Expansion of antigen-specific regulatory T cells with the topical vitamin d analog calcipotriol

Abstract

1,25-Dihydroxyvitamin D(3) is immunosuppressive both in vivo and in vitro. Topical vitamin D analogs such as calcipotriol alter keratinocyte function, but their effects on cutaneous immune responses are less well understood. We demonstrate that exposure of the skin to calcipotriol before transcutaneous immunization with OVA protein and CpG adjuvant prevents Ag-specific CD8(+) T cell priming coincident with Langerhans cell depletion in the skin. Immunization through calcipotriol-treated skin induces CD4(+)CD25(+) regulatory T cells (Treg) that prevent subsequent Ag-specific CD8(+) T cell proliferation and IFN-gamma production. Treg induced by calcipotriol are able to inhibit the induction and the elicitation of protein contact hypersensitivity. Topical calcipotriol treatment also induces RANKL (receptor activator of NF-kappaB ligand) expression by keratinocytes, a TNF family member involved in modulation of skin dendritic cells. UV light B induces Ag-specific tolerance when it is applied before transcutaneous immunization. We suggest that UV light B-induced tolerance is induced via a vitamin D receptor-dependent mechanism as vitamin D receptor (VDR) knockout mice fail to increase FoxP3(+) Treg in their peripheral draining lymph node following irradiation. Additionally, keratinocytes of VDR(-/-) mice fail to induce RANKL upon UV irradiation or calcipotriol treatment. The in vivo expansion of Ag-specific Treg with the topical application of the vitamin D analog calcipotriol followed by transcutaneous immunization is a simple method to augment functional Ag-specific CD4(+)CD25(+)Foxp3(+) Treg populations and mimics Ag-specific UV-induced tolerance.

Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1

Effect of topical calcipotriol upon the priming of CD8+ T cells to cutaneous Ag. CFSE+OT-I CD8+ T cells were transferred into naive B6 mice that were then treated three times daily on the back with 30 mg of calcipotriol ointment 50 μg/g or vehicle (plasticized base). Animals were next topically immunized (TCI) with OVA protein (500 μg) and CpG (500 μg). OT-I cells from the draining LN were assessed for proliferation and IFN-γ production 3 days later as indicated in F. A, Representative histograms and dot plots gated for CD8+ cells. Percentages indicate the proportion of CFSE+ cells that have divided at least once and express IFN-γ, respectively. B, Proliferation and IFN-γ production normalized to OVA-immunized but previously untreated mice. C, Suppressive effect of calcipotriol on OT-I proliferation and IFN-γ production was observed only when subsequent immunization was performed at the same site (back (same site) vs back and abdomen (different site); n = 4). D, Calcipotriol application resulted in depletion of MHC-II+ cells in skin epidermal sheets (n = 2–4). E, Calcipotriol treatment before immunization inhibited induction of OVA protein CHS as measured by ear swelling in mice following OVA application to the ears (n = 6 mice/group; sum of two experiments); all error bars represent SEM.

FIGURE 2

Topical calcipotriol followed by TCI induces Ag-specific CD4+CD25+ Treg. A, The fraction of CD25+ T cells among CD4+ T cells in the skin-draining LN was determined after three daily applications of calcipotriol, followed by OVA immunization (n = 6/group). B, Effect of CD4+ T cell subsets transferred from calcipotriol-treated mice upon CD8+ T cell priming responses in previously naive mice. CD4+CD25+ T cells or CD4+CD25− T cells from calcipotriol-treated and OVA-immunized donors were purified from peripheral LN and spleen 4 days after immunization and were transferred into recipients along with OT-1 CD8+ T cells (as depicted in E). Recipients were next immunized with OVA/CpG. CD4+ CD25+ T cells from calcipotriol and OVA-immunized mice suppressed proliferation of OT-I cells (n = 6/group; ordinate shows percentage control response). C, Ag specificity of CD4+CD25+ cells. No inhibition of CD8+ T cell proliferation or IFN-γ production was noted when 2 × 106 purified CD4+CD25+ T cells from mice treated with calcipotriol and immunized with BSA were transferred (n = 4). D, Calcipotriol-induced Treg efficiently prevented OT-I cell proliferation in vivo. As few as 5 × 104 cells were sufficient to suppress the proliferation of 5 × 106 adoptively transferred OT-I cells.

FIGURE 3

Calcipotriol-induced Treg inhibit Ag-specific CTL priming and CHS elicitation responses in vivo. A and B, Calcipotriol-induced Treg suppressed CHS response in an Ag-specific manner. CD4+CD25+ cells were isolated from the LN and spleen of donor mice (either treated with calcipotriol or untreated) 4 days after OVA or BSA immunization and were transferred into (primed) recipients immunized 4 days previously with either OVA or BSA (as depicted in A). The following day, protein CHS responses were elicited using the same protein that was used for immunization of recipients. Data are representative of two experiments (n = 5 mice/group). C, Calcipotriol-induced Treg suppressed CTL expansion. Donor mice were immunized with OVA after 3 days of topical calcipotriol administration. Four days later, 2 × 106 CD4+CD25+ cells were isolated form peripheral LN and spleen and transferred into naive recipients. Recipients were then immunized with topical OVA 1 day after transfer and reimmunized (boosted) on day 7. Kb OVA-specific CTL in the skin-draining LN were enumerated by tetramer staining on day 13. The fraction of OVA-specific CTL among CD8+ T cells was significantly less in groups receiving Treg compared with controls.

FIGURE 4

Effect of topical calcipotriol and Ag immunization upon CD4+CD25+FoxP3+ T cells. A, Calcipotriol treatment before TCI expands FoxP3+ Treg. Wild-type (B6) or OT-II TCR transgenic mice were either treated with topical calcipotriol for 3 days or untreated and then immunized with OVA and CpG on the following day. Draining LN were then examined for CD4, CD25, and FoxP3 expression on day 4 (n = 3; representative of two experiments). B, OVA Ag-specific Treg are inducible by TCI following calcipotriol treatment. The proportion of CD25+FoxP3+ cells among CD4+ T cells was increased following topical calcipotriol and OVA treatment. This increase was Ag specific in OT-II mice (open bars) and occurred in an Ag-nonspecific fashion in B6 mice (filled bars) (n = 3; representative of two experiments). C, OVA immunization following calcipotriol treatment resulted in a significant increase in the number of Treg compared with calcipotriol treatment alone. The proportion of CD25+FoxP3+ cells among CD4+ T cells in the skin-draining LN was determined 4 days after the topical treatment of B6 mice as indicated. A significant expansion of the proportion of Treg occurred following combined application of calcipotriol and OVA Ag with or without application of topical CpG (n = 4; representative of two experiments). D and E, OVA immunization following calcipotriol treatment up-regulates CTLA-4 in Treg. FoxP3gfp mice were either treated with calcipotriol followed by OVA immunization or left untreated. Four days after immunization, mononuclear cells were isolated from the peripheral LN and spleen, were stained for CTLA-4, and FoxP3-expressing cells were identified by green fluorescence (D). The scatter plot in E includes data from two experiments.

FIGURE 5

Induction of Treg and RANKL is VDR dependent. A, VDR is required for the induction of CD4+CD25+FoxP3+ cells mediated by UVB. Mice were treated with UVB (1000 J/m2) for 4 successive days or were untreated. Three days later, the proportion of CD4+CD25+FoxP3+ cells among CD4+ T cells in the peripheral LN was determined (n = 3; representative of two experiments). B, VDR expression is required for UV induction of RANKL expression. Mice were treated with UVB or calcipotriol. Three days later, skin samples were harvested and RANKL expression was then determined by immunohistochemistry. Calcipotriol induced keratinocyte RANKL expression equivalent to the induction by UV light through VD receptor. Images are representative of three animals per group.