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Resident memory T cells in the skin mediate durable immunity to melanoma

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Science Immunology  14 Apr 2017:
Vol. 2, Issue 10, eaam6346
DOI: 10.1126/sciimmunol.aam6346
  • Fig. 1 CD8 T cells recognizing self-Ags and tumor-specific Ags persist in vitiligo-affected skin and exhibit a TRM cell phenotype.

    (A) Experimental scheme to induce melanoma-associated vitiligo; unaffected mice underwent identical procedures but did not depigment. i.d., intradermally. (B) Mice were treated as in (A), and proportions and absolute numbers of CD8 T cells were detected 65 days after surgery by flow cytometry (gated on live CD45+ cells). (C to E) Naïve Thy1.1+ pmel cells (104) were transferred 1 day before treatment as in (A), and pmel proportions (gated on live CD8+ cells) were quantified 50 days after surgery in (C) vitiligo-affected skin versus unaffected skin; (D) depigmented, perilesional, and pigmented skin sites of vitiligo-affected mice (sites depicted in representative image); and (E) skin versus lymphoid tissues of vitiligo-affected mice. SSC, side scatter; TDLN, tumor-draining lymph node. (F) Expression of CD44, CD62L, CD103, CD69, and CLA on CD8+Thy1.1+ pmel cells in vitiligo-affected mice, treated as in (C); percent CLA+ is reported for the skin (gated on the basis of unstained control). LN, lymph node; MFI, mean fluorescence intensity. (G) Naïve Ly5.1+ OT-I cells (104) were transferred 1 day before treatment according to (A), but with B16-OVA given on day 0; OT-I cells were quantified in the skin and lymphoid tissues (gated on live CD8+) 30 days after surgery. (H) Mice were treated as in (C) or (G), and proportions of pmel or OT-I cells were compared in the skin 30 days after surgery. Symbols represent individual mice; horizontal lines depict means. Significance was determined by t test (B, C, and H), Kruskal-Wallis test (D and G), or one-way ANOVA (E and F); NS (not significant) denotes P > 0.05. Data in each panel are representative of two independent experiments, each with n ≥ 3 mice per group; data in (G) are pooled from two experiments.

  • Fig. 2 Functional melanoma/melanocyte Ag–specific CD8 T cells persist in depigmented hair follicles.

    Mice received 104 naïve Thy1.1+ pmel cells 1 day before treatment as in Fig. 1A, and vitiligo-affected skin was analyzed 30 days later by (A) flow cytometry to detect PD-1 and LAG-3 on CD8+Thy1.1+ pmel cells, with B16 tumor-infiltrating CD8+ cells as a positive control; histograms are representative of n = 8 total mice in two independent experiments. (B) Flow cytometry to detect IFN-γ production by CD8+Thy1.1+ pmel cells from digested skin after 14 hours of ex vivo restimulation with cognate (gp10025–33) or irrelevant (OVA) peptide. Symbols represent individual mice; horizontal lines depict means. Data are representative of two independent experiments, each with n = 4 mice per group; significance was determined by Kruskal-Wallis test (Skin) or one-way ANOVA (LN). (C and D) Perilesional skin sections were analyzed by fluorescence microscopy to determine localization of CD8+ T cells and Thy1.1+CD8+ pmel cells in association with E-cadherin–expressing epidermis and hair follicles containing white or black hairs. (C) Arrowheads indicate pmel cells in hair follicles containing white hairs. Scale bar, 50 μm. (D) Pie charts summarize localization of CD8 T cells (left) and pmel cells (right) in association with the indicated structures, compiled from 89 images from n = 5 mice; percentages are of total counted cells.

  • Fig. 3 Melanoma-specific CD8 T cells maintain residence in vitiligo-affected skin.

    (A to C) Skin graft donor mice received 104 naïve Thy1.1+ pmel cells 1 day before treatment as in Fig. 1A. Fifty days after surgery, vitiligo-affected skin was harvested and grafted onto RAG−/− recipients, which were rested for 50 days before analysis. (A) Quantification of total CD8 T cells (top) and CD8+Thy1.1+ pmel cells (bottom) in RAG−/− skin grafts (Grafted) compared with the skin from time-matched vitiligo-affected control mice (Control). (B) Quantification of CD8 T cells (top) and Thy1.1+ CD8+ pmel cells (bottom) in skin graft–draining lymph nodes from RAG−/− mice. (C) Expression of CD103, CD69, and CLA on Thy1.1+ pmel cells in the skin from mice in (A); percent CLA+ is reported for cells in grafted skin. (D to G) Mice received 104 naïve Thy1.1+ pmel cells 1 day before treatment to induce vitiligo as in Fig. 1A. Vitiligo-affected mice then received FTY720 or no treatment for 35 consecutive days, beginning 30 days after surgery (analyzed 65 days after surgery). (D) Proportions of CD8+ cells in blood. (E and F) Quantification of total CD8+ cells and Thy1.1+ pmel cells in the skin. (G) Phenotype of Thy1.1+ pmel cells in the skin; percent CLA+ is reported for cells in FTY720-treated mice. Symbols represent individual mice; horizontal lines depict means. Significance was determined by t test; NS denotes P > 0.05. Data in each panel are representative of two or more independent experiments, each with n ≥ 4 mice per group.

  • Fig. 4 Host vitiligo is required for the establishment of TRM cells.

    Donor mice were treated as in Fig. 1C, and on the day of surgery, CD8 T cells were harvested from pooled lymph nodes and spleens and transferred into mice treated as shown. (A) Experimental time line. i.v., intravenously. (B) Proportion of tumor-primed pmel cells (gated on CD8+; left) from lymph node on the day of harvest and expression of KLRG1 and CXCR3 (gated on Thy1.1+; middle) and Ki67 (gated on Thy1.1+KLRG1loCXCR3+; right). (C) Thirty days after adoptive transfer as in (A). Pmel cells were detected in the skin of unaffected mice versus vitiligo-affected recipient mice (gated on CD8+). (D) Fractions of recipient mice from each group with any detectable pmel cells within a 2-cm2 patch of the skin on day 30. (E to G) Tumor-primed pmel cells were alternatively transferred as in time line (E). RAG−/− mice received TRP-1 Tg cells 1 day before sham skin surgery to induce vitiligo. RAG−/−TRP-1−/− recipients served as unaffected controls. (F) Quantification of Thy1.1+ pmel cells in unaffected skin versus vitiligo-affected skin 30 days after transfer as in (E) (gated on CD8+). (G) Representative phenotype of pmel cells (gated on Thy1.1+) from the skin in (F). Symbols represent individual mice; horizontal lines depict means. Significance was determined by Mann-Whitney test. Data are representative of two independent experiments, each with n ≥ 5 mice per group, or (D and F) pooled from two experiments, each with n ≥ 3 mice per group.

  • Fig. 5 FucT-VII expression on CD8 T cells promotes skin access, formation of memory, and overt vitiligo development.

    (A and B) Vitiligo incidence was tracked in (A) WT mice versus FucT-VII−/− mice or (B) RAG−/− mice reconstituted with either WT or FucT-VII−/− naïve CD8 T cells, 1 day before treatment as shown in Fig. 1A. Significance was determined by log-rank analysis. Data are (A) representative of or (B) pooled from two independent experiments, each with n ≥ 7 mice per group. (C to F) Mice received 104 naïve, congenically distinct WT and FucT-VII−/− pmel cells, admixed at a 1:1 ratio, 1 day before treatment to induce vitiligo as in Fig. 1A. Flow cytometry was performed to detect relative frequencies of WT versus FucT-VII−/− pmel cells (gated on CD8+ cells) in (C) the skin of vitiligo-affected mice 50 days after surgery, (D) lymph nodes 4 days before surgery, (E) the skin on the day of surgery, and (F) lymph nodes of vitiligo-affected mice 50 days after surgery. Symbols represent individual mice, with lines joining cell populations in the same mouse. Significance was determined by Wilcoxon matched-pairs test (C) or paired t test (D to F). Data in (C) to (F) are combined from two independent experiments, each with n = 3 mice per group. Arrows indicate mean percent difference between WT and FucT-VII−/− population sizes.

  • Fig. 6 CD103 expression on CD8 T cells promotes skin access and TRM cell formation.

    (A and B) Vitiligo (induced as in Fig. 1A) was tracked in (A) WT mice versus CD103−/− mice or (B) CD8−/− mice reconstituted with WT or CD103−/− naïve CD8 T cells. Data are representative of two independent experiments, each with n ≥ 7 mice per group (C and D) Relative proportions of vitiligo-affected mice [from (A) and (B), respectively] with localized vitiligo versus disseminated vitiligo, with dissemination defined by depigmentation extending beyond a 2-cm2 area surrounding the surgical site. (E to J) Mice received 104 naïve, congenically distinct WT and CD103−/− pmel cells, admixed at a 1:1 ratio, 1 day before treatment as in Fig. 1A. Flow cytometry was performed to detect relative frequencies of WT versus CD103−/− pmel cells (gated on CD8+ cells) in (E) the skin of vitiligo-affected mice 45 days after surgery, (F) the skin on the day of surgery, (G) lymph nodes 4 days before surgery, (H) lymph nodes of vitiligo-affected mice 45 days after surgery, and (I) tumors on the day of surgery. (J) Analysis of lymph nodes from (G) to detect IFN-γ production by CD8+Thy1.1+ pmel cells after 5 hours of ex vivo restimulation with cognate (gp10025–33) or irrelevant (OVA) peptide. Symbols represent individual mice. (E to I) Significance was determined by Wilcoxon matched-pairs test, pairing CD103−/− and WT pmel populations in the same mouse (denoted by line-connected points). Arrows indicate mean percent difference between WT and CD103−/− pmel population sizes. Data are pooled from two independent experiments, each with n ≥ 3 mice per group, with the exception of (G), which is representative of three experiments, each with n ≥ 4 mice per group. (J) Significance was determined by Kruskal-Wallis test; data are representative of two independent experiments, each with n = 4 mice per group. NS denotes P > 0.05.

  • Fig. 7 CD103+ CD8 TRM cells are required for long-lived tumor protection.

    Mice were treated as in Fig. 1A, and those that developed vitiligo (right flank) were rechallenged in pigmented skin on the left flank by inoculation with 1.2 × 105 B16 cells 30 days after surgery. (A) Tumor incidence (left) and average diameter of palpable tumors (right) in CD8−/− mice that had been reconstituted with either WT or CD103−/− naïve CD8 T cells. Naïve denotes untreated WT mice. Data are pooled from two independent experiments with n ≥ 8 mice per group. (B) Tumor incidence (left) and average diameter of palpable tumors (right) in vitiligo-affected mice that were either untreated or treated with FTY720 ± anti-CD8 mAb. FTY720 was given daily starting 14 days before tumor challenge and continuing to the end of the experiment. Anti-CD8 was given 1 day before tumor challenge and weekly thereafter. (Left) Data are pooled from two independent experiments, with n ≥ 21 mice per group. (Right) Data are representative of two experiments, with n ≥ 10 mice per group. Significance was determined by Gehan-Breslow-Wilcoxon analysis (left) or two-way ANOVA (right; tumor diameter given as mean ± SEM).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/10/eaam6346/DC1

    Materials and Methods

    Fig. S1. Phenotype of CD8 T cells and OT-I cells in vitiligo-affected skin.

    Fig. S2. IFN-γ production by endogenous TRP-2–specific CD8 T cells in the skin.

    Fig. S3. Appearance of vitiligo-affected skin grafts on RAG−/− mice.

    Fig. S4. Phenotype of skin graft–derived CD8 T cells that enter lymph nodes.

    Fig. S5. CD103 expression by tumor-primed pmel cells.

    Fig. S6. Absence of CLA on FucT-VII−/− T cells.

    Fig. S7. Tumor growth kinetics in FucT-VII−/− mice.

    Fig. S8. Adoptive immunotherapeutic efficacy of CD103-deficient CD8 T cells.

    Fig. S9. CD8 T cell depletion from the skin by anti-CD8 mAb.

    Fig. S10. Effects of local skin depigmentation status on tumor protection.

    Fig. S11. Absence of protection against LLC in mice with vitiligo.

    Fig. S12. Isotype staining controls for flow cytometry.

    Fig. S13. Flow cytometry gating strategies.

    Fig. S14. Representative control staining for immunohistochemistry.

    Table S1. Primary source data.

    Reference (30)

  • Supplementary Materials

    Supplementary Material for:

    Resident memory T cells in the skin mediate durable immunity to melanoma

    Brian T. Malik, Katelyn T. Byrne, Jennifer L. Vella, Peisheng Zhang, Tamer B. Shabaneh, Shannon M. Steinberg, Aleksey K. Molodtsov, Jacob S. Bowers, Christina V. Angeles, Chrystal M. Paulos, Yina H. Huang, Mary Jo Turk*

    *Corresponding author. Email: mary.jo.turk{at}dartmouth.edu

    Published 14 April 2017, Sci. Immunol. 2, eaam6346 (2017)
    DOI: 10.1126/sciimmunol.aam6346

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Phenotype of CD8 T cells and OT-I cells in vitiligo-affected skin.
    • Fig. S2. IFN-γ production by endogenous TRP-2–specific CD8 T cells in the skin.
    • Fig. S3. Appearance of vitiligo-affected skin grafts on RAG-/- mice.
    • Fig. S4. Phenotype of skin graft–derived CD8 T cells that enter lymph nodes.
    • Fig. S5. CD103 expression by tumor-primed pmel cells.
    • Fig. S6. Absence of CLA on FucT-VII-/- T cells.
    • Fig. S7. Tumor growth kinetics in FucT-VII-/- mice.
    • Fig. S8. Adoptive immunotherapeutic efficacy of CD103-deficient CD8 T cells.
    • Fig. S9. CD8 T cell depletion from the skin by anti-CD8 mAb.
    • Fig. S10. Effects of local skin depigmentation status on tumor protection.
    • Fig. S11. Absence of protection against LLC in mice with vitiligo.
    • Fig. S12. Isotype staining controls for flow cytometry.
    • Fig. S13. Flow cytometry gating strategies.
    • Fig. S14. Representative control staining for immunohistochemistry.
    • Table S1. Primary source data.
    • Reference (30)

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