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Fig. 1 | Stem Cell Research & Therapy

Fig. 1

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

Fig. 1

Allo-specific TSCM cells are effectively prepared in vitro. a Protocol for preparation of allo-specific TSCM. The method began with setting up an allo-reactive co-culture by mixing of 2 × 106 E007 cells and 1 × 107 allogeneic PBLs (allo-PBLs) on day 0 (allogeneic activation), to generate allo-specific T cells. The presence of 5 μM TWS119 enriched TSCM in the co-culture (differentiation inhibition). The CFSE diluted cells in the co-culture bulks were sorted by FACS on day 7 (proliferation sorting). The sorted cells were cultured with IL-7 and IL-15 (25 ng/ml each) to expand the TSCM for the next 7 days (cytokine expansion). Then, the cultural bulks were used as the prepared TSCM cells for the tests in vitro and in vivo included in this study. b Gating strategy for identification of CD8+ TSCM subset in the co-culture bulks. Lymphocytes were identified based on SSC versus FSC, and proliferating cells were on a reduced fluorescence intensity of CFSE (CFSEdim). In the gated CFSEdim population, cells expressing CD3 and CD8 were selected for further CD45RA and CD62L analysis. CD8+ TSCM showed the phenotype of CFSEdim CD3+ CD8+ CD45RA+ CD62L+, TCM of CD45RA- CD62L+, TEM of CD45RA- CD62L-, TEF of CD45RA+ CD62L-, and CD95 was further detected to distinguish TSCM from TN, the latter showed the phenotype of CFSE high CD95- in the co-culture bulks, but TSCM of CFSEdim CD95+. The illustration was a representative of co-culture bulks analyzed by FCM on day 7 before sorting, the white peaks in CD95, CCR7, and CD28 plots were isotype controls. The broken arrows indicated the sequential gating strategy. c TSCM numbers increased by 100 folds in the co-culture bulks with TWS119 enrichment for 7 days. Data are represented as mean ± SD of six individual experiments. d E007-specific T cells underwent proliferation in the co-culture, the CFSEdim cells were sorted by FACS with purity above 98%. e The sorted cells were further expanded by IL-7 and IL-15 for the next 7 days, the allo-specific TSCM increased by another 150 folds. Data are represented as mean ± SD of six individual experiments

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