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. 2021 Sep 14;118(37):e2016963118.
doi: 10.1073/pnas.2016963118.

Chronic UV radiation-induced RORγt+ IL-22-producing lymphoid cells are associated with mutant KC clonal expansion

Julia M Lewis  1 , Patrick F Monico  1 , Fatima N Mirza  1 , Suzanne Xu  1 , Sara Yumeen  1 , Jack L Turban  1 , Anjela Galan  1 , Michael Girardi  2
Affiliations

Chronic UV radiation-induced RORγt+ IL-22-producing lymphoid cells are associated with mutant KC clonal expansion

Julia M Lewis et al. Proc Natl Acad Sci U S A. .

Abstract

Chronic ultraviolet (UV) radiation exposure is the greatest risk factor for cutaneous squamous cell carcinoma (cSCC) development, and compromised immunity accelerates this risk. Having previously identified that epidermal Langerhans cells (LC) facilitate the expansion of UV-induced mutant keratinocytes (KC), we sought to more fully elucidate the immune pathways critical to cutaneous carcinogenesis and to identify potential targets of intervention. Herein, we reveal that chronic UV induces and LC enhance a local immune shift toward RORγt+ interleukin (IL)-22/IL-17A-producing cells that occurs in the presence or absence of T cells while identifying a distinct RORγt+ Sca-1+ CD103+ ICOS+ CD2+/- CCR6+ intracellular CD3+ cutaneous innate lymphoid cell type-3 (ILC3) population (uvILC3) that is associated with UV-induced mutant KC growth. We further show that mutant KC clone size is markedly reduced in the absence of RORγt+ lymphocytes or IL-22, both observed in association with expanding KC clones, and find that topical application of a RORγ/γt inhibitor during chronic UV exposure reduces local expression of IL-22 and IL-17A while markedly limiting mutant p53 KC clonal expansion. We implicate upstream Toll-like receptor signaling in driving this immune response to chronic UV exposure, as MyD88/Trif double-deficient mice also show substantially reduced p53 island number and size. These data elucidate key immune components of chronic UV-induced cutaneous carcinogenesis that might represent targets for skin cancer prevention.

Keywords: TP53; carcinogenesis; innate lymphoid cells; interleukin-22; squamous cell carcinoma.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
LC increase UV-induced mutant keratinocytes in association with RORγt+ IL-22+IL-17+ ILC. (A) Average p53 island area is reduced by 28.98% in LC-deficient (DTA) versus LC-intact (NLC) FVB.TCRβ−/−δ−/− T cell–deficient epidermis following 9 wk UVB (400 J/m2, 3×/wk) exposure. Each dot represents one mouse; the line is mean. Representative immunofluorescent images of p53 islands (red) and CD207+ LC (green) in epidermal sheets. (Scale bar, 20 μm.) (B) In LC-intact (NLC) mice, CD207+ LC density is greater in association with p53 islands (assoc: in/within 30 μm of island; distant: > 30 μm from island). Flow cytometric analysis of characteristic ILC transcription factors (C) and cytokines (D) in cell suspensions prepared from unexposed (UT) versus chronic UVB–exposed (400 J/m2, 3×/wk, 9 wk) LC-deficient (DTA) versus LC-intact (NLC) FVB.TCRβ−/−δ−/− mouse skin. Each dot represents one mouse; the line is mean. Representative contour plots are gated on CD45+Thy1+LIN− skin ILC. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 2.
Fig. 2.
The phenotype of UVB-induced skin ILC3 in T cell–deficient mice is distinct: RORγt+ Sca-1+ CD103+ ICOS+ CD2± CCR6+ icCD3ε+ CD4− NKp46− MHCII−. Skin cell suspensions prepared from chronic UVB– (400 J/m2, 3×/wk, 9 wk) exposed B6.TCRβ−/−δ−/− mice were analyzed by flow cytometry for correlation of (A) RORγt expression along with a variety of other markers and (B) intracellular CD3ε expression along with characteristic ILC cytokines. Representative contour plots are gated on CD45+Thy1+LIN− cells. The composite (A) was prepared from five experiments in which RORγt+ ILC ranged from 31.0 to 41.1% of all skin ILC. Additional markers are shown in SI Appendix, Fig. S4. (C) Following chronic UVB exposure, immunofluorescent staining of epidermal sheets from T cell–deficient mice demonstrate intracellular CD3ε+ cells (icCD3; green) and CD207+ LC (blue) in mutant KC p53 islands (red). (D) Immunofluorescent staining of frozen sections from T cell–deficient mouse skin shows increased intracellular CD3ε+ cells (icCD3; red) following chronic UVB exposure. (Scale bar, 20 ��m.)
Fig. 3.
Fig. 3.
Chronic UVB induces distinct populations of IL-22+ IL-17A+ ILC3 and Th cells. T cell–intact, LC-deficient (DTA) versus LC-intact (NLC) B6 mice remained untreated (UT) or were exposed to chronic UVB (400 J/m2, 3×/wk, 9 wk) before skin was harvested for analysis of gene expression (A) or quantification of p53 islands (B). P53 islands in DTA mice are 44.59% smaller than those found in NLC mice. A flow cytometric analysis of CD45+Thy1+LIN− T cells + ILC from untreated (UT) or chronic UVB–exposed (UVB) WT.B6 mice (C) shows increased RORγt and T-bet expression. The UVB-induced RORγt+ cells are Foxp3− and composed of γδ T cells, αβ T cells, and CD45+Thy1+LIN−TCR− ILC (D). Increased RORγt and T-bet expression correlates with increased IL-22, IL-17A, and IFN-γ (E) production following chronic UVB. *P < 0.05, **P < 0.01, ***P < 0.001. This figure is representative of 12 experiments, with each panel repeated two to three times, and utilized 10 UT DTA, 19 UVB DTA, 10 UT NLC, 20 UVB NLC, 27 UT WT.B6, and 32 UVB WT.B6 mice. NLC and DTA mice were analyzed individually; WT.B6 were analyzed in pools of two to four mice/pool.
Fig. 4.
Fig. 4.
Chronic UV–induced epidermal KC p53 mutational burden is dependent on RORγt+ cells. T cell–intact (A and B) and T cell–deficient (C) RORγt-intact versus RORγt-deficient mice were exposed to chronic UVB (400 J/m2, 3×/wk, 10 wk), and epidermal sheets prepared for quantification of p53 islands and CD3+ T cells or, in T cell–deficient mice, intracellular CD3+ ILC. CD3+ cells are more frequently seen in RORγt-intact epidermis and associated with p53 islands (assoc: in/within 30 μm of island; distant: > 30 μm from island). Representative immunofluorescent images (B) of T cell–intact RORγt-intact (WT) versus deficient (RORγt−/−) show CD207+ LC (green), CD3+ cells (blue), and p53 island (red). (Scale bar, 20 μm.) T cell–deficient mice (D and E) remained unexposed or were exposed to chronic UVB (400 J/m2, 3×/wk, 7 wk). During the final 2 wk of exposure, mice were treated topically with vehicle or 1% RORC inhibitor GSK2981278, and then skin was harvested for analysis of gene expression (D) (RQ to unexposed) and quantification of p53 islands (E). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 5.
Fig. 5.
Mutant p53 keratinocyte clonal expansion is dependent on IL-22. T cell–intact (A, B, and D) and T cell–deficient (C) IL-22–intact versus IL-22–deficient mice were exposed to chronic UVB (400 J/m2, 3×/wk, 12 wk), and epidermal sheets prepared for quantification of p53 islands and CD3+ T cells or intracellular CD3+ ILC (assoc: in/within 30 μm of island; distant: greater than 30 μm away from island). Representative immunofluorescent images (B) of T cell–intact IL-22–intact (WT) versus deficient (IL-22−/−) show CD207+ LC (green), CD3+ cells (blue), and p53 island (red). Costaining with Ki67 (D, blue) shows decreased p53 island proliferation in the absence of IL-22. (Scale bar: 20 μm.) *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 6.
Fig. 6.
TLR signaling initiates the immunopathogenesis of UV-induced mutant KC clonal expansion. (A) P53 island density (Left) and average island size (Right) are reduced by 57.20 and 43.21%, respectively, in MyD88−/−Trif−/− (KO) mice compared with C57BL/6 (WT) mice following chronic UVB exposure (400 J/m2, 3×/wk, 9 wk). Each dot represents one mouse. ***P < 0.001. (B) LC line XS106 expresses a variety of DAMP receptors. Gene expression following 40 cycles of qRT-PCR, expressed as 40-Ct (threshold cycle), and shown relative to β-actin (ACTB). Production of ILC3-activating cytokines by XS106 (C) or isolated LC (D) following exposure to DAMP ligands. Cells were cultured in the presence of predetermined optimal concentrations of DAMP ligands for 48 h, and then supernatants were collected for analysis of IL-23p19 or IL-6 by Bio-Plex assay. Concentrations used: 1 μg/mL LPS, ODN1668 and IMQ; 20 μg/mL HMGB1, 25 μg/mL Poly I:C, 1 mM ATP, 10 μg/mL LTA and BD2. nd = not detected, nt = not tested.
Fig. 7.
Fig. 7.
IL-17 and IL-22 expression are increased in human sun-damaged skin and AK. RNA was isolated from FFPE tissue obtained from normal non–sun-damaged (NNS) or sun-damaged (SD) skin, AK, and squamous cell carcinomas (SCC) for analysis of gene expression (RQ to NNS). *P < 0.05.
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