Low-level methylation of CpG residues within the LTR of proviral integrants. (A) Peripheral blood from 4 patients who received MART-1–specific TCR-transduced lymphocytes was harvested 2 months after adoptive transfer. Lymphocytes were isolated and cultured in complete media with low-dose IL-2 (60 IU/mL; MOCK) or with the addition of daily dacitabine (0.3 μmol/L; DAC). Total RNA was isolated after 3 days of ex vivo culture, reverse transcribed, and subjected to real-time PCR for the MART-1 TCR (left panel) or p21 (right panel). Each bar represents an individual patient. (B) Clustering of CpG dinucleotides within the MSCV LTR, where each oval depicts a single CpG site. Numbering is relative to the transcription start site. (C) Genomic DNA from retrovirally transduced lymphocytes of 2 donors was isolated 7 and 21 days after stimulation and bisulfite conversion was performed. A region of clustered CpG residues within the MSCV LTR were amplified by PCR and cloned. Each row represents sequencing of an individual clone, with methylated cytosine residues shown by shaded circles and nonmethylated residues by unshaded circles. (D) Bisulfite sequencing results of lymphocytes from 4 patients at early time points (1-4 weeks) and late time points (2-12 months) in vivo.