Aryl hydrocarbon receptor knockout and antibody blockade of programmed cell death ligand1 increase co-stimulatory molecules on THP-1 and specific cytokine response of human T cells

A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage

“…Results for AhR knockout experiments and establishment of selection protocol are described in more detail elsewhere (Sonnenburg et al 2023 ). In summary, CRISPR/Cas9 editing and selection protocol resulted in a significantly lower percentage of AhR-positive THP-1 cells (median of 33%) in treated samples as compared to unedited controls.…”

“…This seemingly paradox results may be explained by the concurrent action of different mechanisms. We have previously shown that AhR-knockout induces CD86 and CD54 expression on unstimulated THP-1 while PD-L1 blockade does neither induce nor alter this effect (Sonnenburg et al 2023 ). PD-L1 was shown to cis -dimerise on antigen-presenting cells with CD80, another dendritic cell marker that acts similar to CD86, i.e.…”

“…A final combination of CRISPR/Cas9 editing, dibutylphthalate treatment, and magnetic sorting achieved one third less AhR-positive THP-1 cells. Anti-PD-L1 monoclonal antibody was demonstrated to efficiently block binding sites of the receptor in THP-1 (Sonnenburg et al 2023 ). Up to now, to our knowledge no reports exist on knockout of immunoinhibitory molecules such as AhR or B7H molecules in human cells used in immunoassays.…”

“…Cell lines THP-1 and Jurkat were purchased from the German Collection of Microorganisms and Cell Cultures (DMSZ, Braunschweig, Germany), HaCaT cells were used from a stock available at the institute. Cell culture conditions for proliferation are described elsewhere (Sonnenburg et al 2023 ). All cells were kept in medium containing penicillin/streptomycin (10,000 U/mL/10 mg/mL, Merck) for the first several passages.…”

“…Knockout procedure and the developed selection protocol are described in more detail in a previous publication (Sonnenburg et al 2023 ). Briefly, THP-1 cells were seeded into 96-well round bottom plates at a density of 10,000 cells/well and incubated for 48 h in OptiMEM (Sigma) mixed with 400 ng TrueCut Cas9 protein v2, 2.4 pmol sgRNA, 0.9 µL Cas9 Plus reagent, and 0.3 µL CRISPRMAX Lipofectamine to a volume of 10 µL per well.…”