Photodermatology, Photoimmunology & Photomedicine
LETTER TO THE EDITOR
Methyl aminolevulinate photodynamic therapy for actinic keratosis does not affect peripheral regulatory T-cell level or function Eleonora Reginato, Alexandra Gruber-Wackernagel & Peter Wolf
Research Unit for Photodermatology, Department of Dermatology, Medical University of Graz, Graz, Austria.
Correspondence: Peter Wolf, M.D., Department of Dermatology, Medical University of Graz, Auenbruggerplatz 8, A-8036 Graz, Austria. Tel: +43 316 385 12538; +43 316 385 80315 Fax: +43 316 385 13424 e-mail: [email protected]
Accepted for publication: 26 March 2015
Conflict of interests: The authors state no conflict of interest.
Funding sources: This work was supported by the Oesterreichische Nationalbank Anniversary Fund project no. 13279. Eleonora Reginato was supported through the PhD program of Medical University of Graz. The authors thank H.N. Ananthaswamy (Houston, TX, USA) for critical reading and Jude Richard (Austin, TX, USA) for editing of the manuscript.
To the Editor, Topical photodynamic therapy (PDT) mediated with 5aminolevulinic acid (ALA) or its methyl ester (methyl aminolevulinate; MAL) is widely used to treat actinic keratosis (1). Compared with surgery, topical PDT is noninvasive, is highly effective, and gives excellent cosmetic outcomes. However, PDT can induce direct phototoxic effects on illuminated neoplastic cells and affect both innate and adaptive arms of the host’s immune system by triggering the release of various proinflammatory and acute phase response mediators from the treated site [cited in (2)]. Such PDT-induced acute inflammatory responses can cause host innate immune cells to infiltrate, and in turn, remove damaged cells from treated skin. PDT-induced acute inflammation may also induce 274
adaptive antitumor immunity. In a recent experiment in mice, ALA PDT induced specific antitumor responses in cutaneous squamous cell carcinomas (3). However, there is concern that, under certain conditions, ALA PDT might also exert immunosuppressive effects similar to those induced by UV radiation (4), thus limiting its potential as cancer therapy (5). For example, PDT has been shown to suppress the induction of contact or delayed-type hypersensitivity in mice and humans (2). In particular, clinical studies by Damian’s group (6, 7) suggest that topical ALA or MAL PDT may have immunosuppressive properties that can be influenced by reducing the application rate of light or by administering nicotinamide before light delivery. Our recent work in a mouse tumor model has shown that administration of cyclophosphamide before PDT leads to depletion of regulatory T (Treg) cells and potentiates PDT-mediated immunity, leading in turn to long-term survival and memory immunity (8). Preclinical and clinical studies have demonstrated that PDT can affect the innate and adaptive arms of the immune system. In this study, we asked whether PDT can affect the systemic level and function of Treg cells (9) in patients with multiple AK photosensitized with MAL and exposed to visible red light.
PATIENTS AND METHODS Five patients (four men and one woman; age range, 58–74 years) with AK were enrolled and treated in the study. In brief, standard MAL (methyl 5-amino-4oxopentanoat; MetvixTM) 160 mg/g cream was applied topically to AK lesions on the scalp, face, and/or hands and 1 cm of surrounding skin; then, 3 h later, the lesions were illuminated with light from an LED source (Aktilite CL 128; Photocure, Oslo, Norway; narrow emission spectrum ~630 nm) at a dose of 37 J cm 2. The study protocol was approved by the local ethics committee and performed in accordance with the Helsinki Declaration. ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/phpp.12178
Letter to the Editor
CD25
104
Patient 1
3.88
103
102
101
101 87.2 100
104
4.8 101
102
3.11
101
101 3.96
4.22
84.3
101
104
4.68 102
103
104
4.81
102
101
101 86.8 100
104
5.48 101
102
103
104
2.61
103
102
101
101 3.45 101
102
103
100
104
103
104
3.35
3.79 101
102
103
104
5.33
3.62
103
102
90.3 100 100
102
3.21
89.7 100 100 104
3.66
104
3.36 101
103
102
100
103
2.22
91.1 100 100 104
2.89
103
102
3.34
101
101
5.9 101
103
102
88
104
4
102
100
103
4.77
100 10
3.05
100
102
4.98
100
104
4
103
Patient 5
103
3.08 101
103
102
102
3.87
90.1 100 100
102
10
Patient 4
104
104
88.3 100 100 101
Patient 3
103
4.68
103
2.93
103
102
100
Patient 2
7/14 days after PDT * 104
4.16
Gated on CD4+
Before PDT
(a)
87.8 100
3.21 101
102
103
104
(c)
8
350
7
300
6
P = 0.8302
5 4 3 2
% Proliferation
All patients
%CD25+Foxp3+/CD4+
Foxp3 (b)
250
Before PDT 7/14 days after PDT P = 0.8125
200 150 100 50
1 0
0 Before PDT
7/14 d after PDT
0:1
1:1 Treg:Teff
Fig. 1. Quantification and functional assay of regulatory T cells (Treg) in peripheral blood. (a, b) CD4+CD25+FoxP4+ Treg levels in the blood of AK patients before or after PDT. Comparison of Treg levels was carried out by two-tailed Student paired t-test (b). (c) Treg and T effector (Teff) cells were isolated from peripheral blood collected from the AK patients and cocultured at ratios of 0:1 (control) and 1:1. Gray and white bars indicate values of proliferation observed before and after PDT, respectively. Teff proliferation without Treg cells (0:1 ratio) was set to 100%. Proliferation values were compared by paired Wilcoxon signed rank test. All results are from five AK patients, comparing pre-PDT to post-PDT values either at 7 days (patients 2–5*) or 14 days (patient 1*) after treatment. Photodermatol Photoimmunol Photomed 2015; 31: 274–278 ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
275
Letter to the Editor
Before PDT Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Counts
Patient 1
7/14 days after PDT
Q2: Treg
Patient 2
Patient 3
Patient 4
Patient 5
CD127 PE Fig. 2. CD127 staining of gated Treg and CD4+CD25-FoxP3+ cells. Histogram plots indicate that CD4+CD25+FoxP4+ Treg cells gated in the upper right quadrants (Q2) of Fig. 1a are CD127 , as compared to CD4+CD25-FoxP3+ cells (Q3).
276
Photodermatol Photoimmunol Photomed 2015; 31: 274–278 ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Letter to the Editor
Blood samples from the patients were collected in 8ml Falcon tubes supplemented with EDTA as anticoagulant at two time points: immediately before topical application of MAL (pre-PDT) and 7 or 14 days after PDT (post-PDT). For flow cytometry analysis of Treg cells, 400-ll aliquots of fresh blood samples were stained with CD25 FITC (BD, Cat. No. 345796, clone 2A3), CD127 PE (BD, Cat. No. 557938, clone HIL-7R-M21), CD4 PerCP (BD, Cat. No. 345770, clone SK3), and FoxP3 APC antibody (eBioscience, Cat. No. 17-4776, clone PCH101) using the FoxP3 Staining Buffer Set (eBioscience, Cat. No. 00-5523) according to the manufacturer’s instructions. All data were acquired with a FACS Calibur flow cytometer up to a count of 40000 CD4+ cells for each flow and analyzed with Flow Jo software (TreeStar, Ashland, OR, USA). All plots were gated on CD4+. In all CD25 vs. Foxp3 plots, quadrants were defined in such a way to discriminate between the major population of CD4+CD25-Foxp3 (Teff) cells and minor population of CD4+CD25+Foxp3+ (Treg) cells. Further analysis of CD127 staining was performed to confirm that gated Treg cells were CD127 . For Treg immune function assays, lymphocytes were isolated from the blood using Ficoll Lymphoprep (Axisschield, Cat. No. 1114545); stained with CD4 FITC, CD25 PE-Cy7, and CD127 antibodies (BD Pharmigen); and sorted by FACS (BD FACS ARIA) into Treg (CD4+CD25+CD127 ) and Teff (CD4+CD25-CD127+) subsets. Each cell subset was cultured either alone or together at ratio of 1:1 and subjected to immune function assay as described (10).
RESULTS AND DISCUSSION We evaluated the change in peripheral Treg levels in AK patients before and after a single session of MAL PDT. Treg (CD25+FoxP3+CD4+) levels before and after PDT differed interindividually (Fig. 1a), but they did not differ statistically significantly overall (3.88% pre-PDT vs. 3.62% post-PDT, that is, 6.7% overall reduction) (Fig. 1b). We also analyzed the effect of MAL PDT on the immunosuppressive function of Treg cells cocultured with Teff cells at different ratios. Although MAL PDT did cause ambivalent effects in individual patients (i.e., up- or downregulation of Treg function), the differences were not statistically significant (Fig. 1c). As Treg cells are known to be
Photodermatol Photoimmunol Photomed 2015; 31: 274–278 ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
CD127 , the cells gated in Fig. 1a in the upper and lower right quadrants (Q2: Treg cells and Q3: CD4+CD25-Foxp3+, respectively) have been analyzed also using the CD127 marker (Fig. 2). This control was carried out to ensure that Treg cells gated in Q2 were negative for CD127. The present results indicate that MAL PDT does not significantly affect the level and function of Treg cells in patients with AK. At first glance, this seems to contradict our previous findings in patients with esophageal squamous cell carcinoma (ESCC) in which treatment with laser-guided photofrin-PDT downregulated the immunosuppressive function of peripheral Treg cells (10). However, this apparent contradiction is likely explained by notable differences between the two study populations in terms of photosensitizer application route (systemic vs. topical), timing of illumination after photosensitization (48 h vs. 3 h), PDT dosing (80 J cm 2 vs. 37 J cm 2) and fluence (laser, 1.2–1.5 W cm 2 vs. LED light, 68 mW cm 2), anatomical lesion location (esophageal vs. skin), and tumor type (ESCC vs. AK) and stage of disease. Nonetheless, our present finding that PDT did not significantly affect systemic Treg levels in AK patients (Fig. 1) is quite consistent with our previous finding in ESCC patients. As IL-6 is known to inhibit Treg function, we analyzed serum IL-6 levels before and after PDT and saw no significant differences in the AK patients (data not shown). Conversely, significant upregulation of serum IL-6 levels and downregulation of peripheral Treg function were previously seen in the ESCC patients (10). Together, these findings suggest a crucial role for IL-6 in mediating the effects of PDT on Treg function.
ACKNOWLEDGEMENTS The authors thank Gerlinde Mayer and Isabella Bambach for technical support.
AUTHOR CONTRIBUTION PW and AG-W designed the experiments. ER performed most of the experiments. ER and PW analyzed and interpreted the data and wrote the manuscript. All authors revised and approved the final version of the manuscript.
277
Letter to the Editor
REFERENCES 1. Dirschka T, Radny P, Dominicus R et al. Photodynamic therapy with BF200 ALA for the treatment of actinic keratosis: results of a multicentre, randomized, observer-blind phase III study in comparison with a registered methyl-5-aminolaevulinate cream and placebo. Br J Dermatol 2012; 166: 137– 146. 2. Reginato E, Wolf P, Hamblin MR. Immune response after photodynamic therapy increases anti-cancer and antibacterial effects. World J Immunol 2014; 4: 1–11. 3. Wang H, Li J, Lv T, Tu Q, Huang Z, Wang X. Therapeutic and immune effects of 5-aminolevulinic acid photodynamic therapy on UVB-induced squamous cell carcinomas in hairless mice. Exp Dermatol 2013; 22: 362–363. 4. Schwarz A, Navid F, Sparwasser T, Clausen BE, Schwarz T. In vivo
278
reprogramming of UV radiation-induced regulatory T-cell migration to inhibit the elicitation of contact hypersensitivity. J Allergy Clin Immunol 2011; 128: 826– 833. 5. Wolf P, Fink-Puches R, Reimann-Weber A, Kerl H. Development of malignant melanoma after repeated topical photodynamic therapy with 5aminolevulinic acid at the exposed site. Dermatology 1997; 194: 53–54. 6. Frost GA, Halliday GM, Damian DL. Photodynamic therapy-induced immunosuppression in humans is prevented by reducing the rate of light delivery. J Invest Dermatol 2011; 131: 962–968. 7. Matthews YJ, Damian DL. Topical photodynamic therapy is immunosuppressive in humans. Br J Dermatol 2010; 162: 637–641.
8. Reginato E, Mroz P, Chung H, Kawakubo M, Wolf P, Hamblin MR. Photodynamic therapy plus regulatory T-cell depletion produces immunity against a mouse tumour that expresses a self-antigen. Br J Cancer 2013; 109: 2167–2174. 9. Singh TP, Schon MP, Wallbrecht K, Wolf P. 8-Methoxypsoralen plus UVA treatment increases the proportion of CLA+ CD25 + CD4 + T cells in lymph nodes of K5.hTGFbeta1 transgenic mice. Exp Dermatol 2012; 21: 228–230. 10. Reginato E, Lindenmann J, Langner C et al. Photodynamic therapy downregulates the function of regulatory T cells in patients with esophageal squamous cell carcinoma. Photochem Photobiol Sci 2014; 13: 1281–1289.
Photodermatol Photoimmunol Photomed 2015; 31: 274–278 ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
LETTER TO THE EDITOR
Methyl aminolevulinate photodynamic therapy for actinic keratosis does not affect peripheral regulatory T-cell level or function Eleonora Reginato, Alexandra Gruber-Wackernagel & Peter Wolf
Research Unit for Photodermatology, Department of Dermatology, Medical University of Graz, Graz, Austria.
Correspondence: Peter Wolf, M.D., Department of Dermatology, Medical University of Graz, Auenbruggerplatz 8, A-8036 Graz, Austria. Tel: +43 316 385 12538; +43 316 385 80315 Fax: +43 316 385 13424 e-mail: [email protected]
Accepted for publication: 26 March 2015
Conflict of interests: The authors state no conflict of interest.
Funding sources: This work was supported by the Oesterreichische Nationalbank Anniversary Fund project no. 13279. Eleonora Reginato was supported through the PhD program of Medical University of Graz. The authors thank H.N. Ananthaswamy (Houston, TX, USA) for critical reading and Jude Richard (Austin, TX, USA) for editing of the manuscript.
To the Editor, Topical photodynamic therapy (PDT) mediated with 5aminolevulinic acid (ALA) or its methyl ester (methyl aminolevulinate; MAL) is widely used to treat actinic keratosis (1). Compared with surgery, topical PDT is noninvasive, is highly effective, and gives excellent cosmetic outcomes. However, PDT can induce direct phototoxic effects on illuminated neoplastic cells and affect both innate and adaptive arms of the host’s immune system by triggering the release of various proinflammatory and acute phase response mediators from the treated site [cited in (2)]. Such PDT-induced acute inflammatory responses can cause host innate immune cells to infiltrate, and in turn, remove damaged cells from treated skin. PDT-induced acute inflammation may also induce 274
adaptive antitumor immunity. In a recent experiment in mice, ALA PDT induced specific antitumor responses in cutaneous squamous cell carcinomas (3). However, there is concern that, under certain conditions, ALA PDT might also exert immunosuppressive effects similar to those induced by UV radiation (4), thus limiting its potential as cancer therapy (5). For example, PDT has been shown to suppress the induction of contact or delayed-type hypersensitivity in mice and humans (2). In particular, clinical studies by Damian’s group (6, 7) suggest that topical ALA or MAL PDT may have immunosuppressive properties that can be influenced by reducing the application rate of light or by administering nicotinamide before light delivery. Our recent work in a mouse tumor model has shown that administration of cyclophosphamide before PDT leads to depletion of regulatory T (Treg) cells and potentiates PDT-mediated immunity, leading in turn to long-term survival and memory immunity (8). Preclinical and clinical studies have demonstrated that PDT can affect the innate and adaptive arms of the immune system. In this study, we asked whether PDT can affect the systemic level and function of Treg cells (9) in patients with multiple AK photosensitized with MAL and exposed to visible red light.
PATIENTS AND METHODS Five patients (four men and one woman; age range, 58–74 years) with AK were enrolled and treated in the study. In brief, standard MAL (methyl 5-amino-4oxopentanoat; MetvixTM) 160 mg/g cream was applied topically to AK lesions on the scalp, face, and/or hands and 1 cm of surrounding skin; then, 3 h later, the lesions were illuminated with light from an LED source (Aktilite CL 128; Photocure, Oslo, Norway; narrow emission spectrum ~630 nm) at a dose of 37 J cm 2. The study protocol was approved by the local ethics committee and performed in accordance with the Helsinki Declaration. ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/phpp.12178
Letter to the Editor
CD25
104
Patient 1
3.88
103
102
101
101 87.2 100
104
4.8 101
102
3.11
101
101 3.96
4.22
84.3
101
104
4.68 102
103
104
4.81
102
101
101 86.8 100
104
5.48 101
102
103
104
2.61
103
102
101
101 3.45 101
102
103
100
104
103
104
3.35
3.79 101
102
103
104
5.33
3.62
103
102
90.3 100 100
102
3.21
89.7 100 100 104
3.66
104
3.36 101
103
102
100
103
2.22
91.1 100 100 104
2.89
103
102
3.34
101
101
5.9 101
103
102
88
104
4
102
100
103
4.77
100 10
3.05
100
102
4.98
100
104
4
103
Patient 5
103
3.08 101
103
102
102
3.87
90.1 100 100
102
10
Patient 4
104
104
88.3 100 100 101
Patient 3
103
4.68
103
2.93
103
102
100
Patient 2
7/14 days after PDT * 104
4.16
Gated on CD4+
Before PDT
(a)
87.8 100
3.21 101
102
103
104
(c)
8
350
7
300
6
P = 0.8302
5 4 3 2
% Proliferation
All patients
%CD25+Foxp3+/CD4+
Foxp3 (b)
250
Before PDT 7/14 days after PDT P = 0.8125
200 150 100 50
1 0
0 Before PDT
7/14 d after PDT
0:1
1:1 Treg:Teff
Fig. 1. Quantification and functional assay of regulatory T cells (Treg) in peripheral blood. (a, b) CD4+CD25+FoxP4+ Treg levels in the blood of AK patients before or after PDT. Comparison of Treg levels was carried out by two-tailed Student paired t-test (b). (c) Treg and T effector (Teff) cells were isolated from peripheral blood collected from the AK patients and cocultured at ratios of 0:1 (control) and 1:1. Gray and white bars indicate values of proliferation observed before and after PDT, respectively. Teff proliferation without Treg cells (0:1 ratio) was set to 100%. Proliferation values were compared by paired Wilcoxon signed rank test. All results are from five AK patients, comparing pre-PDT to post-PDT values either at 7 days (patients 2–5*) or 14 days (patient 1*) after treatment. Photodermatol Photoimmunol Photomed 2015; 31: 274–278 ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
275
Letter to the Editor
Before PDT Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Q2: Treg
Q3: Foxp3 APC+, CD25 FITC–
Counts
Patient 1
7/14 days after PDT
Q2: Treg
Patient 2
Patient 3
Patient 4
Patient 5
CD127 PE Fig. 2. CD127 staining of gated Treg and CD4+CD25-FoxP3+ cells. Histogram plots indicate that CD4+CD25+FoxP4+ Treg cells gated in the upper right quadrants (Q2) of Fig. 1a are CD127 , as compared to CD4+CD25-FoxP3+ cells (Q3).
276
Photodermatol Photoimmunol Photomed 2015; 31: 274–278 ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Letter to the Editor
Blood samples from the patients were collected in 8ml Falcon tubes supplemented with EDTA as anticoagulant at two time points: immediately before topical application of MAL (pre-PDT) and 7 or 14 days after PDT (post-PDT). For flow cytometry analysis of Treg cells, 400-ll aliquots of fresh blood samples were stained with CD25 FITC (BD, Cat. No. 345796, clone 2A3), CD127 PE (BD, Cat. No. 557938, clone HIL-7R-M21), CD4 PerCP (BD, Cat. No. 345770, clone SK3), and FoxP3 APC antibody (eBioscience, Cat. No. 17-4776, clone PCH101) using the FoxP3 Staining Buffer Set (eBioscience, Cat. No. 00-5523) according to the manufacturer’s instructions. All data were acquired with a FACS Calibur flow cytometer up to a count of 40000 CD4+ cells for each flow and analyzed with Flow Jo software (TreeStar, Ashland, OR, USA). All plots were gated on CD4+. In all CD25 vs. Foxp3 plots, quadrants were defined in such a way to discriminate between the major population of CD4+CD25-Foxp3 (Teff) cells and minor population of CD4+CD25+Foxp3+ (Treg) cells. Further analysis of CD127 staining was performed to confirm that gated Treg cells were CD127 . For Treg immune function assays, lymphocytes were isolated from the blood using Ficoll Lymphoprep (Axisschield, Cat. No. 1114545); stained with CD4 FITC, CD25 PE-Cy7, and CD127 antibodies (BD Pharmigen); and sorted by FACS (BD FACS ARIA) into Treg (CD4+CD25+CD127 ) and Teff (CD4+CD25-CD127+) subsets. Each cell subset was cultured either alone or together at ratio of 1:1 and subjected to immune function assay as described (10).
RESULTS AND DISCUSSION We evaluated the change in peripheral Treg levels in AK patients before and after a single session of MAL PDT. Treg (CD25+FoxP3+CD4+) levels before and after PDT differed interindividually (Fig. 1a), but they did not differ statistically significantly overall (3.88% pre-PDT vs. 3.62% post-PDT, that is, 6.7% overall reduction) (Fig. 1b). We also analyzed the effect of MAL PDT on the immunosuppressive function of Treg cells cocultured with Teff cells at different ratios. Although MAL PDT did cause ambivalent effects in individual patients (i.e., up- or downregulation of Treg function), the differences were not statistically significant (Fig. 1c). As Treg cells are known to be
Photodermatol Photoimmunol Photomed 2015; 31: 274–278 ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
CD127 , the cells gated in Fig. 1a in the upper and lower right quadrants (Q2: Treg cells and Q3: CD4+CD25-Foxp3+, respectively) have been analyzed also using the CD127 marker (Fig. 2). This control was carried out to ensure that Treg cells gated in Q2 were negative for CD127. The present results indicate that MAL PDT does not significantly affect the level and function of Treg cells in patients with AK. At first glance, this seems to contradict our previous findings in patients with esophageal squamous cell carcinoma (ESCC) in which treatment with laser-guided photofrin-PDT downregulated the immunosuppressive function of peripheral Treg cells (10). However, this apparent contradiction is likely explained by notable differences between the two study populations in terms of photosensitizer application route (systemic vs. topical), timing of illumination after photosensitization (48 h vs. 3 h), PDT dosing (80 J cm 2 vs. 37 J cm 2) and fluence (laser, 1.2–1.5 W cm 2 vs. LED light, 68 mW cm 2), anatomical lesion location (esophageal vs. skin), and tumor type (ESCC vs. AK) and stage of disease. Nonetheless, our present finding that PDT did not significantly affect systemic Treg levels in AK patients (Fig. 1) is quite consistent with our previous finding in ESCC patients. As IL-6 is known to inhibit Treg function, we analyzed serum IL-6 levels before and after PDT and saw no significant differences in the AK patients (data not shown). Conversely, significant upregulation of serum IL-6 levels and downregulation of peripheral Treg function were previously seen in the ESCC patients (10). Together, these findings suggest a crucial role for IL-6 in mediating the effects of PDT on Treg function.
ACKNOWLEDGEMENTS The authors thank Gerlinde Mayer and Isabella Bambach for technical support.
AUTHOR CONTRIBUTION PW and AG-W designed the experiments. ER performed most of the experiments. ER and PW analyzed and interpreted the data and wrote the manuscript. All authors revised and approved the final version of the manuscript.
277
Letter to the Editor
REFERENCES 1. Dirschka T, Radny P, Dominicus R et al. Photodynamic therapy with BF200 ALA for the treatment of actinic keratosis: results of a multicentre, randomized, observer-blind phase III study in comparison with a registered methyl-5-aminolaevulinate cream and placebo. Br J Dermatol 2012; 166: 137– 146. 2. Reginato E, Wolf P, Hamblin MR. Immune response after photodynamic therapy increases anti-cancer and antibacterial effects. World J Immunol 2014; 4: 1–11. 3. Wang H, Li J, Lv T, Tu Q, Huang Z, Wang X. Therapeutic and immune effects of 5-aminolevulinic acid photodynamic therapy on UVB-induced squamous cell carcinomas in hairless mice. Exp Dermatol 2013; 22: 362–363. 4. Schwarz A, Navid F, Sparwasser T, Clausen BE, Schwarz T. In vivo
278
reprogramming of UV radiation-induced regulatory T-cell migration to inhibit the elicitation of contact hypersensitivity. J Allergy Clin Immunol 2011; 128: 826– 833. 5. Wolf P, Fink-Puches R, Reimann-Weber A, Kerl H. Development of malignant melanoma after repeated topical photodynamic therapy with 5aminolevulinic acid at the exposed site. Dermatology 1997; 194: 53–54. 6. Frost GA, Halliday GM, Damian DL. Photodynamic therapy-induced immunosuppression in humans is prevented by reducing the rate of light delivery. J Invest Dermatol 2011; 131: 962–968. 7. Matthews YJ, Damian DL. Topical photodynamic therapy is immunosuppressive in humans. Br J Dermatol 2010; 162: 637–641.
8. Reginato E, Mroz P, Chung H, Kawakubo M, Wolf P, Hamblin MR. Photodynamic therapy plus regulatory T-cell depletion produces immunity against a mouse tumour that expresses a self-antigen. Br J Cancer 2013; 109: 2167–2174. 9. Singh TP, Schon MP, Wallbrecht K, Wolf P. 8-Methoxypsoralen plus UVA treatment increases the proportion of CLA+ CD25 + CD4 + T cells in lymph nodes of K5.hTGFbeta1 transgenic mice. Exp Dermatol 2012; 21: 228–230. 10. Reginato E, Lindenmann J, Langner C et al. Photodynamic therapy downregulates the function of regulatory T cells in patients with esophageal squamous cell carcinoma. Photochem Photobiol Sci 2014; 13: 1281–1289.
Photodermatol Photoimmunol Photomed 2015; 31: 274–278 ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
