Fast and Efficient Genome Editing of Human FOXP3+ Regulatory T Cells

Van Zeebroeck L, Arroyo Hornero R, Côrte-Real BF, Hamad I, Meissner TB, Kleinewietfeld M.
Source: Frontiers in Immunology
Publication Date: (2021)
Issue: 12: 655122
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Gene Expression
Basic Research
Molecular Biology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
Culture Media:
4D-Nucleofector® X-Unit

Treg Stimulation
After isolation, Tregs were cultured for six days in 24-well plates at 250.000 cells/well in 1 mL X-vivo (BE02-060F, Lonza) + 5% heat-inactivated fetal bovine serum (FBS) (S1400, Biowest) with 10 µg/mL plate-bound anti-CD3 (555329, BD Biosciences), 1 µg/ mL soluble anti-CD28 (555725, BD Biosciences) and 300 U/mL IL-2 (11147528001, Sigma-Aldrich) or 1500 IU/mL Proleukin® (Novartis). For short term expansion experiments, Tregs were cultured for 24 hours in above-mentioned conditions and underwent subsequent nucleofection without prior re-plating to 6-well plates.

Treg Nucleofection
24 hours prior to nucleofection, cells were cultured in 6-well plates at a density of 250.000 cells/mL in 2 mL X-vivo + 5% FBS and 100 U/mL IL-2 (11147528001, Sigma-Aldrich) or 500 IU/mL Proleukin® (Novartis). For transfection, cells were collected, centrifuged at 90g for 10 minutes at room temperature, and 1 million Tregs were resuspended in 20 µl P3 Primary Cell 4D-Nucleofector X Kit S (V4XP-3032, Lonza). In PCR tubes, 20 pmol Cas9 nuclease (9212-0.25MG, Aldevron) was mixed with
100 pmol sgRNA (Synthego, Table S1) and incubated at 37°C for a minimum of 10 minutes before adding to the cells. For multiplexing, RNP complexes for each sgRNA were generated separately and equal amounts of each sgRNA was added. The cell/RNP mixture was transferred to Nucleofection cuvette strips (4D-Nucleofector X Kit S, Lonza) and cells were electroporated using the 4D-Nucleofector Core Unit (AAF-1002B, Lonza) and X Unit (AAF-1002X, Lonza) with program EO115. After transfection, 80 µl medium at room temperature (X-vivo + 5% FBS + 100 U/mL IL-2 or 500 IU/mL Proleukin®) was added to the wells of the cuvette strip. Cells were collected and plated in 1 mL pre-warmed medium in 24-well plates and incubated at 37° C until read-out. For re-stimulation, cells were activated 2 hours to 4 days after nucleofection with anti-CD3 plate bound mAb (1 – 10 µg/mL) and 1 µg/mL soluble anti-CD28 (555725, BD Biosciences) in the presence of IL-2.


FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor a-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.