Keratinocytes costimulate naive human T cells via CD2: a potential target to prevent the development of proinflammatory Th1 cells in the skin

The interplay between keratinocytes and immune cells, especially T cells, plays an important role in the pathogenesis of chronic inflammatory skin diseases. During psoriasis, keratinocytes attract T cells by releasing chemokines, while skin-infiltrating self-reactive T cells secrete proinflammatory cytokines, e.g., IFNγ and IL-17A, that cause epidermal hyperplasia. Similarly, in chronic graft-versus-host disease, allogenic IFNγ-producing Th1/Tc1 and IL-17-producing Th17/Tc17 cells are recruited by keratinocyte-derived chemokines and accumulate in the skin. However, whether keratinocytes act as nonprofessional antigen-presenting cells to directly activate naive human T cells in the epidermis remains unknown. Here, we demonstrate that under proinflammatory conditions, primary human keratinocytes indeed activate naive human T cells. This activation required cell contact and costimulatory signaling via CD58/CD2 and CD54/LFA-1. Naive T cells costimulated by keratinocytes selectively differentiated into Th1 and Th17 cells. In particular, keratinocyte-initiated Th1 differentiation was dependent on costimulation through CD58/CD2. The latter molecule initiated STAT1 signaling and IFNγ production in T cells. Costimulation of T cells by keratinocytes resulting in Th1 and Th17 differentiation represents a new explanation for the local enrichment of Th1 and Th17 cells in the skin of patients with a chronic inflammatory skin disease. Consequently, local interference with T cell–keratinocyte interactions may represent a novel strategy for the treatment of Th1 and Th17 cell-driven skin diseases.

Effect of siRNA-mediated HLA-DR knock-down in primary human KCs on the expression of CD25 and CD69 on naïve CD4 + T cells cultured for 24 h with either 2 untreated KCs (white bars) or IFNg-pretreated KCs (black and grey bars) loaded with SEB (n = 5 individual T cell donors). Expression was analyzed by flow cytometry. Data is represented as mean ± SEM. ****=p<0.0001.

Gene expression profiling
The nCounter Nanostring GX Human Immunology V2 panel was used to analyze the expression of 579 immune and inflammation associated target genes and 15 reference control genes in naïve CD4 + T cells which were cultured for 4 h with either untreated or IFNg-treated keratinocytes. Before RNA isolation, T cells were isolated using flow cytometry. Total RNA was then extracted from 1*10 6 naïve T cells using Direct-zol RNA Miniprep Kit (Zymo Research). All RNA samples were quantified by using Qubit RNA assay kit and RNA integrity was assessed using Agilent 2100 Bioanalyzer system. 25 µg total RNA (5 µL/sample) was mixed with nCounter® reporter CodeSet (3 µL) and nCounter® capture ProbeSet (2 µL) with hybridization buffer (5 µL) for an overnight hybridization reaction at 65 °C. The reaction was cooled down to 4 °C, the 5 samples were purified and immobilised on a cartridge and data was assessed on the nCounter SPRINT Profiler. The exported data was analysed using NanoString nSolver 4.0.

Flow cytometry
Monoclonal antibodies recognizing the following surface markers and molecules were

Intracellular staining for cytokines and transcription factors
Cells which were already stained for surface markers, were fixed with 1,5 % PFA for 10 min at room temperature. Intracellular staining was performed in FACS buffer (PBS, 0,5 % albumin Fraction V, 0,1 % NaN3) containing 0,1 % saponin. Cells were washed with the same buffer and resuspended in PBS only for measurement.
For staining of transcription factors in differentiated T cells, surface markers were stained for 10 min on ice, before cells were fixed with True-Nuclear 1x Fix Concentration for 1 h at room temperature. T cells were permeabilized and stained for transcription factors (T-bet (4B10), GATA-3 (16E10A23) and RORgt (Q21-559)) in True-Nuclear 1x Perm Buffer for 30 min at room temperature.