Topic Article
Langerhans Cells Are Not Required for Efficient Skin Graft Rejection
Jagdeep S Obhrai, Martin Oberbarnscheidt, Na Zhang, Daniel L Mueller, Warren D Shlomchik, Fadi G Lakkis, Mark J Shlomchik and Daniel H Kaplan
Journal of Investigative Dermatology (2008), 128, 1950-1955. doi:10.1038/jid.2008.52

A Paradigm Shift in the Mechanisms of Graft Rejection

Deborah Zell ¹, Shasa Hu ¹ and Robert Kirsner ¹

Journal of Investigative Dermatology (2008), 128, 1874. doi:10.1038/jid.2008.173

Without systemic immune suppression, allogeneic transplants are rejected via immunologic mechanisms (Kirsner et al., 1993) thought to involve delayed-type hypersensitivity reactions that are mediated by T cells. Foreign antigens (allograft antigens) are “presented” by antigen-presenting cells (APCs) as they activate T cells in lymph nodes and other secondary lymphoid tissue. Among the highly effective APCs are dendritic cells (DCs), which are thought to be essential to allogeneic graft recognition and rejection. At the time of transplantation, donor DCs are normally included in the transplanted organ or tissue (Lechler et al., 2001). These cells have the capacity to migrate to regional lymph nodes where T-cell activation takes place. Specific to skin are the epidermal DCs, or Langerhans cells (LCs), which likewise are transferred from donor to host with allogeneic skin grafting. Donor LCs are also thought to be important in allogeneic skin graft rejection. The correlation of in vitro studies of LC function from mice to humans is complicated by the fact that LCs display differential effects depending on the environment in which they are studied.
Using a murine model lacking LCs, but retaining a full contingent of other DCs, Obhrai et al. (2008) tested the hypothesis that LCs are important in mediating allogeneic skin graft rejection. This is one of several mouse models that have previously been used to study LC function. Results from earlier work using this same animal model questioned the traditional role of LCs in delayed hypersensitivity reactions by finding, paradoxically, that contact sensitization is upregulated in the absence of LCs (Kaplan et al., 2005).
By performing genetic mismatched skin grafting experiments in various strains of mice and various types of mismatching using LC-deficient mice as donors, Obhrai et al. (2008) found that LCs were not required for skin graft rejection; grafts from LC-deficient mice were routinely rejected. This finding suggests that, at least in these mice, non-LC DCs probably play a role in skin graft rejection. More surprising was the finding that in a male–female mismatched experiment in which skin grafts are normally accepted (FVB mice), donor skin obtained from LC-deficient donors was rejected. This later finding suggests that LCs inhibit male antigen–associated skin graft rejection in certain strains of mice and warrants further investigation. The reason for this, and the mechanism by which it occurs, remain to be elucidated.

QUESTIONS

1. What evidence supports the role of LCs in skin graft rejection?

2. How did the animal model previously developed help in studying contact sensitization and graft rejection?

3. What were the major findings of this study?

4. By what mechanisms can LCs from grafts modulate immune response?

5. What may be the clinical implications of this work?

ANSWERS

1. Langerhans cells (LCs) are highly effective immune cells of the skin. Although they are the best studied of the skin’s dendritic cells (DCs), they are only one of the several types that reside there. Most prominently, DCs “present” antigens to selected T cells, thereby initiating cell-mediated immune responses. LCs are unique in that they are spread, within the epidermis, across the skin surface. As residents of the epidermis, they are radioresistant and dependent on transforming growth factor-β1 for their development, localization, and ultimate survival (Udey and Rosenberg, 2008). Other skin-associated DCs are found in the dermis and regional lymph nodes, which also appear to be critical participants in skin-based immune processes.

DCs were thought to be involved in graft rejection when Lechler et al. (2001) found that DCs from donor grafts would migrate to regional lymph nodes. Additional studies have shown that both the host and the donor DCs participate in graft rejection, but the respective roles of DCs from the host and donor have remained unclear (Benichou et al., 1999; Chen et al., 2003; Reed et al., 2003). No evidence has unequivocally demonstrated that LCs are involved in graft rejection.

However, given that LCs are the most prominent DCs in skin, it has been hypothesized that they are important in skin graft rejection. Early studies by Barker and Billingham (1968) showed that LCs migrate out of donor skin grafts, because an intact lymphatic connection was required for immunization and rejection to take place. In addition, skin grafts are more immunogenic than grafts of solid organs, such as the heart; He et al. (2004) have hypothesized that this observation relates to the large number of LCs that are found in the skin.

Thus, the role of DCs, and particularly LCs, in graft rejection remains unclear. On the other hand, the recent development of LC-deficient murine models allows the study of LCs in a variety of skin-dependent immune processes. It is critical, however, to understand certain subtleties in these models before accurate conclusions can be drawn.

Two broad types of LC-deficient mice have been studied: those engineered to be born in a deficient state (constitutively deficient) and those made deficient after birth (conditionally LC deficient). With both types, diphtheria is used to destroy or target LCs. Constitutively deficient mice are created to express the diphtheria toxin subunit A (DTA) in LCs (Kaplan et al., 2005). As a result, DTA causes cell death, and in this model LCs are deleted as soon as the gene is expressed during embryogenesis (Langerin-DTA). Conditionally LC-deficient mice are generated with cDNA encoding the diphtheria toxin receptor (DTR) inserted into the gene specific for LC Langerin by homologous recombination (Langerin-DTR mice). These mice are born with LCs, but complete depletion occurs within 2 days after administration of DT (Bennett et al., 2005; Kissenpfennig et al., 2005).

Obhari et al. (2008) studied the role of LCs in skin graft rejection. Donor skin grafts from constitutively deficient mice and control mice were placed onto healthy recipient mice to determine the role of LCs in skin graft rejection.

2. The Langerin-DTA (constitutively deficient) mice used in this study had no LCs at birth. Under influence of the Langerin promoter, an “LC specific” gene, DTA had deleted the LCs (Obhari et al., 2008). In addition to the timing of LC depletion between the LC-deficient murine models, these models have different effects on a subpopulation of DCs outside of the epidermis that nonetheless do express Langerin. These cells, termed Langerin + DCs, are found in the lymph nodes as well as the dermis and are characterized as CD8α + (Bursch et al., 2007; Ginhoux et al., 2007; Poulin et al., 2007). The conditionally deficient Langerin-DTR mice have neither LCs nor these Langerin + DCs, whereas the constitutively deficient Langerin-DTA mice do not have LCs but do have Langerin + DCs. Langerin + DCs repopulate different anatomic sites with very different rates (Poulin et al., 2007) and are reestablished in the dermis and lymph nodes of Langerin-DTR mice within a few days after DT treatment, whereas epidermal LCs do not normalize in number for months.

These models make it possible to generate a better understanding of the specific role of LCs and other Langerin + DCs in cutaneous immunity, including contact sensitivity and skin graft rejection. Using constitutively deficient Langerin-DTA mice, Kaplan and co-workers (2005) found a twofold increase in contact hypersensitivity compared with controls, suggesting that LCs may be negative regulators of some immune responses, while a study using Langerin-DTR (conditionally LC-deficient) mice found that LCs were required for optimal hapten sensitization (Bennett et al., 2005, 2007). It appears that timing of DT administration in relation to hapten treatment is critical (Bursch et al., 2007; Wang et al., 2008) and that Langerin + dermal DCs, but not LCs, are key for optimal priming with haptens.

3. This study worked with full-thickness skin grafts from Langerin-DTA mice and control mice transplanted onto healthy recipients. To understand the results, in addition to the subtleties of the LC-deficient models, it is important to understand the unique characteristics of certain mouse strains in response to skin transplantation. Several mouse strains were used in this study, including FVB mice. This is important because the response of female mice to skin grafts from male mice is strain dependent; for example, B6 females reject B6 male grafts and FVB females accept FVB male grafts.

To start, grafts from Langerin-DTA (LC-deficient) mice and LC-sufficient littermates (major histocompatibility complex (MHC)-mismatched) were transplanted onto healthy recipient mice. Both sets of grafts were rejected, whether they were obtained from LC-deficient or LC-sufficient animals, suggesting that LCs are not critical for graft rejection; this is not surprising, given the other DCs present in skin. Similar findings were reported when donors and recipients were matched at all MHC loci but were disparate with respect to Y chromosomes and, probably, minor loci as well. These findings also support the conclusion that LCs are not required for initiation of skin graft rejection.

More intriguing—and even less expected—results were seen in sex-mismatched FVB mice. As noted above, FVB females normally accept FVB male skin grafts. Obhrai et al. (2008) found this to be the case only when the FVB male skin grafts contained LCs. Skin grafts from male FVB Langerin-DTA mice were rejected, suggesting that LCs (in this strain of mice and when male skin grafts were transplanted onto female mice) are required for successful skin grafting. Thus, LCs may suppress immune reactions in addition to inducing them.

4. LCs do not appear to be necessary for graft rejection, whereas DCs appear to be critical, as demonstrated in a Langerin-DTA murine model. It is likely that DCs from donors migrate to regional lymph nodes, where they are recognized as “foreign,” allowing T cells to be primed for attack. Although this study reports that grafts lacking LCs can be rejected, under certain situations LCs may be needed for successful skin grafting.

Immunologic differences in the mouse models were evaluated, but few answers were found. Specifically, T-cell responses on day 14 and cytokine production from both the Langerin-DTA mice and controls revealed no significant increase in IL-2 production and little production of IFN-γ and IL-4 in mice that had received skin grafts from the Langerin-DTA mice (Obhari et al., 2008). No differences were found in T-cell cytokine production, activation state, or induction of T cells that could account for the differences in rejection responses.

5. This work demonstrates that non-LC DCs play a role in graft rejection, and that LCs are not required for and may only assist in skin graft rejection. Further experiments using Langerin-DTA mice (used in these experiments) as recipients (rather than donors) of skin grafts from MHC-matched and -mismatched control mice as well as Langerin-DTA mice may help determine the role of LCs in recipient skin in graft rejection. Furthermore, the study of other LC-deficient mouse models, such as in the conditionally deficient Langerin-DTR model, may clarify the role of Langerin + DCs in graft rejection.

From a therapeutic standpoint, treatments such as therapy with UVR or with tumor necrosis factor-α (TNF-α) inhibitors that affect LCs could be applied to these models—and, eventually, to patients—to determine their effect on graft rejection. TNF-α is known to stimulate LC migration (Barbaroux et al., 2008); thus, anti-TNF medications (which would limit migration) and UVR (which can deplete LCs) (Norval et al., 2008) could be given before transplantation to determine their effects on rejection.

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¹ Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, Florida, USA