Commentary: Production and Characterization of Monoclonal Antibodies to Human Interleukin 2: Strategy and Tactics

Although we had successfully created antigen-specific cytolytic T lymphocyte lines (CTLLs) using conditioned media as a source of growth-promoting factors, we had no effective method to determine the relative activities of different batches of conditioned media. Therefore, it was crucial to create a quantitative assay for the activity we termed “T cell growth factor” (TCGF). Fortunately, Torgny Fredrickson and I had already created a bioassay for the red blood cell growth factor, erythropoietin (EPO), usingmurine fetal liver cells that are enriched for EPO-responsive precursors (1, 2). Thus, patterned on the EPO bioassay, it was straightforward to construct a similar assay for TCGF using as target cells our long-term CTLL. The critical elements of the assay were (1) a low density of CTLL target cells and (2) serial twofold dilutions of conditioned media samples, thereby establishing a dose–response curve that allowed comparison of separate conditioned media (3). Of note was the observation that the curve was symmetrically sigmoid when the linear responses of tritiated thymidine incorporation were plotted vs. the logarithm of the conditioned media dilutions. We arbitrarily assigned 1.0U/mL that yielded 50% of maximal growth promotion at a dilution of 1:10. This assay represented the first ever quantitative bioassay for a lymphokine. Thus, armed with a rapid, quantitative bioassay, we next sought to generate T cell clones derived from our antigen-specific CTLL so that we could assess the potential problem of target cell heterogeneity. We tried two established cloning methods: (1) dilute cell suspensions seeded into soft agar containing TCGF-conditioned media and (2) limiting dilution (0.03–0.01 cells/well) in microtiter plates containing TCGF-conditioned media. The limiting dilution technique in suspension culture worked very well, yielding 67–100% plating efficiency. This was the first description of monoclonal antigen-specific cytolytic T cells (4). Accordingly, T cell clones permitted an unambiguous interpretation that TCGFwas acting directly on clonedT cells and not indirectly through an intermediate cell type, e.g., an APC. We submitted our manuscript to Nature, which again rejected it without review [see Ref. (5)], so that we immediately reformatted it and sent it to the J. Exp. Med., which accepted it without changes, so much for non-scientist journalists (Nature) vs. peer scientists (JEM) making informed editorial decisions (6). Other investigators rapidly adopted these cloning methods, in that they not only allowed for separation of the cell clones, but also could be used to grow large numbers of progeny, which could be used for both biological and molecular characterizations. The ability to createmonoclonal functional T cells was as transformative for T cells asmonoclonal antibodies were for B cells.