HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jameson, J. M. Articles by Havran, W. L. Search for Related Content PubMed PubMed Citation Articles by Jameson, J. M. Articles by Havran, W. L.

A Keratinocyte-Responsive TCR Is Necessary for Dendritic Epidermal T Cell Activation by Damaged Keratinocytes and Maintenance in the Epidermis¹

Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
A unique population of T lymphocytes, designated dendritic epidermal T cells (DETC), homes to the murine epidermis during fetal development. DETC express a canonical TCR, V3/V1, which recognizes Ag expressed on damaged, stressed, or transformed keratinocytes. Recently, DETC were shown to play a key role in the complex process of wound repair. To examine the role of the DETC TCR in DETC localization to the epidermis, maintenance in the skin, and activation in vivo, we analyzed DETC in the TCR^-/- mouse. Unlike previous reports in which the TCR^-/- skin was found to be devoid of any DETC, we discovered that TCR^-/- mice have TCR-expressing DETC with a polyclonal V chain repertoire. The DETC are not retained over the life of the animal, suggesting that the TCR is critical for the maintenance of DETC in the skin. Although the DETC can be activated in response to direct stimulation, they do not respond to keratinocyte damage. Our results suggest that a keratinocyte-responsive TCR is necessary for DETC activation in response to keratinocyte damage and for DETC maintenance in the epidermis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although T lymphocytes comprise most of the T cells in the peripheral blood and lymphoid system, T lymphocytes make up the majority in certain epithelial tissues such as the skin (1, 2). A unique population of T lymphocytes, designated dendritic epidermal T cells (DETC),³resides in the murine skin. DETC were first discovered as Thy-1 positive cells (3, 4) and were later found to have a canonical V3J1C1/V1D2J2C TCR with no junctional diversity (5). Because DETC have such a conserved TCR, it has been suggested that they have a limited Ag repertoire. DETC respond to Ag expressed on damaged, stressed, or transformed keratinocytes in vitro by means of their TCR (6, 7). DETC and keratinocytes also interact in vivo, because DETC play roles in wound repair through the production of keratinocyte growth factors (8). The TCR is likely used in the activation of DETC during wound healing, because TCR^-/- mice have defects in wound repair (8). However, the actual role of the TCR in DETC localization to the skin, maintenance in the epidermis, and activation in vivo is still unknown.

During development, T cells go through a programmed rearrangement of the TCR and subsequently migrate to various epithelial tissues (5, 9, 10, 11, 12, 13, 14, 15). The first wave of rearrangements occurs at fetal days 14–16, resulting in V3/V1 TCR-expressing thymocytes that migrate to the epidermis (5, 9). This precisely timed rearrangement and subsequent migration suggests that the TCR may be important in the final localization of T cells to the skin. However, DETC expressing a unique TCR were still detected in the skin of V1^-/- and V3^-/- mice, suggesting that the canonical V3/V1 TCR is not necessary for DETC migration (16, 17). Interestingly, these replacement DETC retained keratinocyte reactivity, and some could still bind the V3/V1-specific Ab, indicating that TCR conformation may be important in the localization of DETC to the skin. Yet V2/V5 TCR transgenic mice have DETC expressing the transgene in the skin, and these DETC are unresponsive to keratinocytes (18). These results suggest that neither the V3/V1 TCR nor a TCR with keratinocyte specificity is necessary for localization to the epidermis.

The importance of the DETC TCR in homing was revisited when it was found that endogenous -chain rearrangement can still occur in TCR transgenic mice (19). Ferrero et al. (20) proposed that the endogenous V1 rearrangement may permit DETC to migrate to the skin even in TCR transgenic mice. They did not detect DETC in TCR^-/- or TCR transgenic TCR^-/- mice and concluded that V1 is necessary for localization to the skin. Clearly, the role of the TCR and TCR specificity in migration to the epidermis is still unresolved.

Wild-type DETC home to the epidermis and reside there over the life of the mouse. It is not known whether keratinocyte Ag recognition by the TCR is necessary for DETC to be maintained in the epidermis over time. When TdT expression was forced early in the fetal thymus, DETC with variable junctional segments were detected in the epidermis of adult mice (21). However, the wild-type invariant V3/V1 DETC had a much greater proliferative advantage in adults, suggesting that DETC need the keratinocyte-responsive TCR to be maintained in the epidermis over time. This is also supported by the findings that skin from V3^-/- or V1^-/- mice still contains DETC with keratinocyte-responsive TCRs (16, 17). Therefore, TCR recognition of the keratinocyte Ag may be important for DETC maintenance in the epidermis.

DETC play a critical role in wound repair (8). The functional response of DETC to keratinocyte damage likely requires the TCR, because TCR^-/- mice have a delay in wound repair, and DETC are known to require the TCR for recognition of damaged keratinocytes in vitro (6, 8). Therefore, the TCR may be used by DETC to become activated during wound-healing processes in vivo. To examine whether DETC reside in the epidermis of TCR^-/- mice, whether they are retained over time, and whether they are activated by damaged keratinocytes during wound repair, we examined DETC in TCR^-/- mice. Our results suggest that a keratinocyte Ag-responsive TCR is not necessary for DETC to home to the epidermis, but is necessary for DETC to become activated during wound repair and to be maintained in the epidermis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

TCR^-/- mice on the C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). Both TCR^-/- and C57BL/6 mice were bred in our facility. All mice were housed in specific pathogen-free conditions at The Scripps Research Institute animal facility. Mice were used at 10–14 wk of age.

Antibodies

The following Abs were used to stain epidermal sheets or epidermal cell suspensions: PE- TCR (GL3), PE-CD3 (145-2C11), PE-CD103 (M290), PE-CD49b (HM-a2), PE-CD49d (R1-2), PE-V2 (B20.1), PE-CD25 (PC61), PE-CD69 (H1.2F3), PE-CD24 (M1/69), PE-CD4 (GK1.5), PE-TNF- (MP6-XT22), FITC- TCR (H57-597), FITC-CD44 (IM7), FITC-CD8 (53-6.7), FITC-Thy1.2 (53-2.1), FITC-V screening panel (catalog no. 0143KK), and allophycocyanin-Thy1.2 (53-2.1) (BD PharMingen, La Jolla, CA). FITC-CD5 (53-70.3) was obtained from eBioscience (San Diego, CA). Anti-FcR chain (FcRI) sera was collected from peptide-inoculated rabbits and used with FITC-goat anti-rabbit secondary Ab (Jackson ImmunoResearch, West Grove, PA). Preimmune sera from the rabbits were collected and used as a control.

Wounding procedure

Mice were anesthetized with isoflurane. The mouse backs were shaved, back skin was pulled up, and a sterile 3-mm punch tool was used to create three sets of full-thickness wounds as previously described (8). Mice were caged individually, and wounds were left uncovered.

Epidermal sheet preparation and immunofluorescent staining

Epidermal sheets were prepared as previously reported (8). Briefly, following hair removal, ears were excised and split into dorsal and ventral halves. The dermal side was exposed to 3.8% ammonium thiocyanate in PBS for 15 min, and epidermal sheets were separated from dermis and then washed in PBS. The epidermal sheets were fixed in acetone and incubated in 1–2 µg/ml Ab for 1 h at 37°C. Sheets stained with cholera toxin B (Sigma-Aldrich, St. Louis, MO) were incubated in 0.0625 µg/ml fluorescein-labeled cholera toxin B. The stained sheets were rinsed in PBS and mounted on slides with anti-fade mounting medium (DAKO, Carpinteria, CA).

Epidermal cell suspensions

Epidermal cells were prepared from the skin of wild-type and TCR^-/- mice (8). Skin from wounded mice was excised including a 3-mm border around the wound. Briefly, skin was incubated on 0.3% trypsin/GNK solution for 1–2 h at 37°C, and the epidermis was separated from the dermis. Epidermis was incubated for 10 min in 0.3% trypsin/GNK solution at 37°C for two sequential treatments. The epidermal preparation was enriched for live cells by centrifugation over lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada), and cells recovered from the interface were either immediately stained for FACS analysis or incubated overnight with or without 5 µg/ml Con A (Sigma-Aldrich).

FACS analysis

Expression of surface markers was examined by staining cells with 0.5 µg/ml Ab in PBS containing 2% FCS and 0.2% NaN₃ for 20 min at 4°C. Intracellular staining was performed using a kit (Caltag, Burlingame, CA) following manufacturer’s directions. Cells were analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences, Franklin Lakes, NJ).

Immunofluorescent microscopy

For the enumeration of DETC, x200 images of at least seven random fields were taken of each epidermal sheet, and at least four were counted in a grid. Three mice were examined from each strain per time point. Mean values ± SD were calculated, and density was expressed as the number of DETC per square millimeter.

To quantify lipid raft polarization, full-thickness wounds were made in each ear with a punch biopsy tool, and epidermal sheets were prepared 15–20 h later. Epidermal sheets were stained with fluorescein-labeled cholera toxin B and Abs specific for the TCR. Images of lipid rafts were taken with a digital fluorescence microscopy system using SPOT 32 software (Diagnostic Instruments, Sterling Heights, MI). Random cells near the wound site were examined at x1000 magnification and recorded as polarized or not polarized. Over 100 cells from at least six wounded ears were counted for each mouse strain. Cells away from the wound site or observed in nonwounded mice were not round and did not have polarized lipid rafts.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR-expressing DETC reside in the TCR^-/- epidermis

To examine whether DETC are present in the epidermis of TCR^-/- mice, epidermal sheets were prepared and stained with Abs specific for the or TCR. As previously reported (22, 23), DETC that express the TCR reside in the epidermis of TCR^-/- mice (Fig. 1). This contradicts findings by Ferrero et al. (20) in which no DETC were detected in the epidermis of TCR^-/- mice. To verify our results, we performed these experiments on two different sets of mice obtained directly from The Jackson Laboratory and detected DETC by both immunofluorescent staining of epidermal sheets (Fig. 1) and flow cytometry of isolated epidermal cell suspensions (Fig. 2). All DETC expressed CD3, and all CD3-expressing DETC were TCR positive (data not shown). TCR^-/- mice have approximately half the number of DETC as compared with wild-type mice. Furthermore, the DETC appear slightly less dendritic in morphology and reside in clumps of cells that do not uniformly cover the epidermis as compared with those of wild-type animals (Figs. 1 and 3).

View larger version (77K): [in this window] [in a new window]   FIGURE 1. DETC reside in the skin of TCR-/- mice. Ear epidermal sheets were prepared from wild-type (left) and TCR-/- (right) skin. The tissue was stained with Abs specific for TCR (top), TCR (second row), CD3 (third row), CD103 (fourth row), and Thy1.2 (bottom). Pictures were taken at x200 magnification.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. DETC express similar surface markers to wild-type DETC. A, Epidermal cells were isolated from TCR-/- skin and stained with CD4, CD8, CD8, and CD3, and gated on CD3-positive events. B, Wild-type (upper panels) or TCR-/- (lower panels) epidermal cells were stained with CD44, heat-stable Ag (HSA), CD103, CD3, Thy1.2, and/or TCR, and gated on CD3 and Thy1.2 double-positive events. C, CD44 is expressed on and DETC. Ear epidermal sheets were isolated, and the tissue was stained with Abs specific for CD44. All CD44-positive cells were also TCR positive (data not shown). Digital images were obtained at x1000 magnification. These results are representative of at least three separate experiments.

View larger version (37K): [in this window] [in a new window]   FIGURE 3. The DETC V repertoire is diverse, and the number of DETC decreases with age in TCR-/- mice. A, Epidermal cells were prepared from TCR-/- skin. The cells were stained simultaneously with fluorescent Abs specific for CD3, Thy1.2, and the mouse V TCR screening panel. Epidermal () and splenic () cells were analyzed by flow cytometry. The Thy1.2+CD3+ lymphocytes were gated, and percentages of V-positive cells were calculated. These experiments were performed on at least seven mice. B, Ear epidermal sheets were stained with Abs specific for V2 or V5. Pictures were taken at x200 magnification. C, Ear epidermal sheets from wild-type (•) and TCR-/- () mice of various ages were stained with Abs specific for CD3. Pictures were obtained at x200 magnification, and DETC were counted on a grid. At least three mice were examined per strain per time point, and at least nine grids were counted per mouse. Mean values ± SD were calculated, and density is expressed as the number of DETC per square millimeter.

DETC are phenotypically similar to wild-type DETC

DETC are phenotypically similar to wild-type DETC as determined by the analysis of epidermal sheets and epidermal cells by immunofluorescence or flow cytometry (Figs. 1 and 2). One interesting difference between the two populations of DETC is the expression of CD8 on a subset of the DETC, whereas DETC are CD8^- (Fig. 2A). CD8 is also commonly expressed on gut IELs (26). Like DETC from wild-type mice, DETC from TCR^-/- mice have constitutive expression of CD44 and CD3, no expression of CD5, and low expression of CD24 (Figs. 1 and 2, B and C; data not shown). Taken together, phenotypic analysis of DETC suggests that they express similar surface molecules to wild-type DETC, except for TCR expression and a subpopulation with CD8 expression.

Unlike wild-type DETC, the DETC TCR repertoire is diverse

We examined V chain usage by DETC isolated from TCR^-/- mice to determine whether they retain a conserved TCR repertoire that may recognize damaged keratinocytes. V chain usage by DETC was examined by flow cytometry in comparison to splenic cells isolated from the same mouse. In all mice except one, individual V chains were expressed proportionally by DETC and splenic T cells (Fig. 3A). This indicates that the DETC V repertoire is as polyclonal and diverse as the spleen repertoire. For example, the major V chains used in the spleen are V8.1 and -8.2, and they are also used most frequently by the DETC isolated from the TCR^-/- mice (Fig. 3A). One mouse demonstrated preferential usage of the V8.1/8.2 family in the epidermis that differed from the spleen (Fig. 3A3). However, this skewing was not detected in any of the other seven mice tested. Therefore, a conserved conformation of TCR is not necessary for the homing of DETC to the epidermis. Furthermore, the highly diverse population of DETC suggests that they do not recognize one conserved keratinocyte Ag like the wild-type DETC.

The keratinocyte-responsive TCR is not necessary for DETC proliferation but is required for DETC maintenance in the epidermis over time

To determine whether DETC can proliferate and be maintained in the epidermis, we analyzed the density of DETC in young and aged mice. The number of DETC per square millimeter was examined by immunofluorescence and counted in a grid. Like wild-type DETC, the DETC in TCR^-/- mice appear in clusters in the epidermis soon after birth. The cells within each cluster express the same TCR (Fig. 3B) and increase in number over the first few months (C). This suggests that the canonical TCR is not necessary for DETC to proliferate in the epidermis, even though it takes longer for the DETC to accumulate, and the number of DETC never reaches that of DETC in wild-type mice (Fig. 3C). Adult TCR^-/- mice have about half the number of DETC as compared with wild-type (Fig. 3C). Thus, initial proliferation allows for the accumulation of DETC; however, the wild-type TCR is necessary for full establishment of the population.

The number of DETC in wild-type mouse skin remained constant in young and aged animals (Fig. 3C). In contrast, the number of DETC in TCR^-/- skin decreased significantly by 1 year of age (Fig. 3C). By immunofluorescence, we detected fewer cells in the clusters and more distance between each cluster. This suggests that, even though the wild-type TCR is not necessary for DETC to localize to the epidermis, it is necessary for DETC to be retained there over the life of the animal. Our results indicate that the maintenance of DETC in the skin may require a TCR that recognizes the Ag expressed by damaged or stressed keratinocytes.

Most DETC express a functional TCR complex

To determine whether the DETC had a functional TCR, we examined the TCR complex and activation potential of DETC. Unlike peripheral T cells, wild-type DETC express FcRI as a homodimer or in association with the -chain of the CD3 complex (27, 28). To find out whether DETC have the same TCR components as wild-type DETC, we stained epidermal cells and epidermal sheets with Abs to FcRI. We discovered that the majority of DETC express this signaling component (Fig. 4). There was a small population of TCR-expressing DETC that did not express FcRI, and a small population of non-CD3-expressing cells that expressed FcRI (Fig. 4). The non-CD3-expressing cells are not dendritic cells (data not shown) and were not detected in wild-type mice. Thus, they make-up a Thy1.2-positive, TCR-negative subset we have previously detected in the TCR^-/- epidermis (data not shown) that may be similar to cells reported in nude mice (29). The majority of DETC were FcRI positive; this suggests that any defect in DETC activation is not due to the lack of TCR-associated signaling proteins on the DETC.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Most DETC express FcRI. A, Epidermal cells were prepared from wild-type and TCR-/- skin. Cells were stained with Abs specific for CD3, Thy1.2, and FcRI. The cells are gated on CD3 and Thy1.2 double-positive cells. Controls with preimmune serum are shown in gray. B, FcRI is expressed by both and DETC. Epidermal sheets were simultaneously stained with Abs specific for FcRI (green) and CD3 (red). These results are representative of at least three separate experiments.

View larger version (30K): [in this window] [in a new window]   FIGURE 5. DETC are activated upon direct stimulation, but not during wound repair. A, Epidermal cells were isolated from wild-type (upper panels) or TCR-/- (lower panels) skin, cultured with or without Con A, and stained with fluorescent Abs specific for CD25 or CD69, or TCR, and Thy1.2. Cells were analyzed by flow cytometry and gated on TCR, Thy1.2 double-positive cells. Resting DETC are represented by a dotted line, and activated DETC are represented by a solid black line. Unstained control cells are represented by a filled gray histogram. B, Epidermal cells were isolated from TCR-/- skin and stimulated with Abs that recognize CD3. The cells were stained simultaneously with Abs specific for TNF-, CD3, and Thy1.2. Double-positive CD3 and Thy1.2 events were gated. C, Wild-type and TCR-/- mice were wounded, and epidermal cells were isolated from the skin 24 h later. Cells were stained with Abs specific for CD25, CD3 (for TCR-/-) or TCR (for wild type), and Thy1.2. Cells were gated on CD3-positive or TCR-positive lymphocytes and analyzed by flow cytometry. These results are representative of at least two separate experiments.

DETC do not recognize damaged keratinocytes during wound repair

Because the DETC express a functional TCR, we examined whether this TCR can be used to recognize and respond to damaged keratinocytes like the wild-type TCR. Epidermal cells were isolated from wild-type and TCR^-/- mice following wounding and examined for up-regulation of the early activation marker CD25. Wild-type DETC up-regulated CD25 in response to keratinocyte damage, most notably at 1 day postwounding, whereas CD25 expression on DETC was unchanged (Fig. 5C). Similar data was obtained at 48-h postwounding; however, there were fewer cells up-regulating CD25 in wild-type mice. As a further measure of activation, cytokine production during wound repair was examined. TNF- production and integrin (CD49b and CD49d) up-regulation were also impaired in DETC (data not shown), as compared with wild-type DETC during wound repair. These results demonstrate that, although the TCR is functional, it cannot be used to recognize damaged keratinocytes.

To analyze the responsiveness of DETC directly in the tissue, we prepared epidermal sheets of wounded ears from wild-type and TCR^-/- mice. Cholesterol-enriched membrane microdomains called lipid rafts are regions used for cell signaling. Lipid rafts form platforms for signaling during T cell activation, which contain the TCR and localize to the site of contact. We examined lipid raft polarization by DETC in response to damaged keratinocytes by wounding ears and staining the wounded epidermis in situ with cholera toxin B, which labels sphingomyelin/cholesterol lipid domains. The wild-type DETC clearly polarized lipid rafts at the wound site in response to the damaged keratinocytes (Fig. 6). The cholera toxin B colocalized with the TCR at these polarized sites. Wild-type DETC away from the wound site did not have polarized lipid rafts. In contrast, the DETC from TCR^-/- mice did not polarize lipid rafts either at or away from the wound site (Fig. 6). This clearly indicates that the DETC do not respond to damaged keratinocytes in vivo. Taken together, these data suggest that DETC need more than a functional TCR to respond to damaged keratinocytes during wound repair. The TCR must also have a conformation that recognizes Ag expressed by damaged keratinocytes in order for the cells to become activated and produce cytokines during wound repair.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. DETC do not polarize lipid rafts in response to damaged keratinocytes. Mouse ears were wounded with a punch biopsy. Epidermal sheets were stained with fluorescent Abs specific for the TCR complex, and digital images were taken. A and B, Wild-type and TCR-/- mice have round DETC at the wound site 24–48 h postwounding as visualized by staining with Abs specific for the (red) or (green) TCRs (x200 magnification; 48 h postwounding). The arrow indicates the wound edge (A and B). C and D, The polarization of lipid rafts at the wound was viewed in ear epidermal sheets 15–20 h postwounding by CD3 and cholera toxin B fluorescein staining (x1000 magnification). The number of polarized rafts was quantitated as a percentage of total. Digital images were obtained at x1000 magnification.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

An alternative mechanism for DETC homing to the epidermis may be expression of integrins that direct DETC to the epidermis. We analyzed expression of CD103, which is an integrin suggested to have a role in the homing of IELs to the gut epithelia (24) and skin (25). We found that both wild-type and DETC express CD103. CD103 is down-regulated upon DETC activation (data not shown). This correlates with our findings that DETC activated by keratinocyte damage in vivo will round-up, which may be due to a decrease in CD103 expression as well (8). Taken together, this may indicate that CD103 may be involved in the attachment and retention of DETC within the epidermis. Because CD103 can bind to E-cadherin, these integrins may have a role together in DETC homing to the skin.

After DETC localize to the epidermis in wild-type mice, they proliferate rapidly and reach a uniform density by 3 wk. We detected an increase in the number of DETC between 3 wk and 3 mo of age, suggesting that the cells can proliferate. The DETC appeared to be proliferating in situ and not simply continually migrating to the skin, because clusters of cells with the same V or V increased in cell number. Thus, the V3/V1 TCR is not necessary for slow proliferation of DETC in the epidermis. However, the DETC do not reach maximal density like wild-type DETC; therefore, a keratinocyte-responsive TCR may be necessary for DETC maintenance in the skin. Once DETC localize to the epidermis in wild-type mice, they maintain a population there for the life of the mouse. In contrast, the number of DETC in the TCR^-/- mice decreases by almost 50% within 1 year of age. It is possible that, due to a lack of TCR stimulation, DETC do not keep up with the loss of epidermal cells over time. This supports the hypothesis that a keratinocyte-responsive TCR may be necessary for the maintenance of DETC in the epidermis. The TCR may provide the stimulus necessary for proliferation and survival in the epidermis.

We analyzed the components of the TCR complex on DETC and found expression of the FcRI chain as seen in the wild-type DETC TCR complex. In rare cases, FcRI has been associated with the TCR in T cells. For example, early TCR expression in some TCR transgenic mice causes double-negative T cells to express the FcRI as part of the CD3 signaling complex (30). The lack of -chain rearrangement may have caused early TCR expression and allowed the FcRI to be a part of the epidermal T cell signaling complex. The DETC can be activated through their TCR and use the same TCR components as wild-type DETC. Therefore, the DETC have a functional TCR complex that can respond to direct stimulation.

In mice lacking V1 or V3, DETC in the epidermis have a surprisingly conserved TCR repertoire, and many cells retain a TCR conformation specific for stressed keratinocytes (16, 17). DETC express a broad range of TCRs and do not become activated by damaged keratinocytes that normally stimulate wild-type V3V1 DETC, suggesting that DETC do not have the TCR conformation used by wild-type DETC. We have previously shown that TCR^-/- mice have defects in wound repair and a lack of keratinocyte growth factor production at the wound site (8). Our results suggest that, although DETC have the ability to become activated like wild-type DETC, without the keratinocyte-responsive TCR they may not be able to respond to keratinocyte damage in vivo. We have a unique epidermal peeling procedure that allows us to examine the DETC as they function in the tissue itself. Lipid rafts polarize to the site of T cell stimulation upon activation by an APC. The TCR and associated complex are translocated into the lipid rafts to strengthen the signal. Using epidermal peels, we were able to identify this early stage of DETC activation as it occurs temporally during wound repair. Translocation of the lipid rafts to the site of keratinocyte damage was defective in DETC from TCR^-/- mouse wounds. Other obvious signs of T cell activation, such as IL-2R up-regulation and cytokine production, were also defective in DETC isolated directly from the wound sites. Therefore, our results suggest that a keratinocyte-responsive TCR is necessary for DETC activation by damaged keratinocytes during wound repair and to be maintained in the epidermis.

DETC have intimate interactions with epithelial cells in vivo and have been shown to play a role in keratinocyte proliferation during wound repair (8). DETC are also involved in the regression of skin tumors (31) and in the suppression of cutaneous graft-vs-host disease (23). DETC are representative of epithelial T cells in other tissues such as the gastrointestinal tract where IELs are important in repair from dextran sodium sulfate-induced colitis (32). We have shown that, although the TCR may not be involved in the homing of DETC to the epidermis, it plays a key role in activation in vivo and maintenance of DETC in the epidermis. Our results establish a role for the DETC TCR in vivo that may be useful in the future design of therapies that involve intraepithelial T lymphocytes.

	Acknowledgments

 
We thank Marieke Svoboda and Dr. Stephanie Rieder for laboratory assistance and manuscript review. We also thank Dr. R. Ulevitch for use of his immunofluorescent microscope and digital camera.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants 5T32AI-07244 (to J.M.J.), AI-32751 (to W.L.H.), and AI-36964 (to W.L.H.), and the Leukemia and Lymphoma Society (to W.L.H. and J.M.J.). This is manuscript number 15543-IMM from The Scripps Research Institute.

² Address correspondence and reprint requests to Dr. Wendy L. Havran, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: havran{at}scripps.edu

³ Abbreviations used in this paper: DETC, dendritic epidermal T cell; IEL, intraepithelial lymphocyte.

Received for publication November 5, 2003. Accepted for publication January 7, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Allison, J. P., D. M. Asarnow, M. Bonyhadi, A. Carbone, W. L. Havran, D. Nandi, J. Noble. 1991. T cells in murine epithelia: origin, repertoire, and function. Adv. Exp. Med. Biol. 292:63.[Medline]
2. Hayday, A. C.. 2000. Cells: a right time and a right place for a conserved third way of protection. Annu. Rev. Immunol. 18:975.[Medline]
3. Bergstresser, P. R., R. E. Tigelaar, J. H. Dees, J. W. Streilein. 1983. Thy-1 antigen-bearing dendritic cells populate murine epidermis. J. Invest. Dermatol. 81:286.[Medline]
4. Tschachler, E., G. Schuler, J. Hutterer, H. Leibl, K. Wolff, G. Stingl. 1983. Expression of Thy-1 antigen by murine epidermal cells. J. Invest. Dermatol. 81:282.[Medline]
5. Asarnow, D. M., W. A. Kuziel, M. Bonyhadi, R. E. Tigelaar, P. W. Tucker, J. P. Allison. 1988. Limited diversity of antigen receptor genes of Thy-1⁺ dendritic epidermal cells. Cell 55:837.[Medline]
6. Havran, W. L., Y.-H. Chien, J. P. Allison. 1991. Recognition of self antigens by skin-derived T cells with invariant antigen receptors. Science 252:1430.[Abstract/Free Full Text]
7. Reardon, C. L., K. Heyborne, M. Tsuji, F. Zavala, R. E. Tigelaar, R. L. O’Brien, W. K. Born. 1995. Murine epidermal V5/V1-T-cell receptor⁺ T cells respond to B-cell lines and lipopolysaccharides. J. Invest. Dermatol. 105:58S.[Medline]
8. Jameson, J., K. Ugarte, N. Chen, P. Yachi, E. Fuchs, R. Boismenu, W. L. Havran. 2002. A role for skin T cells in wound repair. Science 296:747.[Abstract/Free Full Text]
9. Havran, W. L., S. C. Grell, G. Duwe, J. Kimura, A. Wilson, A. M. Kruisbeek, R. L. O’Brien, W. Born, R. E. Tigelaar, J. P. Allison. 1989. Limited diversity of TCR -chain expression of murine Thy-1⁺ dendritic epidermal cells revealed by V3-specific monoclonal antibody. Proc. Natl. Acad. Sci. USA 86:4185.[Abstract/Free Full Text]
10. Nandi, D., J. P. Allison. 1991. Phenotypic analysis and T cell receptor repertoire of murine T cells associated with the vaginal epithelium. J. Immunol. 147:1773.[Abstract]
11. Takagaki, Y., A. DeCloux, M. Bonneville, S. Tonegawa. 1989. Diversity of T-cell receptors on murine intestinal intraepithelial lymphocytes. Nature 339:712.[Medline]
12. Penninger, J., V. A. Wallace, K. Kishihara, T. Molina, H. Krause, T. W. Mak. 1991. Molecular organization, ontogeny and expression of murine and T cell receptors. Exp. Clin. Immunogenet. 8:57.[Medline]
13. Havran, W. L., J. P. Allison. 1988. Developmentally ordered appearance of thymocytes expressing different T cell antigen receptors. Nature 335:443.[Medline]
14. Haas, W., P. Pereira, S. Tonegawa. 1993. Cells. Annu. Rev. Immunol. 11:637.[Medline]
15. Bonneville, M., C. A. Janeway, Jr, K. Ito, W. Haser, I. Ishida, N. Nakanishi, S. Tonegawa. 1988. Intestinal intraepithelial lymphocytes are a distinct set of T cells. Nature 336:479.[Medline]
16. Mallick-Wood, C. A., J. M. Lewis, L. I. Richie, M. J. Owen, R. E. Tigelaar, A. C. Hayday. 1998. Conservation of T cell receptor conformation in epidermal cells with disrupted primary V gene usage. Science 279:1729.[Abstract/Free Full Text]
17. Hara, H., K. Kishihara, G. Matsuzaki, H. Takimoto, T. Tsukiyama, R. E. Tigelaar, K. Nomoto. 2000. Development of dendritic epidermal T cells with a skewed diversity of TCRs in V1-deficient mice. J. Immunol. 165:3695.[Abstract/Free Full Text]
18. Bonneville, M., S. Itohara, E. G. Krecko, P. Mombaerts, I. Ishida, M. Katsuki, A. Berns, A. G. Farr, C. A. Janeway, Jr, S. Tonegawa. 1990. Transgenic mice demonstrate that epithelial homing of T cells is determined by cell lineages independent of T cell receptor specificity. J. Exp. Med. 171:1015.[Abstract/Free Full Text]
19. Sleckman, B. P., B. Khor, R. Monroe, F. W. Alt. 1998. Assembly of productive T cell receptor variable region genes exhibits allelic inclusion. J. Exp. Med. 188:1465.[Abstract/Free Full Text]
20. Ferrero, I., A. Wilson, F. Beermann, W. Held, H. R. MacDonald. 2001. T cell receptor specificity is critical for the development of epidermal T cells. J. Exp. Med. 194:1473.[Abstract/Free Full Text]
21. Aono, A., H. Enomoto, N. Yoshida, K. Yoshizaki, T. Kishimoto, T. Komori. 2000. Forced expression of terminal deoxynucleotidyl transferase in fetal thymus resulted in a decrease in T cells and random dissemination of V3V1 T cells in skin of newborn but not adult mice. Immunology 99:489.[Medline]
22. Itohara, S., P. Mombaerts, J. Lafaille, J. Iacomini, A. Nelson, A. Clarke, M. Hooper, A. Farr, S. Tonegawa. 1993. T cell receptor gene mutant mice: independent generation of T cells and programmed rearrangements of TCR genes. Cell 72:337.[Medline]
23. Shiohara, T., N. Moriya, J. Hayakawa, S. Itohara, H. Ishikawa. 1996. Resistance to cutaneous graft-vs-host disease is not induced in T cell receptor gene-mutant mice. J. Exp. Med. 183:1483.[Abstract/Free Full Text]
24. Schon, M. P., A. Arya, E. A. Murphy, C. M. Adams, U. G. Strauch, W. W. Agace, J. Marsal, J. P. Donohue, H. Her, D. R. Beier, et al 1999. Mucosal T lymphocyte numbers are selectively reduced in integrin _E (CD103)-deficient mice. J. Immunol. 162:6641.[Abstract/Free Full Text]
25. Lefrancois, L., T. A. Barrett, W. L. Havran, L. Puddington. 1994. Developmental expression of the _IEL7 integrin on T cell receptor and T cell receptor T cells. Eur. J. Immunol. 24:635.[Medline]
26. LeFrancois, L.. 1991. Phenotypic complexity of intraepithelial lymphocytes of the small intestine. J. Immunol. 147:1746.[Abstract]
27. Ohno, H., S. Ono, N. Hirayama, S. Shimada, T. Saito. 1994. Preferential usage of the Fc receptor chain in the T cell antigen receptor complex by T cells localized in epithelia. J. Exp. Med. 179:365.[Abstract/Free Full Text]
28. Park, S. Y., H. Arase, K. Wakizaka, N. Hirayama, S. Masaki, S. Sato, J. V. Ravetch, T. Saito. 1995. Differential contribution of the FcR chain to the surface expression of the T cell receptor among T cells localized in epithelia: analysis of FcR -deficient mice. Eur. J. Immunol. 25:2107.[Medline]
29. Nixon-Fulton, J. L., W. A. Kuziel, B. Santerse, P. R. Bergstresser, P. W. Tucker, R. E. Tigelaar. 1988. Thy-1⁺ epidermal cells in nude mice are distinct from their counterparts in thymus-bearing mice: a study of morphology, function, and T cell receptor expression. J. Immunol. 141:1897.[Abstract]
30. Petersson, K., F. Ivars. 2001. Early TCR expression promotes maturation of T cells expressing FcRI containing TCR/CD3 complexes. J. Immunol. 166:6616.[Abstract/Free Full Text]
31. Girardi, M., D. E. Oppenheim, C. R. Steele, J. M. Lewis, E. Glusac, R. Filler, P. Hobby, B. Sutton, R. E. Tigelaar, A. C. Hayday. 2001. Regulation of cutaneous malignancy by T cells. Science 294:605.[Abstract/Free Full Text]
32. Chen, Y., K. Chou, E. Fuchs, W. L. Havran, R. Boismenu. 2002. Protection of the intestinal mucosa by intraepithelial T cells. Proc. Natl. Acad. Sci. USA 99:14338.[Abstract/Free Full Text]

This article has been cited by other articles:

				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]

				Z. Li, A. R. Burns, R. E. Rumbaut, and C. W. Smith {gamma}{delta} T Cells Are Necessary for Platelet and Neutrophil Accumulation in Limbal Vessels and Efficient Epithelial Repair after Corneal Abrasion Am. J. Pathol., September 1, 2007; 171(3): 838 - 845. [Abstract] [Full Text] [PDF]

				J. M. Wands, C. L. Roark, M. K. Aydintug, N. Jin, Y.-S. Hahn, L. Cook, X. Yin, J. Dal Porto, M. Lahn, D. M. Hyde, et al. Distribution and leukocyte contacts of {gamma}{delta} T cells in the lung J. Leukoc. Biol., November 1, 2005; 78(5): 1086 - 1096. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jameson, J. M. Articles by Havran, W. L. Search for Related Content PubMed PubMed Citation Articles by Jameson, J. M. Articles by Havran, W. L.