Memory regulatory T cells reside in human skin

Abstract

Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, are a dynamic and heterogeneous population of cells that control immune responses and prevent autoimmunity. We recently identified a subset of Tregs in murine skin with properties typical of memory cells and defined this population as memory Tregs (mTregs). Due to the importance of these cells in regulating tissue inflammation in mice, we analyzed this cell population in humans and found that almost all Tregs in normal skin had an activated memory phenotype. Compared with mTregs in peripheral blood, cutaneous mTregs had unique cell surface marker expression and cytokine production. In normal human skin, mTregs preferentially localized to hair follicles and were more abundant in skin with high hair density. Sequence comparison of TCRs from conventional memory T helper cells and mTregs isolated from skin revealed little homology between the two cell populations, suggesting that they recognize different antigens. Under steady-state conditions, mTregs were nonmigratory and relatively unresponsive; however, in inflamed skin from psoriasis patients, mTregs expanded, were highly proliferative, and produced low levels of IL-17. Taken together, these results identify a subset of Tregs that stably resides in human skin and suggest that these cells are qualitatively defective in inflammatory skin disease.

Figure 1. Foxp3 expression in human adult and fetal skin.

(A) Expression of Foxp3 on CD3⁺ T cells in PBMCs (Blood) and skin isolated from healthy adults. Scatter plot is gated on CD3⁺CD4⁺ cells. (B and C) Expression of Foxp3 and CD45RO on CD3⁺CD4⁺ T cells in adult PBMCs and in adult skin as well as human fetal skin. Results are combined data from five or more independent experiments. P values were determined using a 2-tailed unpaired Student’s t test.

Figure 2. Tregs in human skin have an activated effector memory phenotype.

(A and B) Expression of activation markers, memory markers, and cytokines from viable CD3⁺CD4⁺CD45RO⁺ T cells in PBMCs and skin isolated from healthy adults. Skin used for cytokine analysis in B was harvested from face or scalp. Scatter plots in B represent the percentage of cytokine-producing cells within Foxp3⁺ and Foxp3^– gates. All gates are based on isotype control staining or unstimulated controls (for cytokine production in B). EC, extracellular. (C) Percentage of demethylation of genomic DNA in intron 1 of the FOXP3 gene (TSDR locus) of mTregs and mTconvs in PBMCs and skin isolated from healthy adults. Each pie chart represents a sorted population purified from a different donor, and numeric values within pie charts represent the percentage of demethylation at the TSDR locus. Numeric values in parentheses below each chart represent the percentage of Foxp3-expressing cells within each purified cell population. Cell sorting strategy is shown in Supplemental Figure 4. Results in A and B are representative data from more than fifteen independent experiments. Results in C are from three or more replicate experiments. P values were determined using a 2-tailed unpaired Student’s t test.

Figure 3. mTregs localize to HFs in human skin.

(A–C) Confocal microscopy of normal human skin. Sections were stained for CD3 (green), Foxp3 (red), CD1a (blue), and DAPI (gray). Scale bars: 100 μm. HFs autofluoresce in blue. Arrows denote CD3⁺Foxp3⁺ cells (yellow). (D) Quantification of Foxp3⁺ cells (within the CD3⁺ gate) in human adult skin by flow cytometry. Low hair density represents skin harvested from anatomical sites with relatively lower HF density (trunk and upper proximal extremities), whereas high hair density represents skin harvested from anatomical locations with relatively higher hair density (scalp and face). Results are representative of more than five replicate experiments. P values were determined using a 2-tailed unpaired Student’s t test.

Figure 4. TCRβ sequencing of mTregs and memory Tconvs in human skin.

(A) Gating strategy for sorting of mTconvs and mTregs from human skin harvested from healthy adults. Cells are pregated on viable CD3⁺CD4⁺ cells. (B) Hive plots showing relative abundance of unique TCRβ sequence clones and relative overlap of unique sequences between the Foxp3⁺ and Foxp3^– pools. Each line represents one unique clone. The width of the lines denotes the relative abundance of each clone in the Foxp3⁺ and Foxp3^– fractions or the pool of both populations. The most abundant clones (i.e., the widest lines) are positioned distally from the center of the hive, whereas the least abundant clones (thinnest lines) are located centrally. Lines that connect the Foxp3⁺ and Foxp3^– fractions represent unique sequences shared between these cell populations. For example, in skin sample 2, the most abundant sequence in the Foxp3^– fraction is present in relatively low frequency in the Foxp3⁺ fraction and represents the most abundant sequence in the entire pool. In this sample, this sequence is the only one shared between the Foxp3^– and Foxp3⁺ cell populations. P values were determined using Monte Carlo simulation for the probability that Foxp3⁺ cells were not a random sampling of the pooled population (see Methods).

Figure 5. mTregs in human skin are nonmigratory.

(A) CCR7 expression on mTregs and mTconvs in human skin harvested from healthy adults. Cells were pregated on viable CD3⁺CD4⁺CD45RO⁺ cells. (B) Human skin was grafted onto immunodeficient NSG mice, and at specific times thereafter, grafted skin, blood, and spleen (not shown) were harvested and human T cells analyzed by flow cytometry. Cells were pregated on viable CD3⁺CD4⁺hCD45⁺ cells. (C) Absolute numbers of CD3⁺CD4⁺hCD45⁺Foxp3⁺ and CD3⁺CD4⁺hCD45⁺Foxp3^– cells in grafted skin and blood 3 and 7 weeks after grafting. Results are pooled data from two of five representative experiments with two or more mice per group. P values were determined using a 2-tailed unpaired Student’s t test.

Figure 6. Functional analysis of cutaneous mTregs in patients with psoriasis.

(A) Percentage and absolute number of T cells and Tregs in nonlesional psoriatic (NL-PSO) and lesional psoriatic (L-PSO) skin from patients (middle and right panels gated on CD4⁺ and CD3⁺ cells, respectively). (B) Percentage of mTregs and mTconvs in L-PSO skin or normal healthy adult skin (Control), pregated on viable CD3⁺ cells. (C) Intracellular IL-17 production from mTregs and mTconvs in NL-PSO and L-PSO skin, gated on viable CD3⁺CD4⁺CD45RO⁺ cells. Middle panel is a representative flow cytometric plot of IL-17 production, and the scatter plot shows percentages of IL-17–producing cells within the Foxp3 gate. (D) Expression of Ki67 in mTregs and mTconvs in L-PSO skin or site-matched control skin, gated on viable CD3⁺CD4⁺CD45RO⁺ cells. (E) Expression of Ki67 in mTregs and mTconvs in L-PSO or control skin, gated on viable CD3⁺CD4⁺CD45RO⁺ cells. Lines represent paired data from a single patient. Mean deltas between Foxp3^– and Foxp3⁺ cells when comparing PSO versus control skin are 8.340 ± 5.05 versus 0.735 ± 0.50, respectively (P = 0.132). (F) MFI of Foxp3 and CD25 expression on Ki67⁺ and Ki67^– mTregs in L-PSO skin, gated on viable CD3⁺CD4⁺CD45RO⁺Foxp3⁺ cells. (G) Foxp3 and CD127 expression on mTregs and mTconvs in L-PSO or site-matched control skin, gated on viable CD3⁺CD4⁺CD45RO⁺cells. Results are combined data from five or more replicate experiments.