Extended Data Fig. 1: Steady state DETC-keratinocyte interactions depend on the Vγ5Vδ1 TCR and Skint1.
From: Normality sensing licenses local T cells for innate-like tissue surveillance
a) Confocal microscopy of CD3+ DETCs (cyan) and total F-actin or that within CD3 mask (yellow) in epidermal sheets from wild-type and FVB.Tac mice. Representative of n=4/group. Scale bar 20μm. b) Neonatal Tcrd-/- (n=13) and FVB.Tac.Tcrd-/- (n=11) pups were reconstituted with wild-type embryonic thymocytes containing mature Skint1-selected DETC precursors. Confocal microscopy of CD3+ DETCs (cyan) and F-actin within CD3 mask (yellow) in epidermal sheets at 8 weeks of age. Scale bar 20μm. Frequency distribution of number of interacting dendrites within Vγ5+ DETCs (CD3- and F-actin-rich aggregates) per cell using mean values per mouse. c) Confocal microscopy of CD3+ DETCs (cyan) and total F-actin or that within CD3 mask (yellow) in epidermal sheets from wild-type C57BL/6 mice pre-treated with Skint1 Ab or isotype control (n=5/group). Scale bar 20μm. Frequency distribution of number of interacting dendrites (CD3- and F-actin-rich aggregates) per cell using mean values per mouse. d) Instant structured illumination microscopy (iSIM) of Skint1 (magenta) or isotype control in whole mount epidermal sheets from wild-type FVB mice. Scale bar 10μm. e) iSIM of CD3 (cyan), TCRδ (white) and Skint1 (magenta) staining in whole mount epidermal sheets from wild-type FVB(n=3), FVB.Tac (n=5) and Tcrd-/- (n=2) mice. Scale bar 10μm. Mean Skint1 MFI quantified within TCRδ (wild-type, FVB.Tac) or CD3 (Tcrd-/-) masks relative to mean image background value. Each dot represents a single cell. f) qPCR for Skint gene expression from epidermis of wild-type FVB (n=11) and Tcrd-/- mice (n=6). Representative images of and cumulative data from 4 (b) or 3 (e) independent experiments. Pooled data from 2 independent experiments (f). Matched two-way (b-c) or ordinary one-way (e) ANOVA with Sidak’s multiple comparisons test, unpaired two-tailed t-test (f). Values in pie charts and error bars on graph represent mean ± SD. ns: not significant.
