Commentary: “The Role of T3 Surface Molecules in the Activation of Human Cells: A Two-Stimulus Requirement for IL-2 Production Reflects Events Occurring at a Pretranslational Level”

In 1982, identifying the T cells antigen receptor was still the elusive “Holy Grail” of immunology. However, the ability to use monoclonal antibodies (mAbs) to identify and characterize molecules on lymphocytes, coupled with the then recent ability to grow long-term antigen-specific T cell clones, provided a strategy to identify the TCR by isolating clone-specific mAbs. We decided to take on this ambitious, but exciting project. We set out to grow allo-reactive human T cell clones with different specificities in order to use one clone as an immunogen and other clones with different specificities as controls. To grow human T cell clones, a source of growth factors to maintain long-term T cell clones was needed and the recently identified interleukin-2 (IL-2) was the best candidate. However, human T cells required human IL-2 for their propagation and it was going to be cumbersome to stimulate large numbers of human peripheral blood T cells for a source of the growth factor. The IL-2 gene had only recently been cloned and recombinant IL-2 was not available. However, work from Gillis and Watson described a human acute lymphoblastic leukemic T cell line called Jurkat that could be stimulated with phytohemagglutinin (PHA), a mitogenic plant lectin reactive with carbohydrates, to produce IL-2 (1). Stimulating large numbers of Jurkat cells to produce IL-2 for T cell cloning purposes offered a simple solution to our dilemma. We were able to obtain the Jurkat line from Kendall Smith and it seemed our problem was solved. PHA could stimulate the line to produce moderate amounts of bioactive IL-2 and the amount that it produced could be boosted by the addition of the tumor promoter, phorbol myristate acetate (PMA). Just as we were getting started on the project, we were dealt a crushing blow by a paper from Meuer et al., who used T cell clone specific mAbs to convincingly identify the TCR (2). They found a clone specific αβ heterodimer on a human T cell clone. The identification of the αβ heterodimer as the TCR was consistent with a tumor-specific structure that had previously been identified by Jim Allison’s group, who had speculated that the tumorspecific heterodimer might potentially represent the TCR (3). Importantly, Meuer et al. also suggested that the clone-specific heterodimer that they identified was associated with the T3 (later named CD3) complex (2), whose expression was previously linked to antigen-specific recognition (4). It had been known for a few years that mAbs against T3, like PHA, were mitogenic and could substitute for antigen in inducing T cell activation (5, 6). As we regrouped, it occurred to us that since Jurkat cells could be activated by PHA, Jurkat might express T3 and even an antigen receptor. Indeed,we found that Jurkat expressed T3 antigens. In parallel studies, we went on to make clone specific mAbs to the Jurkat αβ heterodimer, including the IgM C305 which is a Vβ8 specific mAb commonly used in Jurkat studies today (7). We considered the possibility that Jurkat might be stimulated via its TCR-T3 complex, but stimulation of the cell with only anti-T3 mAb resulted in no detectable secreted IL-2, as assessed by bioassay (note IL-2 was detected by bioassay using the IL2 dependent clone CTLL-20). However, we found that the addition of PMA, which had boosted IL-2 production induced by PHA, converted a negative result into a very robust positive one (8). The IL-2 response was highly specific for mAbs to T3 combined with PMA. Moreover, we found this result to be quite interesting: two stimuli, one putatively involving a component of the TCR complex, were required for the Jurkat line to produce IL-2. We wondered how these two stimuli operated in concert to induce IL-2 production. Fortunately, we had an experienced molecular biologist in the lab, Bob Wiskocil, with whom to collaborate to address this question. Using Northern blot and dot blot analysis, Bob showed that