**2.6 Skin equivalents integrating DC**

*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

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

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*of relative potency: status of validation.* Contact Dermatitis 57:70-75.

*Langerhans cells and dermal dendritic cells*. Tissue Eng 13:2667-2679.

*contact hypersensitivity*. J Cell Biol 169:569-576.

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(2005). *Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate* 

*18 production in a human keratinocyte cell line to discriminate contact sensitizers from irritants and low molecular weight respiratory allergens.* Toxicol In Vitro 23:789-796.

such an *in vitro* skin equivalent (SE) model. Currently, only two groups have succeeded in introducing LC into an EE (Facy et al., 2004; Facy et al., 2005; Regnier et al., 1997; Schaerli et al., 2005), while just one group was able to introduce dendritic cells (DC) into a dermal equivalent (Guironnet et al., 2001). Neither of these groups reconstructed a full-thickness SE, rendering these models unfit to study all processes involved in the development of a hypersensitivity reaction. Three groups have so far succeeded in reconstructing a full thickness SE containing keratinocytes, fibroblasts, and epidermal LC containing Birbeck granules (Bechetoille et al., 2007; Dezutter-Dambuyant et al., 2006; Ouwehand et al., 2011). The model described by Ouwehand et al. is the only one to use cell line derived LC (MUTZ-3 derived LC) which overcomes the logistical problems of using fresh donor derived LC progenitors from peripheral blood or cord blood (Ouwehand et al., 2011). Importantly this model was shown to be functional and suited to study the first four steps in the development of an allergic response since topical exposure to sensitizers resulted in MUTZ-LC maturation and migration into the dermis (Ouwehand et al., 2011). However, no models until date contain T cells, rendering them unsuitable to study the induction of T cell responses to chemicals. In the future these models may progress to co-culture with T cells in the lower compartment of the transwell system. A shortcoming of the models that require fresh blood-derived precursor cells as their source of LC would be the logistics involved in constructing an autologous T cell containing co-culture variant (Dezutter-Dambuyant et al., 2006). The model of Ouwehand contains a human LC cell line (MUTZ-3 derived LC), established from the peripheral blood of a HLA-A2 positive patient with acute myelomonocytic leukemia (Ouwehand et al., 2011). These MUTZ-LC were able to prime specific and fully functional CTL from HLA-A2-matched healthy donors (Santegoets et al., 2008), which reduce logistical problems created by the need for blood of healthy volunteers. For all of these integrated skin models, further research is required to determine whether they will contribute to the battery of assays being developed for risk assessment. In any case, these models are excellent to study DC biology *in vitro*.

#### **3. Conclusions**

As the induction of contact hypersensitivity is the result of a series of parallel and sequential processes, it is thought that a battery of assays based on the *in vivo* events leading up to sensitization will be required to identify potential sensitizers *in vitro*. Such a battery of assays would be expected to reduce the risk of scoring false negatives and positives. There is a general consensus that a combination of several functionally distinct human-based *in vitro* assays will be successful and may eventually even surpass the accuracy of animal methods such as LLNA. Several mechanistically relevant and promising test-strategies are being developed, which need to be further refined and tested before entering formal validation according to the guidelines of the European Centre for the Validation of Alternative Methods to animal testing (ECVAM). Although recent developments are looking very promising, scientists will find it difficult to meet the 2013 European deadline for replacement of animal based methods with *in vitro* alternatives.

#### **4. References**

Ade, N.; Leon, F.; Pallardy, M.; Peiffer, J.L.; Kerdine-Romer, S.; Tissier, M.H.; Bonnet, P.A.; Fabre, I.; Ourlin, J.C. (2009). *HMOX1 and NQO1 genes are upregulated in response to*  *contact sensitizers in dendritic cells and THP-1 cell line: role of the Keap1/Nrf2 pathway.* Toxicol Sci 107:451-460.

Aiba, S.(2007). *Dendritic cells: importance in allergy.* Allergol Int 56:201-208.

80 Contact Dermatitis

such an *in vitro* skin equivalent (SE) model. Currently, only two groups have succeeded in introducing LC into an EE (Facy et al., 2004; Facy et al., 2005; Regnier et al., 1997; Schaerli et al., 2005), while just one group was able to introduce dendritic cells (DC) into a dermal equivalent (Guironnet et al., 2001). Neither of these groups reconstructed a full-thickness SE, rendering these models unfit to study all processes involved in the development of a hypersensitivity reaction. Three groups have so far succeeded in reconstructing a full thickness SE containing keratinocytes, fibroblasts, and epidermal LC containing Birbeck granules (Bechetoille et al., 2007; Dezutter-Dambuyant et al., 2006; Ouwehand et al., 2011). The model described by Ouwehand et al. is the only one to use cell line derived LC (MUTZ-3 derived LC) which overcomes the logistical problems of using fresh donor derived LC progenitors from peripheral blood or cord blood (Ouwehand et al., 2011). Importantly this model was shown to be functional and suited to study the first four steps in the development of an allergic response since topical exposure to sensitizers resulted in MUTZ-LC maturation and migration into the dermis (Ouwehand et al., 2011). However, no models until date contain T cells, rendering them unsuitable to study the induction of T cell responses to chemicals. In the future these models may progress to co-culture with T cells in the lower compartment of the transwell system. A shortcoming of the models that require fresh blood-derived precursor cells as their source of LC would be the logistics involved in constructing an autologous T cell containing co-culture variant (Dezutter-Dambuyant et al., 2006). The model of Ouwehand contains a human LC cell line (MUTZ-3 derived LC), established from the peripheral blood of a HLA-A2 positive patient with acute myelomonocytic leukemia (Ouwehand et al., 2011). These MUTZ-LC were able to prime specific and fully functional CTL from HLA-A2-matched healthy donors (Santegoets et al., 2008), which reduce logistical problems created by the need for blood of healthy volunteers. For all of these integrated skin models, further research is required to determine whether they will contribute to the battery of assays being developed for risk assessment. In any

As the induction of contact hypersensitivity is the result of a series of parallel and sequential processes, it is thought that a battery of assays based on the *in vivo* events leading up to sensitization will be required to identify potential sensitizers *in vitro*. Such a battery of assays would be expected to reduce the risk of scoring false negatives and positives. There is a general consensus that a combination of several functionally distinct human-based *in vitro* assays will be successful and may eventually even surpass the accuracy of animal methods such as LLNA. Several mechanistically relevant and promising test-strategies are being developed, which need to be further refined and tested before entering formal validation according to the guidelines of the European Centre for the Validation of Alternative Methods to animal testing (ECVAM). Although recent developments are looking very promising, scientists will find it difficult to meet the 2013 European deadline for

Ade, N.; Leon, F.; Pallardy, M.; Peiffer, J.L.; Kerdine-Romer, S.; Tissier, M.H.; Bonnet, P.A.;

Fabre, I.; Ourlin, J.C. (2009). *HMOX1 and NQO1 genes are upregulated in response to* 

case, these models are excellent to study DC biology *in vitro*.

replacement of animal based methods with *in vitro* alternatives.

**3. Conclusions** 

**4. References** 


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**Part 4** 

**Allergic Contact Dermatitis to Specific Allergens** 

