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Natural Killer Receptors on CD8 T Cells and Natural Killer Cells from Different HLA-C Phenotypes in Melanoma Patients

Authors' Affiliations: ¹ Immunology Service and ² Dermatology Section, Virgen de la Arrixaca University Hospital; and ³ Department of Bio-Statistics, School of Medicine, University of Murcia, Murcia, Spain

Requests for reprints: Maria Rocío Álvarez-López, Immunology Service, Virgen de la Arrixaca University Hospital, Ctra. Madrid-Cartagena, 30120 El Palmar, Murcia, Spain. Phone: 34-968-36-90-50; Fax: 34-968-36-96-78; E-mail: mdrocio.alvarez{at}carm.es.

	Abstract

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Purpose: Because immune mechanisms involved in cutaneous melanoma have not been fully elucidated, efforts have been made to achieve prognosis markers and potential targets for immune therapies, but they have not been entirely fruitful thus far. Therefore, the goal of this study was to investigate the involvement of early changes in CD8 T cells and CD56 natural killer (NK) cells expressing NK receptors in different HLA-C dimorphism groups of melanoma patients.

Experimental Design: CD8 T cells and CD56 NK cells were analyzed in 41 patients and 39 sex- and age-matched controls with different HLA-C genotypes by flow cytometry. HLA-C dimorphism at position 80 was tested by PCR sequence-specific primers and PCR sequence-specific oligonucleotide to examine whether it could mediate in the emergence of cells expressing killer cell immunoglobulin-like receptors.

Results: Thirty-five of 41 patients had benign sentinel node, and showed an imbalance in the absolute number of CD8⁺DR⁺ or CD8⁺CD161⁺ peripheral blood T cells according to the CD28 coexpression compared with controls. CD8⁺CD28^–CD158a⁺ T and CD56⁺CD158a⁺ NK cells were significantly increased in HLA-C^Lys80 homozygous nonmetastatic patients, whereas only CD56⁺CD158a⁺ NK cells increased in heterozygous ones. An up-regulation of the CD158a KIR receptor was also seen on NK cells but not in T cells of patients at advanced disease stages.

Conclusions: This work provides, for the first time, evidence of immune activation in early stages of cutaneous melanoma, together with an increase of cells expressing CD158a in patients bearing the corresponding HLA-C ligand, which may be important to evaluate the disease progression and to use individualized immune therapeutic approaches.

CD8 T-lymphocytes and natural killer (NK) cells are believed to be important effector cells involved in eliciting a protection against melanoma (10–12). NK cytotoxic activity is regulated by the balance between activating and inhibitory signals, which are mediated by a group of receptors originally described on NK cells (13, 14), but also detected in minor peripheral blood T-cell subsets, mostly of the CD8⁺CD28^–TCRß⁺ phenotype (15, 16).

NK-associated receptors (NKR) include non-HLA class I–specific receptors, such as CD56, CD57, or CD161 (17–20), as well as receptors that recognize HLA class I molecules, such as CD94/NKG2 heterodimers belonging to the C-type lectin receptor family (21, 22), and killer receptors belonging to the immunoglobulin family (KIR), among them KIR2DL1/S1 and KIR2DL2/3/S2 (23–26).

CD94/NKG2 receptors bind the nonclassic HLA-E molecules (27) and have a limited polymorphism. On the contrary, KIRs and their corresponding ligands in the HLA-C and HLA-B loci (21, 24) are highly polymorphic, conferring to KIR and HLA class I molecules a considerable potential as markers of disease susceptibility and progression (28). In the last years, much attention has been focused on the study of HLA-C, whose alleles can be grouped into two major KIR epitope ligands defined by the presence of either asparagine (Asn) or lysine (Lys) at position 80 in the 1 helix (29, 30). Thus, group C1 (HLA-C^Asn80 alleles) includes HLA-Cw*01, HLA-Cw*03, HLA-Cw*07, HLA-Cw*08, HLA-Cw*12, HLA-Cw*14, and HLA-Cw*16 alleles, ligands for inhibitory KIR2DL2/3 and activating KIR2DS2 forms of the CD158b receptor, whereas group C2 (HLA-C^Lys80 alleles) comprises HLA-Cw*02, HLA-Cw*04, HLA-Cw*05, HLA-Cw*06, HLA-Cw*15, HLA-Cw*17, and HLA-Cw*18 alleles, which are ligands for inhibitory KIR2DL1 and activating KIR2DS1 forms of the CD158a receptor.

Recently, CD8 T and CD56 NK cell subsets expressing NKRs have been associated with different pathologies, including autoimmune, infectious, and tumoral diseases (2, 22, 31–35), in which the activity of both types of cells can be modulated upon KIR-ligand interactions (36, 37).

Therefore, the present study aimed at investigating the different peripheral blood CD8 T-cell and CD56 NK cell subpopulations expressing NKRs in patients presenting a nonmetastatic or lymph node metastatic melanoma, and their relationship with HLA-C phenotypes. To this purpose, the number of cells positive for CD158a or CD158b KIR receptors and HLA-C dimorphism were studied. This allowed us to show a status of activation in the early stages of cutaneous primary melanoma, evidenced by changes in the number of CD8⁺DR⁺ or CD8⁺CD161⁺ T-cells according to the CD28 coexpression with respect to healthy individuals. In addition, it was shown that the number of CD56 NK cells was also increased at early disease stages. Concurrently, a selective expansion of peripheral blood T and NK cells positive for CD158a receptor was observed in nonmetastatic patients carrying cognate HLA-C ligands, together with an up-regulation in the expression of this receptor on CD56 NK cells in patients at advanced disease stages.

	Materials and Methods

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Patients and controls. A total of 41 Caucasian patients with histologic diagnosis of cutaneous malignant melanoma were recruited between 2001 and 2005 from the Dermatology Section of Virgen de la Arrixaca University Hospital in the Murcia region, located in the southeast of Spain. The selection and stage adscription of patients was made by direct physical examination and pathologic review supplemented by laboratory data and radiologic examinations according to recommendations of the American Joint Committee on Cancer for cutaneous melanoma (38). The mean age of melanoma patients was 48 ± 2 years (range 24-65 years), 19 of them were men and 22 were women (Table 1 ). Exclusion criteria were history of other malignant, autoimmune, inflammatory or infectious chronic disease, and immunodeficiency, as well as immunosuppressive treatments.

Sample collection and preparation. Blood drawing was obtained from each patient by venous puncture, and these blood samples were collected in EDTA anticoagulated vacutainer tubes (Becton Dickinson, Mountain View, CA). Afterward, a fresh aliquot was used directly for cytometric analysis and another was stored at –80°C until use for DNA extraction and HLA-C genotyping. In parallel, blood samples from randomly selected healthy volunteers were also obtained, processed, and used as controls.

Monoclonal antibodies. For cytometric studies, the following human monoclonal antibodies were used: anti-CD45 (clone 2D1, IgG1) and anti-CD8 (clone SK1, IgG1) PerCP-conjugated; anti-CD14 (clone M

Flow cytometry. Lymphocytes from peripheral blood samples were stained by adding 10 µL of each monoclonal antibody in an appropriate combination to 100 µL of total blood, incubating for 10 minutes at room temperature in the dark, and fixing with 3 mL fluorescence-activated cell sorting lysing solution (Becton Dickinson) for 5 minutes. Labeled samples were centrifuged at 355 x g for 5 minutes at 4°C; then, the cellular pellet was washed with fluorescence-activated cell sorting flow solution (Becton Dickinson) and finally suspended in 0.5 mL PBS solution (BioMérieux, Marcy l'Etoile, France).

A total of 20,000 cells within a gate of lymphocytes were acquired in a FACSCalibur flow cytometer by using CELL QUEST software (Becton Dickinson), and the data were analyzed using PAINT-A-GATE software (Becton Dickinson). For the analysis, a "lymphocyte gate" was defined by forward/side scatter settings corresponding to a cell population expressing >96% CD45 and <1% CD14.

In the analysis of lymphocyte subsets, the term CD8 T-cells expressing NKRs was used for all CD3⁺CD8⁺brightTCRß⁺ T cells expressing CD56, CD57, CD94, CD161, CD158a, or CD158b receptors, and the NK cell population studied was defined by phenotype CD3^–CD56⁺. Data about CD8 T and CD56 NK cells expressing CD158a or CD158b receptors were unavailable for two controls of 39 and 3 patients of 41.

Possible changes in the intensity of expression of CD158a and CD158b receptors on CD8⁺CD28^– T and CD56 NK cells were also analyzed, measuring the variations in the fluorescence intensity, which were expressed as mean fluorescence intensity (MFI).

DNA extraction and HLA-C genotyping. Genomic DNA was extracted by using QIAamp DNA Blood Midi kit (Qiagen, Hilden, Germany), as recommended by the manufacturer. Subsequently, DNA was spectrophotometrically quantified and amplification reactions were carried out in a Thermal Cycler 9600 (Perkin-Elmer Cetus Instruments, Norwalk, CT). The quality of the PCR product was assessed by agarose gel electrophoresis. From the PCR products, HLA-C typing was done by a Fastype class I HLA-C sequence–specific primer typing kit (Bio Synthesis, Lewisville, TX) and a Dynal RELI sequence-specific oligonucleotide HLA-C typing kit (Dynal Biotech ASA, Oslo, Norway). HLA-C sequence-specific oligonucleotide typing was done by using an AutoRELI 48 (Dynal Biotech). Genotyping was done at a level of resolution, which allowed us to distinguish the HLA-C dimorphism at position 80 of the 1 helix.

HLA-C typing data were used to classify patients and controls in three groups were defined according to their homozygosis or heterozygosis at position 80. Group 1 included individuals bearing only HLA-C alleles with Asn⁸⁰ epitope (homozygous for Asn⁸⁰); group 2 included those bearing only Lys⁸⁰ epitope (homozygous for Lys⁸⁰); and group 3 included individuals carrying both Asn⁸⁰ and Lys⁸⁰ epitopes (Asn⁸⁰/Lys⁸⁰ heterozygous).

Statistical analysis. Demographic data and results of the analysis were prospectively collected in a database (Microsoft Access 2.0; Microsoft Corporation, Seattle, WA), and statistical analysis was done using the SPSS 12.0 software (SPSS Inc., Chicago IL). To detect differences regarding age and sex, a two-tailed unpaired t test and a ² test were respectively used. After log-transformation of cell number data, a parametric one-way ANOVA analysis complemented with the Bonferroni post hoc test adjustment, and the nonparametric Kruskal-Wallis test were used to compare differences in the absolute number of T-lymphocytes, NK cells, and in the MFI of studied KIR receptors between groups. Additionally, parametric unpaired or paired two-tailed t tests were also used. Data were expressed as mean ± SEM, and significant differences were set at P < 0.05.

	Results

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Clinical and histologic characteristics of patients with cutaneous malignant melanoma. Of a total of 41 patients, 35 presented a histologic diagnosis of superficial spreading melanoma, four of lentigo maligna melanoma, and two of acral lentiginous melanoma (ALM). Because no patients with evidence of distant metastastic disease were found in the series of this study, the clinical staging was determined considering thickness and ulceration of primary tumor, as well as sentinel lymph node biopsy. The incidence of metastasis in the sentinel lymph node (14.6%) was similar to that previously reported by our own group and to that of other Caucasian melanoma series (9, 39).

Following the above considerations, and as summarized in Table 1, patients were classified as stage I (individuals with a primary tumor thickness 2.0 mm and without ulceration), stage II (patients with primary tumor thickness 1.01-2.0 mm and ulceration, as well as those with primary tumor thickness >2.0 mm), and stage III (patients with regional lymph node metastasis). For the comparative analysis, patients were subgrouped as nonmetastatic melanoma patients (stage I and II) and lymph node metastatic ones (stage III).

Analysis of CD8 T-cell subsets expressing CD56, CD57, CD94, or CD161 receptors. First of all, total CD8 T cells from peripheral blood were analyzed both in nonmetastatic (clinical stages I-II) and lymph node metastatic (clinical stage III) melanoma patients and in healthy individuals. In all of these groups, a similar number of this T-cell population was detected (Table 2 ). However, CD8 T cells in nonmetastatic melanoma patients showed a higher level of activation, because a higher number of these cells expressed the HLA-DR antigen, although this increase did not reach statistical differences with respect to controls (157 ± 19 versus 114 ± 14, respectively).

Regarding the expression of NKRs on CD8 T-cells, no significant changes were detected in peripheral blood from nonmetastatic patients compared with controls, although cells expressing CD56, CD57, or CD94 were more largely represented in this group of patients (see Table 2). When the study of NKRs on CD8 T cells was made according to the CD28 coexpression, it could be confirmed that the expression of NKRs, as previously reported (4, 15), was more frequent on CD8⁺CD28^– T cells than on CD8⁺CD28⁺ T cells, both in patients and in controls.

Only the CD161 molecule was preferentially expressed on CD8⁺CD28⁺ T cells from controls (P < 0.0001, two-tailed paired t test), whereas in nonmetastatic patients CD161 was equally represented in CD8⁺CD28⁺ and CD8⁺CD28^– T-cell subpopulations (P = 0.3, two-tailed paired t test).

In the lymph node metastatic group of patients (clinical stage III), the number of CD8 T cells expressing NKRs was lower than in the nonmetastatic group of patients (clinical stage I-II) and in the control group. In line with these results, the analysis of HLA-DR antigen revealed that the number of CD8⁺CD28^–DR⁺ activated T cells also decreased in patients with lymph node metastasis with respect to nonmetastatic patients or healthy individuals (Table 2).

Study of KIR2D (CD158a and CD158b) receptors on CD8 T and CD56 NK cells. CD158a and CD158b receptors could only be detected on CD8⁺CD28^– T cells from both healthy controls and melanoma patients. Thus, comparisons between nonmetastatic melanoma patients and controls revealed that these CD8⁺CD28^– T cells positive for CD158a or CD158b appeared increased in patients with respect to controls, but without reaching statistical significance (Table 3 ).

When lymphocyte subpopulations positive for KIR2D receptors (CD158a and CD158b) were analyzed in the group of lymph node metastatic patients, we observed a number of CD8⁺CD28^–CD158a⁺ T cells similar to that detected in the nonmetastatic group. This result was in contrast with that obtained for the CD8⁺CD28^–CD158b⁺ T cells, a subset that appeared decreased in lymph node metastatic patients. The study of these two KIR2D receptors (CD158a or CD158b) on CD56 NK cells showed that the number of CD158a-positive NK cells in lymph node metastatic patients was lower than in nonmetastatic ones, although higher than in controls. Conversely, CD158b-positive NK cells were decreased in the lymph node metastatic group of patients with respect to the group of nonmetastatic patients or healthy individuals. Nonetheless, statistical significance was not reached in any of these cases (Table 3).

MFI of CD158a and CD158b receptors on CD8 T and CD56 NK cells. Changes in the CD158a and CD158b expression intensity were also evaluated by comparing their MFI on the CD8⁺CD28^– T and CD56 NK cell subsets (Fig. 1 ). On T cells, CD158a receptor was expressed with the same intensity both in the two groups of patients and in the control group (Fig. 1A). In contrast, as shown in Fig. 1B, when the MFI of CD158a receptor was determined on NK cells, a significant increase could be observed in melanoma patients presenting lymph node metastasis compared with nonmetastatic ones or even to controls (P = 0.04 and P = 0.03, respectively). However, the density of this receptor on NK cells was similar in the nonmetastatic patients and controls. In the case of CD158b, no significant difference in the MFI was found when comparing lymph node metastatic patients and nonmetastatic ones or controls, which was in contrast with the CD158a results (Fig. 1C and D).

View larger version (22K): [in this window] [in a new window]   Fig. 1. MFI of CD158a and CD158b receptors. A, MFI values of CD158a on CD8+CD28– T cells. B, MFI of CD158a on CD56 NK cells. C, MFI values of CD158b on CD8+CD28– T cells. D, MFI of CD158b on CD56 NK cells. Controls (white columns), nonmetastatic (striped columns), and metastatic melanoma patients (black columns). Columns, mean; bars, SEM. P values were determined by one-way ANOVA analysis and Kruskal-Wallis test. P = 0.03, metastatic patients versus controls; P = 0.04, metastatic versus nonmetastatic patients.

View larger version (20K): [in this window] [in a new window]   Fig. 2. CD8+CD28– T cells and CD56 NK cells positive for CD158a or CD158b receptors in different HLA-C phenotypes. A, number of CD8+CD28–CD158a+ T cells. B, number of CD56+CD158a+ NK cells in individuals Lys80 homozygous, Asn80/Lys80 heterozygous, and Asn80 homozygous. Cells positive for CD158b are represented in (C and D), respectively, for CD8+CD28– T cells and CD56 NK cells. Columns, mean; bars, SE. P values were determined by one-way ANOVA analysis and Kruskal-Wallis test: *, P = 0.007, patients homozygous for Lys80 versus those homozygous for Asn80 (n = 7 and 11, respectively); **, P = 0.03, Lys80 versus Asn80 homozygous patients; and by a two-tailed unpaired t test: ***, P = 0.04, patients versus healthy individuals HLA-C heterozygous (n = 17 and 18, respectively). E, representative density plots of patients of the three different HLA-C phenotypes. Top plots, percentage of CD8+CD28– T lymphocyte subset positive for CD158a on CD8 T cells; bottom plots, percentage of CD56+CD158a+ NK cells on CD56 NK cells.

Figure 2E shows the CD158a receptor expression on T and NK cells of representative homozygous and heterozygous patients. As it can be observed, the expression of CD158a receptor on CD8 T cells was restricted to T cells negative for CD28, especially in individuals homozygous for their specific HLA-C ligand (Lys⁸⁰/Lys⁸⁰ patients). This restriction observed for CD8⁺CD158a⁺ T cells in patients homozygous for Lys⁸⁰ was less marked in the case of CD56⁺CD158a⁺ NK cells, because these cells were also represented in heterozygous patients and even in a lower proportion in patients homozygous for Asn⁸⁰.

To evaluate the level of expression of KIR receptors, and given that no changes were observed in cells positive for CD158b, an analysis of the MFI of CD158a was done. This analysis revealed that the MFI of the CD158a on CD8⁺CD28^– T cells and CD56 NK cells was similar in patients as well as in healthy individuals belonging to different HLA-C groups. Additionally, when the MFI of CD158a observed in each HLA-C group of patients was compared with that of the corresponding group of controls, no differences were observed in any case either (data not shown).

	Discussion

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The results of the present work show an increase of CD8⁺CD28^– peripheral blood T cells positive for HLA-DR in melanoma patients at nonmetastatic clinical stages, and confirm the increase of CD8⁺CD28^– T cells expressing CD161 receptor in melanoma disease (4), indicating that in these patients, CD8⁺CD28^– T cells could be activated (see Table 2).

These changes observed in the CD8⁺ T-cell population seem to be associated with a favorable prognosis, because they were not detected in lymph node metastatic melanoma patients, where the number of CD8⁺CD28^– T cells was close to that of healthy individuals, suggesting that in advanced stages of the disease there is less availability for the activation of CD8 T cells (4). In this sense, our data are in agreement with those reported about the decrease of CD8⁺DR⁺ T cells in metastatic patients (12), but they are also able to show that the HLA-DR down-modulation in patients at this stage is specially restricted to the CD8⁺CD28^– T-cell subset. The mechanisms involved in the differentiation and emergence of this T-cell subset have not yet been clarified, although it has been suggested that a chronic antigenic stimulation might regulate these events in vivo (40). Thus, it is plausible that the observed increase of CD8⁺CD28^– T cells in the circulation of patients suffering from melanoma might reflect a persistent antigenic stimulation, at least in the first stages of the disease. In a similar way, an increase of CD8⁺CD28^– cytotoxic effector T cells has also been described in other tumors (41).

On the other hand, the observation of an increase in the absolute number of CD8⁺CD28^–CD161⁺ T cells in nonmetastatic patients points toward a triggering of the cytotoxic activity mediated by CD8⁺CD28^– T cells in the early melanoma stages, which could be of help for the host tumor immune surveillance at these stages of the disease. This can be supported by the fact that CD161, also known as NKR-P1A (19), has been reported as a cytotoxic-activating receptor (20), previously described in murine models in association with the cytotoxic activity mediated by CD8⁺TCRß⁺ T cells against multiple tumor targets, including melanoma cells (42). Nonetheless, it should be taken into account that the human NKR-P1A receptor can also inhibit NK cell–mediated cytotoxicity through the interaction with a novel ligand recently described (43). However, this ligand/receptor pair differentially regulates the NK and T-cell functions (44).

The analysis of CD56, CD57, and CD94 or even CD158a and CD158b NK receptors on the total of CD8 T cells in our series of melanoma patients was unable to prove a clear positive association with disease. This is probably due to the variability in the number of circulating CD8 T cells expressing the above-mentioned NK receptors both in controls and in melanoma patients, which is in accordance with previous results of Speiser et al. (15), showing variable proportions of effector CD8 T-cells expressing NK receptors in circulating lymphocytes from healthy donors and melanoma patients. In contrast, the present data did not seem to confirm those obtained in a shorter and clinically heterogeneous Spanish melanoma series studied by Casado et al. (4), reporting a significant increase of CD8 T cells positive for CD56, CD57, or CD158b receptors in this disease. However, it should be noted that our data represent absolute values, whereas those of the study by Casado et al. are expressed as percentages, which, at least in part, could explain the observed discrepancies.

Regarding the low levels of CD8 T cells expressing NK receptors in metastatic patients compared with the nonmetatastatic ones (see Table 2), consideration could be given to the presence of an impairment of the CD8-mediated immune response against the tumor in the former patients (4).

With regard to NK cells, CD56 NK cell subpopulation was particularly increased in patients at nonmetastic stages (stages I-II), indicating that the accumulation of these cells principally occurs at the earliest stages of the disease, because in the most advanced stages the expansion and/or accumulation of these cells, in our and other series, was less relevant (12). Interestingly, our results, for the first time, allow us to describe that the increase in NK population is mediated by an accumulation of the CD56 NK cell subset expressing KIR2DL1/S1 receptors (see Table 3), because the number of CD56 NK cells positive for KIR2DL2/3/S2 was similar in patients suffering from melanoma and in healthy individuals. These facts, such as it has been recently proposed for endometriosis, suggest that CD56 NK cells positive for KIR2DL1/S1 might contribute to the induction of tolerance against melanoma (45). Moreover, these findings might support the observation of Guillot et al. (46), about the absence of a link between the effect of IFN- on perforin expression in cytotoxic cells and the posttherapeutic disease evolution, because the expression of KIR receptors on cytotoxic NK cell surface could, at least partially, block the antitumoral function of NK cells.

On the other hand, the observation of an increase in the intensity of expression of KIR2DL1/S1 receptors on CD56 NK cells at stage III of the disease (see Fig. 1B) could help to understand the underlying mechanisms that lead to the depressed NK cell activity previously observed in advanced melanoma disease (12).

The results discussed before are further supported by the following two findings: (a) the increase of CD8 T cells and CD56 NK cells positive for KIR2DL1/S1 receptors in nonmetastatic HLA-C^Lys80 homozygous melanoma patients, and (b) the increase of CD56 NK cells expressing KIR2DL1/S1 in heterozygous ones (see Fig. 2). These results were in contrast with those observed in healthy individuals with the same HLA-C phenoypes, where these cell expansions were absent.

Thus, our findings could be considered of interest because group C2 of HLA-C alleles (HLA-C^Lys80) are specific ligands for KIR2DL1/S1 receptors, and because none of those associations had been seen when the repertoire of NK cells expressing KIR receptors was studied in healthy individuals of the three groups of HLA-C, either in our series or in those previously reported (24, 47). Therefore, it is likely that the cytotoxic activity of CD8 T cell and CD56 NK cell populations expressing KIR2DL1/S1 is inhibited in melanoma patients carrying the corresponding HLA-C ligand. This possibility is sustained by the fact that the gene encoding the inhibitory form KIR2DL1 is present in virtually all individuals (48), and that NK receptor–mediated inhibitory signals seem to be dominant over activating ones (28, 29, 34). Thus, KIR receptors could interfere with the effectiveness of cytotoxic cells in the control of melanoma metastatic spread (3, 23, 29, 30, 36, 49), favoring peripheral tolerance against tumor and disease progression. In support of this, it is important to note that a positive association between HLA-C^Lys80 in homozygosis and the presence of lymph node metastasis in melanoma patients had been recently described (9).

In conclusion, to our knowledge, these findings provide, for the first time, an in vivo evidence of the existence of a status of activation in melanoma patients at the nonmetastatic stages of the disease, determined by an imbalance in the absolute number of CD8⁺DR⁺ or CD8⁺CD161⁺ T cells according to the CD28 coexpression, which could be associated with a stimulation of the immune response at the early stages of cutaneous melanoma. Concurrently, a selective expansion of cells expressing KIR2DL1/S1 receptors is also shown in patients carrying the corresponding HLA-C ligand, together with an up-regulation in the expression of these receptors on CD56 NK cells in patients at advanced disease stages, which point to a particular immune regulatory role of cells expressing KIR2DL1/S1 receptors in melanoma. Thus, it is tempting to speculate about the possibility of opening new therapeutic avenues for the control of melanoma progression, taking into consideration these cells as a target for immune intervention by blocking or enhancing their inhibitory/activatory receptors.

	Acknowledgments

 
We thank J.M. Alemany, M.J. Sanchís, M.C. García Calatayud, and M. López for their excellent technical assistance.

	Footnotes

 
Grant support: Seneca Foundation grants PI-400811/FS/01 and AR 1-02632/FS/02, Research Group G03/104 of Redes Temáticas de Investigación, Fondo de Investigación Sanitaria of the Spanish Health Ministry (grant FIS CM0300073, J.A. Campillo), and Caja Murcia Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: J.A. Campillo is a postdoctoral researcher from the Spanish Health Ministry.

Received 1/ 5/06; revised 4/22/06; accepted 6/ 5/06.

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