Cancer Immunol Immunother (1995) 40:125 131 9 Springer-Verlag 1995 Lianne T. M. Balemans 9 Peter A. Steerenberg 9 Bas H. A. Kremer Frank J. Koppenhagen - Pieter H. M. De Mulder 9 Willem Den Otter Specific tumor memory induced by polyethylene-glycol-modified interleukin-2 requires both helper and cytotoxic T cells Received: 29 September 1994 / Accepted 31 October 1994 Abstract Local polyethylene-glycol (PEG)-modified interleukin-2 (IL-2) immunotherapy of the guinea pig Line 10 (L10) tumor was previously demonstrated to evoke long-lasting systemic immunity after cure of the tumor and metastases. T cells, most likely the helper T cell subpopulation, were demonstrated to be crucial to the antitumor effects. Here we show that systemic immunity is induced within 7 days after the start of PEG-IL-2 therapy, indicating a rapid systemic priming of L10-specific T cells. No in vitro cytotoxic activity was detected in cell suspensions obtained from the primary tumor site, the regional lymph node or the spleen when isolated during (days 21 and 28) in- tratumoral treatment with 200000 IU PEG-IL-2. These data confirm our earlier results obtained with 60000 IU PEG-IL-2. Moreover, no cytolytic activity was observed in the chromium-release assay after in vitro restimulation with irradiated tumor cells. Specific L10 immunity can be transferred using spleen cell sus- pensions. Depletion of such a suspension of helper T cells resulted in rejection of the primary tumor in two L. T. M. Balemans1 (EN)" P. A. Steerenberg 9 B. H. A. Kremer F. J. Koppenhagen. W. Den Otter Laboratory for Pathology, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands P. H. M. De Mulder Department of Medicine, Division of Medical Oncology, University Hospital, Nijmegen, The Netherlands L. T. M. Balemans 9 W. Den Otter Department of Pathology, University Hospital, Utrecht, The Netherlands Present address: Department of Cellular Immunology 0710, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany out of four animals, but all the guinea pigs developed lymph node metastases. Removal of the cytotoxic/sup- pressor phenotype caused rejection of the dermal tu- mor in four of eight guinea pigs, but the capacity to prevent lymph node metastases was retained in all animals. Thus, depletion of either subtype reduces, but does not abrogate, the capacity to transfer L10 immun- ity with spleen cells. In conclusion, our data suggest that tumor cell killing through direct T cell cytotoxicity is not the main mode of action in PEG-IL-2-induced L10 tumor regression. PEG-IL-2 therapy induces early systemic immunity, resulting in rejection of a distant tumor, and the transfer of this immunity depends main- ly on the presence of helper T cells, although cytotoxic T cells may also play a role. Key words Locoregional tumor therapy 9 Systemic im- munity 9 T helper cells 9 Adoptive transfer Introduction Many human and animal tumors are infiltrated with leukocytes. The majority of the mononuclear infiltrate consists of T cells [16]. However, these tumor-infiltrat- ing cells (TIL) seem to be anergic, since in many cases no effective response resulting in tumor eradication is initiated. In vitro culture of isolated TIL in the presence of interleukin-2 (IL-2) rendered these cells cytotoxic, and therefore anergic TIL can apparently be activated in vitro. Tumor characteristics seem to determine which cell types are expanded predominantly. For example, melanoma mainly gives rise to CD8 § cells [-25], while CD4 § may be cultured from other tumor types [27]. In addition to the influence of the tumor on the type of TIL, culture conditions seem decisive for the cell type to be generated [26]. Culture of leukocytes in high concentrations of IL-2 mainly gives rise to natural killer/lymphokine-activated killer (NK/LAK) effectors, while a low IL-2 dose stimulates specific T cells.