of January 28, 2019.This information is current as

-Chain CytokinesγCommon Nodes of Melanoma Patients: Role ofTumor-Invaded and Tumor-Free Lymph

T Cells from+Differentiation of CD8

Parmiani and Roberta MortariniAndrea Anichini, Alessia Scarito, Alessandra Molla, Giorgio

http://www.jimmunol.org/content/171/4/2134doi: 10.4049/jimmunol.171.4.2134

2003; 171:2134-2141; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/171/4/2134.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2003 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Differentiation of CD8� T Cells from Tumor-Invaded andTumor-Free Lymph Nodes of Melanoma Patients: Role ofCommon �-Chain Cytokines1

Andrea Anichini, 2* Alessia Scarito,* Alessandra Molla,* Giorgio Parmiani,† andRoberta Mortarini*

Differentiation of CD8� T cells at the tumor site toward effector and memory stages may represent a key step for the efficacy ofantitumor response developing naturally or induced through immunotherapy. To address this issue, CD8� T lymphocytes fromtumor-invaded (n � 142) and tumor-free (n � 42) lymph nodes removed from the same nodal basin of melanoma patients wereanalyzed for the expression of CCR7, CD45RA, perforin, and granzyme B. By hierarchical cluster analysis, CD8� T cells fromall tumor-free lymph nodes and from 56% of the tumor-invaded lymph node samples fell in the same cluster, characterized mainlyby CCR7� CD45RA�/� cytotoxic factor� cells. The remaining three clusters contained only samples from tumor-invaded lymphnodes and showed a progressive shift of the CD8� T cell population toward CCR7� CD45RA�/� perforin � granzyme B�

differentiation stages. Distinct CD8� T cell maturation stages, as defined by CCR7 vs CD45RA and by functional assays, wereidentified even in melanoma- or viral Ag-specific T cells from invaded lymph nodes by HLA tetramer analysis. Culture for 7 daysof CCR7� perforin � CD8� T cells from tumor-invaded lymph nodes with IL-2 or IL-15, but not IL-7, promoted, mainly inCCR7�CD45RA� cells, proliferation coupled to differentiation to the CCR7� perforin � stage and acquisition of melanomaAg-specific effector functions. Taken together, these results indicate that CD8� T cells differentiated toward CCR7� cytotoxicfactor� stages are present in tumor-invaded, but not in tumor-free, lymph nodes of a relevant fraction of melanoma patients andsuggest that cytokines such as IL-2 and IL-15 may be exploited to promote Ag-independent maturation of anti-tumor CD8� Tcells. The Journal of Immunology, 2003, 171: 2134–2141.

T he definitions of functional status and of Ag-induced dif-ferentiation stage of T cells at tumor site are among themain goals for understanding how T cell-mediated anti-

tumor immunity is regulated in cancer patients during tumor pro-gression or after immunotherapy (1–3). For many years these chal-lenges have relied on assessing the frequency and Ag-specificfunctions of T cells isolated from neoplastic lesions (4). More re-cently, new markers of T cell differentiation have been identified(5–9) and a few models of Ag-induced post-thymic developmentof human CD4� and CD8� T cells have been proposed by analysisof peripheral blood T cell response to viral Ags (6–9). Within theCD8� T cell fraction, the models of human T cell differentiationrely on the identification of distinct T cell subsets depending on theexpression of leukocyte common Ag isoforms (CD45RA and RO),chemokine receptors (such as CCR4, CCR5, CCR6, and CCR7),costimulatory molecules (such as CD27 and CD28), L-selectins

(CD62L), and cytotoxic factors (such as perforin and granzymes)and on functional evaluation of Ag-specific responses (6–11).

The available models are not fully concordant on the phenotypicand functional signatures that identify each T cell differentiationstage (7–9). Nevertheless, in all models Ag-induced CD8� T cellmaturation is seen as a linear sequence from the CCR7�

CD45RA� naive (TN)3 stage to the Ag-experienced CCR7�

CD45RA� central memory (TCM), and CCR7� CD45RA� effec-tor memory (TEM) cells and to the CCR7� CD45RA� stage, de-fined either as terminally differentiated cells (7) or, more recently(11), as TEMRA (CD45RA� effector memory cells). Furthermore,emerging evidence, obtained in both the human system and murinemodels, indicates that each step along the differentiation pathwayis part of a continuum, rather than representing distinct cell sub-sets, and some of the stages may be reversible and affected differ-ently by Ag or cytokines sharing the common cytokine receptor�-chain (�c) (7, 10–12).

A few studies have addressed the issue of antitumor CD8� Tcell differentiation in melanoma patients by adopting the existingmodels of post-thymic CD8� T cell development. In some pa-tients, not subjected to immunotherapy, tumor-specific T cells witha CCR7� CD45RA� TEM phenotype (13) or a CCR7� CD45RA�

effector phenotype (14) have been described in the periphery or ina few cases of invaded lymph nodes, and these cells expressedAg-specific effector functions such as IFN-� release and direct exvivo lytic activity without activation. In vaccinated patients, differen-tiation to the CCR7� and/or CD27� stages on CD8� tumor-specificT cells in peripheral blood has been described after therapy (15–17),

*Human Tumor Immunobiology and †Tumor Immunotherapy Units, Department ofExperimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan,Italy

Received for publication March 24, 2003. Accepted for publication June 3, 2003.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by grants from Associazione Italiana per la Ricercasul Cancro (Milan, Italy), Istituto Superiore di Sanita’ (Rome, Italy), Ministry ofHealth (Rome, Italy), Consiglio Nazionale delle Ricerche (Rome, Italy), and Com-pagnia di S. Paolo (Torino, Italy).2 Address correspondence and reprint requests to Dr. Andrea Anichini, Human TumorImmunobiology Unit, Department of Experimental Oncology, Istituto Nazionale perlo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milan, Italy. E-mail address:andrea.anichini@istitutotumori.mi.it

3 Abbreviations used in this paper: TN, T naive; �c, common � chain; TCM, T centralmemory; TEM, T effector memory; TEMRA, T CD45RA� effector memory; TFLN,tumor-free lymph node; TILN, tumor-invaded lymph node.

The Journal of Immunology

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although these cells in some instances did not express cytotoxic fac-tors and required Ag exposure for functional reactivation.

However, despite this evidence, it remains to be elucidated, atthe population level, whether T cell differentiation is promoted atthe tumor site as a result of the natural evolution of immunity tothe disease compared with a tumor-free tissue. The surgical treat-ment of American Joint Committee on Cancer (AJCC) (18) stageIII melanoma patients can provide ideal test and control tissuesamples to address the question of T cell differentiation at thetumor site. In fact, in several patients both tumor-invaded (TILN)and tumor-free (TFLN) lymph nodes are isolated from the samenodal basin. Moreover, the issue of T cell differentiation at the tumorsite is relevant not only for a better understanding of the natural evo-lution of tumor immunity, but to designing improved protocols ofimmunotherapy. This goal is being pursued through Ag-specific vac-cination, an approach that may promote activation of T cells directedto a few different melanoma Ags (1). Nevertheless, the identificationof efficient means to promote Ag-independent differentiation of CD8�

T cells would further expand the repertoire of tumor-specific T lym-phocytes that could be turned into effector cells.

These issues were addressed by investigating the differentiationprofile and the response to cytokines of CD8� T cells in a largepanel of TILN and TFLN from stage III melanoma patients. Herewe show that a considerable fraction of TILN display a CD8� Tcell phenotypic profile characterized by increasing content of lym-phocytes at the CCR7� cytotoxic factor� stage, a phenotype notfound in CD8� T cells from most TFLN. Furthermore, in patientswhose invaded lymph nodes contained a predominant fraction ofCD8� T cells in early maturation stages (i.e., CCR7�

CD45RA�/� cytotoxic factor�), we show that cytokines such asIL-2 and IL-15 can promote Ag-independent differentiation of afraction of CD8� T cells associated with the acquisition of tumorAg-specific effector functions.

Materials and MethodsPatients and donors

Informed consent was obtained from the patients. Lymphocytes were iso-lated from TILN of 142 melanoma patients in AJCC stage III as previouslydescribed (19). In 42 of these patients lymphocytes were also isolated fromTFLN removed from the same nodal basin as TILN. The presence or theabsence of metastatic melanoma cells in the lymph node samples was as-sessed by conventional histochemistry on serial tissue sections and wasconfirmed by flow cytometry with mAbs to high m.w. melanoma Ags (20).All metastases found in TILN were of the massive type. HLA-A*0201subtyping was performed by PCR-sequence-specific oligonucleotide probetyping as previously described (19). None of the patients enrolled in thisstudy had been subjected to chemotherapy, immunotherapy, or any othertherapy with immunosuppressive activity before isolation of lymphocytes.

Flow cytometric analysis

Different combinations of the following mouse anti-human mAbs wereused: anti-CD8 coupled to PerCP or allophycocyanin, FITC- or PE-anti-CD45RA, and PerCP- or PE-anti-CD3 (BD PharMingen, San Diego, CA).To detect CCR7, cells were stained with IgM anti-CCR7 (BD PharMin-gen), followed by biotin-conjugated rat anti-mouse IgM and then by Cy-Chrome-conjugated streptavidin (BD PharMingen). To detect intracellularperforin or granzyme B, cells were permeabilized with Cytofix/Cytoperm(BD PharMingen) and then stained with FITC anti-perforin (BD PharM-ingen) or PE-anti-granzyme B (CLB, Amsterdam, The Netherlands) in thepresence of Perm/Wash solution (BD PharMingen). In some experimentsCCR7� lymphocytes from TILN and PBL were purified by cell sortingafter staining with anti-CCR7 mAb. Sorting, acquisition, and analysis byfour-color flow cytometry were conducted by a dual-laser FACSCaliburcytofluorometer (BD Biosciences, Mountain View, CA) using CellQuestsoftware. PE-labeled tetramers of HLA-A*0201-containing peptides fromMelan-A/Mart-126–35 (21, 22), gp100209–217 (23), MAGE-3271–279 (24),NY-ESO-1157–165 (25), and influenza matrix58–66 peptides (26) were pur-chased from ProImmune (Oxford, U.K.). Tetramers were titrated againstpeptide-specific T cell lines to minimize background staining while pre-

serving the mean fluorescence intensity of positive cells and then were usedat a final dilution of 1/200 of the stock solution. Cells (2 � 106) werestained for 15 min with PE-labeled tetramers at 37°C and then stained for30 min on ice with other cell surface Abs. Negative controls for tetramerstaining included PBL from HLA-A*0201� healthy controls.

Intracellular IFN-� detection

Lymphocytes from peripheral blood, TILN, or short term T cell cultureswere stained with PE-tetramers for 15 min at 37°C and then stimulated inthe presence of autologous PBMC loaded with 2 �M Melan-A/Mart-127–35

or gp100209–217 peptides for 4–6 h. After the first hour, GolgiStop (BDPharMingen) was added. Cell surface staining was then conducted withPerCP-anti-CD8 and/or CyChrome-anti-CCR7, followed by cell permeabi-lization with Cytofix/Cytoperm and staining with allophycocyanin-anti-IFN-� mAb in the presence of Perm/Wash solution. The expression ofIFN-� was then analyzed by flow cytometry after imposing a double gatingfor CD8� and tetramer� T cells (7).

CD69 up-regulation

Lymphocytes from peripheral blood or TILN were cultured with 20 ng/mlPMA (Sigma-Aldrich, St. Louis, MO) and 500 ng/ml ionomycin (Sigma-Aldrich) for 5 h. Cells were then stained with allophycocyanin-anti-CD8,PE-anti-CD45RA, CyChrome-anti-CCR7, and FITC-anti-CD69 Abs (BDPharMingen) and analyzed by flow cytometry. Alternatively, PMA- plus iono-mycin-stimulated lymphocytes were analyzed for intracellular IFN-� produc-tion by staining with PE-anti-CD8, CyChrome-anti-CCR7, FITC-anti-CD45RA, and allophycocyanin-anti-IFN-� Abs after permeabilization.

Cytokines and CFSE proliferation assay

Lymphocytes from TILN were cultured at 1 � 106/ml in RPMI 1640 con-taining 10% pooled human serum in the presence of 50 ng/ml of IL-2(Chiron, Emeryville, CA), IL-7 (PeproTech EC, London, U.K.), or IL-15(PeproTech EC) for up to 7 days. To detect proliferating lymphocytes, cellswere stained with 2 �M CFSE (Molecular Probes, Eugene, OR) for 10 minat 37°C, followed by three washes with cold RPMI 1640. CFSE-stainedlymphocytes were then cultured in the presence of cytokines, or on platesprecoated with anti-CD3 mAb (10 ng/ml) through cross-linking mediatedby 10 �g/ml goat anti-mouse IgG (Sigma-Aldrich).

Cytotoxic assay

Lymphocytes from TILN were cultured at 1 � 106/ml in RPMI 1640 con-taining 10% pooled human serum with or without 50 ng/ml IL-2 (Chiron),IL-7 (PeproTech EC), or IL-15 (PeproTech EC). After 7 days the cytotoxicactivity of these cultures was tested on Melan-A/Mart-1�, gp100� HLA-A*0201� melanomas, preincubated or not with anti-HLA-A2 mAbCR11.351 (27), and on T2 cells loaded with Melan-A/Mart-127–35 orgp100209–217 peptides. Lysis of targets was evaluated by a 4-h 51Cr releaseassay at E:T cell ratios from 20:1 to 2.5:1, as described previously (19).

Statistical analysis

Comparison of TILN and TFLN phenotypes for the proportion of CD3�

CD8� T cells expressing each of the eight phenotypes defined by CCR7 vsCD45RA and by CCR7 vs perforin was evaluated by ANOVA, followedby Student-Newman-Keuls multiple comparison test. Hierarchical clusteranalysis (28) of the CD3� CD8� T cell differentiation phenotype wasconducted using J-Express Pro software (www.Molmine.com). Data werehierarchically clustered by tissue (TILN or TFLN) and phenotype using acomplete linkage as clustering method and the Pearson correlation as thesimilarity measure.

ResultsCD8� T cell differentiation profile in TILN and TFLN frommelanoma patients

CD3� CD8� T cells from TILN of 142 AJCC stage III melanomapatients were assessed for the expression of markers of differen-tiation. In addition, from 42 of such patients, a tissue sample of aTFLN, removed from the same nodal basin as the TILN, was ob-tained and analyzed. Hierarchical cluster analysis was performedto obtain a comprehensive classification of the CD8� T cell phe-notypic profiles of TILN and TFLN on the basis of the eight sub-sets defined by CCR7 vs CD45RA, and by CCR7 vs perforin. Fourmajor CD8� T cell phenotypic clusters were identified (Fig. 1).

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CD8� T cells from all TFLN and 56% of the TILN samples fellinto cluster 1, characterized by frequent CCR7� CD45RA� (TN)and CCR7� CD45RA� (TCM) phenotypes or even CCR7�

CD45RA� (TEM) cells, but lacking perforin in most instances orexpressing it at very low levels and only in TILN samples, not inTFLN (Fig. 1). Increasing expression of perforin in the CCR7�

subset characterized CD8� T cells in clusters 2, 3, and 4, althoughnot all CCR7� cells were perforin�, at either the TEM or TEMRA

stage (see CCR7�/perforin� column in Fig. 1). These three clus-ters contained only T cells from TILN and showed a progressiveincrease in the proportion of CD8� T cells at the CCR7�

CD45RA� (TEM) and CCR7� CD45RA� (TEMRA, or terminallydifferentiated) (7, 11) stages (Fig. 1). Significant differences in thedifferentiation profile between TILN and TFLN were confirmed bythe Student-Newman-Keuls multiple comparison test for seven ofeight phenotypic subsets defined by CCR7 vs CD45RA and CCR7vs perforin (see Fig. 1). In agreement with these results, analysis ofmatched TILN and TFLN samples from the same patient indicated,in several instances, a shift of CD8� T cells from TILN toward theCCR7� CD45RA� TEM stage, with expression of perforin in theCCR7� fraction, compared with the matched TFLN (representa-tive results from seven patients shown in Fig. 2). Such differences

FIGURE 1. Hierarchical cluster analysis of the dif-ferentiation phenotype of CD3� CD8� T cells inTILN and TFLN from AJCC stage III melanoma pa-tients. Rows represent individual lymphocyte samplesfrom TILN or TFLN; columns represent each of theeight possible phenotypes obtained by analysis ofCCR7 vs CD45RA and of CCR7 vs perforin in CD3�

CD8� lymphocytes by four-color flow cytometry. �,Tissue coding: black, TILN; white, TFLN. The fourmajor clusters were ranked from 1–4 according to aprogressive shift of the CD8� T cell maturation pro-files toward the CCR7� CD45RA� perforin� stage.The number of cases (percentage) from TILN orTFLN belonging to each phenotype cluster is shownon the right side. The percentage of positive cells foreach phenotype subset was coded by 10 levels of grayshading, as indicated at the bottom of the figure. TILNand TFLN differed in the proportion of CD3� CD8�

cells expressing the TN (p � 0.001), TCM (p � 0.001),TEM (p � 0.001), and TEMRA (p � 0.05) phenotypesas well as in the proportion expressing the CCR7�/per-forin�, CCR7�/perforin�, and CCR7�/perforin� pheno-types (p � 0.001 in all instances), while the CCR7�/perforin� phenotype was not significantly different.

FIGURE 2. CD8� T cells differentiation profile ofmatched TILN and TFLN samples from seven pa-tients. TILN (F) and TFLN (E) samples were evalu-ated for the expression of CCR7 vs CD45RA andCCR7 vs perforin in CD3� CD8� T cells by four-color flow cytometry. Results are expressed as the per-centage of positive cells for each of the eight possiblephenotypes. Statistical analysis was indicated as fol-lows: �, p � 0.05; ��, p � 0.01.

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between matched TILN and TFLN pairs were less pronouncedwhen the TILN were isolated from patients in cluster 1, as in thisinstance both TILN and TFLN showed the early stages of differ-entiation (data not shown). Further evidence for differentiation ofCD3� CD8� T cells, from a fraction of TILN, to cytotoxic factor�

stage was obtained by comparing CCR7 vs perforin and CCR7 vsgranzyme B phenotypes. This analysis indicated that both cyto-toxic factors were expressed in the CD8� CCR7� T cells of TILNfrom the clusters 3 and 4 (Fig. 3), although, as seen for perforin,even granzyme B was not always expressed in all CCR7� cells(see CCR7�/granzyme B� column in Fig. 3).

In addition, comparison of PBL and TILN for the CD8� T celldifferentiation phenotype was performed in several patients be-longing to the maturation clusters 1–4, but no relationship wasfound between PBL and TILN phenotypes (data not shown).Taken together these results indicate accumulation of CD8� Tcells at CCR7� cytotoxic factor� stages in a large fraction ofTILN, but not in TFLN, from metastatic melanoma patients.

Functional analysis of CD8� T cell differentiation subsetsfrom TILN

Lymphocytes from TILN in the panel of stage III patients wereevaluated for CD69 up-regulation and cytokine secretion in re-sponse to PMA and ionomycin. All four CCR7/CD45RA subsetsof CD8� T lymphocytes from TILN (Fig. 4A) responded similarlyin terms of CD69 up-regulation (Fig. 4B). In contrast, theCCR7�CD45RA� TN subset did not produce IFN-� in response to

PMA plus ionomycin (Fig. 4C), while this cytokine was mainlyproduced by CD8� cells at the TCM and TEM stages (CCR7�

CD45RA� and CCR7� CD45RA�) and to a lesser extent byTEMRA cells (CCR7� CD45RA� subset; Fig. 4C). Furthermore,the proliferative response of CD8� T cells from TILN to immo-bilized anti-CD3 mAb, as evaluated by CFSE staining, was foundmainly in the CCR7� CD45RA� subset and to a lesser extent inthe CCR7� CD45RA� subset (Fig. 4D). These data indicate thatthe TILN CD8� T cell differentiation subsets defined by CCR7 vsCD45RA expression are associated with distinct functional stages.

Differentiation profile of melanoma- and viral Ag-specific CD8�

T cells in TILN

The CCR7 vs CD45RA profile of melanoma-Ag-specific T cellsrecognized by HLA-A*0201 tetramers was investigated in HLA-A*0201 patients found in the panel of 142 TILN from stage IIIpatients. The differentiation phenotype of tetramer� T cells di-rected to melanocyte lineage Ags (Melan-A/Mart-126–35 andgp100209–217), tumor-restricted epitopes (Mage-3271–279 and NY-ESO-1157–165), or influenza matrix58–66 peptide, from TILN incluster 1 (data from two representative patients in cluster 1 areshown in the upper two rows of panels in Fig. 5) indicated a pre-dominant CCR7� CD45RA�, TN phenotype, in agreement withthe frequent profile of the bulk CD8� T cell population. Evidencefor progressive differentiation of tetramer� T cells was obtained inTILN samples from patients in cluster 2, 3, and 4 (lower threerows of panels in Fig. 5). This was indicated by the increasingproportion of tetramer� T cells expressing a TCM phenotype (as inthe representative patient from cluster 2, Fig. 5) or expressing theTEM or TEMRA phenotype (see representative patients from clus-ters 3 and 4, respectively; Fig. 5). Interestingly, the pattern ofdifferentiation of tetramer� T cells directed to influenza matrixpeptide was similar in each phenotype cluster to the phenotype ofT cells directed to the four tumor Ags (Fig. 5). Analysis of TFLNsamples from HLA-A*0201� patients for the CCR7 vs CD45RAphenotype of CD8� T cells directed to melanoma and influenzaepitopes indicated a predominant TN or TCM phenotype, without ex-pression of perforin (data not shown). Taken together, these data sug-gest that melanoma-specific and viral Ag-specific T cells from thesame TILN or TFLN tissue sample may share a similar differentiationprofile, in agreement with analysis at the bulk CD8� T cell level.

IL-2 and IL-15 may promote differentiation of CD8� CCR7� Tcells from TILN

The CD8� T cell maturation profile of a large fraction of TILNsamples in cluster 1 indicated a prevalence of CCR7� CD45RA�,

FIGURE 3. Analysis of perforin and granzyme B expression inCD3�CD8� lymphocytes from TILN. Rows represent individual lympho-cyte samples from TILN. Columns represent each of the eight possiblephenotypes obtained by analysis of CCR7 vs perforin and of CCR7 vsgranzyme B in gated CD3� CD8� lymphocytes. TILN belong to patientsof differentiation clusters 1 and 3–4 as defined in Fig. 1. The percentage ofpositive cells for each of the indicated phenotypes was coded by 10 levelsof gray shading, as indicated in Fig. 1.

FIGURE 4. Functional analysis of CD8� T cell subsets from TILN defined by their CCR7 vs CD45RA phenotype. CD8� T cells from TILN, afterstaining for CD45RA and CCR7 (A), were evaluated for up-regulation of CD69 (B) and IFN-� production (C) in response to PMA plus ionomycin (�)compared with unstimulated cultures (f). The histograms show the response induced by PMA plus ionomycin in each of the four CD8� subsets (identifiedas 1–4 in A–C) gated by the CCR7 vs the CD45RA phenotype. D, CFSE-stained lymphocytes from TILN were cultured on immobilized anti-CD3 mAbfor 3 days. Gated CD8� lymphocytes were analyzed for CFSE profiles according to their CCR7/CD45RA phenotype.

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TN, or CCR7� CD45RA�, TCM cells, lacking cytotoxic factors.To evaluate whether CD8� T cells from cluster 1 could be inducedthrough the differentiation process, CCR7� T lymphocytes weresorted from TILN, labeled with CFSE, and then cultured with �ccytokines that may play a role in T cell differentiation (10, 11, 29).IL-2 and IL-15 induced a proliferative response in the sortedCCR7� CD8� T cells, while the response to IL-7 was minimal(Fig. 6A). Furthermore, while nonproliferating cells remainedCCR7�, this marker was down-modulated in most CD8� T lym-phocytes that could proliferate to IL-2 and IL-15 (Fig. 6A). SortedCCR7� CD8� T cells from PBL of healthy donors showed a sim-ilar response (Fig. 6B). In contrast to IL-2 and IL-15, the sortedCCR7� CD8� T cells from TILN did not down-modulate CCR7

when proliferating to immobilized anti-CD3 mAb (Fig. 6A). Anal-ysis of CD45RA expression of the sorted CCR7� CD8� T cellsfrom TILN confirmed the presence of two subsets, CD45RA�

(TN) and CD45RA� (TCM; Fig. 6C). In the CD8� subset, prolif-eration to IL-2 or IL-15 was observed mainly in the CD45RA�

fraction and to a lesser extent in the CD45RA� fraction (Fig. 6C).By contrast, in the CD4� subset the response to �c cytokines wasmainly found in the CD45RA� subset (Fig. 6C). By gating forCCR7 and CD45RA on CFSE-labeled lymphocytes, the CD8� Tcells that proliferated to �c cytokines expressed, at the end ofculture (day 7), either a CCR7� CD45RA� (TEM) or aCCR7�CD45RA� (TEMRA) phenotype (data not shown). Furtherevidence for cytokine-induced CD8� T cell differentiation was

FIGURE 5. Phenotype of Ag-specific CD8�

T cells from TILN of HLA-A*0201� patients.Four-color flow cytometric analysis of the ex-pression of CCR7 and CD45RA in the bulkCD8� T cell population and in T cells identifiedby tetramers of HLA-A*0201-containing pep-tides from melanocyte lineage Ags (Melan-A/Mart-1 and gp100), tumor-restricted epitopes(MAGE-3 and NY-ESO-1), or influenza matrix.Patients belong to phenotype clusters 1–4 as de-fined in Fig. 1. Each row of six panels shows thephenotype of bulk CD8� cells and tetramer� Tlymphocytes from TILN of one representativepatient belonging to the indicated phenotypecluster. Numbers represent the percentage ofpositive cells in each quadrant.

FIGURE 6. Role of IL-2 and IL-15 in the differentiation of CD8� T cells from TILN. Sorted CCR7� lymphocytes from TILN of patients in cluster 1(A and C) or from peripheral blood of a healthy donor (B) were stained with CFSE and then cultured for 7 days in medium alone or in the presence of IL-2,IL-7, IL-15, or immobilized anti-CD3 mAb and then analyzed for differentiation markers vs CFSE fluorescence. In each dot plot, right and left quadrantsidentify nonproliferating and proliferating cells, respectively.

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obtained in TILN from cluster 1 by looking at the expression ofcytotoxic factors. Freshly isolated CD8� T cells were mostlyCCR7� perforin� (Fig. 7). In contrast, after culture with IL-2,IL-15, or IL-2 plus IL-15, the CD3� CD8� lymphocytes expresseda predominant CCR7� perforin� phenotype (Fig. 7). Culture withIL-7 led to up-regulation of perforin in a fraction of cells, but mostof them remained CCR7� (Fig. 7). Taken together these data in-dicate that differentiation of CCR7� CD8� T cells from TILN canbe promoted in vitro by �c cytokines such as IL-2 and IL-15. Thisprocess involves mainly the proliferation of CCR7� CD45RA�

TCM cells and, to a lesser extent, of CCR7� CD45RA� TN cells,and leads to differentiation to CCR7� perforin� stage.

Ag-independent differentiation of melanoma-specific T cells fromTILN by �c cytokines

The impact of �c cytokines on T cell phenotype and function wasevaluated in Melan-A/Mart-1- or gp100-specific T cells in TILNfrom HLA-A*0201� patients in cluster 1. In these patients, freshlyisolated tetramer� T cells expressed mostly a CCR7� CD45RA�

phenotype (Fig. 8), a phenotype that did not change by culture inmedium without cytokines (data not shown). In contrast, after cul-ture with IL-2, IL-15, or IL-2 plus IL-15, but not IL-7, tetramer�

T cells showed a CCR7� CD45RA� phenotype in up to 50% ofthe cells (Fig. 8) or even a predominant CCR7� CD45RA� phe-notype in some patients (data not shown).

At the functional level, intracellular expression of IFN-� in re-sponse to peptide-loaded APCs was observed by Melan-A/Mart-1-specific T cells after culture of lymphocytes from TILN withIL-2 and IL-15, but not with IL-7 or in freshly isolated cells (Fig.9A). Similar results were obtained with gp100-specific T cells fromTILN (see Fig. 9B for results on IL-2-cultured lymphocytes).However, while most of the tetramer� T cells produced IFN-�

when stimulated with PMA plus ionomycin (Fig. 9B), only a frac-tion of the cytokine-cultured tetramer� T cells became positive forintracellular IFN-� in response to peptide-loaded APCs (Fig. 9, Aand B). This was observed even in instances where most of theAg-specific T lymphocytes were at the CCR7� stage after cytokineculture (Fig. 9B), suggesting heterogeneity for Ag-specific IFN-�production. Similar results, indicating production of IFN-� in re-sponse to peptide-loaded APCs after cytokine culture, were obtainedeven with flu matrix-specific T cells from TILN (data not shown).

Lymphocytes from TILN in cluster 1, after culture for 7 dayswith �c cytokines, were assessed for Ag-specific cytotoxicity. Af-ter culture with IL-2, IL-15, or IL-2 plus IL-15, the T cell culturesfrom TILN exhibited HLA-A2-restricted lysis of HLA-A*0201�,Melan-A/Mart-1�, gp100� autologous melanoma (Fig. 10A) andlysed APCs loaded with Melan-A/Mart-1 peptide and, to a lesserextent, APCs loaded with gp100209–217 peptide (Fig. 10, C–E). Bycontrast, no lytic activity on melanoma cells (Fig. 10A), or onpeptide-loaded APCs (Fig. 10B) was exerted by freshly isolated Tcells from TILN. Taken together these results suggest that �c cy-tokines, such as IL-2 and IL-15, can promote in-vitro Ag-indepen-dent differentiation of melanoma-specific T cells from TILN.

DiscussionComparison of TILN and TFLN from melanoma patients by hier-archical cluster analysis provided evidence for a marked shift ofCD8� T cell differentiation toward CCR7� cytotoxic factor�

stages at the tumor site in a relevant fraction of tissue samples. Infact, in three of four T cell phenotype clusters, representing �44%of the TILN samples, we found that the differentiation profile ofthe CD8� T cell fraction was gradually shifted toward an increas-ing content of CCR7� CD45RA�/� cells containing the cytotoxicfactors perforin and granzyme B. Thus, in these clusters, the CD8�

FIGURE 7. Expression of CCR7 and perforin in freshly isolated TILN from a patient in cluster 1 and after culture for 7 days with �c cytokines. Dotplots were gated on CD3� CD8� cells. The numbers in the dot plots represent the percentage of positive cells in each quadrant.

FIGURE 8. Effect of �c cytokines onCCR7 vs CD45RA phenotype of melanoma-specific CD8� T cells from TILN in cluster1. Lymphocytes from TILN of an HLA-A*0201� patient from cluster 1 were char-acterized for CCR7 vs CD45RA phenotypebefore (fresh TILN) and after culture for 7days with IL-2, IL-7, IL-15, or IL-2 plus IL-15. The maturation phenotype was assessedin Ag-specific T cells recognized by HLA-A*0201/Melan-A/Mart-126–35 (1) and HLA-A*0201/gp100209–216 (2) tetramers. All dotplots were gated on CD8� tetramer� T cells.The numbers in the dot plots represent thepercentage of positive cells in each quadrant.

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T cell differentiation was consistent with a progressive shift towardthe TEM and TEMRA/terminally differentiated stages. The remain-ing TILN samples and all TFLN defined a single cluster in whichCD8� T cell differentiation was mainly characterized by CCR7�

CD45RA�, TN and CCR7� CD45RA�, TCM stages. Moreover,most TILN and TFLN in this cluster lacked perforin or expressedit at very low levels and only in some TILN samples. The distinctCD8� T cell subsets found in TILN and defined by intracellularand extracellular markers reflected distinct functional stages, asindicated by CFSE analysis for proliferative response to immobi-lized anti-CD3 mAb and by IFN-� release in response to PMAplus ionomycin. Taken together these results suggest that tumor-invaded, but not tumor-free, lymph nodes of a relevant fraction ofstage III melanoma patients are involved in a process of CD8� Tcell differentiation beyond TN, TCM, and cytotoxic factor� stages.

Analysis of T cell maturation stages in melanoma-specific Tcells from TILN indicated that the process of T cell differentiationcould involve even tumor-specific T lymphocytes directed to mel-anocyte-lineage Ags or to tumor-restricted epitopes, as indicatedby the phenotype of tetramer� T cells from TILN of clusters 2–4.This may indicate a role of anti-tumor immunity in shaping theCD8� T cell differentiation profile at the tumor site. However,interestingly, maturation along the TCM3TEM3TEMRA pathwaywas documented in the same TILN even for T cells directed to aviral epitope (influenza matrix). Such an effect may be promotedthrough Ag-independent bystander mechanisms (30, 31), and tu-mor-derived factors have a possible role in such process. In fact,some of the cytokines that can play a role in T cell differentiation,such as IL-15, can be produced by tumor cells, including mela-noma (32). Moreover, other factors, such as IL-6 and IL-10, arefrequently expressed by melanoma cells (33, 34). These factorscould exert an indirect effect on T cell differentiation through theiractivity on the expression of IL-2/IL-15R� and �-chains on T cells

(10, 11), thus promoting the response of CD8� T lymphocytes tosoluble regulators of differentiation.

Culture of CCR7� CD8� lymphocytes from TILN of pheno-typic cluster 1 in the presence of �c cytokines IL-2 and/or IL-15promoted differentiation of a fraction of lymphocytes to theCCR7� perforin� stage. This was associated with acquisition ofAg-specific effector functions, such as cytokine release and cyto-toxicity. This cytokine-induced differentiation was restricted to afraction of CD8� lymphocytes that could proliferate in response toIL-2 and IL-15, suggesting differential expression of functionalcytokine receptors in CCR7� CD8� T cells. Indeed, recent evi-dence indicates that IL-15R� and IL-2/IL-15R�-chain expressionis low, although detectable, in CCR7� CD45RA� TN cells andincreases progressively along the TN3TCM3TEM differentiationpathway (10, 11), while �c is similarly expressed in the threestages (11). In agreement, analysis of the expression of differen-tiation markers indicated that the response of CCR7� CD8� Tcells to �c cytokines was due mainly to proliferation of CD45RA�

cells (i.e., TCM), with some response even in the CD45RA� frac-tion (i.e., TN), and led to down-modulation of CCR7. Down-mod-ulation of CCR7 after culture with �c cytokines is in agreementwith the findings in the CD4� subset (10), where the proliferativeresponse has been shown to lead TCM (i.e., CCR7� CD45RA�)cells to down-modulate CCR7 and acquire effector functions, suchas cytokine release, associated with the CCR7� CD45RA� TEM

stage. In the CD8� subset, Geginat et al. (11) found that uponculture of PBL with IL-7 plus IL-15, TN cells maintained theirphenotype, while the TCM fraction gave rise to cells expressingCCR7 and CD45RA in all possible combinations, including TEM

and TEMRA. These latter phenotypes were the two profiles that wefound expressed at the end of culture by CD8� T cells that pro-liferated to IL-2 or IL-15, used as single cytokines. In addition, adirect comparison of IL-15 alone vs IL-7 plus IL-15 has not been

FIGURE 9. Peptide-specific intracellular IFN-� production by CD8� T cells from TILN after culture with �c cytokines. Lymphocytes from TILN of anHLA-A*0201� patient in cluster 1 were cultured for 7 days with the indicated cytokines (A) or with IL-2 (B) and after staining with tetramers to Melan-A/Mart-1(A) or gp100 (B) were evaluated for intracellular IFN-� production induced by autologous PBMC loaded (�peptide) or not (�peptide) with Melan-A/Mart-127–35

(A) or gp100209–217 (B). T cells in B were stimulated with peptide-loaded PBMCs or with PMA plus ionomycin and stained for CCR7.

FIGURE 10. Ag-specific cytotoxic activity byCD8� T cells from TILN after culture with �c cyto-kines. TILN, either fresh or cultured for 7 days with theindicated cytokines, were assessed for: A, lysis of HLA-A*0201� Melan-A/Mart-1� gp100� autologous mela-noma pre-incubated (empty histograms) or not (blackhistograms) with anti-HLA-A2 mAb; B, lysis of T2cells loaded with the indicated peptides; C–E, lysis ofT2 cells either empty (�) or loaded with gp100209–217

(Œ) or Melan-A/Mart-127–35 (F) peptides.

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performed, but it remains possible that CD8� T cell maturationmay be affected differently by culture with single �c cytokines,performed in our study, vs the combination of �c cytokines. Fur-thermore, we observed down-modulation of CCR7 in the prolif-erative response of CD8� T cells to �c cytokines, but not in theresponse to immobilized anti-CD3 mAb. Interestingly, down-mod-ulation of CCR7 has been described in TN (naive) cells upon stim-ulation with immobilized anti-CD3 plus anti-CD28 (10), suggest-ing that T cell proliferation can be coupled, or not, todifferentiation depending on the specificity of signals (i.e., TCRtriggering vs TCR triggering plus costimulation).

The Ag-independent maturation of a fraction of CD8� T cellsfrom TILN suggests that �c cytokines, such as IL-2 and IL-15,may be exploited for promoting the development of antitumor ef-fectors in cancer patients for immunotherapy purposes (35). Thisgoal appears relevant in the light of the frequent identification ofanti-tumor CD8� T cells at early stages of differentiation at thetumor site. Thus, the available evidence suggests that a large frac-tion of melanoma patients may benefit from strategies aimed atpromoting functional maturation of their antitumor T cells. In prin-ciple, Ag-independent T cell differentiation to the effector stagecould reduce the need for targeting specific tumor Ags, thus avoid-ing the risk of tumor resistance due to Ag loss variants. In addition,T cells from TILN may respond to �c cytokines mainly on thebasis of their differentiation stage, which controls the expression offunctional cytokine receptors, and independently from their Agspecificity. If antitumor T cells can be found at the tumor site,although in early stages of maturation, then cytokine-mediated dif-ferentiation would provide a way of activating T cells directedagainst a wide spectrum of tumor Ags. Thus, cytokine-induceddifferentiation of TILN populations, enriched for antitumor T cells,may represent a potential application for improving protocols ofimmunotherapy for cancer.

AcknowledgmentsWe are grateful to the melanoma patients for their generous participation inthis study.

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