Subsets of CD8 T cells can instruct wound healing. (A) Wound healing assay with SNs of human blood-derived CD45RO+CD45RA−PD1+TIGIT+ CD8 T cells, in vitro activated by anti-CD3/CD28 beads in the presence of MRC-5 cells, treated with either human IgG or Cetuximab, representative example. Statistical verification across experiments using background-subtracted AUC (n = 6, one-way ANOVA). More individual donors in Fig. S3. Scale bars = 400 µm; enhanced for improved visibility. (B) Identification of Tnaive (CD45RA+CCR7+), Temra (CD45RA+CCR7−), Tem (CD45RA−CCR7−), and Tcm (CD45RA−CCR7+) in human blood, followed by intracellular flow cytometry to detect AREG, TNF, and IFN-γ following 4 h incubation in the presence of PMA/ionomycin and transport inhibitors (Stim+) or transport inhibitor only (Stim−, one-way ANOVA with n = 4). Additional gating and controls in Fig. S3. (C) Human blood-derived Tnaive, Tcm, and CD45RA+/−CCR7−PD1+TIGIT+ CD8 T cells were either in vitro cultivated (resting) or in vitro cultivated and activated by anti-CD3/CD28 beads (activated), followed by collection of cell-free SN, which was then used in a wound healing assay with HaCaT cells. Representative images show wound density at 0 and 20 h following wounding and application of cell-free SNs. Statistical verification using normalized AUC (n = 5–7, one-way ANOVA). Scale bars = 400 µm; enhanced for improved visibility. (D) Wound healing assay with SNs of human blood-derived and in vitro activated and co-cultured PD1+TIGIT+ or Tnaive CD8 T cells, treated with Cetuximab during wound healing assay (n = 6, unpaired t test), representative images show wound density at 0 and 30 h following wounding and application of SNs. Statistical verification using normalized AUC (n = 5–7, one-way ANOVA). Scale bars = 400 µm; enhanced for improved visibility. (E) Wound healing assay with human recombinant AREG (100 or 5 ng/ml), TNF (5 ng/ml), and IFN-γ (1 ng/ml), representative images of wound density 0 and 40 h after initial wounding shown. Statistical verification using normalized AUC (n = 15–16, one-way ANOVA). Scale bars = 400 µm; enhanced for improved visibility. (F) EGFR expression in HaCaT cells stimulated o/n with SN generated in the three-cell-type system. Results derived from sequencing data from Fig. 2 C. (G) TGFα (left) and AREG (right) level in SN of three-cell-type system as described in Fig. 1 A in the presence of hIgG, anti-hTNF, or no T cells. Cytokine levels determined by ELISA. All data were derived from several independent experiments with the indicated number of donors.
Figure 4.

Subsets of CD8 T cells can instruct wound healing. (A) Wound healing assay with SNs of human blood-derived CD45RO+CD45RA−PD1+TIGIT+ CD8 T cells, in vitro activated by anti-CD3/CD28 beads in the presence of MRC-5 cells, treated with either human IgG or Cetuximab, representative example. Statistical verification across experiments using background-subtracted AUC (n = 6, one-way ANOVA). More individual donors in Fig. S3. Scale bars = 400 µm; enhanced for improved visibility. (B) Identification of Tnaive (CD45RA+CCR7+), Temra (CD45RA+CCR7−), Tem (CD45RA−CCR7−), and Tcm (CD45RA−CCR7+) in human blood, followed by intracellular flow cytometry to detect AREG, TNF, and IFN-γ following 4 h incubation in the presence of PMA/ionomycin and transport inhibitors (Stim+) or transport inhibitor only (Stim−, one-way ANOVA with n = 4). Additional gating and controls in Fig. S3. (C) Human blood-derived Tnaive, Tcm, and CD45RA+/−CCR7−PD1+TIGIT+ CD8 T cells were either in vitro cultivated (resting) or in vitro cultivated and activated by anti-CD3/CD28 beads (activated), followed by collection of cell-free SN, which was then used in a wound healing assay with HaCaT cells. Representative images show wound density at 0 and 20 h following wounding and application of cell-free SNs. Statistical verification using normalized AUC (n = 5–7, one-way ANOVA). Scale bars = 400 µm; enhanced for improved visibility. (D) Wound healing assay with SNs of human blood-derived and in vitro activated and co-cultured PD1+TIGIT+ or Tnaive CD8 T cells, treated with Cetuximab during wound healing assay (n = 6, unpaired t test), representative images show wound density at 0 and 30 h following wounding and application of SNs. Statistical verification using normalized AUC (n = 5–7, one-way ANOVA). Scale bars = 400 µm; enhanced for improved visibility. (E) Wound healing assay with human recombinant AREG (100 or 5 ng/ml), TNF (5 ng/ml), and IFN-γ (1 ng/ml), representative images of wound density 0 and 40 h after initial wounding shown. Statistical verification using normalized AUC (n = 15–16, one-way ANOVA). Scale bars = 400 µm; enhanced for improved visibility. (F) EGFR expression in HaCaT cells stimulated o/n with SN generated in the three-cell-type system. Results derived from sequencing data from Fig. 2 C. (G) TGFα (left) and AREG (right) level in SN of three-cell-type system as described in Fig. 1 A in the presence of hIgG, anti-hTNF, or no T cells. Cytokine levels determined by ELISA. All data were derived from several independent experiments with the indicated number of donors.

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