Isolation of TCR-engineered lymphocytes from peripheral blood. (A) Timeline representing the outline of TCR-transfer clinical protocols and the experimental design for the isolation of cells used in gene-expression analysis. PBMCs are isolated ∼24 days before infusion by apheresis and subsequently stimulated with OKT3 in presence of IL-2 (Stim1). Retroviral transductions take place 23 and 22 days before infusion and a REP is started 2 weeks before administration of the cells. Culture media are supplemented with IL-2 during the ex vivo culture. The resulting cellular product is administered on day 0, and IL-2 is given for the first few days (1-3 days) to support engraftment. Approximately 1 month after infusion PBMCs are isolated by apheresis. Samples included in this study were prepared as follows: preinfusion samples are CD3⁺ cells isolated by negative selection, using magnetic bead sorting, from PBMCs collected at ∼day −24. Infusion samples are aliquots of the final cellular product, isolated from the infusion bag. Postinfusion samples are CD3⁺ mTCRb⁺ cells isolated from PBMC samples 1 month after infusion by FACS sorting. (B-C) FACS sorting of engineered T cells from PBMCs at 1 month postinfusion. (B) Presort staining of CD3 and mTCRb. (C) Postsort analysis showing enrichment of T cells expressing murine TCRs. Gated on lymphoid, single, PI⁻ cells.