Cytotoxic Activity of Cytokine-Induced Killer Cells Correlates with Expression of SAP and SLAM.

SAP is a small protein, consisting of a single SH2 domain which is mutant in humans with X-linked lymphoproliferative disease. Patients with XLP are affected by fatal EBV infections and malignant B cell lymphomas. The increased risk for B cell lymphomas is suggested to result from impaired immunosurveillance of B cell proliferation by T cells. Here, we investigated the role of SLAM/SAP for activation of effector cells with cytotoxic activity (CIK cells), which are generated by unspecific stimulation of the T cell receptor and addition of exogenous IL-2 as described previously. The TCR activation on day +1 resulted not only in a short peak of activated cells, but activation continued and increased in combination with IL-2. We observed a striking peak of SLAM (Signaling Lymphocyte Activation Molecule) on day +6 in form of extracellular detectable CD150 as well as at the level of proteins and mRNA. Interestingly, the cytotoxic activity and the amount of SHP-2 protein showed a similar pattern as the parameters mentioned above but were shifted one day. Comparing these data for correlation, we observed a significant correlation between cytotoxic activity and CD150 expression pattern (P<0.001) and amount of SLAM protein (P<0.02) as well as between amount of SHP-2 protein and SLAM parameters (P<0.03). IL-10 secretion did not correlate with any of the parameters investigated. Secretion of Th1/Th2 cytokines was determined using a cytometric bead assay. There was no change in the amount of IL-6 and TNF-alpha. IL-4 was below the detection threshold and IL-2 could not be analyzed due to exogenous addition.

IFN-gamma levels increased during cultivation, peaked on day +3 and then remained at about 4ng/ml. IL-10 secretion started after stimulation of cells by anti-CD3, peaked on day +3 and then decreased continuously. Between day +6 and day +7, only 0.86 ± 0.02ng/ml/24hrs/3x10⁶cells were secreted. In summary, activation of peripheral blood cells with agonistic anti-CD3 antibody and exogenous IL-2, as used for generation of CIK cells, results in significant SLAM and SAP activation five days after TCR stimulation. This peak correlates with cytotoxic activity against tumor cells. SLAM expression and binding by SAP seems to be important in the activation of cytotoxic effector cells.