 - m m fragments and stirred in the triple-enzymë mixture described above for 2 - 3 h. The cells were then transferred to a lymphocyte M gradient and centrifuged at room temperature for 20 min; the interphase was collected and the erythrocytes lysed by resuspending the cell pellet in 10% buffered amonium chloride solution (NIH Media Section). The cells were then washed three times in HBSS and resuspended in complete medium. Spleen and thymus were excised, crushed with the hub of a syringe, passed through 100-gauge nylon mesh (Nitex), and washed three times after lysing the erythrocytes as described above.
51Cr-release assay of cytotoxicity. Fresh EL4 tumor cells were labeled with 5ICr (New England Nuclear, Boston, Mass) for 1 h at 37 °, washed three times with complete medium and recounted before dilution and incubation in triplicate with effector cell at varying ratios in 96-weil round-bottom plates (Costar), as previously described [7]. To test for ADCC 10 gl]well alloantisera at the appropriate dilution was incubated with the target cells for 2 0 - 3 0 min in 37°C before the addition of effector cells. The optimal concentrations of alloantisera to achieve maximal target lysis were determined in titration experiments (between 1 : 30 and 1 : 80 [7]. Spontaneous release of 51Cr was measured after incubation of cells with complete medium only and total release was measured after incubation of cells in 0.1 M HC1. Plates were harvested as previously described [7] and radioactivity was counted. Spontaneous release was
