Cancer Immunol. Irnmunother. 7, 19-23 (1979) Studies of Lymphocyte Stimulation to Tumor-Associated Antigen ancer mmunolpgyand mmunotherapy © Springer-Verlag 1979 I. Uptake of Lymphocyte Protein and Nucleic Acid Precursors in Response to Autologous and Allogeneic Tumor-associated Antigen Solubilized from Human Tumors J. A. Roth ~ and D. L. Morton 2 Division of Oncology,Department of Surgery, 54-140 CHS, UCLA School of Medicine, University of California, Los Angeles, California90024, USA 2 SurgicalServices, Veterans Administration Hospital, Sepulveda, California91343, USA Summary. Lymphocyte stimulation to 3 M KCl ex- tracts offresh human tumors was studied by measuring the incorporation of 3H-labeled protein and nucleic acid precursors. L ymphocytes from cancer patients and nor- mal donors were incubated with autologous and allo- geneic extracts. Duplicate lymphocyte cultures were la- beled with 3H-leucine (3H-Leu), 3H-uridine (aH-Udr), or 3H-thymidine (3H-Tdr). All patients were sensitized to keyhole limpet hemocyanin (KLH) prior to testing. Of the 29 cancer patients tested, many demonstrated signif- icant uptake of 3H-Udr (90%) and 3H-Leu (62%), but not 3H-Tdr (7%) in response to soluble tumor extracts. However, most patients demonstrated uptake of all three precursors in response to KLH. Lymphocytes from cancer patients did not undergo morphologic blast cell transformation in the presence of tumor extracts. Signif- icant incorporation of 3H-Leu and 3H-Udr was seen after 24-48 h of incubation, while significant 3H-Tdr incorporation was not detected until day 5. Stimulation by KLH was significantly greater for all isotope precur- sors than stimulation in response to tumor extracts. Re- sponses of lymphocytes from normal donors to tumor extracts were noted, although they occurred less fre- quently than in lymphocytes from cancer patients. L ym- phocytes from cancer patients incorporated 3H-Leu and 3H-Udr, but only rarely incorporated 3H-Tdr in re- sponse to 3 M KCl extracts of fresh tumors. Introduction Stimulation of lymphocyte blastogenesis by autologous and allogeneic tumor cells and tumor extracts is a well- established observation (Dean et al., 1975; Gainor et al., Reprint requests should be addressed to: J. A. Roth 1975; Hsu and Cooperband, 1971; Jehn et al., 1970; Levin et al., 1975; Mavligit et al., 1973a and b, 1974; Savel, 1969; Silva et al., 1976; Stjernsward et al., 1973; Vanky et al., 1974, 1975). However, there is wide varia- tion in the response of cancer patients' lymphocytes to tumor antigen. Some investigators have been unable to demonstrate lymphocyte stimulation to tumor antigen or have only noted responses in a small fraction of sam- ples tested (Dean et al., 1975; Hsu and Cooperband, 1971; Savel, 1969; Vanky et al., 1975). Others found lymphocyte stimulation in 36%-100% of the patients tested (Gainor et al., 1975; Jehn et al., 1970; Mavligit et al., 1973a, 1974; Silva et al., 1976; Stjernsward et al,, 1973). The cause for this variability is unknown. Our study was undertaken to delineate those factors in- fluencing lymphocyte responsiveness to autologous and allogeneic tumor-associated antigens (TAA). Previous studies of lymphocyte stimulation concen- trated on measuring lymphocyte proliferation, as de- tected by 3H-Tdr incorporation. However, 3H-Tdr incor- poration represents a relatively late event following ini- tial lymphocyte-TAA contact. We have shown that stimulation of protein synthesis (SPS) as measured by 3H-Leu incorporation, occurs early in the course of lym- phocyte-TAA interaction (Roth et al., 1975a and b, 1976). Failure of lymphocytes to synthesize RNA and/or protein is a possible cause of variability in lym- phocyte DNA synthesis in response to TAA. To investi- gate this hypothesis, we simultaneously measured syn- thesis of RNA, protein, and DNA, using tritium and 14C-labeled protein and nucleic acid precursors. Our purpose was to measure lymphocyte RNA, protein, and DNA synthesis in response to autologous and allogeneic tumor cell extracts prepared from fresh surgical speci- mens. The kinetics of these responses were determined, and the responses to TAA were compared with re- sponses to a nontumor antigen (keyhole limpet hemo- cyanin, KLH). 0340-7004/79/0007/0019/$ 01.00