American Society of Hematology

Fig. 9.

$Fig. 9. Lentiviral vector-mediated gene transfer of LCR-containing vectors in human T lymphocytes. / (A) SIV-based lentiviral vectors carrying EGFP as the reporter gene are schematically shown; details on vector construction are available on request. The 2.1-kb LCR element was cloned in the antisense orientation in lentiviral vectors carrying either the parental MLV LTR (LTR) or an LTR lacking the viral enhancer (Δ) as the internal promoter. The titer of each vector, measured as the transducing unit (TU) per milliliter viral supernatant on NIH-3T3 cells, is indicated. (B) Anti-CD3–activated human T lymphocytes were transduced with the set of lentiviral vectors shown in panel A and were analyzed for CD3 and EGFP expression 72 hours later by FACS. Inclusion of the CD2 LCR 5′-upstream of the enhancer-deleted MLV LTR was associated with a relative reduction of the EGFP+ fraction expressing the marker at arbitrarily defined low levels (left box, quadrant 2) compared with cells expressing EGFP at intermediate levels (right box, quadrant 2) (panels Δ and Δ-LCR). This phenomenon was detected, but less markedly, in cells transduced by the lentiviral vectors carrying the unmodified LTR (panels LTR and LCR-LTR). Percentage values relative to each box and MFI of the entire EGFP+ cell population are indicated. (C) Effects of CD2 LCR on long-term gene expression. Anti-CD3–activated human T lymphocytes were transduced with either the parental (LTR) or the LCR-containing (Δ-LCR) vector and were sorted by FACS. EGFP expression was analyzed by FACS either immediately after sorting (day 0 panels) or after 6-week in vitro culture of the T lymphocytes in the presence of IL-2 (100 U/mL). Down-modulation of EGFP expression was detected in both samples.$

Lentiviral vector-mediated gene transfer of LCR-containing vectors in human T lymphocytes.

(A) SIV-based lentiviral vectors carrying EGFP as the reporter gene are schematically shown; details on vector construction are available on request. The 2.1-kb LCR element was cloned in the antisense orientation in lentiviral vectors carrying either the parental MLV LTR (LTR) or an LTR lacking the viral enhancer (Δ) as the internal promoter. The titer of each vector, measured as the transducing unit (TU) per milliliter viral supernatant on NIH-3T3 cells, is indicated. (B) Anti-CD3–activated human T lymphocytes were transduced with the set of lentiviral vectors shown in panel A and were analyzed for CD3 and EGFP expression 72 hours later by FACS. Inclusion of the CD2 LCR 5′-upstream of the enhancer-deleted MLV LTR was associated with a relative reduction of the EGFP⁺ fraction expressing the marker at arbitrarily defined low levels (left box, quadrant 2) compared with cells expressing EGFP at intermediate levels (right box, quadrant 2) (panels Δ and Δ-LCR). This phenomenon was detected, but less markedly, in cells transduced by the lentiviral vectors carrying the unmodified LTR (panels LTR and LCR-LTR). Percentage values relative to each box and MFI of the entire EGFP⁺ cell population are indicated. (C) Effects of CD2 LCR on long-term gene expression. Anti-CD3–activated human T lymphocytes were transduced with either the parental (LTR) or the LCR-containing (Δ-LCR) vector and were sorted by FACS. EGFP expression was analyzed by FACS either immediately after sorting (day 0 panels) or after 6-week in vitro culture of the T lymphocytes in the presence of IL-2 (100 U/mL). Down-modulation of EGFP expression was detected in both samples.

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