Human CIK Maintain Their In Vitro and In Vivo Anti-Tumor Ability after Cryopreservation.

Adoptive cellular therapy holds promise for improving the outcome of hematopoietic cell transplantation (HCT). At present, donor lymphocyte infusion post-HCT is efficacious for only a limited number of diseases, yet can induce significant graft versus host disease (GVHD). To improve the outcome of this approach, it would be beneficial to identify populations of T cells that retain graft versus tumor (GVT) effects with reduced propensity for GVHD. We have previously described studies of murine expanded Cytokine Induced Killer (CIK) cells which are ex vivo activated and expanded T cells that express both T and NK markers. CIK cells mediate cytotoxicity both in vivo and in vitro in a non- MHC restricted NKG2D dependent manner. Human CIK cells were expanded from PBMC from 9 healthy donors, cultured with IFNg, CD3 and IL-2 and maintained in AastromRepliCell® biochambers for 21–28 days. We aimed to determine whether cryopreservation of the CIK affects viability, cytotoxicity and phenotype. Cells were cryopreserved immediately after harvest at 10x106/ml and stored in liquid nitrogen vapor phase. CIK viability was not compromised with cryopreservation and cells thawed at 1, 2, 4, 8, 10 and 28 weeks after freezing were 96% viable (range 95%–99%). Immediately upon thawing, CIK cells showed diminished cytotoxicity against the B cell lymphoma cell lines DB and SUDHL4 with 6–10% killing at the 40:1 E:T ratio. However, thawed CIK cells regained their pre-freeze cytotoxic activity against these targets within 5 hours of being placed in reactivation medium containing IL-2 at 300 IU/ml. Reactivation of the CIK cells was extended up to 48 hours but showed no further increase in cytotoxicity beyond that attained at 5 hours; nor did increasing the IL-2 concentration to 1500 IU/ml in the reactivation medium improve CIK cell activity over the same time course. Cell viability declined during reactivation, decreasing from an average 96% upon thawing to 60% over 48 hours. Thawed CIK cells placed in reactivation medium maintained their cytotoxic activity up to 14 days in vitro. The cytotoxicity of reactivated CIK cells was assessed in vivo using SCID mice inoculated IP with 1x10⁶ human ovarian cancer UCI-101 cells expressing the firefly luciferase gene. The mice were treated weekly with 2x10⁷ cryopreserved and thawed human CIK cells that were re-cultured for 5 hours before injection. Following each administration of CIK cells, there was a reduction of tumor signal. Weekly treatments resulted in a better survival outcome for the mice receiving CIK cells as compared to PBS control mice. This study demonstrates that human CIK cells may be reactivated after cryopreservation and regain their cytotoxic potential. These finding have important implications for the application of these cells as adoptive cellular therapy.