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Research ArticleDermatologyOncology
Open Access | 
10.1172/JCI183274
1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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1Center for Cancer Immunology and Cutaneous Biology Research Center, Krantz Family Center for Cancer Research and Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA.
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
3Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
4VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
5VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA.
6Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
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Published January 2, 2025 - More info
Published in Volume 135, Issue 1 on January 2, 2025
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Abstract
Cutaneous squamous cell carcinoma (cSCC) incidence and deaths continue to rise, underscoring the need for improved cSCC prevention. Elimination of actinic keratosis (AK) precursor lesions is a major strategy to prevent cSCC. Topical calcipotriol and 5-fluorouracil (5-FU) have been shown to eliminate AKs and reduce the risk of cSCC development, but the mechanism was undefined. In this issue of the JCI, Oka et al. demonstrate that type 2 immunity is necessary and sufficient for the elimination of premalignant keratinocytes and cSCC prevention. Paired biopsies from AK lesions and unaffected skin revealed that only keratinocytes from AKs produced thymic stromal lymphopoietin (TSLP) and damage-associated molecular patterns, resulting in selective recruitment of Th2 cells to the AK lesion. In mouse models of skin carcinogenesis, TSLP was necessary to recruit Th2 cells and trigger IL-24–mediated keratinocyte cell death. These findings suggest that the TSLP/Th2/IL-24 axis is a potential therapeutic target for SCC prevention.
Authors
Matthew D. Vesely, Sean R. Christensen
× AbstractThe continuous rise in skin cancer incidence highlights an imperative for improved skin cancer prevention. Topical calcipotriol-plus–5-fluorouracil (calcipotriol-plus–5-FU) immunotherapy effectively eliminates precancerous skin lesions and prevents squamous cell carcinoma (SCC) in patients. However, its mechanism of action remains unclear. Herein, we demonstrate that calcipotriol-plus–5-FU immunotherapy induces T helper type 2 (Th2) immunity, eliminating premalignant keratinocytes in humans. CD4+ Th2 cells were required and were sufficient downstream of thymic stromal lymphopoietin cytokine induction by calcipotriol to suppress skin cancer development. Th2-associated cytokines induced IL-24 expression in cancer cells, resulting in toxic autophagy and anoikis followed by apoptosis. Calcipotriol-plus–5-FU immunotherapy was dependent on IL-24 to suppress skin carcinogenesis in vivo. Collectively, our findings establish a critical role for Th2 immunity in cancer immunoprevention and highlight the Th2/IL-24 axis as an innovative target for skin cancer prevention and therapy.
Graphical Abstract
Introduction
Despite significant advances in cancer therapeutics led by innovations in immunotherapy, current cancer therapeutics have prohibitive side effects and costs for use in cancer prevention. Hence, effective strategies for cancer immunoprevention are urgently needed. Cutaneous squamous cell carcinoma (SCC) is the second most common cancer, which can cause substantial morbidity, mortality, and economic burden (1). SCC is a highly immunogenic cancer that is immune-regulated from its inception (2). Importantly, actinic keratosis (AK), a precursor to SCC, can be identified and treated to prevent SCC. Patients with multiple AKs have a relatively high cumulative skin cancer risk (3). Current AK field treatments include topical 5-fluorouracil (5-FU), photodynamic therapy, imiquimod, and tirbanibulin (4–7). Although they can eliminate AKs, only 5-FU has been proven to reduce the risk of SCC within 1 year after treatment, and this benefit is no longer apparent 2 years after treatment (8). The impact of other AK field treatments on SCC prevention is unknown. By contrast, AK immunotherapy with the aim of SCC immunoprevention provides an innovative, attainable strategy.
We have previously demonstrated the high efficacy of topical calcipotriol-plus–5-FU therapy for eliminating AKs through a randomized, double-blind clinical trial (9). Calcipotriol is an FDA-approved low-calcemic vitamin D analog used for psoriasis treatment (10). Calcipotriol induces the expression of thymic stromal lymphopoietin (TSLP) in keratinocytes (9, 11, 12). 5-FU synergizes with calcipotriol to generate an immune-mediated modality for AK clearance. Calcipotriol-plus–5-FU treatment promotes the induction of TSLP in AK keratinocytes, leading to massive T cell infiltration and tissue-resident memory T (TRM) cell formation in AKs (9, 13). Importantly, topical calcipotriol-plus–5-FU treatment has shown efficacy in preventing SCC within 3 years after treatment (13). Because calcipotriol-plus–5-FU treatment activates the adaptive immune system to eliminate AKs and helps prevent SCC (9, 13), this topical immunotherapy highlights an innovative therapeutic strategy for AK treatment distinct from current cytotoxic treatments for AK. However, the precise nature of the antitumor immunity induced by calcipotriol-plus–5-FU treatment and its effector mechanism(s) in humans remain unknown.
Herein, we investigated the mechanism of calcipotriol-plus–5-FU immunotherapy in eliminating premalignant keratinocytes. Through an open-label clinical trial, we found that CD4+ T helper 2 (Th2) cells were the dominant immune cells infiltrating AKs after calcipotriol-plus–5-FU treatment. Th2-polarized CD4+ T cells responding to TSLP played a seminal role in tumor suppression in vivo. Calcipotriol-plus–5-FU immunotherapy induced cell death in AKs via toxic autophagy and anoikis followed by apoptosis. IL-24 (melanoma differentiation–associated gene-7 or MDA-7) was a downstream effector molecule induced by Th2-associated cytokines, IL-4 and IL-13 (14–16), that promoted toxic autophagy and apoptosis in cancer cells (14, 15, 17). IL-24 was essential for calcipotriol-plus–5-FU immunotherapy to mediate skin cancer protection in vivo. Our findings highlight Th2 immunity as a promising target for skin cancer prevention and treatment.
ResultsCalcipotriol-plus–5-FU immunotherapy trial. We performed an open-label clinical trial to investigate the mechanism of calcipotriol-plus–5-FU immunotherapy. Eighteen patients with AKs who met the eligibility criteria were enrolled in the study (Supplemental Figure 1A and Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/JCI183274DS1). All the participants applied 0.0025% calcipotriol-plus–2.5% 5-FU field treatment to the entirety of their qualified anatomical sites, face, scalp, right upper extremity (RUE) and/or left upper extremity (LUE), twice daily for 6 days (Supplemental Figure 1B). All the participants completed the treatment course and underwent clinical evaluation and AK/normal skin biopsies before treatment (day 0), 1 day after the last treatment (day 7), and 8 weeks after treatment (Figure 1A and Supplemental Figure 1, A and B). Topical calcipotriol-plus–5-FU immunotherapy led to a mean reduction in the number of AKs of 95% on the face, with 7 out of 10 participants showing complete clearance, 82% on the scalp, 65% on the RUE, and 68% on the LUE 8 weeks after treatment (Supplemental Figure 1, C and D). Calcipotriol-plus–5-FU immunotherapy induced profound erythema centered around the AKs after treatment, followed by a resolution of skin erythema and elimination of AKs by week 8 (Figure 1B). All skin reactions were resolved by week 4 after treatment.
Figure 1Calcipotriol-plus–5-FU immunotherapy induces robust Th2 immunity in AKs associated with TSLP and DAMP upregulation in keratinocytes. (A) Schematic diagram of calcipotriol-plus–5-FU immunotherapy open-label trial. (B) Representative clinical photographs of skin treated with calcipotriol-plus–5-FU. Photographs were taken before (day 0), and after treatment (day 7 and week 8). (C) Representative H&E-stained AKs before (day 0) and after (day 7) calcipotriol-plus–5-FU treatment. (D) Representative images of CD4/CD8-stained AKs before (day 0) and after (day 7) calcipotriol-plus–5-FU treatment. Note that CD4+ and CD8+ cells are CD3+ T cells. (E–H) Quantification of CD4+ T cells in AKs (E), CD8+ T cells in AKs (F), CD4+ T cells in normal skin (G), and CD8+ T cells in normal skin (H) before (day 0) and after (day 7) calcipotriol-plus–5-FU treatment. (I) Representative images of CD4/GATA3-stained AKs before (day 0) and after (day 7) calcipotriol-plus–5-FU treatment. Note that GATA3+CD4+ cells are CD3+ T cells. (J and K) Quantification of GATA3+CD4+ T cells (J) and Foxp3+CD4+ T cells (K) in AKs before (day 0) and after (day 7) calcipotriol-plus–5-FU treatment. (L) Representative images of TSLP-stained AKs before (day 0) and after (day 7) calcipotriol-plus–5-FU treatment. (M) Quantification of TSLP+ cells as percentage DAPI+ keratinocytes in AKs before (day 0) and after (day 7) calcipotriol-plus–5-FU treatment. (N) Representative images of ANXA1-stained AKs before (day 0) and after (day 7) calcipotriol-plus–5-FU treatment. (O–Q) Quantification of ANXA1+ cells (O), CALR+ cells (P), and HMGB1+ cells (Q) as percent DAPI+ keratinocytes in AKs before (day 0) and after (day 7) calcipotriol-plus–5-FU treatment. (R) Representative images of HLA-II–stained AKs before (day 0) and after (day 7) calcipotriol-plus–5-FU treatment. (S) Quantification of HLA-II+ cells as percentage DAPI+ cells in AKs. Each dot represents an AK or normal skin sample. n = 18 participants at each time point; paired t test. Dashed lines mark the epidermal basement membrane in immunofluorescence images. Scale bars: 100 μm.
Calcipotriol-plus–5-FU immunotherapy induces robust Th2 immunity in AKs. Calcipotriol-plus–5-FU treatment induced massive immune cell infiltration in AKs, dominated by CD4+ T and, to a lesser extent, CD8+ T cells (Figure 1, C–F). By contrast, calcipotriol-plus–5-FU immunotherapy did not cause T cell infiltration into the normal skin (Figure 1, G and H, and Supplemental Figure 2, A and B). GATA3+CD4+ Th2 cells were markedly increased in AKs after treatment (Figure 1, I and J). Foxp3+CD4+ regulatory T cell number was not changed in AKs after treatment (Figure 1K and Supplemental Figure 2C). Calcipotriol-plus–5-FU immunotherapy did not affect GATA3+CD4+ Th2 or Foxp3+CD4+ regulatory T cell numbers in the normal skin (Supplemental Figure 2, D and E). The other CD4+ T cell subsets, T-bet+CD4+ Th1 and RORγt+CD4+ Th17 cells, were rare in AKs before and after treatment (Supplemental Figure 2, F and G). Calcipotriol-plus–5-FU immunotherapy also induced CD103+CD4+ resident memory T cell formation in AKs (Supplemental Figure 2H) (18). IHC staining further confirmed that GATA3+CD4+ Th2 cells were the dominant cell type in AKs treated with calcipotriol-plus–5-FU while cytotoxic molecules, Perforin and Granzyme B, SLAMF7, a cytotoxicity-related transcription factor, and T-bet were rarely detected (Supplemental Figure 2I). These results indicate that Th2 cells are the primary effector T cells activated by calcipotriol-plus–5-FU immunotherapy in AKs.
Calcipotriol-plus–5-FU immunotherapy upregulates TSLP and damage-associated molecular patterns in the premalignant keratinocytes. To determine the upstream activators of Th2 immunity in AKs, we investigated the immune factors induced by calcipotriol-plus–5-FU treatment in AK keratinocytes. Calcipotriol-plus–5-FU immunotherapy upregulated TSLP in premalignant keratinocytes but did not affect TSLP expression in the normal skin (Figure 1, L and M, and Supplemental Figure 3, A and B) (19). Interestingly, TSLP was detectable in the plasma of 3 out of 4 participants who treated their face, scalp, RUE, and LUE with calcipotriol-plus–5-FU (Supplemental Figure 3C). As calcipotriol induced TSLP expression in AKs but not in the normal human skin, we investigated whether calcipotriol specifically induced TSLP in malignant keratinocytes. Calcipotriol induced TSLP expression only in SCC cells but not in the normal keratinocyte cell lines (Supplemental Figure 3, D–G). Damage-associated molecular patterns (DAMPs) released in response to cellular stress and death are potent immune activators (20). Among them, Annexin A1 (ANXA1), calreticulin (CALR), and High Mobility Group Box 1 (HMGB1) were highly upregulated in premalignant keratinocytes after calcipotriol-plus–5-FU treatment (Figure 1, N–Q, and Supplemental Figure 3, H and I). Although human leukocyte antigen class I (HLA-I) expression was not affected by calcipotriol-plus–5-FU immunotherapy (Supplemental Figure 3, J and K), HLA-II was highly upregulated in AKs after calcipotriol-plus–5-FU treatment (Figure 1, R and S). HLA-II expression in AK keratinocytes was induced by calcipotriol-plus–5-FU therapy compared with 5-FU monotherapy (Supplemental Figure 3, L and M). The induction of HLA-II, DAMPs, and TSLP in premalignant keratinocytes after calcipotriol-plus–5-FU immunotherapy provides a robust axis for Th2 cell activation in AKs.
T cell immunity induced by calcipotriol-plus–5-FU treatment persists over 5 years. We have shown that calcipotriol-plus–5-FU immunotherapy lowers the risk of SCC development within 3 years after treatment (13). To determine whether T cell immunity induced by calcipotriol-plus–5-FU immunotherapy against AKs persists long-term, we collected AK and normal skin biopsies from the participants in the randomized trial comparing calcipotriol-plus–5-FU versus Vaseline-plus–5-FU for AK treatment over 5 years after the completion of the trial (9). We collected 5 pairs of AK and normal skin biopsies from participants who had a history of Vaseline-plus–5-FU treatment in the randomized clinical trial and never received calcipotriol-plus–5-FU since (Figure 2A). We collected 11 pairs of AK and normal skin biopsies from participants who received calcipotriol-plus–5-FU treatment either in the randomized clinical trial or open-label trial but never since (Figure 2A). Significantly more CD3+ T, CD4+ T, CD103+CD3+ TRM, and CD103+CD4+CD3+ TRM cells infiltrated AKs of the participants who had a history of calcipotriol-plus–5-FU treatment compared with the participants who had a history of Vaseline plus 5-FU treatment (Figure 2, B–G, and Supplemental Figure 4A). GATA3+CD4+ T cells were increased in AKs of the participants who had a history of calcipotriol-plus–5-FU treatment compared with the participants who had a history of Vaseline-plus–5-FU treatment (Figure 2, H and I). Increased T cell infiltration in AKs was not observed in the normal skin of participants with a history of calcipotriol-plus–5-FU treatment (Supplemental Figure 4, B–F). To investigate which immune factors could be responsible for CD4+ T cell activation in AKs that developed in participants with a history of calcipotriol-plus–5-FU treatment, we evaluated ANXA1 and HLA-II expression in AK versus normal skin. AK keratinocytes showed higher expression of ANXA1 and HLA-II compared with normal skin (Figure 2, J–M). These results suggest that T cell immunity, originally induced by calcipotriol-plus–5-FU treatment, can be activated during AK development long after the treatment is completed, which may explain the reduced risk of SCC observed in calcipotriol-plus–5-FU–treated patients (Figure 2N) (13).
Figure 2T cell immunity induced by calcipotriol-plus–5-FU immunotherapy persists over 5 years. (A) Time from Vaseline-plus–5-FU versus calcipotriol-plus–5-FU treatment to biopsy for each participant in the follow-up clinical study. (B) Representative images of CD4/CD3-stained AKs from participants who had a history of (H/o) Vaseline-plus–5-FU versus calcipotriol-plus–5-FU treatment. (C and D) Quantification of CD3+ T cells (C) and CD4+ T cells (D) in AKs from participants who had a history of Vaseline-plus–5-FU versus calcipotriol-plus–5-FU treatment. Each dot represents cell counts from a high power field (hpf) image. 3 hpf images are included per sample (n = 5 participants in Vaseline-plus–5-FU group, n = 11 participants in calcipotriol-plus–5-FU group, Mann-Whitney U test). (E) Representative images of CD4/CD103-stained AKs from participants who had a history of Vaseline-plus–5-FU versus calcipotriol-plus–5-FU treatment. (F and G) Quantification of CD103+CD3+ T cells (F) and CD103+CD4+ T cells (G) in AKs from participants who had a history of Vaseline-plus–5-FU versus calcipotriol-plus–5-FU treatment. Each dot represents cell counts from an hpf image. 3 hpf images are included per sample (n = 5 participants in Vaseline-plus–5-FU group, n = 11 participants in calcipotriol-plus–5-FU group, Mann-Whitney U test). (H) Representative images of CD4/GATA3-stained AKs from participants who had a history of Vaseline-plus–5-FU versus calcipotriol-plus–5-FU treatment. (I) Quantification of GATA3+CD4+ T cells in AKs from participants who had a history of Vaseline-plus–5-FU versus calcipotriol-plus–5-FU treatment. Each dot represents cell counts from an hpf image. 3 hpf images are included per sample (n = 5 participants in Vaseline-plus–5-FU group, n = 11 participants in calcipotriol-plus–5-FU group, Mann-Whitney U test). (J) Representative images of ANXA1-stained AKs and normal skin. (K) Quantification of ANXA1+ cells in the epidermis of AKs and normal skin. (n = 16 participants for AK and normal skin samples, paired t test). (L) Representative images of HLA-II-stained AKs and normal skin. (M) Quantification of HLA-II+ cells in AKs and normal skin. (n = 16 participants for AK and normal skin samples, paired t test). (N) Schematic diagram depicting the mechanism by which calcipotriol-plus–5-FU immunotherapy induces Th2 immunity against AKs. Bar graphs show mean + SD. Dashed lines mark the epidermal basement membrane in immunofluorescence images. Scale bars: 100 μm.
CD4+ T cells are required for the tumor protective effect of calcipotriol-plus–5-FU immunotherapy. To determine the role of CD4+ T cells in mediating the efficacy of calcipotriol-plus–5-FU immunotherapy for skin cancer suppression in vivo, we studied the spontaneous chemical skin carcinogenesis model in mice (21). WT mice received 7,12-dimethylbenz(a)anthracene (DMBA) once on the back skin, followed by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) application twice a week for 20 weeks to induce skin tumor development (Figure 3A). Topical EtOH-plus–0.5% 5-FU cream, 20 nmol calcipotriol-plus–control cream, or 20 nmol calcipotriol-plus–0.5% 5-FU treatment was applied to animals’ back skin 3 times a week from week 6 to 9 after DMBA (Figure 3A). Notably, calcipotriol-plus–5-FU treatment led to significantly delayed tumor onset and reduced tumor counts over time compared with EtOH-plus–5-FU and calcipotriol-plus–control cream treatment (Figure 3, B–D). CD4+ T cell depletion during topical calcipotriol-plus–5-FU treatment significantly diminished the efficacy of calcipotriol-plus–5-FU immunotherapy in WT mice (Figure 3, B–D, and Supplemental Figure 5A). These findings demonstrate that topical calcipotriol-plus–5-FU immunotherapy efficacy for skin cancer prevention depends on CD4+ T cell activation.
Figure 3Calcipotriol-plus–5-FU immunotherapy prevents skin cancer development in a Th2 cell–dependent manner. (A) Schematic diagram of a 3-week topical therapy and CD4+ T cell depletion during the skin cancer development in WT mice on the FVB background undergoing DMBA/TPA skin carcinogenesis protocol. (B) Representative photographs of mouse back skin treated with EtOH-plus–5-FU, calcipotriol-plus–control cream, calcipotriol-plus–5-FU, and calcipotriol-plus–5-FU combined with anti-CD4 (α-CD4) antibody at week 20 after DMBA. Black arrows point to skin tumors. (C and D) Time to skin tumor onset (C, log-rank test) and the number of tumors per mouse over time (D, 2-way ANOVA with Dunnett’s multiple comparison test) in WT mice treated (Tx) with EtOH-plus–5-FU, calcipotriol-plus–control cream, calcipotriol-plus–5-FU, and calcipotriol-plus–5-FU combined with α-CD4 antibody. All groups are compared with EtOH-plus–5-FU group unless otherwise indicated. (E) Quantification of Tslp mRNA expression in murine skin treated with control vehicle, 5-FU, calcipotriol, or calcipotriol-plus–5-FU for 3 consecutive days. A day after last treatment, mRNA was isolated from the treated skin for analysis. Each dot represents a mouse (n = 5 in control vehicle and 5-FU groups, n = 6 in calcipotriol and calcipotriol-plus–5-FU groups, Kruskal-Wallis test with Dunn’s multiple comparison test). (F) Schematic diagram of adoptive T cell transfer and DMBA/TPA skin carcinogenesis in mice on the BALB/c background with different genotypes. (G) Representative photographs of mouse back skin at week 19 after DMBA. Black arrows point to skin tumors. (H and I) Time to tumor onset (H, log-rank test) and number of tumors per mouse over time (I, 2-way ANOVA with Dunnett’s multiple comparison test) in DMBA/TPA-treated WT, Rag1KO, Rag1KO + CD4+ T cell, Tslptg, Tslptg Rag1KO, and Tslptg Rag1KO + CD4+ T cell groups. All groups are compared with the WT group. (J and K) Time to tumor onset (J, log-rank test) and number of tumors per mouse over time (K, 2-way ANOVA with Dunnett’s multiple comparison test) in DMBA/TPA-treated WT, Il4rKO, Tslptg, and Tslptg Il4rKO mice. All groups are compared with the WT group unless otherwise indicated. Please note that WT and Tslptg groups are common in H–K. Bar graphs show mean + SD. Scale bars: 1 cm. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.
CD4+ T cells are responsible for TSLP-mediated tumor protection in the skin. Topical calcipotriol treatment alone or in combination with 5-FU induced TSLP expression in the skin (Figure 3E) (12, 22, 23). We have previously shown that the antitumor function of calcipotriol is TSLP dependent (9, 24–27). To investigate the mechanism by which TSLP induction in the skin leads to tumor protection, we subjected K14-Tslptg/+ (Tslptg) mice that overexpress TSLP in skin keratinocytes to a skin carcinogenesis protocol (Figure 3F). DMBA/TPA-treated Tslptg mice did not develop skin tumors (Figure 3, G–I) (
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