Human epidermal Langerhans cells maintain immune homeostasis in skin by activating skin resident regulatory T cells

Abstract

Recent studies have demonstrated that the skin of a normal adult human contains 10-20 billion resident memory T cells, including various helper, cytotoxic, and regulatory T cell subsets, that are poised to respond to environmental antigens. Using only autologous human tissues, we report that both in vitro and in vivo, resting epidermal Langerhan cells (LCs) selectively and specifically induced the activation and proliferation of skin resident regulatory T (Treg) cells, a minor subset of skin resident memory T cells. In the presence of foreign pathogen, however, the same LCs activated and induced proliferation of effector memory T (Tem) cells and limited Treg cells' activation. These underappreciated properties of LCs, namely maintenance of tolerance in normal skin, and activation of protective skin resident memory T cells upon infectious challenge, help clarify the role of LCs in skin.

Figure 1. A subset of skin resident memory T cells proliferates in presence of autologous LC

(A) Proliferation of skin resident memory T cells labeled with CFSE and cultured alone, or with autologous DDC or LC at a ratio 2:1 after 6 days, or with conditioned medium from LC or DDC. Proliferation as measured by CFSE dilution, from a representative experiment. (B) Numbers indicate the percentage of proliferating CD3⁺ T cells. (C) Percentage of proliferating CD3⁺ skin resident memory T cells, each sign represents a single subject, two-tailed, unpaired t-test. (D) CFSE labeled skin resident memory T cells were co-cultured with autologous LC, DDC or alone. Proliferation as measured by CFSE dilution, from a representative experiment at different days. (E) Graph shows percentage of proliferating CD3⁺ skin-resident memory T cells. Results are the mean +SD. .

Figure 2. Proliferating skin-resident memory T cells are preferentially CD4⁺ T lymphocytes

(A) Proliferation of CD3⁺ CFSE labeled skin resident memory T cells stimulated with autologous LC. At day 6 of co-culture, skin-resident memory T cells were stained for CD4 and CD8 and proliferative and non-proliferative skin resident memory T cells subsets were analyzed. Numbers in quadrants indicate percent cells (representative experiment). (B) Percentage of CD4⁺ and CD8⁺ T cells in the proliferative population. The graph shows mean +/− SD. Data are representative of 4 independent experiments (two-tailed, unpaired t-test).

Figure 3. Proliferating CD4⁺ skin resident memory T cells in presence of autologous LC are functional regulatory T cells

(A) CFSE labeled skin resident memory T cells were stimulated with autologous LC. At day 6, CD3⁺ skin resident memory T cells were stained for CD4, CD25, CD127 and FoxP3. Numbers in quadrants represent percentage. (B) Percent of CD4⁺CD25⁺FoxP3⁺CD127⁻ regulatory T cells (skin resident memory Treg cells) and skin resident memory T cells non-Treg in the proliferative population assessed for different donors. The graph shows mean +/− SD. Data are representative of 5 independent experiments (two-tailed, unpaired t-test). (C) Figure represents fold amplification of CD4⁺CD25⁺FoxP3⁺CD127⁻ Treg cells after 6 days co-culture with autologous LC relative to Treg cells present at day 0. Each sign represent independent experiments, bar represent mean. (D) The depletion of CD25⁺ skin resident memory T cells prior to co-culture with autologous LC decreases the proliferation of skin resident memory T cells. CFSE labeled skin resident memory T cells or CFSE labeled CD25⁻ skin resident memory T cells were co-cultured with autologous LC for 6 days. Proliferation was measured by the dilution of CFSE. Numbers indicate percentage of proliferative cells. (E) Expanded skin resident memory Treg cells from 6 day co-cultures were isolated and then cultured with autologous CFSE-labeled skin resident Tem cells in presence of anti-CD3 (1μg/ml) and irradiated Peripheral Blood Mononuclear Cells (iPBMCs). CFSE dilution was analyzed at day 5. These data are representative of three independent experiments with cells from different subjects. (F) Expanded Treg cells were cultured with skin resident Tem cells in the presence of anti-CD3 (1μg/ml) and iPBMCs. After 4 days of coculture, [³H]thymidine was added to each well. The cells were harvested after 16 hours and radioactivity was measured. The graph shows mean +/− S.D. Data are representative of two independent experiments with triplicate (two-tailed, unpaired t-test).

Figure 4. Expansion of skin resident memory Treg cells is dependent on MHC class II, CD80-86, and IL-2 or IL-15

(A) Proliferation of CFSE labeled skin resident memory T cells co-cultured with autologous LC in the presence of the indicated neutralizing antibodies or isotype-matched, non-reactive antibodies as control. Proliferation of CD3⁺ T cells was measured by CFSE dilution. Numbers represent the percent of divided cells for a representative donor. (B) Percentage of skin resident memory T cells proliferating in presence of neutralizing antibodies compared to matching isotype control. The graph shows means +/− SD. Data are representative of 3 independent experiments (two-tailed, unpaired t-test). (C) Proliferation of CD3⁺ T cells measured by CFSE dilution in the presence of different concentrations of pan-class II blocker. (D) Proliferation of CFSE labeled skin resident memory T cells co-cultured with autologous LC in the presence of the indicated neutralizing antibodies or isotype-matched control. Graph shows means +/− S.D and data are representative of 2 independent experiments with duplicate.

Figure 5. Skin resident memory Treg cells are found in the epidermis near Langerhans cells, and express the proliferation marker Ki67

(A) Immunofluorescence of skin showing expression of FoxP3 (green) and CD3 (red), FoxP3, CD3 double–positive regulatory T cells (Treg) are found mainly in the epidermis and the papillary dermis. (B) FoxP3⁺ skin resident memory Treg cells are found in close proximity to CD1a⁺ LC (red). (C) A proportion of FoxP3⁺ (green) Treg cells co-express Ki67 (red), resulting in a yellow overlay. Right panel, histogram shows the percentage of FoxP3/Ki67 double positive cells in the FoxP3⁺ population in epidermis/papillary dermis or dermis. The graph shows mean +/− S.D and data are representative of 6 independent donors (two-tailed, unpaired t-test). Scale Bar: bottom left 50μm, bottom right 10μm.

Figure 6. Skin resident memory Treg cells are abundant in epidermis

Epidermal and dermal layers from skin explants were separated using dispase treatment. (A) Epidermal cells contained abundant CD3⁺ skin resident memory T cells and are enriched for FoxP3⁺ skin resident memory Treg cells. CD1a⁺ LC were also abundant in epidermis. (B) Skin resident memory T cells were abundantly present in dermis with fewer skin-resident memory Treg cells than epidermis. No substantial CD1a⁺ LC were found in dermis. Numbers in quadrant indicate percent cells.

Figure 7. During infection, proliferation of skin resident memory T cells demonstrates a mixed population composed of pathogen specific skin resident Tem cells and skin resident memory Treg cells

CFSE labeled skin resident memory T cells were co-cultured with autologous LC in the presence or absence of heat inactivated C.albicans for 6 days. (A) Skin resident memory T cells were then stained for CD3. Numbers in quadrant show percentage of expanded T cells. (B) Data from 10 independent experiments (two tailed, unpaired t test). (C) CFSE labeled skin resident memory T cells stimulated with autologous LC in the presence of heat-inactivated C.albicans. At day 6, CD3⁺ skin resident memory T cells were stained for CD4, CD25, CD127 and FoxP3. Numbers in quadrants represent percent cells in proliferative and non-proliferative populations. Data are representative of 3 independent experiments. (D) The graph shows percent skin resident memory Treg cells and skin resident Tem cells in the proliferative populations when skin resident memory T cells and LC were co-cultured with different ratio of C.albicans. Data are representative of 2 independent experiments with duplicate. (E) Depletion of CD25⁺ increased the proliferation of skin resident Tem cells in the presence of pathogen. CFSE labeled T cells or CD25⁻ T cells were co-cultured with autologous LC for 6 days in the presence of C.albicans. Proliferation was measured by the dilution of CFSE. Numbers indicate the percent of divided cells for a representative donor.