Human Langerhans cells induce distinct IL-22-producing CD4+ T cells lacking IL-17 production

Abstract

IL-22 is a cytokine that acts mainly on epithelial cells. In the skin, it mediates keratinocyte proliferation and epidermal hyperplasia and is thought to play a central role in inflammatory diseases with marked epidermal acanthosis, such as psoriasis. Although IL-22 was initially considered a Th17 cytokine, increasing evidence suggests that T helper cells can produce IL-22 even without IL-17 expression. In addition, we have shown the existence of this unique IL-22-producing T cell in normal skin and in the skin of psoriasis and atopic dermatitis patients. In the present study, we investigated the ability of cutaneous resident dendritic cells (DCs) to differentiate IL-22-producing cells. Using FACS, we isolated Langerhans cells (LCs; HLA-DR(+)CD207(+) cells) and dermal DCs (HLA-DR(hi)CD11c(+)BDCA-1(+) cells) from normal human epidermis and dermis, respectively. Both LCs and dermal DCs significantly induced IL-22-producing CD4(+) and CD8(+) T cells from peripheral blood T cells and naive CD4(+) T cells in mixed leukocyte reactions. LCs were more powerful in the induction of IL-22-producing cells than dermal DCs. Moreover, in vitro-generated LC-type DCs induced IL-22-producing cells more efficiently than monocyte-derived DCs. The induced IL-22 production was more correlated with IFN-gamma than IL-17. Surprisingly, the majority of IL-22-producing cells induced by LCs and dermal DCs lacked the expression of IL-17, IFN-gamma, and IL-4. Thus, LCs and dermal DCs preferentially induced helper T cells to produce only IL-22, possibly "Th22" cells. Our data indicate that cutaneous DCs, especially LCs, may control the generation of distinct IL-22 producing Th22 cells infiltrating into the skin.

Fig. 1.

Sorting strategy and surface molecule expression of LCs and dermal DCs. (A) LCs were sorted as HLA-DR⁺CD207⁺ cells from epidermal single cell suspensions, whereas dermal DCs were sorted as HLA-DR^hiCD11c⁺BDCA-1⁺ cells from single cell suspension of dermal emigres. (B) Flow cytometric analysis of surface molecules on LCs and dermal DCs. HLA-DR⁺CD207⁺ cells from epidermis and HLA-DR^hiCD11c⁺BDCA-1⁺ cells from dermis were subjected to the analysis of cell surface expression of CD40, CD80, CD83, and CD86. Blue lines indicate isotype controls. Data are representative of four independent experiments.

Fig. 2.

LCs and dermal DCs activate CD4⁺ T cells, leading to cell proliferation and IL-22 production. (A) Allogeneic whole peripheral T cells were labeled with CFSE and cultured for 7 days with sorted LCs or dermal DCs. Proliferation was determined by the dilution of CFSE analyzed by flow cytometry. Live, CD4⁺ cells were first gated and then plotted as CFSE vs. CD3. Percentage of live, proliferating CD4⁺ T cells is indicated in gate. Data are representative of four independent experiments. (B) Intracellular IL-22 vs. IL-17, IFN-γ, and IL-4 detected in proliferating CD4⁺ T cell population following 7-day coculture of whole peripheral T cells and sorted allogeneic LCs or dermal DCs. T cells stimulated by LCs or dermal DCs were restimulated with PMA and ionomycin in the presence of BFA for 4 h and subsequently analyzed for the production of indicated cytokines by flow cytometry. Live, CD4⁺ proliferating T cells were first gated and then cytokine production profile was analyzed. Numbers in quadrants indicate percentage of gated cells in each. Data are representative of six independent experiments. (C) Frequency of the cells producing IL-22 among CD4⁺ T cells before culture and that among proliferating CD4⁺ T cells after 7-day MLR assay. Horizontal axis indicates stimulators of T cells, and “No DC” group represents T cells cultured for 7 days without DCs. Asterisks (*) indicate statistical significance (P < 0.05). Data represent the mean (±SD) of six independent experiments.

Fig. 3.

Unique IL-22-producing cells represent a major subset among IL-22-producing CD4⁺ T cells. (A) FACS gating used in the analysis of IL-22-producing T cells stimulated by LCs or dermal DCs. (B) Frequency of Th1, Th2, and Th17 cells among IL-22-producing CD4⁺ T cells. Horizontal axis indicates stimulators of T cells. Data represent the mean (±SD) of six independent experiments.

Fig. 4.

LCs and dermal DCs induce Th22 differentiation from naive CD4⁺ T cells. (A) Allogeneic naive CD4⁺ T cells were labeled with CFSE and cultured for 7 days with sorted LCs or dermal DCs. Proliferation was determined by the dilution of CFSE analyzed by flow cytometry. Live, CD4⁺ cells were first gated and then plotted as CFSE vs. CD3. Percentage of live, proliferating CD4⁺ T cells is indicated in gate. Data are representative of four independent experiments. (B) Intracellular IL-22 vs. IL-17, IFN-γ, and IL-4 detected in proliferating, CD4⁺ T cell population following 7-day coculture of naive CD4⁺ T cells and sorted allogeneic LCs or dermal DCs. Naive CD4⁺ T cells stimulated by LCs or dermal DCs were restimulated with PMA and ionomycin in the presence of BFA for 4 h and subsequently analyzed for the production of indicated cytokines by flow cytometry. Live, CD4⁺ proliferating T cells were first gated and then cytokine production profile was analyzed. Numbers in quadrants indicate percentage of gated cells in each. Data are representative of six independent experiments. (C) Frequency of the cells producing IL-22 among CD4⁺ T cells before culture and that among proliferating CD4⁺ T cells after 7-day MLR assay. Horizontal axis indicates stimulators of T cells, and “No DC” group represents T cells cultured for 7 days without DC. Asterisks (*) indicate statistical significance (P < 0.05). Data represent the mean (±SD) of six independent experiments. (D) Frequency of Th1, Th2, and Th17 cells among IL-22-producing CD4⁺ T cells. Analysis was performed as described in Fig. 3 A and B. Horizontal axis indicates stimulators of T cells. Data represent the mean (±SD) of six independent experiments.

Fig. 5.

LCs are more efficient than dermal DCs at inducing IL-22-producing CD4⁺ T cells. (A) Whole peripheral T cells were cultured with LCs or dermal DCs for 7 days and subsequently restimulated with PMA and ionomycin in the presence of BFA for 4 h. The frequency of IL-22-producing cells among proliferating CD4⁺ T cells stimulated by LCs or dermal DCs was examined by intracellular cytokine staining. (B) Naive CD4⁺ T cells were cultured with LCs or dermal DCs for 7 days and subsequently restimulated with PMA and ionomycin in the presence of BFA for 4 h. The frequency of IL-22-producing cells among proliferating CD4⁺ T cells was examined by intracellular cytokine staining. Asterisks (*) indicate statistical significance (P < 0.05).