Figure 3.

Analyses of sequential biopsies and peripheral blood samples during treatment. (a) Biopsies from each patient were obtained during colonoscopy and endoscopy procedures at the indicated time points after adoptive transfer of autologous peripheral blood lymphocytes (PBL) transduced to express a carcinoembryonic antigen (CEA) reactive T cell receptor (TCR). DNA was extracted from these specimens, and quantitative PCR was then performed to determine the amount of retroviral DNA present in biopsy specimens. The number of retroviral DNA sequences present in each sample was normalized to the amount of β-actin DNA and expressed as a percent relative to that present in the adoptively transferred PBL using the comparative C_T method. In addition, nonquantitative PCR was also performed on samples from patient 1 using specific distal 5′ (α chain V region) and 3′ (β chain C region) primers to amplify the full-length TCR αβ construct encoding the murine CEA-reactive TCR. The PCR products were then separated using a precast 2% agarose ethidium bromide gel (Invitrogen) (top panel inset). Txd, transduced; UT, untransduced; Esoph, esophagus; Stom, stomach; Duod, duodenum. (b) Biopsies from patient 3 were obtained during colonoscopy and endoscopy procedures 7 days after adoptive transfer of autologous PBL transduced to express the CEA reactive TCR. Biopsy specimens were disrupted using a BD Medimachine and/or passage through a 40 µm nylon filter to obtain single cell suspensions. These were then stained with anti-CD3, -CD8, -CD4, and –murine TCR β chain constant region antibodies and analyzed by fluorescence-activated cell sorting. The results shown were gated on CD3+ T cells, and the numbers indicate the percentages of cells in each quadrant. (c) Interferon (IFN)-γ protein levels in consecutive serum samples from each patient were determined by enzyme-linked immunosorbent assay. APC, antigen presenting cells; FITC, fluorescein isothiocyanate.