The Journal of Experimental Medicine
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 205 No. 10 2221-2234
More than 100,000 squamous cell carcinomas
(SCCs) of the skin are diagnosed each year in
the United States ( 1 ). Nonmelanoma skin can-
cer, of which SCC is the second most frequent
type, is the fi fth most costly cancer, accounting
for 4.5% of all Medicare cancer costs ( 2 ). The
premalignant precursors to SCC, actinic kera-
toses, are the third most frequent reason in the
United States for consulting a dermatologist ( 3 ).
More than 5.2 million physician visits are made
each year for the treatment of actinic keratoses
at a cost of more than 900 million dollars annu-
ally ( 4 ).
Solid organ transplant recipients on immuno-
suppressive medications frequently develop mul-
tiple and aggressive SCCs ( 5 ). These individuals
have a 65 – 250-fold increased risk of SCCs, nearly
10% of these tumors metastasize, and the major-
ity of these patients die as a result ( 5, 6 ). The
development of SCCs in transplant recipients is
linked to the use of medications that suppress
T cell activity ( 6 ). T cell function therefore
appears critical to the immunological control of
SCCs. We present our fi ndings that SCCs from
Rachael A. Clark:
Abbreviations used: CCL, CC
chemokine ligand; CLA, cuta-
neous lymphocyte antigen; hpf,
high power fi eld; iNOS, induc-
ible nitric oxide synthase; PDC,
plasmacytoid DC; SCC, squa-
mous cell carcinoma; TLR,
Toll-like receptor; T reg, regu-
The online version of this article contains supplemental material.
Human squamous cell carcinomas evade
the immune response by down-regulation
of vascular E-selectin and recruitment
of regulatory T cells
Rachael A. Clark , 1 Susan J. Huang , 1 George F. Murphy , 2 Ilse G. Mollet , 3
Dirkjan Hijnen , 4 Manoj Muthukuru , 1 Carl F. Schanbacher , 1
Vonetta Edwards , 5 Danielle M. Miller , 1 Jenny E. Kim , 1 Jo Lambert , 3
and Thomas S. Kupper 1
1 Harvard Skin Disease Research Center and the Department of Dermatology, and 2 Department of Pathology,
Brigham and Women ’ s Hospital, Boston, MA 02115
3 Department of Dermatology, Ghent University Hospital, B-9000 Ghent, Belgium
4 Department of Dermatology, Utrecht University Medical Center, 3508 GA Utrecht, Netherlands
5 University of Maryland, Program in Molecular and Cell Biology, College Park, MD 20742
Squamous cell carcinomas (SCCs) of the skin are sun-induced skin cancers that are particu-
larly numerous in patients on T cell immunosuppression. We found that blood vessels in
SCCs did not express E-selectin, and tumors contained few cutaneous lymphocyte antigen
(CLA) + T cells, the cell type thought to provide cutaneous immunosurveillance. Tumors
treated with the Toll-like receptor (TLR)7 agonist imiquimod before excision showed induc-
tion of E-selectin on tumor vessels, recruitment of CLA + CD8 + T cells, and histological
evidence of tumor regression. SCCs treated in vitro with imiquimod also expressed vascular
E-selectin. Approximately 50% of the T cells infi ltrating untreated SCCs were FOXP3 +
regulatory T (T reg) cells. Imiquimod-treated tumors contained a decreased percentage of T
reg cells, and these cells produced less FOXP3, interleukin (IL)-10, and transforming growth
factor (TGF)- ? . Treatment of T reg cells in vitro with imiquimod inhibited their suppressive
activity and reduced FOXP3, CD39, CD73, IL-10, and TGF- ? by indirect mechanisms. In vivo
and in vitro treatment with imiquimod also induced IL-6 production by effector T cells. In
summary, we fi nd that SCCs evade the immune response at least in part by down-regulat-
ing vascular E-selectin and recruiting T reg cells. TLR7 agonists neutralized both of these
strategies, supporting their use in SCCs and other tumors with similar immune defects.
© 2008 Clark et al. This article is distributed under the terms of an Attribu-
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IMMUNE EVASION IN SQUAMOUS CELL CARCINOMAS OF THE SKIN | Clark et al.
expression (not depicted). Few SCC T cells were Th2 biased
as demonstrated by the lack of two independent markers for
Th2 bias, ST2L and the ? -IFN receptor ? chain ( Fig. 1 A )
( 14 – 16 ). Analysis of cytokine production by intracellular fl ow
cytometry confi rmed that most T cells from SCCs were Th1
biased ( Fig. 1 C ).
T cells resident in human skin have a diverse T cell reper-
toire, consistent with their role in immunosurveillance against
a variety of pathogens and tumors ( 9 ). T cells in cervical can-
cers have a more biased repertoire, refl ecting local expansion
of tumor-specifi c T cell clones ( 17 ). We analyzed the TCR
repertoire of T cells infi ltrating SCCs by fl ow cytometry for
V ? TCR subfamilies and found that these cells were diverse,
without detectable V ? bias ( Fig. 1 D ).
Blood vessels of invasive SCCs in skin do not
E-selectin is a ligand for CLA that is expressed on postcapil-
lary venules in the skin. Vascular E-selectin is up-regulated
with infl ammation and supports the entry of CLA + T cells
into the skin under both normal and infl amed conditions ( 11,
12, 18 ). We examined tumor vessels for E-selectin expression
by immunohistochemistry and found that blood vessels in
areas surrounding the tumor (peritumoral areas) expressed
E-selectin, but that blood vessels within the tumor parenchyma
did not. ( Fig. 2, A – C ). Staining for CD31 was included to
both healthy and immunocompromised individuals evade the
immune response, at least in part, by down-regulation of vas-
cular E-selectin, exclusion of skin-homing memory T cells, and
recruitment of regulatory T (T reg) cells.
SCCs are infi ltrated by diverse noncutaneous central
memory T cells
SCCs of the skin commonly have associated T cell infi ltrates,
but the clinical persistence of the cancer suggests that these
T cells are unable to destroy the tumor ( 7 ). We isolated T cells
from human invasive SCCs and compared them with T cells
from normal human skin, the population thought to provide
immunosurveillance ( Fig. 1 ) ( 8, 9 ). Peripheral tissue eff ector
T cells express tissue-specifi c homing receptors and preferen-
tially recirculate through the tissue in which they fi rst en-
countered their antigens ( 10, 11 ). Skin resident T cells express
the skin addressins cutaneous lymphocyte antigen (CLA) and
CCR4, which bind to E-selectin and CC chemokine ligand
(CCL) 22 on skin endothelium ( 9, 11, 12 ). T cells from SCCs
did not express CLA and CCR4 ( Fig. 1 A ) and instead ex-
pressed l- selectin and CCR7, markers of central memory T
cells that are normally found only in the blood or lymph nodes
( 13 ). Studies of cryosections confi rmed that T cells from SCCs
lack the skin addressin CLA ( Fig. 1 B ). T cells from SCCs
developing in transplant recipients also lacked CLA and CCR4
Figure 1. T cells infi ltrating SCCs are noncutaneous central memory T cells. (A) T cells isolated from SCCs were memory (CD45RO + ) Th1-biased T
cells that lacked expression of skin-homing addressins (CLA, CCR4) and instead expressed markers characteristic of central memory T cells (L-selectin/
CCR7), a cell type that is usually restricted to blood or lymph nodes. In contrast, T cells from normal human skin expressed high levels of the skin-homing
addressins CLA and CCR4 and most lacked CCR7/L-selectin coexpression. Similar results were observed in three additional SCC samples. (B) Confi rmation
that T cells infi ltrating SCCs lack CLA expression. Frozen sections of invasive SCCs were stained for CD3 (red) and CLA (green). Only a small number of T
cells infi ltrating SCCs were skin-homing T cells (CD3 + CLA + , yellow). A higher magnifi cation of the same fi eld is also shown. (C) Intracellular cytokine analy-
sis of CD4 + T cells from two SCC tumors demonstrated a Th1 bias with few Th2 or Th17 T cells present. Unstimulated cells produced no detectable cyto-
kines (not depicted). (D) Analysis of T cells from SCCs by fl ow cytometry for TCR V ? expression demonstrated signifi cant TCR diversity. Analysis of two
additional tumors showed comparable diversity. Bars, 100 μ m.
JEM VOL. 205, September 29, 2008
ients on immunosuppressive medications also lacked vascular
E-selectin within the tumors (not depicted).
Imiquimod-treated SCCs express vascular E-selectin
and are infi ltrated by CLA + skin-homing T cells
Imiquimod is a topical Toll-like receptor (TLR)7/8 ago-
nist that is eff ective in the treatment of a wide range of skin
identify blood vessels. This fi nding was observed in all SCCs
studied; we performed immunohistochemical stains on tu-
mors from four patients and conducted similar studies using
three-color immunofl uorescence analysis on tumors from fi ve
additional patients. Hoechst nuclear stain was used to con-
fi rm the presence of invasive tumor in immunofl uorescence
studies. Two additional SCCs excised from transplant recip-
Figure 2. Blood vessels in areas of invasive SCCs do not express E-selectin, but imiquimod treatment induces vascular E-selectin expression
and normalization of T cell homing. (A) Serial sections of SCCs demonstrated the presence of CD31 + blood vessels in areas of invasive SCCs, but
these vessels lack E-selectin expression. (B) A second example is shown, in which tumor and peritumoral areas were present in a single stained section.
(C) Immunofl uorescence studies on a third sample stained for CD31 (green) and E-selectin (red), showing no E-selectin expression on tumor vessels. (D) SCCs
treated with imiquimod before excision were heavily infi ltrated with CLA + (skin-homing) cytotoxic T cells. (E) A subset of tumor vessels in imiquimod-
treated SCCs expressed E-selectin. (F) A second sample showing E-selectin expression on a subset of vessels embedded within a tumor nodule after im-
iquimod treatment. Large atypical keratinocytes forming a tumor nodule are demonstrated by Hoechst stain (blue). (G) In vitro treatment of SCC tumor
tissue with imiquimod up-regulated E-selectin on a subset of tumor vessels. 2-mm bread loaf sections of untreated SCC tumor were incubated for 24 h in
control medium, TNF- ? , or imiquimod. Samples were then frozen, sectioned, and stained for CD31 and E-selectin. In all studies, Hoechst nuclear stain was
used to identify areas of invasive tumor. Bars: (B, C, E, and F) 100 μ m.
IMMUNE EVASION IN SQUAMOUS CELL CARCINOMAS OF THE SKIN | Clark et al.
immunocompromised patients had greatly increased numbers
of FOXP3 + T reg cells when compared with the population
found in normal skin ( Fig. 4 A ). SCC FOXP3 + T reg cells
were CD4 + , lacked expression of addressins found on skin
resident T reg cells (CLA, CCR4, and CCR6) ( 20 ), and in-
stead expressed markers of central memory T cells ( Fig. 4 B ,
l -selectin/CCR7 + ). FOXP3 is expressed at high and con-
stant levels by T reg cells, but expression of FOXP3 is also tran-
siently increased in activated non – T reg cells ( 20, 21, 24 ). Our
earlier work has shown that although activated T cells in-
crease expression of FOXP3, this transient increase was
small and did not obscure the identifi cation of true T reg cells,
which expressed FOXP3 at levels a log higher than activa-
ted non – T reg cells ( 20 ). CD127 negativity has recently been
reported to discriminate between T reg cells and effector
malignancies, including basal cell carcinomas and SCCs ( 19 ).
We studied invasive SCCs treated with imiquimod from
10 to 14 d before tumor excision (mean 12.7 d). All treated
tumors exhibited areas of fi brosis surrounding the tumors,
consistent with stromal changes of tumor regression (not de-
picted). Imiquimod-treated SCCs contained > 80% CD8 +
cytotoxic T cells and, in contrast to untreated tumors, the
majority of these T cells expressed CLA ( Fig. 2 D ). The ma-
jority also lacked l- selectin and CCR7 coexpression, sug-
gesting a shift toward eff ector memory cells, the type normally
found within the skin ( 9 ). T cells also expressed CCR6, but
most lacked CCR4.
In contrast to untreated tumors, vessels of imiquimod-
treated SCCs expressed E-selectin on a subset of tumor
vessels ( Fig. 2, E and F ). Human endothelial cells have not
previously been shown to respond to imiquimod. To study
the ability of imiquimod to induce E-selectin expression
ex vivo, we cultured portions of an untreated SCC tumor
with imiquimod or TNF- ? in vitro. Imiquimod and TNF- ?
had similar eff ects; both induced E-selectin expression on a
subset of tumor vessels, likely representing postcapillary ven-
ules ( Fig. 2 G ).
Imiquimod indirectly up-regulates E-selectin on human
To determine if endothelial cells were the direct targets of
imiquimod, we measured the expression of TLR7 and TLR8
in dermal microvascular endothelial cells by immunostaining
and real-time PCR. Blood vessels in SCCs expressed TLR7
and TLR8 by immunostaining ( Fig. 3 A ). However, TLR
antibodies can be cross-reactive, so expression was con-
fi rmed by real-time PCR analysis of purifi ed cultures of dermal
microvascular endothelial cells ( Fig. 3 B ). Although purifi ed
endothelial cells expressed TLR7 and TLR8, they did not
up-regulate E-selectin when treated in vitro with imiquimod
( Fig. 3 C ). In contrast, inclusion of activated APCs in the co-
cultures or overnight treatment with TNF- ? resulted in a
robust induction of E-selectin.
To determine if SCC tumor cells could suppress endo-
thelial expression of E-selectin, we co-cultured endothelial
cells with the SCC cell line SCC13 in the presence or absence
of imiquimod and TNF- ? ( Fig. 3 D ). We found no eff ect on
the baseline or induced levels of endothelial E-selectin, sug-
gesting that SCC tumor cells themselves do not directly sup-
press vascular E-selectin expression.
SCCs are infi ltrated by FOXP3 + T reg cells
We further characterized the central memory T cells infi l-
trating SCCs and found they contained a large population of
CD25 hi CD69 lo T cells, a phenotype similar to that of natural
T reg cells ( Fig. 4 A ) ( 20 ). Natural T reg cells, which develop
as a separate lineage within the thymus, can be distinguished
from other T cells by their constant and high expression of the
transcription factor FOXP3 ( 21 – 23 ). We and others have found
excellent correlation of high FOXP3 expression with suppre-
ssive ability ( 20 ). T cells isolated from SCCs of both normal and
Figure 3. Imiquimod up-regulates E-selectin on endothelial cells
by an indirect mechanism. (A) Immunofl uorescence studies on frozen
sections of SCC tumor stained for the vascular marker CD31 (green)
and TLR7 or TLR8 (red). (B) Confi rmation of TLR7 and TLR8 expression
by real-time quantitative PCR. Cultured dermal microvascular endo-
thelial cells were analyzed for TLR7 and TLR8 expression and com-
pared with T cell – depleted peripheral blood mononuclear cells (APCs).
(C) Imiquimod does not directly induce E-selectin on endothelial cells.
Purifi ed human endothelial cells were cultured with imiquimod or acti-
vated APC for 3 d or TNF- ? for 12 h, and then harvested and assayed
by fl ow cytometry for the expression of CD31 and E-selectin. Experi-
ments using two additional endothelial cell donors produced similar
results. (D) SCC tumor cells do not suppress baseline or induced
E-selectin expression on endothelial cells. SCC13 tumor cells were co-
cultured with endothelial cells in control medium (SCC), with imiqui-
mod (SCC+imiquimod) or with TNF- ? (SCC+TNF- ? ). No changes in
basal or induced levels of endothelial E-selectin were observed. Experi-
ments using two additional endothelial donors produced similar re-
sults. Bar, 100 μ m.
JEM VOL. 205, September 29, 2008
receptors ( Fig. 1 ). We therefore confi rmed our fi ndings
using primary SCC tumor tissue. We used three-color
immunofl uorescence staining of frozen sections of SCC tu-
mors to enumerate non – T reg (FOXP3 ? CD3 + ) and T reg
(FOXP3 + CD3 + ) cells in areas of invasive SCCs. Nearly 50%
of the T cells infi ltrating SCCs from both normal and immuno-
suppressed patients were FOXP3 + T reg cells ( Figs. 4 E and
5 C ). Approximately 50 – 60% of the total T cells in SCCs
were CD4 + ( Fig. 1 A ), suggesting that the vast majority
of CD4 + T cells in SCCs are actually FOXP3 + T reg cells.
cell populations in humans ( 25 – 27 ); we found that SCC
FOXP3 + T cells lacked expression of CD127, suggesting
that they do not represent recently activated non – T reg cells
(Fig. S1, available at http://www.jem.org/cgi/content/full/
Our method of T cell isolation from skin depends upon
the ability of T cells to migrate out of the skin in response to
chemokines produced by dermal fi broblasts ( 28 ). T cells with
central memory markers may not migrate effi ciently to skin
cell chemokines because they express diff erent chemokine
Figure 4. SCCs are heavily infi ltrated by FOXP3 + T reg cells recruited from blood. (A) T cells isolated from SCCs developing in normal individuals
and transplant recipients contained many CD25 hi FOXP3 + T reg cells. (B) T reg cells isolated from SCCs were CD4 + central memory T cells (L-selectin/CCR7 + )
and were distinct from cutaneous T reg cells found in normal skin as shown by their lack of expression of key skin-homing addressins (CLA, CCR4). The
last two graphs are gated to show only CD3 + FOXP3 + T cells. (C) Direct study of FOXP3 + T reg cells in areas of invasive SCCs using immunofl uorescence
staining of frozen sections. SCCs were stained for CD3 (red) and FOXP3 (green). Two FOXP3 + T reg cells are shown at the top of the left image, and a
FOXP3 ? nonregulatory T cell is shown on the bottom. A larger fi eld is shown in the right image. (D) A lower magnifi cation image of another SCC, demon-
strating that large numbers of FOXP3 + T reg cells (red cells with green nuclei) surround nodules of invasive tumor, which appear as pools of green sec-
ondary to nonspecifi c staining of tumor keratin. (E) Enumeration of T reg cells in frozen sections of SCCs. The number of T reg cells and nonregulatory T
cells were counted in 10 high power (40X) fi elds in SCCs from normal patients (Immunocompetent) and transplant recipients (Transplant rcp) and the
results were compared with that of normal skin. Shown are the mean and SD of counts from 10 fi elds. (F) FOXP3 + T reg cells are not locally expanded
within SCCs. SCC sections were costained for FOXP3 and Ki-67, a marker of cell proliferation. Proliferative and nonproliferative FOXP3 + T reg cells were
counted in 5 hpf for each donor; the mean and SD for each tumor are shown. SCC9 and 10 are from immunocompetent individuals; SCC11 is from a
transplant recipient. Bars: (C, left) 10 μ m; (C, right, and D) 100 μ m.
IMMUNE EVASION IN SQUAMOUS CELL CARCINOMAS OF THE SKIN | Clark et al.
these cells was reduced to roughly 10%, similar to that found
in normal human skin ( Fig. 5, B and C ).
Imiquimod inhibits T reg cell function
TLR8 agonists can block the ability of T reg cells to suppress
T cell responses, suggesting that imiquimod may have an ef-
fect on tumor-associated T reg cells ( 29 ). We isolated T reg
cells from normal human skin and studied the eff ect of imiqui-
mod on these cells. In vitro treatment of purifi ed skin T cells
with imiquimod for 1 wk had no direct eff ect on the viability
of FOXP3 + T reg cells, suggesting that T reg cells are not
depleted by imiquimod ( Fig. 5 D ). Our previous studies dem-
onstrate that skin-resident natural T reg cells proliferate in re-
sponse to culture with dermal fi broblasts and IL-15 ( 20 ). We
found that imiquimod only slightly reduced proliferation
of FOXP3 + T reg cells under these conditions ( Fig. 5 E ).
To study T reg cell function, we cultured skin explants with
To determine if T reg cells are recruited from the blood or
locally expanded within the tumor, we stained tumors for ex-
pression of Ki-67, an antigen expressed by dividing cells. We
observed very few proliferating T reg cells in SCCs, suggesting
that recruitment of these cells from the blood is the predomi-
nant mechanism for their accumulation within SCCs ( Fig. 4 F ).
Imiquimod treatment is associated with decreased
percentages of FOXP3 + T reg cells
T cells isolated from SCC tumors treated with imiquimod
contained decreased percentages of FOXP3 + T reg cells ( Fig.
5 A ). We counted the absolute number of T reg cells in the
cryosections of imiquimod-treated tumors and found that this
drop in the percentage of T reg cells resulted from a marked
infl ux of non – T reg cells into the tumor, most of which were
cytotoxic CD8 + T cells ( Figs. 5 B and 2 D ). FOXP3 + T reg
cells were still present in treated tumors, but the percentage of
Figure 5. Imiquimod-treated SCCs contain decreased percentages of FOXP3 + T reg cells and imiquimod treatment in vitro blocks the
ability of T reg cells to suppress. (A) T cells isolated from imiquimod-treated SCCs contain few detectable FOXP3 + T reg cells. (B) Direct enumera-
tion of FOXP3 + T reg cells in sections of imiquimod-treated SCCs (tx). Counts from untreated SCCs (untx) are shown for comparison. Mean and SD are
shown. (C) Percentage of FOXP3 T reg cells infi ltrating normal human skin (nml), untreated SCC tumors (untx, triangles; SCCs from healthy individ-
uals, circles; SCCs from transplant recipients) and imiquimod-treated SCCs (tx). Bars indicate the SD of the percentage of T reg cells from 10 hpf.
(D) Imiquimod does not affect the viability of nonregulatory T cells (FOXP3 ? ) and FOXP3 + T reg cells (FOXP3 + ) isolated from human skin. Viability was
assessed after 1 wk of incubation in either control medium or imiquimod. (E) Imiquimod only slightly inhibits the proliferation of skin-derived T reg
cells. T cells from human skin were labeled with CFSE and cultured with dermal fi broblasts and IL-15 for 1 wk in the presence or absence of imiqui-
mod. Cells were then stained for FOXP3 expression. FOXP3 + T reg cells that have proliferated are shown in the top left quadrant of each histogram.
(F) Imiquimod treatment paralyzes regulatory T cell function. T cells isolated from human skin were cultured for three days in control medium (untx,
gray bars) or imiquimod (tx, black bars), and then separated into T reg cells, enriched CD25 hi T cells (CD25 hi ), and responder CD25 lo T cells (CD25 lo ).
Cells were stimulated with soluble anti-CD3 and – CD28 and proliferation was assayed by incorporation of [ 3 H]thymidine. Untreated CD25 hi sup-
pressed CD25 lo T cell proliferation, but pretreatment of CD25 hi cells with imiquimod blocked suppression. Suppression of imiquimod-treated CD25 lo
was restored by adding untreated CD25 hi T cells, demonstrating that the suppressive defect was is the CD25 hi subset. (G) At least three days of im-
iquimod pretreatment is required for loss of suppressive function. Skin T cells were cultured in control medium (squares) or imiquimod (circles) for
the indicated length of time, and then cells were sorted and analyzed for suppressive ability. Bars indicate the SD of experiments from two different
JEM VOL. 205, September 29, 2008
reg cells (not depicted). These fi ndings suggested that imiqui-
mod may aff ect both T reg and non – T reg cells.
Imiquimod indirectly up-regulates IL-6 production
by effector T cells
In our suppression assays, imiquimod was added to skin ex-
plant cultures during the fi nal period before T cell collection.
We could not determine from these experiments if the eff ects
of imiquimod on T cells were direct or were mediated by
factors produced by other cells within the skin.
Imiquimod induces the production of IL-6 from human
monocytes, plasmacytoid DCs (PDCs), and keratinocytes,
and the production of IL-6 by T cells or nearby DCs has
been shown in mice to render T cells resistant to suppression
by T reg cells ( 30 – 34 ). T cell expression of IL-6 in response
to imiquimod has not been previously reported. We found
that the addition of imiquimod to skin explant cultures for
1 wk induced IL-6 expression in 22% (SD of 0.2) of skin
T cells compared with a baseline expression of 5.5% (SD of
2.5; Fig. 6, A and B ). We then examined T cells isolated from
SCCs treated in vivo with imiquimod and found that 53%
(SD of 0.64) of T cells from treated tumors produced IL-6,
either imiquimod or control medium for 3 – 5 d. We then col-
lected T cells from explant cultures, isolated enriched popula-
tions of FOXP3 + T reg cells by high speed fl ow cytometry
sorting for CD3 + CD4 + CD25 hi CD69 lo T cells, and tested the
ability of these cells to suppress the proliferation of T cells iso-
lated from the same skin sample, as described previously ( 20 ).
When suppression assays, which lasted 6 d, were performed in
the absence of imiquimod, we found that CD25 hi T reg cells
pretreated for 3 d with imiquimod failed to suppress T cell
proliferation, whereas untreated CD25 hi T reg cells from the
same sample of skin did suppress T cell responses ( Fig. 5 F ).
The reciprocal experiment showed that pretreatment of both
CD25 lo responder cells and CD25 hi T reg cells with imiqui-
mod showed no suppression, but the addition of untreated
CD25 hi T cells restored suppression. This eff ect depended on
the pretreatment of T reg cells with imiquimod; 5 d of pre-
treatment with imiquimod produced similar results, but 2 d
of treatment induced only a partial loss of suppressive ability,
suggesting that inactivation of T reg cells required at least 3 d
of imiquimod treatment ( Fig. 5 G ). When imiquimod was
additionally added to suppression assays, we found that eff ec-
tor T cells were less susceptible to suppression by untreated T
Figure 6. Imiquimod induces IL-6 production by effector T cells and reduces FOXP3 and production of IL-10 and TGF- ? by T reg cells.
(A) Imiquimod treatment induces IL-6 production by T cells in vitro and in vivo. Imiquimod or control medium was added to explant cultures of nor-
mal human skin for 1 wk. T cells were then isolated from these cultures and examined for IL-6 production. Imiquimod treatment of purifi ed skin T
cells alone had no effect. T cells were isolated from SCCs that were either untreated (SCC16) or treated in vivo with topical imiquimod (SCC8, 17, 18)
and analyzed for IL-6 production. Histograms of SCC T cells are gated to show only CD3 + T cells. (B) IL-6 production from multiple donors after in
vitro (skin T cells) or in vivo (SCC; 8, 17, 18) treatment with imiquimod. (C) FOXP3 expression as assayed by mean fl uorescence intensity (MFI) under
identical staining conditions in T cells from normal skin explant cultures (NS) treated for 1 wk with control medium (triangle) or imiquimod (circle,
mean and SD of three determinations are shown) or from untreated SCCs (16; triangle) or SCCs treated in vivo with imiquimod (circles; 8, 16, 18).
(D) IL-10 and TGF- ? production as determined by intracellular cytokine analysis of FOXP3 + T reg cells isolated from untreated (SCC16) or imiquimod
treated SCCs (SCC17). (E) IL-10 and TGF- ? production by FOXP3 + T reg cells isolated from normal skin explant cultures (NS) treated for 1 wk with
control medium (white bars) or imiquimod (black bars) and by FOXP3 + T reg cells isolated from untreated SCCs (16) or SCCs treated in vivo with
imiquimod (8, 17, 18).
IMMUNE EVASION IN SQUAMOUS CELL CARCINOMAS OF THE SKIN | Clark et al.
reduced the expression of both CD39 and CD73 on T reg cells
(Fig. S2, available at http://www.jem.org/cgi/content/full/jem
.20071190/DC1). Treatment of purifi ed skin T cells with im-
iquimod did not aff ect T reg cell surface marker expression or
cytokine production, arguing for an indirect mechanism of ef-
fect. In summary, we fi nd that imiquimod decreases T reg cell
FOXP3, CD39, and cytokine production and that this eff ect is
dependent on the presence of other cells resident in skin.
compared with 10% from an untreated SCC ( Fig. 6, A and B ).
In contrast, culture of purifi ed skin T cells with imiquimod
did not induce IL-6 production (not depicted), suggesting
that IL-6 induction occurs by an indirect mechanism.
Imiquimod indirectly decreases expression of FOXP3, IL-10,
TGF- ? , CD39, and CD73
Our suppression assays demonstrate that imiquimod inhibits the
ability of T reg cells to suppress. To study this further, we exam-
ined the eff ect of imiquimod on T cells isolated from human skin.
When imiquimod was added to explant cultures for 1 wk before
T cell isolation, expression of the FOXP3 protein by T reg cells
was signifi cantly decreased ( Fig. 6 C ). FOXP3 protein expression
by T reg cells correlates with their ability to suppress T cell re-
sponses ( 21 ). We then examined FOXP3 expression in T reg cells
isolated from SCCs and found that although levels of FOXP3 in
imiquimod-treated tumors tended to be lower than in untreated
SCCs, this diff erence was not signifi cant ( Fig. 6 C ).
T reg cells can suppress T cell responses by the production of
cytokines such as IL-10 and TFG- ? , and by cell-contact mech-
anisms shown recently to involve, in part, the action of CD39
and CD73 on T reg cells ( 35 ). Imiquimod added to skin explant
cultures decreased the production of both IL-10 and TFG- ? by
FOXP3 + T reg cells ( Fig. 6 E ). Moreover, in addition to being
present at fi vefold lower numbers, T reg cells isolated from SCCs
treated in vivo with imiquimod produced less IL-10 and TFG- ?
( Fig. 6, D and E ). Lastly, we found that CD39 and CD73 were
preferentially expressed by FOXP3 + T reg cells versus FOXP3 ?
eff ector T cells, and that treatment in vitro with imiquimod
Figure 7. SCCs contain immature and iNOS + DCs. Cryosections of untreated and imiquimod-treated SCCs were immunostained for the DC marker
CD11c and markers of DC maturation CD83 (A) and DC-LAMP (B). Numerous immature DCs were present in untreated SCCs, whereas treated SCCs con-
tained signifi cant numbers of mature DC. Untreated SCCs also contained a population of cells that expressed high levels of iNOS (C, indicated by arrow),
but these cells were not detected in imiquimod-treated SCCs. There was also some staining of tumor cells (T) in both untreated and treated tumors.
A concentration-matched isotype control for iNOS staining is shown in the fi rst image. Bars, 100 μ m.
Figure 8. Imiquimod treated tumors contain expanded populations
of TCR-biased cytotoxic T cells. T cells were isolated from untreated
and imiquimod-treated SCCs and analyzed for V ? expression by fl ow
cytometry. Data are presented as the absolute number of CD8 + T cells of
each V ? family in 100 hpf. Imiquimod-treated tumors contained larger
numbers of cytotoxic T cells, and these T cells had clearly biased V ? reper-
toires, consistent with local expansion of tumor-specifi c T cells.
JEM VOL. 205, September 29, 2008
Eff ector memory T cells preferentially migrate through
the peripheral tissue in which they fi rst encountered antigen
( 10, 11 ). This tissue-specifi c migration ensures that tissues are
populated by T cells specifi c for pathogens likely to be en-
countered again in that tissue. Such T cells can migrate pref-
erentially because they express addressins that bind to specifi c
counterreceptors on the endothelium of a particular tissue.
For example, CLA on skin-homing T cells binds to E-selec-
tin expressed on cutaneous postcapillary venules, supporting
the entry of T cells into the skin under both normal and in-
fl amed conditions ( 11, 12, 18 ). SCC-specifi c T cells should
express CLA because they fi rst encounter antigen within the
skin-draining lymph nodes. We have found that SCCs do
not express E-selectin on tumor vessels and are not infi ltrated
by CLA + skin-homing T cells. The tumor therefore excludes
the population of skin-homing memory T cells expected to
contain tumor-specifi c T cells. We observed a lack of vascular
E-selectin and exclusion of CLA + T cells from SCCs arising
in both normal and immunosuppressed individuals, suggest-
ing that aberrant T cell homing occurs in tumors from both
Impaired T cell homing also occurs in other types of hu-
man cancer. Reduced expression of adhesion molecules on
blood vessels has been described in human breast, gastric, and
lung cancer ( 41, 42 ). Melanoma metastases express low levels
of the addressins E-selectin, P-selectin, and ICAM-1, and this
is associated with low numbers of T cells within the meta-
static tumor nodules ( 43 ). Because of its readily accessible lo-
cation, cutaneous SCC is a model cancer in which to study
the defective T cell homing that may underlie poor immune
responses to several human cancers. Additional studies of the
nature of endothelial cells within SCCs will be critical to un-
derstanding how these tumors regulate the expression of vas-
T reg cells can suppress the activation, cytokine produc-
tion, and proliferation of other T cells, and they are crucial to
the development and maintenance of self-tolerance ( 21, 44,
45 ). We have found that up to 50% of the T cells infi ltrating
cutaneous SCCs from both normal and immunosuppressed
individuals are FOXP3 + T reg cells. These cells form a dense
infi ltrate surrounding tumor nests and are well positioned to
impair the responses of eff ector T cells that gain access to the
tumor. T reg cells from SCCs lack CLA and CCR4 and are
therefore distinct from the T reg cells that populate normal
skin ( 20 ). Instead, T reg cells from SCCs coexpress l- selectin
and CCR7. This phenotype is similar to that of central mem-
ory T cells, a type of cell found normally only in the blood or
lymph nodes. We observed no local proliferation of T reg cells
in tumors ( Fig. 4 F ), and thus recruitment from the blood
may be the primary mechanism for enrichment of these
cells in tumors. T reg cells are recruited to ovarian carcinoma
by the interaction of tumor CCL22 with CCR4 on T reg
cells, and recruitment of T reg cells to Hodgkin lymphomas
also involves CCR4 ( 46 ). However, CCR4 is highly expressed
by T cells that provide immunosurveillance of the skin ( 9 ), and
recruitment of this subset would likely be detrimental to tumor
SCCs are infi ltrated by immature and inducible nitric oxide
synthase (iNOS) + DCs
We have found that two important eff ects of imiquimod, the
induction of vascular E-selectin and immunomodulation of
T reg and eff ector T cells, appear to occur via indirect mech-
anisms. Human dermis contains macrophages and dermal
DCs, and SCCs also contain PDCs ( 36, 37 ); these cell types
have been reported to respond directly to imiquimod. We
analyzed SCCs by immunostaining and found that these tu-
mors contain large numbers of CD11c + DCs ( Fig. 7, A and B ,
DC). However, most DCs were immature, as demonstrated
by their lack of DC-LAMP and CD83 expression. In con-
trast, imiquimod-treated SCCs contained mature DCs ex-
pressing both CD83 and DC-LAMP, consistent with reports
that imiquimod induces maturation of DCs ( 31, 38 ). Addi-
tionally, a population of cells expressing high levels of iNOS
was present in untreated but not imiquimod-treated SCCs
( Fig. 7 C ). These cells co-stained weakly for CD11c, but
were negative for CD34 (not depicted), suggesting that they
represent DCs as opposed to myeloid-derived suppressor
cells ( 39, 40 ).
Imiquimod-treated tumors contain expanded clonal
populations of cytotoxic T cells
Our fi ndings suggest that imiquimod treatment allows the
entry of tumor-specifi c T cells into SCCs and may also block
the ability of T reg cells to suppress the activity and prolifera-
tion of these cells once they enter the tumor. Expansion of
tumor-specifi c T cells would be expected to produce a skew-
ing of the T cell repertoire in imiquimod-treated tumors.
To evaluate this, we analyzed the TCR repertoire of CD8 +
T cells isolated from untreated and imiquimod-treated tu-
mors using flow cytometry for TCR V ? families ( Fig. 8 ).
Untreated tumors were infi ltrated by small numbers of di-
verse CD8 T cells, but imiquimod-treated tumors contained
larger numbers of CD8 + T cells with a markedly skewed V ?
repertoire. This skewed repertoire, together with the histo-
logical changes of tumor regression seen in treated tumors,
suggests successful proliferation and infi ltration of tumor-spe-
cifi c CD8 + T cells in treated but not in untreated tumors.
Cutaneous immunosurveillance is an invisible process when
it is functioning properly. Normal human skin contains 1 mil-
lion memory T cells/cm 2 , and there are nearly twice as many
T cells resident in normal skin than are present in the entire
circulation ( 9 ). This suggests that immune surveillance of the
skin is a high priority for the immune system. The suscepti-
bility of individuals on T cell – immunosuppressant medications
to SCCs suggests that T cells may play a role in controlling
these tumors. We were intrigued by the fact that SCCs are
heavily infi ltrated by T cells that nonetheless fail to control
tumor growth. We fi nd that SCCs from both healthy and
immunocompromised individuals exclude skin-homing mem-
ory T cells, and instead recruit a population of T reg cells
normally restricted to the blood and lymph nodes.
IMMUNE EVASION IN SQUAMOUS CELL CARCINOMAS OF THE SKIN | Clark et al.
that distinguished progressively growing tumors from those
that spontaneously regressed ( 61 ). Thus, the dilution of tu-
mor T reg cells by recruitment or local expansion of CD8 + T
cells may tip the balance toward immunological destruction.
We hypothesized that imiquimod may inhibit tumor-as-
sociated T reg cells, given that TLR8 ligation was recently
shown to block the suppressive ability of T reg cells ( 29 ). In-
deed, we found that treatment of T reg cells with imiquimod
in vitro blocked their ability to suppress T cell proliferation
without reducing viability. Imiquimod treatment decreased
the expression of FOXP3 and production of the cytokines
IL-10 and TGF- ? in T reg cells isolated from human skin.
CD39 and CD73 have been recently implicated in contact-
dependent suppression by T reg cells in mice; we found that
both CD39 and CD73 were preferentially expressed by T reg
cells and that expression was down-regulated after treatment
with imiquimod ( 35 ). These eff ects were observed if imiqui-
mod was added to the skin explant cultures before T cell iso-
lation, but direct treatment of purifi ed skin T cells had no
eff ect, suggesting an indirect mechanism mediated by another
cell type present in skin. Candidate responsive cells in skin
include PDCs, macrophages, and keratinocytes, each of which
has been shown to respond directly to imiquimod ( 30 – 32 ).
T reg cells isolated from SCCs treated in vivo with imiquimod
also had decreased IL-10 and TGF- ? production, confi rming
that the eff ects we see in vitro are also present in vivo.
In addition to the eff ect on T reg cells, imiquimod in-
duced the production of IL-6 by eff ector T cells, albeit by an
indirect mechanism. To our knowledge, imiquimod-stimu-
lated IL-6 production by T cells has not been reported previ-
ously. IL-6 production by murine T cells renders them
resistant to suppression by T reg cells, and early reports suggest
a similar eff ect occurs in human psoriatic T cells ( 34, 62, 63 ).
Thus, TLR agonists such as imiquimod both decrease the
suppressive activity of T reg cells and increase the resistance
of eff ector cells to suppression. Again, this eff ect was indirect
and required the presence of other cells in skin. Over 50%
of effector T cells isolated from imiquimod-treated SCCs
produced IL-6, compared with 10% in an untreated tumor,
confi rming the in vivo relevance of this fi nding. Studies to
identify the imiquimod-responsive cells in skin and the sig-
nals that mediated endothelial and T cell responses to imiqui-
mod are ongoing in our laboratory.
The indirect nature of imiquimod ’ s eff ects on endothelial
and T cells highlights the critical role of innate immune cells
such as DCs in tumor responses. We found large numbers
of immature DCs in untreated SCCs, whereas mature DCs
were evident only after imiquimod treatment. Immature DCs
within tumors can prevent proper antigen presentation and
can induce the formation of T reg cells ( 64 ). We also ob-
served a population of iNOS-expressing DCs in untreated,
but not treated, SCCs. Nitric oxide (NO) impairs the ability
of human endothelial cells to express E-selectin in vitro ( 65,
66 ). Exogenously produced NO down-regulates the expression
of MAdCAM-1 on gut vessels, decreases lymphocyte rolling,
and has been proposed as a possible therapy for infl ammatory
survival. In fact, we see no expression of CCR4 on the T reg
cells infi ltrating SCCs, suggesting another addressin must be
responsible for recruiting these and other central memory
T cells into tumors. Preliminary studies in our laboratory
have shown that SCC vessels do not express peripheral node
addressin, a group of l- selectin ligands that recruit memory
and naive l- selectin – expressing T cells into lymph nodes, nor do
they express the CCR7 ligands CCL19 and CCL21 (unpub-
lished data) ( 47 ). This is consistent with the lack of CD45RA +
naive T cells in tumors. Ongoing studies in our laboratory
are focused on identifying the vascular ligands expressed by
SCC tumor vessels that support the recruitment of central
memory T cells.
Imiquimod is a topical immune response modifi er that is
eff ective in the treatment of basal cell carcinomas, SCCs, and
SCC precursor lesions actinic keratoses ( 19 ). Imiquimod in-
duces tumor regression via both immunological and nonim-
munological mechanisms by activating TLR7 and TLR8 and
binding to adenosine receptors ( 48 – 51 ). Imiquimod stimu-
lates blood mononuclear cells to produce a variety of infl am-
matory cytokines, including IFN- ? , TNF- ? , IL-1, IL-12,
IL-6, IL-8, and IL-10 ( 32, 52 – 56 ). Clinical response to topi-
cal imiquimod has been associated with the migration of
PDCs into the skin and subsequent cytokine production ( 57,
58 ). It has been suggested that SCCs may be infi ltrated by
Th2-biased T cells, and that clearance of these tumors after
imiquimod therapy might be a result of a shift from Th2- to
Th1-biased immunity ( 59 ). However, we found very few
Th2-biased T cells in SCCs ( Fig. 1 A ). Thus, lack of tumor
destruction is not likely a result of Th-2 bias among tumor-
infi ltrating T cells.
Our results show that treatment of SCC with imiquimod
is associated with induction of E-selectin on tumor vessels,
infi ltration by CLA + skin-homing CD8 + cytotoxic T cells,
and histological evidence of tumor regression. In vitro treat-
ment of SCCs with imiquimod induced E-selectin on tumor
vessels, suggesting that this medication may act to restore nor-
mal T cell homing, allowing CLA + skin-homing T cells access
to the tumor, where they can initiate tumor destruction.
We have found that SCC blood vessels and dermal microvas-
cular endothelial cells express TLR7 and TLR8, but do not
respond directly to imiquimod. Vascular responses to im-
iquimod therefore require the presence of APCs or other
imiquimod-responsive cell types. To respond directly to
TLR7/8 agonists, a cell must take up and deliver agonists to
the endosomal compartment where TLR7/8 are located, and
endosomes must subsequently undergo acidifi cation and mat-
uration ( 60 ). Thus, human endothelial cells may not respond di-
rectly to TLR7/8 agonists because they are nonphagocytic or
lack endosomal maturation.
In addition to its eff ect on E-selectin expression, imiqui-
mod treatment of SCC results in a fi vefold reduction in the
percentage of tumor-infi ltrating FOXP3 + T reg cells. Treated
tumors contain vastly increased numbers of CD8 + T cells, in
essence diluting out tumor T reg cells. In a mouse model of
sarcoma, it was the relative percentage of FOXP3 + T reg cells
JEM VOL. 205, September 29, 2008
secondary antibody was added (1:100 dilution) for 30 min, followed by
three rinses. Sections were stained with 0.5 μ g/ml Hoechst stain for 2 min,
rinsed briefl y in PBS/1% BSA, and then mounted using Prolong anti-fade
mounting medium (Invitrogen) and examined immediately by immuno-
fl uorescence microscopy. Antibodies were obtained from the following:
BD Biosciences (CLA, CD3, CD8, CD31, E-selectin, and Ki-67), Imgenex
(TLR8 clone 44C143, TLR7, CD83, and DC-LAMP), and R & D Systems
(CD11c). In all studies, Hoechst nuclear stain was used to confi rm the pres-
ence of invasive tumor. Sections were photographed using a microscope
(Eclipse 6600; Nikon) equipped with a 40 × /0.75 objective lens (Plan Fluor;
Nikon). Images were captured with a camera (SPOT RT model 2.3.1;
Diagnostic Instruments) and were acquired with SPOT 4.0.9 software
Cytokine analysis. T cells from SCC tumors were stimulated with either
control medium or 50 ng/ml PMA and 750 ng/ml ionomycin for 6 h;
10 μ g/ml Brefeldin A (Calbiochem) was added after 1 h. Cells were stained
for surface markers, fi xed, permeabilized, stained with anticytokine anti-
bodies, and examined by fl ow cytometry.
TCR diversity analysis of tumor-infi ltrating T cells. T cells were iso-
lated from SCC tumors via 1 wk explant cultures and examined by fl ow cytom-
etry for V ? expression, CD3, CD4, and CD8. V ? staining was performed
using the IOtest Beta Mark TCR V ? Repertoire kit (Beckman Coulter) as
per manufacturer ’ s instructions. For Fig. 6 , the number of CD8 T cells in
100 high power fi elds (hpf) was calculated for each V ? family by the follow-
ing formula: (percentage of V ? expression) × (mean number of T cells in
1 hpf from Fig. 4 E and Fig. 5 B ) × (100).
Immunohistochemical studies. For detection of CD31 and E-selectin
on SCC blood vessels, 5- μ m sections were cut and stored as described in
Immunofl uorescence studies. Sections were fi xed in ? 20 ° C acetone for
5 min, air dried, and incubated with 4 μ g/ml primary antibody or 4 μ g/ml
of mouse IgG as a negative control for 1 h at room temperature. Sections
were washed in PBS three times for 5 min, and then incubated with a 1:200
dilution of secondary antibody at room temperature for 30 min. Sections
were washed three times in PBS, incubated with ABC-peroxidase at room
temperature for 30 min, and then washed three times in PBS. The substrate
reaction was performed for 30 s (CD31) or 2.5 min (E-selectin). Sections
were then counterstained with hematoxylin (Gill ’ s No. 1; Thermo Fisher
Scientifi c). Primary anti-CD31 was obtained from Dako, anti – E-selectin
was purchased from R & D Systems, and secondary antibody was biotinyl-
ated horse anti – mouse IgG (Vector Laboratories). The Vectastain Elite stan-
dard ABC-peroxidase kit and NovaRED substrate were obtained from
Real-time quantitative PCR for endothelial cell expression of TLR7
and TLR8. Human adult dermal microvascular endothelial cells were pur-
chased from Cambrex Corporation and cultured in endothelial basal me-
dia (Clonetics Corp.) supplemented with 25 μ g/ml dibutyryl cyclic AMP
(Sigma-Aldrich), 1 μ g/ml hydrocortisone acetate (Sigma-Aldrich), 20% heat-
inactivated FCS, 100 U/ml penicillin, and 100 μ g/ml streptomycin. RNA
was isolated from second passage cultures. For comparison, identical studies
were performed on T cell – depleted PBMCs, isolated from peripheral blood
by Ficoll density centrifugation followed by T cell depletion using the pan –
T cells isolation kit (Miltenyi Biotech) and AutoMACS instrument (Miltenyi
Biotech). For RNA extraction and cDNA synthesis, cells were placed in
RNALater RNA-stabilizing reagent (QIAGEN) and frozen at ? 80 ° C for
later use. Frozen cells were then lysed and homogenized, and total RNA
was extracted using QIAGEN RNeasy mini kits as specifi ed by the manu-
facturer. Avian RT fi rst-strand kits (Sigma-Aldrich) were used to synthesize
cDNA from total RNA. The concentration of total RNA was determined
at the optical density at 260 nm (OD 260 ), and discrepancies in the amount
of total RNA extracted were corrected by loading the same amount and
concentration of RNA for cDNA synthesis. The purity of cDNA was
bowel disease ( 67 ). We are currently investigating the possi-
bility that NO production suppresses E-selectin expression
on tumor vessels.
We have evidence for three novel mechanisms of action
for imiquimod that may initiate and sustain the immunological
destruction of SCC. First, imiquimod up-regulates E-selectin
on tumor vessels. This E-selectin expression is associated with
an infl ux of CLA + cytotoxic T cells into the tumor, there by
delivering potentially tumor-reactive T cells to the cancer
while at the same time diluting out tumor T reg cells. Second,
imiquimod inhibits the suppressive activity of T reg cells,
decreasing the levels of FOXP3 protein and surface molecules
associated with contact inhibition, as well as reducing the
production of immunosuppressive cytokines. Lastly, imiquimod
induces the production of IL-6 by eff ector T cells that may
render them resistant to suppression by T reg cells.
Impaired T cell homing and recruitment of regulatory T
cells are features of many human cancers. We fi nd that the
TLR7 agonist imiquimod neutralizes both of these defenses,
supporting the use of this medication in SCCs and in other tu-
mors that use similar strategies to evade the immune response.
MATERIALS AND METHODS
SCC samples. Tumor samples consisted of curetted tumor removed before
taking the fi rst Mohs section during Mohs micrographic excision of biopsy-
proven SCCs. Acquisition of tumor samples and all studies were approved
by the Institutional Review Board of the Dana Farber Cancer Institute and
were performed in accordance with the Declaration of Helsinki. Tumors
were divided into bread loaf sections. Adjacent sections were used for (a)
immunohistochemical or immunofl uorescence studies to confi rm the pres-
ence of invasive tumor cells and (b) T cell isolation, as described in the fol-
lowing section. The SCC13 SCC cell line was provided by J. Rheinwald
(Brigham and Women’s Hospital, Boston, MA).
Isolation of T cells from SCC tumors and normal skin. The clinically
evident portions of biopsy-proven invasive SCC tumors were obtained. The
tumors were divided into bread loaf sections. One section was histologically
examined to confi rm the presence or absence of invasive SCC tumor. Sec-
tions without evidence of tumor were designated peritumoral. Tumor-infi l-
trating T cells were isolated from sections adjacent to those studied by
histology. T cells were isolated from SCC tumors in the absence of exoge-
nous cytokines, as previously described ( 28 ). For normal skin studies, T cells
were isolated from skin discarded after plastic surgery procedures from 3 wk
explant cultures ( 28 ). Skin was provided by T. Cochran (Boston Center for
Plastic Surgery, Boston, MA) and E. Eriksson (Brigham and Women’s Hos-
pital, Boston, MA).
Flow cytometry studies. Flow cytometry analysis of T cells was performed
using directly conjugated monoclonal antibodies obtained from: BD Bio-
sciences (CD3, CD4, CD8, CD25, CD69, CD45RO, and IL-10), BD
PharMingen (CLA, CCR4 [1G], CD73, and CCR6), Abcam (CD39),
Beckman Coulter (L-selectin), R & D Systems (TGF- ? , CCR7, and CD127),
and eBioscience (FOXP3, clone PCH101). Analysis of fl ow cytometry sam-
ples was performed on Becton Dickinson FACScan or FACSCanto instru-
ments, and data were analyzed using FACSDiva software (V5.1).
Immunofl uorescence studies. SCC tumors were embedded in OCT,
frozen, and stored at ? 80 ° C until use. 5- μ m cryosections were cut, air dried,
fi xed for 5 min in acetone, rehydrated in PBS, and blocked with 20 μ g/ml
of human IgG (Jackson ImmunoResearch Laboratories) for 15 min at
room temperature. Sections were incubated with primary antibody for
30 min, and then rinsed three times in PBS/1% BSA for 5 min. If necessary,
IMMUNE EVASION IN SQUAMOUS CELL CARCINOMAS OF THE SKIN | Clark et al.
FOXP3, and analyzed by fl ow cytometry. Percentage of survival was calcu-
lated as the number of T cells (imiquimod-treated)/(control medium-treated) ×
100 for both CD3 + FOXP3 ? (non-T reg cell) and CD3 + FOXP3 + (T reg
cell) T cells. To assess the eff ect on proliferation, T cells from normal human
skin were labeled with 0.5 ? M CFSE (Invitrogen) per manufacturer ’ s direc-
tions and cultured for 1 wk on dermal fi broblast monolayers and IL-15
(20 ng/ml; R & D Systems) either with or without 3 ? M imiquimod. T cells
were stained for CD3 and FOXP3 and analyzed by fl ow cytometry. For
regulatory cell functional assays, 3 ? M imiquimod was added to explant cul-
tures during the last 2 – 5 d before T cell collection, and cells were then har-
vested and assayed for regulatory activity.
Functional assays of regulatory T cell activity. T cells were isolated
from untreated or imiquimod-treated explant cultures and assayed for regu-
latory T cell functional, as previously described ( 20 ). If included, imiquimod
was present at 3 ? M concentration.
Online supplemental material. In Fig. S1. FOXP3 + T reg cells were
isolated from SCC via explant cultures and stained for surface CD127 and
nuclear FOXP3 as described in Materials and methods. In Fig. S2, for ex-
pression of CD39 and CD73, imiquimod or control medium was added to
explant cultures of normal human skin for the last 7 d before T cell iso-
lation. T cells were then collected from explant cultures, stained for surface
CD39 and CD73 and for nuclear FOXP3, and examined by fl ow cytometry.
The online version of this article is available at http://www.jem.org/cgi/
Dr. Thomas Cochran of the Boston Center for Plastic Surgery and Dr. Elof Eriksson
of Brigham and Women ’ s Hospital generously provided normal human skin
samples. Dr. James Rheinwald of Brigham and Women ’ s Hospital kindly provided
the SCC13 cell line The authors thank Drs. Robert Fuhlbrigge, Richard Miller, Carsten
Weishaupt, and Adam Calarese and for helpful comments on the manuscript.
This research was supported by National Institutes of Health (NIH) grant
1K08AI060890-01A1, a Translational Research Award from the Leukemia and
Lymphoma Society (to R.A. Clark), a Pilot & Feasibility grant from the Harvard Skin
Disease Research Center (to R.A. Clark from NIH grant P30 AR-42689-11, to
T.S. Kupper), a Developmental project from the SPORE in Skin Cancer (to R.A. Clark,
from NIH grant P50 CA-93683-04, to T.S. Kupper), and a Clinical Investigator Award
from the Damon Runyon Cancer Research Foundation (to R.A. Clark).
The authors have no confl icting fi nancial interests.
Submitted: 12 June 2007
Accepted: 9 July 2008
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determined by the OD 260 /OD 280 ratio. For primer design, nucleotide se-
quences were determined from PubMed (National Center for Biomedical
Information), and the primers were custom designed using primer3 soft-
ware. Primer pairs were as follows: TLR7, 5 ? -TGGAAATTGCCCTC-
GTTGTT-3 ? and 5 ? -GTCAGCGCATCAAAAGCATT-3 ? ; TLR8,
5 ? -CTTCGATACCTAAACCTCTCTAGCAC-3 ? and 5 ? -AAGATC-
CAGCACCTTCAGATGA-3 ? . Real-time RT-PCR analysis was per-
formed using the iCycler (Bio-Rad Laboratories) with SYBR Green kits
(Bio-Rad Laboratories) and mRNA quantifi cation by the standard curve
method, as previously described ( 68, 69 ). In brief, for each transcript ana-
lyzed, a standard curve with predetermined concentrations and serial diluted
respective PCR amplifi cation products from 0.1 to 0.00001 ng was con-
structed. This approach allows the standards to be amplifi ed in the same way
as the template cDNA in the unknown samples because the product sequence
and size are identical. Levels of Cyclophilin A mRNA served as an internal
control to normalize samples for variations in sample volume loading, pres-
ence of inhibitors, and nucleic acid recovery during extraction and cDNA
synthesis procedures. The normalized initial concentration of each transcript
in every sample was converted to the initial copy number by using the fol-
lowing formula: Amount (copies/ ? l) = 6 × 1,023 (copies/mole) × concen-
tration (grams/microliter)/molecular mass (grams/mole), where the mean
molecular weight of double-stranded DNA equals the number of base pairs ×
660 Daltons/base pair. All analyses were performed in triplicate.
In vitro treatment of endothelial cells with imiquimod. Human
endothelial cells (Lonza Group) from two diff erent donors were expanded
with EGM-2 BulletKit growth media (Lonza Group). Cells were cultured
on RepCell temperature-responsive plates (CellSeed). 3 μ M imiquimod or
10 ng/ml TNF- ? was added for indicated lengths of time, either alone or in
combination with APC. APCs were isolated from human blood by fi coll
density centrifugation and depletion of T cells using the Pan-T isolation kit,
followed by MACS separation (Miltenyi Biotech). 4.5 million APCs were
added to each well of a 6-well plate (CellSeed), and the combined culture
was maintained in EGM-2 medium. APCs cultured in EGM-2 endothelial
media became activated, produced infl ammatory cytokines, and induced
endothelial cell E-selectin in the presence or absence of imiquimod. On the
day of FACS analysis, plates were cooled to room temperature to promote
spontaneous release of endothelial cells. Released cells were stained with di-
rectly conjugated antibodies to CD31 and E-selectin (BD Biosciences) and
analyzed by fl ow cytometry. For experiments with SCC13, SCC13 cells
(provided by J. Rheinwald) were cultured with endotheilial cells for 3 d, and
TNF- ? (if present) was added for the last 12 h.
In vitro treatment of SCC tumor with TNF- ? or imiquimod. Freshly
excised SCC tumor was divided into 2-mm-thick slices. Slices were incu-
bated for 24 h in control medium (Iscove ’ s modifi ed medium [Mediatech]
with 20% heat-inactivated FBS [Sigma-Aldrich], penicillin and streptomy-
cin, and 3.5 μ l/liter ? -mercaptoethanol) alone or with the addition of 1 ng/ml
TNF- ? or 3 ? M concentration of imiquimod. 10,000 × (30 mM) im-
iquimod stocks were made by solubilizing imiquimod cream in DMSO.
Stocks were then diluted 1:10 in culture medium, and 1 μ l of this 1,000 ×
stock was added to each milliliter of culture medium. For control medium
samples, an equivalent amount of DMSO was added to the control culture
medium (a 1:10,000 dilution). After 24 h, the SCC slices were embedded in
OCT, frozen in liquid nitrogen, and stored at ? 80 ° C until sectioning. Sec-
tions were then cut, stained, and photographed as described in Immuno-
fl uorescence studies.
Imiquimod treatment of skin T cells. To study the eff ect of imiquimod
on skin T cell viability, T cells were isolated from normal skin as described
in Isolation of T cells, and cultured for 1 wk on monolayers of feeder human
dermal fi broblasts in either control medium (Iscove ’ s modifi ed medium with
20% heat inactivated FBS, penicillin and streptomycin, and 3.5 μ l/liter
? -mercaptoethanol, with 1:10,000 DMSO) or medium containing 3 ? M
imiquimod. T cells were then harvested, counted, stained for CD3 and
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