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Blood, Vol. 112, Issue 6, 2261-2271, September 15, 2008
Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells
Blood Till et al.
112: 2261
Supplemental materials for: Till et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 76.5 KB)
- Table S1. Clonality of T cells produced by limiting dilution and by bulk culture (PDF, 73.5 KB)
- Table S2. Absolute CD20+ B-cell count of treated patients (PDF, 43.2 KB)
- Table S3. Absolute CD3+ T-cell count of treated patients (PDF, 43.2 KB)
- Figure S1. Immune response to modified T cells (JPG, 353 KB)
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(A) The presence of cellular immune responses to infused T cells was assessed by using serially collected patient PBMC in chromium-51 release assays in which target cells were either the modified T cells expressing the scFvFc:ζ and NeoR gene products (solid lines) or pre-infusion autologous PBMC (interrupted lines) at E:T ratios of 20:1 (for patients A and B) or 100:1 (for patients D, F, G, and H). No evidence of a cellular immune response was detected in any patient. (B) Post-infusion native T cells retain the ability to respond to antigen, as evidenced by cytotoxic activity against allogeneic LCLs as tested in the first two patients (results for patient A shown). (C) Humoral immune response assays for reactivity to infused scFvFc:ζ gene products were performed using ELISA assays in which patient serum collected at serial timepoints after T-cell infusions was placed in wells coated with Leu-16 murine anti-human CD20 Ab, followed by a primary biotinylated Leu-16 anti-CD20 Ab, followed by secondary HRP-conjugated avidin. A standard curve generated with goat anti-mouse IgG Fab-specific Ab was used to quantify antibody concentration in test samples, and baseline patient serum was used as a negative control. No evidence of reactive Ab was detected in the serum samples. Data shown represent the mean of triplicate assays (± 1 SD).
- Figure S2. Humoral immune response assay by flow cytometry (JPG, 117 KB)
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Patient serum from 2 weeks and 3 months after T-cell infusions was incubated with Jurkat cells expressing the CD20-specific cTCR, followed by a secondary FITC-conjugated goat anti-human F(ab′)2 Ab and subsequent FACS analysis. Patient serum collected prior to T-cell infusions was used as a negative control, and Daudi B-cell lymphoma cells were used as a positive control. No evidence of humoral immune responses to the cTCR was observed.
- Figure S3. Expression of the CD20-specific scFvFc:ζ cTCR (JPG, 45.4 KB)
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T cells selected for re-infusion into patients were co-incubated with FITC-conjugated polyclonal goat anti-mouse IgG Fab-specific Ab or FITC-conjugated murine IgG1 isotype control Ab and cells were analyzed using flow cytometry. The results demonstrate a single population of cells expressing a low amount of cTCR that is expressed in a typical Gaussian distribution (open histograms), producing a unimodal peak, with the low-expression end of the spectrum overlapping the negative control distribution (filled histograms). The percentages shown represent estimates of the proportion of cTCR-positive cells as determined by measuring the cells with a fluorescence intensity greater than 97.5% of the negative control. The unimodal configuration of the histograms of the transfected cells suggests that some cells express the cTCR at a level below the detection limit of the assay; however, the existence of a subset of truly cTCR-negative cells cannot be excluded.
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