Breaking of inhibitory signals in immune cell-based sensitisation assays

To overcome the immune-inhibitory signals interfering between different cells types of our eLCSA, the molecular approach of genome editing was used in this study. As dendritic cells interact with both keratinocytes and T cells which are mechanistically involved in the pathogenesis of skin sensitisation (Peiser et al. 2012), the expression of immune-inhibitory molecules PD-L1 and AhR was experimentally blocked and altered on dendritic cell-similar THP-1. 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. However, in AhR+/− mice lacking exon 2 on a C57BL/6 background, a loss of AhR induced decreased expression of anti-inflammatory cytokine IL-10 in LPS-induced macrophages, thereby providing further evidence on the physiological role of AhR in regulating the inflammatory response (Zhu et al. 2018). This fits well with our data provided here that vice versa AhR knockout may enhance inflammatory response induced by specific stimuli such as skin sensitisers.

Tier 1: modulation of dendritic cell markers enhanced by AhR-k.o. and anti-PD-L1

As hypothesised, in this study AhR-k.o. combined with anti-PD-L1 strongly increased the response of THP-1 to the allergen DNCB as shown for the marker CD54 (Fig. 1). In contrast, treatment of AhR-k.o. THP-1 with or without additional PD-L1 blockade with the irritant SLS significantly reduced levels of CD54 after coculture with HaCaT keratinocytes. Expression of CD86 was also slightly reduced in these cells after SLS treatment. Since CD54 acts as adhesion molecule for the binding of T cells, reduced CD54 expression on irritant stimulated AhR-k.o. THP-1 should hamper subsequent T cell activation in our test system.

These results could be regarded as a proof-of-principle for enhancing the level of response to allergens by modulating inhibitory molecules. Thus, AhR-k.o. and anti-PD-L1 could also be proposed to enhance sensitivity in different regulatory sensitisation assays including OECD endorsed h-CLAT. In a mouse model, AhR expression in C3H/HeOuJ mice was triggered by treatment with TCDD followed by sensitisation to peanut extract (Schulz et al. 2013). While the absolute number of dendritic cells nearly doubled, expression of CD54 was not increased. This is in accordance with our results showing increase CD54 after allergen exposure if not AhR stimulation but knockout was applied.

Tier 1: AhR-knockout and PD-L1 blockade allow discrimination of sensitisers from irritant by IL-8 secretion of THP-1

The pro-inflammatory cytokine IL-8 is produced by epithelial cells like keratinocytes as well as by monocytes (Baggiolini and Clark-Lewis 1992). Our results for IL-8 secretion into culture media of tier 1 indicate that secretion of IL-8 by THP-1 is regulated by AhR and PD-L1 (Fig. 2).

Involvement of AhR in IL-8 regulation was previously shown by another working group in the myeloid cell line KBM-7 and primary monocyte-derived macrophages (Zablocki-Thomas et al. 2020). Our experiments show that blockade of PD-L1 additionally reduces IL-8 production by AhR-k.o. THP-1. Interestingly, stimulation with a strong allergen like DNCB overcame inhibition of IL-8 production as evidenced by similar concentrations in respective cocultures as compared to w.t. THP-1 cocultures. Therefore, relative induction of IL-8 was higher in knockout cocultures than in w.t. cocultures leading to a wider dynamic range for this parameter. However, MBT failed to break AhR-knockout and PD-L1 blockade mediated inhibition of IL-8. In contrast, in cocultures with PD-L1 blocked AhR-k.o. cells stimulated with SLS relative IL-8 concentrations were even lower as compared to respective control cocultures (although not statistically significant). This again may contribute to a clearer distinction between allergens and irritants in the eLCSA compared with conventional methods. However, further investigations including detection of other keratinocyte activation markers are needed to fully introduce a read-out for KE1 in the eLCSA.

Tier 2: AhR-k.o. and anti-PD-L1 enhance sensitiser response of CD3

Previous work in our working group showed the functional integration of T cells as immune effectors into a cell-based sensitisation assay (Frombach et al. 2018). Here, we propose CD3 on Jurkat cells as an additional predictive marker. As indispensable part of the functional T cell receptor (TCR) complex, CD3 is needed for signal transduction by phosphorylation of cytoplasmatic ITAM regions upon binding of TCR (Dong et al. 2019). In cocultured Jurkat cells, DNCB-treated AhR-k.o. THP-1 increased the level of CD3 in the eLCSA as compared to cocultures with untreated controls (Fig. 4). This effect was even more pronounced in cocultures with additional PD-L1 blockade. Moreover, Jurkat in coculture with MBT-treated PD-L1-blocked AhR-k.o. THP-1 also increased CD3 expression. This increase was not observed for irritant SLS in any coculture and not at all in cocultures with w.t. THP-1. In contrast, prior treatment with sensitisers of only PD-L1 blocked THP-1 led to significantly reduced CD3 expression in cocultivated Jurkat T cells. Thus, combination of PD-L1 blockade and AhR-knockout in THP-1 acted over-additive on the CD3 expression of cocultivated Jurkat.

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. activates T cells, while dimerisation with PD-L1 inhibits PD-L1/PD-1 interaction (Sugiura et al. 2019). Considering parallel down-regulation of B7 molecules CD86 and CD80 in AhR-activated moDC (Wang et al. 2014), it is reasonable to assume that CD80 is also upregulated in AhR-knockout THP-1 [as was shown for CD86 in our previous study, (Sonnenburg et al. 2023)] enhancing dimerisation with PD-L1. Thus, several activating mechanisms would be enhanced in AhR-k.o. cells with additionally blocked PD-L1. In cells only blocked for PD-L1 expression of CD80 and CD86 would be significantly lower but still elevated by sensitiser stimulus leading to activation of other inhibitory mechanisms in activated T cells such as expression of CTLA4. This receptor collocates with CD28, which is the binding partner of CD86 and CD80, and exerts regulatory action (Linsley et al. 1992). Since only around 10% of the cocultuture’s medium still contained test substance after transferral of treated THP-1 to Jurkat culture, it is safe to assume that Jurkat stimulation was by direct signal transduction from activated THP-1 rather than contact with test substances.

In conclusion, for a possible read-out both modifications (PD-L1 blockade alone or in combination with AhR-knockout) proved useful to induce a measurable sensitiser-mediated alteration of a T cell marker.

AhR-k.o. and anti-PD-L1 increase sensitiser-induced response of MIP-3α, MIP-1β, TNF-α, and IL-8

Among a panel of 12 cytokines analysed (not shown), MIP-3α, MIP-1β, TNF-α, and IL-8 were found to be affected in the supernatant of eLCSA by the two modifications of THP-1 cells. In general, in this study the identification of the source of the soluble factors, that could be keratinocytes, THP-1 or T cells, was not a primary goal. However, for potency derivation and further regulatory purposes rather the kinetics and range of release than the definition of the source might be relevant.

In comparison to cocultures with w.t. THP-1, MIP-3α concentrations were elevated if THP-1 were treated with MBT and when the AhR-k.o. system was used (Fig. 5). In contrast, previous SLS treatment of AhR-k.o. THP-1 led to a reduction of MIP-3α production down to control levels. In addition, DNCB treatment induced MIP-3α secretion. MIP-3α (CCL20) is known to be involved in pathogenesis of allergies of the upper respiratory tract (Ahrens et al. 2015; Post et al. 2015) and in steady state migration of dendritic cells in the skin (Charbonnier et al. 1999). We propose MIP-3α as an additional allergy marker for the application in skin sensitisation assays.

Regulation of a further inflammatory chemokine, MIP-1β (CCL4), was observed in response to MBT and DNCB and was enhanced in cocultures with PD-L1-blocked THP-1 (Fig. 6). MIP-1β was detected on mRNA and protein level after stimulation by DNCB or MBT by another working group (Hirota and Moro 2006). In a further study with THP-1 cells, all sensitising chemicals tested, including DNCB, induced release of MIP-1β and non-sensitizers with one exception were truly negative (Lim et al. 2008). In a sensitisation assay of MUTZ-3-derived Langerhans cells, HaCaT and primary dermal fibroblasts, MIP-1 β was used in combination with further biomarkers GM-CSF and IL-8 to improve the detection of pre- and pro-haptens (Lee et al. 2018). However, until now application of blocking for immunoregulatory molecules and its effect on MIP-1β were not reported.

TNF-α is released at the early stage of sensitisation and also in the effector phase after allergen re-exposure by sensitised epithelial barriers and dendritic cells. (Ahmad et al. 2018). For TNF-α, in our study blocking of PD-L1 increased response to MBT (Fig. 7). Only few studies investigated the effect of PD-L1 blocking on immune cells. Two recent reports found enhanced release of TNF-α in macrophages and moDCs (Bar et al. 2020; Lu et al. 2019).

Endogenous ligands for activation of AhR, FICZ or ITE, were shown to inhibit the production TNF-α in moDCs (Wang et al. 2014) which may indicate a regulatory role of AhR for TNF-α. This is supported by our previous findings where cocultures with AhR-k.o. THP-1 contained higher levels of TNF-α than respective w.t. cocultures (Sonnenburg et al. 2023). The stronger the sensitising potency of the test substance, the higher were the measured relative TNF-α concentrations in cocultures with AhR-k.o. THP-1. Hence, SLS treatment reduced TNF-α concentrations as compared to untreated controls but DNCB treatment induced higher TNF-α concentrations in the cocultures of tier 2.

A role of TNF-α in sensitisation in vivo and in vitro was previously shown (Ahmad et al. 2018) and our data indicate that modification of monocytic cells such as THP-1 helps to enhance distinction between allergen and an irritant. Thus, a combination of detecting TNF-α in sensitisation assays and applying PD-L1 blocking and AhR knockout as proposed in this study is plausible.

In addition to CD86, IL-8 was established to be the best validated marker for in vitro sensitisation assays using dendritic cells or dendritic cell-related cells such as THP-1 or MUTZ-3. For example, IL-8 is analysed in the reporter gene assay or IL-8 Luc assay as recommended by OECD TG442E (OECD 2018b). Therefore, IL-8 was used as an allergy marker with known predictivity to validate eLCSA. Detection of IL-8 was optimised in PD-L1 blocked and AhR-k.o. cocultures for DNCB and MBT, respectively, again providing much better distinction from vehicle controls and irritant SLS than it was seen in cocultures with w.t. THP-1 (Fig. 8).