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

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.

Natural variants of α-gliadin peptides within wheat proteins with reduced toxicity in coeliac disease.

The only generally accepted treatment of coeliac disease (CD) is a lifelong gluten-free diet. Wheat gluten proteins include gliadins, low and high molecular weight glutenins. However, we have found significant structural variations within these protein families among different cultivars. To determine which structural motifs might be less toxic than others, we assessed five variants of α-gliadin immunodominant CD-toxic peptides synthesised as 16mers in CD T cell stimulation assays with gluten-sensitive T cell lines generated from duodenal biopsies from CD-affected individuals. The peptides harboured the overlapping T cell epitopes DQ 2.5-glia-α-2 and naturally occurring variants that differed in certain amino acids (AA). The results revealed that introduction of two selected AA substitutions in α-gliadin peptides reduced immunogenicity. A peptide with three AA substitutions involving two glutamic acids (E) and one glutamine residue (G) revealed the peptide was negative in 5:5 samples. We used CD small-intestinal organ culture to assess CD toxicity that revealed two peptides with selected substitution of both glutamic acid (E) and proline (P) residues abrogated evidence of CD toxicity.

High Salt Inhibits Tumor Growth by Enhancing Anti-tumor Immunity.

Excess salt intake could affect the immune system by shifting the immune cell balance toward a pro-inflammatory state. Since this shift of the immune balance is thought to be beneficial in anti-cancer immunity, we tested the impact of high salt diets on tumor growth in mice. Here we show that high salt significantly inhibited tumor growth in two independent murine tumor transplantation models. Although high salt fed tumor-bearing mice showed alterations in T cell populations, the effect seemed to be largely independent of adaptive immune cells. In contrast, depletion of myeloid-derived suppressor cells (MDSCs) significantly reverted the inhibitory effect on tumor growth. In line with this, high salt conditions almost completely blocked murine MDSC function in vitro. Importantly, similar effects were observed in human MDSCs isolated from cancer patients. Thus, high salt conditions seem to inhibit tumor growth by enabling more pronounced anti-tumor immunity through the functional modulation of MDSCs. Our findings might have critical relevance for cancer immunotherapy.