As the implantation and long-term existence of tumor-specific T cells in host are the prerequisite for adoptive immunotherapy, memory stem T cells (TSCM) with self-renewal and differentiation capacity show the greatest potential to implant and long-term exhibit function in vivo, compared with other T cells of differentiation stages. Hence, tumor-specific TSCM have become potential candidate for adoptive T cell therapy of cancer. Here, we reported a protocol to generate allogeneic antigen-specific CD8+ TSCM cells from human PBLs. To prepare allogeneic antigen-specific CD8+ TSCM, we used an LCL named E007 of defined HLA allotyping as simulator, a co-culture of E007 and allogeneic PBLs was carried out in the presence of differentiation inhibitor TWS119 for 7 days. Sorting of proliferation cells ensured the E007-specificity of the prepared TSCM cells. The sorted lymphocytes underwent further expansion by cytokines IL-7 and IL-15 for further 7 days, making the E007-specific CD8 + TSCM expanded in number. The stem cell and T memory cell properties of the prepared CD8+ TSCM were observed in NOD-SCID mice. Our protocol began with 1 × 107 PBLs and resulted in 2 × 107 E007-specific CD8+ TSCM cells in 2 weeks of preparation. The prepared TSCM cells exhibited a proliferative history and rapid differentiation into effector cells upon the E007 re-stimulation. Importantly, the prepared TSCM cells were able to exist long and reconstitute other T cell subsets in vivo, eradicating the E007 cells effectively after transferred into the LCL burden mice. This protocol was able to prepare allogeneic antigen-specific CD8+ TSCM cells from human PBLs. The prepared TSCM showed the properties of stem cells and T memory cells. This study provided a reference method for generation of antigen-specific TSCM for T cell adoptive immunotherapy.

We measured the killing activities of cytokine-induced killer cells (CIK) cells after hypothermal storage of such cells to identify an effective freezing solution. Fourteen days after induction of CIK cells from cultured umbilical cord blood, cells were frozen and stored in a new storage solution (10% [v/v] dimethyl sulphoxide [DMSO]; 10% [w/v] Dextran; and lymphocytes, but without serum) and traditional cell freezing solution (15% [v/v] DMSO; 10% [w/v] human albumin; and lymphocytes, also without serum). Cells were thawed and cultured after 180 days, and cell morphology and proliferation were investigated. Flow cytometric measurement (FCM) was used to define the immune phenotype. The lactate dehydrogenas ( LDH) method was employed to test the ability of stored and freshly induced CIK cells to kill The myelogenous leukemia cell line K562. The morphologies and immune phenotypes of freshly induced and stored (in either solution) cells were similar. The proliferative activity and killing capacity of fresh CIK cells and those stored in the new solution were similar, and higher than those of cells stored in the traditional solution.