Target Cell Recognition by Cytolytic T Cells: Different Requirements for the Formation of Strong Conjugates or for Proceeding to Lysis

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Abstract
Conjugates between nonadherent peritoneal exudate lymphocytes from alloimmune mice, and neutral redlabeled or unlabeled tumor cell targets, were assayed by a procedure involving co-sedimentation of lymphocytes and target cells, vigorous resuspension, then visualization under phase-contrast or normal optics. The requirements for obtaining such conjugates, and for maintaining them in a state resistant to shear force, were compared to those for cytolysis in the usual 51Cr release procedure. The following discrepancies were noted between the requirements for formation and visualization of such conjugates, and the requirements for the recognition stage of the cytolytic assay: 1) although in both cases divalent cations were required, more Mg++ was necessary to obtain suspended conjugates than to obtain lysis; 2) although cytochalasin A added before cell contact was inhibitory in both cases, the post-recognition steps leading to lysis were not affected, whereas preformed conjugates were affected; 3) glucose was required to obtain suspended conjugates, whereas the hexose requirement in the 51Cr release assay was restricted to a post-recognition state leading to lysis. The discrepancies between the two assays were explained by the observation that under all the inhibitory conditions tested (addition of EDTA, deoxyglucose, or cytochalasin A) preformed conjugates, although not immediately dissociated, were rendered labile to shear force. The resuspension step, necessary to visualize conjugates, imposed more stringent requirements on the T cell-target cell interaction than did the lytic assay using pelleted cells. The results suggest that the establishment of a tenacious linke between a cytolytic T lymphocyte and a target cell requires secondary, nonimmunologic interactions that follow the initial specific recognition event, and that these strengthening interactions are not necessary for cytolysis. The T cell receptor itself might only have a weak affinity, or be readily detached from the membrane, so that it has more of a “reading” than a “binding” role. A practical conclusion is that the metabolic requirements for T cell “recognition” are not fixed, but are imposed by the experimental conditions under which recognition is tested.