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

Abstract

Tissue-resident memory T (T_RM) cells have been widely characterized in infectious disease settings; however, their role in mediating immunity to cancer remains unknown. We report that skin-resident memory T cell responses to melanoma are generated naturally as a result of autoimmune vitiligo. Melanoma antigen-specific T_RM cells resided predominantly in melanocyte-depleted hair follicles and were maintained without recirculation or replenishment from the lymphoid compartment. These cells expressed CD103, CD69, and CLA (cutaneous lymphocyte antigen), but lacked PD-1 (programmed cell death protein-1) or LAG-3 (lymphocyte activation gene-3), and were capable of making IFN-γ (interferon-γ). CD103 expression on CD8 T cells was required for the establishment of T_RM cells in the skin but was dispensable for vitiligo development. CD103⁺ CD8 T_RM cells were critical for protection against melanoma rechallenge. This work establishes that CD103-dependent T_RM cells play a key role in perpetuating antitumor immunity.

Figure 1. CD8 T cells recognizing tumor/self- and tumor-specific antigens persist in vitiligo-affected skin, and exhibit a T_RM cell phenotype

(A) Experimental scheme to induce melanoma-associated vitiligo; unaffected mice underwent identical procedures but did not depigment. (B) Mice were treated as in A, and proportions and absolute numbers of CD8 T cells were detected 65d post-surgery by flow cytometry; gated on live CD45⁺ cells. (C–E) 10⁴ naïve Thy1.1⁺ pmel cells were transferred 1d prior to treatment as in A, and pmel proportions (gated on live CD8⁺ cells) were quantified 50d post-surgery in (C) vitiligo-affected 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. (F) Expression of CD44, CD62L, CD103, CD69, and CLA on CD8⁺Thy1.1⁺ pmel cells in vitiligo-affected mice, treated as in panel C; percent CLA⁺ is reported for skin, gated based on unstained control. (G) 10⁴ naïve Ly5.1⁺ OT-I cells were transferred 1d prior to treatment according to A, but with B16-OVA given on d0; OT-I cells were quantified in skin and lymphoid tissues (gated on live CD8⁺) 30d post-surgery. (H) Mice were treated either as in C or G, and proportions of pmel or OT-I cells were compared in skin 30d post-surgery. Symbols represent individual mice; horizontal lines depict means. Significance was determined by t test (for B, C, and H), Kruskal-Wallis test (for D and G), or one-way ANOVA (for E and F); NS denotes p > 0.05. Data in each panel are representative of 2 independent experiments, each with n ≥3 mice/group; data in G are pooled from two experiments.

Figure 2. Functional melanoma/melanocyte Ag-specific CD8 T cells persist in depigmented hair follicles

Mice received 10⁴ naïve Thy1.1⁺ pmel cells 1d prior to treatment as in Fig. 1A, and vitiligo-affected skin was analyzed 30d 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 2 independent experiments. (B) Flow cytometry to detect IFN-γ production by CD8⁺Thy1.1⁺ pmel cells from digested skin, following 14h ex vivo restimulation with cognate (gp100_25–33) or irrelevant (OVA) peptide. Symbols represent individual mice; horizontal lines depict means. Data are representative of 2 independent experiments each with n = 4 mice/group; significance was determined by Kruskal-Wallis test (skin) or one-way ANOVA (LN). (C–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) Arrows 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.

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

(A–C) Skin graft donor mice received 10⁴ naïve Thy1.1⁺ pmel cells 1d prior to treatment as in Fig. 1A. Fifty days post-surgery, vitiligo-affected skin was harvested and grafted onto RAG^−/− recipients, which were rested for 50d prior to analysis. (A) Quantification of total CD8 T cells (top) and CD8⁺Thy1.1⁺ pmel cells (bottom) in RAG^−/− skin grafts (Grafted) compared to 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 skin from mice in A; percent CLA⁺ is reported for cells in grafted skin. (D–G) Mice received 10⁴ naïve Thy1.1⁺ pmel cells 1d prior to treatment to induce vitiligo as in Fig. 1A. Vitiligo-affected mice then received FTY720, or no treatment, for 35 consecutive days, beginning 30d post-surgery (analyzed 65d post-surgery). (D) Proportions of CD8⁺ cells in blood. (E–F) Quantification of (E) total CD8⁺ cells and (F) Thy1.1⁺ pmel cells, in skin. (G) Phenotype of Thy1.1⁺ pmel cells in 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 ≥ 2 independent experiments, each with n ≥4 mice/group.

Figure 4. Host vitiligo is required for the establishment of T_RM 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 timeline; (B) Proportion of tumor-primed pmel cells (gated on CD8⁺; left) from LN on the day of harvest; expression of KLRG1 and CXCR3 (gated on Thy1.1⁺; middle), and Ki67 (gated on Thy1.1⁺KLRG1^loCXCR3⁺; right). (C) Thirty days post-adoptive transfer as shown in A; pmel cells were detected in skin of unaffected vs. 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 skin, on d30. (E–G) Tumor-primed pmel cells were alternatively transferred as in timeline (E); RAG^−/− mice received TRP-1 Tg cells 1d prior to sham skin surgery to induce vitiligo; RAG^−/−TRP-1^−/− recipients served as unaffected controls. (F) Quantification of Thy1.1⁺ pmel cells in unaffected vs. vitiligo-affected skin 30d post-transfer as in E (gated on CD8⁺). (G) Representative phenotype of pmel cells (gated on Thy1.1⁺) from skin in panel F. Symbols represent individual mice; horizontal lines depict means. Significance was determined by Mann-Whitney test; Data are representative of 2 independent experiments each with n ≥5 mice/group, or (D, F) pooled from 2 experiments each with n ≥3 mice/group.

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

(A–B) Vitiligo incidence was tracked in (A) wild-type (WT) mice vs. FucT-VII^−/− mice or (B) RAG^−/− mice reconstituted with either WT or FucT-VII^−/− naïve CD8 T cells, 1d prior to treatment as shown in Fig. 1A. Significance was determined by log-rank analysis. Data are (A) representative of, or (B) pooled from 2 independent experiments, each with n ≥ 7 mice group. (C–F) Mice received 10⁴ naïve, congenically distinct WT and FucT-VII^−/− pmel cells, admixed at a 1:1 ratio, 1d prior to treatment to induce vitiligo as in Fig. 1A. Flow cytometry was performed to detect relative frequencies of WT vs. FucT-VII^−/− pmel cells (gated on CD8⁺ cells) in (C) skin of vitiligo-affected mice 50d post-surgery, (D) lymph nodes 4d prior to surgery, (E) skin on the day of surgery, and (F) lymph nodes of vitiligo-affected mice 50d post-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–F). Data in C–F are combined from 2 independent experiments, each with n = 3 mice/group. Arrows indicate mean percent difference between WT and FucT-VII^−/− population sizes.

Figure 6. CD103 expression on CD8 T cells promotes skin access and T_RM cell formation

(A–B) Vitiligo (induced as in Fig. 1A) was tracked in (A) wild-type (WT) mice vs. CD103^−/− mice or (B) CD8^−/− mice reconstituted with WT or CD103^−/− naïve CD8 T cells. Data are representative of 2 independent experiments, each with n ≥ 7 mice/group (C–D) Relative proportions of vitiligo-affected mice (from A and B, respectively) with localized vs. disseminated vitiligo; with dissemination defined by depigmentation extending beyond a 2 cm² area surrounding the surgical site. (E–J) Mice received 10⁴ naïve, congenically distinct WT and CD103^−/− pmel cells, admixed at a 1:1 ratio, 1d prior to treatment as in Fig. 1A. Flow cytometry was performed to detect relative frequencies of WT vs. CD103^−/− pmel cells (gated on CD8⁺ cells) in (E) skin of vitiligo-affected mice 45d post-surgery, (F) skin on the day of surgery, (G) lymph nodes 4d prior to surgery, (H) lymph nodes of vitiligo-affected mice 45d post-surgery, and (I) tumors on the day of surgery. (J) Analysis of lymph nodes from panel G, to detect IFN-γ production by CD8⁺Thy1.1⁺ pmel cells, following 5h ex vivo restimulation with cognate (gp100_25–33) or irrelevant (OVA) peptide. Symbols represent individual mice. (E–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 2 independent experiments, each with n ≥ 3 mice/group, with the exception of G, which is representative of 3 experiments each with n ≥ mice/group. (J) Significance was determined by Kruskal-Wallis test; data are representative of 2 independent experiments each with n = 4 mice/group. NS denotes p > 0.05.

Figure 7. CD103⁺ CD8 T_RM cells are required for long-lived tumor protection

Mice were treated as in Fig. 1A, and those that developed vitiligo (right flank) were re-challenged in pigmented skin on the left flank by inoculation with 1.2×10⁵ B16 cells 30d post-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 wild-type mice. Data are pooled from 2 independent experiments with n ≥ 8 mice 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 prior to tumor challenge and continuing to the end of the experiment. Anti-CD8 was given 1d prior to tumor challenge and weekly thereafter. (Left) data are pooled from 2 independent experiments with n ≥21 mice/group; (right) data are representative of 2 experiments with n ≥10 mice/group. Significance was determined by Gehan-Breslow-Wilcoxon analysis (left panels), or two-way ANOVA (right panels; tumor diameter given as mean +/− SEM).