IntroductionCytokine-induced killer (CIK) cells are ex vivo-activated lymphocytes that can be obtained in large numbers within 3 weeks of culture from either human peripheral blood or BM, or cord blood mononuclear cells by the sequential addition of IFN-␥, anti-CD3 Ab (OKT3), and high doses of recombinant human IL-2 (rhIL-2). 1-4 CIK cells represent an heterogeneous cell population, including a large majority of CD3 ϩ CD56 ϩ cells and minor fractions of typical T cells (CD3 ϩ CD56 Ϫ ) and NK cells (CD3 Ϫ CD56 ϩ ). 5,6 CIK cells can lyse a broad array of tumor targets, including acute myeloid leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia by a non-MHC-restricted, natural killer (NK)-like mechanism. 3,6 Interestingly, in mice, CIK cells display negligible alloreactivity and cause minimal GVHD compared with allogeneic splenocytes, when infused after allogeneic BM transplantation in murine models. 7,8 CIK cells express several chemokine receptors and can migrate to the site of tumors after intravenous administration, as shown by models in vivo. [9][10][11][12][13] Because CIK cells could be produced by a simple approach and displayed antitumor activity in vitro, they appeared to be suitable candidates for cell therapy in solid and hematopoietic tumor treatment. Indeed, both autologous and allogeneic CIK cells have been used in phase 1 and 2 clinical trials for the treatment of different tumor types. In these trials, they displayed limited toxicity, whereas evidence has been obtained that they exert antitumor activity. [14][15][16][17][18][19][20] The molecular structures that account for tumor recognition and killing by CIK cells are only partially understood. Previous observations suggested a possible involvement of the NKG2D and lymphocyte function-associated antigen-1 (LFA-1) molecules [21][22][23] ; however, little is known on the role of other activating NK receptors, including DNAX accessory molecule-1 (DNAM-1), NKp30, NKp44, NKp46, as well as on the involvement of TCR/CD3 complex in the cytolytic activity of CIK cells.Our previous studies clarified that CD3 ϩ CD56 ϩ CIK cells have phenotypic characteristics typical of terminally differentiated CD8 ϩ effector memory T cells (T EMRA ; CCR7 Ϫ , CD45RA ϩ , CD62L low , CD11a ϩ , CD27 ϩ , CD28 Ϫ ). We also showed that CIK cells originate in vitro from CD56 Ϫ CD8 ϩ T-cell progenitors that strongly expand on culture in the presence of IL-2 and acquire CD56 antigen. 5 In addition, CIK cells share several characteristics with NK cells, such as the large granular lymphocyte structure, the capacity to kill the HLA class I-negative cell line K562, and the surface expression of high densities of CD56 and NKG2D. Different from NK cells, however, CIK cells express low densities of NKp30, whereas they do not express NKp44 and NKp46, and the inhibitory killer immunoglobulin-like receptors, NKG2A and CD94. 5 In the present study, we investigated in detail the receptors involved in the NK-like cytolytic activity of CIK cells and analyzed whether they ...