Polyclonality of genetically modified T-cell populations. (A,C) Immunoscope study of Vβ T lymphocytes from P8 and P2, 11 months and 45 months after gene therapy, respectively, and age-matched controls (gray-shaded outline). Vβ usage (black) is similar to normal controls (gray). (B) In vivo 5′LTR integration site analysis of P1, P2, P4, P5, P6, P7, P8, and P9 T-cell clones in 8 patients. Ten to 200 ng of DNA from sorted T cells (CD3⁺) was analyzed by using LAM-PCR at different time points (5 to 41 months) after gene therapy. The electrophoresed amplification product displays a restriction length polymorphism of integration sites, with each band indicating the presence of a different insertion locus in the assayed material. Presence of a polyclonal population of gene-modified CD3 cells was detected in each patient. Numbers denote months after transplantation. The arrow indicates the internal vector 3′LTR amplification product. A total of 1.0 μg nontransduced human leukocyte DNA was used as a negative control (-C); first lane, 100-bp ladder. After chemotherapy: integration site analysis after chemotherapy in P4 and P5. LAM-PCR was performed on 100 ng of DNA isolated from peripheral blood leukocytes 3 months after induction chemotherapy. Clonality analysis reveals the recurrence of multiple clones contributing to lymphopoiesis.