**2.4 DC maturation and migration**

Human skin accommodates immature cutaneous dendritic cells, called LC and DDC. Upon allergen exposure of the skin, DCs recognize and internalize the hapten-protein complex, thereby losing their potential to capture new antigens and gain the potential to present the hapten-protein complex to naïve T cells. These changes are commonly known as DC maturation. Simultaneously, under the influence of epidermal cytokines (e.g.: IL-1α, IL-1β, TNF-α, IL-18) and fibroblast-, blood endothelial-, and lymph endothelial-chemokines (CXCL12, CCL19, CCL21), maturing LC migrate from the epidermis to the dermis and then to the draining lymph nodes where they can present the antigen to T-cells (Antonopoulos et al., 2008; Cumberbatch et al., 2003a; Cumberbatch et al., 2001; Cumberbatch et al., 2003b; Enk and Katz, 1992; MartIn-Fontecha et al., 2003; Ouwehand et al., 2008; Villablanca and Mora, 2008). DC activation upon allergen exposure also leads to functional changes in DC, such as alterations in cytokine (TNF-α IL-1β) and chemokine secretion (De Smedt et al., 2002; Toebak et al., 2006a; Toebak et al., 2006b), and to upregulation of chemokine receptors expression (CCR7, CXCR4) (Jugde et al., 2005; Rustemeyer et al., 2003; Staquet et al., 2004). Many co-stimulatory molecules and intercellular adhesion molecules (e.g.: HLA-DR, HLA-ABC, CD40, CD80, CD83, CD86, ICAM-1/CD54) are also upregulated during DC maturation (Aiba et al., 1997; Ozawa et al., 1996; Rambukkana et al., 1995). Together these changes in biomarker expression and migratory properties have been tested in *in vitro* DC based assays in order to distinguish a sensitizer from a non-sensitizer.

*DC maturation assays:* The maturation of DC upon exposure to contact allergens has been extensively reviewed by Dos Santos et al. (Dos Santos *et al.*, 2009). Both DC derived from fresh blood cell progenitors and DC-like cell lines have been tested. Currently, the secretion of IL-8 (also known as CXCL8) by allergen activated maturing DC appears to be the most promising read-out in order to distinguish a sensitizer from a non-sensitizer in both DC derived from fresh blood cell progenitors and DC-like cell lines (Fig. 3). With the exception of several false positive and negative results, also CD54, CD86 and p38 mitogen activated protein kinase (MAPK) upregulation could distinguish most sensitizers from nonsensitizers. Based on these studies, the h-CLAT (Ashikaga et al., 2010; Ashikaga et al., 2006) and MUSST *in vitro* assays (Klein and Reek, 2000), which measure surface marker expression in THP-1 and U-937 cells, respectively, have entered pre-validation. When comparing with the U-937 and THP-1 ring studies each involving 2 laboratories testing 6

*The KeratinoSens assay:* This assay measures gene-induction events at sub-cytotoxic concentrations, based on the fact that the majority of skin sensitizers induce the Nrf2-Keap1- ARE regulatory pathway (Ade et al., 2009; Andreas et al., 2011; Natsch and Emter, 2008; Vandebriel et al., 2010). The KeratinoSens reporter cell line was made by transfection of human HaCaT keratinocytes with the antioxidant response element (ARE) from the human AKR1C2 gene, which was inserted in front of a SV40 promoter and placed upstream of a luciferase gene. Induction of luciferase in this cell line was used to screen for skin sensitizers, resulting in both qualitative (sensitizer/ non-sensitizer categorization) and quantitative (concentration for significant gene induction) reproduciblity between laboratories (Emter et al., 2010). This extensive KeratoSens ring study involving 5 laboratories tested 28 substances and had an accuracy ranging between 85.7 and 92.9 % in

Human skin accommodates immature cutaneous dendritic cells, called LC and DDC. Upon allergen exposure of the skin, DCs recognize and internalize the hapten-protein complex, thereby losing their potential to capture new antigens and gain the potential to present the hapten-protein complex to naïve T cells. These changes are commonly known as DC maturation. Simultaneously, under the influence of epidermal cytokines (e.g.: IL-1α, IL-1β, TNF-α, IL-18) and fibroblast-, blood endothelial-, and lymph endothelial-chemokines (CXCL12, CCL19, CCL21), maturing LC migrate from the epidermis to the dermis and then to the draining lymph nodes where they can present the antigen to T-cells (Antonopoulos et al., 2008; Cumberbatch et al., 2003a; Cumberbatch et al., 2001; Cumberbatch et al., 2003b; Enk and Katz, 1992; MartIn-Fontecha et al., 2003; Ouwehand et al., 2008; Villablanca and Mora, 2008). DC activation upon allergen exposure also leads to functional changes in DC, such as alterations in cytokine (TNF-α IL-1β) and chemokine secretion (De Smedt et al., 2002; Toebak et al., 2006a; Toebak et al., 2006b), and to upregulation of chemokine receptors expression (CCR7, CXCR4) (Jugde et al., 2005; Rustemeyer et al., 2003; Staquet et al., 2004). Many co-stimulatory molecules and intercellular adhesion molecules (e.g.: HLA-DR, HLA-ABC, CD40, CD80, CD83, CD86, ICAM-1/CD54) are also upregulated during DC maturation (Aiba et al., 1997; Ozawa et al., 1996; Rambukkana et al., 1995). Together these changes in biomarker expression and migratory properties have been tested in *in vitro* DC

*DC maturation assays:* The maturation of DC upon exposure to contact allergens has been extensively reviewed by Dos Santos et al. (Dos Santos *et al.*, 2009). Both DC derived from fresh blood cell progenitors and DC-like cell lines have been tested. Currently, the secretion of IL-8 (also known as CXCL8) by allergen activated maturing DC appears to be the most promising read-out in order to distinguish a sensitizer from a non-sensitizer in both DC derived from fresh blood cell progenitors and DC-like cell lines (Fig. 3). With the exception of several false positive and negative results, also CD54, CD86 and p38 mitogen activated protein kinase (MAPK) upregulation could distinguish most sensitizers from nonsensitizers. Based on these studies, the h-CLAT (Ashikaga et al., 2010; Ashikaga et al., 2006) and MUSST *in vitro* assays (Klein and Reek, 2000), which measure surface marker expression in THP-1 and U-937 cells, respectively, have entered pre-validation. When comparing with the U-937 and THP-1 ring studies each involving 2 laboratories testing 6

based assays in order to distinguish a sensitizer from a non-sensitizer.

the different laboratories (Andreas et al., 2011).

**2.4 DC maturation and migration** 

sensitizers, 3 non-sensitizers and combined assessment of CD86 and CD54, the THP-1 assay showed a 100 % inter-laboratory accuracy and the U-937 assay showed a 67 % interlaboratory accuracy (Sakaguchi et al., 2006).

Fig. 3. Dendritic cell biomarker assay. DC (cell line MUTZ-3) are exposed for 24 hours to sensitizer NiSO4 or non-sensitizer SDS. The relative amount of IL-8 secreted after NiSO4 exposure as compared to its solvent control increases with increasing chemical concentrations, while no increase in IL-8 secretion is observed after SDS exposure (Ouwehand et al. 2010b).

*DC migation assay:* Migration of LC is associated with an increase in CXCR4 and a decrease in CCR1/ CCR3/ CCR5 receptors (Lin et al., 1998; Neves et al., 2008) on the maturing LC's combined with an increase in the secretion of the chemokine CXCL12 (ligand for CXCR4) in the dermis from fibroblasts (Fig. 4). The increase in CXCL12 secretion by fibroblasts is a general stress signal since it is induced by TNF-α (Ouwehand et al., 2008) and in burns (Avniel et al., 2006) and is therefore not restricted to sensitizers. The mature LC's eventually travel in a CXCR4 / CCR7 dependent manner to the lymph nodes where they may prime T cells resulting in sensitization (Villablanca and Mora, 2008). In contrast to sensitizer mediated LC migration via the CXCR4 / CXCL12 axis, non-sensitizer (irritant) mediated LC migration is mediated by maintained CCR1/ CCR3/ CCR5 (not decreased) expression and low CXCR4 expression on immature LC together with upregulated CCL5 secretion by dermal fibroblasts. Increased levels of CCL5 result in drawing CCR1/ CCR3/ CCR5 expressing LC from the epidermis into the dermis (Ouwehand et al., 2010a). The DC migration assay is based on the differential chemokine receptor expression on LC after exposure to sensitizers (CXCR4) or non-sensitizers (CCR1, CCR3 and/or CCR5) and their ability therefore to migrate preferentially to CXCL12 or CCL5 respectively (Fig. 4). This

Progress on the Development of Human *In Vitro* Assays for

the three laboratories (Rees et al. tox. In Vitro. In press).

carboxyfluorescein succinimidyl ester (CFSE).

**2.5 Induction of a T cell response** 

**2.6 Skin equivalents integrating DC** 

Assessment of the Sensitizing Potential of a Compound: Breaking Down the *In Vivo* Events 79

assay uses MUTZ-3 cell line derived LCs (MUTZ-LC). MUTZ-LC exposed to non-cytotoxic concentrations of sensitizers (including pro-haptens) in the upper chamber of a transwell migrate towards CXCL12 in the lower transwell chamber, whereas MUTZ-LC exposed to non-sensitizers in the upper chamber of a transwell migrate towards CCL5 in the lower transwell chamber (Ouwehand et al., 2010b). The read-out of the assay is the relative number of fluorescent MUTZ-LC entering the lower chamber expressed as a ratio of CXCL12 : CCL5. A ratio > 1 indicates that the chemical is a sensitizer whereas a ratio < 1 indicates that the chemical is a non-sensitizer. This assay has been tested in a ring-study, whereby three different European laboratories have shown that the assay is transferable. Intra-laboratory and inter-laboratory variation with regards to MUTZ-3 progenitor culture, differentiation to MUTZ-LC, maturation and assay implementation showed that results were consistent between the laboratories, and the dose response data was reproducible in

*T cell assays:* The induction of T cell responses to chemicals, drugs and protein allergens is the key event that decides whether sensitization will lead to manifestation of disease. Therefore, T cell assays despite their complexity can support and improve risk assessment and hazard identification strategies. Test chemicals can be added either directly to the T cells or as haptenprotein conjugates. Alternatively, chemicals can also be used to modify DC, which in turn present the hapten protein-complex to T cells. The current status on T cell assays has recently

1. T cell proliferation using radioactive labeling or dilution fluorochromes such as

2. T cell activation markers, such as CD137, CD154 (CD40L), and IFN-γ to identify

In order to increase the sensitivity of the system, these methods may be combined with a polyclonal T cell amplification step before the addition of specific allergen (Geiger et al., 2009). These assays could help in predictive risk assessment and hazard identification as they may address the induction of innate immune responses. Because of the need to work with primary cells in an autologous setting, due to differences in individual TCR repertoires, these assays are not expected to be used for high throughput screening, but may be suitable

*Full thickness skin equivalent integrating LC in the epidermis:* The five crucial steps involved in sensitization could in principle be investigated using freshly excised human skin, as this is physiologically the most relevant model to study the human skin immune defence system. However, a major drawback of using excised skin for screening purposes is the regular need for large amounts of fresh skin, which potentially creates logistical obstacles and high donor variation. Alternatively, an *in vitro* fully integrated model containing defined cell types (e.g. keratinocytes, melanocytes, fibroblasts, LC, and T cells) from allogeneic sources or cell lines, would reduce the donor variability and logistical obstacles experienced by using freshly excised human skin for in vitro assays. Several attempts have been made in order to develop

been reviewed (Martin et al., 2010). Typically, two readout systems are used:

antigen-activated T cells (Frentsch et al., 2005; Wehler et al., 2008).

a tiered approach, in order to predict the immunological effect of a chemical.


Fig. 4. A: Mechanism of Langerhans cell migration after sensitizer (hapten) or irritant exposure. Full thickness skin was exposed to NiSO4 (sensitizer), or SDS (irritant) for 16 h. Cryosections were prepared and immuno-stained for the characteristic LC marker CD1a (Magnification 200x). Upon exposure to both sensitizer and irritant a decrease in LC (red stained cells) number could be observed in the epidermis and a subsequent increase in LC number was found in the dermis, as compared to the vehicle control (Ouwehand et al., 2008; 2010a).

B: Protocol for DC migration assay. The number of MUTZ-LC migrating towards either rhCXCL12 or rhCCL5 in the transwell assay system is expressed as a CXCL12/CCL5 ratio. A CXCL12/CCL5 ratio above 1 is indicative of a sensitizer, whereas a CXCL12/CCL5 ratio below 1 is indicative of a non-sensitizer (Ouwehand et al., 2010b).

assay uses MUTZ-3 cell line derived LCs (MUTZ-LC). MUTZ-LC exposed to non-cytotoxic concentrations of sensitizers (including pro-haptens) in the upper chamber of a transwell migrate towards CXCL12 in the lower transwell chamber, whereas MUTZ-LC exposed to non-sensitizers in the upper chamber of a transwell migrate towards CCL5 in the lower transwell chamber (Ouwehand et al., 2010b). The read-out of the assay is the relative number of fluorescent MUTZ-LC entering the lower chamber expressed as a ratio of CXCL12 : CCL5. A ratio > 1 indicates that the chemical is a sensitizer whereas a ratio < 1 indicates that the chemical is a non-sensitizer. This assay has been tested in a ring-study, whereby three different European laboratories have shown that the assay is transferable. Intra-laboratory and inter-laboratory variation with regards to MUTZ-3 progenitor culture, differentiation to MUTZ-LC, maturation and assay implementation showed that results were consistent between the laboratories, and the dose response data was reproducible in the three laboratories (Rees et al. tox. In Vitro. In press).
