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The Majority of Human Peripheral Blood CD4⁺CD25^highFoxp3⁺ Regulatory T Cells Bear Functional Skin-Homing Receptors¹

Harvard Skin Disease Research Center, Department of Dermatology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115

	Abstract

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CD4⁺CD25⁺ T regulatory cells (Treg) are thought to be important in the peripheral tolerance. Recent evidence suggests that human peripheral blood CD4⁺CD25⁺ T cells are heterogeneous and contain both CD4⁺CD25^high T cells with potent regulatory activity and many more CD4⁺CD25^low/med nonregulatory T cells. In this study, we found that virtually all peripheral blood CD4⁺CD25^highFoxp3⁺ Treg expressed high levels of the chemokine receptor CCR4. In addition, 80% of Treg expressed cutaneous lymphocyte Ag (CLA) and 73% expressed CCR6. These molecules were functional, as CLA⁺ Treg showed CD62E ligand activity and demonstrable chemotactic responses to the CCR4 ligands CCL22 and CCL17 and to the CCR6 ligand CCL20. The phenotype and chemotactic response of these Treg were significantly different from those of CD4⁺CD25^med nonregulatory T cells. We further demonstrated that blood CLA⁺ Treg inhibited CD4⁺CD25^– T cell proliferation induced by anti-CD3. Based on homing receptor profile, CLA⁺ Treg should enter normal skin. We next isolated CD4⁺CD25^high T cells directly from normal human skin; these cells suppressed proliferation of skin CD4⁺CD25^– T cells. Therefore, the majority of true circulating Treg express functional skin-homing receptors, and human Treg may regulate local immune responses in normal human skin.

	Introduction

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The active maintenance of peripheral immune tolerance is important to prevent autoimmune diseases and to dampen potentially damaging local immune responses in peripheral tissues such as skin. Because of its role as a critical interface with the external environment, a sophisticated cutaneous immune surveillance system has developed, wherein effective recruitment of skin-homing memory T cells from blood into skin is important (1). The role played by T regulatory cells (Treg)⁴ in cutaneous immunity is not well understood. There is accumulating evidence that human CD4⁺CD25⁺ Treg normally mediate peripheral tolerance by active immune suppression, and that altered function of CD4⁺CD25⁺ Treg may be an important factor in a wide range of human autoimmune and inflammatory diseases, including inflammatory skin diseases (2, 3, 4, 5). Very recently, it has become clear that human peripheral blood CD4⁺CD25⁺ T cells are heterogeneous and contain both CD4⁺CD25^high cells (2–3% of CD4⁺ T cells) with potent Treg activity, as well as many more CD4⁺CD25^med nonregulatory cells (6). These blood CD4⁺CD25^high Foxp3⁺ Treg mediate suppression in a contact-dependent manner, and neither IL-10 nor TGF- are involved in this suppression (6, 7). Although unfractionated CD4⁺CD25⁺ T cells from blood have been studied extensively, the phenotypical and functional heterogeneity of this population have proved confounding. In particular, the chemokine receptor, selectin-selectin ligand, and integrin expression on purified and functional CD4⁺CD25^high Treg has not been well characterized to date.

Differential expression of adhesion molecules and chemokine receptors determine specific migration of leukocytes into distinct tissues and microenvironments. Skin-associated memory T cells, for example, are defined by expression of the cutaneous lymphocyte Ag (CLA) (8, 9, 10) and CCR4 (11, 12). It has been reported that majority of human peripheral blood CD4⁺CD25^high Treg express CD62L (6) and CCR7 (13). This combination should allow Treg to enter from blood into secondary lymphoid tissues, suggesting a role for Treg in controlling systemic immunity as central memory T cells (14) at secondary lymphoid organs. In contrast, it has also been reported that unfractionated blood CD4⁺CD25⁺ T cells express variable levels of peripheral tissue-homing receptors such as skin-associated CLA (4, 15), CCR4 (16), and gut-associated ₄₇ integrin (17). In addition, Iellem et al. (15) reported Treg express skin- vs gut-skewed homing receptors, describing that 30% of CD4⁺CD25⁺ T cells express CLA. However, the peripheral tissue-homing specificity of the true CD4⁺CD25^high Treg remains obscure.

In this study, we showed that, surprisingly, the vast majority of the pure CD4⁺CD25^high Foxp3⁺ Treg circulating in peripheral blood express skin-associated homing receptors CLA, uniformly high levels of CCR4, and abundant CCR6. In addition, we will also show that skin T cells from normal human skin include many CD4⁺CD25^high Treg. Our results shed light upon a role of human Treg in cutaneous immune surveillance in terms of maintaining peripheral tolerance in normal skin.

	Materials and Methods

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Abs and reagents

mAbs to human CD3 (HIT3a and UCHT1), CD4 (RPA-T4, L200), CD11a (G-25.2), CD25 (M-A251), CD28 (CD28.2), CD45RA (HI100), CD45RO (UCHL1), CD49d (9F10), CD62L (SK11), CD69 (L78), CCR4 (1G1), CD195 (2D7/CCR5), CCR6 (11A9), CD183 (1C6/CXCR3), CD184 (12G5), CLA (HECA-452), and ₇ integrin (FIB504) and the isotype controls to these Abs were purchased from BD Biosciences. mAbs to human CD4 (11830), CCR7 (150503), CCR8 (191704), CXCR5 (51505.111), and CXCR6 (56811) were purchased from R&D Systems. mAbs to Foxp3 (PCH101) and CD25 (BC96) and the isotype controls to these Abs were purchased from eBioscience. Recombinant human CCL17, CCL19, CCL20, CCL21, CCL22, CD62E/Fc chimera, CD62P/Fc chimera, IL-2, and IL-15 were purchased from R&D Systems.

Cell isolation from blood

All experiments using human materials were done in compliance with local institutional review board policy. PBMCs were prepared by density gradient separation (Histopaque; Sigma-Aldrich) of peripheral blood or of cells collected during platelet pheresis of normal donors. CD4⁺ T cells were isolated using magnetic beads (CD4⁺ T cell isolation kit II; Miltenyi Biotec). The CD4⁺ T cells were incubated with anti-CD4-PE Cy5, anti-CD25-PE, and anti-CLA FITC Abs, and subpopulations of CD4⁺ T cells were isolated by using FACSAria (BD Biosciences). T cell-depleted accessory cells were obtained by incubating PBMC with anti-CD3 microbeads (Miltenyi Biotec) and irradiated at 3000 rad.

T cell proliferation assay

Peripheral blood CD4⁺CD25^– T cells (2500 cells/well) were cocultured with an indicated subpopulation of CD4⁺ T cells (1250 cells/well, otherwise stated) in the presence of accessory cells (25,000 cells/well) in U-bottom 96-well plates (Costar; Corning). The cells were stimulated either with 1–100 ng/ml soluble anti-CD3 (HIT3a) plus 100 ng/ml soluble anti-CD28 or with 300 ng/ml immobilized anti-CD3 (UCHT1). All cells were cultured in triplicate and in a final volume of 200 µl of RPMI 1640 medium with L-glutamine supplemented with 5 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen Life Technologies), 1 mM sodium pyruvate, nonessential amino acids (Cellgro; Mediatech), and 5% human AB serum (Fisher Scientific). After 5 days of culture, 1 µCi [³H]thymidine was added to each well. The cells were harvested after 16 h, and radioactivity was measured using a liquid scintillation counter (Wallac). The results are expressed as the mean cpm ± SEM of triplicate wells.

Flow cytometric analysis

To detect surface chemokine receptors, a mixed solution of fluorescence-conjugated anti-CD4, anti-CD25, and anti-chemokine receptor Abs was added to whole blood and incubated for 30 min at room temperature. RBC were depleted and the remaining cells were fixed immediately after the lysis using lysing and fixative reagents (R&D Systems) according to the manufacturer’s instruction. To detect other surface molecules than chemokine receptors, T cells were isolated from PBMC using magnetic beads (Pan T Cell Isolation kit II; Miltenyi Biotec) and stained with a mixed solution of fluorescence-conjugated anti-CD4, anti-CD25, and specific Abs to the various kinds of molecules. Thirty minutes later, the cells were fixed. Cells were analyzed with FACScan. To detect intracellular Foxp3 expression, PE anti-human Foxp3 staining set (eBioscience) was used. Briefly, T cells were incubated with anti-CD4 PerCP, anti-CD25 allophycocyanin, and anti-CLA FITC. The cells were fixed, permeabilized, and stained with anti-Foxp3 PE or PE-labeled control Ab according to the manufacturer’s instruction. Cells were analyzed with FACSCanto (BD Bioscience).

Detection of CD62E and CD62P ligands

Peripheral blood CD4⁺ T cells purified as described were incubated with 10 µg/ml CD62E/Fc chimera or CD62P/Fc chimera in HBSS supplemented with 2 mM calcium, 5% FCS, and 1 mM HEPES. To test calcium dependency of the binding, HBSS supplemented with 5 mM EDTA instead of calcium was used. After incubation for 30 min at 4°C, the cells were gently washed and incubated with biotin-conjugated goat F(ab')₂ anti-human IgG (BD Bioscience) for 30 min at 4°C. The cells were washed and incubated with streptavidin-PerCP for 30 min at 4°C. The cells were subsequently washed and stained with anti-CD4 allophycocyanin, anti-CD25 PE, and anti-CLA FITC. The cells were analyzed with FACSCalibur (BD Bioscience).

Chemotaxis assay

Chemotaxis assays were performed using Transwell plates with 5-µm pores (Corning). T cells were prepared from PBMC by using magnetic beads (Pan T Cell Isolation kit II; Miltenyi Biotec). Upper wells were loaded with 2 x 10⁵ T cells per well and the cells were allowed to chemotaxis for 3 h at 37°C. After chemotaxis, migrated cells in bottom wells were collected and stained with anti-CD4 and anti-CD25. The number of viable migrated cells and input cells were counted by flow cytometry using quantification beads, and the percentage of net migrated cells was calculated. The results are expressed as the mean ± SEM of duplicate wells.

Skin T cell assay

Skin T cells were prepared by three-dimensional skin explant cultures as previously described (18, 19). Skin obtained from cosmetic surgery procedures was minced into explants 2 mm x 2 mm x 2 mm in size. The skin explants were placed on the surface of cellfoam matrices (Cytomatrix) (20) that had been preincubated in a solution of 100 µg/ml rat tail collagen I (BD Biosciences). The culture was maintained in the presence of 25 ng/ml IL-2 and 20 ng/ml IL-15, which were added to expand skin-resident memory T cells, in IMDM (Mediatech) with 10% FBS (Sigma-Aldrich), 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME in a 24-well plate. After 3 wk, T cells that had migrated out of explants into the culture wells were collected.

The skin T cells were incubated with anti-CD4-PE Cy5, anti-CD25-PE, and anti-CD69-FITC, and subpopulations of CD4⁺ T cells were isolated by using FACSAria. Irradiated T cell-depleted PBMC were prepared from a blood donor who was different from the skin donor and used as accessory cells. Skin CD4⁺CD25^– T cells (2500 cells/well) were cocultured with a selected population of skin CD4⁺ T cells (1250 cells/well) in the presence of the accessory cells (25,000 cells/well). The cells were stimulated with 10 ng/ml soluble anti-CD3 (HIT3a) plus 100 ng/ml soluble anti-CD28. The cell proliferation was measured as earlier described.

	Results

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CD4⁺CD25^high T cells from peripheral blood are functional human Treg

At the outset we wished to confirm the observation that human peripheral blood CD4⁺CD25⁺ cells contain both CD4⁺CD25^high regulatory and CD4⁺CD25^low/med nonregulatory cells (6). We isolated highly pure CD4⁺CD25^–, CD4⁺CD25^med, and CD4⁺CD25^high T cells (Fig. 1A) from peripheral blood of a healthy donor and conducted a T cell proliferation assay. We set the gate for the isolation of CD4⁺CD25^high cells so that cells in the gate may represent 2–3% of total CD4⁺ T cells. CD4⁺CD25^– T cell proliferation induced by anti-CD3 and anti-CD28 stimulation was strongly inhibited when the cells were cocultured with CD4⁺CD25^high T cells but not with CD4⁺CD25^med T cells or CD4⁺CD25^– T cells (Fig. 1B). CD4⁺CD25^high T cells did not proliferate to the CD3 stimulation in the single culture, suggesting that the CD4⁺CD25^high T cells were anergic. The inhibitory effect of CD4⁺CD25^high T cells on CD4⁺CD25^– T cell proliferation was dependent on the number of CD4⁺CD25^high T cells added to the wells (Fig. 1C). We also found that CD4⁺CD25^high T cells could not inhibit strong CD4⁺CD25^– T cell proliferation induced by immobilized anti-CD3 (data not shown). These results were consistent with previous findings about human Treg (6, 7), and we concluded that CD4⁺CD25^high T cells, but not CD4⁺CD25^med T cells, are true human Treg.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Human peripheral blood CD4+CD25high T cells, but not CD4+CD25med T cells, inhibited CD4+CD25– T cell proliferation. A, Isolated CD4+ T cells were stained with anti-CD4 and anti-CD25 Abs. Gated areas are used to sort CD4+CD25–, CD4+CD25med, and CD4+CD25high T cells. B, CD4+CD25high T cells (top) were cultured alone () or cocultured with CD4+CD25– (•), CD4+CD25med (), and CD4+CD25high () T cells at a 2:1 ratio (solid line). Single culture (bottom) of CD4+CD25– (), CD4+CD25med (), and CD4+CD25high () T cells (1250/well) was also conducted. These cells were stimulated with or without soluble anti-CD3 and anti-CD28. C, A constant number of CD4+CD25– T cells were cocultured with the number of cells/well and stimulated with soluble anti-CD3 and anti-CD28 (100 ng/ml each).

We next investigated the expression profile of tissue-homing receptors on CD4⁺CD25^high Treg and compared it with the profile on CD4⁺CD25^med and CD4⁺CD25^– T cells. We found that virtually all CD4⁺CD25^high Treg expressed high levels of CD45RO and CCR4. With regard to adhesion molecules and other chemokine receptors, 80% (range from 68 to 90%) of Treg expressed CLA and 73% (range from 62 to 84%) expressed CCR6 (Fig. 2). No significant expression of CCR8 was noted. Consistent with the notion that skin-associated CLA and gut-associated ₄₇ integrin are reciprocally expressed by memory T cells, only 6% (range from 2 to 9%) of CD4⁺CD25^high Treg expressed ₇ integrin. Although nearly all Treg expressed CD62L, only 60% expressed CCR7. In contrast, 57% of CD4⁺CD25^med nonregulatory T cells expressed CCR4, and only 37% expressed CLA, and 38% expressed CCR6. In addition to CLA and CCR4, an important role of CCR6 on skin-homing memory T cells has also been described (21, 22). In contrast, relatively few peripheral blood CD4⁺CD25^high Treg expressed ₄₇ integrin, one of the receptors that would permit them access to lamina propria in the gut. Therefore, the highly purified CD4⁺CD25^high Treg were strikingly enriched in putative skin-homing T cells.

View larger version (45K): [in this window] [in a new window]   FIGURE 2. Majority of CD4+CD25high Treg-expressed skin-homing receptors. Human peripheral blood T cells were isolated and stained with anti-CD4, anti-CD25, and mAbs specific to the indicated molecules. A, Data are expressed as histograms for CD4+CD25– and CD4+CD25med T cells but as dot plots for CD4+CD25high T cells because the number of CD4+CD25high Treg was too small to be presented as a histogram. Cells were stained with specific mAb (filled histograms) or isotype control (open histograms). A parting line in the dot plots is based on isotype control. The numbers indicate percentages of positive cells. B, FACS profiles of the three T cell subpopulations are summarized (n = 6). Each circle shows individual data; bar, mean value; SSC, side scatter.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Differential expression of Foxp3 and CLA on CD4+CD25high, CD4+CD25med, and CD4+CD25– T cells. Peripheral blood T cells were costained with anti-CD4, and anti-CD25 and with anti-CLA, and anti-Foxp3. Three gates were set based on the intensity of CD25 by CD4+ T cells to identify CD4+CD25high, CD4+CD25med, and CD4+CD25– T cells. One representative from identical results is shown. Values shown are the percentage of positive cells in each quadrant.

We next investigated the functional activity of CLA, CCR4, and CCR6 expressed by Treg. We tested whether CLA expression by Treg was associated with acquisition of binding activity to CD62E as judged by flow cytometry. We found a strong and significant correlation between CLA expression and CD62E-binding activity (Fig. 4). Virtually all CLA⁺ Treg showed CD62P-binding activity as well. In contrast, CD4⁺CD25^med T cells showed less binding activity to CD62E and CD62P in proportion to lower numbers of CLA⁺ cells. There were some CLA⁺ CD4⁺ T cells that did not show binding activity to CD62E, which we attribute to the very low affinity of the CD62E chimera. The binding to CD62E and CD62P was calcium dependent, as no binding activity was detected in medium containing EDTA. No positive binding was detected when control human IgG instead of the chimeras was used (data not shown).

View larger version (35K): [in this window] [in a new window]   FIGURE 4. CLA+ CD4+CD25high T cells showed binding activity to CD62E and CD62P. Blood CD4+ T cells were incubated with CD62E/Fc or CD62P/Fc chimeras in the presence of either calcium or EDTA. The cells were costained with anti-CD4, anti-CD25, and anti-CLA. One representative result gating on CD4+CD25high and CD4+CD25med T cell subsets is shown. Values shown are the percentage of positive cells in each quadrant.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. CD4+CD25high T cells showed demonstrable chemotactic responses toward CCR4 and CCR6 ligands. Peripheral blood T cells were allowed to migrate toward CCL17, CCL21 (CCR4 ligands) (upper), CCL20 (CCR6 ligand) (middle), CCL19, and CCL21 (CCR7 ligands) (lower) for 3 h in Transwell plates. After migration, cells were collected and stained with anti-CD4 and anti-CD25. The net migrations of CD4+CD25high Treg to CCL17 (•), CCL22 (), CCL20 (), CCL19 (), and CCL21 () and those of CD4+CD25med T cells to CCL17 (), CCL22 (), CCL20 (), CCL19 (), and CCL21 () are depicted.

To rule out the unlikely possibility that true Treg are included only in CD4⁺CD25^high CLA^– T cells, we further separated CD4⁺CD25^high T cells into CLA⁺ and CLA^– cell subpopulations and assessed their regulatory function. CD4⁺CD25^high CLA⁺ and CLA^– T cells equally inhibited CD4⁺CD25^– T cell proliferation induced by soluble anti-CD3 and anti-CD28 stimulation (Fig. 6A), a phenomenon that was dependent on the number of Treg added to the coculture (Fig. 6B). In keeping with their identity as true Treg, neither of these two subpopulations inhibited CD4⁺CD25^– T cell proliferation induced by immobilized anti-CD3 (Fig. 6A). Thus, authentic Treg were included in both CLA⁺ and CLA^– subpopulations of CD4⁺CD25^high T cells. Overall, analysis on peripheral blood Treg revealed that the large majority of true human Treg with potent regulatory activity express high levels of functional skin-homing receptors, implying that they may normally traffic to skin.

View larger version (36K): [in this window] [in a new window]   FIGURE 6. Both CD4+CD25high CLA+ and CD4+CD25high CLA– T cells inhibited CD4+CD25– T cell proliferation. A, CD4+CD25– T cells were cultured alone () or cocultured with CD4+CD25– (•), CD4+CD25high CLA+ (), or CD4+CD25high CLA– () T cells at a 2:1 ratio, in which the cells were stimulated with or without soluble anti-CD3 and anti-CD28. The cells were also stimulated with immobilized anti-CD3; graph (right) shows the proliferation of CD4+CD25– T cells alone () or those cocultured with CD4+CD25– (), CD4+CD25high CLA+ (), or CD4+CD25high CLA– () T cells are indicated. B, A constant number of CD4+CD25– T cells were cocultured with the indicated number of cells/well and stimulated with soluble anti-CD3 and anti-CD28 (100 ng/ml each).

To determine whether Treg were present in skin, we isolated T cells directly from normal human skin. We cultured explants of skin on specialized matrices that have previously been found to support the survival of skin-resident T cells without altering their phenotype (18, 19). Skin T cells prepared by this method were 78% positive for CLA and 89% positive for CCR4 (18). In the present study, skin T cells were stained with anti-CD4, anti-CD25, and anti-CD69 Abs. CD69 is known as an activation marker for T cells. Many CD4⁺CD25^high T cells, with or without concurrent CD69 expression, were found among skin T cells (Fig. 7A). Because activated nonregulatory T cells expressing CD69 might be contaminating our CD4⁺CD25^high T cells, we isolated CD4⁺CD25^–, CD4⁺CD25^med, CD4⁺CD25^highCD69⁺, and CD4⁺CD25^highCD69^– T cells, and conducted a functional assay to assess the regulatory activity of each T cell subpopulation. Stimulation with soluble anti-CD3 and anti-CD28 Abs induced skin CD4⁺CD25^– T cell proliferation. Surprisingly, coculturing the cells with each of these skin T cell subpopulations, with the exception of CD4⁺CD25^– T cells, significantly inhibited the skin CD4⁺CD25^– T cell proliferation (Fig. 7B). Interestingly, only CD4⁺CD25^– T cells alone among the four subpopulations proliferated in response to CD3 stimulation in a single culture, suggesting these other T cell subsets were anergic (data not shown). These results suggest that the majority of skin CD4⁺CD25^med/high T cells have Treg activity, and that it is independent of their CD69 expression.

View larger version (38K): [in this window] [in a new window]   FIGURE 7. Skin CD4+CD25med/high T cells from normal human skin inhibited skin CD4+CD25– T cell proliferation. A, Skin T cells harvested from skin explants culture were stained with anti-CD4, anti-CD25, and anti-CD69 Abs. Gates are used to sort CD4+CD25–, CD4+CD25med, CD4+CD25highCD69+, and CD4+CD25+CD69– T cells. B, Skin CD4+CD25– T cells were cultured alone () or cocultured with skin CD4+CD25– (), CD4+CD25med (), CD4+CD25highCD69+ (), or CD4+CD25highCD69– () T cells at a 2:1 ratio, in which the cells were stimulated with (+) or without (–) soluble anti-CD3 (10 ng/ml) and anti-CD28 (100 ng/ml).

	Discussion

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We were initially surprised at the high proportion of CD4⁺CD25^high T cells in peripheral blood that expressed both CCR4 and CLA, indicating that they had the potential to gain access to normal skin. The expression of skin-skewed homing receptors by peripheral blood CD4⁺CD25⁺ T cells was previously reported by the other investigators (4, 15); 30% of CD4⁺CD25⁺ T cells expressed CLA and 80% expressed CCR4. These studies, however, did not fractionate CD4⁺CD25⁺ into their functionally distinct components. Our results revealed that the vast majority of pure CD4⁺CD25^high Foxp3⁺ Treg strongly expressed these skin-homing receptors; 80% of Treg expressed CLA and 96% expressed CCR4. In addition, 73% of these cells also expressed CCR6, a chemokine receptor that mediates chemotaxis to the antimicrobial peptide human defensin expressed abundantly in skin (25). The great majority of circulating CLA⁺ T cells in peripheral blood coexpress CCR4 (11), whereas 56% of CLA⁺ T cells coexpress CCR6 (19). Expression of CCR6 may facilitate CLA⁺ Treg migration to skin, but CCR6 does not seem to be a functional marker for Treg because both CCR6⁺ CD4⁺CD25^high Treg and CCR6^– CD4⁺CD25^high Treg from human PBMC show equivalent suppressive activity (26). Also it is unlikely that CLA is a marker for Treg or involved in the mechanism of suppression because both CLA⁺ and CLA^– CD4⁺CD25^high Treg inhibited CD4⁺CD25^– T cell proliferation similarly. It is plausible that CLA^– Treg, which were a relatively minor subpopulation in peripheral blood CD4⁺CD25^high T cells, are serving as systemic Treg that travel into lymph nodes using CD62L and CCR7 and mediate suppression in secondary lymphoid tissues; however, at least some CLA⁺ Treg also express CD62L and CCR7.

The skin-homing molecules expressed by CD4⁺CD25^high Treg cells were all functional. A significant number of CLA⁺CD4⁺CD25^high Treg showed CD62E-binding activity and demonstrable chemotactic responses to CCR4 and CCR6 ligands. Constitutive homing of memory T cells with skin-homing markers into human skin has been recently reviewed (1), and we have reported the coexpression of CD62E, CCL17, and ICAM-1 by dermal vessels in noninflamed human skin (27). Schmuth et al. (28) reported constitutive expression of CCL20 mRNA in normal human dermis and epidermis, so that known ligands for CLA, CCR4, and CCR6 exist on dermal vessels in normal skin. Indeed, we recently reported that vast majority of skin T cells isolated from normal human skin express CLA, CCR4, and CCR6 and that significant numbers of CD4⁺CD25⁺CD69^– T cells with regulatory activity were included among the skin resident T cells (19).

We then analyzed Treg extracted directly from normal human skin by isolating several subsets of CD4⁺CD25⁺ T cells. In contrast to peripheral blood, functional regulatory activity in skin included not only CD4⁺CD25^high T cells, but also CD4⁺CD25^med T cells. The regulatory activity of these cells was not different between populations of cells with and without CD69, which has been regarded as a marker to discriminate activated effector/memory T cells from Treg. It was somewhat surprising that both CD4⁺CD25^high T cells and CD4⁺CD25^med T cells showed Treg activity when added to stimulated CD4⁺CD25^– skin T cells, whereas in peripheral blood, only CD4⁺CD25^high T cells had Treg activity. As the overall intensity of CD25 expression was higher in skin CD4⁺ T cells than blood CD4⁺ T cells, CD4⁺CD25^med skin T cells might in fact correspond more closely to CD4⁺CD25^high blood T cells. It is also conceivable that skin CD4⁺CD25^med T cells are a unique Treg population residing in human skin. Cavani et al. (4) demonstrated regulatory activity of skin CD4⁺CD25⁺ T cells, which presumably contain both CD4⁺CD25^high and CD4⁺CD25^med T cells, isolated from negative patch tests to nickel in healthy individuals. These results imply that skin may serve as a great reservoir of Treg with CD4⁺CD25^med/high phenotype.

The reasons for skin-homing biased profile of blood Treg is unclear; however, it is possible to speculate. Like all human Treg thus far reported, both blood and skin Treg effectively suppressed weak to moderate immune responses (e.g., soluble anti-CD3/CD28), and were less effective at suppressing strong immune stimulation (e.g., immobilized anti-CD3). In its role as our principal interface with the environment, the skin is constantly exposed to subclinical traumatic insults, including many of an infectious nature. In addition, the skin has a varied and abundant normal bacterial, viral, and fungal flora, much of which does not provoke a clinically obvious immunologic response. Treg may mediate suppression to weakly or moderately activated effector T cells in skin, not only to maintain peripheral tolerance to autoantigens but also to prevent exuberant inflammatory responses to nonpathogenic resident normal flora.

In conclusion, we have described a strikingly skin-tropic phenotype of CD4⁺CD25^high Treg from peripheral blood, and in parallel many Treg isolated from normal human skin. Taken together, these observations suggest a role for human Treg in cutaneous immune surveillance and in the maintenance of peripheral tolerance to autoantigen and foreign Ag in normal skin. Our results are consistent with the idea that an imbalance in recruitment and/or function of effector and Treg may be a crucial pathological factor in immune-based T cell-mediated skin diseases like psoriasis.

	Acknowledgments

 
We thank Dr. Clare Baecher-Allan for technical advice on the T regulatory cell functional assay and Dr. Natasha Barteneva for assistance in cell sorting.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grant AI-41707 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (to T.S.K.).

² Current address: Biological Research Laboratories, Sankyo Company, Ltd, 2–58 Hiromachi 1-chome, Shinagawa-ku, 140-8710 Tokyo, Japan.

³ Address correspondence and reprint requests to Dr. Thomas S. Kupper, Harvard Skin Disease Research Center, Department of Dermatology, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: tkupper{at}partners.org

⁴ Abbreviations used in this paper: Treg, T regulatory cell; CLA, cutaneous lymphocyte Ag.

Received for publication January 27, 2006. Accepted for publication July 7, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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