Th1 Polarization and Apoptosis-Inducing Activity of CD4 + T -Cells in Cytokine-Induced Killers Might Favor the Antitumor Cytotoxicity of Cytokine-Induced Killers In Vivo

Objective: The cytokines induced killers (CIKs) treatment is an emerging adoptive immunotherapy with greater antitumor activity than lymphocyte-activated killers (LAKs). Our previous study suggested that CD4⁺ T-cells in CIKs (CD4⁺ CIKs) might contribute to the CIK-mediated therapy in vivo. In this experiment, we studied the mechanisms that might be involved. Methods: Fresh CD4⁺ CIKs were purified and proportions of Th1- and Th2-type cells were determined by intracellular cytokine staining. Cytokine secretion and mRNA synthesis were measured by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative reverse transcriptase—polymerase chain reaction (RT-PCR), respectively. Direct cytolysis and apoptosis-inducing activity were examined by the lactate dehydrogenase (LDH) method and Annexin-V staining, respectively.Results: The Th1 polarization in CD4⁺ CIKs was identified, characterized with the enhanced frequency of Th1 subset, and a dramatic increase of the levels of interleukin-2 (IL-2) and interferon gamma (IFN-γ) in the culture supernatant. The elevation in synthesis of Th1-type cytokines could be maintained without any exogenous cytokine supplement, as implied by the results from quantitative RT-PCR. Although no tumor lysis occurred, an early stage of apoptosis was detected in Raji cocultured with CD4⁺ CIKs after 4 hours of incubation. This apoptosis-inducing activity of CD4⁺ CIKs elevated along with the incubation time and depended on the cell contact through the Fas/FasL pathway. Conclusions: CD4⁺ CIK is a subset that might favor the antitumor cytotoxicity of CIKs in vivo by producing an advantageous Th1-dominance microenvironment and inducing tumor apoptosis though the Fas/FasL pathway.