Abstract
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 α-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.
Highlights
CD4+FOXP3+ regulatory T cells (Tregs) are essential for maintaining immune homeostasis and peripheral tolerance [1, 2]
Tregs were isolated from peripheral blood mononuclear cells (PBMC) and stimulated for six days with plate-bound anti-CD3 and soluble anti-CD28 monoclonal Ab (mAb) in the presence of IL-2
Since it has been observed that high cell densities negatively affect transfection efficiency [38], cells were rested for 24 hours prior to transfection in 6-well plates in the presence of IL-2 but without further T cell receptor (TCR) stimulation
Summary
CD4+FOXP3+ regulatory T cells (Tregs) are essential for maintaining immune homeostasis and peripheral tolerance [1, 2]. They have numerous modes of action: e.g. secretion of immunosuppressive cytokines [IL-10 [3], TGF-b [4], IL-35 [5]], induction of Genome Editing of Human Tregs cell death by perforin and granzyme [6], IL-2 deprivation [7], CTLA-4-regulated downregulation of CD80/86 on APCs [8] and depletion of extracellular ATP and adenosine generation by CD39 [9, 10] Due to their immunosuppressive characteristics, numerous studies are currently exploring the potential of Treg cellular therapy for the induction of tolerance to autoantigens and alloantigens in the context of autoimmunity and transplant rejection [1, 2, 11,12,13]. Adoptive Treg therapy using geneticallyengineered Tregs with enhanced stability in the presence of proinflammatory IL-6 environments could be a promising treatment in the context of autoimmune and infectious diseases
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