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Background  As the implantation and long-term existence of tumor-specific T cells in host are the prerequisite for adoptive immunotherapy, memory stem T cells (T SCM ) 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 T SCM  have become potential candidate for adoptive T cell therapy of cancer. Here, we reported a protocol to generate allogeneic antigen-specific CD8+ T SCM  cells from human PBLs.    Methods  To prepare allogeneic antigen-specific CD8+ T SCM , 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 T SCM  cells. The sorted lymphocytes underwent further expansion by cytokines IL-7 and IL-15 for further 7 days, making the E007-specific CD8 + T SCM  expanded in number. The stem cell and T memory cell properties of the prepared CD8+ T SCM  were observed in NOD-SCID mice.    Results  Our protocol began with 1 × 10 7  PBLs and resulted in 2 × 10 7  E007-specific CD8+ T SCM  cells in 2 weeks of preparation. The prepared T SCM  cells exhibited a proliferative history and rapid differentiation into effector cells upon the E007 re-stimulation. Importantly, the prepared T SCM  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.    Conclusions  This protocol was able to prepare allogeneic antigen-specific CD8+ T SCM  cells from human PBLs. The prepared T SCM  showed the properties of stem cells and T memory cells. This study provided a reference method for generation of antigen-specific T SCM  for T cell adoptive immunotherapy.    Electronic supplementary material  The online version of this article (10.1186/s13287-018-1080-1) contains supplementary material, which is available to authorized users.

Antigen-specific CD8+ memory stem T cells generated from human peripheral blood effectively eradicate allogeneic targets in mice