The Journal of Clinical Investigation http://www.jci.org Volume 124 Number 3 March 2014
Memory regulatory T cells
reside in human skin
Robert Sanchez Rodriguez,1 Mariela L. Pauli,1 Isaac M. Neuhaus,1 Siegrid S. Yu,1
Sarah T. Arron,1 Hobart W. Harris,2 Sara Hsin-Yi Yang,3 Bryan A. Anthony,4 Francis M. Sverdrup,5
Elisabeth Krow-Lucal,6 Tippi C. MacKenzie,7 David S. Johnson,8 Everett H. Meyer,8 Andrea Löhr,8
Andro Hsu,9 John Koo,1 Wilson Liao,1 Rishu Gupta,1 Maya G. Debbaneh,1 Daniel Butler,1
Monica Huynh,1 Ethan C. Levin,1 Argentina Leon,1 William Y. Hoffman,10 Mary H. McGrath,10
Michael D. Alvarado,2 Connor H. Ludwig,1 Hong-An Truong,1 Megan M. Maurano,1 Iris K. Gratz,1
Abul K. Abbas,3 and Michael D. Rosenblum1
1Department of Dermatology, 2Department of Surgery, and 3Department of Pathology, UCSF, San Francisco, California, USA.
4Department of Medicine, Washington University School of Medicine, Saint Louis, Missouri, USA. 5Center for World Health and Medicine,
Saint Louis University, Saint Louis, Missouri, USA. 6Division of Experimental Medicine, Department of Medicine, and
7Division of Pediatric Surgery, UCSF, San Francisco, California, USA. 8GigaGen Inc., San Francisco, California, USA.
9Syapse Inc., Palo Alto, California, USA. 10Division of Plastic Surgery, UCSF, San Francisco, California, USA.
Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, are a
dynamic and heterogeneous population of cells that control immune responses and prevent autoimmunity.
We recently identified a subset of Tregs in murine skin with properties typical of memory cells and defined this
population as memory Tregs (mTregs). Due to the importance of these cells in regulating tissue inflammation
in mice, we analyzed this cell population in humans and found that almost all Tregs in normal skin had an
activated memory phenotype. Compared with mTregs in peripheral blood, cutaneous mTregs had unique cell
surface marker expression and cytokine production. In normal human skin, mTregs preferentially localized
to hair follicles and were more abundant in skin with high hair density. Sequence comparison of TCRs from
conventional memory T helper cells and mTregs isolated from skin revealed little homology between the two
cell populations, suggesting that they recognize different antigens. Under steady-state conditions, mTregs
were nonmigratory and relatively unresponsive; however, in inflamed skin from psoriasis patients, mTregs
expanded, were highly proliferative, and produced low levels of IL-17. Taken together, these results identify
a subset of Tregs that stably resides in human skin and suggest that these cells are qualitatively defective in
inflammatory skin disease.
Foxp3-expressing regulatory T cells (Tregs) play an indispensable
role in establishing and maintaining immune homeostasis. It was
originally thought that Tregs are a relatively homogenous popu-
lation generated exclusively in the thymus. However, subsequent
studies revealed that an additional subset was derived from cells
induced to become Tregs outside of the thymus (1), adding to the
complexity of the ontogeny of this cell population. Emerging data
suggest the existence of even more complexity, as multiple Treg
subsets are being defined with specialized functions and unique
cell fates. Perhaps the most distinct subsets of Tregs are those that
reside in peripheral tissues. In the gastrointestinal tract, a popu-
lation of Tregs is induced by microbial flora and is specialized to
secrete IL-10 (2). In visceral adipose tissue (VAT), a population of
Tregs preferentially expresses the peroxisome proliferator–activated
receptor γ (PPARγ), which confers highly specialized functions,
including the expression of genes involved in lipid metabolism (3).
We recently characterized a distinct population of Tregs in
murine skin (4, 5). Using an inducible model of cutaneous self-anti-
gen expression, we found that upon induction of antigen, thymus-
derived Tregs are activated and accumulate in skin. A subset of
these cells is maintained in the tissue for relatively long periods in
the absence of antigen and has an enhanced capacity to suppress
cutaneous autoimmunity when antigen is reexpressed. These cells
fit stringent criteria for effector memory cells and were named
memory Tregs or mTregs. Consistent with an effector memory
phenotype, mTregs require IL-7 for their maintenance in skin (4).
Although studies characterizing specialized Treg subsets in
murine tissues are beginning to emerge, very little is known about
Tregs in human tissues. Given the limited accessibility of fresh
human tissue, functional characterization of Tregs in humans has
largely been limited to peripheral blood. Comprehensive analysis
of Tregs in blood reveals a heterogeneous population composed
of resting Tregs with a “naive” phenotype, “activated” Tregs with
characteristics of memory cells, and Foxp3-expressing cells that
secrete effector cytokines and lack suppressive capacity (6).
It is important to elucidate the fundamental biology of Tregs in
human peripheral tissues in order to define abnormalities in these
cells in inflammatory diseases and to exploit Tregs for treating
such disorders. Tregs are thought to mediate the majority of their
functions in the tissues in which they reside (7), and optimal ther-
apeutic approaches directed at either augmenting or inhibiting
Tregs will most likely require strategies that target specific subsets,
in an attempt to efficiently treat disease and limit systemic side
effects. In this report, we phenotypically and functionally char-
acterize Tregs in human skin. Similar to the mTreg population
Authorship note: Robert Sanchez Rodriguez and Mariela L. Pauli contributed
equally to this work. Michael D. Rosenblum and Abul K. Abbas contributed equally
to this work.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2014;124(3):1027–1036. doi:10.1172/JCI72932.
1028 The Journal of Clinical Investigation http://www.jci.org Volume 124 Number 3 March 2014
identified in mice, almost all Tregs in human skin have an effector
memory phenotype. We show that mTregs in human skin have
distinct cell surface marker expression, cytokine production, in
situ localization, TCR expression, and functional capacity in both
normal and diseased skin. These results reveal a unique subset of
Tregs resident in human skin and suggest that these cells may be
qualitatively defective in inflammatory skin disease.
Tregs in normal human skin. A comprehensive analysis of immune
cell populations in human skin has been hampered by technical
challenges in obtaining adequate numbers of cells from relatively
harsh digestion protocols. Consequently, the majority of studies
published to date rely on either immunohistochemical analyses
or assays in which skin cells are cultured for several days, some-
times in the presence of growth factors (8, 9). In order to objec-
tively study Tregs in human skin, we optimized a chemical diges-
tion method for isolating leukocytes from this tissue. Surgically
discarded samples of clinically normal-appearing skin were gently
digested overnight (in the absence of exogenous growth factors),
and single-cell suspensions were immediately analyzed by 12-color
flow cytometry. In order to control for potential artifacts in the
isolation process, human PBMCs were processed in a manner
identical to that used for skin. Adult human skin contains read-
ily detectable Foxp3-expressing CD4+ T cells (Figure 1A). Whereas
approximately 5% of CD4+ T cells consistently express Foxp3 in
adult peripheral blood, approximately 20% of CD4+ T cells in adult
skin express Foxp3, with considerably more variability when com-
pared with that found in blood. Interestingly, greater than 95% of
CD4+Foxp3+ cells in the skin express CD45RO, whereas 75%–80%
of skin CD4+Foxp3– cells express this marker (Figure 1B). These
results indicate that almost all of the CD4+Foxp3+ cells in adult
skin have previously seen antigen (outside of the thymus), consis-
tent with a memory T cell phenotype. Interestingly, the percent-
age of CD4+Foxp3+ cells in human fetal skin is markedly reduced
when compared with that in adult skin, with a significantly lower
percentage of these cells expressing CD45RO (Figure 1C). As
expected, CD45RO expression is also reduced on CD4+Foxp3– cells
in fetal skin when compared with its expression in adult skin (Sup-
plemental Figure 1; supplemental material available online with
this article; doi:10.1172/JCI72932DS1). Taken together, these data
support the notion that Tregs encounter skin-associated antigens
over time and gradually accumulate in this tissue.
Tregs in human skin are activated memory cells. mTregs in murine
skin express markers of prior activation as well as markers typ-
ical of effector memory T cells (4, 5). The fact that greater than
95% of Tregs in normal human skin express CD45RO suggests
that this population is a bona fide memory T cell population. To
explore this further, we examined these cells for both activation
and effector memory T cell markers (Figure 2A). Compared with
CD45RO+CD4+Foxp3+ cells in adult peripheral blood (hereaf-
ter referred to as mTregs), the equivalent cell population in skin
expressed significantly higher levels of the Treg activation markers
CTLA-4 (extracellular), CD25, and ICOS (Figure 2A). Cutaneous
mTregs also expressed higher levels of Foxp3 when compared with
mTregs in peripheral blood (Supplemental Figure 2). With respect
to memory markers, mTregs in the skin expressed high levels of
CD27 and BCL-2 (Figure 2A). Interestingly, mTregs in human skin
did not express high levels of IL-7Rα (CD127). This is in contrast to
mTregs in murine skin, which have increased expression of CD127
(relative to Tregs in skin-draining lymph nodes) and require IL-7
for their maintenance in skin in the absence of antigen (4).
Approximately 24 hours after initiation of TCR stimulation in
vitro, human CD4+ T cells begin to show increased Ki67 expression
(10). In the presence of persistent TCR stimulation, the percentage
of Ki67-expressing cells peaks at 72 hours and steadily declines to
approximate baseline levels 5 days later. In addition, the majority
of naive human CD45RO– Tregs convert to CD45RO+ cells within
4 days of TCR stimulation (11). If similar kinetics apply in vivo,
expression of Ki67 and CD45RO can be used to approximate the
window of time that a T cell has encountered antigen in humans.
It follows that the majority of cells that express both Ki67 and
CD45RO are likely to have seen antigen 5 days or less prior to har-
vesting the tissue, whereas the majority of cells that express only
CD45RO (i.e., Ki67– cells) are likely to have seen antigen greater than
5 days before harvest. Consistent with previous reports (11, 12),
approximately 10%–20% of CD45RO+ Tregs in peripheral blood
are actively cycling, as evidenced by intracellular Ki67 expression
(Figure 2A). In contrast, a significantly lower percentage of mTregs
in the skin are cycling. This suggests that in the steady state, a larger
percentage of mTregs in peripheral blood have recently seen anti-
gen compared with mTregs in skin, despite the fact that cutaneous
mTregs express higher levels of Treg activation markers.
Lack of cytokine expression and demethylation of genomic DNA
in intron 1 of the FOXP3 gene (TSDR locus) strongly correlate with
Foxp3 expression in human adult and fetal skin. (A) Expression of
Foxp3 on CD3+ T cells in PBMCs (Blood) and skin isolated from healthy
adults. Scatter plot is gated on CD3+CD4+ cells. (B and C) Expression
of Foxp3 and CD45RO on CD3+CD4+ T cells in adult PBMCs and in
adult skin as well as human fetal skin. Results are combined data from
five or more independent experiments. P values were determined using
a 2-tailed unpaired Student’s t test.
The Journal of Clinical Investigation http://www.jci.org Volume 124 Number 3 March 2014
Tregs in human skin have an activated effector memory phenotype. (A and B) Expression of activation markers, memory markers, and cytokines
from viable CD3+CD4+CD45RO+ T cells in PBMCs and skin isolated from healthy adults. Skin used for cytokine analysis in B was harvested from
face or scalp. Scatter plots in B represent the percentage of cytokine-producing cells within Foxp3+ and Foxp3– gates. All gates are based on
isotype control staining or unstimulated controls (for cytokine production in B). EC, extracellular. (C) Percentage of demethylation of genomic DNA
in intron 1 of the FOXP3 gene (TSDR locus) of mTregs and mTconvs in PBMCs and skin isolated from healthy adults. Each pie chart represents
a sorted population purified from a different donor, and numeric values within pie charts represent the percentage of demethylation at the TSDR
locus. Numeric values in parentheses below each chart represent the percentage of Foxp3-expressing cells within each purified cell population.
Cell sorting strategy is shown in Supplemental Figure 4. Results in A and B are representative data from more than fifteen independent experi-
ments. Results in C are from three or more replicate experiments. P values were determined using a 2-tailed unpaired Student’s t test.
1030 The Journal of Clinical Investigation http://www.jci.org Volume 124 Number 3 March 2014
the suppressive function of mTregs isolated from human blood (6).
When compared with mTreg cells in peripheral blood, mTregs iso-
lated from skin expressed significantly less IL-2 and IL-17, with no
difference in IFN-γ or IL-10 expression (Figure 2B). Consistent with a
phenotype of stably differentiated Tregs, cutaneous mTregs are fully
demethylated at the TSDR region of the FOXP3 gene (Figure 2C).
Taken together, these results suggest that Tregs in normal adult
skin are a highly activated, stable effector memory Treg population.
mTregs in human skin preferentially localize to hair follicles. In order
to define the anatomic localization of mTregs, we performed
Treg-specific immunofluorescence microscopy on normal human
skin. Consistent with findings in mice (4, 13), we observed that
Tregs in human skin preferentially local-
ized to hair follicles (HFs) (Figure 3, A–C).
We observed very few CD4+Foxp3+ cells
in the interfollicular dermis, as most cells
were localized in close proximity to the
follicular epithelium. In contrast, CD4+
Foxp3– conventional T cells (Tconvs) dis-
played a more diverse distribution with
little predilection for HFs. Consistent
with these findings, flow cytometric quan-
tification showed that skin with high hair
density (i.e., scalp and face) had signifi-
cantly higher percentages of Tregs when
compared with skin with low hair den-
sity (Figure 3D). We found no significant
difference in Treg percentages when data
were stratified by gender and age (data not
shown); however, only adult skin (>19 years
of age) was available for analysis.
mTregs in human skin express unique TCRs.
Discovering the nature of the antigens
that T cells recognize in tissues is of fun-
damental importance in elucidating the
biology of these cells. This is especially true for human tissue, in
which antigen-specific T cell–directed therapies are being imple-
mented to treat autoimmune diseases and cancer (14). An essen-
tial question that remains to be answered is whether Tconvs and
Tregs found in the same tissue recognize the same or different
antigens. To begin to answer this question, we performed deep
sequencing of the TCR β chain (TCRβ) from mTregs and mem-
ory CD4+ Tconvs (mTconvs) purified from normal human skin.
TCR sequence diversity in the CDR3 region is an indirect measure
of TCR specificity (15, 16). Thus, we compared TCRβ sequences
between mTconvs and mTregs, with the rationale that sequences
shared between these two cell populations suggest the possibility
that they recognize the same antigens, whereas nonoverlapping
sequences suggest that they recognize different antigens. We chose
sequencing of TCRβ, because this chain of the TCR contains the
most sequence diversity (15). Surprisingly, we found that very few
TCRβ sequences were shared between mTconvs and mTregs (Fig-
ure 4 and Table 1), suggesting that these two T cell populations
predominantly recognize different antigens. Although sequencing
depth was limited by the number of cells we could extract from
skin for these analyses, we used a series of Monte Carlo statistical
simulations (see Methods) in four separate experimental replicates
to determine that the mTreg population is qualitatively different
from the mTconv population. Taken together, these results indi-
mTregs localize to HFs in human skin. (A–C) Confocal microscopy of
normal human skin. Sections were stained for CD3 (green), Foxp3
(red), CD1a (blue), and DAPI (gray). Scale bars: 100 μm. HFs auto-
fluoresce in blue. Arrows denote CD3+Foxp3+ cells (yellow). (D) Quan-
tification of Foxp3+ cells (within the CD3+ gate) in human adult skin by
flow cytometry. Low hair density represents skin harvested from ana-
tomical sites with relatively lower HF density (trunk and upper proximal
extremities), whereas high hair density represents skin harvested from
anatomical locations with relatively higher hair density (scalp and face).
Results are representative of more than five replicate experiments.
P values were determined using a 2-tailed unpaired Student’s t test.
TCRβ sequencing of mTregs and memory Tconvs in human skin. (A) Gating strategy for sorting
of mTconvs and mTregs from human skin harvested from healthy adults. Cells are pregated on
viable CD3+CD4+ cells. (B) Hive plots showing relative abundance of unique TCRβ sequence
clones and relative overlap of unique sequences between the Foxp3+ and Foxp3– pools. Each
line represents one unique clone. The width of the lines denotes the relative abundance of each
clone in the Foxp3+ and Foxp3– fractions or the pool of both populations. The most abundant
clones (i.e., the widest lines) are positioned distally from the center of the hive, whereas the least
abundant clones (thinnest lines) are located centrally. Lines that connect the Foxp3+ and Foxp3–
fractions represent unique sequences shared between these cell populations. For example, in
skin sample 2, the most abundant sequence in the Foxp3– fraction is present in relatively low
frequency in the Foxp3+ fraction and represents the most abundant sequence in the entire pool.
In this sample, this sequence is the only one shared between the Foxp3– and Foxp3+ cell pop-
ulations. P values were determined using Monte Carlo simulation for the probability that Foxp3+
cells were not a random sampling of the pooled population (see Methods).
The Journal of Clinical Investigation http://www.jci.org Volume 124 Number 3 March 2014
cate that mTregs in the skin possess a distinct TCR repertoire,
suggesting that they recognize different antigens than those rec-
ognized by cutaneous mTconvs.
mTregs in human skin are nonmigratory. Based on chemokine recep-
tor expression patterns, it has been postulated that the majority of
T cells in human skin are resident in this tissue and are thus non-
migratory (9). To a large extent, this conclusion is based on the lack
of CCR7 expression seen on T cells isolated from skin. Given that
CCR7 mediates the migration of memory T cells to secondary lym-
phoid organs (17), we analyzed the expression of this chemokine
receptor on cutaneous mTregs and mTconvs. Consistent with
previous reports (9), the majority of T cells isolated from human
skin lacked CCR7 expression (Figure 5A). However, we observed
a marked dichotomy within the small fraction of T cells that did
express CCR7. This chemokine receptor was almost exclusively
expressed on mTconvs (Figure 5A). These results suggested that
mTregs are a more skin-resident (i.e., nonmigratory) cell popula-
tion when compared with mTconvs. To test this, we used an in vivo
model of human T cell migration. When human skin is grafted
onto immunodeficient mice, a functional human microcircula-
tion develops, allowing for the migration of human and murine
cells to and from the grafted tissue (18). To determine the relative
migratory potential of cutaneous mTregs and mTconvs in vivo,
we grafted normal human skin onto immunodeficient NSG mice
and assayed for these cell populations in the blood and spleen 3
and 7 weeks after grafting. We performed the analyses 3 or more
weeks after grafting to allow inflammation and wound healing
to resolve and to allow human dermal microvessels to anasto-
mose with murine blood vessels (18). Consistent with the relative
differences in CCR7 expression, we readily detected mTconvs in
blood and spleen at all time points analyzed, whereas mTregs were
undetectable in the blood and spleen and were found only in skin
grafts (Figure 5B). Interestingly, we found that mTconvs prolifer-
ated in grafted skin, as evidenced by intracellular Ki67 expression;
in contrast, mTregs maintained the basal prolifera-
tion levels (~5% Ki67-expressing cells) we observed in
nongrafted normal skin (Figure 2A). Taken together,
these results suggest that mTregs in human skin are
nonmigratory and relatively anergic when compared
with cutaneous mTconvs.
mTregs proliferate and produce IL-17 in psoriatic skin.
The studies outlined above phenotypically and func-
tionally characterize mTregs in normal human skin.
Because it is important to know how these cells func-
tion in both homeostatic and inflammatory contexts,
we set out to functionally define these cells in the
skin of patients with psoriasis. Due to inherent dif-
ficulties in obtaining adequate numbers of cells for
functional analyses (from relatively small skin biopsy specimens),
previous studies examining Tregs in psoriasis patients have relied
on analyzing peripheral blood and/or performing semiquantita-
tive microscopic analyses on tissue sections (19, 20). It has become
increasingly clear from both mouse models and studies of human
disease that the secondary lymphoid organs and peripheral blood
may not accurately represent the pathologic processes in tissues.
We optimized our skin digestion protocol to functionally char-
acterize mTregs from 4-mm skin punch biopsies obtained from
patients with clinically active psoriasis. In the majority of these
studies, we compared lesional skin with nonlesional skin (defined
as >10 cm away from a psoriatic plaque harvested from the same
anatomical site) obtained from the same patient in an attempt to
TCRβ sequencing of mTregs and mTconvs in normal human skin
P < 0.0001
P < 0.0001
P < 0.0001
P < 0.0001
Quantification of unique TCRβ sequence clones after deep sequencing of genomic DNA
from mTregs and mTconvs purified as described. Data shown are results from skin sam-
ples obtained from four different healthy donors.
mTregs in human skin are nonmigratory. (A) CCR7 expression on
mTregs and mTconvs in human skin harvested from healthy adults.
Cells were pregated on viable CD3+CD4+CD45RO+ cells. (B) Human
skin was grafted onto immunodeficient NSG mice, and at specific times
thereafter, grafted skin, blood, and spleen (not shown) were harvested
and human T cells analyzed by flow cytometry. Cells were pregated on
viable CD3+CD4+hCD45+ cells. (C) Absolute numbers of CD3+CD4+
hCD45+Foxp3+ and CD3+CD4+hCD45+Foxp3– cells in grafted skin and
blood 3 and 7 weeks after grafting. Results are pooled data from two
of five representative experiments with two or more mice per group.
P values were determined using a 2-tailed unpaired Student’s t test.
1032 The Journal of Clinical Investigation http://www.jci.org Volume 124 Number 3 March 2014
Functional analysis of cutaneous mTregs in patients with psoriasis. (A) Percentage and absolute number of T cells and Tregs in nonlesional
psoriatic (NL-PSO) and lesional psoriatic (L-PSO) skin from patients (middle and right panels gated on CD4+ and CD3+ cells, respectively).
(B) Percentage of mTregs and mTconvs in L-PSO skin or normal healthy adult skin (Control), pregated on viable CD3+ cells. (C) Intracellular IL-17
production from mTregs and mTconvs in NL-PSO and L-PSO skin, gated on viable CD3+CD4+CD45RO+ cells. Middle panel is a representative
flow cytometric plot of IL-17 production, and the scatter plot shows percentages of IL-17–producing cells within the Foxp3 gate. (D) Expression
of Ki67 in mTregs and mTconvs in L-PSO skin or site-matched control skin, gated on viable CD3+CD4+CD45RO+ cells. (E) Expression of Ki67
in mTregs and mTconvs in L-PSO or control skin, gated on viable CD3+CD4+CD45RO+ cells. Lines represent paired data from a single patient.
Mean deltas between Foxp3– and Foxp3+ cells when comparing PSO versus control skin are 8.340 ± 5.05 versus 0.735 ± 0.50, respectively
(P = 0.132). (F) MFI of Foxp3 and CD25 expression on Ki67+ and Ki67– mTregs in L-PSO skin, gated on viable CD3+CD4+CD45RO+Foxp3+ cells.
(G) Foxp3 and CD127 expression on mTregs and mTconvs in L-PSO or site-matched control skin, gated on viable CD3+CD4+CD45RO+cells.
Results are combined data from five or more replicate experiments.
The Journal of Clinical Investigation http://www.jci.org Volume 124 Number 3 March 2014
internally control for variability between patients and between
anatomic locations. However, in situations where this was not
possible, we compared cells obtained from psoriatic skin with site-
matched skin from normal healthy controls. As expected, when
compared with nonlesional skin, psoriatic skin had an increased
percentage of T cells (Figure 6A). We found that increased per-
centages and absolute numbers of Foxp3-expressing T cells were
present in psoriatic skin (Figure 6A). The percentage of Foxp3+
cells that coexpressed CD45RO was similar between lesional and
nonlesional skin (Figure 6B), suggesting that the increased Foxp3-
expressing cells we observed in lesional skin were not secondary
to an influx of naive cells. When compared with nonlesional
skin, CD45RO+CD4+Foxp3+ cells in lesional skin produced more
IL-17 (Figure 6C). We have observed that the frequency of IL-17–
producing T cells in normal skin varies with anatomic location.
Normal facial/scalp skin contains a higher percentage of IL-17–
producing T cells when compared with normal trunk skin (Sup-
plemental Figure 3). Interestingly, lesional skin harvested from
the trunk of psoriasis patients contained a higher percentage of
IL-17–producing mTregs when compared with either nonlesional
trunk skin (harvested from the same patient) or facial/scalp skin
harvested from normal healthy controls (Supplemental Figure 3).
There was also a trend toward increased IFN-γ production from
CD45RO+CD4+Foxp3+ cells isolated from lesional psoriatic skin
compared with that seen in nonlesional skin; however, this was not
statistically significant (Supplemental Figure 4). Interestingly, in
contrast to both nonlesional skin and normal skin grafted onto
immunodeficient mice (described above), CD45RO+CD4+Foxp3+
cells in psoriasis lesions were actively proliferating, with approxi-
mately 15% to 35% of cells expressing Ki67 compared with baseline
levels of 5% in the skin of healthy controls (Figure 6D). A higher
percentage of Foxp3+ cells were cycling compared with Foxp3– cells
in 4 of 5 patients (Figure 6E). It is known that Foxp3 is transiently
expressed in recently activated human CD4+ Tconvs (21, 22). Thus,
it is possible that increased Foxp3-expressing cells in psoriatic skin
represent a recently activated, proliferating Tconv population.
However, human Tconvs that transiently express Foxp3 express
this protein at lower levels compared with activated Tregs (23). In
addition, Foxp3-expressing Tconvs express lower levels of CD25
and do not repress CD127 expression or IL-2 production (23). In
psoriatic skin, CD45RO+CD4+Foxp3+ cells have a phenotype that
is completely different from that of Foxp3-expressing Tconvs. Pro-
liferating Foxp3+ cells in psoriatic skin express high levels of Foxp3
and CD25 (Figure 6F), maintain low levels of CD127 expression
(Figure 6G), and do not produce IL-2 (Supplemental Figure 4).
Thus, our data suggest that Foxp3+ cells proliferating in psori-
atic skin are bona fide mTregs and are not transiently activated
Tconvs. These results raise the intriguing possibility that excessive
proliferation, perhaps associated with some production of effector
cytokines, is a property of mTregs in inflammatory diseases such
as psoriasis. Whether these putative Treg abnormalities are the
cause or the consequence of inflammation is, of course, a question
of fundamental importance for understanding the role of these
cells in the disease process.
In the studies described herein, we phenotypically and function-
ally characterize Tregs in human skin. We define this population
as being bona fide effector memory cells with distinct cell surface
marker expression, cytokine production, in situ localization, TCR
expression, and functional capacity in two different biological con-
texts. Because of these attributes, we propose that this subset of
Tregs is a unique cell population resident in human skin.
Our data are in agreement with previous reports showing that
Tregs comprise approximately 10% of all T cells in normal human
skin (8). We define this population as being highly activated mem-
ory cells similar to the “activated Treg” or “aTreg” population pre-
viously described in human blood (6). However, compared with
aTregs in blood, we observed that mTregs in skin expressed higher
levels of Treg activation markers, contained a lower percentage of
cycling cells, and produced less IL-2 and IL-17 (Figure 2). These
findings suggest that mTregs may be further along the Treg differ-
entiation pathway than aTregs and perhaps represent the final stage
in Treg differentiation, a stage present only in peripheral tissues.
Sakaguchi and colleagues’ characterization of Tregs in human
blood and our studies in transgenic mice support a model in
which the life cycle of a Treg involves activation of naive cells by
antigen, followed by proliferation and differentiation to become
more potent suppressor cells (5, 6). We have previously shown that
murine Tregs activated by self-antigen expressed in skin progres-
sively accumulate in this tissue (5). Here, we show that human
fetal skin has significantly less Tregs than adult skin, with fewer
CD45RO-expressing cells (Figure 1). This directly supports a model
in which throughout the life of an individual, Tregs are continually
exposed to cutaneous antigens and progressively accumulate in
skin. The nature of the antigens recognized by mTregs (i.e., self-pro-
teins expressed in the postnatal environment, foreign pathogens,
and/or commensal microbes) remains to be determined.
It is intriguing that in both mice and humans, mTregs preferen-
tially localize to the epithelium of HFs (Figure 3 and refs. 4, 13). This
may be a consequence of the antigens that mTregs recognize and/or
a unique niche provided by these highly specialized and dynamic
organelles. HFs are constantly cycling, with waves of new protein
expression at each stage of the cycle that change over time (24). In
addition, HFs are colonized by a multitude of microbes (25). Thus,
it is likely that many HF-associated antigens are not expressed in
the thymus, making peripheral tolerance mechanisms essential to
prevent autoimmune attack. In addition, epidermal stem cells reside
in a specific HF niche, providing another important reason to pre-
vent autoimmunity directed at these structures (26). It is possible
that HFs recruit the services of Tregs for this purpose. Consistent
with the idea that Tregs play an important role in regulating HF-
associated autoimmunity are recent studies of alopecia areata (AA).
AA is one of the most common autoimmune diseases in humans
and results from a T cell–mediated autoimmune attack on HFs (27).
Interestingly, the largest genome-wide association study in patients
with AA shows a strong correlation with polymorphisms in CTLA4,
CD25, and EOS (28), all of which play essential roles in Treg function
(29, 30). A subsequent study directly correlates polymorphisms in
the FOXP3 promoter with AA (31).
Our comparison of TCRβ sequences between mTconvs and
mTregs in the skin shows very little overlap, suggesting that these
two populations recognize different antigens (Figure 4 and Table 1).
Previous studies examining TCR repertoires between Tconvs
and Tregs in human blood and murine lymphoid organs have
revealed various degrees of overlap (32–35). However, few studies
have examined these T cell populations in tissues. Perhaps the
best-characterized tissue-specific Tregs to date are those that reside
in VAT (3, 36). Consistent with our data, TCR repertoires between
Tregs and Tconvs in murine VAT show very little overlap (36).
1034 The Journal of Clinical Investigation http://www.jci.org Volume 124 Number 3 March 2014
Thus, it seems that tissues are enriched for T cells that have a
narrow TCR repertoire when compared with blood and second-
ary lymphoid organs and that TCR specificities in tissues differ
between Tregs and other CD4+ T cell populations. It is intriguing
to speculate that in normal healthy tissue, Tregs recognize self-an-
tigens and/or commensal microbes, whereas Tconvs recognize
foreign pathogens. To the best of our knowledge, this is the first
report of TCR sequencing of cells purified from peripheral tissue
in humans. Determining the antigens that Tregs and Tconvs rec-
ognize in tissues may have a profound impact on our ability to spe-
cifically manipulate these cell populations for therapeutic benefit.
Examination of mTregs in human skin grafted onto immunode-
ficient mice revealed this population to be nonmigratory and rel-
atively anergic compared with cutaneous Tconvs (Figure 5). This
is consistent with recent studies performed in murine skin, where
Tregs were relatively nonmigratory in the steady state when com-
pared with other T cell populations in skin (13). In our humanized
mouse model, it is unknown whether Tconvs cycle in response to
lymphopenia or to recognition of xenoantigen and whether entry
into the cell cycle contributes to their migratory potential. Fur-
thermore, although differences in CCR7 expression could predict
differences in migratory capacity, the extent to which this chemok-
ine receptor mediates Tconv migration out of grafted skin in this
model is not known. Nevertheless, grafting human skin onto
immunodeficient mice represents one of the best in vivo models
for studying human cutaneous T cell migration. Our assay clearly
reveals mTregs to have markedly less migratory potential com-
pared with that of Tconvs. It is intriguing to speculate that mTregs
in skin have evolved to become tissue-resident cells as a result of a
variety of essential functions they play within the tissue, making
their continued presence essential for their tissue-specific func-
tion. Indeed, VAT Tregs in mice (and not VAT Tconvs) have been
shown to play a role in both glucose and lipid metabolism that
may be independent of their ability to suppress inflammation (3).
Although mTregs minimally proliferate in normal skin, we found
that these cells actively cycle in inflamed lesions of psoriasis (Figure 6).
Despite increasing in percentage relative to Tconvs, mTregs are
unable to resolve disease. Thus, we observed that mTregs in psori-
atic skin appear to be qualitatively defective in controlling inflam-
mation. This is supported by previous work showing that local
production of IL-6 impairs Treg function in patients with psoriasis
(20). In this study, DCs, ECs, and T cells in psoriatic skin expressed
high levels of IL-6, and Tregs in lesional skin expressed IL-6R.
Peripheral blood–derived DCs from psoriatic patients inhibited
Treg-mediated suppression in vitro in an IL-6–dependent fashion,
with no effect on Treg proliferation. The inability of mTregs to
resolve disease may also in part be a result of aberrant IL-17 pro-
duction by this cell population. A small but significantly increased
percentage of mTregs in psoriatic skin produced IL-17 compared
with mTregs in nonlesional skin (Figure 6C), and expression of
this cytokine was normally suppressed in mTregs when compared
with aTregs in peripheral blood (Figure 2B). Consistent with this,
a recent report showed that peripheral blood–derived Tregs from
patients with psoriasis more readily differentiate into IL-17A–pro-
ducing cells upon stimulation ex vivo (19).
The magnitude and nature of Treg defects in autoimmune dis-
eases remain fundamental and largely unresolved issues. In order
to address this, it is essential to first define the properties of these
cells in healthy individuals. Because of the emerging idea that
immune regulation occurs largely in tissues, we chose to address
these questions in an accessible human tissue, the skin. The stud-
ies described herein are initial experiments attempting to define
long-lived Tregs in healthy human skin. Our preliminary studies
of psoriasis patients have already raised the possibility of unex-
pected Treg defects associated with this disease. Further elucidat-
ing the fundamental biology of Tregs in tissues and the factors
that influence their maintenance and functions in health and dis-
ease is an essential next step in our attempt to understand how
immune responses are regulated in humans.
Human specimens. Normal human skin was obtained from patients at
UCSF undergoing elective surgery, in which healthy skin was discarded
as a routine procedure. Skin was obtained from psoriasis patients (female
and male; age range 20–76 years) who had been off all systemic therapy for
greater than 3 weeks prior to surgery. All patients had greater than 10%
body surface area involvement, with psoriasis severity index scores (PASI)
ranging from 10 to 40. Four-millimeter punch biopsies were obtained from
psoriatic plaques (lesional) and from clinically normal-appearing skin
(nonlesional) at least 10 cm away from the lesional skin biopsies (i.e., in
the same anatomic location).
Mice. All animal studies were performed in compliance with the US
Department of Health and Human Services guidelines for the care and
use of laboratory animals and were approved by the Laboratory Animal
Resource Center of UCSF. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were
obtained from The Jackson Laboratory.
Skin digestion and flow cytometry. Skin samples were stored at 4°C in a
sterile container with PBS and gauze until the time of digestion. Sub-
cutaneous fat and hair were removed, and skin was minced finely with
dissection scissors and mixed in a 6-well plate with 3 ml of digestion
buffer consisting of 0.8 mg/ml Collagenase Type 4 (4188; Worthington),
0.02 mg/ml DNAse (DN25-1G; Sigma-Aldrich), 10% FBS, 1% HEPES, and
1% penicillin/streptavidin in RPMI medium. Samples were incubated over-
night in 5% CO2 and harvested with wash buffer (2% FBS, 1% penicillin/
streptavidin in RPMI medium), then double filtered through a 100-μm
filter, centrifuged, and counted. Human PBMCs were prepared by Ficoll-
Hypaque gradient centrifugation. Fresh, thawed PBMCs were incubated
overnight with digestion buffer, similarly to the skin samples. For detec-
tion of intracellular cytokine production, skin cells and PBMCs were stim-
ulated with 70 ng/ml PMA and 700 ng/ml ionomycin in the presence of
brefeldin A (Sigma-Aldrich) for 4 to 5 hours. The following antibodies
were used for flow cytometry: anti–hBCL-2-PE (BD Biosciences); anti–
hCCR7-PE (BioLegend); anti–hCD3 (-Alexa700, -APC, -APC-eFluor780,
-eFluor605, -FITC, or -PE from eBioscience or -PE-Cy7 from BioLegend);
anti–hCD4 (-PE-Cy7 from BD or -PerCP from BioLegend); anti–hCD25-
PE-Cy7 (BD); anti–hCD27-APC-eFluor780 (eBioscience); anti–hCD45RO-
FITC (eBioscience); anti–hCD127-Brilliant Violet 650 (BioLegend);
anti–hCTLA-4-PE (eBioscience); anti–hFoxp3 (-eFluor 450 or -APC
from eBioscience); anti-hICOS-APC (eBioscience); anti–hIFN-γ (-APC
from eBioscience or -Alexa700 from BioLegend); anti–hIL-2-Alexa700
(BioLegend); anti–hIL-10-APC (BD); anti–hIL17A -PE (eBioscience); anti–
Ki67 (-Alexa647 or -PE-Cy7 from BD); anti–hTNF-α-PE-Cy7 (eBiosci-
ence); and LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies).
Data were acquired by an LSRFortessa flow cytometer (BD Biosciences)
and analyzed using FlowJo software (Tree Star Inc.).
FOXP3 gene DNA methylation. Multiplex quantitative PCR was used to
analyze the methylation status of specific CpG dinucleotides in the TSDR
region of the human FOXP3 gene as described previously (37). PCR primers
and probes used in these assays were: GGTTTGTATTTGGGTTTTGTTGT-
TATAGT (forward); CTATAAAATAAAATATCTACCCTCTTCTCTTCCT
The Journal of Clinical Investigation http://www.jci.org Volume 124 Number 3 March 2014
random subsamplings, we calculated the Bhattacharyya coefficient (BC)
for each subsample and pool pair, which quantifies the similarity between
each subsample and the pool pair and is calculated as:
where fj,1 and fj,2 are the frequencies of clonotype j in samples 1 and 2,
respectively, and n is the number of unique clonotypes present across sam-
ples 1 and 2. The distribution of BC scores in the Monte Carlo simulation
were then used to calculate a P value for the probability that the FoxP3+
cells were not a random sampling of the full skin TCRβ cell population.
Skin grafting. Normal human skin (~1 cm × 1 cm) was grafted onto the
backs of NSG mice as previously described (38). Briefly, healthy skin
was dermatomed to a thickness of 0.4 mm and transplanted using an
absorbable tissue seal (Vetbond; 3M). Bandages were maintained for
1 week after grafting. Transplanted human skin, peripheral blood, and
spleens were harvested, processed, and analyzed by flow cytometry 3 and
7 weeks after grafting.
Statistics. Analysis of the TCR sequencing data is described above.
Other group-versus-group data comparisons were performed using a
2-tailed unpaired Student’s t test. P values of less than 0.05 were con-
Study approval. Studies using human tissue were approved by the UCSF
Committee on Human Research (study number 10-02830) and by the IRB
of UCSF. All patients provided written informed consent prior to biopsies.
Fetal skin samples were obtained from the UCSF Medical Center and San
Francisco General Hospital (San Francisco, California, USA). Samples were
from second trimester skin that would have otherwise been discarded at
the time of the procedure. Blood samples were obtained from healthy adult
volunteers (study number 12-09489). Animal experiments were approved
by the IACUC of the UCSF.
M.D. Rosenblum is supported by an NIH K08 grant
(1K08AR062064-01), a Burroughs Wellcome Career Award for
Medical Scientists (CAMS), the Scleroderma Research Founda-
tion, and the UCSF Department of Dermatology. This work was
partially funded through NIH grants P01 AI35297 (to A.K. Abbas),
R01 AI73656 (to A.K. Abbas), U19 AI56388 (to A.K. Abbas), NIH
R01AR065174 (to W. Liao), and K08AR057763 (to W. Liao).
Received for publication September 3, 2013, and accepted in
revised form November 21, 2013.
Address correspondence to: Michael D. Rosenblum, Assistant
Professor of Dermatology, UCSF Department of Dermatology,
1701 Divisadero Street, 3rd Floor, San Francisco, California
94115, USA. Phone: 415.353.7800; Fax: 415.353.7870; E-mail:
Rosenblummd@derm.ucsf.edu. Or to: Abul K. Abbas, Profes-
sor and Chair, Department of Pathology, UCSF, M590, 505 Par-
nassus Avenue, San Francisco, California 94143, USA. Phone:
415.514.0681; Fax: 415.502.4563; E-mail: Abul.Abbas@ucsf.edu.
(reverse); VIC-TGGTGGTTGGATGTGTTG-MGBNFQ (unmethylated
probe); and 6FAM-CGGTCGGATGCGTC-MGBNFQ (methylated probe).
The sorting strategy for mTreg and mTconv purification is outlined in Sup-
plemental Figure 5. Briefly, viable CD3+CD4+CD45RO+CD25hiCD27hi cells
and CD3+CD4+CD45RO+CD25–CD27– cells were FACS purified from dis-
carded skin harvested from normal healthy males. In addition, viable CD3+
CD4+CD45RO+CD25hiCD27hi cells, CD3+CD4+CD45RO–CD25hiCD27hi
cells, and CD3+CD4+CD45RO+CD25–CD27– cells were purified from
healthy male human PBMC samples. Foxp3 staining on sorted cell popu-
lations verified that CD25hiCD27hi cells were greater than or equal to 75%
Foxp3+ and that CD25–CD27– cells were less than or equal to 8.3% Foxp3+.
Immunofluorescence microscopy. Skin specimens were immediately placed
in Tissue-Tek OCT Compound (Fisher Scientific), frozen on dry ice, and
stored at –80°C. Cryosections (6-mm) were fixed in 100% acetone and
stained for Foxp3 (rat IgG2a, clone PCH101, dilution 1:50; eBioscience),
CD3 (rabbit IgG, polyclonal, dilution 1:500; Abcam), and CD1a (mouse
IgG1, clone 7A7, dilution 1:100; Abcam). Secondary antibodies recogniz-
ing rat (donkey IgG, Alexa Fluor 488), rabbit (donkey, Alexa Fluor 555) and
mouse (goat, Alexa Fluor 647) were used at 1:1,000 and obtained from Life
Technologies, as was the DAPI used for nuclear staining. Confocal images
were acquired using a Nikon C1si spectral confocal microscope.
TCR sequencing. Genomic DNA was isolated from FACS-purified CD3+
CD4+CD45RO+Foxp3+ and CD3+CD4+CD45RO+Foxp3– subsets. TCRβ
repertoire sequencing was performed using GigaMune Rep-Seq molec-
ular kits and ClonoByte repertoire analysis software (GigaGen). Briefly,
genomic DNA was amplified by PCR with a set of 45 primers targeting
the TCRβ V genes paired with 13 primers targeting the TCRβ J genes.
This set of 58 primers amplifies the CDR3 region of TCRβ and also intro-
duces universal priming sites to the amplicons. A second round of PCR
was performed on the resulting amplicons using universal primers. Each
sample was indexed with a unique 6-nucleotide tag, allowing demulti-
plexing of samples after sequencing. Samples were then sequenced on a
MiSeq sequencer (Illumina) to a length of 150 bp. The sequencing reads
for each sample were analyzed using ClonoByte TCRβ repertoire analysis
software (GigaGen). ClonoByte aligns each sequence to the set of TCRβ V
and TCRβ J genes and identifies the conserved cysteine and phenylalanine
that form the boundaries of the CDR3. For quality purposes, reads that
did not have a uniquely identifiable V gene, were out of frame, as defined
by the conserved cysteine and phenylalanine, or contained a stop codon
or a sequencing error in the form of an uncalled base, were discarded. All
other nucleotide sequences were translated into their amino acid equiva-
lent. To avoid distorting the diversity of the TCRβ CDR3 repertoire by the
tail of low-abundance clones caused by PCR and sequencing errors, we ran
a no-template negative control (NTC) alongside each batch of samples for
every stage in the process, including sequencing. In the rare cases in which
the NTC showed a strong spurious CDR3 signal, we removed those clones
from all corresponding samples. Additionally, we removed all reads map-
ping to clones whose frequency was less than 0.1% of the most abundant
clone’s frequency and then normalized the remaining frequencies to the
total number of remaining reads. This conservative criterion minimizes
the chance of unique clones likely to have arisen due to PCR and sequenc-
ing errors being treated as real. A Monte Carlo simulation was conducted
for each skin sample, wherein a simulated pool of Foxp3+ and Foxp3– cells
was created, followed by a random subsampling of clones equal to the
number of clones in the Foxp3 population for that sample. After 10,000
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