Surgical Oncology 1993; 2: 241-249 Production of immunosuppressive factors by a cultured tumour cell line and their effect on lymphocyte proliferation and cell cycle response I. BROTHERICK, B. K. SHENTON, J. A. KIRBY, K. M. RIGG, J. M. PALMER*, S. J. YEAMAN* AND T. W. J. LENNARD Depertment of Surgery, & *Department of Biochemistry and Genetics, The Medical School, University of Newasde UpOnTytw, Newcastle upon Tyne, NE2 4HH, UK lmmunosuppression observed in patients with malignancy may be due to factors released by tumour cells. Medium conditioned by COLO 205 cells was found to inhibit mitogen-stimulated lymphocyte proliferation. Examination of CD25 and Class II MHC induction on PBMC incubated in complete, or COLO205 conditioned, medium was not significant. The prevalence of lymphocytes in the S-phase of the cell cycle was enhanced after mitogenic stimulation and addition of COLO 205 conditioned medium. This was balanced by a concomitant fall in proportion of cells in the G,,/G, phases of the cell cycle. The immunosuppressive properties of COLO 205 conditioned medium was abrogated by heating to 60°C for 30 min and by digestion with trypsin. Fractionation of the medium by gel filtration yielded two immunosuppressive fractions with relative molecular weights of 66,000 and below 20,666. It was concluded that cultured COLO 205 cells produce immunosuppressive protein/peptide factors which block cell proliferation during DNA synthesis. These factors fail to prevent upregulation of membrane-associated markers of cell activation. Surgical Oncology 1993; 2: 241-246. Keywords: cell cycle, immunosuppression, lymphocyte, proliferation, tumour. INTRODUCTION It has long been recognized that patients with malig- nant tumours have depressed immune responses [I, 21. Furthermore it has been shown, in vitro, that similar immunosuppressive phenomena can be produced by ceil-free extracts of solid tumours [3], by solid tumour products [4], by sera from cancer patients [5] and by tumour cell culture supernatants (61. Indeed immunosuppressive activity has also been demonstrated by incubating lymphocytes with the suspected inhibitory agent and assessing their response to mitogens [7, 81. Further evidence for immunosuppresive activity has been obtained from conventional assays of both NK [9] and LAK cell function [lo, 111. Correspondence: Dr 1. Brotherick, Deparhent of Surgery, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK. Isolation of tumour-associated immunosuppres- sive factors has to some extent enabled charac- terization of their biochemical properties. Some proposed inhibitory agents include serum-derived suppressive peptides with relative molecular weight of between 5000 and 7000 [5], ‘unknown factors’ from culture medium [lo, 111 and TGFjI [12-141. Further possibilities include plasma fibronectin [15], products of ~53 positive tumour cell lines [16] and, the suppressive action of Mycoplasma spp. or their products within culture experiments [I 71. Incubation of lymphocytes with an activating agent, such as the mitogen PHA, converts the cells from a non-dividing quiescent state referred to as G,, to one of active proliferation [18]. On entering the G,-phase of the cell cycle, the proteins essential for DNA synthesis are produced. Mitogenic stimula- tion also induces expression of CD25, the IL-2 receptor [ 191, Class II MHC molecules and transferrin receptors [20, 211. In order for cell division to