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Imiquimod Enhances IFN-c Production and Effector Function of T Cells Infiltrating Human Squamous Cell Carcinomas of the Skin Susan J. Huang1, Dirkjan Hijnen2, George F. Murphy3, Thomas S. Kupper1, Adam W. Calarese1, Ilse G. Mollet4, Carl F. Schanbacher1, Danielle M. Miller1, Chrysalyne D. Schmults1 and Rachael A. Clark1

Squamous cell carcinomas (SCCs) are sun-induced skin cancers that are particularly numerous and aggressive in patients taking T-cell immunosuppressant medications. Imiquimod is a topical immune response modifier and Toll-like receptor 7 (TLR7) agonist that induces the immunological destruction of SCC and other skin cancers. TLR7 activation by imiquimod has pleiotropic effects on innate immune cells, but its effects on T cells remain largely uncharacterized. Because tumor destruction and formation of immunological memory are ultimately T-cell-mediated effects, we studied the effects of imiquimod therapy on effector T cells infiltrating human SCC. SCC treated with imiquimod before excision contained dense T-cell infiltrates associated with tumor cell apoptosis and histological evidence of tumor regression. Effector T cells from treated SCC produced more IFN-g, granzyme, and perforin and less IL-10 and transforming growth factor-b (TGF-b) than T cells from untreated tumors. Treatment of normal human skin with imiquimod induced activation of resident T cells and reduced IL-10 production but had no effect on IFN-g, perforin, or granzyme, suggesting that these latter effects arise from the recruitment of distinct populations of T cells into tumors. Thus, imiquimod stimulates tumor destruction by recruiting cutaneous effector T cells from blood and by inhibiting tonic anti-inflammatory signals within the tumor. Journal of Investigative Dermatology (2009) 129, 2676–2685; doi:10.1038/jid.2009.151; published online 11 June 2009

INTRODUCTION Recipients of solid organ transplants on immunosuppres- More than 100,000 squamous cell carcinomas (SCC) of the sive medications have a 65- to 250-fold increased risk of SCC skin are diagnosed in the United States each year (Diepgen (Berg and Otley, 2002). SCCs in these patients are aggressive; and Mahler, 2002). The treatment of non-melanoma skin nearly 10% of these tumors metastasize and the majority of cancer, of which SCC is the second most frequent type, patients die as a result (Berg and Otley, 2002; Euvrard et al., accounts for 4.5% of all Medicare cancer costs (Housman 2003). The development of SCC in transplant recipients is et al., 2003). Actinic keratoses (AKs), the premalignant linked to the use of medications that suppress T-cell function precursors to SCC, are the third most common reason for (Euvrard et al., 2003). consulting a dermatologist (Feldman et al., 1998). More than We have found that blood vessels in human SCC lack 5.2 million physician visits are made annually for the E-selectin, allowing the tumor to exclude skin-homing treatment of AKs, at a cost of over $900 million (Warino effector T cells, while at the same time recruiting regulatory et al., 2006). T cells (Treg cells), a cell type that can suppress immune responses (Sakaguchi et al., 1995; Clark et al., 2008). We 1Harvard Skin Disease Research Center and the Department of Dermatology, found that the TLR7 agonist, imiquimod, acted indirectly to Brigham and Women’s Hospital, Boston, Massachusetts, USA; 2Department address both of these mechanisms of immune evasion, of Dermatology, Utrecht University Medical Center, Utrecht, The inducing E-selectin on blood vessels and allowing the influx 3 Netherlands; Department of Pathology, Brigham and Women’s Hospital, of skin homing T cells, as well as reducing the number and Boston, Massachusetts, USA and 4Department of Dermatology, Ghent University Hospital, Gent, Belgium activity of tumor Treg cells (Clark et al., 2008). Correspondence: Dr Rachael A. Clark, Department of Dermatology, Brigham Effector T cells are critical for both the destruction of SCC and Women’s Hospital, EBRC Room 505A, 221 Longwood Avenue, Boston, and the establishment of long-term, protective immunologi- Massachusetts 02115, USA. cal memory. Despite their importance, comprehensive E-mail: [email protected] studies of the phenotype and function of these cells in SCC Abbreviations: AK, actinic keratosis; CLA, cutaneous lymphocyte antigen; are lacking. Similarly, imiquimod has been shown to induce SCC, squamous cell carcinoma; TGF-b, transforming growth factor-b; TLR7, Toll-like receptor 7; Treg cells, regulatory T cells IFN-g production in vitro from mouse and human T cells, but Received 23 July 2008; revised 9 March 2009; accepted 3 April 2009; the effect of this medication on tumor-resident T cells has not published online 11 June 2009 been studied thoroughly (Wagner et al., 1999). We present

2676 Journal of Investigative Dermatology (2009), Volume 129 & 2009 The Society for Investigative Dermatology SJ Huang et al. Effects of Imiquimod on T Cells in Human SCC

here our findings that imiquimod therapy significantly specimens, fibrosis was uniform across the base, suggesting enhances the number and effector function of T cells that fibrosis was not the result of scarring from a prior biopsy infiltrating human SCC of the skin. procedure. Dense infiltrates of CD8 þ T cells surrounded nests of residual tumor, and their presence was associated RESULTS with tumor cell apoptosis as evidenced by TUNEL staining þ Imiquimod-treated SCCs are infiltrated by CD8 þ T cells that (Figure 1d). Untreated SCCs were infiltrated by fewer CD8 are associated with tumor cell apoptosis and histological T cells and they lacked both fibrotic regression and tumor cell evidence of tumor regression apoptosis (data not shown). We have reported earlier that untreated SCCs are infiltrated by equal numbers of CD4 þ and CD8 þ T cells, whereas Production of IFN-c, perforin, and granzyme is markedly tumors treated with imiquimod before excision contain enhanced in T cells from imiquimod-treated SCC 490% CD8 þ cytotoxic T cells (Clark et al., 2008). To study We isolated the T cells from untreated and imiquimod-treated the distribution and characteristics of these cells, we co- SCC and examined the production of cytokines and proteins stained cryosections of imiquimod-treated SCC for CD8 þ associated with cytotoxic activity. Approximately 50% of and for markers of tumor cell proliferation and apoptosis T cells infiltrating untreated SCC are FOXP3 þ Treg cells (Figure 1). We found that imiquimod-treated SCC had three (Clark et al., 2008). For these studies, FOXP3 þ co-staining clear histological zones. First, an area of residual tumor cells, was used to exclude Treg cells from these analyses. Non- many of which were proliferating, was evident superficially regulatory CD4 þ and CD8 þ T cells from imiquimod-treated (Figure 1a and b). A dense, band-like infiltrate of CD8 þ SCC produced markedly increased levels of IFN-g (Figure 2a T cells was present beneath the tumor and a zone of fibrotic and b). IFN-g was produced by 69% of CD4 þ T cells regression was present beneath the T-cell infiltrates. The zone (SD ¼ 3.1, N ¼ 3) and 79% of CD8 þ T cells (SD ¼ 7.4, N ¼ 3) of fibrotic regression was always present at the base of the from treated tumors, compared with 8.2% of CD4 þ specimen and its width varied with the amount of residual (SD ¼ 1.4, N ¼ 3) and 21% of CD8 þ T cells (SD ¼ 13, tumor; tumors that had almost fully regressed had the thickest N ¼ 3) from untreated tumors. Few T cells from either treated zones of fibrosis and those in the earlier stages of destruction or untreated SCC produced IL-17 (Figure 2a and b). The had fibrosis limited to the deeper portions of the dermis. In all percentages of T cells producing granzyme and perforin, two

1

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Hoechst TUNEL Merge

Figure 1. Imiquimod treated SCCs are heavily infiltrated by CD8 þ T cells that are associated with tumor regression and apoptosis of tumor cells. (a) Cryosections of SCC treated with imiquimod were stained for CD8 þ (green) and Ki-67 (orange) to identify proliferating tumor cells. Treated SCCs had three clear histological zones: (1) residual tumor, (2) a dense CD8 þ T-cell infiltrate, and (3) a deeper fibrotic zone of tumor regression. (b) A higher power view is shown of the interface between the zones of T-cell infiltration and tumor regression. Residual tumor (arrow) is surrounded by a dense T-cell infiltrate (T) beneath which is a zone of fibrotic regression (R). (c) An additional imiquimod-treated tumor stained with Hoechst nuclear dye is shown. Residual tumor (arrow) can be identified by the large atypical keratinocyte nuclei. A zone of dense T-cell infiltrate (T) and an area of fibrotic regression (R) are also evident. (d) CD8 þ T cells infiltrates are associated with tumor cell apoptosis. Hoechst nuclear stain delineates a tumor nodule (arrow) surrounded by T cells (T). TUNEL staining shows widespread tumor cell apoptosis in areas of dense T-cell infiltrates. Three additional treated tumors showed similar results. Bar ¼ 100 mm.

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Untreated SCC Imiquimod-treated SCC Untreated SCC Imiquimod-treated SCC 100 100 P<0.01 90 P<0.0001 90 80 80 70 70 60 60 T cells T cells + + 50 50 40 40 30 30 % CD8 % CD4 20 20 10 10 0 0 IL-17 IFN-γ IL-17 IFN-γ

CD4+ T cells CD8+ T cells Untreated Imiquimod tx Untreated Imiquimod tx

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Untreated SCC Untreated SCC Imiquimod-treated SCC Imiquimod treated SCC 100 100 P<0.01 P<0.001 90 P<0.005 P<0.0005 90 80 80 70 70 60 60 T cells T cells + + 50 50 40 40 30 30 % CD8 % CD4 20 20 10 10 0 0 Granzyme Perforin Granzyme Perforin

Figure 2. Production of IFN-c, perforin, and granzyme is markedly enhanced in T cells from SCC treated with imiquimod. IFN-g is produced by the majority of both (a) CD4 þ and (b) CD8 þ T cells in SCC treated with imiquimod, compared with lower production in untreated tumors. IL-17 production was low in both treated and untreated tumors. (c) CD4 þ and CD8 þ T cells from SCC had increased co-expression of perforin and granzyme after imiquimod therapy. Study of additional treated tumors confirmed enhanced production of perforin and granzyme by both (e) CD4 þ and (f) CD8 þ T cells.

molecules associated with T-cell cytotoxicity, were also already present within the tumor. To determine whether increased in imiquimod-treated SCC (Figure 2c–f). Overall CD8 þ T cells proliferate locally within imiquimod-treated 46% of CD4 þ T cells from imiquimod-treated SCC produced SCC, we co-stained cryosections of treated SCC with CD8 þ granzyme (SD ¼ 11, N ¼ 3) and 43% produced perforin and the proliferation antigen, Ki-67 (Figure 3). Although (SD ¼ 5.1, N ¼ 3). Of CD8 þ T cells from treated SCC, 81% proliferating cells were readily detectable in stained sections, produced granzyme (SD ¼ 6.1, N ¼ 3) and 73% produced these perforin (SD ¼ 9.4, N ¼ 3). were almost invariably tumor cells. Less than 5% of the CD8 þ T cells present in imiquimod-treated SCC had recently CD8 þ T cells do not proliferate locally within imiquimod- proliferated, suggesting that the increased number of CD8 þ treated tumors cells results from enhanced recruitment from the blood We have observed a shift in the CD4 þ /CD8 þ T-cell ratio in (Figure 3c). untreated tumors from 1:1 in untreated tumors to 1:10 in imiquimod-treated tumors, and this shift is associated with a Non-regulatory T cells from imiquimod-treated SCC produce three-fold increase in the total number of T cells infiltrating less IL-10 and TGF-b tumors (Clark et al., 2008). Increased numbers of CD8 þ T We reported earlier that imiquimod treatment is associated cells could result from either an influx of T cells from the with decreased production of IL-10 and transforming growth blood or from local expansion of tumor-specific T cells factor-b (TGF-b) by FOXP3 þ Treg cells present in SCC (Clark

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a b differential effects of recruitment versus local T-cell modula- tion, we studied the effects of imiquimod on the population of T cells resident in normal human skin. In order to focus on non-regulatory T cells, FOXP3 þ Treg cells were excluded from all analyses. For these studies, imiquimod was added to cultures of normal skin for 1 week before T-cell isolation (Clark et al., 2006). T cells from the imiquimod-treated skin had increased expression of the early activation antigen CD69 but decreased levels of CD25 expression (Figure 6a). We found that a large population of T cells from normal human skin Proliferative Non-proliferative produced IL-10 (Figure 6b). Imiquimod treatment of normal c 15.0 100 skin T cells reduced the production of IL-10 but had no effect 90 12.5 80 on the production of IFN-g and IL-17 (Figure 6c and d). In 10.0 70 contrast to a prior report that in vitro treatment with 60 imiquimod induced perforin production in human CD8 þ T 7.5 50 CD8 T cells CD8 CD8 T cells CD8 + – 40 cells (Ambach et al., 2004), we observed no change in 5.0 30 perforin and granzyme production in CD3 þ T cells after 2.5 20 % Ki67 % Ki67 10 imiquimod treatment (data not shown). TGF-b was produced 0.0 0 at baseline by o2% of skin-resident effector T cells; we were SCC8 SCC13 SCC17 SCC8 SCC13 SCC17 therefore unable to study the reduction of TGF-b production Figure 3. CD8 þ T cells do not proliferate locally within imiquimod-treated after treatment with imiquimod. The addition of imiquimod tumors. (a) Co-staining of cryosections of imiquimod-treated SCC with to purified skin T cells removed first from the skin CD8 þ (green) and Ki-67 (orange) showed proliferation of tumor cells but few microenvironment had no effect on the expression of CD8 þ T cells. (b) Higher power view of a second tumor with similar findings. þ activation antigens or on cytokine production, confirming (c) Quantification of CD8 T-cell proliferation in three treated tumors. earlier work that showed that the effect of imiquimod on Bar ¼ 100 mm. T cells is indirect and requires the presence of other, imiquimod-responsive cell types (Wagner et al., 1999). et al., 2008). We studied the production of IL-10 and TGF-b by non-regulatory T cells and found that in untreated SCC, DISCUSSION significant amounts of IL-10 were produced by both FOXP3 þ The increased incidence of SCC in patients on T-cell- regulatory and FOXP3 non-regulatory T cells (Figure 4a). immunosuppressive medications suggests that T cells have Non-regulatory T cells from tumors treated with imiquimod a critical function in controlling these tumors. T cells are produced less IL-10 (Figure 4b–d, mean 43%, SD ¼ 2.1, N ¼ 3 present within SCC but do not control tumor growth. We from untreated tumors compared with mean 20%, SD ¼ 4.1, þ found earlier that SCCs exclude CLA skin homing T cells N ¼ 3 from treated tumors). TGF-b was also produced by a þ and instead recruit FOXP3 Treg cells (Clark et al., 2008). mean 12% of non-regulatory T cells in untreated SCC and Thus, tumors persist, in part, because of aberrant T-cell production was decreased to 2% in imiquimod-treated homing. tumors (Figure 4e, SD ¼ 1.5, N ¼ 3 from untreated tumors Imiquimod is a topical immune response modifier that is and SD ¼ 1.1, N ¼ 3 from treated tumors). These results effective in the treatment of basal cell carcinomas, SCCs, and indicate that non-regulatory T cells are also a significant AKs (Hurwitz et al., 2003; Schon et al., 2003; Patel et al., source of immunosuppressive cytokines in SCC and that 2006; Peris et al., 2006). Imiquimod induces tumor regression production of these cytokines is decreased by treatment with through both immunological and non-immunological me- imiquimod. chanisms by serving as a TLR7 agonist and by binding to IL-10 has been reported to be produced by SCC tumor adenosine receptors (Hemmi et al., 2002; Jurk et al., 2002; cells (Kim et al., 1995). We immunostained cryosections of Lee et al., 2003; Schon et al., 2003; Gorden et al., 2005). SCC and did observe detectable IL-10 production by tumor Cells of the innate immune system are clearly critical to cells (Figure 5a). Also, intracellular cytokine analysis of the initiation of immune reactions in response to TLR agonists the SCC cell line, SCC13, demonstrated IL-10 production such as imiquimod. Clinical response to topical imiquimod (Figure 5b). therapy has been associated with the migration of plasmacy- toid dendritic cells into the skin and subsequent cytokine Treatment of normal human skin with imiquimod enhances production (Palamara et al., 2004; Urosevic et al., 2005). We T-cell activation, and reduces IL-10 production but does not have found that the effects of imiquimod on endothelial and affect the production of IFN-c T cells are all indirect and require the presence of other, Imiquimod could alter the biology of T cells in SCC by imiquimod-responsive cell types (Clark et al., 2008). recruiting different T-cell populations into the tumor, by Despite the importance of innate cells in the first stages altering the activity or function of T cells within the tumor, or of TLR7 agonist responses, tumor destruction and the by a combination of these two mechanisms. To separate the subsequent formation of immune memory are ultimately

www.jidonline.org 2679 SJ Huang et al. Effects of Imiquimod on T Cells in Human SCC

Untreated SCC Imiquimod-treated SCC

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104 104 104 104 PerCP PerCP PerCP PerCP + + + 103 103 103 + 103 CD3 CD3 CD3 CD3 102 102 102 102

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Untreated SCC Untreated SCC Imiquimod-treated SCC Imiquimod-treated SCC 55 18 P<0.001 50 P<0.001 16 45 14 40 35 12 30 10 25 8 20 β -producing Teff 6 15 4 IL-10-producing Teft

10 TGF 5 2 0 0 Figure 4. Effector T cells from imiquimod-treated SCC produce less IL-10 and TGF-b. (a) Non-regulatory T cells in untreated SCC are a significant source of IL-10. IL-10 is also produced by tumor FOXP3 þ Treg cells. (b and c) IL-10 production by non-regulatory T cells is reduced in tumors treated with imiquimod. (d) Summarized data from treated and untreated tumors. (e) TGF-b production by non-regulatory T cells is also reduced after imiquimod therapy.

T-cell-mediated events. It is therefore critical to understand (Figure 1). We found little proliferation of CD8 þ T cells in the downstream effects of TLR agonists such as imiquimod on SCC, suggesting that the increased number of these cells is a the activity and effector functions of T cells. result of enhanced recruitment from the blood (Figure 3). This Imiquimod therapy induced marked shifts in the T-cell result also suggests that proliferation of tumor-specific T cells populations infiltrating SCC. Untreated SCCs are populated may occur instead in the draining lymph nodes. Immature by non-cutaneous central memory T cells, a cell type not dendritic cells are numerous in untreated SCCs, but usually found in the skin (Clark et al., 2008). These T cells are imiquimod induces dendritic cell maturation and likely unlikely to be tumor-specific and are aberrantly recruited into stimulates migration to the lymph nodes (Clark et al., the tumor by unknown mechanisms. Topical therapy with 2008). Imiquimod therapy may therefore enhance antigen imiquimod induced a massive recruitment of CLA þ skin- presentation and proliferation of tumor-specific T cells in the homing CD8 þ effector T cells into SCC. Observation of draining lymph nodes. multiple tumors at various stages of immune destruction T cells from imiquimod-treated tumors produced large allowed us to observe that these T cells formed a dense, amounts of IFN-g, a Th1 cytokine critical for antitumor band-like infiltrate that originated in the deep dermis and immune responses (Nakajima et al., 2001). T cells from migrated up, inducing tumor cell death at the leading edge imiquimod-treated tumors also showed enhanced production and leaving behind areas of fibrotic tumor regression of perforin and granzyme, two proteins that mediate killing

2680 Journal of Investigative Dermatology (2009), Volume 129 SJ Huang et al. Effects of Imiquimod on T Cells in Human SCC

Control Anti-IL-10

105 61 39

104 PE 103

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3 4 5 –650 01010 10 IL-10 APC

Figure 5. Human SCC tumor cells also produce IL-10. (a) Immunostaining of untreated SCC shows that tumor cells, identified by their large, atypical nuclei on Hoechst stain (lower panels, blue), synthesize IL-10. The left panel (control) shows staining with secondary and tertiary antibodies alone and the right panel also includes the primary anti-IL-10 antibody. (b) The SCC cell line, SCC13, produces IL-10. SCC13 tumor cell lines were analyzed for IL-10 production by intracellular cytokine analysis. Bar ¼ 100 mm.

Control medium Imiquimod 5 6 1 105 49 13 105 73010 104 104 104 PerCP

+ 3 103 103 10 CD8 CD25 FITC CD25 FITC 0 2 2 10 10 66 26 21 59 –749 102 103 104 105 102 103 104 105 102 103 104 105 –1340 CD69 PE-Cy7 CD 69 PE-Cy7 IL-10 APC

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% producing IL-10 5 10 0 0 CD4+ CD8+ IL-17 IFN-γ IL-17 IFN-γ CD4+ CD8+

Figure 6. Treatment of skin effector T cells in vitro with imiquimod enhances activation and reduces IL-10 production but has no effect on IL-17 and IFN-c. (a) T cells isolated from human skin treated for 1 week with imiquimod expressed increased CD69 and decreased CD25. Imiquimod treatment of purified skin T cells removed first from the skin microenvironment had no effect (not shown). (b) IL-10 is produced by both CD4 þ and CD8 þ non-regulatory T cells from normal human skin. (c) Non-regulatory T cells isolated from imiquimod-treated skin produced less IL-10 but (d) production of IL-17 and IFN-g was unaffected. For all parts of this figure, only non-regulatory T cells are shown; Treg cells were removed by gating out FOXP3 þ Treg cells before analysis. Mean and s.d. of three donors are shown. by cytotoxic T cells. Our results in SCC agree with prior gene treated tumors. Untreated SCC contain B50% Treg cells and profiling studies in basal cell carcinomas showing a marked half of these Treg cells produce IL-10 (Clark et al., 2008). upregulation of genes associated with cytotoxic T-cell Approximately 45% of non-regulatory T cells in SCC also function after an initial phase of innate cell activation (Panelli produce IL-10, suggesting that nearly half of the T cells in et al., 2007). untreated SCC produce IL-10 and that contributions from In addition to these pro-inflammatory T-cell changes, non- Treg cells and non-regulatory T cells are roughly equivalent. regulatory T cells from imiquimod-treated SCC also had The tumor cells of SCC have also been reported to produce reduced production of the immunosuppressive cytokines, IL-10 (Kim et al., 1995). We observed IL-10 production by IL-10 and TGF-b (Figure 4). Non-regulatory T cells were a tumor cells as shown by immunostaining of SCC tumors and significant source of IL-10 and TGF-b in untreated SCC, and intracellular cytokine analysis of the cell line SCC13 this production was significantly decreased in imiquimod- (Figure 5). Thus, IL-10 is produced by at least three cell types

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Untreated SCC Imiquimod: consequences Imiquimod:indirect local on homing T-cell modulation

− E-selectin E-selectin+ Tumor-cell killing

Teff Treg 50% FOXP3+ ↑ IL-6 ↓ IL-10, TGF-β ↓ Treg IFN-γ Dilution of IL-10 ↓ CD39, CD73 perforin tumor Treg ↓ TGF-β ↓ FOXP3 granzyme − CLA non-cutaneous T cells Influx of skin homing CLA+ T cells

Figure 7. The effects of imiquimod on tumor-associated T cells: consequences on T-cell recruitment versus local modulation of T-cell function. A summary of the current studies and our prior work (Clark et al., 2008) is shown. (a) Untreated SCCs lack vascular expression of E-selectin and are populated by non- cutaneous central memory T cells, 50% of which are FOXP3 þ Treg cells. (b) Imiquimod treatment of SCC induces vascular E-selectin and the recruitment of tumor-specific CLA þ skin-homing T cells. These cells dilute out Treg cells resident in the tumor and their activation within the tumor leads to production of IFN-g, perforin, granzyme, and to tumor cell destruction. (c) Imiquimod also acts locally on tumor T cells, indirectly inducing the production of IL-6 by non-regulatory effector T cells (Teff), likely rendering them resistant to suppression. Imiquimod also reduces Teff production of IL-10 and TGF-b, thereby reducing tonic inhibitory signals within the tumor. Imiquimod also acts on to regulatory T cells (Treg cells) to reduce their ability to suppress through cytokine production (IL-10, TGF-b) and contact suppression (CD39, CD73).

in human SCC: tumor-infiltrating lymphocytes (both regula- the potential to inhibit local immune response to the tumor tory and non-regulatory T cells) and the tumor cells (Sakaguchi et al., 1995; Hori et al., 2003). Imiquimod- themselves. mediated activation of the endothelium leads to an influx Imiquimod has marked effects on T-cell recruitment into of CD8 þ T cells, diluting the percentage of tumors, but it may also locally affect the phenotype and tumor Treg cells to levels found in normal skin. In addition function of T cells within the tumor. To separate out the to this recruitment-mediated effect, imiquimod also differential effects of recruitment versus local T-cell modula- inhibits the ability of Treg cells to suppress T-cell responses. tion, we studied the effects of imiquimod on T cells isolated Imiquimod-treated Treg cells produce less IL-10 and from normal skin samples treated in vitro with imiquimod. TGF-b, and have lower levels of expression of molecules Imiquimod enhanced T-cell activation as evidenced by critical for suppressive function, including FOXP3 þ , increased expression of CD69 (Figure 6a). We found that CD39, and CD73 (Clark et al., 2008). Imiquimod also acts over 25% of FOXP3 CD4 þ T cells from normal human skin locally to induce IL-6 production by non-regulatory T cells, expressed IL-10 (Figure 6b and c). This population of IL-10- likely rendering them resistant to suppression (Kono et al., producing T cells has not been observed earlier and may play 1994; Gibson et al., 1995, 2002; Pasare and Medzhitov, a role in dampening immune responses in normal skin. 2003; Korn et al., 2007). We report here that imiquimod has Imiquimod treatment significantly decreased IL-10 produc- additional effects on non-regulatory T cells, inducing tion by both CD4 þ and CD8 þ T cells, but had no effect on their activation and inhibiting tonic expression of IL-10. the production of IFN-g or IL-17 (Figure 6c and d). Imiquimod Imiquimod therefore acts locally on both Treg cells and may therefore alter the inflammatory tone in normal skin by non-regulatory T cells to block tolerogenic signals within inhibiting the tonic production of anti-inflammatory cyto- the tumor. kines such as IL-10. The lack of effect on IFN-g production Imiquimod can induce the immunological destruction of suggests that the differences we observe in tumors treated basal cell carcinomas, AKs, and Bowen’s disease (Gupta in vivo reflect the recruitment of Th1-biased T cells into the et al., 2005; Schulze et al., 2005; Patel et al., 2006). In tumor. addition to treating existing AKs, imiquimod decreased the On the basis of our observations, we have separated the number of new and recurrent lesions in the year following effects of imiquimod on tumor-infiltrating T cells into those therapy, suggesting a prolonged enhancement of immunolo- reflecting differential migration versus local modulation of gical memory (Krawtchenko et al., 2007). Imiquimod is T-cell activity (Figure 7). In this model, activation of the increasingly being reported as an effective therapy for tumor endothelium plays a critical role in the recruitment of invasive SCC in normal or in immunosuppressed patients skin-homing CLA þ T cells, which contain the subset of T who are poor surgical candidates (Nouri et al., 2003; Hengge cells specific for SCC (Clark et al., 2008). These T cells and Schaller, 2004; Martin-Garcia, 2005; Palungwachira produce IFN-g, granzyme, and perforin, leading to tumor cell et al., 2005; Konstantopoulou et al., 2006; Peris et al., 2006). destruction. Prior to imiquimod therapy, B50% of the T cells Our studies support the use of imiquimod as an adjunctive infiltrating SCC are FOXP3 þ Treg cells (Clark et al., 2008). therapy to reduce tumor size before Mohs’ surgery, to treat Treg cells can suppress the activation, cytokine production, residual disease after surgery, or as a monotherapy for and cytotoxic activity of other T cells, and their presence has unresectable tumors.

2682 Journal of Investigative Dermatology (2009), Volume 129 SJ Huang et al. Effects of Imiquimod on T Cells in Human SCC

Squamous cell carcinomas evade immune responses, at Adjacent sections were used for (a) immunofluorescence studies to least in part, by excluding skin-homing effector T cells and confirm the presence of invasive tumor cells and (b) T-cell isolation, by recruiting Treg cells (Clark et al., 2008). Imiquimod as described below. Imiquimod-treated tumors were obtained from neutralizes both of these defenses, restoring appropriate patients treated daily with topical imiquimod for 10–14 days before T-cell homing and reducing the percentage and suppressive excision. ability of Treg cells. Reduced vascular addressin expression and impaired T-cell homing has been described in human Immunofluorescence studies melanoma, breast, gastric and lung cancers (Piali et al., 1995; Freshly excised SCC tumors were embedded in OCT (optimal cutting Madhavan et al., 2002; Weishaupt et al., 2007). Treg cells temperature), frozen, and stored at 80 1C until use. Cryosections of inhibit immune responses to a large number of human 5 mm were cut, air-dried, fixed for 5 minutes in acetone, rehydrated cancers, including melanoma, lung, breast, ovarian and renal in phosphate-buffered saline, and blocked with 20 mgml1 of human cell cancers (Zou, 2006). Imiquimod’s efficacy in the IgG (Jackson, Bar Harbor, ME) for 15 minutes at room temperature. treatment of SCC suggests that TLR7 agonists may have a The sections were incubated with a primary antibody for 30 minutes, role in the treatment of other malignancies that use similar then rinsed three times in phosphate-buffered saline/1% BSA for mechanisms of immune evasion. 5 minutes. The sections were stained with 0.5 mgml1 Hoechst stain Although two short-term trials have found that imiquimod for 2 minutes, rinsed briefly in phosphate-buffered saline/1% BSA, is safe and effective in the prevention of SCC in transplant mounted using Prolong anti-fade mounting medium (Invitrogen, recipients, the long-term effects of imiquimod on organ Carlsbad, CA), and examined immediately by immunofluorescence tolerance are unknown (Brown et al., 2005; Ulrich et al., microscopy. Antibodies were obtained from BD (Becton Dickinson, 2007). Concerns about inducing widespread immune activa- Franklin Lakes, NJ), (CD8 þ FITC, 1:20 dilution; Ki-67 PE, 1:5 tion and graft rejection continue to limit the use of this dilution). IL-10 staining: rat anti-human IL-10 (BD, 1:5 dilution), medication in the treatment of transplant recipients in whom secondary FITC goat anti-rat IgG (Invitrogen, 1:50), and tertiary SCCs are a major cause of morbidity and mortality. The rabbit polyclonal anti-FITC conjugated to Alexa Fluor 488 (Mole- effects of imiquimod on vascular addressin expression and cular Probes, Eugene, OR, 1:50). In all studies, Hoechst nuclear stain T-cell function are indirect (Wagner et al., 1999; Clark et al., was used to confirm the presence of invasive tumor. TUNEL staining 2008). Once the cytokines or other molecules that mediate was performed on sections from untreated and treated SCC using the imiquimod’s effects have been identified, these agents could ApopTag Red In situ Apoptosis Detection Kit (Chemicon, Billarica, be incorporated into delayed release delivery systems and MA) according to the manufacturer’s instructions. Following the final administered directly to tumors. Local release of these agents wash step, the sections were stained with Hoechst dye and mounted within the tumor microenvironment could induce immuno- as described above. The sections were photographed using a Nikon logical destruction of tumors without the risk of widespread Eclipse 6600 microscope (Nikon, Melville, NY) equipped with a immune activation engendered by TLR agonists such as Nikon 40/0.75 Plan Fluor objective lens. Images were captured imiquimod. These agents would also obviate the requirement with a SPOT RT model 2.3.1 camera (Diagnostic Instruments, for TLR7 responder cells, such as plasmacytoid dendritic Sterling Heights, MI) and were obtained using SPOT 4.0.9 software cells, to be present within tumors. A lack of plasmacytoid (Diagnostic Instruments). dendritic cells in tumors correlated with the lack of response to imiquimod in human basal cell carcinoma (Urosevic et al., Isolation of T cells from SCC tumors and normal skin 2005). The clinically evident portions of biopsy-proven invasive SCC The skin is an accessible site in which human tumor tumors were obtained. The tumors were divided into bread loaf immunity can be analyzed because immune reactions in the sections. One section was histologically examined to confirm the skin are visible, easily sampled, and can be manipulated with presence or absence of invasive SCC tumor. Sections without topical medications. By studying SCC, we can observe, evidence of tumor were designated peritumoral. Tumor-infiltrating sample, and modify the human immune response to cancer. T cells were isolated from sections adjacent to those studied by Insights gained from these studies will likely be applicable to histology. T cells were isolated from SCC tumors in the absence of the treatment of both cutaneous and non-cutaneous malig- exogenous cytokines as described previously (Clark et al., 2006). For nancies. normal skin studies, T cells from the skin discarded after plastic surgery procedures were isolated from 3-week explant cultures MATERIALS AND METHODS maintained in the presence of 100 U ml1 IL-2 (R&D Systems, SCC samples Minneapolis, MN) and 20 ng ml1 of IL-15 (R&D Systems) as Tumor samples consisted of curetted tumor removed before excision described previously (Clark et al., 2006). of the first Mohs’ section during Mohs’ micrographic excision of biopsy-proven SSCs. Acquisition of tumor samples and all studies Flow cytometry studies were approved by the Institutional Review Board (IRB) of the Flow cytometry analysis of T cells was carried out using directly Partners Human Research Committee and were performed in conjugated monoclonal antibodies purchased from BD Biosciences accordance with the Declaration of Helsinki Principles. Patient (CD3 þ , CD4 þ , CD8 þ , CD25, and CD69) and eBioscience (San consent was not required because the IRB determined that collection Diego, CA) (FOXP3 þ , clone PCH101). Staining for FOXP3 þ and of discarded SCC tumors did not meet the definition of human activated NF-kB p65 was performed as per the manufacturer’s subjects research. Tumors were divided into bread loaf sections. instructions. Analysis of flow cytometry samples was carried out on

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Becton Dickinson FACScan or FACSCanto instrument, and data were Berg D, Otley CC (2002) Skin cancer in organ transplant recipients: epidemiology, analyzed using FACSDiva software (V5.1). pathogenesis, and management. J Am Acad Dermatol 47:1–17 Brown VL, Atkins CL, Ghali L, Cerio R, Harwood CA, Proby CM (2005) Safety Analyses of cytokine, granzyme, and perforin production and efficacy of 5% imiquimod cream for the treatment of skin dysplasia 1 in high-risk renal transplant recipients: randomized, double-blind, T cells were stimulated with 50 ng ml PMA (phorbol 12-myristate placebo-controlled trial. Arch Dermatol 141:985–93 1 13-acetate; Sigma, St Louis, MO) and 750 ng ml ionomycin Clark RA, Chong BF, Mirchandani N, Yamanaka K, Murphy GF, Dowgiert RK 1 (Invitrogen) for 6 hours, with the inclusion of 10 mgml brefeldin- et al. (2006) A novel method for the isolation of skin resident A (Calbiochem) for the last 5 hours of incubation. SCC13 tumor cells T cells from normal and diseased human skin. J Invest Dermatol 126: (kindly provided by Dr James Rheinwald) were cultured and 1059–70 brefeldin-A was added to the cultures 12 hours before analysis. Clark RA, Huang SJ, Murphy GF, Mollet IG, Hijnen D, Muthukuru M et al. (2008) Human squamous cell carcinomas evade the immune response The cells were stained for surface marker expression, fixed, by down-regulation of vascular E-selectin and recruitment of regulatory permeabilized, and stained with anti-cytokine, granzyme, or T cells. J Exp Med 205:2221–34 perforin antibodies (IL-17, IL-10, IFN-g, TGF-b, granzyme, and Diepgen TL, Mahler V (2002) The epidemiology of skin cancer. Br J Dermatol perforin, all from BD Pharmingen, Franklin Lakes, NJ), and examined 146(Suppl 61):1–6 by flow cytometry. We observed loss of cytokine staining in Euvrard S, Kanitakis J, Claudy A (2003) Skin cancers after organ transplanta- unstimulated samples lacking PMA and ionomycin (but treated with tion. N Engl J Med 348:1681–91 brefeldin-A) and in samples treated with PMA and ionomycin but Feldman SR, Fleischer AB Jr, McConnell RC (1998) Most common lacking brefeldin-A, suggesting that T-cell stimulation was required dermatologic problems identified by internists, 1990–1994. Arch Intern Med 158:726–30 for cytokine production and that cytokines synthesized by T cells Gibson SJ, Imbertson LM, Wagner TL, Testerman TL, Reiter MJ, Miller RL et al. were secreted. (1995) Cellular requirements for cytokine production in response to the immunomodulators imiquimod and S-27609. J Interferon Cytokine Res Imiquimod treatment of normal skin T cells 15:537–45 Imiquimod stocks of 10,000 (30 mM) were prepared by solubiliz- Gibson SJ, Lindh JM, Riter TR, Gleason RM, Rogers LM, Fuller AE et al. (2002) ing imiquimod cream in DMSO. The stocks were then diluted 1:10 Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod. Cell Immunol in culture medium and 1 ml of this 1,000 stock was added to each 218:74–86 microliter of culture medium (end concentration was 3 mM). For Gorden KB, Gorski KS, Gibson SJ, Kedl RM, Kieper WC, Qiu X et al. (2005) control medium samples, an equivalent amount of DMSO was Synthetic TLR agonists reveal functional differences between human added to the control culture medium (a 1:10,000 dilution). Normal TLR7 and TLR8. J Immunol 174:1259–68 1 skin explant cultures were set up and maintained in 100 U ml of Gupta AK, Davey V, McPhail H (2005) Evaluation of the effectiveness of IL-2 (R&D Systems) and 20 ng ml1 IL-15 (R&D Systems) to produce imiquimod and 5-fluorouracil for the treatment of actinic keratosis: an expanded population of skin-resident T cells as described Critical review and meta-analysis of efficacy studies. J Cutan Med Surg 9:209–14 previously (Clark et al., 2006). Imiquimod or control DMSO was Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K et al. (2002) added to 2-week skin explant cultures and replenished with feeds for Small anti-viral compounds activate immune cells via the TLR7 MyD88- 1 week; cells were harvested on week 3. In separate experiments, dependent signaling pathway. Nat Immunol 3:196–200 T cells were harvested from the 2-week skin explant cultures (grown Hengge UR, Schaller J (2004) Successful treatment of invasive squamous cell in IL-2 and IL-15 for 2 weeks) and thereafter cultured separately with carcinoma using topical imiquimod. Arch Dermatol 140:404–6 IL-2 and IL-15 and with or without imiquimod for 1 week. Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T cell development by the transcription factor FOXP3 þ . Science 299:1057–61 CONFLICT OF INTEREST Housman TS, Feldman SR, Williford PM, Fleischer AB Jr, Goldman ND, The authors state no conflict of interest. Acostamadiedo JM et al. (2003) Skin cancer is among the most costly of all cancers to treat for the Medicare population. J Am Acad Dermatol 48:425–9 ACKNOWLEDGMENTS The authors thank Dr Thomas Cochran of the Boston Center for Plastic Hurwitz DJ, Pincus L, Kupper TS (2003) Imiquimod: a topically applied Surgery and Dr Elof Eriksson of Brigham and Women’s Hospital for link between innate and acquired immunity. Arch Dermatol generously providing normal human skin samples; Dr Richard Miller and 139:1347–50 3M Pharmaceuticals for kindly providing imiquimod cream and powdered Jurk M, Heil F, Vollmer J, Schetter C, Krieg AM, Wagner H et al. (2002) imiquimod; and Dr James Rheinwald for generously providing the SCC13 cell Human TLR7 or TLR8 independently confer responsiveness to the line. This research was supported by NIH Grant 1K08AI060890-01A1, a antiviral compound R-848. Nat Immunol 3:499 Translational Research Award from the Leukemia and Lymphoma Society (to Kim J, Modlin RL, Moy RL, Dubinett SM, McHugh T, Nickoloff BJ et al. (1995) R.A.C.), a Pilot & Feasibility grant from the Harvard Skin Disease Research IL-10 production in cutaneous basal and squamous cell carcinomas. A Center (to R.A.C. from NIH Grant P30 AR-42689-11, to T.S.K.), a mechanism for evading the local T cell immune response. J Immunol Developmental project from the SPORE in Skin Cancer (to R.A.C., from 155:2240–7 NIH Grant P50 CA-93683-04, to T.S.K.), a Clinical Investigator Award from the Damon Runyon Cancer Research Foundation (to R.A.C.), and a Howard Kono T, Kondo S, Pastore S, Shivji GM, Tomai MA, McKenzie RC et al. (1994) Hughes Medical Institute Medical Research Training Fellowship (to S.J.H.). Effects of a novel topical immunomodulator, imiquimod, on keratinocyte cytokine gene expression. Lymphokine Cytokine Res 13:71–6 Konstantopoulou M, Lord MG, Macfarlane AW (2006) Treatment of invasive squamous cell carcinoma with 5-percent imiquimod cream. 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