Efficient immunomagnetic selection of LV'VFas-modified T cells using anti-LNGFR⁺ microbeads.

(A) LV'VFas-modified T cells were isolated by immunomagnetic selection using either anti-LNGFR mAb and anti-IgG1–conjugated microbeads (indirect method, n = 27) or anti-LNGFR–coated microbeads (direct method, n = 26). The number of viable cells was assessed by trypan blue exclusion. The frequency of the ΔLNGFR-expressing cells in cultures prior to selection (■) or after selection (░) was examined by flow cytometry using anti-LNGFR and anti-CD3 mAbs. ▪ denotes the yield of the selection procedure. Means and standard deviations are displayed. (B-C) Representative flow cytometry analysis of ΔLNGFR expression in macaque T cells either unmodified (B) or transduced with the LV'VFas retroviral vector (C). Macaque PBLs were stimulated with anti-CD3 and anti-CD28 and exposed to LV'VFas retroviral supernatant. T cells were then stained with anti–LNGFR-PE and anti-CD3 FITC mAbs and analyzed by flow cytometry. The percentage of cells positive for both ΔLNGFR and CD3 are as indicated. These data are representative of 16 experiments with macaque (n = 7) or human (n = 9) T cells. (D-E) LV'VFas⁺ T cells were then enriched with anti-LNGFR⁺ microbeads, as described in “Materials and methods.” The enriched fraction (D) and the depleted fraction (E) of T cells were evaluated for expression of ΔLNGFR and CD3.