Established in vitro assays for regulatory testing of skin sensitisation partly suffer from only moderate sensitivity, specificity, and predictivity when testing specific groups of chemicals. This may be due to limited biomarker response in vitro in cell types that interact as crucial players of in vivo skin sensitisation pathogenesis. Here, we propose a molecular approach to overcome this limitation. In our model, we apply genome editing and blocking of immunoregulatory molecules to increase the range of biomarker modulation by sensitising chemicals. To this end, aryl hydrocarbon receptor (AhR) knockout was done by CRISPR/Cas9 technology in THP-1 cells and combined with Programmed Cell Death-Ligand (PD-L)1 blockade. AhR-knockout THP-1 in coculture with HaCaT keratinocytes showed increased CD54 expression compared to wild type cells after stimulation with 10 µmol/L dinitrochlorobenzene (DNCB) that was further enhanced by anti-PD-L1. After stimulation of AhR-knockout THP-1 with 200 µmol/L mercaptobenzothiazol or 10 µmol/L DNCB, cocultivated Jurkat T cells significantly increased expression of T cell receptor-associated CD3. No such increase was detected after prior treatment of THP-1 with 150 µmol/L of irritant sodium lauryl sulphate. Additionally, higher levels of inflammatory cytokines MIP-3α, MIP-1β, TNF-α, and IL-8 were found in supernatants of enhanced loose-fit co-culture based sensitisation assay (eLCSA) after substance treatment. Hence, eLCSA allowed to discriminate between sensitisers and non-sensitisers. Thus, inhibition of immunoinhibitory pathway signalling by combining AhR knockout and PD-L1 antibody blockage into an assay involving main acting cell types in skin sensitisation may increase sensitivity and specificity of such assays and allow potency derivation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00204-023-03506-3.