Springer Semin Immunopathol (1992) 13: 315-331

Springer Seminars in Immunopathology 9 Springer-Verlag 1992

Epidermal T lymphocytes-ontogeny, features and function Elisabeth Payer, Adeiheid Elbe, and Georg Stingl Division of Cutaneous Immunobiology,Departmentof DermatologyI, University of Vienna Medical School, Vienna International Research Cooperation Center, Brunner Strasse 59, A-1235 Vienna, Austria

Introduction In the not-so-distant past the skin was generally viewed as a passive target for immune-mediated injury. However, work in the last decade revealed that skin cells per se can exert immunologic functions. Langerhans cells, for instance, are potent antigen-presenting cells for class II-restricted T cell responses. Various cell types within the skin (e. g., keratinocytes, melanocytes, endothelial cells, fibroblasts) can be stimulated to synthesize and secrete a variety of cytokines that play a crucial role in the modulation of immunologic and inflammatory reactions. The more recent demonstration of T lymphocytes within both the rodent and human epidermis led to the idea that the epidermis may even function as a self-sustaining lymphoid tissue. In this chapter, we give a detailed description of ontogeny, phenotype and function of T cells in the mammalian epidermis and, by doing so, construct a model concerning their role in cutaneous immune reactions.

Epidermal T cells in mice Discovery and characterization of Thy-1 + dendritic epidermal cells In 1983, two independent groups of investigators [7, 80] discovered a cell population within the murine epidermis which is characterized by the presence of abundant surface Thy-1 antigen. In all mouse strains investigated, Thy-1 + cells of the ear and trunk epidermis are evenly and densely distributed and exhibit a pronounced dendritic shape (Fig. 1A). These cells have been termed Thy-1 + (dendritic) epidermal cells (Thy-1 + DEC or Thy-1 + EC). Thy-1 + EC in tail and footpad epidermis are lower in frequency and generally exhibit a round to polygonal shape with only small dendrites [7, 68, 73, 80]. Immunohistologic analysis of semithin sections of murine skin revealed that Thy-1 + DEC are located in the basal layer of the epidermis [64]. Depending upon the strain (C57BL/6 > C3H/He > AKR/J > BALB/c) and body region (ear/trunk > tail/footpad) investigated, strongly Thy-1 + cells comprise 0 . 8 % - 2 . 7 % of all EC as

Correspondence to: E. Payer

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Fig. 1A, B. Thy-1 and TCR V~3 expression on dendritic epidermal T cells. Sheets of mouse ear epidermis from C57BL/6 mice were incubated with biotinylated-anti-TCR Vv3 monoclonal antibodies (mAb) followed by incubation with anti-Thy-l.2-FITC together with Texas Red Streptavidin. Note that all Thy-1 § cells (A) also react with the anti-TCR V~3 mAb (B). A, Bx250

determined by anti-Thy-1 immunolabeling studies with single EC suspensions [80]. Ultrastructurally, these cells are characterized by a slightly lobulated nucleus, vimentin-type intermediate-size filaments, a villous surface and membrane-bound granules with an electron-dense core and/or small internal vesicles. These cells lack keratin filaments, desmosomes, melanosomes and Birbeck granules and exhibit neither anti-Ia- nor DOPA-reactivity. Thus, Thy-1 + DEC are a unique EC population which differs from keratinocytes, mature melanocytes, Merkel cells and Langerhans cells [7, 64, 65, 80]. Studies with Thy-l-disparate radiation bone marrow chimeras [8, 15, 33, 65], as well as the demonstration of Ly-5/T200/CD45 determinants on Thy-1 + DEC [44, 80] established the bone marrow derivation of these cells. Among hemopoietic cells, Thy-1 antigens are predominantly, if not exclusively, expressed on T cells and natural killer (NK) cells, which indicates that Thy-1 + DEC belong to one of these cell lineages. The further observation that Thy-1 + epidermal leukocytes do not react with anti-CD4, anti-CD8 or anti-CD5 mAb (Table 1) [7, 64, 80] showed that Thy-1 + DEC are a homogeneous cell system completely different from most mature T cells. Evidence for the NK cell nature of Thy-1 + DEC came from the finding that they express asialo GM 1 glycoprotein and contain membranebound granules with an electron-dense core and/or small internal vesicles similar to those found in large granular lymphocytes enriched for NK cells [64]. It should be mentioned that the asialo GM1 glycoprotein is not a NK cell-specific marker but is also expressed on thymocytes, certain peripheral T cells and macrophages. Subsequent in vitro studies showed that similarities between Thy-1 + DEC and certain T cells or NK cells are not limited to their phenotypic features but also occur at the functional level. In 1986, Nixon-Fulton et al. [52] induced

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Table 1. Comparison of the phenotype of resident and cultured dendritic epidermal T ceils (DETC) and resident and cultured epidermal T cells of nude mice (NETC)

CD45 Thy- 1 CD3 TCR CD5 CD4 CD8 Asialo GM1 Fc3,RII Ia

Resident D E T C

D E T Clines

Resident N E T C

N E T Clines

+ + + W3 . . + . .

+ + + Vv3

n.d. + + Not Vv3

+ + + Not V~3

-(+) +

-(+) +/-

. .

. .

. .

+/. .

. .

. .

n.d., Not determined proliferative activity in Thy-1 + DEC-enriched ( 2 0 % - 6 0 % ) EC suspensions using a combination of the T cell mitogen concanavalin A (Con A) and the T cell growth factor IL-2. This strategy regularly resulted in the outgrowth of CD45 +, Thy-1 § CD5 , C D 4 - , C D 8 - (with some cells transiently expressing CD8), Ia , M a c - l - and asialo GM1 § or asialo G M - 1 - cells (Table 1) [17, 54, 81]. Further propagation of these cells is dependent upon the addition of IL-2 to the culture system and results in cell lines/clones exhibiting the essential phenotypic features of resident Thy-1 + DEC. Studies in our laboratory using EC suspensions enriched up to 90 % Thy-1 § DEC yielded similar results. Our additional observation that growth stimuli for peripheral T cells [e. g., phorbol esters (PMA) + ionomycin] failed to induce proliferation in the Thy-1 § DEC population (Table 2) [81], showed that Thy-1 § DEC differ from most peripheral T cells in both phenotype and function. Evidence suggesting an N K cell nature for Thy-1 § DEC came from the observation that short-term cultured Thy-1 § DEC and Thy-1 § DEC-derived lines/clones display cytotoxic activity against NKsensitive YAC-1 target cells [54, 79]. However, for several reasons this finding cannot be accepted as formal p r o o f of the N K cell nature of Thy- 1 + DEC. First, in other experiments using freshly isolated Thy-1 § DEC, short-term bulk cultures and various Thy-1 + DEC clones, the cells exhibit only very low, to not detectable, YAC-1 killing [64, 79, 85]. Second, similar to lymphokine-activated killer (LAK) cells, certain Thy-1 § DEC clones kill NK-resistant targets like EL-4, J774A. 1 or P815 cells [58, 79]. Third, severe combined immunodeficient (scid) mice, which lack T and B cells due to an enzymatic defect, are devoid of Thy-1 § DEC, but contain normal numbers of functional N K cells [53]. Thy-1 + dendritic epidermal cells are a distinct subset o f T cells

The polymorphic T cell receptor (TCR) is the critical antigen-recognition moiety of T lymphocytes. While the B cell antigen receptor (surface-bound immunoglobulins) has long been recognized (reviewed in [83]), the molecular nature of the T C R was only defined in the mid 1980s (reviewed in [3]). This structure occurs in two different configurations which are never simultaneously expressed on the

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T a b l e 2. Activation requirements for the in vitro proliferation of DETC Supplement added to the medium Con A Con A + P M A + Iono IL-2 Con A + I L - 2 Con A + I L - 2 + I L - 1 PMA P M A + Iono P M A + IL-2 Iono + IL-2 P M A + Iono + IL-2 P M A + Iono + IL-2 + IL- 1

Proliferative response

+++ + + + +

+ + +

Cells were cultured for 4 - 5 days in the presence of 2.5 ~tg/ml concanavalin A (Con A), 10 ng/ml phorbol myristate acetate (PMA), 500 ng/ml ionomycin (Iono), 100 U recombinant h u m a n interleukin (rhlL)-2, and 5 U rhlL-1

same cell. The majority of functional T cells in the thymus and the peripheral organs of adult mice express TCR composed of a 42- to 45-kDa ot chain and a 42- to 44-kDa 13 chain (TCR or//3) (reviewed in [3, 19]). These heterodimers recognize antigens in context with products of genes encoded for by the major histocompatibility complex (MHC). The more recently identified TCR 3"/6 heterodimers are present on early fetal thymocytes and on a small fraction of adult thymocytes and peripheral T cells (reviewed in [2]). These TCR 3"/f-bearing cells recognize a heterogeneous array of ligands, including classical and nonclassical MHC antigens, bacterial heat-shock proteins and various self proteins. Both TCR species are noncovalently linked to the CD3 complex which consists of five transmembrane molecules and transduces activation signals from the TCR to the interior of the cell [3]. Since the expression of TCR defines the T cell nature of a given cell, several groups of investigators began to search for the presence of TCR gene transcripts and/or proteins in Thy-1 § DEC-derived cell lines. Using Northern blot analysis, Stingl et al. [75] found that Thy-1 § DEC-derived lines contain transcripts for various TCR genes, thus providing strong evidence for the assumption that Thy-1 + DEC belong to the T cell lineage. The further observation that most Thy-1 § DEC-derived cell lines contain full-sized TCR 3' chain mRNA but no, or only incomplete, TCR oLand/3 chain transcripts indicated that TCR expressed by Thy-1 § DEC are of the 3"/6 rather than the od/3 type [40, 75]. Definitive experimental proof for this contention came from immunoprecipitation experiments with anti-CD3 antibodies or anti-C3'l,2 antisera raised against an oligopeptide within the transmembrane region of the TCR 7 chain (synthesized on the basis of the nucleotide sequence). These reagents precipitated a 80- to 90-kDa molecule from most Thy-1 § DEC-derived cell lines which consists of two disulfide-linked chains, i. e., the 35-kDa 3' chain and the 45-kDa 6 chain [12, 38, 40, 76]. Treatment of these precipitates with N-glycosidase F and subsequent analysis on SDS-PAGE revealed core protein sizes of 31-kDa for the TCR 3"-chain and of 34-kDa for the TCR 6 chain [12, 38]. These data obtained with Thy-1 §

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DEC-derived cell lines were found to be truly representative of the in vivo situation: (i) immunolabeling studies showed that all Thy-1 + cells within epidermal sheet preparations express the CD3 complex [74] and that most of these cells also react with an anti-C2/1,2 antiserum [74]; (ii) immunoprecipitation of the TCR/CD3 complex from Thy-1 + DEC-enriched EC suspensions gave the same 35- to 45-kDa heterodimer as that obtained from the cell lines [74]. The finding that Thy-1 + DEC uniformly express surface-bound CD3 antigens associated predominantly, if not exclusively, with TCR 2//6 heterodimers strongly implied that resident Thy-1 + DEC are functional T cells and these cells were, therefore, renamed dendritic epidermal T cells (DETC) [74]. Sequencing of cDNA derived from freshly isolated DETC and from DETC-derived lines and clones revealed that most, if not all, DETC express TCR V~3/JT1/Cvl and V~I/D~2/ J~2/C~ mRNA, and that almost all in-frame junctions within these gene segments were identical (canonical sequences) [4, 5]; for defining the variable 2/-chain genes the Garman nomenclature [25] will be used in this article. Together with the observation that the TCR Vv3-specific mAb 536 reacts with DETC lines [30], as well as with the overwhelming majority (86 % - 9 8 % depending on the mouse strain) of freshly isolated DETC [31] (Fig. 1), these data strongly suggested that most, if not all, DETC express nonpolymorphic 35- to 45-kDa Vv3/V~I TCR heterodimers. It should be emphasized that, with the exception of a small portion ( < 2 %) of mammary gland T cells [63], DETC are the only T cells in adult mice that express TCR Vv3/V~I heterodimers [31, 78]. With respect to the apparent homogeneity of DETC, one should note that stimulation of EC suspensions with Con A and IL-2 occasionally yielded cell lines expressing CD3-associated TCR other than V~3/V~I [39, 48, 75]. Several candidates for the cellular source of these lines exist. First, it is conceivable that they are derived from single-positive peripheral T cells "contaminating" the EC suspensions. This is rather unlikely because the EC-derived cell lines do not express CD4 or CD8 antigens. Second, C D 4 - / C D 8 - (double-negative), CD3 +/TCR Vv3- cells might reside within the epidermis and, upon stimulation, give rise to Thy-1 + epidermal T cell lines expressing TCR other than Vv3-encoded TCR heterodimers. This contention is supported by the following observations: (i) occasionally, minute numbers of CD3+/TCR 2/(non-W3)/6 + and CD3+/TCR or//3+ cells can be identified in epidermal sheet preparations of adult euthymic animals (A. Elbe and E. Payer, unpublished observation); and (ii) the epidermis of 6- to 12-month-old athymic nude mice contains appreciable numbers of Thy- 1 +, asialo GM 1 +, CD3 +, CD4-, CD5-, CDS- cells (up to 3 % of Thy-1 + cells) which express various types of TCR heterodimers (E. Payer, S. Strohal, R. Kutil, A. Elbe, and G. Stingl, J. Immunol., in press), and it is quite conceivable that similar cells also exist in euthymic mice. A third explanation for the propagation of certain Thy-1 + DEC lines that express TCR heterodimers other than V~3/V~I is that the cell culture conditions chosen (Con A+IL-2) induce rearrangement of various TCR genes and surface expression of the corresponding TCR in Thy-1 +, CD3- epidermal cells. Such cells are present in minute numbers in the epidermis of normal mice (A. Elbe and E. Payer, unpublished observation), but are the major population of Thy-1 + cells in fetal [22] and athymic nude mice [55]. At the present time, the ontogenic relationship

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of these CD45 +, Thy-1 + cells which are CD3- or CD3+/TCR c~//3+ or CD3+/TCR (non-Vv3)/6+ to TCR V~3+ DETC is not clear. Therefore, we will use the term DETC only for TCR Vv3+ dendritic epidermal T cells.

The fetal period is crucial for the development of DETC Certain observations suggest that the fetal or perinatal period is critical for both the maturation and expansion of DETC. Small numbers of Thy-1 + cells can first be detected in epidermal sheets prepared from fetal mice at day 17 of gestation [22, 66] or in cell suspensions prepared from the skin of 16-day-old fetuses (A. Elbe, R. Strohal, O. Kilgus, E. Payer, S. Schreiber, and G. Stingl, submitted for publication). As opposed to DETC, these cells are usually round in shape and do not express a TCR/CD3 complex [22]. Within the next 2 - 3 weeks a pronounced heterogeneity develops in the increasing population of CD45 +, Thy-1 + cells. While the relative proportion of round, Thy-1 +, CD3- cells gradually decreases, there is a continuous increase in Thy-1 +, CD3 + dendritic cells. At 14 days of age, the epidermis contains peak levels of dendritic Thy-1 +, CD3 +/TCR W3+ lymphocytes (DETC) ([22] and A. Elbe, unpublished observation). From the 2nd to the 6th week after birth the DETC density decreases slightly and then remains relatively stable for the next 6 - 1 2 months [22, 68, 73]. The mechanism(s) governing the homeostasis of the DETC population is (are) still unclear. Theoretically, these cells may have a self-renewing capacity and/or may be replaced by bone marrow-derived immigrants. While there exists some evidence for in-situ proliferation of DETC (see below), a series of experiments has clearly shown that a meaningful repopulation of DETC from the bone marrow does not occur in adult life. First, when DETC are depleted from the adult murine epidermis (e. g., by UV treatment), they do not reappear [1]. Second, intravenous injection of Thy-l-disparate, congenic or MHC-compatible adult bone marrow cells into lethally irradiated recipient mice results in the immigration of Thy-1 +, CD3-, but never of Thy-1 +, CD3 + donor-type cells into the recipients' epidermis (Fig. 2) [33]. Third, transplantation of adult or newborn skin onto Thy-l-disparate normal adult mice results in the immigration of Thy-1 +, CD3-, but not of Thy-1 +, CD3 + recipient-type cells into the graft (E. Payer, unpublished observation).

Ontogeny of DETC While these findings supported the contention that DETC maturation occurs early in life rather than in the adult animal, the important question remained whether this is a thymus-dependent or a thymus-independent event.

Arguments for thymic maturation of DETC. The detection of predominantly round Thy-1 § CD3-, but not of Thy-1 § CD3 § dendritic cells in the epidermis of athymic mice [55] was originally taken as a strong argument for the thymus dependence of DETC. However, this does not prove their thymic derivation since the skin of athymic nu/nu mice is grossly abnormal and may not be able to provide the appropriate microenvironment for DETC maturation. Allison and co-workers later reported that the first TCR-bearing fetal thymocytes (day 14 of gestation)

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Fig. 2A, B. Donor-type Thy-1§ cells in adult bone marrow chimeric animals [C57BL/6 (Thy-1.2)-- B6PL-Thy-1a (Thy-1.1)] fail to express CD3 antigens. Epidermal sheets of mice killed 1 year after chimerizationwere first incubated with biotinylatedanti-Thy-1.2 mAb followed by antiCD3e-FITC and Texas Red Streptavidin. Donor-type Thy-1.2+ cells (A) do not react with the antiCD3e mAb (B), while recipient-type dendritic cells (Thy-l.2-) were CD3 § (B). A, B x 250 express TCR V~3, and that the entire TCR configuration of these fetal thymocytes is essentially indistinguishable from that expressed by freshly isolated DETC and DETC-derived clones or hybridomas [4, 5, 35, 42]. The further observation that the disappearance of TCR Vv3§ cells from the fetal thymus directly precedes their first appearance in the epidermis (day 17 - 19 of gestation) led Havran and Allison [28] to the idea that TCR Vv3 § thymocytes leave the thymus, migrate to the epidermis, and become DETC. To test this assumption, they transplanted fetal and newborn thymic lobes into either syngeneic athymic nude mice or Thy-l-disparate euthymic newborn mice. In keeping with their hypothesis, they found that several weeks after implantation of day 14 fetal thymic lobes, but not of day 2 postpartum thymic lobes, CD3+/TCR Vy3 § cells were readily detectable in the epidermis [29]. We have obtained a similar result using a slightly different approach. Our experiments showed that a single intravenous injection of day 16 fetal thymocytes into Thy-l-disparate nude mice results in the appearance of distinct clusters of donor-type CD3§ W 3+ DEC in the recipients' epidermis (Fig. 3). In contrast, the injection of either adult thymocytes or of day 16 fetal thymocytes which had been depleted of C D 3 § Vv3 + cells was not followed by the emergence of the DETC population [59]. While after injection of unseparated day 16 fetal thymocyte preparations (including Thy-1 § CD3+/TCR Vv3 + cells), donor-type DETC were regularly found in the recipients' epidermis, no donor-derived cells could be detected in the spleen and lymph nodes of these animals [59]. These data directly show that fetal TCR Vv3 § (but not TCR Vv3-) thymocytes selectively migrate to the skin and home to the epidermis. Thereafter, these cells acquire a dendritic shape and undergo vigorous proliferation as evidenced by the dramatic and continuous increase in the cellular

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density of the clusters [59]. The virtually exclusive use of TCR V~3/V~I specificities by adult DETC, as well as the selective homing of TCR W 3 § fetal thymocytes to the epidermis, led to the idea that the TCR V~3 may be the homing receptor for the skin/epidermis. This hypothesis was dismissed after it had been demonstrated that TCR C~4 § and TCR V~2/V~5§ cells migrate into the epidermis of C~4 and W2/V~5 transgenic mice, respectively [11, 23]. The selective presence of TCR V~3§ cells in the epidermis of adult mice cannot only be explained by a selective migration of the relevant precursor cells to the skin but may also be due to a selective survival of these cells within the epidermis. The latter concept is based on the finding that in early fetal life (shortly after the first appearance of TCR V~3 § cells in the thymus) TCR Vv3§ cells can be detected in the fetal gut and liver [16, 41], but not in these organs in adult animals [16, 78]. One explanation for these data is that TCR V~3+ cells which " r a n d o m l y " migrate from the fetal thymus to the fetal liver and gut cannot survive, while the TCR V~3§ cells that reach the epidermis are rescued from cell death due to the interaction with a putative self ligand (similar to the intrathymic positive selection event of TCR odfl + cells).

Evidence for extrathymic maturation of DETC. As mentioned above, the first lymphocytes populating the fetal epidermis (day 1 6 - 17 of gestation) carry the CD45 +, Thy-1 +, CD3- phenotype [22]. The further observation that these cells are then gradually outnumbered by Thy-1 +, CD3 + cells raised questions about their ultimate fate. While it is conceivable that the Thy-1 +, CD3- fetal epidermal cells die or leave the epidermis, the attractive possibility remained that they mature into DETC upon receipt of educational stimuli from the fetal skin microenvironment. To test this assumption we transplanted full-thickness grafts from body wall skin of day 16 fetal C57BL/6 (Thy-1.2) mice onto full-thickness wound beds of B6PL-Thy-la (Thy-1.1) mice. Analysis of the grafts at various time points after

Fig. 3. Visualization of a cluster of FITC-anti-CD3e-reactivedendritic cells within an epidermal sheet ofa C3H nu/nu mouse 12 weeksafter the injectionof syngeneicday 16 fetal thymocytes, x 150

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transplantation revealed the presence of steadily increasing numbers of donor-type Thy-1 § CD3 § dendritic cells which uniformly reacted with anti-TCR V~3 mAb (A. Elbe, R. Strohal, O. Kilgus, E. Payer, S. Schreiber, and G. Stingl, J. Immunol., in press). Transplantation of day 16 fetal skin onto Thy-l-disparate athymic nude mice gave the same results, thus indicating that thymic factors are not necessary for the intraepidermal maturation and proliferation of DETC. There are two explanations for the development of a dense network of TCR Vv3§ cells in the transplant. First, minute numbers of thymus-derived Thy-1 +, CD3§ Vv3§ cells might have been present in the fetal skin graft and, upon proliferation, gave rise to the DETC network. Several observations argue against this concept: (i) we consistently failed to detect CD3 + cells in day 16 fetal skin, (ii) stimulation of fetal skin cells with Con A and IL-2 did not result in the outgrowth of CD3 § cells, and (iii) discrete clusters of DETC, which should be visible upon proliferation of only a few scattered TCR Vv3+ cells, were not present in the epidermal sheet preparations at various times after grafting. The second explanation for the derivation of CD3§ V~3+ cells in the transplant is that CD45 § , Thy-1 § CD3- cells (the only lymphocytes within the fetal skin graft) are the relevant DETC precursors. In fact, we have found that these cells, although not maturing into CD3§ V~3+ cells, can transcribe CD3~ and TCR C~, C~I or C~4 genes upon in vitro stimulation. This shows that Thy-1 +, CD3- cells belong to the T cell lineage and one may speculate that upon receipt of appropriate stimuli from the fetal skin microenvironment they undergo both differentiation and proliferation. At this point several remarks concerning DETC proliferation are warranted. It is now established that both fetal and adult DETC [49, 50, 59] can undergo proliferation. The factor(s) promoting this event has (have) yet to be clarified. While IL-2 is critical for the in vitro proliferation of DETC [52, 81], our recent observation that IL-2-deficient mice have normal numbers of DETC excludes IL-2 as an essential growth factor in vivo (A. Elbe and T. Hfinig, unpublished observation). One should not forget that the obviously conflicting data about the role of the thymus in DETC development have been obtained in artificial experimental systems. Thus, it remains to be shown if one or both DETC developmental pathways are operative under physiologic conditions. In either case, recent in vitro and in vivo experiments strongly indicate that the ultimate precursor is derived from the fetal liver [34, 56]. According to the "thymic theory" of DETC development, the fetal stem cells programmed to rearrange V~3 gene segments properly migrate to the thymus where the rearrangement of the TCR genes takes place. Cells expressing TCR W3/V61 heterodimers encoded by the canonical Vv3/Jvl/CT1 and V61/D62/J~2/C6 sequences may then be positively selected [36, 41] and equipped with the homing receptor for the epidermis. Alternatively, the "extrathymic concept" would imply that fetal stem cells directly migrate into the fetal epidermis and there undergo TCR rearrangement and putative positive selection.

Function of DETC Until very recently the function of DETC was entirely unknown, since there existed no information about the physiologic ligand of their TCR Vv3/V~I. The observa-

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tion that the TCR repertoire of TCR 3//6-bearing cells in nonlymphoid organs is uniform at a given anatomical site, but differs from one location to another (e. g., TCR V~3/V~I in the epidermis, TCR V~4/V~I in reproductive organs [37] and TCR V~5/V~4 in the gut [9]) led to the suggestion that each TCR 3//6 set is designed to recognize antigens most likely to be present in that particular environment. In support of this concept, two groups of investigators recently obtained evidence that DETC recognize an epidermal antigen. Havran et al. [32] reported that the addition of irradiated keratinocytes (but not of irradiated fibroblasts) to either freshly isolated DETC or long-term DETC lines leads to IL-2 secretion by DETC. The keratinocyte/DETC interaction is MHC nonrestricted and can be blocked by anti-TCR W 3 or CD3, mAb. Using a slightly different approach, Lewis and Tigelaar [45] found that DETC lines respond to heat-shocked, but not to untreated, keratinocytes. These data indicate that the TCR Vv3/V~I recognizes "stress proteins" of keratinocytes, and that the binding of these proteins leads to the activation of DETC. The exact nature of these proteins and the occurrence of an in vivo DETC response specifically induced by EC injury have yet to be demonstrated. The presented data strongly argue against the assumption that DETC are the natural targets of Langerhans cells that present antigenic peptides in the context of MHC class I and class II proteins. Other experimental systems have indicated a role for DETC in the regulation of T cell responses. While the intravenous injection of haptenated Langerhans cells sensitizes the recipient animal and leads to a marked ear-swelling response after challenge, the intravenous injection of haptenated DETC does not lead to a similar event [18, 77]. Instead, the intravenous injection (or footpad injection) of haptenconjugated DETC into naive mice results in a down-regulation of contact hypersensitivity (CHS) responses as manifested by a marked reduction of the ear-swelling response in the recipient mouse after sensitization and challenge with the relevant hapten [ 18, 77, 84]. The induction of this immune tolerance by haptenated DETC is antigen specific but not H-2 restricted [84]. The mechanism by which these cells interfere with the development of a CHS response is not yet clear. It has been hypothesized that haptenated DETC either activate suppressor cell circuits or, alternatively, directly kill hapten-specific T helper cells [57, 84]. Evidence for the latter mechanism comes from in vitro experiments showing that activated DETC can exhibit MHC-nonrestricted NK-like killing [54, 79]. Although the above experiments may not necessarily represent the in situ situation, one may assume that the expression of murine CHS is regulated by two opposing events, i.e., Langerhans cell-derived sensitizing signals and DETC-derived tolerizing signals. Recently, Shiohara et al. [72] have proposed that one of the functions of DETC is to protect the epidermis against damage by autoreactive T cells. This concept was based on the observations that (i) the epidermis of mice that had recovered from graft versus host disease (GVHD) contains much higher numbers of Thy-1 + cells than normal mice, and (ii) that these Thy-1 + cells protect the epidermis against a second attack of GVHD effector T cell clones. Since it is not yet clear whether the Thy-1 + EC mediating this protective effect express TCR V~3/V~I, the actual role of DETC in this phenomenon remains elusive.

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Epidermal T cells in the rat The epidermis of adult rats contains a population of dendritic CD45 +, CD3 +, Thy-1 , Ia- lymphocytes (A. Elbe, O. Kilgus, S. Schreiber, T. Htinig, and G. Stingl, manuscript in preparation). Similar to murine DETC, these rat dendritic T cells do not express CD5, CD4, and CD8 antigens. Although the TCR repertoire of the rat epidermal T lymphocytes has not yet been determined because of the lack of appropriate reagents, the presence of some TCR od/3+ cells indicates that rat epidermal T lymphocytes are more heterogeneous than mouse DETC.

Epidermal T cells in humans It has been shown that clinically normal-appearing adult human epidermis contains a minor but routinely detectable population of CD2 +, CD3 +, CD5 + T cells. These intraepidermal T cells comprise 2 % of all skin T cells and 0.5 % - 1% of all EC [13, 20, 24]. In sharp contrast to the mouse system, the vast majority of human epidermal T cells bear TCR od/3 rather than TCR 7/6 heterodimers [14, 20, 24, 26]. So far, there exists no definitive information about the diversity of their TCR od/3 and TCR 3~/6 repertoires. Among human epidermal T cells CD4-, CD8 + cells outnumber CD4 +, CD8- cells; in addition there exists a population of CD3 +, CD4-, CD8- cells carrying TCR er rather than TCR 3'/6 specificities [27]. Approximately 80 % of intraepidermal T cells stain with the antiCD45RO mAb UCHL-1 [24], implying that they are memory cells. The derivation of these intraepidermal memory cells can be explained in two ways. First, they could have been generated by a Langerhans cell-mediated antigenic stimulation of naive T cells indigenously residing within the epidermis. Alternatively, they could have entered the skin/epidermis in an already sensitized state. Because the ontogenetic development of human epidermal T cells has not yet been carefully studied, no meaningful statement can currently be made about the validity of the former concept. In contrast, there exist some experimental data, mainly derived from studies with the mAb HECA-452, which support the latter theory. This antibody reacts with 10 % - 2 0 % of peripheral blood T cells and with 85 % of lymphocytes in inflammatory skin lesions [60]. HECA-452-reactive cells have the CD45RA ~~ CD44 high phenotype and, thus, qualify as memory cells. Cell attachment experiments have shown that memory T cells, but not resting T cells, adhere to cells expressing the endothelial lymphocyte adhesion molecule-1 (ELAM-1 [69]) and that the HECA-452 + T cell subset is the predominant ELAM-l-binding population among circulating lymphocytes [61]. Furthermore, it has been shown that ELAM-1 is preferentially expressed on endothelial cells of venules within inflammatory skin lesions and that these venules are associated with a predominantly HECA-452 + T cell infiltrate [61]. These data strongly suggest that the interaction between HECA-452-reactive molecules and ELAM-1 mediates the adhesion of memory T cells to the endothelial cells of the dermal microvasculature and, thus, promotes the migration of memory T cells into the dermis. The further migration of these cells into the epidermis involves various cytokines, chemotactic factors and adhesion molecules. Primarily on the basis of

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in vitro studies, one would assume that IL-1 [67], IL-8 [43], as well as the IFN~-induced peptide 10 (IP-10) [46] are the most important keratinocyte-derived T cell chemotactic signals. In vitro, IFN--y and TNF-o~ are capable of inducing IP-10 and IL-8 production by keratinocytes [6, 46] and presumably play an important role in T cell recruitment to the epidermis. Additionally, these cytokines trigger the expression of the intercellular adhesion molecule 1 (ICAM-1) by keratinocytes [6, 21], rendering these cells capable of binding lymphocyte function-associated antigen-1 (LFA-1)-bearing T cells [47]. These data support the concept that the epidermotropic migration of dermal T cells is initiated by certain keratinocytederived cytokines (the production of which can also be induced or enhanced by secretory products of activated T cells themselves, e.g., IFN-'y) and that their attachment to and, finally, their entrance into the epidermis can be mediated by the LFA-1/ICAM-1 interaction [51, 70, 71]. In summary, we favor the concept that human epidermal T cells (at least in their vast majority) are not the descendants of naive, resting T cells indigenously residing in the epidermis, but rather represent the progeny of activated T cells which had entered the skin and epidermis in the course of an inflammatory reaction.

Conclusions

The murine epidermis contains a network of Thy-1 § dendritic T cells. These T cells arise from early fetal stem cells and differentiate in the fetal or neonatal thymic or epidermal microenvironment. Their lack of expression of CD5, CD4, and CD8 antigens, as well as their virtually exclusive expression of a CD3/TCR V~3/V61 complex, distinguishes DETC from the bulk of peripheral T cells. The early appearance of TCR 3,/6 cells in ontogeny, the lack of expression of CD4 and CD8 antigens, and the relative paucity of 3/and 6 genes compared to a and/3 genes, indicates that 7/6 T cells provide a phylogenetically primitive, broadly acting, and poorly discriminating immunologic defense system. In this system, recognition of antigen is not restricted by classical MHC class I and class II antigens, but may occur in the context of relatively nonpolymorphic restricting elements, such as Qa [82], Tla [10] or CD1 [62]. This rather primitive immune system provided by DETC may serve to protect the epidermal integrity. Upon recognition of self proteins released following epidermal injury, DETC may become activated and assist in the removal of altered cells. In this limited fashion, the epidermis may be an independently competent immunologic system. However, the fact that the TCR repertoire of DETC does not allow for the recognition of antigenic peptides in conjunction with MHC moieties excludes the possibility that the diverse immune response elicited by topical contact with foreign antigens is mediated by DETC. Whether this statement also applies to the human epidermis cannot be answered at the present time. Let us consider a few plausible concepts concerning derivation and function of human epidermal T cells. First, one could postulate that in early ontogeny, the human epidermis harbors a small, indigenous population of naive T lymphocytes with monomorphic TCR representing an analogue to murine DETC. These cells could function in a manner similar to that proposed for murine

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D E T C . T h e y m a y even p e r s i s t into adult life, so far u n d e t e c t e d b e c a u s e they w o u l d b e o u t n u m b e r e d b y i m m i g r a t i n g p o l y m o r p h i c T cells f r o m p e r i p h e r a l l y m p h o i d organs. S e c o n d , it is c o n c e i v a b l e that the h u m a n e p i d e r m i s contains an i n d i g e n o u s p o p u l a t i o n o f n a i v e T l y m p h o c y t e s with a p o l y m o r p h i c T C R r e p e r t o i r e r e p r e s e n t ing a p h y l o g e n e t i c a l l y a d v a n c e d a n a l o g u e to m u r i n e D E T C . A l t h o u g h e q u i p p e d with T C R a l l o w i n g antigen r e c o g n i t i o n in the context o f M H C , their d e n s i t y is p r o b a b l y too l o w to m a k e t h e m an effective host defense s y s t e m against the multitude o f e n v i r o n m e n t a l antigens p r e s e n t e d b y L a n g e r h a n s cells. O n e could rather a s s u m e that they p r o l i f e r a t e u p o n r e c o g n i t i o n o f s e l f antigens o c c u r r i n g in a p e r t u r b e d e p i d e r m i s . T h e a u t o r e a c t i v i t y o f these cells m a y not n e c e s s a r i l y be beneficial. F i n a l l y , the fact that the entry o f circulating H E C A - 4 5 2 + m e m o r y cells into the skin is d e p e n d e n t u p o n the i n j u r y - i n d u c e d E L A M - 1 e x p r e s s i o n b y e n d o t h e l i a l cells o f the d e r m a l m i c r o v a s c u l a t u r e c o u l d indicate that all T cells p r e s e n t in adult h u m a n e p i d e r m i s are r e c r u i t e d u p o n alteration o f the skin. F o l l o w ing this r e a s o n i n g , the h u m a n e p i d e r m i s should not b e r e g a r d e d as a c o m p l e t e , self-sustaining i m m u n o l o g i c o r g a n but rather as a h o m i n g site for and a target o f l y m p h o c y t e s a n t i g e n i c a l l y sensitized in p e r i p h e r a l l y m p h o i d o r g a n s .

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