Commensal papillomavirus immunity preserves the homeostasis of highly mutated normal skin

Abstract

Immunosuppression commonly disrupts the homeostasis of mutated normal skin, leading to widespread skin dysplasia and field cancerization. However, the immune system's role in maintaining the normal state of mutated tissues remains uncertain. Herein, we demonstrate that T cell immunity to cutaneotropic papillomaviruses promotes the homeostasis of ultraviolet radiation-damaged skin. Mouse papillomavirus (MmuPV1) colonization blocks the expansion of mutant p53 clones in the epidermis in a CD8⁺ T cell-dependent manner. MmuPV1 activity is increased in p53-deficient keratinocytes, leading to their specific targeting by CD8⁺ T cells in the skin. Sun-exposed human skin containing mutant p53 clones shows increased epidermal beta-human papillomavirus (β-HPV) activity and CD8⁺ T cell infiltrates compared with sun-protected skin. The expansion of mutant p53 clones in premalignant skin lesions associates with β-HPV loss. Thus, immunity to commensal HPVs contributes to the homeostasis of mutated normal skin, highlighting the role of virome-immune system interactions in preserving aging human tissues.

Keywords: CD8(+) T cell immunity; aging skin; clonal mutation; cutaneotropic papillomavirus; virome.

Figure 1.. Papillomavirus skin colonization protects against the expansion of mutant p53 clones in the UV-damaged skin.

(A) Representative low, medium, and high magnification images of hematoxylin and eosin (H&E)-stained SKH-1 back skin colonized with MmuPV1 versus VLP followed by UV skin treatment. (B) Representative immunofluorescent (IF) images of MmuPV1- and VLP-colonized SKH-1 skin stained with Ki67, a proliferation marker, and keratin 6 (K6), a marker for epidermal hyperplasia. (C) Epidermal thickness of SKH-1 skin colonized with MmuPV1 (n = 10) versus VLP (n = 10) followed by UV skin treatment. Epidermal thickness was measured in 9 to 10 randomly selected high power field (HPF) images of each skin. Each dot represents an average of three measurements in one HPF image. (D) Representative low, medium, and high magnification images of H&E-stained SKH-1 back skin colonized with MPyV versus its VLP followed by UV skin treatment. (E) Representative IF images of Ki67/K6-stained MPyV- versus VLP-infected SKH-1 skin after UV skin treatment. (F) Epidermal thickness of SKH-1 skin colonized with MPyV (n = 7) versus VLP (n = 9) followed by UV skin treatment. Epidermal thickness was measured in 8 to 10 randomly selected HPF images of each skin. Each dot represents an average of three measurements in one HPF image. (G) Representative immunohistochemical (IHC) images of p53-stained SKH-1 skin colonized with MmuPV1 versus VLP after UV skin treatment. Arrows point to mutant p53 clones in the epidermis. (H-J) Mutant p53 clones’ number per 1 cm of the epidermis (H), thickness (I), and size (J) in SKH-1 skin colonized with MmuPV1 (n = 10) versus VLP (n = 10) followed by UV skin treatment. Each dot represents a mouse (H) or a mutant p53 clone (I,J). (K) Representative IHC images of p53-stained SKH-1 skin infected with MPyV versus VLP after UV skin treatment. Arrows point to mutant p53 clones in the epidermis. (L-N) Mutant p53 clones’ number per 1 cm of the epidermis (L), thickness (M), and size (N) in SKH-1 skin colonized with MPyV (n = 7) versus VLP (n = 9) followed by UV skin treatment. Each dot represents a mouse (L) or a mutant p53 clone (M,N). Dashed lines in IF images mark the epidermal basement membrane. Two-tailed unpaired t-test (C,F,I,J,M,N), two-tailed Mann-Whitney U test (H,L), ns: not significant, bar graphs show mean + s.d., scale bars: 100 μm. See also Figure S1.

Figure 2.. CD8⁺ T cell immunity blocks the expansion of mutant p53 clones in MmuPV1-colonized epidermis.

(A) Representative IF images of CD103/CD8/p53-stained SKH-1 skin colonized with MmuPV1 versus VLP after UV skin treatment. Note that epidermal CD8⁺ cells are CD3⁺ T cells. (B-E) Quantification of epidermal CD8⁺ T cells (B), CD103⁺CD8⁺ T cells (C), CD137⁺CD8⁺ T cells (D) and Tox⁻CD8⁺ T cells (E) as the percentage of total DAPI⁺ epidermal cells within the mutant p53 clones (p53 clone) versus nonclonal epidermis (Nonclone) of MmuPV1- (n = 10) and VLP-colonized (n = 10) mice at the completion of UV skin treatment. Each dot represents the T cell count in a mutant clone or a randomly selected nonclonal epidermis of comparable size. (F) Representative IF images of CD8/p53-stained SKH-1 skin infected with MmuPV1 versus VLP and undergone UV skin treatment together with anti-CD8-IR700 antibody (αCD8) or control IgG-IR700 antibody (IgG) treatment protocol (Figure S4C). (G-I) Mutant p53 clones’ number per 1 cm of the epidermis (G), thickness (H), and size (I) in the skin of SKH-1 mice that received MmuPV1 plus IgG (n = 6), MmuPV1 plus αCD8 (n = 7), VLP plus IgG (n = 10), or VLP plus αCD8 (n = 9) at the completion of UV skin treatment. Each dot represents a mouse (G) or a mutant p53 clone (H,I). Dashed lines in IF images mark the epidermal basement membrane. Two-tailed unpaired t-test (B-E,H,I), two-tailed Mann-Whitney U test (G), ns: not significant, bar graphs show mean + s.d., scale bars: 100 μm. See also Figures S2–S4.

Figure 3.. MmuPV1-specific CD8⁺ T cell immunity blocks the expansion of MmuPV1-colonized mutant p53 clones.

(A) Schematic diagram of the experimental design to examine the function of virus-specific memory T cells in targeting virus-colonized mutant p53 clones in UV-treated B6 nude mice. (B) Representative back skin images of B6 nude mice that received MmuPV1-induced CD44⁺CD8⁺ memory T cells followed by MmuPV1 (n = 10) versus VLP (n = 10) back skin infection. Note the development of warts on the back skin of B6 nude mice infected with MmuPV1 after receiving MPyV-induced CD44⁺CD8⁺ memory CD8⁺ T cells (n = 2) or PBS control (n = 2). (C) Representative IF images of mutant p53 clones in the back skin of MmuPV1- versus VLP-infected B6 nude mice that received MmuPV1-induced memory CD8⁺ T cells followed by UV skin treatment for 18 weeks. (D and E) Mutant p53 clones’ number per 1 cm of the epidermis (D) and size (E) in the MmuPV1- (n = 10) versus VLP-infected (n = 10) skin of B6 nude mice that received MmuPV1-induced memory CD8⁺ T cells and UV skin treatment. Each dot represents a mouse (D) or a mutant p53 clone (E). Dashed lines in IF images mark the epidermal basement membrane. Two-tailed Mann-Whitney U test (D) and two-tailed unpaired t-test (E), ns: not significant, bar graphs show mean + s.d., scale bars: 1 cm (B) and 100 μm (C). See also Figure S5.

Figure 4.. p53 loss upregulates MmuPV1 activity in keratinocytes leading to their elimination by CD8⁺ T cells.

(A and B) Relative MmuPV1 DNA (A) and E1^E4 RNA (B) levels in p53^KO and WT PAM212 keratinocytes infected with MmuPV1 (n = 6) versus VLP (n = 3). The values were normalized to the MmuPV1-infected WT group. These data were verified in three independently generated p53^KO keratinocyte cell lines. (C and D) Relative MPyV DNA (C) and large T antigen RNA (D) levels in p53^KO and WT PAM212 keratinocytes infected with MPyV (n = 9) versus VLP (n = 3) normalized to MPyV-infected WT group. (E and F) Relative MmuPV1 DNA (E) and E1^E4 RNA (F) levels in p53^KO PAM212 keratinocytes transduced with empty (control) versus p53^R155C-expressing vector and WT keratinocytes transduced with empty vector followed by MmuPV1 infection for three days (n = 6 in each group). The values were normalized to WT control group. (G) Representative images of H&E-stained tumors formed by p53^KO keratinocytes infected with MmuPV1 versus VLP and injected subcutaneously into syngeneic mice colonized with MmuPV1 or VLP (Figure S6D). Note the absence of any tumors at the site of most MmuPV1-infected p53^KO keratinocyte injections in MmuPV1-colonized mice. (H) Quantification of tumor weights formed by p53^KO keratinocytes (p53^KO Kr) infected with MmuPV1 versus VLP and injected into mice colonized with MmuPV1 or VLP at day 21 post-injection (n = 10 tumors per group). (I) Representative IF images of CD8/CD3-stained tumors formed by p53^KO keratinocytes infected with MmuPV1 versus VLP and injected into mice colonized with MmuPV1 or VLP. A rare tumor that formed by MmuPV1-infected p53^KO keratinocytes in a MmuPV1-colonized mouse is shown. (J) Quantification of tumor-infiltrating CD8⁺ T cells as the percentage of total DAPI⁺ cells in the tumor. T cells were counted in up to five randomly selected HPF images from each tumor. Each dot indicates the T cell count in one HPF image. The three MmuPV1-infected p53^KO keratinocyte-derived tumors in MmuPV1-colonized mice were used for quantification. (K and L), Representative images of H&E-stained tumors (K) and tumor weights (L) formed by MmuPV1-infected p53^KO keratinocytes in MmuPV1-colonized mice that received anti-CD8 (αCD8) versus IgG control antibody at day 21 post-keratinocyte injection (n = 10 tumors per group). Note the absence of any tumors at the site of most MmuPV1-infected p53^KO keratinocytes injections in MmuPV1-colonized mice that received IgG control antibody. Two-tailed Mann-Whitney U test, ns: not significant, bar graphs show mean + s.d., scale bars: 100 μm. See also Figure S6.

Figure 5.. CD8⁺ T cells preferentially target MmuPV1-colonized mutant p53 clones in the epidermis.

(A) Schematic diagram of UV treatment followed by MmuPV1 or VLP infection in SKH-1 mice to investigate the specific targeting of MmuPV1-infected mutant p53 clones by CD8⁺ T cells. (B) Representative IF images of CD8/p53-stained SKH-1 back skin treated with UV followed by MmuPV1, VLP back skin infection, or MmuPV1 abdominal skin infection. Note that epidermal CD8⁺ cells are CD3⁺ T cells. (C) The ratio of epidermal CD8⁺ T cells in mutant p53 clones to the nonclonal epidermis in mice infected with MmuPV1 on the back skin (n = 7), VLP on the back skin (n = 6), or MmuPV1 on the abdominal skin (Abd MmuPV1, n = 5). Each dot represents the ratio of T cells count per DAPI⁺ epidermal cells in one mutant p53 clone to the average number of T cells per DAPI⁺ epidermal cells across 4–10 HPF images of nonclonal epidermis. Dashed lines in IF images mark the epidermal basement membrane. Two-tailed Mann-Whitney U test, ns: not significant, bar graphs show mean + s.d., scale bar: 100 μm.

Figure 6.. β-HPV activity is increased in sun-damaged normal human skin.

(A) Representative images of p53 IHC (top) and β-HPV RNA ISH using probes that detect 25 β-HPV types (bottom) on human facial and truncal skin. Arrow points to a mutant p53 clone in facial skin, which was absent in sun-protected truncal skin. Arrowheads point to positive RNA ISH signals (red dots). (B) Quantification of β-HPV RNA signals per 100 keratinocytes in facial (n = 31) and truncal skin (n = 30). (C) Representative IF images of CD8/CD3/p53-stained human facial and truncal skin. Dashed lines in IF images mark the epidermal basement membrane. (D and E) Epidermal CD3⁺ T cell (D) and CD8⁺ T cell (E) counts as the percentage of total DAPI⁺ epidermal cells in mutant p53 clones (p53 clone) versus nonclonal epidermis (Nonclone) in facial (n = 31) and truncal skin (n = 30). Each dot indicates the T cell count in a mutant clone or a randomly selected nonclonal epidermis of comparable size. Two-tailed unpaired t-test (B,D,E), ns: not significant, bar graphs show mean + s.d., scale bars: 100 μm. See also Figure S7.

Figure 7.. Precancerous skin lesions that are marked by expanded mutant p53 clones have reduced β-HPV activity compared with normal skin.

(A) Representative images of β-HPV RNA ISH-stained AK and adjacent normal skin in immunocompetent patients. Arrowheads point to β-HPV RNA signals (red dots). (B) Quantification of β-HPV RNA signals per 100 keratinocytes in AKs and adjacent normal skin (n = 30). (C) Representative images of β-HPV RNA ISH-stained AKs from immunosuppressed and immunocompetent patients. Arrowheads point to β-HPV RNA signals (red dots). (D) Quantification of β-HPV RNA signals per 100 keratinocytes in AKs from immunosuppressed (n = 30) and immunocompetent (n = 30) patients. (E) Representative IF images of CD8/p53-stained human AKs from immunosuppressed and immunocompetent patients. Note that CD8⁺ cells in the epidermis are CD3⁺ T cells. Arrows point to epidermal CD8⁺ T cells. Dashed lines mark the epidermal basement membrane. (F) Quantification of epidermal CD8⁺ T cells as the percentage of total DAPI⁺ epidermal cells in the expanded mutant p53 clones of AKs from immunosuppressed (n = 30) and immunocompetent (n = 30) patients. Each dot indicates the T cell count in a mutant p53 clone. Paired t-test (B), two-tailed unpaired t-test (D,F), bar graphs show mean + s.d., scale bars: 100 μm. See also Figure S7.