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Blood, Vol. 110, Issue 8, 2793-2802, October 15, 2007
Co-expression of cytokine and suicide genes to enhance the activity and safety of tumor-specific cytotoxic T lymphocytes
Blood Quintarelli et al.
110: 2793
Supplemental materials for Quintarelli et al, Vol. 110, Issue 8, 2793-2802.
Files in this Data Supplement:
- Table S1. Repertoire analysis of EBV-CTL lines generated from 3 representative donors (PDF, 45.2 KB)
- Figure S1. Proliferation of IL-2 transgenic CTLs is mediated by IL-2 (PDF, 10.7 KB). -
EBV-CTLs transduced with the iC.  CD34/IL-2v vector were cultured (105 cells/well) with irradiated autologous LCL at a 4:1 responder-to-stimulator ratio with or without the addition of antibodies blocking the IL-2 cytokine or the IL-2 receptor (CD25). After 72 hours, the cells were pulsed with 1 µCi methyl-tritiated (3H)-thymidine (Amersham Biosciences) and cultured for an additional 15 hours. The cells were then harvested onto filters and dried, and cpm were measured in a  -scintillation counter (TriCarb 2500 TR; PerkinElmer, Meriden, CT). The experiments were performed in triplicate. The addition of either blocking IL-2 or CD25 antibodies inhibited the proliferation of IL-2 transgenic CTLs. Proliferation was unaffected by the addition of isotype control antibodies. Data are means, with SD error bars.
- Figure S2. IL-2 or IL-15 transgenic CD4+ EBV-CTLs expanded after stimulation with EBV-LCLs (PDF, 48.2 KB). -
(A) CD4+ EBV-CTLs were transduced with iC. CD34/IL-2v, iC. CD34/IL-15v, or CD34v vectors, maintained in culture, and stimulated once a week with autologous LCLs (E:T ratio 1:1) without exogenous cytokines. Control EBV-CTLs transduced with the CD34v vector were also maintained in culture by adding rhIL-2 (50 U/mL). CD4+ EBV-CTLs transgenic for IL-2 or IL-15 significantly proliferated in response to EBV-LCLs. (B) In contrast, control CTLs proliferated only in the presence of antigen and exogenous rhIL-2 .
- Figure S3. Cytotoxic activity of EBV-CTLs transduced with iC. ÄCD34/IL-2v, iC. ÄCD34/IL-15v, or ÄCD34v vectors (PDF, 27.6 KB). -
The figure illustrates the standard chromium-51 (51Cr) release assay of two representative CTL lines transduced with iC. CD34/IL-2v, iC. CD34/IL-15v, or CD34v vectors against autologous LCLs, allogeneic LCLs, HSB-2, and K562 cell lines at multiple E:T ratios. IL-2 or IL-15 transgenic CTLs maintained cytotoxic activity against autologous LCLs compared to control CTLs growing with exogenous rhIL-2. Data show means and SD error bars.
- Figure S4. Tumor free survival of mice treated with EBV-CTLs transduced with iC. ΔCD34/IL-2v, iC. ΔCD34/IL-15v, or iC. ΔCD34v vectors (PDF, 21.2 KB).
- Figure S5. CD34 expression allows efficient selection of transgenic CTLs (PDF, 79.7 KB). -
EBV-CTLs transduced with iC. CD34/IL-2v, iC. CD34/IL-15v, or iC. CD34v vectors were stained with CD34-PE antibody and analyzed by FACS before (upper panels) and after (lower panels) selection using CD34 microbeads. CD34 selection allowed efficient enrichment of CD34+ cells.
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