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T Cells and NK Cells: Relevance for Tumor Surveillance
Institute of Cell Biology, University of Bern, Bern, Switzerland
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Abstract |
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T cells in normal skin express CCR8 and produce proinflammatory cytokines. In this study we examined other subsets of cutaneous lymphocytes, focusing on those with potential function in purging healthy tissue of transformed and stressed cells. Human dermal cell suspensions contained significant populations of V
1+ 
T cells and CD56+CD16– NK cells, but lacked the subsets of V2+ T cells and CD56+CD16+ NK cells, which predominate in peripheral blood. The skin-homing receptors CCR8 and CLA were expressed by a large fraction of both cell types, whereas chemokine receptors associated with lymphocyte migration to inflamed skin were absent. Neither cell type expressed CCR7, although T cells up-regulated this lymph node-homing receptor upon TCR triggering. Stimulation of cutaneous V1+ T cell lines induced secretion of large amounts of TNF-, IFN-, and the CCR8 ligand CCL1. In contrast to cutaneous T cells, both cell types had the capacity to produce intracellular perforin and displayed strong cytotoxic activity against melanoma cells. We therefore propose that T cells and NK cells are regular constituents of normal human skin with potential function in the clearance of tumor and otherwise stressed tissue cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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In contrast, we are just beginning to understand the migration properties and function of T cells and other lymphocytes within healthy tissue (10, 11). We recently identified the chemokine receptor CCR8 as a specific marker of T cells present in normal skin and demonstrated that CCL1/I-309, the unique ligand for CCR8, was constitutively produced at strategic sites in noninflamed skin (12). These cutaneous T cells secreted large amounts of proinflammatory cytokines and may thus contribute to memory responses after contact with previously encountered Ags. However, T cells are unlikely candidates for surveillance against tumors and stressed tissue cells (12). Therefore, we turned our attention to T cells and NK cells. In contrast to T cells, T cells recognize Ags without the need for processing and presentation by classical MHC molecules (13, 14). The major subset of T cells in human peripheral blood expresses the V2 gene segment and recognizes nonpeptide Ags of mostly microbial origin. In contrast, most T cells in epithelial tissues express the V1 gene segment and respond to stress-associated factors, including heat shock proteins and the MHC-related molecules MHC-I-related protein A (MICA) and MHC-I-related protein B (MICB), which are induced on tumor and virus-infected cells (13, 14, 15, 16). NK cells similarly recognize abnormal cells using a repertoire of receptors that detect stress-associated molecules as well as the loss of surface MHC protein (17).
Murine skin harbors a major population of V1+ T cells, known as dendritic epidermal T cells (DETCs), which appear to play a critical role in tumor surveillance, as evidenced in T cell-deficient mice (18, 19). Surprisingly, however, a counterpart of DETCs does not exist in human epidermis (1), and it is thus unclear what types of lymphocyte mediate equivalent immune surveillance functions in human skin. In this study we demonstrate that normal human dermis contains distinct populations of T cells and NK cells, both of which express receptors for homing to noninflamed skin and for recognition of allogeneic tumor cells. Together, these cells are likely to make a unique contribution to the elimination of cutaneous tumor and otherwise stressed cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Fluorochrome-conjugated Abs to human proteins were purchased from the following sources: rat Ab to CLA (HECA-452), mouse Abs to CD3 (UCHT1), CD16 (3G8), CD45 (HI30), CD56 (B159), pan-TCR- (11F2), V2 (B6.1), perforin (dG9), CCR4 (1G1), and CCR6 (11A9) were obtained from BD Pharmingen. Mouse Ab to V1 (TS8.2) was purchased from Endogen (Perbio Sciences), and mouse Abs to CD94 (DX22) and NKG2D (1D11) were obtained from eBioscience. Rat Ab to CCR7 (3D12; M. Lipp, Department of Tumor Genetics and Immunogenetics, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany) and rabbit Ab to CCR8 (12) were detected using biotinylated donkey anti-rat IgG or biotinylated donkey anti-rabbit IgG, respectively (Jackson ImmunoResearch Laboratories), followed by streptavidin-PE (DakoCytomation) or streptavidin-allophycocyanin (BD Biosciences). Isotype-matched control mAbs were purchased from BD Biosciences. Perforin staining was performed after fixation with 2% paraformaldehyde and permeabilization with 0.5% saponin. Flow cytometric analysis was performed on a FACSCalibur (BD Biosciences), using propidium iodide to exclude dead cells. OptEIA ELISA kits for the detection of IFN- and TNF- were obtained from BD Pharmingen and performed according to the manufacturer’s instructions. The CCL1 ELISA was performed as described previously (12).
Cell preparations
Split skin was obtained using a dermatome and normal skin from patients undergoing abdominoplastic or mammareduction surgery. For most experiments, skin was digested in dispase II (1.25 U/ml; Roche) and collagenase D (1 mg/ml; Roche) for 15–30 min at 37°C, followed by removal of the epidermis and trypsin/EDTA digestion (30 min, 37°C) and gentle homogenization of remaining dermal fragments. Cells were filtered and either analyzed immediately or further purified by magnetic isolation of CD45+ leukocytes using the MACS system (Miltenyi Biotec). For the isolation of cells for chemotaxis or the analysis of molecules sensitive to dispase and trypsin (CCR4 and CCR6), skin was digested in collagenase D only. Skin T cell clones were generated by first enriching bulk dermal cells using a T cell isolation kit (Miltenyi Biotec), followed by sorting single TCR-+ cells into each well of round-bottom, 96-well plates containing 5 x 104 irradiated (40 Gy) allogeneic PBMC, 5 x 103 irradiated (70 Gy) EBV-transformed B cells, 500 ng/ml PHA, and 400 U/ml IL-2. Growing clones were expanded in 200 U/ml IL-2 and restimulated every 
3 wk. The T cell clones have been described previously (12).
Functional assays
Four-hour Transwell chemotaxis assays were performed using a 5-µm pore size polycarbonate filter (Corning Costar) using cells recovered at 37°C for 2 h before the assay. Migrated cells were collected from the lower compartment and stained and quantified by flow cytometry relative to a fixed number of polystyrene beads. Calcein release cytotoxicity assays were performed as previously described (20). The cytokine production capacity of T cell clones was determined after stimulation with PMA (20 ng/ml) and ionomycin (1 µM) for 24 h at a cell concentration of 1.6 x 106/ml.
Statistical analysis
Data given in the text are presented as the mean ± SEM. Statistical significance was assessed using unpaired Student’s t test, and p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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1+ T cells and CD16– NK cells in normal human skin
Normal skin tissue from patients undergoing mammareduction or abdominoplastic surgery was digested to release the dermal cells into suspension, which was subsequently analyzed by flow cytometry. Among the CD45+ leukocytes, populations of T cells and NK cells could be readily identified (Fig. 1a). On the average, from six skin samples, 3.8 ± 1.1% of skin leukocytes were (CD3+-TCR+) T cells, whereas 10.6 ± 0.9% were (CD3–CD56+) NK cells. T cells represented 4.7 ± 1.1% of the total cutaneous CD3+ T cells (Fig. 1b), similar to previous estimates based on immunohistochemical studies (1). The V1+ subset of T cells predominated in normal human skin, because an Ab to the V1 chain, but not an Ab to the V2 chain, stained virtually identical numbers of cells, as detected with an Ab to pan-TCR-.
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A subpopulation of cutaneous NK cells expresses receptors for homing to noninflamed skin
Based on our previous study, CCR8 and its chemokine ligand CCL1 were proposed to provide a selective homing mechanism for the entry of T cells to the skin under steady-state conditions (12). As illustrated in Fig. 2a, a population of skin NK cells also expressed CCR8. These cells differed from the majority of CCR8– NK cells by their low side-scatter profile, suggesting that the CCR8+ NK cells have reduced granularity. These two NK cell populations also differed in the expression of CLA, which was coexpressed with CCR8 on NK cells with low side scatter. The significance of the inverse relationship between CCR8 and CLA expression, on the one hand, and side scatter characteristics, on the other hand, is presently unclear. All skin NK cells, regardless of side scatter characteristics, lacked the lymph node (LN)-homing chemokine receptor CCR7. CCR8 on NK cells was functional, because it enabled freshly isolated cells to migrate toward CCL1 in a dose-dependent manner (Fig. 2b). This is in clear contrast to NK cells in peripheral blood, which completely lack CCR8 (Fig. 2c), but a subset of which express CCR7 (22).
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T cells display skin-homing properties
Peripheral blood T cells demonstrate inflammatory migration features and, thus, differ from the majority of blood T cells, which express CCR7 for continuous recirculation through secondary lymphoid tissues (23). In human skin, however, we found that the two T cell subsets largely shared a selection of tissue-homing receptors. Flow cytometric analysis (Fig. 3a) revealed that approximately half (53.1 ± 6.9%) the T cells in normal skin express CCR8, and almost all cells (88.9 ± 7.7%) were positive for CLA. In contrast, CCR7 was not detectable, and two chemokine receptors associated with T cell homing to inflamed skin (CCR4 and CCR6) (10, 11, 24) were also absent. As an internal control (12), the majority of cutaneous T cells were positive for CCR8 and CLA, and only minor fractions, or none at all, expressed CCR4, CCR6, or CCR7 (Fig. 3a). In comparison with skin, T cells in peripheral blood lacked CCR8, and only a few cells were positive for CLA (Fig. 3b), underscoring the unique nature of skin-resident T cells.
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T cells
The yield of primary T cells isolated from noninflamed skin tissue did not allow a detailed investigation; therefore, functional and phenotypic studies were performed with a panel of skin-derived T cell clones. Similar to freshly isolated skin T cells, approximately half the clones expressed readily detectable levels of CCR8 (>20% CCR8 positivity; Fig. 4a, left panel). Under the present culture conditions, CCR8– clones did not up-regulate the expression of this chemokine receptor, whereas CCR8+ clones maintained their respective levels, suggesting that CCR8 is a stable feature in distinct cutaneous T cells. As with T cells (12), the conditions required for induction of CCR8 expression in CCR8– T cells are not known. Chemotaxis assays confirmed that CCR8 on receptor-positive T cell clones was functional, as shown previously for T cell clones (12) (data not shown). In contrast, only one of the 21 clones examined expressed CCR4 (>20% CCR4 positivity; Fig. 4a, right panel), which is in keeping with the lack of skin-associated inflammatory chemokine receptors on fresh isolated cutaneous T cells. Interestingly, PHA activation of T cell clones resulted in a transient loss of cell surface CCR8 (Fig. 4b). This suggests that T cells temporarily alter their homing properties after local activation and may no longer be retained in the skin environment, but instead gain responsiveness to alternative chemokines (25). In keeping with this concept, CCR7 was strikingly up-regulated on day 3 after activation (Fig. 4b), followed by complete down-regulation as cells returned to a resting state (not shown). This observation suggests that locally activated cutaneous T cells can assist adaptive immune responses after relocation to the draining LNs, as proposed recently for blood T cells (26).
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(7–43 ng/ml) and IFN- (94–1485 ng/ml) after stimulation with PMA and ionomycin (Fig. 4c). Additionally, they secreted large amounts of CCL1 (6–81 ng/ml), suggesting that CCL1 was responsible in part for the transient loss of cell surface CCR8.
Skin T cells and NK cells have the capacity to lyse melanoma cells
To address the potential role of cutaneous T cells and NK cells in tumor surveillance, their ability to mount cytolytic responses against melanoma cells was examined. First, skin-derived T cell clones were tested in cytotoxicity assays using heterologous melanoma cell lines as targets. Of seven clones tested, five exerted robust cytotoxic responses against the melanoma cell line SK-Mel2, resulting in up to 90% target cell death at an E:T cell ratio of 25:1, whereas two clones failed to do so (Fig. 5a, left panel). Similar results were observed with the melanoma cell line HS-294 (not shown). In control experiments using skin-derived T cell clones (six CD8+ clones and four CD4+ clones), eight clones completely lacked lytic activity against SK-Mel2 and HS-294 cells (not shown), and only two clones showed borderline activity (Fig. 5a, right panel). These data are not surprising, because the mode of Ag recognition differs strikingly between T cells and T cells (13, 14), and the frequency of memory CD8+ T cells with alloreactivity is relatively low. Major mechanisms contributing to lymphocyte-mediated cytotoxicity involve perforin, which is released at the contact zone between effector and target cells (27), and activating NK receptors, such as NKG2D/CD314, which recognizes MHC-I-related MICA and MICB as well as other stress-related molecules (15, 17). Perforin was not detected in freshly isolated skin T cells (Fig. 5b). However, all 12 T cell clones tested contained detectable levels of perforin, whereas only two of 26 skin-derived T cell clones were positive (Fig. 5c). The lack of perforin in skin T cell clones is in line with their proposed involvement in the control of memory responses rather than tissue cell lysis (12). In contrast to T cells, freshly isolated T cells were uniformly positive for NKG2D and maintained this activating receptor during subsequent cell culture (Fig. 5d). NKG2D signaling in NK cells and T cells leads to direct cell activation that may result in target cell lysis, whereas cytotoxicity in CD8+ T cells is largely controlled by their clonal TCRs recognizing specific tumor Ags (28, 29). Collectively, these data suggest that T cells present in normal skin have the potential to eliminate stressed cells, including tumor cells.
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T cells, freshly isolated skin NK cells completely lacked this lytic marker (Fig. 6b, left panel). Remarkably, and in clear contrast to T cells, NKG2D could not be detected on freshly isolated skin NK cells, and its induction of expression required previous NK cell culture in the presence of IL-2. Obviously, NK cells in healthy human skin are devoid of immediate lytic activities, which is in keeping with a previous study demonstrating that the CD16– subset of NK cells within LNs fully depends on activation for mounting lytic activity (30).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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T cells and NK cells that express receptors for homing to normal skin and, in relation to their state of activation, receptors for tumor cell killing. Although the presence of V1+ T cells in the human dermis has been previously documented (1), their function and migration properties have remained elusive. Their unique TCR repertoire indicates that they are not a random sample from circulating T cells (31), a view supported by our finding that cutaneous, but not blood, T cells expressed the skin-homing receptors CCR8 and CLA. Human dermal T cells uniformly expressed the activating receptor NKG2D for killing of tumor or otherwise stressed cells (17). Although freshly isolated cutaneous T cells lacked perforin, they acquired it upon cell activation, in keeping with the observation that murine DETCs require activation to develop cytotoxic function against melanoma cells (32). In many respects, dermal T cells resemble V1+ T cells in human intestinal epithelium, which also killed heterologous intestinal tumor cells by a process involving MHC-related MICA and MICB and probably other stress-related molecules (15, 33, 34).
Similar to T cells, the nature and function of NK cells present in normal human skin are ill defined. We show in this study that these cells constitute a significant fraction of dermal leukocytes. Of note and in clear contrast to NK cells in peripheral blood and LNs (17, 30), NKG2D is completely absent in cutaneous NK cells, but is strongly induced upon in vitro culture. Similarly, perforin is present in cultured, but not freshly isolated NK cells. Obviously, healthy human dermis is devoid of NK cells with the capacity for immediate target cell lysis. The majority of cutaneous NK cells with a low side-scatter profile expressed both CCR8 and CLA, but lacked CCR7 and therefore displayed a homing phenotype highly reminiscent of cutaneous and T cells, but clearly different from that of NK cells in peripheral blood (21, 22, 30). The major subset of CD16+ NK cells in blood expresses CXCR1 and CX3CR1 for homing to inflammatory sites and contains perforin for immediate cytotoxic effector function. In contrast, the minor subset of CD16– NK cells expresses CD62L and CCR7 for recirculation through LNs and has no intracellular perforin. Cutaneous NK cells also lack CD16 and perforin expression, but differ from blood CD16– NK cells with respect to migration properties. Interestingly, normal small-intestinal epithelium was also recently shown to harbor a population of NK cells (35). Thus, in analogy to memory T cells (10, 11, 24), subsets of CD16– NK cells with individual homing preferences for normal peripheral tissues may exist.
In contrast to the rapid recruitment of effector lymphocytes to sites of skin inflammation (9, 24), the mechanisms controlling the steady-state traffic of memory T cells and their maintenance within healthy tissue are not well understood (10, 11). CCL1, the single ligand for CCR8, is produced in normal healthy skin, notably by vascular and perivascular cells within superficial dermal plexus and by epidermal Langerhans cells and melanocytes (12). This chemokine system may regulate the recruitment and cutaneous positioning of blood CCR8+ T cells and, as proposed in this study, a large fraction of cutaneous T cells and NK cells. Yet, we still have little knowledge about the traffic parameters and the life span of cutaneous lymphocytes (36, 37). The scarcity of CCR8+ lymphocytes within peripheral blood may indicate that the skin is their primary site of residence. In preliminary coculture experiments, we have noticed that dermal fibroblasts supported the slow proliferation of freshly isolated cutaneous T cells and NK cells (L. M. Ebert and B. Moser, unpublished observations). Skin is a rich source of growth factors and, in analogy to bone marrow (38), may support homeostatic expansion in local lymphocytes. It is possible that skin lymphocyte numbers are maintained by several factors, including rates of skin recruitment and exit as well as the rates of local survival and proliferation.
Distinct lymphocyte populations within healthy skin are likely to play a crucial role in local immune surveillance. Skin-tropic memory T cells, also referred to as peripheral immune surveillance T cells (11, 12), may contribute to memory responses against previously encountered pathogens. However, cytotoxic peripheral immune surveillance T cells with selectivity for skin tumors appear to be rare, suggesting that they may not participate in tumor surveillance. Our present study has characterized for the first time populations of human skin-homing T cells and NK cells with the potential to elaborate lytic functions, which may involve molecules commonly associated with situations of tissue stress, such as those present on transformed or virally infected cells (13, 14, 15, 16, 17). Furthermore, we have shown that these three distinct subsets of cutaneous lymphocytes share a common address code, enabling their colocalization within noninflamed skin. Due to their specialization in Ag recognition and function, these diverse cell types may cooperate in cutaneous immune surveillance against a wide variety of challenges.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 Current address: Ludwig Institute for Cancer Research (Melbourne Branch), Heidelberg, Victoria, 3084 Australia. 
2 Address correspondence and reprint requests to Dr. Bernhard Moser, Institute of Cell Biology, University of Bern, Baltzerstrasse 4, CH-3012 Bern, Switzerland. E-mail address: bernhard.moser{at}izb.unibe.ch
3 Abbreviations used in this paper: DC, dendritic cell; DETC, dendritic epidermal T cell; LN, lymph node; MICA, MHC-I-related protein A; MICB, MHC-I-related protein B.
Received for publication August 23, 2005. Accepted for publication January 17, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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bearing cells in normal human skin. J. Invest. Dermatol. 94: 37-42. [Medline] cells: a right time and a right place for a conserved third way of protection. Annu. Rev. Immunol. 18: 975-1026. [Medline] T cells: functional plasticity and heterogeneity. Nat. Rev. Immunol. 2: 336-345. [Medline] T cells. Science 279: 1737-1740. T cells: a nonredundant system in the immune-surveillance against cancer. Trends Immunol. 23: 14-18. [Medline] T cells. Science 294: 605-609. T-cell involvement in humoral immunity. Blood 102: 3693-3701. T cells. Science 309: 264-268. 2V2 T cells enhances their antigen-dependent effector function. Immunity 15: 83-93. [Medline] repertoire in normal human skin is restricted and distinct from the TCR- repertoire in the peripheral blood. J. Invest. Dermatol. 116: 275-280. [Medline] T cells. J. Immunol. 169: 1236-1240. This article has been cited by other articles:
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R. E. Mills, K. R. Taylor, K. Podshivalova, D. B. McKay, and J. M. Jameson Defects in Skin {gamma}{delta} T Cell Function Contribute to Delayed Wound Repair in Rapamycin-Treated Mice J. Immunol., September 15, 2008; 181(6): 3974 - 3983. [Abstract] [Full Text] [PDF]
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J. Holderness, L. Jackiw, E. Kimmel, H. Kerns, M. Radke, J. F. Hedges, C. Petrie, P. McCurley, P. M. Glee, A. Palecanda, et al. Select Plant Tannins Induce IL-2R{alpha} Up-Regulation and Augment Cell Division in {gamma}{delta} T Cells J. Immunol., November 15, 2007; 179(10): 6468 - 6478. [Abstract] [Full Text] [PDF]
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P. Schaerli, L. M. Ebert, and B. Moser Comment on "The Vast Majority of CLA+ T Cells Are Resident in Normal Skin" J. Immunol., August 1, 2006; 177(3): 1375 - 1376. [Full Text] [PDF]
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), and total migrated NK cells (CD3– CD56+) were enumerated using flow cytometry relative to a standard number of polystyrene beads. c, PBMC were analyzed by flow cytometry, gating on CD3–CD56+ NK cells (n = 4).
