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Functional Redundancy of Langerhans Cells and Langerin+ Dermal Dendritic Cells in Contact Hypersensitivity

      The relative roles of Langerhans cells (LC), dermal dendritic cells (DC), and, in particular, the recently discovered Langerin+ dermal DC subset in the induction and control of contact hypersensitivity (CHS) responses remain controversial. Using an inducible mouse model, in which LC and other Langerin+ DC can be depleted by injection of diphtheria toxin, we previously reported impaired transport of topically applied antigen to draining lymph nodes and reduced CHS in the absence of all Langerin+ skin DC. In this study, we demonstrate that mice with a selective depletion of LC exhibit attenuated CHS only upon sensitization with a low hapten dose but not with a high hapten dose. In contrast, when painting a higher concentration of hapten onto the skin, which leads to increased antigen dissemination into the dermis, CHS is still diminished in mice lacking all Langerin+ skin DC. Taken together, these data suggest that the magnitude of a CHS reaction depends on the number of skin DC, which have access to the hapten, rather than on the presence or absence of a particular skin DC population. LC and (Langerin+) dermal DC thus seem to have a redundant function in regulating CHS.

      Abbreviations

      ACD
      allergic contact dermatitis
      CHS
      contact hypersensitivity
      DC
      dendritic cell(s)
      DNFB
      2,4-dinitrofluorobenzene
      DT
      diphtheria toxin
      DTR
      diphtheria toxin receptor
      LC
      Langerhans cell(s)
      LN
      lymph node(s)
      WT
      wild type

      Introduction

      Dendritic cells (DC) are a heterogenous family of antigen-presenting cells critical for the induction and control of T cell immunity and tolerance. Upon antigen encounter at epithelial borders to the environment, DC migrate to local lymph nodes (LN), where they activate naive T cells to become effector cells (
      • Banchereau J.
      • Steinman R.M.
      Dendritic cells and the control of immunity.
      ;
      • Banchereau J.
      • Briere F.
      • Caux C.
      • et al.
      Immunobiology of dendritic cells.
      ;
      • Steinman R.M.
      • Hawiger D.
      • Liu K.
      • et al.
      Dendritic cell function in vivo during the steady state: a role in peripheral tolerance.
      ). In mouse skin, at least three phenotypically distinct DC populations can be distinguished: Langerhans cells (LC) expressing the C-type lectin Langerin/CD207 form a contiguous network in the epidermis, whereas Langerin+ and Langerinneg DC subsets reside in the dermis (
      • Bursch L.S.
      • Wang L.
      • Igyarto B.
      • et al.
      Identification of a novel population of Langerin+ dendritic cells.
      ;
      • Ginhoux F.
      • Collin M.P.
      • Bogunovic M.
      • et al.
      Blood-derived dermal langerin+ dendritic cells survey the skin in the steady state.
      ;
      • Poulin L.F.
      • Henri S.
      • de Bovis B.
      • et al.
      The dermis contains langerin+ dendritic cells that develop and function independently of epidermal Langerhans cells.
      ). LC can be further separated from Langerin+ dermal DC by their expression or lack of EpCam and CD103, respectively (
      • Nagao K.
      • Ginhoux F.
      • Leitner W.W.
      • et al.
      Murine epidermal Langerhans cells and langerin-expressing dermal dendritic cells are unrelated and exhibit distinct functions.
      ). Whether the different skin DC subsets exert specific functions in the regulation of cutaneous immune responses remains elusive (
      • Romani N.
      • Ebner S.
      • Tripp C.H.
      • et al.
      Epidermal Langerhans cells--changing views on their function in vivo.
      ,
      • Romani N.
      • Clausen B.E.
      • Stoitzner P.
      Langerhans cells and more: langerin-expressing dendritic cell subsets in the skin.
      ;
      • Clausen B.E.
      • Kel J.M.
      Langerhans cells: critical regulators of skin immunity?.
      ).
      Allergic contact dermatitis (ACD), one of the most common occupational diseases, leads to considerable health-related quality of life impairment in affected patients and its prevention therefore is of high socioeconomic impact. ACD, also referred to as contact hypersensitivity (CHS), is a T cell-mediated skin inflammation caused by repeated exposure to contact allergens, i.e., low-molecular-weight chemicals called haptens (
      • Vocanson M.
      • Hennino A.
      • Rozieres A.
      • et al.
      Effector and regulatory mechanisms in allergic contact dermatitis.
      ). During the asymptomatic sensitization phase, haptens penetrating the skin bind to host proteins, which are then collected by resident DC that migrate to the LN to activate naive hapten-specific T cells. In sensitized individuals, re-exposure to the hapten initiates the effector phase and clinical expression of ACD, characterized by recruitment and activation of specific T cells at the site of hapten challenge. In mice, painting of a sensitizer onto the skin initiates production of proinflammatory cytokines that activate skin DC to prime hapten-specific T cells in draining LN (
      • Grabbe S.
      • Schwarz T.
      Immunoregulatory mechanisms involved in elicitation of allergic contact hypersensitivity.
      ). Re-exposure to the same hapten onto the ear induces a transient ear swelling reaction mediated by IFNγ-producing CD8+ T cells and controlled by CD4+ T cells secreting IL-4 and IL-10 (
      • Gorbachev A.V.
      • Fairchild R.L.
      CD4+ T cells regulate CD8+ T cell-mediated cutaneous immune responses by restricting effector T cell development through a Fas ligand-dependent mechanism.
      ). LC have been critically linked to the initiation of contact sensitivity responses (
      • Toews G.B.
      • Bergstresser P.R.
      • Streilein J.W.
      Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB.
      ), however, recent advances in the pathophysiology of CHS have revisited the dogma that LC are mandatory for T-cell priming.
      The essential contribution of LC to hapten sensitization has recently been challenged in different LC ablation models (
      • Bennett C.L.
      • van Rijn E.
      • Jung S.
      • et al.
      Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity.
      ;
      • Kaplan D.H.
      • Jenison M.C.
      • Saeland S.
      • et al.
      Epidermal langerhans cell-deficient mice develop enhanced contact hypersensitivity.
      ,
      • Kaplan D.H.
      • Kissenpfennig A.
      • Clausen B.E.
      Insights into Langerhans cell function from Langerhans cell ablation models.
      ;
      • Kissenpfennig A.
      • Henri S.
      • Dubois B.
      • et al.
      Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells.
      ;
      • Bennett C.L.
      • Clausen B.E.
      DC ablation in mice: promises, pitfalls, and challenges.
      ). On one hand, knock-in mouse strains have been independently generated by two laboratories, in which expression of the high-affinity human diphtheria toxin (DT) receptor (DTR) is driven by the endogenous langerin promotor. In these Langerin-DTR mice, LC can be depleted for at least 2–4 weeks after a single injection of DT (
      • Bennett C.L.
      • van Rijn E.
      • Jung S.
      • et al.
      Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity.
      ). Initial experiments demonstrated a similar and reduced CHS responses in the French (
      • Kissenpfennig A.
      • Henri S.
      • Dubois B.
      • et al.
      Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells.
      ) and Dutch (
      • Bennett C.L.
      • van Rijn E.
      • Jung S.
      • et al.
      Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity.
      ) Langerin-DTR mice, respectively, as compared with wild type (WT). Later, a population of Langerin+ dermal DC distinct of epidermal LC was discovered (
      • Bursch L.S.
      • Wang L.
      • Igyarto B.
      • et al.
      Identification of a novel population of Langerin+ dendritic cells.
      ;
      • Ginhoux F.
      • Collin M.P.
      • Bogunovic M.
      • et al.
      Blood-derived dermal langerin+ dendritic cells survey the skin in the steady state.
      ;
      • Poulin L.F.
      • Henri S.
      • de Bovis B.
      • et al.
      The dermis contains langerin+ dendritic cells that develop and function independently of epidermal Langerhans cells.
      ) and it was established in the French mice that these recover from the toxin treatment within 1–2 weeks after DT (
      • Bursch L.S.
      • Wang L.
      • Igyarto B.
      • et al.
      Identification of a novel population of Langerin+ dendritic cells.
      ). Using the French Langerin-DTR mice, it was then shown that CHS responses were indeed decreased when all Langerin+ skin DC were absent, but restored to WT levels once the Langerin+ dermal DC subset had returned (
      • Bursch L.S.
      • Wang L.
      • Igyarto B.
      • et al.
      Identification of a novel population of Langerin+ dendritic cells.
      ). In contrast, CHS was still impaired in the Dutch Langerin-DTR mice 4 weeks after DT (
      • Bennett C.L.
      • Noordegraaf M.
      • Martina C.A.
      • et al.
      Langerhans cells are required for efficient presentation of topically applied hapten to T cells.
      ).
      On the other hand, transgenic mice expressing the toxic subunit of DT under control of the human langerin promotor constitutively lack epidermal LC from birth, whereas other Langerin+ DC populations, in particular, Langerin+ dermal DC are not affected (
      • Kaplan D.H.
      • Jenison M.C.
      • Saeland S.
      • et al.
      Epidermal langerhans cell-deficient mice develop enhanced contact hypersensitivity.
      ). These Langerin-DTA mice mount exaggerated CHS reactions suggesting that LC play a regulatory rather than a stimulatory role in this model. Therefore, to date the requirement of LC, Langerin+ and Langerinneg dermal DC during the sensitization of CHS remains controversial or unknown.
      In this study, we take advantage of the differential repopulation kinetics between epidermal LC and Langerin+ dermal DC and assess CHS responses in mice selectively lacking LC and mice lacking all Langerin+ DC. In particular, we compare different haptens, hapten concentrations and, for the first time, the two inducible LC depletion mouse models side-by-side. Our data demonstrate that in both Langerin-DTR strains the magnitude of a CHS reaction depends on the antigen dose and the number of skin DC that have access to the hapten, rather than the presence or function of one of the three skin DC subsets.

      Results

      Rapid steady state repopulation of dermal Langerin+ DC

      The skin contains at least three subsets of DC, namely epidermal LC and Langerin+ and Langerinneg dermal DC. In Langerin-DTR mice, all Langerin+ cells are rapidly depleted upon injection with DT. We previously described that steady state LC reconstitution of the epidermis takes at least 2–4 weeks (
      • Bennett C.L.
      • van Rijn E.
      • Jung S.
      • et al.
      Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity.
      ), whereas Langerin+ LN DC recover faster from the toxin treatment (
      • Bennett C.L.
      • Noordegraaf M.
      • Martina C.A.
      • et al.
      Langerhans cells are required for efficient presentation of topically applied hapten to T cells.
      ). These data were obtained before the recent discovery of a distinct subset of Langerin+ DC in the dermis, and in this study we further characterize the steady state repopulation of dermal Langerin+ cells. Langerin-DTR mice were injected with DT and the repopulation kinetics of Langerin+ cells in epidermis, dermis, and LN was assessed (Figure 1). As expected, 2 days after toxin treatment all Langerin+ DC in epidermis, dermis, and LN were efficiently depleted. Already after 1 week, Langerin+ cells were reappearing in the dermis and in skin-draining LN, and continued to increase in numbers over time, whereas the epidermis essentially remained devoid of LC. Nonetheless, we observe only a partial recovery of the Langerin+ CD103+ DC population in the dermis and skin-draining LN (Figure 1b). In fact, the rare Langerin+ major histocompatibility complex II+ (MHCII+) DC present in the dermis 2 weeks after DT are difficult to make out in cross-sections of ear skin (Figure 1a, right panel, white arrowhead). Analysis of Langerin+ cells among whole ear skin crawl-outs analyzed 15 days after DT confirms the profound LC deficiency and incomplete reconstitution of Langerin+ CD103+ dermal DC (Figure 1c). Notably, the French and Dutch Langerin-DTR mice show a similar Langerin+ dermal DC recovery of 44.7 and 29.2%, respectively (calculated from absolute numbers of migratory cells, Figure 1c). Consequently, topical application of FITC 2 or 10 days after DT results in antigen transport to draining LN by Langerinneg dermal DC or both Langerin+ and Langerinneg dermal DC subsets (Supplementary Figure S1 online). This difference in repopulation kinetics between LC and Langerin+ dermal DC after toxin treatment enables us to study CHS responses in the absence of all Langerin+ cells or in the absence of LC alone (see Table 1 for an overview).
      Figure thumbnail gr1
      Figure 1Steady state repopulation of Langerin+ dendritic cell (DC) populations. (a) Anti-Langerin (red fluorescence) and anti-major histocompatibility complex II (MHCII) (green fluorescence) staining of ear cross-sections of wild type (WT) (left panel) and Langerin-diphtheria toxin receptor (DTR) mice (“Dutch mice”) injected with diphtheria toxin (DT) at 48hours (middle panel) or 14 days (right panel) earlier. The arrowhead identifies one of the rare Langerin+ dermal DC (yellow fluorescence) that had recovered from the toxin at 2 weeks. Bars = 50μm. (b) Recovery of Langerin+ cells in epidermis, dermis, and cutaneous lymph node (LN). Mice were injected with DT at day 0. Single-cell suspensions were prepared at different time points and analyzed for the presence of Langerin+ cells. Values of untreated mice (day 0) were set equal to 100%. The graph shows the percentages of MHCII+ Langerin+ cells (mean±SD). In dermal suspensions, epidermal Langerhans cells (LC) in transit and Langerin+ dermal DC were distinguished by differential expression of EpCam and CD103 (not shown). Only numbers of CD103+EpCamneg dermal Langerin+ cells are considered in the columns for dermis. Four mice per time point were analyzed. (c) At 15 days after injection of phosphate-buffered saline (PBS) or DT, whole-skin explants were prepared from the ears of French or Dutch Langerin-DTR mice, and further cultured for 4 days. Migratory cells from 4–6 skin explants were pooled and stained for CD11c, Langerin and CD103. At least 3,000 live migratory cells were acquired by fluorescence activated cell sorting. Dot plots display migratory cells gated for Langerin expression. Percentages represent the proportions of CD11c+ CD103neg LC and CD11c+ CD103+ Langerin+ dermal DC. Results are representative of two independent experiments.
      Table 1Low- and high-dose CHS in the presence or absence of distinct skin DC subsets
      Composition of skin DC compartmentLow-dose CHSHigh-dose CHS
      WT+++++
      Lack of epidermal LC only (DT day -10 or -15)Similar (++)
      Lack of all Langerin+ cells (DT day -1 or -2)
      Abbreviations: CHS, contact hypersensitivity; DC, dendritic cell; DT, diphtheria toxin; LC, Langerhans cells; WT, wild type. + Degree of ear swelling; ↓ reduction in ear swelling as compared with WT mice.

      Low-dose CHS is decreased in the absence of both all Langerin+ DC and LC alone

      We have previously shown that in the absence of all Langerin+ cells CHS responses are diminished due to inefficient hapten transport to local LN for the priming of naive antigen-specific T cells (
      • Bennett C.L.
      • van Rijn E.
      • Jung S.
      • et al.
      Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity.
      ,
      • Bennett C.L.
      • Noordegraaf M.
      • Martina C.A.
      • et al.
      Langerhans cells are required for efficient presentation of topically applied hapten to T cells.
      ). In this study, we investigated the differential roles (if any) of epidermal LC versus Langerin+ and Langerinneg dermal DC in mediating CHS. Taking advantage of the slow return of the epidermal LC in relation to dermal Langerin+ DC, we injected mice with DT 2 days before inducing CHS to deplete all Langerin+ cells, or 10 days before inducing CHS to selectively deplete epidermal LC only (Table 1). On the basis of our observation that a reduced dose of hapten preferentially targets LC in the epidermis (
      • Bennett C.L.
      • Noordegraaf M.
      • Martina C.A.
      • et al.
      Langerhans cells are required for efficient presentation of topically applied hapten to T cells.
      ), we sensitized mice with 0.5% oxazalone on the shaved abdomen and challenged with 0.25% of oxazalone on one ear 5 days later. Consistent with our previous findings, CHS responses are significantly reduced in Langerin-DTR mice, when they lack all Langerin+ cells. Similarly, mice in which dermal Langerin+ cells have recovered from the toxin, exhibit a diminished CHS response at this low oxazolone concentration as compared with WT (Figure 2a).
      Figure thumbnail gr2
      Figure 2Contact hypersensitivity reactions to low-dose hapten. Wild type (WT) or Langerin-diphtheria toxin receptor (DTR) mice (“Dutch mice”) injected with diphtheria toxin (DT) at day -2 or -10 (n=6–8) were (a) sensitized on the abdomen with 0.5% oxazolone and challenged 5 days later with 0.25% oxazolone on one ear, or (b) sensitized with 0.5% 2,4-dinitrofluorobenzene (DNFB) and challenged with 0.25% DNFB. Ear swelling was measured as the difference between before and after challenge. One representative experiment out of three is shown; symbols represent individual mice. Ear swelling responses were compared using a Student's t-test; WT versus DTR + DT: *P≤0.05. LC, Langerhans cells.
      To exclude any bias of a particular hapten (oxazolone) on the results of this study, we also induced low-dose CHS with 2,4-dinitrofluorobenzene (DNFB). As depicted in Figure 2b, upon sensitization with 0.5% followed by challenge with 0.25% DNFB, CHS responses are significantly impaired 2 days as well as 10 days after injection of DT. These data indicate that at low-hapten concentrations, CHS is reduced both in the absence of all Langerin+ DC and of LC alone, irrespective of the type of hapten.

      High-dose CHS is only diminished in mice lacking all Langerin+ DC

      To address the contribution of Langerin+ dermal DC to CHS reactions, we painted a higher concentration of hapten onto the skin leading to increased antigen dissemination into the dermis. We sensitized and challenged the mice with 2 and 0.5% oxazolone or 1 and 0.5% DNFB, respectively. As expected, mice lacking all Langerin+ cells still develop significantly diminished CHS to both haptens (Figure 3a and b, left panels). In contrast, when only epidermal LC are absent, CHS responses are comparable with WT mice, and when they are reduced, this difference often is not significant (Figure 3, right panels). These findings suggest that once a hapten efficiently reaches the dermis, LC are dispensable and (Langerin+) dermal DC are sufficient for the induction of an effective CHS response.
      Figure thumbnail gr3
      Figure 3Contact hypersensitivity responses elicited by high-dose hapten. Wild type (WT) or Langerin-diphtheria toxin receptor (DTR) mice (“Dutch mice”) injected with diphtheria toxin (DT) at day -2 or -10 (n=6–8) were (a) sensitized on the abdomen with 2% oxazolone and challenged 5 days later with 0.5% oxazolone on one ear, or (b) sensitized with 1% 2,4-dinitrofluorobenzene (DNFB) and challenged with 0.5% DNFB on one ear. Ear swelling was measured as the difference between before and after challenge. One representative experiment out of three is shown; symbols represent individual mice. Ear swelling responses were compared using a Student's t-test; WT versus DTR+DT: *P≤0.05. LC, Langerhans cells.

      Similar reduction of low- and high-dose CHS in French and Dutch Langerin-DTR mice

      Performing similarly timed DT treatments, using the French Langerin-DTR mice,
      • Bursch L.S.
      • Wang L.
      • Igyarto B.
      • et al.
      Identification of a novel population of Langerin+ dendritic cells.
      ) reported diminished CHS in the absence of all Langerin+ DC, but no reduction in CHS when only LC are absent from the skin. Therefore, it was important to compare low- and high-dose CHS responses in the French and Dutch Langerin-DTR mice immediately or 10 days after DT. As shown in Figure 4, following a low-dose protocol of inducing CHS to oxazolone both Langerin-DTR mice mount attenuated ear swelling reactions in the absence of all Langerin+ DC (Figure 4a) and LC alone (Figure 4b). These results demonstrate that when tested in the same experiment, under conditions that limit hapten delivery preferentially to the epidermis, the French and Dutch Langerin-DTR mice develop equally reduced CHS after ablation of all Langerin+ DC, but also in the selective absence of LC. In addition, when analyzed side-by-side both Langerin-DTR mutants respond similarly in high-dose CHS (Figure 5).
      Figure thumbnail gr4
      Figure 4Comparison of low-dose contact hypersensitivity in French and Dutch Langerin-diphtheria toxin receptor (DTR) mice. Wild type (WT), French or Dutch Langerin-DTR mice injected with diphtheria toxin (DT) at (a) day -1 or (b) day -10 (n=6–9) were sensitized on the abdomen with 0.5% oxazolone and challenged 5 days later with 0.25% oxazolone on one ear. Swelling was measured as the difference between challenged (right) and unchallenged (left) ear. The summary of 2–3 experiments is shown; symbols represent individual mice. Ear swelling responses were compared using a Student's t-test; WT versus DTR+DT: *P≤0.05. LC, Langerhans cells.
      Figure thumbnail gr5
      Figure 5High-dose contact hypersensitivity in French and Dutch Langerin-diphtheria toxin receptor (DTR) mice. Wild type (WT), French or Dutch Langerin-DTR mice injected with diphtheria toxin (DT) at (a) day -1 or (b) day -10 (n=8–10) were sensitized on the abdomen with 2% oxazolone and challenged 5 days later with 0.5% oxazolone on one ear. Ear swelling was measured as the difference between challenged (right) and unchallenged (left) ear. The summary of two experiments is shown; symbols represent individual mice. Ear swelling responses were compared using a Student's t-test; WT versus DTR+DT: *P≤0.05. LC, Langerhans cells.

      Discussion

      CHS, in the form of occupational ACD to low-molecular-weight chemicals (haptens), leads to a considerable impairment of the quality of life in affected patients (
      • Vocanson M.
      • Hennino A.
      • Rozieres A.
      • et al.
      Effector and regulatory mechanisms in allergic contact dermatitis.
      ). Initiation of disease is critically linked to the migration of skin DC to local LN and activation of naive hapten-specific T cells during the asymptomatic sensitization phase. Re-exposure to the relevant hapten induces the effector phase and clinical expression of ACD. DC are a heterogenous family of antigen-presenting cells and as such key regulators of T cell immunity and tolerance (
      • Banchereau J.
      • Steinman R.M.
      Dendritic cells and the control of immunity.
      ;
      • Banchereau J.
      • Briere F.
      • Caux C.
      • et al.
      Immunobiology of dendritic cells.
      ;
      • Steinman R.M.
      • Hawiger D.
      • Liu K.
      • et al.
      Dendritic cell function in vivo during the steady state: a role in peripheral tolerance.
      ;
      • Steinman R.M.
      • Banchereau J.
      Taking dendritic cells into medicine.
      ). Therefore, a better understanding of DC function in CHS may open improved immunotherapeutic approaches to treat patients with ACD.
      The skin contains at least three phenotypically distinct DC populations (
      • Merad M.
      • Ginhoux F.
      • Collin M.
      Origin, homeostasis and function of Langerhans cells and other langerin-expressing dendritic cells.
      ). A network of Langerin+ EpCam+ CD103neg LC is located in the epidermis and Langerin+ EpCamneg CD103+ and Langerinneg prototypic tissue DC reside in the dermis (
      • Nagao K.
      • Ginhoux F.
      • Leitner W.W.
      • et al.
      Murine epidermal Langerhans cells and langerin-expressing dermal dendritic cells are unrelated and exhibit distinct functions.
      ). All three skin DC subsets can migrate to draining LN in response to local inflammation, but whether they have specific roles in inducing or regulating an immune response is still largely unknown. LC have long been considered mandatory for CD8+ T-cell priming in CHS, however, this paradigm has recently been challenged in experiments using different LC-deficient mouse models (
      • Bennett C.L.
      • Clausen B.E.
      DC ablation in mice: promises, pitfalls, and challenges.
      ;
      • Kaplan D.H.
      • Kissenpfennig A.
      • Clausen B.E.
      Insights into Langerhans cell function from Langerhans cell ablation models.
      ).
      To date, two types of LC ablation models have been generated and analyzed in CHS. On one hand, LC-deficient Langerin-DTA transgenic mice, which constitutively lack LC from birth, whereas other Langerin-expressing DC populations, in particular, Langerin+ dermal DC are not affected (
      • Kaplan D.H.
      • Jenison M.C.
      • Saeland S.
      • et al.
      Epidermal langerhans cell-deficient mice develop enhanced contact hypersensitivity.
      ). On the other hand, two Langerin-DTR knock-in strains, which enable the inducible depletion of LC and other Langerin+ DC by the injection of DT (
      • Bennett C.L.
      • van Rijn E.
      • Jung S.
      • et al.
      Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity.
      ;
      • Kissenpfennig A.
      • Henri S.
      • Dubois B.
      • et al.
      Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells.
      ). Against common expectation from the ‘LC paradigm’ (
      • Wilson N.S.
      • Villadangos J.A.
      Lymphoid organ dendritic cells: beyond the Langerhans cells paradigm.
      ), CHS reactions were not abolished in any of these LC ablation models indicating that dermal DC can contribute to the priming of hapten-specific CD8+ T cells in CHS (
      • Bennett C.L.
      • Clausen B.E.
      DC ablation in mice: promises, pitfalls, and challenges.
      ;
      • Kaplan D.H.
      • Kissenpfennig A.
      • Clausen B.E.
      Insights into Langerhans cell function from Langerhans cell ablation models.
      ). The reasons why Langerin-DTA mice show enhanced CHS are not clear. It has been interpreted to suggest that LC may have a regulatory function in vivo (
      • Kaplan D.H.
      • Jenison M.C.
      • Saeland S.
      • et al.
      Epidermal langerhans cell-deficient mice develop enhanced contact hypersensitivity.
      ). Alternatively, one may speculate that exaggerated T cell immunity in Langerin-DTA mice results from the constitutive LC deficiency and failing peripheral tolerance mechanisms during development of the immune system. Therefore, whether or not LC are, e.g., required for efficient skin graft rejection remains controversial (
      • Merad M.
      • Hoffmann P.
      • Ranheim E.
      • et al.
      Depletion of host Langerhans cells before transplantation of donor alloreactive T cells prevents skin graft-versus-host disease.
      ;
      • Obhrai J.S.
      • Oberbarnscheidt M.
      • Zhang N.
      • et al.
      Langerhans cells are not required for efficient skin graft rejection.
      ).
      By injecting DT after sensitization, but before hapten challenge into the two inducible LC depletion models, it was consistently found that LC (and Langerin+ dermal DC) have no role in regulating the effector T cell response in CHS. In contrast, when DT was given during or shortly before sensitization CHS reactions were reported to be similar (
      • Kissenpfennig A.
      • Henri S.
      • Dubois B.
      • et al.
      Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells.
      ) or diminished (
      • Bennett C.L.
      • van Rijn E.
      • Jung S.
      • et al.
      Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity.
      ) as compared with WT. In 2007, it was discovered that not all Langerin+ cells in the dermis were transmigrating LC, but that a second phenotypically distinct Langerin+ DC subset was located in the dermis (
      • Bursch L.S.
      • Wang L.
      • Igyarto B.
      • et al.
      Identification of a novel population of Langerin+ dendritic cells.
      ;
      • Ginhoux F.
      • Collin M.P.
      • Bogunovic M.
      • et al.
      Blood-derived dermal langerin+ dendritic cells survey the skin in the steady state.
      ;
      • Poulin L.F.
      • Henri S.
      • de Bovis B.
      • et al.
      The dermis contains langerin+ dendritic cells that develop and function independently of epidermal Langerhans cells.
      ). Thus, the initial CHS experiments in Langerin-DTR mice were in fact performed in the absence of both Langerin+ skin DC subsets. After it was described in the French mice that the dermal Langerin+ DC recover faster from the toxin treatment than the LC, these discordant CHS results were reconciled by documenting reduced CHS in the absence of all Langerin+ skin DC, whereas CHS was indistinguishable from WT when only LC were absent (
      • Bursch L.S.
      • Wang L.
      • Igyarto B.
      • et al.
      Identification of a novel population of Langerin+ dendritic cells.
      ). From this it was proposed that dermal Langerin+ DC, but not LC, are critical to mediate CHS.
      In this study, we establish that also in the Dutch Langerin-DTR mice a fraction of dermal Langerin+ DC has returned already 2 weeks after DT, when the epidermis is still devoid of LC. This confirms earlier observations (
      • Nagao K.
      • Ginhoux F.
      • Leitner W.W.
      • et al.
      Murine epidermal Langerhans cells and langerin-expressing dermal dendritic cells are unrelated and exhibit distinct functions.
      ) and is in line with the repopulation kinetics of DC depleted from CD11c-DTR mice, which fully recover to pre-DT levels within 1 week (
      • Jung S.
      • Unutmaz D.
      • Wong P.
      • et al.
      In vivo depletion of CD11c(+) dendritic cells abrogates priming of CD8(+) T cells by exogenous cell-associated antigens.
      ). Although it is not clear why in the skin there is only partial (<50%) reconstitution of LC and Langerin+ dermal DC in the steady state (Figure 1b and (
      • Bennett C.L.
      • van Rijn E.
      • Jung S.
      • et al.
      Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity.
      ;
      • Bursch L.S.
      • Wang L.
      • Igyarto B.
      • et al.
      Identification of a novel population of Langerin+ dendritic cells.
      )), the two inducible Langerin-DTR models show similar depletion and repopulation dynamics of Langerin+ (skin) DC populations (Figure 1c).
      During our earlier experiments, we observed that differences in ear swelling between DT-treated and -untreated mice were less significant at standard concentrations as compared with low concentrations of hapten, e.g., trinitrochlorobenzene (
      • Bennett C.L.
      • van Rijn E.
      • Jung S.
      • et al.
      Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity.
      ) and oxazolone (
      • Bennett C.L.
      • Noordegraaf M.
      • Martina C.A.
      • et al.
      Langerhans cells are required for efficient presentation of topically applied hapten to T cells.
      ) and proposed that LC are mainly required for efficient induction of CHS when antigen dissemination into the dermis is limited. This observation was anticipated by
      • Bacci S.
      • Alard P.
      • Dai R.
      • et al.
      High and low doses of haptens dictate whether dermal or epidermal antigen-presenting cells promote contact hypersensitivity.
      , who also suggested that the requirement for LC in CHS was only given at low doses of hapten. In elegant syngeneic grafting experiments, the authors demonstrate that epidermal grafts obtained from ear skin treated with DNFB at a low dose sensitized the recipient mice. In contrast, dermal grafts failed to induce CHS when the donor skin received an epicutaneous application of a low dose of DNFB, whereas vigorous CHS was detected by dermal grafts prepared from skin treated with a high dose of DNFB. This hypothesis of antigen diffusion/access is in agreement with the requirement of LC for effective tumor immunotherapy after epicutaneous immunization with low dose of antigen (
      • Stoitzner P.
      • Green L.K.
      • Jung J.Y.
      • et al.
      Tumor immunotherapy by epicutaneous immunization requires langerhans cells.
      ), however, our previous studies were carried out before the Langerin+ dermal DC were described. To determine whether the magnitude of a CHS reaction is a function of antigen dose or the presence of Langerin+ dermal DC, we assessed both low- and high-hapten dose CHS in mice lacking all Langerin+ DC or selectively depleted of epidermal LC (Figures 2 and 3). Consistent with our previous data and with
      • Bursch L.S.
      • Wang L.
      • Igyarto B.
      • et al.
      Identification of a novel population of Langerin+ dendritic cells.
      , CHS responses are attenuated in the absence of all Langerin+ cells applying both a low and a higher dose of oxazolone or DNFB onto the skin. In contrast, when only epidermal LC are absent CHS is still diminished at a low-hapten dose, but restored to WT at a higher concentration of both oxazolone and DNFB (summarized in Table 1). Notably, low-dose CHS is similarly reduced in DT-treated French Langerin-DTR mice irrespective of the presence or absence of Langerin+ dermal DC (Figure 4). Furthermore, both Langerin-DTR models equally respond in high-dose CHS (Figure 5), which excludes any ambiguity concerning the different Langerin-DTR strains.
      While this paper was under revision, a study by
      • Honda T.
      • Nakajima S.
      • Egawa G.
      • et al.
      Compensatory role of Langerhans cells and langerin-positive dermal dendritic cells in the sensitization phase of murine contact hypersensitivity.
      using the French Langerin-DTR model reported reduced CHS to DNFB in the absence of all Langerin+ skin DC, whereas CHS was similar to WT in mice lacking either LC or Langerin+ dermal DC. In agreement with the present study, this suggests that both Langerin+ skin DC subsets play compensatory roles in the sensitization phase of CHS. However, it remains elusive why the authors fail to detect the essential role of LC for efficient sensitization at a low antigen dose as described in this study and observed by others (
      • Bacci S.
      • Alard P.
      • Dai R.
      • et al.
      High and low doses of haptens dictate whether dermal or epidermal antigen-presenting cells promote contact hypersensitivity.
      and
      • Stoitzner P.
      • Green L.K.
      • Jung J.Y.
      • et al.
      Tumor immunotherapy by epicutaneous immunization requires langerhans cells.
      ). Since we have assessed the same antigen (DNFB), depletion timing (DT day -1 and day -10) and LC ablation model (French Langerin-DTR mice) as used by Honda et al., a difference in antigen concentration seems to be the only remaining explanation for this discordant observation.
      Taken together, we demonstrated that the population dynamics of, in particular, Langerin+ dermal DC is similar in the Dutch and French Langerin-DTR mice and that both strains mount comparable CHS responses when tested side-by-side. These data strongly suggest that the magnitude of a CHS reaction depends on the concentration of a given hapten and the number of DC present in the skin, which activate naive hapten-specific T cells in the LN. This confirms and extends classical observations on the role of LC in CHS (
      • Toews G.B.
      • Bergstresser P.R.
      • Streilein J.W.
      Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB.
      ;
      • Bacci S.
      • Alard P.
      • Dai R.
      • et al.
      High and low doses of haptens dictate whether dermal or epidermal antigen-presenting cells promote contact hypersensitivity.
      ). In particular, the degree of CHS is not dependent on a specific skin DC subset, i.e., Langerin+ dermal DC. Therefore, LC and (Langerin+) dermal DC exert redundant functions in mediating CHS.

      Materials and Methods

      Mice

      WT C57Bl/6 mice were obtained from Harlan. Generation of the Dutch Langerin-DTR knock-in mice expressing the high-affinity human DTR has been described previously (
      • Bennett C.L.
      • van Rijn E.
      • Jung S.
      • et al.
      Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity.
      ). Mice were housed in the animal facilities of the Academic Medical Center (Animal Research Institute Amsterdam, University of Amsterdam) or Innsbruck Medical University, except for the French Langerin-DTR mice (
      • Kissenpfennig A.
      • Henri S.
      • Dubois B.
      • et al.
      Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells.
      ), which were only kept in Innsbruck. All animal experimentation was in compliance with EU as well as national laws and approved by the local ethical committees.

      Toxin treatment

      As reported previously, mice were intraperitoneally injected with 400ng DT (Sigma-Aldrich, Zwijndrecht, the Netherlands) in phosphate-buffered saline (
      • Bennett C.L.
      • van Rijn E.
      • Jung S.
      • et al.
      Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity.
      ). For the inducible depletion of all Langerin+ DC, mice were treated with DT 2 days prior to induction of CHS. To specifically deplete epidermal LC, mice were injected 14 days before hapten sensitization.

      FITC painting

      FITC (Sigma-Aldrich) was dissolved in DMSO and diluted to 1% in 1/1 acetone/dibutylphthalate (Sigma-Aldrich). A volume of 25μl was painted on the dorsal side of both ears. After 48hours, draining LN were collected and DC were isolated and analyzed by flow cytometry.

      Induction of CHS

      To elicit CHS, WT and DT-treated Langerin-DTR mice were sensitized with 50μl of either 0.5 or 2% oxazolone (Sigma) in acetone:olive oil (4:1) on the shaved abdominal skin. Five days later, mice were challenged with 20μl of 0.25 or 0.5% oxazolone on the right ear. Ear swelling was measured at 24 and 48hours post challenge. CHS responses were calculated as the difference between ear thickness before and after challenge. Alternatively, mice were sensitized with 50μl of either 0.5 or 1% DNFB (Sigma-Aldrich) on day 0 and challenged with 20μl of 0.25 or 0.5% DNFB on the right ear 5 days later. In the experiments depicted in Figures 4 and 5, the mice were sensitized with 50μl of 0.5% or 2% oxazolone and challenged with 20μl of 0.25% or 0.5% oxazolone on both sides of one ear. Swelling was measured as the difference of challenged (right) and unchallenged (left) ear.

      Isolation of DC

      Epidermal and dermal cells were prepared from the ears of C57BL/6 and DT-treated Langerin-DTR mice. Dorsal and ventral sides of ears were floated on a solution of 1.2Uml−1 dispase (Roche Applied Bioscience, Almere, the Netherlands) in RPMI for 90minutes at 37°C, in order to separate dermis and epidermis. Next, the epidermis was incubated in 0.5% trypsin in RPMI and the dermis in 500Uml−1 collagenase D (Worthington Biochemicals, Lakewood, NJ) in RPMI for 45minutes at 37°C. Epidermis and dermis were then mechanically scraped and passed through a 70μm filter before the cells were washed and resuspended in fluorescence activated cell sorting buffer. Skin-draining LN were harvested and incubated with collagenase D (Worthington Biochemicals) and EDTA to facilitate release of DC.

      Whole-skin explant culture

      Briefly, ears of mice previously treated with phosphate-buffered saline or DT were cut off at the base, and ear skin was split into dorsal and ventral halves (
      • Stoitzner P.
      • Holzmann S.
      • McLellan A.D.
      • et al.
      Visualization and characterization of migratory Langerhans cells in murine skin and lymph nodes by antibodies against Langerin/CD207.
      ). The dorsal halves were cultured in 24-well plates (one per well) in complete culture medium, without additional chemokines. After 4 days, emigrant DC were pooled from four to six individual ear skin explants, and investigated by flow cytometry.

      Flow cytometry

      For flow cytometric analysis, cells were incubated with Fc Block (2.4G2, BD Pharmingen, Rotterdam, the Netherlands) and stained with anti-CD11c-PE, (N418, eBioscience, Frankfurt, Germany), anti-MHCII-PE (M5/114, BD Pharmingen), anti-EpCam-PerCp-Cy5.5 (G8.8, Biolegend, Uithoorn, the Netherlands) and anti-CD103-Alexa647 (2E7, Biolegend). For intracellular staining with anti-Langerin-Alexa488 or anti-Langerin-biotin (929.F3, Dendritics, Lyon, France), cells were fixed in 2% paraformaldehyde and stained in 0.5% saponin buffer. PE-Cy7-conjugated streptavidin (BD Pharmingen) was used to visualize biotin-conjugated antibody. Experiments were performed on a FACSCanto and analyzed using FlowJo software (Becton Dickinson, Breda, the Netherlands).

      Immunofluorescence on ear cryosections

      Cross-sections from ears were fixed in acetone and stained according to standard procedures with anti-Langerin (929.F3, Dendritics) followed by anti-rat-Cy3 (Jackson Immunoresearch Laboratories, Suffolk, UK) and anti-MHCII-Alexa488 (M5/114, BD Pharmingen). Images were viewed using a DMRA fluorescence microscope (Leica, Rijswijk, the Netherlands). Pictures were taken with a Kx14 camera (Apogee Instruments, Roseville, CA).

      ACKNOWLEDGMENTS

      We thank Sonja Zahner, Junda Kel, and Mathilde Girard-Madoux for many helpful discussions, Inge Brouwers-Haspels for expert technical assistance, Bernard Malissen for kindly providing French Langerin-DTR mice, Niki Romani for his continuous encouragement and support, and Jon Laman and Nikolaus Romani for critically reading of the manuscript. This work was supported by grants from The Netherlands Organization for Scientific Research (NWO, VIDI 917.76.365) and the Landsteiner Foundation for Blood Transfusion Research (LSBR, 0414F) to BEC. PS is the recipient of research grants from the Innsbruck Medical University (MFI-9442) and the Austrian Science Fund (FWF-21487).

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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