Generation of Cytotoxic T Lymphocytes in Vitro

Abstract

It is well established that the immune response to alloantigens is characterized by the formation of effector cells which specifically destroy target cells carrying the sensitizing alloantigens in the absence of antibody and/or complement (for references, see 1). These effector cells belong to the thymus-derived (T) lymphocyte series and are generally referred to as cytotoxic T lymphocytes (CTL)1With the development of a precise and reproducible cytotoxic assay system (2), it has been possible to follow the appearance of CTL during the primary and secondary response of mice to normal or tumor allografts. In certain instances, such studies have shown an earlier appearance and a higher peak of cytotoxic activity in spleens of mice injected twice with allogeneic tumor cells as compared to that observed after primary immunization, suggesting the existence of an anamnestic response in T-cell-mediated immunity (3-4).Recently, it has been found that mouse CTL can be generated in vitro using mixed leukocyte cultures (MLC). Following the work of H~yry and Defendi (5), several studies have established the usefulness of the MLC system in providing an in vitro experimental model to analyze the series of events resulting in the appearance of CTL (6-10).The present study was undertaken to investigate the effect of in vivo immunization on CTL generation in MLC. Using improved tissue culture conditions, we found that cell-mediated cytotoxic responses were significantly higher when the responding cells were obtained from immune (MLC-Imm) rather than normal spleens. At the peak of the response, MLC-Imm populations were five times more cytotoxic than MLC populations. Physical and immunological characterization of the effector cells generated in MLC-Imm gave results simi-

Characterization of a monoclonal antibody to L1210 leukaemia

“…Two to three weeks after fusion, 100 ,l of culture supernatant from wells with growing hybrids, as seen under the microscope, were collected for preliminary screening. Cytotoxic antibodies in hybridoma cultures were detected by the 51Cr-release assay, using 5lCr-labelled L1210/DTIC cells as targets (Cerottini et al, 1974). Briefly, 25 ,ul containing 106 target cells/ml were exposed to 25 1l culture supernatant for 30 min at 37°C.…”

“…As shown in 398 1( t NK activity was tested in a 4 h 51Cr-release assay with 5lCr-labelled YAC-1 as target cells. Data are presented as % specific lysis, according to the formula and methods described by Cerottini (1974).…”

Generation of Cytotoxic T Lymphocytes in Vitro

“…Primary MLC were established by mixing either 5 X 106 or 25 X 106 viable C57BL/6 responding spleen cells with an equal number of irradiated (1,000 fads) DBA/2 stimulating spleen cells (5). Secondary cultures were established by mixing 0.4 × 106 viable MLC ceils with an equal number of irradiated (1,000 rads) DBA/2 normal spleen cells in a final vol of 0.8 ml of MLC medium in 12 × 75 mm round-bottomed tubes (no.…”

“…Cytotoxic Assay.--For the cytotoxic assay, various numbers of viable MLC cells were incubated with 10 × 103 51Cr-labeled target cells in 0.4 ml DMEM-5% FBS-10 mM Hepes for 3 h at 37°C, in a shaking water bath (5). To terminate the assay, 0.6 ml cold phosphate-buffered saline was added to each tube.…”

“…Cultured cells to be used as target cells in the cytotoxic assay were labeled with 51Cr as described previously (5), and suspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS, Flow Laboratories, Inc., Rockville, Md. ), and 10 mM N-Hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) pH 7.2.…”