1. Vγ4 γδ T Cells Provide an Early Source of IL-17A and Accelerate Skin Graft Rejection 
Yashu Li, Zhenggen Huang, Rongshuai Yan, Meixi Liu, Yang Bai, Guangping Liang, Xiaorong Zhang, Xiaohong Hu, Jian Chen, Chibing Huang, Baoyi Liu, Gaoxing Luo, Jun Wu, Weifeng He 
Journal of Investigative Dermatology 
Volume 137, Issue 12, Pages (December 2017)
DOI: /j.jid
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2. Figure 1
Vγ4 T cells promoted skin graft rejection in the early stages of transplantation. (a) Skins grafts were harvested from C57BL/6WT male mice and transplanted onto age-matched WT and TCRδ−/− female mice (n = 10 each). (b) Skin grafts were harvested from WT or TCRδ−/− male mice and transplanted onto WT females (n = 10 each). (c) WT female mice were injected i.v. with Vγ1-specific Ab, Vγ4-specific Ab, or isotype Ab (n = 10/group) 3 days before transplantation of skin grafts from WT male mice. (d) Female TCRδ−/− mice were reconstituted with Vγ4 or Vγ1 T cells sorted from WT female mice (1 × 105 cells/mouse, n = 5/group) immediately after transplantation. (e) Recipients were injected i.v. with Vγ4-specific Ab (n = 10 each) 3 days before, or 10 days after, transplantation of skin grafts from WT male mice. All data represent at least three independent experiments. **P < 0.01; ***P < Ab, antibody; WT, wild-type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
IL-17A was essential for Vγ4-mediated skin graft rejection. (a) Skin grafts were harvested from WT male mice and transplanted onto WT females, followed by injection of IFN-γ, IL-17A neutralizing antibodies, or control isotype antibody (n = 10/group) on days 0, 2, and 4. (b) Female TCRδ−/− mice were reconstituted with either WT, IFN-γ−/−, or IL-17A−/−Vγ4 T cells sorted from WT, IFN-γ−/−, or IL17A−/− female mice, respectively (1 × 105 cells/mouse, n = 5/group). Skin grafts from WT male mice were transplanted onto the mice with reconstituted cells. (c, d) Skin grafts were harvested from WT male mice and grafted onto age-matched WT female mice. Skin grafts (c) and host skin (including epidermis and dermis) (d) around grafts from three to five mice were obtained 3, 5, and 7 days after transplantation, and the expression of IL-17A (17 kD) and GAPDH (36 kD) was detected by western blotting. (e) Immunochemistry analysis for IL-17A expression in grafts and host epidermis around grafts from WT and Vγ4D recipients. Black dotted lines show the boundary between grafts and host skin. Left scale bar = 200 μm, right scale bar = 100 μm. (f) Cell suspensions were prepared from normal skin, skin grafts, and host epidermis around grafts from three to five mice each, and cells were stained with anti-Vγ4 and anti-TCRγδ antibodies. Data represent at least three independent experiments. All error bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean; WT, wild-type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 2
IL-17A was essential for Vγ4-mediated skin graft rejection. (a) Skin grafts were harvested from WT male mice and transplanted onto WT females, followed by injection of IFN-γ, IL-17A neutralizing antibodies, or control isotype antibody (n = 10/group) on days 0, 2, and 4. (b) Female TCRδ−/− mice were reconstituted with either WT, IFN-γ−/−, or IL-17A−/−Vγ4 T cells sorted from WT, IFN-γ−/−, or IL17A−/− female mice, respectively (1 × 105 cells/mouse, n = 5/group). Skin grafts from WT male mice were transplanted onto the mice with reconstituted cells. (c, d) Skin grafts were harvested from WT male mice and grafted onto age-matched WT female mice. Skin grafts (c) and host skin (including epidermis and dermis) (d) around grafts from three to five mice were obtained 3, 5, and 7 days after transplantation, and the expression of IL-17A (17 kD) and GAPDH (36 kD) was detected by western blotting. (e) Immunochemistry analysis for IL-17A expression in grafts and host epidermis around grafts from WT and Vγ4D recipients. Black dotted lines show the boundary between grafts and host skin. Left scale bar = 200 μm, right scale bar = 100 μm. (f) Cell suspensions were prepared from normal skin, skin grafts, and host epidermis around grafts from three to five mice each, and cells were stained with anti-Vγ4 and anti-TCRγδ antibodies. Data represent at least three independent experiments. All error bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean; WT, wild-type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 3
Vγ4 T cells infiltrated into skin grafts via the CCR6-CCL20-dependent pathway. (a) Grafts from WT male mice grafted onto WT female mice with CCL20 neutralizing antibody or isotype IgG treatment (n = 5/group). Three days after transplantation, FACS was used to analyze the expression of CCR6 on skin-infiltrating Vγ4 T cells in transplantation areas. (b, d) Three days after transplantation, the expression of CCL20 (11 kD) (b) IL-17A (17 kD) (d) and GAPDH (36 kD) in whole skin grafts and host epidermis around grafts collected from three to five mice per group was detected by western blotting. Representative western blots are shown in the top panel, and the quantification of protein levels using densitometry is shown in the bottom panel. (c) Skin grafts from WT male mice transplanted onto WT females with CCL20 neutralizing antibody or isotype IgG treatment (n = 10/group). All error bars represent mean ± SEM. **P < 0.01, ***P < CCL20, chemokine (C-C motif) ligand 20; CCR6, chemokine receptor 6; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean; WT, wild-type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

6. Figure 4
IL-23 and IL-1β induced IL-17A production from infiltrating Vγ4 T cells. (a) Skin grafts were harvested from C57BL/6 WT male mice and grafted onto age-matched WT female mice (n = 5 each). IL-1β (17 kD), IL-23 (20 kD), and GAPDH (36 kD) expression in skin graft and host epidermis around grafts was detected by western blotting. Normal skin and epidermis from three mice were used as controls. (b, c) Graft recipient mice were subcutaneously injected with IL-1β and IL-23 neutralizing antibody (IL-1β/23 Ab) or control isotype antibody (Control) on days 0, 2, and 4 after transplantation. (b) The expression of IL-17A (17 kD) and GAPDH (36 kD) in skin grafts and host epidermis around grafts from three mice was detected by western blotting 3 days after transplantation. (c) Representative survival curve of recipients (n = 10 each) with the indicated treatment. (d) Recipients with Vγ4-specific Ab treatment 3 days before transplantation were treated with IL-1β and IL-23 neutralizing antibodies or control isotype antibody (n = 10/group) on days 0, 2, and 4 after transplantation. The graphs show one representative of three independent experiments. All error bars represent mean ± SEM. **P < 0.01, ***P < Ab, antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean; WT, wild-type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

7. Figure 5
Infiltrating Vγ4 T-cell-derived IL-17A promoted the accumulation of DCs in dLNs to activate αβ T cells. (a, b) Age-matched C57BL/6 WT female mice were injected i.v. with Vγ4-specific Ab or isotype control Ab (n = 10/group) 3 days before transplantation of skin grafts from WT male mice. On day 5 after transplantation, the number of MHCIIhighCD86+CD11+ DCs (a) and IL-17A-positive CD4 αβ T cells (b) in draining lymph nodes was determined by FACS. (c, d) Female TCRδ−/− mice were reconstituted with WTVγ4 T cells sorted from WT female mice (n = 5/group) or IL-17A−/− Vγ4 T cells sorted from IL-17A−/− female mice. Skin grafts from WT male mice were transplanted onto the recipients with reconstituted WT or IL-17A−/−Vγ4 T cells. Five days after transplantation, the number of MHCIIhighCD86+CD11+DCs (c) and IL-17A-positive CD4 αβT cells (d) in draining lymph nodes was determined by FACS. All data represent at least three individual experiments. All error bars represent mean ± SEM. ***P < Ab, antibody; DC, dendritic cell; dLN, draining lymph node; MHCII, major histocompatibility complex, II; SEM, standard error of the mean; WT, wild-type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions