**5. Regulatory mechanisms in allergic contact dermatitis**

Both sensitization to chemicals and the effector phase of contact allergy are highly regulated events. This task is guaranteed by multiple mechanisms, including antigen-presenting cells and effector T cells apoptosis, production and release of anti-inflammatory mediators and action of a specialized subset of T lymphocytes with down-regulatory properties known as regulatory T cells.

Regulatory T cells (Treg) are a critical CD4 T cell subset involved in the control of immune homeostasis and in regulation of inflammation (Sakaguchi et al., 2008). Treg represent about 5-10% of the whole T cell compartment and are characterized by the preferential expression of several molecules including CD25, the alpha chain of IL-2 receptor (Sakaguchi et al., 1995), and FOXP3, a transcription factor which is necessary for Treg development and function (Fontenot et al., 2003).

CD4+CD25+ Treg cells have been implicated in the control CHS responses to haptens in mice. The first evidence of such a role for Treg cells came from a study investigating the mechanisms responsible for the "oral tolerance" phenomenon (Dubois et al., 2003). It was known that DNFB oral administration prior to sensibilisation could induce a tolerant state and prevent the development of ACD (Garside et al., 2001). Desvignes and coworkers showed that tolerance induction was dependent on the presence of CD4 T cells (Desvignes et al., 1996 ; Desvignes et al., 2000) suggesting the implication of a CD4 T cell subset with regulatory characteristics. Using in vivo models of adoptive transfer and antibody depletion of CD4+CD25+ cells, Dubois et al. demonstrated that naturally occurring CD4+CD25+ T cells are instrumental for orally induced tolerance and control hapten-specific CD8+ T cell responses mediating skin inflammation (Dubois et al., 2003).

Animal Models of Contact Dermatitis 33

showed that ICOS expressing Treg during ACD were hapten-specific, activated Treg cells

Fig. 5. FACS analysis of skin infiltrating CD4+ FOXP3+ Treg cells in a murine model of primary irritative contact dermatitis. (A, C) Gating strategy for Treg identification in draining lymph nodes (A) and ear skin (C). (B, D) ICOS and CD103 expression on

CD4+FOXP3- conventional T cells and CD4+FOXP3+ Regulatory T cells from (B) draining lymph nodes and (D) ear skin. FSC-A, forward scatter; SSC-A, side scatter; CTRL, control.

proliferating in response to their cognate antigen in vivo.

Further studies confirmed the role of Treg cells in classical ACD reaction. Depletion of CD4+ CD25+ T cells by in vivo treatment of mice with an anti-CD25 mAb at the time of sensitization led to an increased CD8+ T cell priming and an enhanced ACD reaction (Kish et al., 2005). Conversely, IL-2-IgG2b fusion protein treatment of mice induces a decreased ACD reaction associated with an increase in the CD4+ CD25+ Treg cell numbers (Ruckert et al., 2002).

Importantly, a role for CD4+ CD25+ Treg cells in maintaining immune tolerance to skin allergens has been confirmed in humans. Cavani et al. showed that CD4+ T cells isolated from the peripheral blood of six healthy nonallergic individuals showed a limited capacity to proliferate in response to nickel in vitro, but responsiveness was strongly augmented by CD25+ Treg depletion (Cavani et al. 2003).

Mechanisms involved in Treg maintenance of skin tolerance still remain to be fully elucidated. As IL-10 has been shown to mediate in vivo Treg suppression in other murine models of disease, it has been evaluated in the context of ACD. IL-10 has been shown to participate in restoring oral tolerance to haptens induced by CD4+ T cells (Dubois et al., 2003). Subsequently Ring et al. showed that Treg cells injection into sensitized mice at the time of local hapten challenge significantly inhibited influx of effector T cells into inflamed skin tissue, but that this effect was abrogated when CD4+CD25+ Treg cells isolated from IL-10–/– mice were transferred (Ring et al., 2006). More recently, Rudensky and coworkers generated mice in which Treg cell-specific ablation of a conditional IL-10 allele was induced by Cre recombinase knocked into the *Foxp3* gene locus (Rubtson et al., 2008). These mice were more prone to skin hypersensivity reaction induced by DNFB, thus formally demonstrating the implication of IL-10 in Treg mediated control of CHS.

Another potential mechanism through which Treg could inhibit effector migration to inflamed skin site, has been proposed by Ring and coworkers who found that Treg cell– derived adenosine plays a major role in preventing the elicitation of CHS reactions by blocking the interaction of effector T cells with the vascular endothelium (Ring et al., 2009).

Independently from the mechanism involved, CD4+CD25+Foxp3+ regulatory T cells require to be activated in order to develop their full suppressive capacity. Recently, studies from our and other groups better defined Treg activation in murine models of CHS. Analysing the expression kinetics of CD62L, CD69 and CD44 expression at Treg surface, Ring et al. showed that during the sensitization phase of CHS reactions, Treg get activated in the draining lymph nodes while Treg get activated in the blood during the elicitation phase. (Ring et aI., 2010). Employing the isolation protocol resumed in Fig. 4, we were able to extend the analysis of Treg activation to the skin tissue site in a model of primary allergic dermatitis (Simonetta et al., 2010). We confirmed that Treg were activated in lymph nodes after skin application of a strong hapten, as revealed by the surface upregulation of the activation molecules ICOS and CD103 (Fig. 5). Surprisingly when we extended the analysis to the skin tissue, we found high levels of ICOS and CD103 expression at surface of skin infiltrating Treg of both primed and non-primed animals (Fig. 5), indicating that skin Treg under normal physiological conditions are already in an activated state. A study performed by Vocanson and coworkers futher extended our results on ICOS expression on Treg revealing that these cells present superior suppressive activity and express IL-10, IL-17, and IFN- (Vocanson et al., 2010). More importantly for ACD comprehension, the authors

Further studies confirmed the role of Treg cells in classical ACD reaction. Depletion of CD4+ CD25+ T cells by in vivo treatment of mice with an anti-CD25 mAb at the time of sensitization led to an increased CD8+ T cell priming and an enhanced ACD reaction (Kish et al., 2005). Conversely, IL-2-IgG2b fusion protein treatment of mice induces a decreased ACD reaction associated with an increase in the CD4+ CD25+ Treg cell

Importantly, a role for CD4+ CD25+ Treg cells in maintaining immune tolerance to skin allergens has been confirmed in humans. Cavani et al. showed that CD4+ T cells isolated from the peripheral blood of six healthy nonallergic individuals showed a limited capacity to proliferate in response to nickel in vitro, but responsiveness was strongly augmented by

Mechanisms involved in Treg maintenance of skin tolerance still remain to be fully elucidated. As IL-10 has been shown to mediate in vivo Treg suppression in other murine models of disease, it has been evaluated in the context of ACD. IL-10 has been shown to participate in restoring oral tolerance to haptens induced by CD4+ T cells (Dubois et al., 2003). Subsequently Ring et al. showed that Treg cells injection into sensitized mice at the time of local hapten challenge significantly inhibited influx of effector T cells into inflamed skin tissue, but that this effect was abrogated when CD4+CD25+ Treg cells isolated from IL-10–/– mice were transferred (Ring et al., 2006). More recently, Rudensky and coworkers generated mice in which Treg cell-specific ablation of a conditional IL-10 allele was induced by Cre recombinase knocked into the *Foxp3* gene locus (Rubtson et al., 2008). These mice were more prone to skin hypersensivity reaction induced by DNFB, thus formally

Another potential mechanism through which Treg could inhibit effector migration to inflamed skin site, has been proposed by Ring and coworkers who found that Treg cell– derived adenosine plays a major role in preventing the elicitation of CHS reactions by blocking the interaction of effector T cells with the vascular endothelium (Ring et al., 2009). Independently from the mechanism involved, CD4+CD25+Foxp3+ regulatory T cells require to be activated in order to develop their full suppressive capacity. Recently, studies from our and other groups better defined Treg activation in murine models of CHS. Analysing the expression kinetics of CD62L, CD69 and CD44 expression at Treg surface, Ring et al. showed that during the sensitization phase of CHS reactions, Treg get activated in the draining lymph nodes while Treg get activated in the blood during the elicitation phase. (Ring et aI., 2010). Employing the isolation protocol resumed in Fig. 4, we were able to extend the analysis of Treg activation to the skin tissue site in a model of primary allergic dermatitis (Simonetta et al., 2010). We confirmed that Treg were activated in lymph nodes after skin application of a strong hapten, as revealed by the surface upregulation of the activation molecules ICOS and CD103 (Fig. 5). Surprisingly when we extended the analysis to the skin tissue, we found high levels of ICOS and CD103 expression at surface of skin infiltrating Treg of both primed and non-primed animals (Fig. 5), indicating that skin Treg under normal physiological conditions are already in an activated state. A study performed by Vocanson and coworkers futher extended our results on ICOS expression on Treg revealing that these cells present superior suppressive activity and express IL-10, IL-17, and IFN- (Vocanson et al., 2010). More importantly for ACD comprehension, the authors

demonstrating the implication of IL-10 in Treg mediated control of CHS.

numbers (Ruckert et al., 2002).

CD25+ Treg depletion (Cavani et al. 2003).

showed that ICOS expressing Treg during ACD were hapten-specific, activated Treg cells proliferating in response to their cognate antigen in vivo.

Fig. 5. FACS analysis of skin infiltrating CD4+ FOXP3+ Treg cells in a murine model of primary irritative contact dermatitis. (A, C) Gating strategy for Treg identification in draining lymph nodes (A) and ear skin (C). (B, D) ICOS and CD103 expression on CD4+FOXP3- conventional T cells and CD4+FOXP3+ Regulatory T cells from (B) draining lymph nodes and (D) ear skin. FSC-A, forward scatter; SSC-A, side scatter; CTRL, control.

Animal Models of Contact Dermatitis 35

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