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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Immunity. Author manuscript; available in PMC Jul 19, 2013.
Published in final edited form as:
PMCID: PMC3716276
NIHMSID: NIHMS483165
Human Epidermal Langerhans Cells Maintain Immune Homeostasis in Skin by Activating Skin Resident Regulatory T Cells
Julien Seneschal,1,2 Rachael A. Clark,1 Ahmed Gehad,1 Clare M. Baecher-Allan,1 and Thomas S. Kupper1
1Department of Dermatology/Harvard Skin Disease Research Center, Brigham and Women’s Hospital, Boston, MA 02115
2Department of Dermatology and Pediatric Dermatology/ University of Bordeaux, INSERM U1035 and CIRID UMR 5164, Bordeaux, France
Corresponding author: Thomas S. Kupper Harvard Skin Disease Research Center and Department of Dermatology, Room 671 Brigham and Women’s Hospital 77 Avenue Louis Pasteur Boston, MA, 02115 ; tkupper/at/partners.org
Recent discoveries indicate that the skin of a normal individual contains 10-20 billion resident memory T cells ( which include various T helper, T cytotoxic, and T regulatory subsets, that are poised to respond to environmental antigens. Using only autologous human tissues, we report that both in vitro and in vivo, resting epidermal Langerhan cells (LC) selectively and specifically induced the activation and proliferation of skin resident regulatory T cells (Treg), a minor subset of skin resident memory T cells. In the presence of foreign pathogen, however, the same LC activated and induced proliferation of effector memory T (Tem) cells and limited Treg cells activation. These underappreciated properties of LC: namely maintenance of tolerance in normal skin, and activation of protective skin resident memory T cells upon infectious challenge, help clarify the role of LC in skin.
It was recently demonstrated that there are an estimated 20 billion T cells in the skin of an adult human, with virtually all of them bearing markers that identify them as skin homing memory T cells (CD45RO/CLA/CCR4) (Clark et al., 2006a). Not only are there twice as many T cells in skin as in blood, but more than 20-fold more memory T cells with a skin homing phenotype can be found in skin rather than blood. In mouse models of skin infection (Jiang et al., 2012) and presumably in humans, these cells accumulate over time and are thought to represent a compendium of T cell memory to antigens encountered through skin (Clark, 2010). Recent studies from human CTCL patients treated with alemtuzumab demonstrate an abundant population of nonmalignant normal skin resident effector memory T cells (Tem) that do not readily recirculate, and appear to protect the host from infection locally(Clark et al., 2012). Skin dendritic cells can readily present antigen to skin resident memory T cells, a process that can occur immediately after pathogen invasion of skin and requiring no cellular recruitment or migration from blood (Heath and Carbone, 2009; Wakim et al., 2008). However, most studies on the role of dermal DC or LC have examined their capacity to stimulate naïve T cells (Fujita et al., 2009; Furio et al., 2010; Klechevsky et al., 2008; Mathers et al., 2009), an interaction that can occur only after DC or LC have left skin, migrated through afferent lymphatics and ultimately entered the T cell-rich area of the skin draining lymph node. Given their respective strategic locations, from a purely stochastic perspective, it is far more likely that skin resident DC’s will encounter skin resident memory T cells in skin, rather than a naïve T cell in lymph node. However, because of the relatively recent discovery of skin resident memory T cells, studies on the interaction of skin resident DC and skin resident memory T cells from the same individual have not yet been published.
Human skin also contains a resident population of regulatory T cells (Treg) that represent between 5 and 10% of the total resident skin T cells (Clark et al., 2008; Clark and Kupper, 2007). To date, most human Treg cells studies analyzing their phenotype or function have been performed on rare (1-4%) blood-derived effector CD45RO+FoxP3hi (Miyara et al., 2009; Sakaguchi et al., 2010; Wing and Sakaguchi, 2010). These effector CD45RO+ Treg can suppress the proliferation and cytokine production of effector T cells. Although these effector Treg express CD45RO, which is the marker of conventional memory T cells, no evidence for memory and recall responses by these Treg cells has been previously demonstrated in mouse experiments until recently. Indeed, effector memory Treg cells have been shown to persist in skin and induce tolerance to a novel transgenically expressed “autoantigen” in a mouse model (Rosenblum et al., 2011). However little is known about the function of effector CD45RO+ skin resident memory Treg cells which populate peripheral human tissues such as skin. We have previously demonstrated that human skin Treg cells can proliferate in an antigen-independent manner when cultured with IL-15 and dermal fibroblasts (Clark and Kupper, 2007), conditions that can be seen in inflamed tissue (Rappl et al., 2001). Moreover, another study showed that skin human skin-resident memory Treg cells can locally expand during cutaneous inflammation (Vukmanovic-Stejic et al., 2008) and can serve as a brake to regulate the inflammation in peripheral tissue (Dudda et al., 2008). More recently, it was shown in a human model that glucocorticosteroids can induce proliferation of Treg cells in skin(Stary et al., 2011)., Under normal conditions, however, the function and homeostasis of human skin resident memory Treg cells remain uncharacterized.
Normal human skin contains also distinct populations of dendritic cells (DC): Langerhans Cells (LC) expressing CD1a and Langerin/CD207 (Valladeau et al., 2000), and dermal resident DC (DDC) expressing CD1c, which is also known as blood dendritic cell antigen (BDCA1) (Zaba et al., 2007; Zaba et al., 2009). LC mainly populate the epidermis, and are traditionally considered as the first line of defense against exogenous pathogen invasion (Merad et al., 2008). More recently, LC function has become more controversial, with some studies suggesting their true role is not immune stimulation but rather immunoregulation (Kaplan, 2010; Lutz et al., 2010). Studies in transgenic mice have revealed a role for LC in the induction of tolerance. Data using LC-deficient mouse models indicated that LC might actually dampen inflammation and mediate tolerance, especially in contact hypersensitivity reactions (Bobr et al., 2010; Igyarto et al., 2009; Kaplan et al., 2005). At the same time, human LC have been found to be the most powerful DC subset to induce the in vitro differentiation and the polarization of naïve CD4+ T cells into, respectively, either Th2 (Furio et al., 2010), Th17 (Mathers et al., 2009) or more recently Th22 (Fujita et al., 2009) effector cell phenotypes. Moreover, human LC can also prime and cross-prime naïve CD8+ T cells (Klechevsky et al., 2008). Thus, there is substantial controversy regarding the physiologic role of LC with regard to immunoregulation versus immunostimulation (Stoitzner, 2010).
This paradox has been difficult to reconcile experimentally, in part because of certain apparent differences between human and murine skin. Langerin/CD207, an integral glycoprotein of Birbeck granules that define human LC (Valladeau et al., 2000), is expressed by a subset of dermal DC in mice that is not observed in human skin (Bursch et al., 2007). Moreover, many resident T cells in murine skin are TCRγδ+ and have a limited antigenic repertoire (Nanno et al., 2007), while T cells resident to human skin are predominantly TCRαβ and have great TCR diversity (Clark et al., 2006a). To further complicate matters, most human studies of LC have involved stimulation of allogeneic, rather than autologous, naïve T cells from peripheral blood, thus introducing a potential artifact into the system. These and other differences make it difficult to directly compare experimental results on human and murine LC biology, or to extrapolate experimental results involving murine LC to human skin immunity. Increasing, the trend among immunologists has been to address questions relevant to human immunology using human models(Davis, 2008, 2012), as we have chosen to do here.
In the present study, we demonstrate that LC induced the proliferation of a subset of autologous skin resident memory T cells in vitro. These proliferating T cells were exclusively CD4+CD25+FoxP3+CD127 skin resident Treg cells, and their function was demonstrated by suppression of proliferation of autologous skin resident Tem cells. The LC mediated proliferation of these skin resident memory Treg cells could be completely blocked by antibodies to Class II MHC, as well as CD80-86, suggesting antigen presentation. Moreover, the proliferation could be blocked with antibodies to LC derived IL-15 activity or T cell derived IL-2. Immunohistochemical analysis and FACS analysis of normal human skin revealed that skin resident memory Treg were predominantly located within or near the epidermis or follicular epithelium, always proximity to LC. A subset of these skin resident memory Treg were positive for Ki67 in situ, a cell cycle antigen specific for recent or ongoing proliferation (Scholzen and Gerdes, 2000), suggesting that what we observed in vitro is occurring in vivo at steady state. Interestingly, when the skin pathogen C. albicans was added to the co-cultures in vitro, an additional subset of T cells proliferated. Further analysis showed that not only skin resident memory Treg cells, but also antigen-specific skin resident Tem cells subsets proliferated under these conditions, and that the skin resident Tem cells produced the effector cytokines IFNγ and IL-17. These data suggest that under steady state conditions in skin, LC mediate tolerance to self antigens, but can also be activated by infection to mediate protective immunity. Thus, their capacity to negatively or positively modify immune response depends upon context.
Langerhans cells induce proliferation of skin resident CD4+ T cells in the absence of exogenous antigen
We carefully extracted and purified LC, dermal DC, and skin-resident memory T cells from the skin of multiple individual donors (Figure S1). We first isolated skin resident memory T cells from normal human skin as previously described (Clark et al., 2006a). Skin T cells are composed of CD45RO+ T cells expressing skin homing addressins CLA and CCR4 and lacking co-expression of CCR7 and CD62L (Clark, 2010). In parallel, we isolated cutaneous DC from the same donor. Skin DC contained epidermal HLA-DR+CD207+ LC and dermal HLA-DRhiBDCA1+ DC (DDC) (Zaba et al., 2007; Zaba et al., 2009). Both LC and DDC expressed significant levels of CD86, CD83 and DC-LAMP (Figure S2) as described in previous studies (Bond et al., 2009; Klechevsky et al., 2008). Skin resident memory T cells were then stained with carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured in presence of purified autologous skin DC. After 6 days of co-culture, T cell proliferation was assessed by flow cytometry. We observed a significant expansion of skin resident memory T cells in the presence of LC, but no significant proliferation in the presence of DDC. Medium conditioned by LC or DDC had no effect on skin resident memory T cells proliferation (Figure 1A,B). Thus, epidermal LC were strong inducers of skin resident memory T cell proliferation whereas DDC produced negligible proliferation (Figure 1C and Figure S3). Skin resident memory T cells proliferation induced by LC became observable between day 4 and 6 (Figure 1D,E).
Figure 1
Figure 1
A subset of skin resident memory T cells proliferates in presence of autologous LC
We next explored the phenotype of the proliferative skin resident memory T population. Skin resident memory T cells were cultured alone or in the presence of LC. After 6 days, Skin resident memory T cells were isolated and stained with antibodies directed against CD3, CD4, CD8. Strikingly, the proliferative skin resident memory T cells were overwhelmingly CD4+ T cells (96.7% ± 2.7% for four donors; (Figure 2A,B), whereas the non proliferative cells were composed of a more substantial fraction of CD8+ T cells. Therefore, LC but not DDC induced the proliferation of skin resident memory T cells, a process which required cell contact. CD4+ T cells were preferentially stimulated.
Figure 2
Figure 2
Proliferating skin-resident memory T cells are preferentially CD4+ T lymphocytes
The proliferating skin-resident memory T cells are Treg cells
In normal human skin, resident memory T cells contain a population of skin-resident memory Treg cells that are CD4+CD25+FoxP3+CD127, and we then asked whether the expansion of T cells induced by LC in vitro might involve skin-resident memory Treg cells. To address this issue, skin isolated CFSE labeled skin resident memory T cells were again cultured with purified autologous LC. At day 6, T cells were analyzed with antibodies directed against CD4, CD25, FoxP3 and CD127 by flow cytometry. Strikingly, in the expanded proliferative T cell population, 84.8% ± 8% of the T cells were CD4+CD25+FoxP3+CD127 , CTLA-4+, GITR+ and CD69 compatible with a Treg phenotype (Figure 3A,B and Figure S4A,B), whereas the fraction of skin resident memory Treg cells in the non-proliferative, skin-resident memory T cells subset was comparable with the fraction observed in the control population. Moreover, an actual absolute expansion of Treg cells with a range of amplification from 1.5 to 3.0 fold was observed after co-culture with autologous LC (Figure 3C). Deletion of CD25+ skin resident memory T cells subset prior to co-culture with autologous LC significantly reduced the expansion of skin-resident memory Treg cells (Figure 3D). Because phenotypic markers do not measure Treg function, we next assessed the ability of the expanded skin resident memory Treg population to suppress T cell proliferation in a secondary MLR. The CD4+CD25+CD127 skin resident memory Treg cells were harvested and isolated after 6 days of co-culture and then added to a MLR composed of CFSE labeled skin resident Tem cells and irradiated PBMC’s as stimulators. The addition of this activated population of skin resident memory Treg reproducibly and significantly inhibited autologous skin resident Tem cells proliferation in this assay (Figure 3E ), and showed that there was >65% suppression of such responses at a 1:1 ratio. These results were confirmed in additional experiments in using a [3H]thymidine incorporation assay (Figure 3F). Again, isolated CD4+CD25+CD127 skin resident memory Treg cells strongly inhibited the proliferation of skin resident Tem cells. Therefore, LC specifically induced the proliferation of skin resident memory Treg cells, but not skin-resident Tem cells, under steady state conditions and these expanded skin resident memory Treg cells showed potent suppressive activity. Taken together, these results suggest that Treg cells, a minor population among skin resident memory T cells, were being specifically induced to proliferate by LC. These Treg cells demonstrated a classical phenotype (CD4+CD25+FoxP3+CD127), and mediated significant suppression of proliferation of skin resident Tem cells.
Figure 3
Figure 3
Proliferating CD4+ skin resident memory T cells in presence of autologous LC are functional regulatory T cells
LC mediated skin resident memory Treg cells proliferation is antigen-specific
We demonstrated above that LC have the unique capacity to induce proliferation of functional skin resident memory Treg cells. To further understand which signals are involved in the interaction between LC and skin resident memory T cells, we co-cultured CFSE labeled skin resident memory T cells with autologous LC in presence of different blocking antibodies: anti-MHC class I and/or II, CD80-86, CD1a, IL-2 and IL-15Rα. At day 6, expansion of skin CD3+ skin resident memory T cells was assessed by dilution of CFSE using flow cytometry (Figure 4A,B). Blocking the MHC class II presentation and more specifically HLA-DR and DP, significantly reduced the proliferation of skin resident memory T cells (Figure 4C,D). Similarly, blocking the costimulatory signal CD80-86 or the addition of cytokine antibodies directed against IL-2 or IL-15Rα significantly decreased the induction of skin resident memory T cell proliferation (Figure 4B). In contrast, the proliferation of skin skin resident memory Treg cells was not dependent of CD1a or MHC Class I presentation. These data strongly suggest that proliferation of skin skin resident memory T cells by resident LC proceeds in an antigen dependent manner.
Figure 4
Figure 4
Expansion of skin resident memory Treg cells is dependent on MHC class II, CD80-86, and IL-2 or IL-15
Skin resident memory Treg cells are located in the epidermis or at the papillary dermal-epidermal junction, and proliferate in situ
We next asked if we could detect interactions between LC and skin resident memory Treg cells in vivo. We examined multiple specimens of freshly obtained human skin, using immunofluoresence microscopy. As we have previously described, abundant CD3+ skin resident memory T cells could be identified in uninflamed human skin, and a subset of CD3+ skin resident memory T cells co-expressed nuclear FoxP3 and CD25 (Figure S5A), identifying them as skin resident memory Treg cells (Figure 5A). Interestingly, these skin resident memory Treg cells appeared to be localized in or near epidermis and follicular epithelium, in contact or close proximity to CD1a+ epidermal and follicular LC (Figure 5B and Figure S5C). We next asked if these skin resident memory Treg cells showed evidence for proliferation by examining expression of the marker Ki67, which is expressed through all phases of the cell cycle, but is absent in cells in G0. CD3+FoxP3+ skin resident memory T cells that expressed Ki67 could be readily observed in skin sections, while Ki67+ CD3+FoxP3 skin resident Tem cells were undetectable. The CD3+FoxP3+Ki67+ skin resident memory T cells were predominantly located in epidermis, follicular epithelium, or upper papillary dermis, rather than in the deeper papillary or reticular dermis (Figure 5C and Figure S5B,C). Moreover we observed that CD1c/BDCA1+ DDC were found close to FoxP3+ skin-resident memory Treg cell in a resting state (Figure S5D). The strict association of Ki67 expression with cellular proliferation leads us to conclude that in vivo as well as in vitro, epidermal LC induce the proliferation of skin resident memory Treg cells, consistent with a tolerogenic role for LC at steady state. These results showed that Treg cells and LC co-localized in epidermis and follicular epithelium, and that a discernable population of these Treg cells appeared be entering the cell cycle, as judged by Ki67 expression. This in vivo data is completely consistent with the observations of LC induced Treg cells proliferation in vitro
Figure 5
Figure 5
Skin resident memory Treg cells are found in the epidermis near Langerhans cells, and express the proliferation marker Ki67
Abundant skin resident memory T cells, including skin resident memory Treg cells, are present in the epidermis by FACS analysis
To confirm that skin resident memory T cells were indeed present in epidermis, we subjected skin explants to dispase treatment, which cleaves skin specifically at the dermal epidermal junction without disrupting epidermal cell-cell interactions. Dermis and epidermis were then separated, enzymatically digested, and analyzed independently. Skin resident memory T cells were readily found in the epidermis (3% all cells), and 17% of these CD3+ cells were FoxP3+ skin resident memory Treg cells. LC were similarly abundant (1.2% of all cells), and no cells with DDC markers were observed in the epidermal fraction, confirming the specificity of the separation (Figure 6A). Dermis contained a smattering of CD1a+ LC (0.1%), and again skin resident memory T cells were abundant (3.6% of all cells). About 70% fewer of these skin resident memory T cells were FoxP3+ skin resident memory Treg cells (6%), suggesting that skin resident memory Treg cells are enriched in the epidermal compartment (Figure 6B). Thus, our immunofluoresence histology observations were validated by FACS analysis of the dermal and epidermal compartments, that is, LC and skin resident memory Treg cells can be found together in the epidermal compartment.
Figure 6
Figure 6
Skin resident memory Treg cells are abundant in epidermis
During infection, LC induce proliferation of both skin resident memory Treg cells and skin pathogen-specific Tem cells
Our data strongly suggest that human LC have the ability to maintain peripheral tolerance by inducing local proliferation of skin resident memory Treg cells. However, recent studies (Fujita et al., 2009; Furio et al., 2010; Klechevsky et al., 2008; Mathers et al., 2009) on the biology and function of human LC showed their capacity to induce differentiation and expansion of naïve CD4+ T cells into potent Tem cells cells rather than Treg cells, in apparent contrast with our data. To further assess the function of LC in human skin, we analyzed the proliferation of skin resident memory T cells induced by autologous LC in the presence of Candida albicans (C.albicans). Most immunocompetent individuals can manifest a delayed type hypersensitivity response to C. albicans, a ubiquitous human skin commensal which can cause skin inflammation in the setting of barrier disruption (Romani, 2000). C. albicans applied to the surface of intact skin provokes no response, but when injected intradermally, can induce T cell mediated inflammation which is evident by 24-48 hours (Esch and Buckley, 1988). We have previously shown that most individuals have skin resident memory T cells that can respond in vitro to C. albicans, both via proliferation and IL-17 and IFNγ production. We asked whether LC would present C. albicans antigens to skin resident memory T cells in an immunogenic or tolerogenic fashion. In 9 out of 10 donors, addition of C. albicans antigen led to substantially increased proliferation of skin-resident memory T,cells (Figure 7A,B) and proliferating skin resident memory T cells included both FoxP3+CD127 and FoxP3 CD127+ T cells, indicating that both skin resident memory Treg cells and skin resident Tem cells were proliferating (Figure 7C). Interestingly, low doses of C. albicans antigen favored Treg cells proliferation, while higher doses favored Tem cells proliferation (Figure 7D). This is consistent with the observation that Treg cells are most effective at lower levels of antigen. Moreover, depletion of CD25+ Treg cells is associated with increased proliferation in the presence of C. albicans suggesting the absence of Treg cells inhibition on specific C.albicans Tem cells expansion (Figure 7E).
Figure 7
Figure 7
During infection, proliferation of skin resident memory T cells demonstrates a mixed population composed of pathogen specific skin resident Tem cells and skin resident memory Treg cells
The proliferation of skin resident memory T cells to C. albicans could be blocked completely by antibodies to Class II MHC and CD80/86, as well as antibodies to IL-2 and IL-15Rα (Figure S6). Proliferating cells produced IL-17 and IFNγ (Figure S7A), and were enriched for Vβ2, Vβ3, and Vβ8 αβTcr T cells (Figure S7B). Interestingly, there was a highly reproducible >50% inhibition of proliferation with a blocking antibody to CD1a, which had no effect on skin resident memory Treg cells proliferation in the absence of C. albicans. Taken together, these data suggest that after pathogenic antigen exposure, LC induced proliferation of pathogen specific Tem cells as well as Treg cells. Thus, depending upon the biological context, LC can mediate tolerance or immunity.
There are several features of our study which have not been previously reported, and these are discussed below. First, we have examined the function of LC in normal human skin, and most importantly the interaction of LC with skin resident memory T cells, a population first described in 2006 (Clark et al., 2006a). These studies are made possible by a technical advance; specifically, our ability to extract large numbers of skin resident memory T cells from human skin, as we have described previously (Clark et al., 2006b). Virtually all skin resident memory T cells have memory (CD45RO) and skin homing (CLA, CCR4) markers, most have a Tem phenotype, and all are thought to be derived from prior antigen encounters in skin draining lymph nodes (Clark, 2010). Indeed, recent mouse models have indicated that it is these skin resident memory T cells, rather than circulating T cells or antibodies, that mediate host defense upon microbial challenge (Gebhardt et al., 2009; Liu et al., 2010). More recently, this was validated in a model of human disease(Clark et al., 2012). Thus, the interaction between skin resident dendritic cells (including LC) and skin resident memory T cells is likely to be essential for rapid recall or memory responses in skin to proceed. In contrast, most recent papers describe human LC interactions with naïve T cells (Fujita et al., 2009; Furio et al., 2010; Klechevsky et al., 2008; Mathers et al., 2009), a process requiring that LC exit the epidermis and migrate down afferent lymphatics to lymph node, and most relevant to primary immune responses. We do not suggest that these studies are not important, only that they describe the biology of primary rather than memory responses.
Second, we describe the capacity of LC, but not dermal DC, to induce the proliferation of a subset of skin resident memory T cells, specifically CD4+CD25+FoxP3+CD127 T cells that have the phenotype of Treg cells (Liu et al., 2006). This proliferation appears to be dependant upon antigen presentation, as it can be inhibited completely by antibodies to either Class II MHC or CD80-86. In contrast, antibodies to MHC Class I or CD1a had no affect. The proliferation could also be blocked by antibodies to IL-2 and IL-15 activity, cytokines derived from T cells (Waldmann, 2006) or LC (Munz et al., 2005; Ratzinger et al., 2004), respectively. These data are consistent with LC presenting antigen to skin resident memory Treg cells. While it is tempting to speculate that it is self-antigen that is being presented, we cannot rule out the possibility that there is some carryover of normal skin flora antigens in our culture conditions. However, a recent report in a K5-ovalbumin expressing murine model demonstrated that self-reactive skin resident memory Treg cells could be generated and persist in skin. These memory Treg cells could reduce and ultimately suppress an autoimmune T cell mediated skin disease (Rosenblum et al., 2011).
Supporting the idea that epidermal LC and skin resident memory Treg cells have ample opportunity to interact in vivo, our FACS analysis of dermal and epidermal compartments confirm that skin resident memory T cells in general are abundant in epidermis and dermis and that skin-resident memory Treg cells in particular are more abundant in epidermis than dermis. Immunohistochemical studies demonstrate that skin resident memory Treg cells are localized to either the epidermis or the dermal epidermal junction, and overlap with CD1a+ LC. A reproducible fraction (5%) of these skin resident memory Treg cells is positive to Ki67. Ki67 is generally considered a proliferation marker, and is present during all phases of the cell cycle, but is absent in resting (G0) cells (Scholzen and Gerdes, 2000). This in vivo finding is completely consistent with our in vitro observation that LC selectively induce proliferation of the skin resident memory Treg cells subset of skin resident T cells. While the immunostaining data show that skin resident memory Treg cells are the most abundant skin resident T cells to interact with LC in vivo, the in vitro proliferation data indicate that even when skin resident memory Treg cells are a minority population, they are still preferentially induced to proliferate by LC from normal skin.
Phenotype does not prove function (Sakaguchi et al., 2010), and thus we felt it necessary to establish whether our CD3+CD4+FoxP3+CD127 could suppress Tem cell immune responses. We felt that the most relevant responder Tem cells would be the autologous skin resident Tem cells derived from the same sample of skin. We were able to demonstrate quite clearly that the addition of skin resident memory Treg cells substantially blocked the proliferation of autologous skin resident Tem cells to allogeneic antigen, using as a read-out either CFSE dilution or [3H]thymidine incorporation. Thus, LC induced expansion of skin resident memory Treg cells that can suppress autologous skin resident Tem cell responses. Given the localization of these cells in the epidermis and dermis, it is tempting to speculate that LC are activating skin resident memory Treg cells to maintain tolerance to self antigens (Morelli et al., 2001; Morelli and Thomson, 2007). It is also possible that LC could present antigens from normal skin resident microbial flora to skin resident memory Treg cells (Kubo et al., 2009). Further studies will determine which of these potential roles is more important.
Most studies of human LC have focused upon their potent ability to activate and differentiate naïve T cells in vitro. Preferential generation of Th2 (Furio et al., 2010), Th17 (Mathers et al., 2009), and most recently Th22 (Fujita et al., 2009) cells by LC has been noted, as has the cross presentation of antigen to CD8+ effector cells (Banchereau et al., 2009; Klechevsky et al., 2008). As noted above, all such interactions occur physiologically in lymph nodes, the only possible site where LC and naïve T cells will encounter each other. Given the paradox that LC are immunoregulatory within skin in the absence of exogenous antigen (our data), and the ample evidence that LC that have migrated to LN are profoundly immunostimulatory, we asked whether LC could present antigen in an immunostimulatory way to skin resident Tem cells, a population that a LC would certainly encounter during a recall immune response prior to migration to LN.
Candida antigen, via skin test, has been used clinically as a measure of immunocompetence. Typically, >75% of humans will have a positive skin test to C. albicans 48 hours after intradermal injection (Esch and Buckley, 1988). This time point is consistent with a memory immune response, and our unpublished data suggests that it is mediated by skin resident Tem cells specific for C. albicans. Thus, we reasoned that most individuals would have skin resident Tem cells that respond to C. albicans. We next asked whether LC could present C. albicans to skin skin resident Tem cells. The addition of C. albicans to cultures of autologous LC and skin resident memory T cells induced significantly more T cell proliferation than in its absence. Under these circumstances, two populations of T cells proliferated. First was the CD4+CD25+FoxP3+CD127 population that proliferated in the absence of C. albicans antigen. But in the presence of C. albicans, a second population of T cells proliferated; these were FoxP3lo, CD127+, and produced the effector cytokines IFNγ and IL-17 (Acosta-Rodriguez et al., 2007). The cells that proliferated were also of a phenotype that suggested C. albicans specificity by TCR Vβ (Devore-Carter et al., 2008; Walsh et al., 1996). This latter population consists of skin resident Tem cells specific for C. albicans, and if their proliferation was blunted by the concurrent proliferation of skin-resident memory Treg cells, it did not occur to a substantial degree.
Given the abundance of skin resident memory T cells in normal skin, many of them specific for normal skin microbial flora (Clark, 2010; Clark et al., 2006a), it stands to reason that the activation of these cells must be tightly controlled. Our data are consistent with a model wherein LC in normal intact skin favor constitutive local proliferation and activation of skin resident memory Treg cells, thus providing a brake on inappropriate activation of skin resident Tem cells in normal skin, whether to autoantigens of foreign antigens. However, this brake appears to be readily cast aside when a pathogen is encountered, as would occur when the physical epidermal barrier is breached or disrupted and innate immune danger signals are provided. Under these conditions, LC are then capable of presenting antigen in an immunogenic fashion to skin resident Tem cells. Our data also suggest that there is a threshold of antigen below which LC induce Treg cells proliferation, but above which LC induce Tem cells activation and proliferation. Thus, depending upon the biological context, LC can function to maintain peripheral tolerance, but also have the capacity to activate protective skin resident memory T cells that can mount an effective host response to pathogen.
Isolation of cells from Skin
Acquisition of skin samples and all scientific studies were approved by the Institutional Review Board of the Partners Human Research Committee. Samples of normal adult human skin were obtained as discarded human tissue from cutaneous surgeries. T cells were isolated from skin using short term explant (5 days) cultures as previously described (Clark et al., 2006a). No exogenous cytokines were added. LC and dermal DC were purified from normal human skin (Zaba et al., 2007). Subcutaneous fat was excised, and remaining tissue was washed with PBS. The dermal layer was heavily scored with a scalpel and incubated with 1mg/ml type 1 collagenase (Invitrogen), 1mg/ml dispase (Invitrogen), and penicillin-streptomycin solution overnight at 37°C. Epidermal and dermal sheets were separated and placed in RPMI 1640 supplemented with 10% pooled human serum (Cellgro Mediatech), 0.1% gentamicin reagent solution (Invitrogen), and 1% 1M HEPES buffer (Sigma-Aldrich). After 48h at 37°C, cells were collected and filtered with 40μm cell strainers (BD Biosciences) and then enriched with a Ficoll-diatrizoate gradient (GE Healthcare Bio-Sciences). LC were further purified by positive selection using anti-CD1a MACS (Miltenyi-Biotec). Dermal DC were purified by positive selection using BDCA1+ isolation kit (Miltenyi-Biotec).
Antibodies
All antibodies used for immunofluorescence and FACS are listed in Supplementary Table 1.
Immunofluorescence
5μm-thick sections of formalin-fixed, paraffin-embedded tissues from normal human skin, were cut, deparaffinized, and subjected to a heat-induced epitope retrieval step.. Slides were rinsed in cool running water and washed in Tris-buffered saline, pH 7.4, before incubation with primary antibodies and then incubated appropriate secondary antibodies: Alexa Fluor® 488-conjugated anti-mouse or rat or Alex-Fluor® 594-conjugated anti-mouse. Additional negative controls were performed by adding isotype mouse antibodies as primary antibodies. After subsequent washing, the sections were mounted with Prolong Gold antifade reagent with DAPI (Invitrogen) and covered with cover slip. Immunofluoresecence reactivity was viewed on an Olympus BX51/52 system microscope coupled to a Cytovision System (applied Imaging).
Candida albicans
C. albicans were seeded in RPMI medium plus 10% (vol/vol) FCS in 24-well plate. Plate was incubated for 2h at 65°C to heat inactivated yeast, and was cooled before to be used for experiments.
Analysis of T cell proliferation
For recall responses, skin isolated T cells were stained with 2 μM CFSE (Molecular Probes) at 37°C for 3 min(Parish et al., 2009). CFSE labeled T cells were washed 3 times and then were cultured (5×104/well) in 96-well plates in RPMI medium supplemented with 10% pooled human serum, to which autologous LC or dermal DC (2.5×104/well) were added. In some experiments heat-killed C.albicans (5×104) was added to the co-culture.
Blocking Experiments
Purified anti-CD80, CD86, HLA DR-DP-DQ, HLA DR, HLA DP and HLA DQ, HLA A,B,C, CD1a, IL-2 and IL-15Rα or isotypes were added to skin DC and the plate were kept for 30 min at 4°C and then washed before T cells were added.
Regulatory T Cell Assay
Regulatory T cells were isolated after 6 days of co-culture with LC, by using the CD4+CD25+CD127dim/ regulatory T cell isolation kit II (Miltenyi Biotec). T responders CD4+CD25 were isolated from normal skin explant culture. CFSE labeled CD4+CD25 (effectors) and CD4+ CD25+ CD127 (suppressors) T cells (2.5 × 103/well) were stimulated with plate-bound anti-CD3 (clone UCHT1, BD Pharmingen) used at a concentration of 1μg/ml, coated in PBS for 2-4 h at 37°C, in presence of irradiated T cells depleted PBMCs (2.5 × 104/well). All cells were cultured in a final volume of 200μl. Proliferation was monitored by CFSE dilution on day 5 or by pulsing (18h) each well with 1μCi of [3H]thymidine (Perkin Elmer) as described previously (Baecher-Allan and Hafler, 2006).
Statistics
Statistical significance was calculated with two-tailed Student’s t test.
HIGHLIGHTS
  • Langerhans Cell (LC) selectively and specifically interact with skin resident memory T cells.
  • LC induce activation and proliferation of skin-resident memory Treg cells at steady state, and by doing so control the steady state activation of skin resident effector memory T cells (Tem) .
  • In the presence of pathogen antigen, LC induce the proliferation of protective skin resident Tem cells, while limiting proliferation of skin-resident memory Treg cells.
  • This helps resolve an ongoing controversy about whether LC are tolerogenic or immunogenic by revealing that these functions are influenced by biological context.
Supplementary Material
Suppl Data
Suppl Figures
ACKNOWLEDGMENTS
We thank Dr Thomas Cochran of the Boston Center for Plastic Surgery and Dr Elof Eriksson of Brigham and Women’s Hospital for kindly providing adult human skin samples. We thank Dr George F. Murphy and Dr Qian Zhan of Brigham and Women’s hospital for technical advice on immunofluorescence assay. We thank Dr Alain Taieb, Dr Patrick Blanco and Dr Jean-François Moreau from University of Bordeaux for helpful discussion. This work was supported by grants from the NIH (RO-1 AI 025082 ). J.S was supported by grants from Fondation René Touraine (European Fellowships 2008), Ministère Français des Affaires Etrangères, and Centre Hospitalier Universitaire de Bordeaux, France.
Footnotes
AUTHOR CONTRIBUTIONS J.S designed the experiments, analyzed, interpreted the data and drafted the paper; R.A.C provided intellectual input, technical advice, and helped edit the manuscript. A.G performed experiments in Figure 7. C.M.B.-A provided intellectual input, technical advice. T.S.K conceived the project, helped plan the experiments, analyzed and interpreted the data, and contributed to drafting and editing the manuscript.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial interest
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