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. 2006 Jan;13(1):151-9.
doi: 10.1016/j.ymthe.2005.07.688. Epub 2005 Sep 2.

High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation

Yangbing Zhao  1 , Zhili Zheng, Cyrille J Cohen, Luca Gattinoni, Douglas C Palmer, Nicholas P Restifo, Steven A Rosenberg, Richard A Morgan
Affiliations

High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation

Yangbing Zhao et al. Mol Ther. 2006 Jan.

Abstract

The use of nonviral gene transfer methods in primary lymphocytes has been hampered by low gene transfer efficiency and high transfection-related toxicity. In this report, high gene transfection efficiency with low transfection-related toxicity was achieved by electroporation using in vitro-transcribed mRNA. Using these methods, >90% transgene expression with >80% viable cells was observed in stimulated primary human and murine T lymphocytes transfected with GFP or mCD62L. Electroporation of unstimulated human PBMCs or murine splenocytes with GFP RNA yielded 95 and 56% GFP+ cells, respectively. Electroporation of mRNA for NY-ESO-1, MART-1, and p53 antigen-specific TCRs into human T lymphocytes redirected these lymphocytes to recognize melanoma cell lines in an MHC-restricted manner. The onset of gene expression was rapid (within 30 min) and durable (up to 7 days postelectroporation) using both GFP and TCR-mediated recognition of target cells. There was no adverse effect observed on the T lymphocytes subjected to RNA electroporation evaluated by cell growth rate, annexin-V staining of apoptotic cells, BrdU incorporation, tumor antigen-specific recognition or antigen-specific TCR affinity. The results of this study indicate that mRNA electroporation provides a powerful tool to introduce genes into both human and murine primary T lymphocytes.

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Figures

FIG. 1
FIG. 1
GFP expression in electroporated human PBLs. (A) 5 × 106 stimulated human PBLs were electroporated with increasing amounts of in vitro-transcribed (IVT) GFP RNA at 0 (electroporation without RNA, filled histogram), 10 (open histogram with solid line), 20 (open histogram with dotted line), and 40 μg (open histogram with dark solid line). Viability is the percentage of cells that excluded PI in the total population. GFP expression and mean fluorescence intensity (MFI) of the viable cells were measured on the days indicated. (B) Two times dilution of GFP RNA (starting from 1 μg) as indicated was electroporated into 1 × 106 (in 50-μl volume) stimulated PBLs. 24 h postelectroporation, GFP expression was detected by flow cytometry. (C) Stimulated PBLs were electroporated and immediately cultured in prewarmed culture medium (CM). At various time points as indicated, the samples were collected and fixed in 2% paraformaldehyde at 4°C and subjected to flow cytometry analysis to detect the GFP expression.
FIG. 2
FIG. 2
Effects of electroporation on T cell growth and function. (A) 5 × 106 stimulated PBLs were electroporated with increasing amounts of IVT GFP RNA as indicated. Samples were collected at different times postelectroporation and the cell numbers were counted and growth curves plotted. (B) PBLs were electroporated as in A. Three days after electroporation, the numbers of annexin-V- and 7-AAD-positive cells (% of total cells) were determined. The results shown were plotted without gating (representative of two experiments). (C) The melanoma tumor antigen gp100-specific TCR-transduced PBL line (gp100-APB) or nontransduced PBLs as control (PBL) were electroporated with GFP (gp100-APB/GFP RNA) and compared to PBLs electroporated without RNA (gp100-APB/NO EP). Serially diluted gp100 peptide-pulsed T2 cells were cocultured with the electroporated PBLs. IFN-γ secretion was detected by ELISA (representative of two experiments).
FIG. 3
FIG. 3
Tumor antigen-specific recognition by TCR RNA-electroporated T cells. (A) PBLs were stimulated with OKT3 for 3 days. CD4 T cells were depleted and continued in culture for an additional 3 days; the cells were harvested and electroporated with mRNA of both α and β TCR for NY-ESO-1 (ESO class I) or MART-1 (MART-1 class I). After overnight culture, the cells were stained with CD8 Ab and Ab against Vβ8 (NY-ESO-1) or Vβ12 (MART-1). (B) PBLs were electroporated as described above with MART-1 TCR RNA (MART-1 TCR), or NY-ESO-1 TCR RNA (ESO TCR). After 2 h culture in CM, the cells were cocultured with peptide-pulsed T2 cells. IFN-γ secretion was detected by ELISA. (C) Tumor recognition of TCR RNA-electroporated T cells. PBLs were stimulated with OKT3 for 2 days and cultured in CM with IL-2 for an additional 18 days before electroporation of NY-ESO-1 TCR RNA or GFP RNA. Four hours postelectroporation, the electroporated PBLs or nonelectroporated PBLs were cocultured with panels of melanoma cell lines described under Materials and Methods. IFN-γ secretion was measured from the culture supernatant by ELISA after overnight coculture. (D) PBLs were stimulated with OKT3 for 3 days and electroporated with p53-specific TCR (α and β chains) or GFP RNA. TCR expression was detected by flow cytometry at different days after electroporation as indicated (representative of three experiments).
FIG. 4
FIG. 4
RNA electroporation of murine splenocyte T cells. (A) 2.5 × 106 freshly prepared C57BL/6 splenocytes were electroporated with 5 μg GFP RNA. GFP expression in T lymphocytes was detected by flow cytometry after 24 h culture. The numbers indicate the percentage of cells within each quadrant. The numbers in parentheses represent the percentage of GFP expression cells in CD3+ and CD3− lymphocytes. (B) ConA-stimulated splenocytes from C57BL/6 mice were electroporated with GFP RNA, and GFP expression was detected 24 h postelectroporation. The numbers in parentheses represent the percentage of GFP expression cells in CD4 (60%) or CD8 (78%) T cells. (C) GFP or mCD62L was electroporated into gp100 peptide-stimulated splenocytes from Pmel TCR transgenic mice (Pmel) or Pmel TCR transgenic/CD62L knockout mice (Pmel/CD62L KO). Twenty-four hours after electroporation, the cells were stained for antibody against murine CD62L and the expression of CD62L and GFP was detected by FACS (representative of three experiments).
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