- Email: [email protected]
NKG2 Subtypes on Tumor-Infiltrating Lymphocytes in Primary and Metastatic Melanoma
Expression of CD94/NKG2 Subtypes on Tumor-In®ltrating Lymphocytes in Primary and Metastatic Melanoma Claudia S. Vetter, Per thor Straten,* Patrick Terheyden, Jesper Zeuthen,* Eva-B. BroÈcker, and JuÈrgen C. Becker Department of Dermatology, University of WuÈrzburg, Germany; *Department of Tumor Cell Biology, Danish Cancer Society, Denmark
Natural killer receptors are expressed both on natural killer populations and subpopulations of T cells, mainly a/b TCR+CD8+ T cells. We have characterized the expression of the C-type lectin natural killer receptor CD94/NKG2 on tumor-in®ltrating lymphocytes in primary and metastatic melanoma lesions. By immunohistochemistry, 5±10% of the tumorin®ltrating lymphocytes, both in primary and metastatic lesions, expressed CD94. More than 95% of these CD94+ cells coexpressed CD8 and the percentage of CD94 expression within the CD8+ cell population ranged from 5 to 20% with a higher expression in metastatic lesions. CD94/NKG2 exists both in an inhibitory and an activating form; thus, it was necessary to determine whether the inhibitory CD94/NKG2-A/B, the activating CD94/NKG2-C/E, or both were expressed on tumor-in®ltrating lymphocytes. Reverse transcription±polymerase chain
reaction using speci®c primers for NKG2-A/B and C/E mRNA revealed the presence of NKG2-C/E in all primary and metastatic lesions. In contrast, the inhibitory NKG2-A/B was only present in 50% of primary tumors whereas 80% of tumor-in®ltrating lymphocytes in metastatic lesions expressed these transcripts. In healthy humans, the mean number of inhibitory natural killer receptors is higher than that of activating receptors, but the opposite was true for tumor-in®ltrating lymphocytes in melanoma. The reversal of the ratio of inhibitory to activating natural killer receptors among tumor-in®ltrating lymphocytes suggests a regulated event due to either speci®c factors within the tumor microenvironment, preferential homing of T cell subsets, or certain stages of T cell activation. Key words: cytotoxic T lymphocyte/natural killer receptor/T cell response. J Invest Dermatol 114:941±947, 2000
T
structural groups of natural killer receptors have been characterized. The ®rst group includes type I integral membrane proteins of the immunoglobulin superfamily, e.g., p50, p58.1, p58.2, p70, p140, LAIR-1, or NKp44 (Colonna and Samaridis, 1995; Pende et al, 1996; Meyaard et al, 1997; Vitale et al, 1998); whereas, the second group includes type II integral membrane proteins of the C-type lectin superfamily (Chang et al, 1995). The C-type lectin-like receptors are more abundant than immunoglobulin-like receptors (Lopez-Botet et al, 1998). The initial name of these receptors, killer inhibitory receptor, is presumptive regarding their functional activity, as most natural killer receptors exist in two forms: one mediating activation whereas the other sustains inhibition of natural killer cell function (Moretta et al, 1998). The difference between activating and inhibiting receptors is based on the presence or absence of an ITIM (Isakov, 1997). Activating isoforms, lacking the immunoreceptor tyrosine-based inhibitory motif (ITIM), noncovalently associate with the adapter molecules such as DAP10 or DAP12, which pass the killing signal on to the downstream signaling pathway (Lanier et al, 1998). The CD94 molecule belongs to the C-type lectin superfamily and is covalently bound to another type II membrane protein, NKG2 (Lopez-Botet et al, 1998). The functional capacity of the CD94/NKG2 heterodimer to mediate activation or inhibition is determined by the NKG2 subtype: NKG2-A and NKG2-B subtypes generate an inhibitory receptor, whereas NKG2-C and NKG2-E in conjunction with CD94 results in an activating complex (Lanier et al, 1998). Recent molecular genetic studies suggest that the NKG2-D has distinct features from the other NKG2 molecules, including its position within another functional
wo general systems of cellular immunity have developed during evolution: innate (natural immunity) and acquired (adaptive immunity). Only recently, it was recognized that two of the major players of these systems, natural killer and T cells, share an essential group of receptors termed natural killer receptors (Moretta et al, 1996; Lanier, 1998). Initially, the cytolytic activity mediated by natural killer cells was regarded as non-major histocompatibility complex (MHC)-restricted as susceptibility to natural killer lysis appeared to be unrelated to the MHC haplotype of the target cell. Over the last years, however, it became evident that, whereas the natural killer cell-mediated cytotoxicity is not MHC restricted, it is still regulated by the expression of MHC class I molecules A better understanding of this phenomenon began in 1990 when Ljunggren and KaÈrre (1990) proposed the ``missing self hypothesis'', which implies that a reduced expression of MHC class I molecules on target cells stops the otherwise present inhibition of natural killer cell functions, thus permitting natural killing. This hypothesis stimulated the search and subsequent discovery of the very complex and still expanding system of natural killer cell receptors (Lanier, 1997). To date, two
Manuscript received November 9, 1999; revised January 25, 2000; accepted for publication February 1, 2000. Reprint requests to: Priv. Doz. Dr JuÈrgen C. Becker, Department of Dermatology, Julius-Maximilians-University, Josef-Schneider-Str. 2, D97080 WuÈrzburg, Germany. Email: [email protected] Abbreviations: PBL, peripheral blood lymphocytes; TIL, tumorin®ltrating lymphocytes. 0022-202X/00/$15.00
´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 941
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gene. Furthermore, the degree of homology of NKG2-D to the other NKG2 family members, including NKG2-A, NKG2-C, and NKG2-E, is similar to other unrelated C-type lectins (21±24% identity), suggesting that NKG2-D may not belong to the NKG2 family (Ho et al, 1998). It is well established that CD94/NKG2-A speci®cally interacts with HLA-E and that this interaction is dependent on the association of HLA-E with peptide. Moreover, no interaction between CD94/NKG2-A and classical HLA class I molecules has been described; however, class I leader sequence peptides bind to and stabilize HLA-E (Borrego et al, 1998; Lee et al, 1998; Brooks et al, 1999). The genomic information for CD94 and the NKG2 maps to the human ``natural killer gene complex'' on chromosome 12p12.3±p13.1 (Plougastel et al, 1996). It should be noted, that NKG2-A and NKG2-B are products of one gene, whereas NKG2-C and NKG2-E are encoded by different genes (Glienke et al, 1998; Sobanov et al, 1999). Natural killer cells use inhibitory natural killer receptors to monitor target cells for reduced or absent expression of self MHC class I molecules. The accuracy of detection is facilitated by overlapping subsets of natural killer cells expressing different natural killer receptors. This enables the detection of the loss of expression of a single allele of one MHC locus. As mentioned above natural killer receptors are also expressed on a small subset of T cells and in¯uence T cell receptor (TCR) mediated functions (Mingari et al, 1996). Upon interaction of the TCR with MHC class I natural killer receptors inhibit the cytolytic function of T cellsÐas demonstrated both for redirected as well as antigen-speci®c lysis (Noppen et al, 1998). Furthermore, the inhibition of lysis of solid tumors, in particular melanoma, has been reported (Ikeda et al, 1997; Speiser et al, 1999a). Thus, the induction of natural killer receptors on tumor-in®ltrating lymphocytes (TIL) has been discussed as some form of immunescape. If lost, reduced or aberrant expression of MHC class I molecules on tumor cells, as well as the existence of activating natural killer receptors and their interaction with destruction tags, are taken in consideration, however, the scenario becomes more complex (Bauer et al, 1999). Indeed, the expression of natural killer receptors on T cells may serve as a valuable instrument for the regulation of T cell responses. In order to obtain more information on the role of natural killer receptors in anti-tumor responses, we scrutinized the expression of CD94/NKG2 subtypes on melanoma in®ltrating lymphocytes in situ and in vitro. MATERIALS AND METHODS Tumor samples Fresh tumor samples from 18 primary and 16 metastatic melanomas were obtained by surgical excision following informed patient consent. The tumors were divided in two parts one of which was snap frozen in liquid nitrogen immediately after surgery and stored at ±80°C. The remaining part, which included the thickest portion of the tumor, was used for routine histology and diagnosis. All metastatic lesions were cutaneous metastases. Immunohistochemistry Five micron sections of tumor lesions were ®xed in cold acetone and air dried for 30 min. Slides were incubated for 30 min with the indicated speci®c primary monoclonal antibody (MoAb) at predetermined optimal dilutions. After two washes, the sections were incubated with biotinylated goat anti-rat or anti-mouse immunoglobulin (DAKO, Hamburg, Germany) for 20 min, followed by another two washes. Staining was revealed with streptavidin±horseradish peroxidase and 3-amino-9-ethylcarbazole (AEC)-substrate. The reaction was stopped by washing after 25 min. For double staining, slides were incubated with rabbit serum at a ®nal dilution of 1:5, followed by the second primary MoAb. After a 40 min incubation and two washes, the species speci®c alkaline phosphatase (AP)-conjugated secondary antibodies were added and after additional washing steps staining with alkaline phosphatase-anti-alkaline phosphatase and Fast Blue substrate was performed. For single staining, slides were subjected to haÈmalaun nucleus staining. All antibodies are commercially available: CD3 (DAKO, clone UCHT1), CD4 (DAKO, clone MT310), CD8 (DAKO, clone C8/144B), CD56 (DAKO, clone MOC-1), and CD94 (Pharmingen, Hamburg, Germany; clone HP-3D9).
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Reverse transcription±polymerase chain reaction (reverse transcription±PCR) RNA was extracted using the Purescript Isolation Kit (Gentra Systems Inc., Minneapolis, MN) cDNA synthesis was carried out using M-MLV SuperScript II reverse transcriptase (GibcoBRL, Life Technologies Inc., Gaithersburg, MD) in a total volume of 50 ml 1 3 buffer provided by the manufacturer containing 10 mM dithiothreitol. Incubations were performed at 37°C for 30 min, 42°C for 30 min, 50°C for 30 min, and 72°C for 5 min cDNA were ampli®ed with primers for GAPDH (GAP-371p; 5¢-AGGGGGGAGCCAAAAGGG-3¢, GAP-891m; 5¢-AGGAGT GGGTGTCGCTGTTG-3¢; size 540 bp), NKG2-AB (AB391p; 5¢-TGGGAATTATCTGTC TTATCTTAA-3¢, AB-868m; 5¢CAAATGCAAACGCTTTACC-3¢, size for NKG2-A; 496 bp, NKG2B; 442 bp), and NKG2-CE (CE-108p, 5¢-CCGAACAGGAAATATTCCAAG-3¢, CE-294m 5¢-AAGAATTGTTCTGCTCCAGGA-3¢, size for both NKG-C and NKG-E transcripts; 207 bp). Ampli®cations were carried out in a total volume of 25 ml containing 1 3 PCR buffer [5 mM KCl, 20 mM Tris pH 8.4, 2.0 mM MgCl2, 0.2 mM cresol red, 12% sucrose, 0.005% (wt/vol) bovine serum albumin (Boehringer-Mannheim, Mannheim, Germany)], 2.5 pmol of each primer, 40 mM dNTP (Pharmacia LKB, Uppsala, Sweden) and 1.25 units of AmpliTaq polymerase (Perkin Elmer Cetus Corporation, Emeryville, CA). The parameters used for ampli®cation were 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s for 32 (GAPDH) or 38 (NKG2) cycles taking advantage of a ``touch down'' setting going from 65°C to 60°C over the ®rst nine cycles. Taq polymerase and dNTP were added to the reaction tube at an 80°C step between the denaturation and annealing steps of the ®rst cycle. Isolation of TIL and in vitro culture Melanoma in®ltrating lymphocytes were obtained by mincing freshly resected tumor biopsies from which all adjacent nonmalignant tissues had been removed. The resulting small pieces were digested for 2.5 h in RPMI 1640 with 0.25% collagenase D (Boehringer Mannheim) and 100 mg per ml deoxyribonuclease type I (Boehringer Mannheim) followed by a passage through a steel sieve. After three washes, cells were either used immediately for analysis or expanded in bulk cultures at 1 3 105 cells per ml in RPMI 1640, supplemented with 10% heat-inactivated human AB-plasma (Transfusion Medicine Center, University WuÈrzburg), 1% penicillin/ streptomycin; and 1% sodium pyruvate, 2 mM L-glutamine, 100 IU per ml interleukin-2 (Aldesleukin, Chiron; Ratingen, Germany) and 10% lymphocyte-conditioned medium, which was generated as previously described (Becker et al, 1995). Flow cytometry For cell labelling, cells were washed in phosphatebuffered saline, resuspended with 0.1% bovine serum albumin in phosphate-buffered saline at 1 3 105 per 100 ml and incubated with nonconjugated primary MoAb for 30 min at 4°C. After two washes, ¯uorescein isothiocyanate-conjugated secondary antibodies (Dianova, Hamburg, Germany) were added for 30 min. With a washing step inbetween a phycoerythrin-conjugated MoAb directed against the second antigen was added. Isotype-matched murine MoAb served as controls. Dead cells were identi®ed by propidium iodide staining (5 mg per ml). Fluorescence was analyzed using a FACScan (Becton Dickinson, Heidelberg, Germany). All MoAb are commercially available: CD4, CD8, or CD56 (Becton Dickinson) and CD94 (Clone HP-3D9, 315±020; Ancell, Bayport, MN).
RESULTS CD94 expression on TIL in primary and metastatic melanoma The frequent expression of natural killer receptors on activated T cells and in particular the report that melanomaspeci®c T cell clones express C-type lectin-like receptors prompted us to scrutinize the expression of CD94 on TIL. To identify populations of cells expressing CD94, we analyzed 18 primary tumors and 16 cutaneous metastases by immunohistochemistry (Fig 1). Initial studies using single staining for either CD3, CD4, CD8, CD56, or CD94, demonstrated that in most primary (14 of 18), but all metastatic tumors (16 of 16), CD94 was detectable. The percentage of CD94+ cells of all lymphocytes varied from patient to patient, but a trend towards a larger CD94+ population for metastatic lesions was obvious (data not shown); however, no correlation of qualitative or quantitative CD94 expression and prognostic parameters of primary tumors, e.g., tumor thickness, was present (Table I). Subsequently, we performed a series of double stainings including CD4CD94, CD8CD94, and CD56CD94 in order to characterize the CD94+ population in more detail (Fig 2).
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This analysis revealed that all CD56+ natural killer cells bore CD94. The percentage of CD56+ cells, however, as detected by immunohistology using either single or double staining never exceeded 1% of TIL. It was previously reported that the expression of C-type lectin-like receptors among T cells in peripheral blood is con®ned to cytotoxic T lymphocytes. We con®rmed this ®nding for TIL in melanoma: the major part of the CD94+ cells
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coexpressed CD8; CD4CD94 double positive cells were not detected in either primary or metastatic lesions. The CD8/CD94 double staining allowed for a more accurate quantitative analysis than the CD94 single staining (Fig 3). Within the primary tumors, the CD94+ cells comprised up to 7% of the CD8+ T cells with a median of 4%; for metastatic lesions, however, the CD94+ cells constituted up to 17% (median: 7%) of CD8+ cells. This difference was statistically signi®cant (t test, p < 0.01). Notably, up to 4% of TIL expressed CD94 in the absence of CD8. Predominance of CD94/NKG2-C/E on TIL in melanoma Although natural killer receptors-mediated inhibitory signals were suggested to be dominant over inhibitory signals, it was necessary to resolve whether the inhibitory CD94/ NKG2-A/B, or the activating CD94/NKG2-C/E, or both are expressed on TIL. This differentiation was performed by reverse transcription±PCR by means of speci®c primers for NKG2-A/B and C/E, respectively. Using this approach, we were able to demonstrate the presence of activating NKG2-C/E mRNA in all primary and metastatic lesions. In contrast, the inhibitory NKG2A/B was only present in 50% of primary tumors. Its frequency, however, increased to 80% in metastatic lesions. Semi-quantitative analysis con®rmed the differential expression of NKG2-A/B in primary tumors and metastases (Fig 4). Even if the limitations of quantitative reverse transcription±PCR analysis are taken into account, these data corroborate the qualitative PCR data, suggesting a pronounced expression of the inhibitory NKG2-A/ B in metastatic melanoma as compared with primary tumors. It should be noted that this form of analysis does not provide any information whether NKG2-A/B and NKG2-C/E are expressed in the same or in different cells. Preliminary results from single cell PCR of cultured TIL, however, indicated that both scenarios occur. Kinetics of CD94/NKG2-A/B and CD94/NKG2-C/E expression on TIL in vitro We next tested whether T cells expressing NKG2-A/B or NKG2-C/E would display a differential behavior in vitro. We ®rst characterized CD94/NKG2-A/B and NKG2-C/E expression on TIL bulk cultures obtained from three primary melanomas over a period of 4 wk. Phenotypic analysis immediately after isolation by ¯ow cytometry using CD3CD94, CD4CD94, CD8CD94, and CD56CD94 double staining con®rmed the in situ ®ndings that the majority of CD94 positive cells coexpressed CD8, and that virtually all CD56 positive cells expressed CD94. The percentage of CD56 expressing cells, however, ranged between 2% and 6% of total lymphocytes; thus it was higher than anticipated by immunohistology (data not shown). Over the course of several weeks the expression pattern of CD94 on CD8+ T and CD56+ natural killer cells remained remarkable stable (Fig 5A). Reverse transcription±PCR of bulk cultures immediately after isolation con®rmed the expression of the NKG2-A/B and NKG2-C/E mRNA in TIL. Analysis of mRNA expression weekly over a period of 4 wk revealed that only one of three cultures showed a decreased expression of NKG2-A/B (Fig 5B). Notably, the cytotoxic capacity of cultures showing a differential NKG2-A/B expression against the autologous tumor seemed not to be correlated with the level of this expression (data not shown). DISCUSSION
Figure 1. Expression of CD94 on TIL. Sections of primary (A) and metastatic melanoma (B, C) were subjected to staining with anti-CD94 (A, B), and anti-CD56 (C) monoclonal antibodies. Scale bars: (A) 100 mm; (B, C) 25 mm.
There is a general agreement that melanoma cells are subject to an immunologic response, and insight into the nature of anti-tumor T cell responses has accumulated extensively during the past years. Notably, a high number of peptide epitopes derived from melanoma-associated antigens were identi®ed as targets for cytotoxic lymphocytes (De-Smet et al, 1997). Activation of T cells through antigen recognition is mediated by ligation of the TCR to HLA/peptide complexes (Chien and Davies, 1993). Conversely, natural killer cell mediated cytotoxicity is in general suppressed upon recognition of class I molecules (Lanier, 1998). Hence,
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natural killer cells may plug the gap in the immune response against MHC class I de®cient tumors. Lately it has been shown that the expression of natural killer receptors is not restricted to natural killer cells, but that T cells may also express natural killer receptors, and that these natural killer receptors in¯uence the outcome of TCR/HLA ligation. Thus, it has been suggested that the expression of inhibitory natural killer receptors by melanoma-speci®c T cells could suppress anti-tumor cytotoxicity and accomplish the escape of melanoma cells from immune surveillance (Ikeda et al, 1997; Speiser et al, 1999a). In line with these ®ndings, we were able to demonstrate that CD94/NKG2 is widely expressed by TIL in situ, both on natural killer and T cells. Immunohistochemistry revealed that 5±10% of TIL express the CD94 molecule and the majority of the receptors appear to be expressed by CD8+ T cells. These data corroborate the reports of Speiser et al (1999a, b) describing that the number of T cells expressing natural killer receptors in peripheral
blood are higher in melanoma and vitiligo patients compared with healthy donors. In addition, we scrutinized this CD94/NKG2 expression by determining if activating, inhibitory or both CD94/NKG2 heterodimers were present on TIL. Using reverse transcription± PCR, speci®cally amplifying NKG2-A/B and NKG2-C/E transcripts, we demonstrate the perceptible predominance of the activatory CD94/NKG2-C/E dimers. Surprisingly, the inhibitory NKG2-A/B was only detected in 50% of the primary lesions whereas the activating NKG2-C/E could be detected in all lesions. Recent studies have shown that in healthy human donors the mean number of inhibitory natural killer receptors is higher than the mean number of activating receptors (Valiante et al, 1997). For the melanoma lesions characterized here, this seems not to be the case, indicating a regulated event. The expression of the stress inducible molecules like MICA/B on melanoma cells may be such a feedback
Table I. Characteristics of primary melanoma Age
Sex
Type
Thicknessa
In®ltrateb
CD8+
CD8+/CD94+
CD94+
37 80 50 51 67 63 42 69 64 32 69 50 65 77 73 69 69 94
M M M M F M F F M F M F F M F M F F
in situ SSM SSM SSM SSM SSM SSM SSM NMM SSM NMM SSM NMM SSM SSM NMM SSM LMM
0.2 0.5 0.7 0.9 1.4 1.8 1.9 1.97 2.0 2.0 2.15 2.3 2.6 2.6 2.8 3.6 6.0
(+) (+) ++ (+) ++ + (+) ++ + ++ ++ (+) + + ++ ++ + ++
90.0§ 94.2 92.5 89.7 96.2 91.7 95 95.5 94.9 98.6 98.3 92.9 95.6 100.0 93.1 100.0 98.1 93.7
7.5 5.4 5.7 9.2 3.4 7.5 4.0 4.0 4.1 0.9 1.3 6.1 4.0 0.0 6.4 0.0 1.9 5.3
2.5 0.4 1.8 1.1 0.4 0.8 1.1 0.5 1.0 0.4 0.4 1.0 0.4 0.0 0.5 0.0 0.0 1.0
aBreslow's bThe
tumor thickness is given in mm. in¯ammatory in®ltrate is classi®ed according to the criteria of Elder and Clark as ++, brisk; +, non brisk; (+), dim; é, absent. are given as a percentage of the total of stained cells.
cResults
Figure 2. Predominant expression of CD94 on CD8+ T cells. Sections of metastatic melanoma were subjected to double staining with monoclonal antibodies against CD4CD94 (A), CD8CD94 (B, C), or CD56CD94 (D). CD4, CD8, and CD56 positive cells are depicted by a blue, CD94 positive by a red, and double positive by a dark purple color. Scale bars: 25 mm.
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mechanism (Groh et al, 1999; Bauer et al, 1999). Hence, the reduced expression of such destruction tags may result in an ineffective activation of NKG2-C/E expressing TIL. The capacity of inhibitory natural killer receptors to inhibit T cell cytotoxicity against melanoma cells has been demonstrated in vitro, but it is not known whether this mechanism is a widespread inhibitor of melanoma cell killing in vivo (Ikeda et al, 1997; Noppen et al, 1998; Speiser et al, 1999a). If so, our data indicate that the phenomenon is likely to take place in metastatic tumors. Although
Figure 3. Relative expression of CD94 among CD8+ TIL in primary and metastatic melanoma. Numbers of CD94+ cells are given as a percentage of all stained cells of a CD8CD94 double staining with CD8 single positive cells depicted in gray, CD8CD94 double positive in black, and CD94 single positives in white.
Figure 4. Predominance of CD94/NKG2-C/E in primary melanoma. Samples of primary tumors (A) and metastases (B) were analyzed by reverse transcription±PCR with speci®c primers for NKG2-A/B and NKG2-C/E as described in Materials and Methods. Details on patient and tumor characteristics are given in Table I.
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only 50% of the primary lesions showed the presence of inhibitory NKG2 transcripts, it is evident that these were present in the majority of metastatic lesions analyzed. The question whether NKG2-A/B and NKG2-C/E are either coexpressed by the same cells or expressed by separate populations of cells remains elusive. It has been shown, however, that in vitro established T cell clones may express two or more functional natural killer receptors, and it is likely that this is the case in vivo as well. Furthermore, several reports demonstrated that inhibitory signals overrule activating signals (Lopez-Botet and Bellon, 1999). Thus, it is possible that the expression of both NKG2-A/B and NKG2-C/E molecules leaves only an inhibitory function. It is well known that in vitro culture of TIL induces dramatic changes in the T cell population through cell death and oligoclonal expansion (Faure et al, 1998), which prompted us to test whether the expression of CD94 would change during in vitro culture. Surprisingly, this appeared to be the case only for one of three cultures in which a decrease in the expression of NKG2-A/B mRNA was detected. This observation suggests that (i) the expression of inhibitory natural killer receptors does not necessarily re¯ect a reduced capacity of TIL to be maintained in vitro (thorStraten et al, 1999), and (ii) the culture conditions used did not differentially in¯uence the expression of NKG2 molecules. The molecular mechanism for the regulation of expression of natural killer receptors by T cells remains unknown, but several lines of evidence suggest that these receptors are expressed as a consequence of T cell activation (Mingari et al, 1995, 1998; Phillips et al, 1995). In vitro expression of natural killer receptors, however, is not easily induced and even activated virus-speci®c T cells do not express natural killer receptors. A possible candidate of induction of CD94 on TIL in melanoma is transforming growth factor-b. Transforming growth factor-b has been demonstrated to be secreted by several melanoma cell lines in vitro as well as being expressed within melanoma lesions in situ (Wagner et al, 1998). This cytokine is a strong inducer of CD94 expression on T cells in vitro (Bertone et al, 1999). CD94 expression on TIL may also be induced as a consequence to the loss of MHC class I molecules on melanoma cells. Loss of MHC class I molecules in both primary and metastatic melanoma has been repeatedly reported (Ferrone and Marincola, 1995). Approximately 15% of primary and 50% of metastatic melanoma display reduced MHC expression; thus, loss of MHC class I expression and increased CD94/NKG2-A/B expression seem to be linked to similar tumor stages; however, we did not analyze the possibility of such a correlation in detail. It is also conceivable that CD94 expression on TIL is a marker of activation and expansion stage of T cells (Hamann et al, 1999). It was shown repeatedly that in situ expanded clonotypic T cells within the TIL population are not capable of further growth in vitro (thor-Straten et al, 1999). The features of these cells include a reduction in telomere length, as well as a low or absent expression of CD28. Recently, a correlation between the lack of CD28 and the presence of natural killer receptors, such as CD94/NKG2, has been demonstrated (Speiser et al, 1999b). Within these CD28±/ natural killer receptor positive T cell populations, clonally expanded T cells are dominant (Mingari et al, 1996). These ®ndings suggest that lack of CD28 and expression of natural killer receptors are signs of a particular activation stage of T cell, i.e., highly differentiated, nonproliferating effector cells. The expression of natural killer receptors by T cells offers a new perspective to the regulation of T cell responses, and it is still a matter of debate whether the presence of natural killer receptors on TIL is harmful to the host (Mingari et al, 1998). The absence of inhibitory natural killer receptors on TIL in 50% of primary melanoma and the presence of both inhibitory and activating receptors in most metastatic lesions indicates that there is no simple answer to this question. It is tempting to speculate that natural killer receptors are regulators of T cell responses involving TCR of low af®nity such as those for self-antigens in conjunction with stress
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Figure 5. Kinetics of CD94/NKG2-A/B and CD94/NKG2-C/E expression on TIL in vitro. (A) Surface expression of CD94 on CD8+ TIL single cell suspensions immediately after isolation or subsequent to a culture period of 4 wk was analyzed by ¯ow cytometry. Representative results are given. (B) Reverse transcription±PCR analysis for the presence of NKG2-A/B and NKG2-C/E mRNA in TIL single cell suspensions immediately after isolation (lanes 1, 3, and 5) or subsequent to a 4 wk culture period (lanes 2, 4, and 6) was performed.
inducible molecules, e.g., MICA/B, which serve as destruction tags (Bauer et al, 1999; Raulet, 1999). The expression of activating natural killer receptors on responding T cells would enable the fast clearance of these cells from the host. Once the T cell response is unsuccessful for prolonged periods, however, it is suppressed by the upregulation of inhibitory natural killer receptors (Halary et al, 1997). Thus, natural killer receptors would act as a safeguard to avoid long-lasting autoimmune responses, but at the same time allow killing, even of cells expressing self-antigens like melanoma differentiation antigens if danger signals are present (Matzinger, 1994; Pardoll, 1999). This hypothesis would explain the destruction of normal melanocytes in close vicinity of a primary melanoma in patients not developing generalized vitiligo (Becker et al, 1999). We express our appreciation to Claudia Siedel and Tina Seremet for their excellent technical assistance. We would like to give special thanks to all patients for their enthusiastic collaboration. We also thank Dr. Alex McLellan for critical discussions during the preparation of the manuscript. This work was partly funded by the BMFT (IZKF Wuerzburg B12) and the Danish Cancer Society.
REFERENCES Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, Spies T: Activation of NK cells and T cells by NKG2D, a receptor for stress- inducible MICA. Science 285:727±729, 1999 Becker JC, Brabletz T, Kirchner T, Conrad CT, Brocker EB, Reisfeld RA: Negative transcriptional regulation in anergic T cells. Proc Natl Acad Sci USA 92:2375± 2378, 1995 Becker JC, Guldberg P, Zeuthen J, Brocker EB, thor-Straten P: Accumulation of identical T cells in melanoma and vitiligo-like leukoderma. J Invest Dermatol 113:1033±1038, 1999 Bertone S, Schiavetti F, Bellomo R, Vitale C, Ponte M, Moretta L, Mingari MC: Transforming growth factor-beta-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes. Eur J Immunol 29:23±29, 1999 Borrego F, Ulbrecht M, Weiss EH, Coligan JE, Brooks AG: Recognition of human histocompatibility leukocyte antigen (HLA) -E complexed with HLA class I signal sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-mediated lysis. J Exp Med 187:813±818, 1998 Brooks AG, Borrego F, Posch PE, et al: Speci®c recognition of HLA-E, but not classical, HLA class I molecules by soluble CD94/NKG2A and NK cells. J Immunol 162:305±313, 1999 Chang C, Rodriguez A, Carretero M, Lopez-Botet M, Phillips JH, Lanier LL: Molecular characterization of human CD94: a type II membrane glycoprotein related to the C-type lectin superfamily. Eur J Immunol 25:2433±2437, 1995 Chien YH, Davis MM: How alpha beta T-cell receptors ``see'' peptide/MHC complexes. Immunol Today 14:597±602, 1993 Colonna M, Samaridis J: Cloning of immunoglobulin-superfamily members
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associated with HLA-C and HLA-B recognition by human natural killer cells. Science 268:405±408, 1995 De-Smet C, Lurquin C, De-Plaen E, et al: Genes coding for melanoma antigens recognised by cytolytic T lymphocytes. Eye 11:243±248, 1997 Faure F, Even J, Kourilsky P: Tumor-speci®c immune response: current in vitro analyses may not re¯ect the in vivo immune status. Crit Rev Immunol 18:77±86, 1998 Ferrone S, Marincola FM: Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional signi®cance and clinical relevance. Immunol Today 16:487±494, 1995 Glienke J, Sobanov Y, Brostjan C, et al: The genomic organization of NKG2C, E, F, and D receptor genes in the human natural killer gene complex. Immunogenetics 48:163±173, 1998 Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH, Spies T: Broad tumorassociated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB. Proc Natl Acad Sci USA 96:6879±6884, 1999 Halary F, Peyrat MA, Champagne E, et al: Control of self-reactive cytotoxic T lymphocytes expressing gamma delta T cell receptors by natural killer inhibitory receptors. Eur J Immunol 27:2812±2821, 1997 Hamann D, van Roos MT, Lier RA: Faces and phases of human CD8 T-cell development. Immunol Today 20:177±180, 1999 Ho EL, Heusel JW, Brown MG, Matsumoto K, Scalzo AA, Yokoyama WM: Murine Nkg2d and Cd94 are clustered within the natural killer complex and are expressed independently in natural killer cells. Proc Natl Acad Sci USA 95:6320± 6325, 1998 Ikeda H, Lethe B, van Lehmann F, et al: Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor. Immunity 6:199±208, 1997 Isakov N: ITIMs and ITAMs. The Yin and Yang of antigen and Fc receptor-linked signaling machinery. Immunol Res 16:85±100, 1997 Lanier LL: Natural killer cells: from no receptors to too many. Immunity 6:371±378, 1997 Lanier LL: NK cell receptors. Annu Rev Immunol 16:359±393, 1998 Lanier LL, Corliss B, Wu J, Phillips JH: Association of DAP12 with activating CD94/NKG2C NK cell receptors. Immunity 8:693±701, 1998 Lee N, Llano M, Carretero M, Ishitani A, Navarro F, Lopez-Botet M, Geraghty DE: HLA-E is a major ligand for the natural killer inhibitory receptor CD94/ NKG2A. Proc Natl Acad Sci USA 95:5199±5204, 1998 Ljunggren HG, Karre K: In search of the ``missing self'': MHC molecules and NK cell recognition. Immunol Today 11:237±244, 1990 Lopez-Botet M, Bellon T: Natural killer cell activation and inhibition by receptors for MHC class I. Curr Opin Immunol 11:301±307, 1999 Lopez-Botet M, Carretero M, Bellon T, Perez-Villar JJ, Llano M, Navarro F: The CD94/NKG2 C-type lectin receptor complex. Curr Top Microbiol Immunol 230:41±52, 1998 Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 12:991± 1045, 1994 Meyaard L, Adema GJ, Chang C, Woollatt E, Sutherland GR, Lanier LL, Phillips JH: LAIR-1, a novel inhibitory receptor expressed on human mononuclear leukocytes. Immunity 7:283±290, 1997 Mingari MC, Vitale C, Cambiaggi A, Schiavetti F, Melioli G, Ferrini S, Poggi A: Cytolytic T lymphocytes displaying natural killer (NK) -like activity: expression of NK-related functional receptors for HLA class I molecules (p58 and CD94) and inhibitory effect on the TCR-mediated target cell lysis or lymphokine production. Int Immunol 7:697±703, 1995
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Mingari MC, Schiavetti F, Ponte M, et al: Human CD8+ T lymphocyte subsets that express HLA class I-speci®c inhibitory receptors represent oligoclonally or monoclonally expanded cell populations. Proc Natl Acad Sci USA 93:12433± 12438, 1996 Mingari MC, Moretta A, Moretta L: Regulation of KIR expression in human T cells: a safety mechanism that may impair protective T-cell responses. Immunol Today 19:153±156, 1998 Moretta A, Bottino C, Vitale M, Pende D, Biassoni R, Mingari MC, Moretta L: Receptors for HLA class-I molecules in human natural killer cells. Annu Rev Immunol 14:619±648, 1996 Moretta A, Sivori S, Ponte M, Mingari MC, Moretta L: Stimulatory receptors in NK and T cells. Curr Top Microbiol Immunol 230:15±23, 1998 Noppen C, Schaefer C, Zajac P, et al: C-type lectin-like receptors in peptide-speci®c HLA class I-restricted cytotoxic T lymphocytes: differential expression and modulation of effector functions in clones sharing identical TCR structure and epitope speci®city. Eur J Immunol 28:1134±1142, 1998 Pardoll DM: Inducing autoimmune disease to treat cancer. Proc Natl Acad Sci USA 96:5340±5342, 1999 Pende D, Biassoni R, Cantoni C, et al: The natural killer cell receptor speci®c for HLA-A allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140-kD disulphide-linked dimer. J Exp Med 184:505±518, 1996 Phillips JH, Gumperz JE, Parham P, Lanier LL: Superantigen-dependent, cellmediated cytotoxicity inhibited by MHC class I receptors on T lymphocytes. Science 268:403±405, 1995 Plougastel B, Jones T, Trowsdale J: Genomic structure, chromosome location, and alternative splicing of the human NKG2A gene. Immunogenetics 44:286±291, 1996 Raulet DH: Does a low level of expression of HLA molecules engender autoimmunity? N Engl J Med 340:314±315, 1999 Sobanov Y, Glienke J, Brostjan C, Lehrach H, Francis F, Hofer E: Linkage of the NKG2 and CD94 receptor genes to D12S77 in the human natural killer gene complex. Immunogenetics 49:99±105, 1999 Speiser DE, Pittet MJ, Valmori D, et al: In vivo expression of natural killer cell inhibitory receptors by human melanoma-speci®c cytolytic T lymphocytes. J Exp Med 190:775±782, 1999a Speiser DE, Valmori D, Rimoldi D, et al: CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients. Eur J Immunol 29:1990±1999, 1999b thor-Straten P, Becker JC, Guldberg P, Zeuthen J: In situ T cells in melanoma. Cancer Immunol Immunother 48:386±395, 1999 Valiante NM, Uhrberg M, Shilling HG, et al: Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity 7:739±751, 1997 Vitale M, Bottino C, Sivori S, et al: NKp44, a novel triggering surface molecule speci®cally expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis. J Exp Med 187:2065± 2072, 1998 Wagner SN, Schultewolter T, Wagner C, Briedigkeit L, Becker JC, Kwasnicka HM, Goos M: Immune response against human primary malignant melanoma: a distinct cytokine mRNA pro®le associated with spontaneous regression. Lab Invest 78:541±550, 1998
Natural killer receptors are expressed both on natural killer populations and subpopulations of T cells, mainly a/b TCR+CD8+ T cells. We have characterized the expression of the C-type lectin natural killer receptor CD94/NKG2 on tumor-in®ltrating lymphocytes in primary and metastatic melanoma lesions. By immunohistochemistry, 5±10% of the tumorin®ltrating lymphocytes, both in primary and metastatic lesions, expressed CD94. More than 95% of these CD94+ cells coexpressed CD8 and the percentage of CD94 expression within the CD8+ cell population ranged from 5 to 20% with a higher expression in metastatic lesions. CD94/NKG2 exists both in an inhibitory and an activating form; thus, it was necessary to determine whether the inhibitory CD94/NKG2-A/B, the activating CD94/NKG2-C/E, or both were expressed on tumor-in®ltrating lymphocytes. Reverse transcription±polymerase chain
reaction using speci®c primers for NKG2-A/B and C/E mRNA revealed the presence of NKG2-C/E in all primary and metastatic lesions. In contrast, the inhibitory NKG2-A/B was only present in 50% of primary tumors whereas 80% of tumor-in®ltrating lymphocytes in metastatic lesions expressed these transcripts. In healthy humans, the mean number of inhibitory natural killer receptors is higher than that of activating receptors, but the opposite was true for tumor-in®ltrating lymphocytes in melanoma. The reversal of the ratio of inhibitory to activating natural killer receptors among tumor-in®ltrating lymphocytes suggests a regulated event due to either speci®c factors within the tumor microenvironment, preferential homing of T cell subsets, or certain stages of T cell activation. Key words: cytotoxic T lymphocyte/natural killer receptor/T cell response. J Invest Dermatol 114:941±947, 2000
T
structural groups of natural killer receptors have been characterized. The ®rst group includes type I integral membrane proteins of the immunoglobulin superfamily, e.g., p50, p58.1, p58.2, p70, p140, LAIR-1, or NKp44 (Colonna and Samaridis, 1995; Pende et al, 1996; Meyaard et al, 1997; Vitale et al, 1998); whereas, the second group includes type II integral membrane proteins of the C-type lectin superfamily (Chang et al, 1995). The C-type lectin-like receptors are more abundant than immunoglobulin-like receptors (Lopez-Botet et al, 1998). The initial name of these receptors, killer inhibitory receptor, is presumptive regarding their functional activity, as most natural killer receptors exist in two forms: one mediating activation whereas the other sustains inhibition of natural killer cell function (Moretta et al, 1998). The difference between activating and inhibiting receptors is based on the presence or absence of an ITIM (Isakov, 1997). Activating isoforms, lacking the immunoreceptor tyrosine-based inhibitory motif (ITIM), noncovalently associate with the adapter molecules such as DAP10 or DAP12, which pass the killing signal on to the downstream signaling pathway (Lanier et al, 1998). The CD94 molecule belongs to the C-type lectin superfamily and is covalently bound to another type II membrane protein, NKG2 (Lopez-Botet et al, 1998). The functional capacity of the CD94/NKG2 heterodimer to mediate activation or inhibition is determined by the NKG2 subtype: NKG2-A and NKG2-B subtypes generate an inhibitory receptor, whereas NKG2-C and NKG2-E in conjunction with CD94 results in an activating complex (Lanier et al, 1998). Recent molecular genetic studies suggest that the NKG2-D has distinct features from the other NKG2 molecules, including its position within another functional
wo general systems of cellular immunity have developed during evolution: innate (natural immunity) and acquired (adaptive immunity). Only recently, it was recognized that two of the major players of these systems, natural killer and T cells, share an essential group of receptors termed natural killer receptors (Moretta et al, 1996; Lanier, 1998). Initially, the cytolytic activity mediated by natural killer cells was regarded as non-major histocompatibility complex (MHC)-restricted as susceptibility to natural killer lysis appeared to be unrelated to the MHC haplotype of the target cell. Over the last years, however, it became evident that, whereas the natural killer cell-mediated cytotoxicity is not MHC restricted, it is still regulated by the expression of MHC class I molecules A better understanding of this phenomenon began in 1990 when Ljunggren and KaÈrre (1990) proposed the ``missing self hypothesis'', which implies that a reduced expression of MHC class I molecules on target cells stops the otherwise present inhibition of natural killer cell functions, thus permitting natural killing. This hypothesis stimulated the search and subsequent discovery of the very complex and still expanding system of natural killer cell receptors (Lanier, 1997). To date, two
Manuscript received November 9, 1999; revised January 25, 2000; accepted for publication February 1, 2000. Reprint requests to: Priv. Doz. Dr JuÈrgen C. Becker, Department of Dermatology, Julius-Maximilians-University, Josef-Schneider-Str. 2, D97080 WuÈrzburg, Germany. Email: [email protected] Abbreviations: PBL, peripheral blood lymphocytes; TIL, tumorin®ltrating lymphocytes. 0022-202X/00/$15.00
´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 941
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gene. Furthermore, the degree of homology of NKG2-D to the other NKG2 family members, including NKG2-A, NKG2-C, and NKG2-E, is similar to other unrelated C-type lectins (21±24% identity), suggesting that NKG2-D may not belong to the NKG2 family (Ho et al, 1998). It is well established that CD94/NKG2-A speci®cally interacts with HLA-E and that this interaction is dependent on the association of HLA-E with peptide. Moreover, no interaction between CD94/NKG2-A and classical HLA class I molecules has been described; however, class I leader sequence peptides bind to and stabilize HLA-E (Borrego et al, 1998; Lee et al, 1998; Brooks et al, 1999). The genomic information for CD94 and the NKG2 maps to the human ``natural killer gene complex'' on chromosome 12p12.3±p13.1 (Plougastel et al, 1996). It should be noted, that NKG2-A and NKG2-B are products of one gene, whereas NKG2-C and NKG2-E are encoded by different genes (Glienke et al, 1998; Sobanov et al, 1999). Natural killer cells use inhibitory natural killer receptors to monitor target cells for reduced or absent expression of self MHC class I molecules. The accuracy of detection is facilitated by overlapping subsets of natural killer cells expressing different natural killer receptors. This enables the detection of the loss of expression of a single allele of one MHC locus. As mentioned above natural killer receptors are also expressed on a small subset of T cells and in¯uence T cell receptor (TCR) mediated functions (Mingari et al, 1996). Upon interaction of the TCR with MHC class I natural killer receptors inhibit the cytolytic function of T cellsÐas demonstrated both for redirected as well as antigen-speci®c lysis (Noppen et al, 1998). Furthermore, the inhibition of lysis of solid tumors, in particular melanoma, has been reported (Ikeda et al, 1997; Speiser et al, 1999a). Thus, the induction of natural killer receptors on tumor-in®ltrating lymphocytes (TIL) has been discussed as some form of immunescape. If lost, reduced or aberrant expression of MHC class I molecules on tumor cells, as well as the existence of activating natural killer receptors and their interaction with destruction tags, are taken in consideration, however, the scenario becomes more complex (Bauer et al, 1999). Indeed, the expression of natural killer receptors on T cells may serve as a valuable instrument for the regulation of T cell responses. In order to obtain more information on the role of natural killer receptors in anti-tumor responses, we scrutinized the expression of CD94/NKG2 subtypes on melanoma in®ltrating lymphocytes in situ and in vitro. MATERIALS AND METHODS Tumor samples Fresh tumor samples from 18 primary and 16 metastatic melanomas were obtained by surgical excision following informed patient consent. The tumors were divided in two parts one of which was snap frozen in liquid nitrogen immediately after surgery and stored at ±80°C. The remaining part, which included the thickest portion of the tumor, was used for routine histology and diagnosis. All metastatic lesions were cutaneous metastases. Immunohistochemistry Five micron sections of tumor lesions were ®xed in cold acetone and air dried for 30 min. Slides were incubated for 30 min with the indicated speci®c primary monoclonal antibody (MoAb) at predetermined optimal dilutions. After two washes, the sections were incubated with biotinylated goat anti-rat or anti-mouse immunoglobulin (DAKO, Hamburg, Germany) for 20 min, followed by another two washes. Staining was revealed with streptavidin±horseradish peroxidase and 3-amino-9-ethylcarbazole (AEC)-substrate. The reaction was stopped by washing after 25 min. For double staining, slides were incubated with rabbit serum at a ®nal dilution of 1:5, followed by the second primary MoAb. After a 40 min incubation and two washes, the species speci®c alkaline phosphatase (AP)-conjugated secondary antibodies were added and after additional washing steps staining with alkaline phosphatase-anti-alkaline phosphatase and Fast Blue substrate was performed. For single staining, slides were subjected to haÈmalaun nucleus staining. All antibodies are commercially available: CD3 (DAKO, clone UCHT1), CD4 (DAKO, clone MT310), CD8 (DAKO, clone C8/144B), CD56 (DAKO, clone MOC-1), and CD94 (Pharmingen, Hamburg, Germany; clone HP-3D9).
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Reverse transcription±polymerase chain reaction (reverse transcription±PCR) RNA was extracted using the Purescript Isolation Kit (Gentra Systems Inc., Minneapolis, MN) cDNA synthesis was carried out using M-MLV SuperScript II reverse transcriptase (GibcoBRL, Life Technologies Inc., Gaithersburg, MD) in a total volume of 50 ml 1 3 buffer provided by the manufacturer containing 10 mM dithiothreitol. Incubations were performed at 37°C for 30 min, 42°C for 30 min, 50°C for 30 min, and 72°C for 5 min cDNA were ampli®ed with primers for GAPDH (GAP-371p; 5¢-AGGGGGGAGCCAAAAGGG-3¢, GAP-891m; 5¢-AGGAGT GGGTGTCGCTGTTG-3¢; size 540 bp), NKG2-AB (AB391p; 5¢-TGGGAATTATCTGTC TTATCTTAA-3¢, AB-868m; 5¢CAAATGCAAACGCTTTACC-3¢, size for NKG2-A; 496 bp, NKG2B; 442 bp), and NKG2-CE (CE-108p, 5¢-CCGAACAGGAAATATTCCAAG-3¢, CE-294m 5¢-AAGAATTGTTCTGCTCCAGGA-3¢, size for both NKG-C and NKG-E transcripts; 207 bp). Ampli®cations were carried out in a total volume of 25 ml containing 1 3 PCR buffer [5 mM KCl, 20 mM Tris pH 8.4, 2.0 mM MgCl2, 0.2 mM cresol red, 12% sucrose, 0.005% (wt/vol) bovine serum albumin (Boehringer-Mannheim, Mannheim, Germany)], 2.5 pmol of each primer, 40 mM dNTP (Pharmacia LKB, Uppsala, Sweden) and 1.25 units of AmpliTaq polymerase (Perkin Elmer Cetus Corporation, Emeryville, CA). The parameters used for ampli®cation were 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s for 32 (GAPDH) or 38 (NKG2) cycles taking advantage of a ``touch down'' setting going from 65°C to 60°C over the ®rst nine cycles. Taq polymerase and dNTP were added to the reaction tube at an 80°C step between the denaturation and annealing steps of the ®rst cycle. Isolation of TIL and in vitro culture Melanoma in®ltrating lymphocytes were obtained by mincing freshly resected tumor biopsies from which all adjacent nonmalignant tissues had been removed. The resulting small pieces were digested for 2.5 h in RPMI 1640 with 0.25% collagenase D (Boehringer Mannheim) and 100 mg per ml deoxyribonuclease type I (Boehringer Mannheim) followed by a passage through a steel sieve. After three washes, cells were either used immediately for analysis or expanded in bulk cultures at 1 3 105 cells per ml in RPMI 1640, supplemented with 10% heat-inactivated human AB-plasma (Transfusion Medicine Center, University WuÈrzburg), 1% penicillin/ streptomycin; and 1% sodium pyruvate, 2 mM L-glutamine, 100 IU per ml interleukin-2 (Aldesleukin, Chiron; Ratingen, Germany) and 10% lymphocyte-conditioned medium, which was generated as previously described (Becker et al, 1995). Flow cytometry For cell labelling, cells were washed in phosphatebuffered saline, resuspended with 0.1% bovine serum albumin in phosphate-buffered saline at 1 3 105 per 100 ml and incubated with nonconjugated primary MoAb for 30 min at 4°C. After two washes, ¯uorescein isothiocyanate-conjugated secondary antibodies (Dianova, Hamburg, Germany) were added for 30 min. With a washing step inbetween a phycoerythrin-conjugated MoAb directed against the second antigen was added. Isotype-matched murine MoAb served as controls. Dead cells were identi®ed by propidium iodide staining (5 mg per ml). Fluorescence was analyzed using a FACScan (Becton Dickinson, Heidelberg, Germany). All MoAb are commercially available: CD4, CD8, or CD56 (Becton Dickinson) and CD94 (Clone HP-3D9, 315±020; Ancell, Bayport, MN).
RESULTS CD94 expression on TIL in primary and metastatic melanoma The frequent expression of natural killer receptors on activated T cells and in particular the report that melanomaspeci®c T cell clones express C-type lectin-like receptors prompted us to scrutinize the expression of CD94 on TIL. To identify populations of cells expressing CD94, we analyzed 18 primary tumors and 16 cutaneous metastases by immunohistochemistry (Fig 1). Initial studies using single staining for either CD3, CD4, CD8, CD56, or CD94, demonstrated that in most primary (14 of 18), but all metastatic tumors (16 of 16), CD94 was detectable. The percentage of CD94+ cells of all lymphocytes varied from patient to patient, but a trend towards a larger CD94+ population for metastatic lesions was obvious (data not shown); however, no correlation of qualitative or quantitative CD94 expression and prognostic parameters of primary tumors, e.g., tumor thickness, was present (Table I). Subsequently, we performed a series of double stainings including CD4CD94, CD8CD94, and CD56CD94 in order to characterize the CD94+ population in more detail (Fig 2).
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This analysis revealed that all CD56+ natural killer cells bore CD94. The percentage of CD56+ cells, however, as detected by immunohistology using either single or double staining never exceeded 1% of TIL. It was previously reported that the expression of C-type lectin-like receptors among T cells in peripheral blood is con®ned to cytotoxic T lymphocytes. We con®rmed this ®nding for TIL in melanoma: the major part of the CD94+ cells
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coexpressed CD8; CD4CD94 double positive cells were not detected in either primary or metastatic lesions. The CD8/CD94 double staining allowed for a more accurate quantitative analysis than the CD94 single staining (Fig 3). Within the primary tumors, the CD94+ cells comprised up to 7% of the CD8+ T cells with a median of 4%; for metastatic lesions, however, the CD94+ cells constituted up to 17% (median: 7%) of CD8+ cells. This difference was statistically signi®cant (t test, p < 0.01). Notably, up to 4% of TIL expressed CD94 in the absence of CD8. Predominance of CD94/NKG2-C/E on TIL in melanoma Although natural killer receptors-mediated inhibitory signals were suggested to be dominant over inhibitory signals, it was necessary to resolve whether the inhibitory CD94/ NKG2-A/B, or the activating CD94/NKG2-C/E, or both are expressed on TIL. This differentiation was performed by reverse transcription±PCR by means of speci®c primers for NKG2-A/B and C/E, respectively. Using this approach, we were able to demonstrate the presence of activating NKG2-C/E mRNA in all primary and metastatic lesions. In contrast, the inhibitory NKG2A/B was only present in 50% of primary tumors. Its frequency, however, increased to 80% in metastatic lesions. Semi-quantitative analysis con®rmed the differential expression of NKG2-A/B in primary tumors and metastases (Fig 4). Even if the limitations of quantitative reverse transcription±PCR analysis are taken into account, these data corroborate the qualitative PCR data, suggesting a pronounced expression of the inhibitory NKG2-A/ B in metastatic melanoma as compared with primary tumors. It should be noted that this form of analysis does not provide any information whether NKG2-A/B and NKG2-C/E are expressed in the same or in different cells. Preliminary results from single cell PCR of cultured TIL, however, indicated that both scenarios occur. Kinetics of CD94/NKG2-A/B and CD94/NKG2-C/E expression on TIL in vitro We next tested whether T cells expressing NKG2-A/B or NKG2-C/E would display a differential behavior in vitro. We ®rst characterized CD94/NKG2-A/B and NKG2-C/E expression on TIL bulk cultures obtained from three primary melanomas over a period of 4 wk. Phenotypic analysis immediately after isolation by ¯ow cytometry using CD3CD94, CD4CD94, CD8CD94, and CD56CD94 double staining con®rmed the in situ ®ndings that the majority of CD94 positive cells coexpressed CD8, and that virtually all CD56 positive cells expressed CD94. The percentage of CD56 expressing cells, however, ranged between 2% and 6% of total lymphocytes; thus it was higher than anticipated by immunohistology (data not shown). Over the course of several weeks the expression pattern of CD94 on CD8+ T and CD56+ natural killer cells remained remarkable stable (Fig 5A). Reverse transcription±PCR of bulk cultures immediately after isolation con®rmed the expression of the NKG2-A/B and NKG2-C/E mRNA in TIL. Analysis of mRNA expression weekly over a period of 4 wk revealed that only one of three cultures showed a decreased expression of NKG2-A/B (Fig 5B). Notably, the cytotoxic capacity of cultures showing a differential NKG2-A/B expression against the autologous tumor seemed not to be correlated with the level of this expression (data not shown). DISCUSSION
Figure 1. Expression of CD94 on TIL. Sections of primary (A) and metastatic melanoma (B, C) were subjected to staining with anti-CD94 (A, B), and anti-CD56 (C) monoclonal antibodies. Scale bars: (A) 100 mm; (B, C) 25 mm.
There is a general agreement that melanoma cells are subject to an immunologic response, and insight into the nature of anti-tumor T cell responses has accumulated extensively during the past years. Notably, a high number of peptide epitopes derived from melanoma-associated antigens were identi®ed as targets for cytotoxic lymphocytes (De-Smet et al, 1997). Activation of T cells through antigen recognition is mediated by ligation of the TCR to HLA/peptide complexes (Chien and Davies, 1993). Conversely, natural killer cell mediated cytotoxicity is in general suppressed upon recognition of class I molecules (Lanier, 1998). Hence,
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natural killer cells may plug the gap in the immune response against MHC class I de®cient tumors. Lately it has been shown that the expression of natural killer receptors is not restricted to natural killer cells, but that T cells may also express natural killer receptors, and that these natural killer receptors in¯uence the outcome of TCR/HLA ligation. Thus, it has been suggested that the expression of inhibitory natural killer receptors by melanoma-speci®c T cells could suppress anti-tumor cytotoxicity and accomplish the escape of melanoma cells from immune surveillance (Ikeda et al, 1997; Speiser et al, 1999a). In line with these ®ndings, we were able to demonstrate that CD94/NKG2 is widely expressed by TIL in situ, both on natural killer and T cells. Immunohistochemistry revealed that 5±10% of TIL express the CD94 molecule and the majority of the receptors appear to be expressed by CD8+ T cells. These data corroborate the reports of Speiser et al (1999a, b) describing that the number of T cells expressing natural killer receptors in peripheral
blood are higher in melanoma and vitiligo patients compared with healthy donors. In addition, we scrutinized this CD94/NKG2 expression by determining if activating, inhibitory or both CD94/NKG2 heterodimers were present on TIL. Using reverse transcription± PCR, speci®cally amplifying NKG2-A/B and NKG2-C/E transcripts, we demonstrate the perceptible predominance of the activatory CD94/NKG2-C/E dimers. Surprisingly, the inhibitory NKG2-A/B was only detected in 50% of the primary lesions whereas the activating NKG2-C/E could be detected in all lesions. Recent studies have shown that in healthy human donors the mean number of inhibitory natural killer receptors is higher than the mean number of activating receptors (Valiante et al, 1997). For the melanoma lesions characterized here, this seems not to be the case, indicating a regulated event. The expression of the stress inducible molecules like MICA/B on melanoma cells may be such a feedback
Table I. Characteristics of primary melanoma Age
Sex
Type
Thicknessa
In®ltrateb
CD8+
CD8+/CD94+
CD94+
37 80 50 51 67 63 42 69 64 32 69 50 65 77 73 69 69 94
M M M M F M F F M F M F F M F M F F
in situ SSM SSM SSM SSM SSM SSM SSM NMM SSM NMM SSM NMM SSM SSM NMM SSM LMM
0.2 0.5 0.7 0.9 1.4 1.8 1.9 1.97 2.0 2.0 2.15 2.3 2.6 2.6 2.8 3.6 6.0
(+) (+) ++ (+) ++ + (+) ++ + ++ ++ (+) + + ++ ++ + ++
90.0§ 94.2 92.5 89.7 96.2 91.7 95 95.5 94.9 98.6 98.3 92.9 95.6 100.0 93.1 100.0 98.1 93.7
7.5 5.4 5.7 9.2 3.4 7.5 4.0 4.0 4.1 0.9 1.3 6.1 4.0 0.0 6.4 0.0 1.9 5.3
2.5 0.4 1.8 1.1 0.4 0.8 1.1 0.5 1.0 0.4 0.4 1.0 0.4 0.0 0.5 0.0 0.0 1.0
aBreslow's bThe
tumor thickness is given in mm. in¯ammatory in®ltrate is classi®ed according to the criteria of Elder and Clark as ++, brisk; +, non brisk; (+), dim; é, absent. are given as a percentage of the total of stained cells.
cResults
Figure 2. Predominant expression of CD94 on CD8+ T cells. Sections of metastatic melanoma were subjected to double staining with monoclonal antibodies against CD4CD94 (A), CD8CD94 (B, C), or CD56CD94 (D). CD4, CD8, and CD56 positive cells are depicted by a blue, CD94 positive by a red, and double positive by a dark purple color. Scale bars: 25 mm.
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mechanism (Groh et al, 1999; Bauer et al, 1999). Hence, the reduced expression of such destruction tags may result in an ineffective activation of NKG2-C/E expressing TIL. The capacity of inhibitory natural killer receptors to inhibit T cell cytotoxicity against melanoma cells has been demonstrated in vitro, but it is not known whether this mechanism is a widespread inhibitor of melanoma cell killing in vivo (Ikeda et al, 1997; Noppen et al, 1998; Speiser et al, 1999a). If so, our data indicate that the phenomenon is likely to take place in metastatic tumors. Although
Figure 3. Relative expression of CD94 among CD8+ TIL in primary and metastatic melanoma. Numbers of CD94+ cells are given as a percentage of all stained cells of a CD8CD94 double staining with CD8 single positive cells depicted in gray, CD8CD94 double positive in black, and CD94 single positives in white.
Figure 4. Predominance of CD94/NKG2-C/E in primary melanoma. Samples of primary tumors (A) and metastases (B) were analyzed by reverse transcription±PCR with speci®c primers for NKG2-A/B and NKG2-C/E as described in Materials and Methods. Details on patient and tumor characteristics are given in Table I.
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only 50% of the primary lesions showed the presence of inhibitory NKG2 transcripts, it is evident that these were present in the majority of metastatic lesions analyzed. The question whether NKG2-A/B and NKG2-C/E are either coexpressed by the same cells or expressed by separate populations of cells remains elusive. It has been shown, however, that in vitro established T cell clones may express two or more functional natural killer receptors, and it is likely that this is the case in vivo as well. Furthermore, several reports demonstrated that inhibitory signals overrule activating signals (Lopez-Botet and Bellon, 1999). Thus, it is possible that the expression of both NKG2-A/B and NKG2-C/E molecules leaves only an inhibitory function. It is well known that in vitro culture of TIL induces dramatic changes in the T cell population through cell death and oligoclonal expansion (Faure et al, 1998), which prompted us to test whether the expression of CD94 would change during in vitro culture. Surprisingly, this appeared to be the case only for one of three cultures in which a decrease in the expression of NKG2-A/B mRNA was detected. This observation suggests that (i) the expression of inhibitory natural killer receptors does not necessarily re¯ect a reduced capacity of TIL to be maintained in vitro (thorStraten et al, 1999), and (ii) the culture conditions used did not differentially in¯uence the expression of NKG2 molecules. The molecular mechanism for the regulation of expression of natural killer receptors by T cells remains unknown, but several lines of evidence suggest that these receptors are expressed as a consequence of T cell activation (Mingari et al, 1995, 1998; Phillips et al, 1995). In vitro expression of natural killer receptors, however, is not easily induced and even activated virus-speci®c T cells do not express natural killer receptors. A possible candidate of induction of CD94 on TIL in melanoma is transforming growth factor-b. Transforming growth factor-b has been demonstrated to be secreted by several melanoma cell lines in vitro as well as being expressed within melanoma lesions in situ (Wagner et al, 1998). This cytokine is a strong inducer of CD94 expression on T cells in vitro (Bertone et al, 1999). CD94 expression on TIL may also be induced as a consequence to the loss of MHC class I molecules on melanoma cells. Loss of MHC class I molecules in both primary and metastatic melanoma has been repeatedly reported (Ferrone and Marincola, 1995). Approximately 15% of primary and 50% of metastatic melanoma display reduced MHC expression; thus, loss of MHC class I expression and increased CD94/NKG2-A/B expression seem to be linked to similar tumor stages; however, we did not analyze the possibility of such a correlation in detail. It is also conceivable that CD94 expression on TIL is a marker of activation and expansion stage of T cells (Hamann et al, 1999). It was shown repeatedly that in situ expanded clonotypic T cells within the TIL population are not capable of further growth in vitro (thor-Straten et al, 1999). The features of these cells include a reduction in telomere length, as well as a low or absent expression of CD28. Recently, a correlation between the lack of CD28 and the presence of natural killer receptors, such as CD94/NKG2, has been demonstrated (Speiser et al, 1999b). Within these CD28±/ natural killer receptor positive T cell populations, clonally expanded T cells are dominant (Mingari et al, 1996). These ®ndings suggest that lack of CD28 and expression of natural killer receptors are signs of a particular activation stage of T cell, i.e., highly differentiated, nonproliferating effector cells. The expression of natural killer receptors by T cells offers a new perspective to the regulation of T cell responses, and it is still a matter of debate whether the presence of natural killer receptors on TIL is harmful to the host (Mingari et al, 1998). The absence of inhibitory natural killer receptors on TIL in 50% of primary melanoma and the presence of both inhibitory and activating receptors in most metastatic lesions indicates that there is no simple answer to this question. It is tempting to speculate that natural killer receptors are regulators of T cell responses involving TCR of low af®nity such as those for self-antigens in conjunction with stress
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 5. Kinetics of CD94/NKG2-A/B and CD94/NKG2-C/E expression on TIL in vitro. (A) Surface expression of CD94 on CD8+ TIL single cell suspensions immediately after isolation or subsequent to a culture period of 4 wk was analyzed by ¯ow cytometry. Representative results are given. (B) Reverse transcription±PCR analysis for the presence of NKG2-A/B and NKG2-C/E mRNA in TIL single cell suspensions immediately after isolation (lanes 1, 3, and 5) or subsequent to a 4 wk culture period (lanes 2, 4, and 6) was performed.
inducible molecules, e.g., MICA/B, which serve as destruction tags (Bauer et al, 1999; Raulet, 1999). The expression of activating natural killer receptors on responding T cells would enable the fast clearance of these cells from the host. Once the T cell response is unsuccessful for prolonged periods, however, it is suppressed by the upregulation of inhibitory natural killer receptors (Halary et al, 1997). Thus, natural killer receptors would act as a safeguard to avoid long-lasting autoimmune responses, but at the same time allow killing, even of cells expressing self-antigens like melanoma differentiation antigens if danger signals are present (Matzinger, 1994; Pardoll, 1999). This hypothesis would explain the destruction of normal melanocytes in close vicinity of a primary melanoma in patients not developing generalized vitiligo (Becker et al, 1999). We express our appreciation to Claudia Siedel and Tina Seremet for their excellent technical assistance. We would like to give special thanks to all patients for their enthusiastic collaboration. We also thank Dr. Alex McLellan for critical discussions during the preparation of the manuscript. This work was partly funded by the BMFT (IZKF Wuerzburg B12) and the Danish Cancer Society.
REFERENCES Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, Spies T: Activation of NK cells and T cells by NKG2D, a receptor for stress- inducible MICA. Science 285:727±729, 1999 Becker JC, Brabletz T, Kirchner T, Conrad CT, Brocker EB, Reisfeld RA: Negative transcriptional regulation in anergic T cells. Proc Natl Acad Sci USA 92:2375± 2378, 1995 Becker JC, Guldberg P, Zeuthen J, Brocker EB, thor-Straten P: Accumulation of identical T cells in melanoma and vitiligo-like leukoderma. J Invest Dermatol 113:1033±1038, 1999 Bertone S, Schiavetti F, Bellomo R, Vitale C, Ponte M, Moretta L, Mingari MC: Transforming growth factor-beta-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes. Eur J Immunol 29:23±29, 1999 Borrego F, Ulbrecht M, Weiss EH, Coligan JE, Brooks AG: Recognition of human histocompatibility leukocyte antigen (HLA) -E complexed with HLA class I signal sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-mediated lysis. J Exp Med 187:813±818, 1998 Brooks AG, Borrego F, Posch PE, et al: Speci®c recognition of HLA-E, but not classical, HLA class I molecules by soluble CD94/NKG2A and NK cells. J Immunol 162:305±313, 1999 Chang C, Rodriguez A, Carretero M, Lopez-Botet M, Phillips JH, Lanier LL: Molecular characterization of human CD94: a type II membrane glycoprotein related to the C-type lectin superfamily. Eur J Immunol 25:2433±2437, 1995 Chien YH, Davis MM: How alpha beta T-cell receptors ``see'' peptide/MHC complexes. Immunol Today 14:597±602, 1993 Colonna M, Samaridis J: Cloning of immunoglobulin-superfamily members
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associated with HLA-C and HLA-B recognition by human natural killer cells. Science 268:405±408, 1995 De-Smet C, Lurquin C, De-Plaen E, et al: Genes coding for melanoma antigens recognised by cytolytic T lymphocytes. Eye 11:243±248, 1997 Faure F, Even J, Kourilsky P: Tumor-speci®c immune response: current in vitro analyses may not re¯ect the in vivo immune status. Crit Rev Immunol 18:77±86, 1998 Ferrone S, Marincola FM: Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional signi®cance and clinical relevance. Immunol Today 16:487±494, 1995 Glienke J, Sobanov Y, Brostjan C, et al: The genomic organization of NKG2C, E, F, and D receptor genes in the human natural killer gene complex. Immunogenetics 48:163±173, 1998 Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH, Spies T: Broad tumorassociated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB. Proc Natl Acad Sci USA 96:6879±6884, 1999 Halary F, Peyrat MA, Champagne E, et al: Control of self-reactive cytotoxic T lymphocytes expressing gamma delta T cell receptors by natural killer inhibitory receptors. Eur J Immunol 27:2812±2821, 1997 Hamann D, van Roos MT, Lier RA: Faces and phases of human CD8 T-cell development. Immunol Today 20:177±180, 1999 Ho EL, Heusel JW, Brown MG, Matsumoto K, Scalzo AA, Yokoyama WM: Murine Nkg2d and Cd94 are clustered within the natural killer complex and are expressed independently in natural killer cells. Proc Natl Acad Sci USA 95:6320± 6325, 1998 Ikeda H, Lethe B, van Lehmann F, et al: Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor. Immunity 6:199±208, 1997 Isakov N: ITIMs and ITAMs. The Yin and Yang of antigen and Fc receptor-linked signaling machinery. Immunol Res 16:85±100, 1997 Lanier LL: Natural killer cells: from no receptors to too many. Immunity 6:371±378, 1997 Lanier LL: NK cell receptors. Annu Rev Immunol 16:359±393, 1998 Lanier LL, Corliss B, Wu J, Phillips JH: Association of DAP12 with activating CD94/NKG2C NK cell receptors. Immunity 8:693±701, 1998 Lee N, Llano M, Carretero M, Ishitani A, Navarro F, Lopez-Botet M, Geraghty DE: HLA-E is a major ligand for the natural killer inhibitory receptor CD94/ NKG2A. Proc Natl Acad Sci USA 95:5199±5204, 1998 Ljunggren HG, Karre K: In search of the ``missing self'': MHC molecules and NK cell recognition. Immunol Today 11:237±244, 1990 Lopez-Botet M, Bellon T: Natural killer cell activation and inhibition by receptors for MHC class I. Curr Opin Immunol 11:301±307, 1999 Lopez-Botet M, Carretero M, Bellon T, Perez-Villar JJ, Llano M, Navarro F: The CD94/NKG2 C-type lectin receptor complex. Curr Top Microbiol Immunol 230:41±52, 1998 Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 12:991± 1045, 1994 Meyaard L, Adema GJ, Chang C, Woollatt E, Sutherland GR, Lanier LL, Phillips JH: LAIR-1, a novel inhibitory receptor expressed on human mononuclear leukocytes. Immunity 7:283±290, 1997 Mingari MC, Vitale C, Cambiaggi A, Schiavetti F, Melioli G, Ferrini S, Poggi A: Cytolytic T lymphocytes displaying natural killer (NK) -like activity: expression of NK-related functional receptors for HLA class I molecules (p58 and CD94) and inhibitory effect on the TCR-mediated target cell lysis or lymphokine production. Int Immunol 7:697±703, 1995
CD94 EXPRESSION ON TIL
947
Mingari MC, Schiavetti F, Ponte M, et al: Human CD8+ T lymphocyte subsets that express HLA class I-speci®c inhibitory receptors represent oligoclonally or monoclonally expanded cell populations. Proc Natl Acad Sci USA 93:12433± 12438, 1996 Mingari MC, Moretta A, Moretta L: Regulation of KIR expression in human T cells: a safety mechanism that may impair protective T-cell responses. Immunol Today 19:153±156, 1998 Moretta A, Bottino C, Vitale M, Pende D, Biassoni R, Mingari MC, Moretta L: Receptors for HLA class-I molecules in human natural killer cells. Annu Rev Immunol 14:619±648, 1996 Moretta A, Sivori S, Ponte M, Mingari MC, Moretta L: Stimulatory receptors in NK and T cells. Curr Top Microbiol Immunol 230:15±23, 1998 Noppen C, Schaefer C, Zajac P, et al: C-type lectin-like receptors in peptide-speci®c HLA class I-restricted cytotoxic T lymphocytes: differential expression and modulation of effector functions in clones sharing identical TCR structure and epitope speci®city. Eur J Immunol 28:1134±1142, 1998 Pardoll DM: Inducing autoimmune disease to treat cancer. Proc Natl Acad Sci USA 96:5340±5342, 1999 Pende D, Biassoni R, Cantoni C, et al: The natural killer cell receptor speci®c for HLA-A allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140-kD disulphide-linked dimer. J Exp Med 184:505±518, 1996 Phillips JH, Gumperz JE, Parham P, Lanier LL: Superantigen-dependent, cellmediated cytotoxicity inhibited by MHC class I receptors on T lymphocytes. Science 268:403±405, 1995 Plougastel B, Jones T, Trowsdale J: Genomic structure, chromosome location, and alternative splicing of the human NKG2A gene. Immunogenetics 44:286±291, 1996 Raulet DH: Does a low level of expression of HLA molecules engender autoimmunity? N Engl J Med 340:314±315, 1999 Sobanov Y, Glienke J, Brostjan C, Lehrach H, Francis F, Hofer E: Linkage of the NKG2 and CD94 receptor genes to D12S77 in the human natural killer gene complex. Immunogenetics 49:99±105, 1999 Speiser DE, Pittet MJ, Valmori D, et al: In vivo expression of natural killer cell inhibitory receptors by human melanoma-speci®c cytolytic T lymphocytes. J Exp Med 190:775±782, 1999a Speiser DE, Valmori D, Rimoldi D, et al: CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients. Eur J Immunol 29:1990±1999, 1999b thor-Straten P, Becker JC, Guldberg P, Zeuthen J: In situ T cells in melanoma. Cancer Immunol Immunother 48:386±395, 1999 Valiante NM, Uhrberg M, Shilling HG, et al: Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity 7:739±751, 1997 Vitale M, Bottino C, Sivori S, et al: NKp44, a novel triggering surface molecule speci®cally expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis. J Exp Med 187:2065± 2072, 1998 Wagner SN, Schultewolter T, Wagner C, Briedigkeit L, Becker JC, Kwasnicka HM, Goos M: Immune response against human primary malignant melanoma: a distinct cytokine mRNA pro®le associated with spontaneous regression. Lab Invest 78:541±550, 1998