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. 2012 Apr 1;188(7):2972-6.
doi: 10.4049/jimmunol.1100887. Epub 2012 Mar 5.

Cutting edge: dendritic epidermal γδ T cell ligands are rapidly and locally expressed by keratinocytes following cutaneous wounding

H Kiyomi Komori  1 , Deborah A Witherden, Ryan Kelly, Kevin Sendaydiego, Julie M Jameson, Luc Teyton, Wendy L Havran
Affiliations

Cutting edge: dendritic epidermal γδ T cell ligands are rapidly and locally expressed by keratinocytes following cutaneous wounding

H Kiyomi Komori et al. J Immunol. .

Abstract

TCR-specific activation is pivotal to dendritic epidermal T cell (DETC) function during cutaneous wound repair. However, DETC TCR ligands are uncharacterized, and little is known about their expression patterns and kinetics. Using soluble DETC TCR tetramers, we demonstrate that DETC TCR ligands are not constitutively expressed in healthy tissue but are rapidly upregulated following wounding on keratinocytes bordering wound edges. Ligand expression is tightly regulated, with downmodulation following DETC activation. Early inhibition of TCR-ligand interactions using DETC TCR tetramers delays wound repair in vivo, highlighting DETC as rapid responders to injury. To our knowledge, this is the first visualization of DETC TCR ligand expression, which provides novel information about how ligand expression impacts early stages of DETC activation and wound repair.

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Figures

Fig. 1
Fig. 1. DETC tetramers bind cell surface ligands on keratinocyts
(A) Tetramer staining of the PAM 212 cell line. (B) TCR specific binding of DETC tetramers. DETC sTCR tetramers were incubated with anti-Vγ2 or anti-Vγ3 Abs prior to cell surface staining. Data are representative of ≥3 experiments.
Fig. 2
Fig. 2. Keratinocytes express DETC TCR ligands at sites of tissue damage
Epidermal sheets isolated from wounded B6 mice stained with DAPI (blue), anti-CD3 (green) and either DETC or G8 tetramer (red). Wound edges delineated by the white dashed line. Scale bar represents 50 μm. Data are representative of ≥3 experiments.
Fig. 3
Fig. 3. DETC TCR Ag is up-regulated prior to DETC activation, and down-modulated following DETC activation
Epidermal sheets were isolated and stained with DAPI (blue), anti-CD3 (green), and DETC TCR tetramers (red) at multiple times following wounding. (A) Morphology of DETC (>2, 1-2, or 0 dendrites) within 300 μm of the wound edge was assessed at each time point. A minimum of 225 cells from ≥3 experiments were counted for each time point. (B) Epidermal sheets from non-wounded skin. Staining of wounded epidermal sheets was observed 1 h (C), 2 h (D), 3 h (E), or 19 h (F) post-wounding. (C-F) Images were acquired at the wound edge, delineated by white dashed lines, and DETC morphology indicated by arrowheads. Scale bar represents 50 μm. (G) Distance of DETC tetramer staining extending from the wound edge was measured at each of the observed time points. Error bars represent SD. Data are representative of ≥3 experiments.
Fig. 4
Fig. 4. DETC tetramers bind the cell surface of keratinocytes
PAM 212 keratinocytes were stained with DETC (A) or G8 (B) tetramers (red), anti-CD29 (green) and DAPI (blue). Data is representative of >3 experiments.
Fig. 5
Fig. 5. DETC tetramers bind E13.5 fetal, but not adult, thymic epithelial cells
Thymus from E13.5 (A, B) or 5 week old (C, D) animals stained with either DETC (A, C) or anti-Vγ3 blocked DETC (B, D) tetramer (red), anti-CD3 (green), and counterstained with DAPI (blue). Sections were visualized by confocal microscopy. Data are representative of 3 experiments.
Fig. 6
Fig. 6. DETC tetramers delay wound closure in vivo
Full thickness wounds on B6 and TCRδ-/- mice were treated with DETC tetramers, G8 tetramers, or streptavidin and wound closure monitored over time. p values were determined by student t-test, and error bars represent SEM. **p<0.01. Data shown represent the mean of ≥6 wounds from 3 mice per treatment and are representative of 2 experiments.
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