ORIGINAL ARTICLE

Reduced Number and Impaired Function of Circulatingcd T Cells in Patients with Cutaneous Primary Melanoma

KatyArgentati,1 Francesca Re,1 Stefano Serresi,n Maria G. Tucci,n Beatrice Bartozzi, Giovanni Bernardini,and Mauro ProvincialiLaboratory of Tumor Immunology, Immunology Center, I.N.R.C.A. Research Department, Ancona, Italy; nDermatology Unit, I.N.R.C.A. Hospital,Ancona, Italy

We studied the peripheral representation, in vitro expan-sion, cytokine production, and cytotoxicity of cd Tlymphocytes from 23 patients with cutaneous primarymelanoma and 28 healthy subjects. We demonstratedthat the absolute number and the percentage of circulat-ing cdþ Tcells were signi¢cantly reduced in melanomapatients in comparison with healthy subjects. The de-crease was due to a reduction of Vd2 T cells, whereasthe number of Vd1 Tcells was not a¡ected. As a conse-quence, the Vd2/Vd1 ratio was inverted in melanomapatients. A lower percentage of cdþ T cells producingtumor necrosis factor-a or interferon-c was found inmelanoma patients. After a 10 d in vitro culture, both

the percentage and the expansion index of cd T cells,and in particular of Vd2 subset, were signi¢cantly re-duced in melanoma patients in comparison withhealthy subjects. The cytotoxicity of sorted cd T cellsagainst tumor cell lines and the percentage of cd Tcellsproducing perforins were preserved in melanoma pa-tients. The numerical and functional impairment of cdT cells could contribute to the inadequate immune re-sponse found in melanoma patients and o¡ers thepotentiality for the planning of new approaches ofimmune therapy of malignant melanoma. Key words:£uorescence-activated cell sorter/human/melanoma/tumorimmunity/cd Tcells J Invest Dermatol 120:829 ^834, 2003

Various clinical and experimental observations pointto the existence of an immunologic host defense incutaneous malignant melanoma. CD3þ T cell re-ceptor (TCR) a/b-expressing lymphocytes are con-sidered the prevailing lymphocyte subset in primary

as well as secondary malignant melanoma (Strohal et al, 1994).Immunomodulatory therapies with cytokines or adoptive trans-fer of T cells have accomplished complete or partial tumor re-gression in melanoma patients. Nevertheless, the immuneresponse is in most cases inadequate to control tumor growth astumor progression often occurs (Lee et al, 1999; Dudley et al,2001). Hence, the coexistence of a cellular immune response inmelanoma lesions (Kammala et al, 1999), demonstrated by thepresence of clonally expanded T cells, and the inability of mela-noma-speci¢c killer T cells to arrest tumor growth, remainsa major paradox of tumor immunology (Thor Straten et al, 1999;Nielsen et al, 2000).T lymphocytes bearing the gdTCR represent a minor popula-

tion of human peripheral lymphocytes (1^10%) the majority ofthem expressing the Vd9/Vd2 TCR and the CD3þCD4^CD8^

phenotype (Groh et al, 1989; Poccia et al, 1998). The second mostfrequent subset of peripheral blood gd Tcells expresses Vd1 in as-sociation with various Vg elements. The ability of gd T cells torespond to nonprocessed and nonpeptidic phosphoantigens in amajor histocompatibility complex (MHC)-unrestricted manner

are important features distinguishing them from ab T cells(Brenner et al, 1987; Bukowski et al, 1995). gdTcells are activated bymycobacterial antigens, such as isopentenyl pyrophosphate (IPP)and related phenyl pyrophosphate derivatives (Costant et al, 1994;Tanaka et al, 1995; Burk et al, 1995), stress-associated heat shockproteins (Fisch et al, 1999), as well as by several cytokines, such asinterleukin (IL)-2 (Kjeldsen-Kragh et al, 1993), IL-12 (Fujimiyaet al, 1997), or tumor necrosis factor (TNF)-a (Ueta et al, 1996;Lahn et al, 1998). Other signals, such as MHC class I-related chainsA and B, are able to engage the activating receptor, NKG2D, pre-sent on Vd2 T cells, substantially enhancing the TCR-dependentVd2 T cell response to nonpeptide antigens (Das et al, 2001; Wuet al, 2002). Although little is known about the physiologic sig-ni¢cance of gd T cells, their marked reactivity toward mycobac-terial and parasitic antigens, as well as tumor cells, suggests thatgd T cells play a part in the anti-infectious and anti-tumoral im-mune surveillance (Poccia et al, 1998; Zheng et al, 2001). gdTcellsmay contribute to the immune defense against cancer, having atleast two important functions, i.e., reactivity to tumor cells, andregulatory interactions with abT cells (Kabelitz et al, 1999). gd Tcells strongly react against certain lymphoma cells, such as Daudicells, suggesting a cross-reactivity between microbial and tumor-associated antigens (Kunzmann et al, 2000). Furthermore, gd Tcells have been identi¢ed among tumor-in¢ltrating lymphocytesin various cancer types (Kabelitz et al, 2000). Once activated, gdTcells produce high levels of cytokines and, mainly, IFN-g andTNF-a (Poccia et al, 1997). Mainly because of their cytokine pro-duction, gdTcells have been proposed to be involved in co-ordi-nating the interplay between innate and acquired immunity and,in particular, to guide the establishment of acquired immunitycontributing to select appropriate antigens and the strategies fortheir elimination, and to the de¢nition of ab T cell responses1Equally contributed to this work.

Reprint requests to: Mauro Provinciali, MD, Laboratory of TumorImmunology, Immunology Center, INRCA Gerontol. Res. Departmentof Via Birarelli 8, 60121 Ancona, Italy. Email: m.provinciali@inrca.it

Manuscript received October 8, 2002; revised November 27, 2002;accepted for publication December 19, 2002

0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc.

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toward T helper (Th) 1 or Th2 phenotype (Fearon and Locksley,1996; Mak and Ferrick, 1998). The strict relationship between gdand ab T cells is bidirectional, as demonstrated by the fact thatgd T cell function may depend on ab T cells (Vila et al, 1995;Burns et al, 1996).The role of gd T cells in the early anti-tumor defense against

melanoma has been suggested by the evidence that gdTcells mayconstitute up to 25% of lymphocyte in¢ltrate in cutaneousprimary melanomas, whereas they are not present in metastaticmelanoma (Bachelez et al, 1992). These cells exert potent cytotoxicactivity against autologous tumor cells (Bachelez et al, 1992;Nanno et al, 1992). In another study, the survival of patients withnecrotizing choroidal melanomas was increased with evidenceof Vg1 and Vd1 TCRþ cells (Bialasiewicz et al, 1999).On the basis of the pivotal role that gd T cells may have in the

immune response against melanoma acting directly on tumorcells and, secondarily, by modulating the phenotype of T cell re-sponses, we evaluated the peripheral representation and the in vitroexpansion, cytokine production, and cytotoxicity of gd T cellsfrom melanoma patients, comparing the results with those ob-tained in healthy controls. This study demonstrated for the ¢rsttime an alteration of circulating gd T cells in melanoma patients,with a signi¢cant decrease of the number of gd Tcells in the per-ipheral blood, an altered pattern of cytokine production, and animpaired in vitro expansion of these cells. The knowledge aboutthe deterioration of gd T cells could account for the melanoma-related alterations of T cell-mediated adoptive responses and maybe helpful for the planning of new approaches of immunetherapy in melanoma patients.

MATERIALS AND METHODS

Cell preparation and stimulation Human peripheral blood wasobtained from 23 melanoma patients (mean age7SD 59.6716.1 y;median: 64.5 y; range 32^80) and 28 healthy subjects (mean age7SD57.5718.9 y; median: 67.0 y; range 32^79). Healthy subjects werevolunteers in good and stable clinical condition, and had laboratoryparameters in the physiologic range. Melanoma patients have beenadmitted to the Dermatology Unit of the I.N.R.C.A. Hospital of Ancona(Italy). Melanoma patients were in good health other than for the existenceof melanoma as checked on the basis of clinical and laboratory parameters.Melanoma patients and healthy subjects were equally distributed accordingto sex and the percentage of male and female inside each group was aboutthe same. The investigations were performed after approval by a localinstitutional review board. A written informed consent was obtainedfrom each subject. Diagnosis of melanoma was histologically con¢rmed.All patients brought cutaneous primary nonmetastatic melanoma andwere staged according to the new American Joint Committee on Cancerstaging system for cutaneous melanoma (Balch et al, 2000). In detail, thenumber of patients and the relative tumor thickness was as follows: p1.0mm (n¼15), 1.01^2.0 mm (n¼ 4), 2.01^4.0 mm (n¼ 2), and p4.0 mm(n¼ 2). A blood drawing was taken before the surgical excision.Peripheral blood mononuclear cells (PBMC) were fractionated on

Ficoll-Paque (Pharmacia, Uppsala, Sweden) and separated by densitygradient centrifugation (400� g, 30 min). Cells from the interface ofthe gradients were washed twice with Ca2þ and Mg2þ -free phos-phate-bu¡ered saline (Gibco, Life Technologies, Gaithersburg, MD) andresuspended in RPMI 1640 supplemented with 10% heat-inactivated fetalbovine serum, penicillin (100 U per ml) and streptomycin (100 mg per ml)(all from Life Technologies, complete medium) at a concentration of1�106 per ml. The viability was always greater than 98% as determinedby trypan blue exclusion. Lymphocytes from both melanoma patients andhealthy donors were tested fresh. In each experiment a similar number ofmelanoma patients and healthy donors was examined. Each donor wastested once and all the tests were carried out with a single blood sample.Mononuclear cells were cultured in the complete medium supplementedwith 100 U per ml of IL-2 (Chiron Milano, Italia). Phosphoantigen-speci¢c stimulation of gd T cells was performed using the nonpeptidicantigen isopentenylpyrophosphate (30 mg per ml, IPP; Sigma, St Louis,MO). After 1 wk of culture, the volume corresponding to half the culturemedium was replaced by fresh medium. On day 10 of culture, viable cellswere counted and used for £uorescence-activated cell sorter analysis andcytotoxicity. The expansion of gd T cells was followed by cytometricanalysis through double staining of stimulated cells with anti-CD3

[phycoerythrin (PE)] and anti-pan gd, anti-Vd1, or anti-Vd2 T cells[£uorescein isothiocyanate (FITC)] monoclonal antibody (MoAb). Theabsolute number of gd T cells in each culture was calculated as follows:(percentage of gd T cells among total cells) � (total cell count)/100. Thegd T cell expansion index was then calculated by dividing the absolutenumber of gd T cells in stimulated cultures by the absolute number of gdTcells before culture (Poccia et al, 1997).

MoAb and £uorescence-activated cell sorter analysis The PE-conjugated MoAb anti-CD3 was purchased from EuroClone (Devon,Wetherby, West York, UK). The FITC-conjugated anti-pan-TCR gd, anti-TCR Vd1, and anti-TCR Vd2 were purchased from Endogen (Boston,MA). IgG1 (Becton Dickinson Milano, Italy) was used as isotype control.PBMC (1�106) were labeled with 10 ml of anti-CD3 or anti-TCRVd1

MoAb or 5 ml of anti-pan-TCR gd or anti-TCR Vd2 MoAb in a ¢nalvolume of 150 ml of RPMI 1640 with 10% fetal bovine serum for 30 minin ice. At the end of the incubation, cells were washed in PBS, resuspendedin Isoton II (Coulter, Euro Diagnostics, Hialeah, FL) and immediatelyanalyzed with a Coulter XL £ow cytometer.

Intracellular detection of IFN-g,TNF-a, and perforin Mononuclearcells were stimulated with IPP and IL-2 for 18 h, and GolgiPlug (a proteintransport inhibitor containing brefeldin A; PharMingen, Milton Keynes,U.K.) was added during the last 12 h of culture to block intracellulartransport processes and allow cytokine accumulation. Stimulated cells(1�106) were stained with the anti-pan-TCR gd MoAb for 30 min at41C. Fixation^permeabilization of cells was performed in PBS/2%paraformaldehyde for 15 min at 41C, followed by incubation for 30 minat room temperature in the dark with PE-conjugated anti-human IFN-gMoAb or anti-human TNF-a MoAb diluted in PBS, 1% bovine serumalbumin, and 0.05% saponin. Cells were ¢nally washed twice in PBS, 1%bovine serum albumin, and 0.01% saponin and analyzed on a XL £owcytometer (Coulter). Perforins were measured after 10 d of incubationusing an anti-human perforin-PE (clone dG9, BD PharMingen) at thesame conditions above reported. The controls for unspeci¢c stainingincluded incubation with isotype-matched MoAb (PharMingen).

Isolation of gd T lymphocytes and cytotoxic assay gd Tlymphocytes, in vitro expanded with IPP and IL-2 for 10 d, were isolatedthrough cyto£uorimetric cell sorting (Vantage, Becton Dickinson). Thepurity of gd T cells, assessed by cyto£uorimetric analysis, was greater than95%.Cytotoxic assay was performed by a £uorimetric method as recently

reported (Provinciali et al, 1992). The natural killer resistant cell line Daudiand the natural killer cell sensitive K562 cell line were used as target cells.Daudi is a human lymphoblastoid B cell line derived from a Burkittlymphoma, which constitutively expresses antigens recognized by Vd9Vd2 T cells. K562 is a human myeloid cell line derived from chronicmyelogenous leukemia. The £uorescence was read with a 1420 VICTOR2

multilabel counter (Wallac,Turku, Finland).The percentage of speci¢c lysiswas calculated as follows:

% specific lysis ¼ ½ðFmed � FexpÞ=Fmed��100

where F represent the £uorescence of the solubilized cells after thesupernatant has been removed; med¼ F from target incubated inmedium alone; exp¼ F from target incubated with e¡ector cells.Lytic units (LU20/10

7 cells) were calculated by using a computationalmethod (Bryant et al, 1992). One LU corresponded to the number ofe¡ector cells required to produce 20% of speci¢c lysis.

Statistical analysis Statistical analysis was performed using parametric(Student’s t test) or nonparametric (Mann^Whitney rank sum test) testson the basis of the distribution of the data. Di¡erence between meanswere considered signi¢cant at po0.05. Correlation between gd T cellnumber or expansion and tumor thickness was compared by simple linearregression analysis. The statistical analysis was performed with SigmaStatsoftware version 1.03 (Jandel Scienti¢c, Erkrath, Germany).

RESULTS

Ex vivo analysis of cd T lymphocytes Peripheral bloodlymphocytes from 23 melanoma patients and 28 healthy subjectswere analyzed for the percentage and the absolute number of gdT cells through double staining with anti-CD3 and anti-gdMoAb. As shown in Table I and Fig 1(A), the absolute numberof gd T cells was signi¢cantly reduced in melanoma patients incomparison with healthy subjects (66.1733.8 vs 92.9732.6;

830 ARGENTATI ETAL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

po0.01). The absolute number of peripheral lymphocytes wassimilar in the two groups of subjects (Table I). As shown inFig 1(B), the percentage of CD3þ gd T cells in peripheralblood was signi¢cantly lower in melanoma patients than inhealthy donors (mean7SD, 3.272.1 vs 4.871.7; po0.01). Asshown in Table I, the absolute number of Vd1 T cells did notshow signi¢cant di¡erence in the two groups of donors(38.5713.1 and 33.1719.4, in healthy subjects and melanomapatients, respectively). Di¡erently, the absolute number of Vd2T cells was signi¢cantly reduced in melanoma patients incomparison with healthy subjects (31.7713.7 vs 68.2735.8;po0.01). The Vd2 and Vd1 subsets were di¡erently representedin the two groups: in healthy controls the Vd2 subset waspredominant (Vd2/Vd1 ratio¼1.8), whereas in melanomapatients the proportion of Vd2 and Vd1 subsets was similar(Vd2/Vd1 ratio¼ 0.9) (Table I). No signi¢cant correlation wasfound plotting the number of gd T cells and the tumorthickness of the respective patient.

Cytokine production by cd T lymphocytes As it has beendemonstrated that activated gd T cells produce IFN-g and TNF-a, we studied the intracellular production of these lymphokinesin 1 d stimulated gd T cells from healthy subjects and melanomapatients. As shown in Figs 2 and 3, the percentage of gd T cellsproducing IFN-g was signi¢cantly reduced in melanoma patientsin comparison with healthy controls (mean7SD, 9.375.5 vs18.379.5; po0.01). In a similar way, the percentage of gd T cellsproducing TNF-a was lower in melanoma patients than inhealthy controls (11.573.8 vs 17.977.7 for melanoma patients andhealthy subjects, respectively; po0.02).

Expansion of cd T lymphocytes The expansion of gd T cellsfrom healthy controls and melanoma patients was evaluated after10 d of culture in the presence of IPP and low-dose IL-2. Boththe proportion of gd T cells, evidenced by double staining£uorescence-activated cell sorter analysis, and their relativeincrease in comparison with the gd T cell number found on day0 (expansion index) were evaluated. As shown in Fig 4, theproportion of gd T cells reached on day 10 was signi¢cantlylower in melanoma patients than in healthy donors (mean7SD,11.978.4 vs 36.5717.6; po0.01). In a similar way, the expansionindex of gd T cells on day 10 vs day 0 was signi¢cantly reducedin melanoma patients in comparison with healthy controls(4.473.8 vs 8.676.7; po0.02) (Fig 4). As shown in Table II, theexpansion index of the Vd2 subset was signi¢cantly lower inmelanoma patients than in healthy donors (3.373.6 vs 5.874.4;po0.03).

Cytotoxicity of cd T lymphocytes In order to evaluate thecytotoxic potential of gd T cell cultures after in vitro expansion,we tested the cell activity of gd T cells against Daudi and K562tumor cell lines and their perforin content through £owcytometry. Puri¢ed cultures of activated gd T cells (10 dincubation), obtained through cyto£uorimetric cell sorting, werecytotoxic against Daudi tumor cells. A great heterogeneity ofcytotoxic activity was found in both groups considered. Meanlevels of cytotoxicity were similar between healthy subjects andmelanoma patients (438.27141.7 vs 402.57359.2, data not shown).The cytotoxic activity of gd T cells was also tested against theK562 tumor cell line, with a similar distribution of cytotoxicityin normal donors and melanoma patients (data not shown).The percentage of gd T cells producing perforins among total

gdþ T cells was not signi¢cantly di¡erent in melanoma patientsin comparison with healthy subjects (42.8737.6 vs 51.478.8, datanot shown).

DISCUSSION

In this study we have demonstrated for the ¢rst time the existenceof numerical and functional alterations of gd Tcells from patientswith cutaneous primary melanomas. The number of circulatinggd T cells, the percentage of cells producing IFN-g or TNF-a,and their in vitro expansion, were all decreased in melanoma pa-tients when compared with healthy age-matched subjects.Both the percentage and the absolute number of circulating gd

T cells were signi¢cantly reduced in patients with cutaneous pri-mary melanoma, thus suggesting a speci¢c de¢cit for this lym-phocyte population. The reduction of gd T cell number well

Table I. Absolute number of lymphocytes, cdTcells,Vd1 Tcells, and Vd2 Tcells, in healthy subjects and melanoma patients

Absolute no. (n�mm3)

Donors Lymphocytes gd T Cells Vd1 T Cells Vd2 T Cells Vd2/Vd1 ratio

Healthy subjects 21597118a 92.9732.6 38.5713.1 68.2735.8 1.8Melanoma patients 22687539 66.1733.8n 33.1719.4 31.7713.7n 0.9

aData are expressed as mean7SD.npo0.01 vs healthy subjects.

Figure1. Absolute number and percentage of cd T cells in the per-ipheral blood of melanoma patients and healthy donors. Freshly iso-lated PBMC from patients with cutaneous primary melanomas (n¼ 23) orhealthy subjects (n¼ 28) were double stained with MoAb anti-pan gd(FITC) and anti-CD3 (PE) and analyzed by £ow cytometry. (A) The abso-lute number of gd Tcells, and (B) the percentage of gd Tcells among totalCD3þ T cells, were both signi¢cantly reduced in melanoma patients incomparison with healthy subjects (po0.01).

GD T CELLS IN MELANOMA 831VOL. 120, NO. 5 MAY 2003

correlated with the decrease of theVd2 Tcell subset, i.e., the mostfrequent subset of circulating gd T cells (Groh et al, 1989; Pocciaet al, 1998). The Vd2 population is involved in the reactivity to-ward microbial antigens and tumor cell antigens (Poccia et al,1998; Zheng et al, 2001). The role of Vd2 T cells in the immunedefense against cancer has been demonstrated on the basis of theirreactivity against certain lymphoma cells, such as Daudi cells(Kunzmann et al, 2000), and for their presence among tumor in-¢ltrating lymphocytes in various cancer types (Kabelitz et al,2000). On this basis, our data strongly suggest that an impairedgd T cell potential may contribute to a lower immune defenseagainst melanoma. Both Vd1 and Vd2 T cell populations havebeen reported to contribute to anti-tumor immunity because oftheir cytotoxic and Th1-type cytokines producing activities (Wuet al, 2002). Furthermore, both gdT cell subsets have been foundable to respond to stress-induced expression of the MHC class I-related chains A and B (Wu et al, 2002). MHC class I-relatedchains function as ligands for NKG2D, an activating receptorcomplex that triggers natural killer cells, costimulates CD8 aband Vg9d2 gd T cells, and is required for stimulation of Vd1 Tcells (Groh et al, 1999; Das et al, 2001; Girardi et al, 2001;Wu et al,2002). The role of gd T cells in the early anti-tumor defenseagainst melanoma has been previously suggested by the evidencethat a signi¢cant accumulation of Vd1 T cells was found in threeof 11 primary cutaneous melanomas, with proportions of gd Tcells ranging from 15 to 25% of lymphocyte in¢ltrate, whereasgd T cells were not present in eight metastatic melanomas

(Bachelez et al, 1992). In our study, we did not ¢nd numericalchanges of the Vd1 T cell population in melanoma patients incomparison with controls, whereas the Vd2 subset was signi¢-cantly reduced in melanoma patients. Almost half of our melano-ma patients had an absolute number of circulating gd T cellssimilar or lower than half of the mean number found in controls(Fig 1). Phenotypic analysis of the tumor in¢ltrating gd T cellsrevealed that they use the products of the Vd9 and Vd2 genes ina way similar to most of circulating TCR gd. It is noteworthythat gd lines or clones developed from lymphocytes in¢ltratingprimary melanoma display a potent autologous MHC-unrest-ricted tumor cell cytotoxicity (Bachelez et al, 1992; Nanno et al,1992). In a more recent study, the presence of gd T cells well cor-related with the survival of patients with necrotizing choroidalmelanoma (Bialasiewicz et al, 1999). In our study, not only thenumber but also the function of gd Tcells was altered in melano-ma patients.The in vitro expansion of gd Tcells, that represent oneof the most relevant functional parameters for gdTcells, was sig-ni¢cantly reduced in patients with cutaneous primary melano-mas. This would imply that gd T cells from melanoma patientshave a decreased proliferative capacity in comparison withhealthy subjects. Under normal conditions, gdT cells respond toantigen challenge by secreting large quantities of TNF-a andIFN-g (Poccia et al, 1997; Kabelitz et al, 2000), which contributeto the activation of both speci¢c and aspeci¢c immune responses.In this study, we show that the percentage of gd Tcells producingeither TNF-a or IFN-g is signi¢cantly reduced in melanoma

Figure 2. Analysis of cytokine production by cdTcells in melanoma patients and healthy donors. PBMC from patients with cutaneous primarymelanomas or healthy subjects, were stimulated for 18 h in the presence of IPP (30 mg per ml) and IL-2 (100 U per ml). The last 12 h of culture wereperformed in the presence of GolgiPlug, a protein transport inhibitor containing brefeldin. Single-cell analysis of cytokine synthesis in gd T cells from arepresentative healthy subject or melanoma patient was performed following dual staining with cell surface anti-gd (FITC) MoAb and intracellular anti-IFNgor anti- TNF-a (PE) MoAb. Numbers in brackets indicate the percentages of gd cells synthesizing a given cytokine among total gd T lymphocytes.

832 ARGENTATI ETAL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

patients. Instead, the lytic activity of gd Tcells, evidenced by theircytotoxicity toward Daudi and K562 tumor cells and their produc-tion of perforins, seems to be well preserved in melanoma patients.On the basis of the data reported in this paper, we suggest that

the impairment of gd Tcell number and function may contribute

to the ine⁄cacious immune defense against melanoma with atleast two di¡erent mechanisms. One of these is based on the low-er gd T cell potential, determined by the reduced number andexpansion of gd T cells, and particularly of Vd2 subset, found inmelanoma patients. In this case, the impairment of gd Tcells mayrepresent a new mechanism by which melanoma tumor cells es-cape an e⁄cacious immune response. The second mechanism isbased on the regulatory interactions between gdT cells and ab Tcells. Several studies have demonstrated that the type and thefunctioning of the speci¢c lymphocyte response is dependent onsignals provided by the innate recognition system (Fearon andLocksley, 1996; Medzhitov and Janeway, 1997). Indeed, even ifthe recognition of foreign or nonself proteins is controlled bythe rearranging genes that encode speci¢c receptors, the func-tional outcome of most responses to pathogens and the initiationof the response itself is determined by the type of innate immuneresponse they elicit (Fearon and Locksley, 1996; Medzhitov andJaneway, 1997). Recent evidence has suggested that one of the cru-cial players involved in the regulation of innate and acquired im-munity is the gdTcell population (Mak and Ferrick, 1998). Thereis substantial cross-talk between gd and ab T cells. It has beendemonstrated that the proliferative response of human peripheralblood gdTcells towards microbial antigens or Daudi tumor cells,depends on helper signals provided by CD4þ ab T cells (Burnset al, 1996a,b). Moreover, some gdT cell function may depend onab T cells, as, for example, the gdT cell proliferation in responseto activated CD4þ ab T cells (Burns et al, 1996a,b), and the anti-gen presentation by CD4þ ab Tcells to gdTcells (Vila et al, 1995;Collins et al, 1998). On the other hand, substantial evidence hassuggested that gd T cells regulate certain ab T cell-mediated im-mune responses, pointing the de¢nition of ab T cell responsestoward a Th1 or Th2 phenotype (Fearon and Locksley, 1996;Mak and Ferrick, 1998; Kabelitz et al, 2000). This fact is particu-larly related by the cytokines secreted by gdT cells that, in turn,are able to mediate both innate and acquired immunity (Ferricket al, 1995). We propose that, in analogy with what has beenrecently demonstrated by us in aged people and centenarians(Argentati et al, 2002), the numerical and functional impairmentof gd T cells demonstrated in this study could determine aderangement in the establishment of acquired immunity, withconsequent di⁄culties in selecting appropriate antigens and thestrategies for their elimination. Further studies will be performedto investigate the speci¢c causes involved in the impairment of gdT cells in primary cutaneous melanoma patients. At present, wemay only suggest that an impairment of gd T cells may deter-mine a lower immune defense against melanoma which, in turn,may predispose to melanoma. The healthy and stable conditionsof melanoma patients examined in this study make improbablethat the gd T cell defect is secondary to the malignancy, even ifthis possibility may not be de¢nitively excluded. Finally, the factthat approximately 10% of patients with melanoma have familyhistories of the disease, suggesting a genetic susceptibility (Platzet al, 1997), raises the question on whether the di¡erences we havefound might be attributable to the genetic background of themelanoma patients.We do not think this is the case, as melanomapatients and controls enrolled in our study were identi¢ed in the

Figure 3. Percentage of cd Tcells producing cytokines in melanomapatients and healthy donors. gdþ T cells from patients with cutaneousprimary melanomas or healthy subjects were activated as reported in Fig 2and analyzed for cytokine production after dual staining with cell surfaceanti-pan gd (FITC) MoAb and intracellular anti-IFN-g or anti-TNF-a (PE)MoAb.The mean percentage of gd Tcells producing either IFN-g orTNF-a was signi¢cantly lower in melanoma patients in comparison with healthysubjects (po0.01 and po0.02 for IFN-g or TNF-a, respectively).

Figure 4. Evaluation of cd T cell expansion in melanoma patientsand healthy donors. PBMC from patients with cutaneous primary mela-nomas or healthy subjects were stimulated for 10 d in the presence of IPP(30 mg per ml) and IL-2 (100 U per ml). The expansion index (left panel)and the percentage (right panel) of in vitro expanded gdþ T cells amongtotal CD3þ Tcells were signi¢cantly lower in melanoma patients in com-parison with healthy subjects (po0.02 and po0.01).

Table II. Expansion index of cdTcells and Vd2 Tcells inhealthy subjects and melanoma patients.

Expansion Indexa

Donors gdT cells Vd2 T cells

Healthy subjects 8.676.7b 5.874.4Melanoma patients 4.473.8n 3.373.6nn

aThe expansion index was calculated by dividing the absolute number of gd Tcells in stimulated cultures by the absolute number of gd T cells before culture.

bData are expressed as mean7SD.npo0.02 and nnpo0.03 vs healthy subjects.

GD T CELLS IN MELANOMA 833VOL. 120, NO. 5 MAY 2003

same geographical area (Ancona area in Italy), and melanoma pa-tients did not have family histories of melanoma.In conclusion, the demonstration of a numerical and functional

derangement of gd Tcells, which we have found in patients withcutaneous primary melanomas, make them a potentially usefultool for the planning of new approaches of immune therapy inmalignant melanoma.

This work was supported in part by Ministero della Sanita' Ricerca Finalizzata 1998and 2000 (M.P.).

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