Adoptive cell transfer (ACT) and RNA-LPX treatment

Day of adoptive cell transfer (ACT)

• Irradiate tumor bearing or non-tumor bearing WT mice 4 h prior ACT with sublethal dose of 2.5-5 Gy using an XRAD320 irradiator and a dose range of 0.44 Gy/min. No irradiation is needed for NSG mice.

ACT of murine T cells (alternative I):

• Harvest murine transduced T cells from cell culture vessels into appropriate tubes.

• Wash T cells with murine T cell medium once.

• Layer carefully 5 volumes transduced T cells to 3 volumes Ficoll-Paque PREMIUM (1.084 g/mL).

• Perform density gradient centrifugation with reduced acceleration and strongly reduced break for 20 min, 800 ×g to remove debris.

• Harvest cells in lymphocyte layer and wash cells twice with PBS to remove remaining serum proteins.

• Determine cell count of total viable cells.

• Take 0.2-0.5 ×10⁶ cells for transgene (CAR or GFP) expression analysis by flow cytometry.

• Calculate the total amount of transgene (CAR or GFP) positive cells in total viable cells.

• Adjust T cell numbers to transgene positive T cells/200 µL with PBS (e.g. 1 ×10⁶ CAR expressing T cells/200 µL/ mouse).

• Inject 200 µL of cell solution carefully into the retrobulbar venous plexus of anesthetized WT mice (e.g. by 2.5 % Isofluran inhalation anesthesia) with 30 G syringe (e.g. BD Micro-Fine).

ACT of human T cells (alternative II):

• Harvest human transduced T cells directly from cell culture vessels into appropriate tubes after transduction process. Alternatively thaw frozen transduced T cells at 37°C and transfer cells into 10 volumes of human T cell medium supplemented with 2 ng/mL DNAseI and invert carefully.

• Wash cells twice with PBS to remove remaining serum proteins.

• Determine cell count of total viable cells.

• Take approximately 0.2-0.5 ×10⁶ cells for transgene (CAR surface) expression analysis by flow cytometry.

• Calculate the total amount of transgene (CAR or GFP) positive cells in total viable cells.

• Adjust T cell numbers to transgene positive T cells/200 µL with PBS (e.g. 1 ×10⁶ CAR expressing T cells/ 200 µL/ mouse).

• Inject 200 µL of cell solution carefully intravenously into the retrobulbar venous plexus of anesthetized NSG (e.g. by 2.5 % Isofluran inhalation anesthesia) with 30 G syringe.

Day of RNA-Lipoplex (RNA-LPX) treatment

• Work under RNase-free conditions.

• Bring the following stock solutions to room temperature: in vitro transcribed RNA (refer to RNA), nuclease-free water, 1.5 M sodium chloride and liposomes. F12 Liposomes contain 66.6 mol cationic DOTMA per 33.3 mol helper lipid DOPE in a ratio 2:1 – according to WO/2013/143555 and Kranz et al. 2016.

• Calculate needed volumes of RNA, sodium chloride, liposomes and water for complexation. Thereby consider a charge ratio of 1.3 to 2 of cationic DOTMA and RNA for lipoplex mediated delivery into secondary lymphoid organs. The final concentration of sodium chloride should be 150 mM in generated RNA-LPXs. 

• Mix thoroughly calculated volumes of nuclease-free water, 1.5 M sodium chloride, 20-40 µg RNA (encoding for e.g. tumor-antigen or T cell epitopes) in a nuclease-free tube. Add liposomes at last and vortex immediately for 10 seconds.

• Incubate generated RNA-LPX 10 min at room temperature.

• Inject RNA-LPX carefully intravenously into the retrobulbar venous plexus of anesthetized NSG (e.g. by 2.5 % Isofluran inhalation anesthesia) with 30 G syringe.

Solutions:

Murine T cell medium

RPMI1640-GlutaMAX supplemented with 10% (v/v) heat-inactivated FBS, 1x non-essetial amino acids, 1 mM sodium pyruvate, 10 mM HEPES, 50 μM β-Mercaptoethanol, 50 IU/mL Penicillin and 50 μg/mL Streptomycin.

Human T cell medium

X-VIVO 15 medium (Lonza) supplemented with 5% (v/v) human serum.

1.5 M NaCl solution

Dilute 1 mL sterile and RNase-free 5 M sodium chloride solution from e.g. Ambion with 2.33 mL nuclease-free water. Scale-up accordingly.

Reference

Kranz, Lena M.; Diken, Mustafa; Haas, Heinrich; Kreiter, Sebastian; Loquai, Carmen; Reuter, Kerstin C. et al. (2016): Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. In: Nature 534 (7607), S. 396–401. DOI: 10.1038/nature18300.

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