Differentiation of CD8⁺ T Cells from Tumor-Invaded and Tumor-Free Lymph Nodes of Melanoma Patients: Role of Common γ-Chain Cytokines¹

The definitions of functional status and of Ag-induced differentiation stage of T cells at tumor site are among the main goals for understanding how T cell-mediated antitumor immunity is regulated in cancer patients during tumor progression or after immunotherapy (1, 2, 3). For many years these challenges have relied on assessing the frequency and Ag-specific functions of T cells isolated from neoplastic lesions (4). More recently, new markers of T cell differentiation have been identified (5, 6, 7, 8, 9) and a few models of Ag-induced post-thymic development of human CD4⁺ and CD8⁺ T cells have been proposed by analysis of peripheral blood T cell response to viral Ags (6, 7, 8, 9). Within the CD8⁺ T cell fraction, the models of human T cell differentiation rely on the identification of distinct T cell subsets depending on the expression 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, 7, 8, 9, 10, 11).

The available models are not fully concordant on the phenotypic and functional signatures that identify each T cell differentiation stage (7, 8, 9). Nevertheless, in all models Ag-induced CD8⁺ T cell maturation is seen as a linear sequence from the CCR7⁺ CD45RA⁺ naive (T_N)³ stage to the Ag-experienced CCR7⁺ CD45RA⁻ central memory (T_CM), and CCR7⁻ CD45RA⁻ effector memory (T_EM) cells and to the CCR7⁻ CD45RA⁺ stage, defined either as terminally differentiated cells (7) or, more recently (11), as T_EMRA (CD45RA⁺ effector memory cells). Furthermore, emerging evidence, obtained in both the human system and murine models, indicates that each step along the differentiation pathway is part of a continuum, rather than representing distinct cell subsets, and some of the stages may be reversible and affected differently by Ag or cytokines sharing the common cytokine receptor γ-chain (γc) (7, 10, 11, 12).

A few studies have addressed the issue of antitumor CD8⁺ T cell differentiation in melanoma patients by adopting the existing models of post-thymic CD8⁺ T cell development. In some patients, not subjected to immunotherapy, tumor-specific T cells with a CCR7⁻ CD45RA⁻ T_EM phenotype (13) or a CCR7⁻ CD45RA⁺ effector phenotype (14) have been described in the periphery or in a few cases of invaded lymph nodes, and these cells expressed Ag-specific effector functions such as IFN-γ release and direct ex vivo lytic activity without activation. In vaccinated patients, differentiation to the CCR7⁻ and/or CD27⁻ stages on CD8⁺ tumor-specific T cells in peripheral blood has been described after therapy (15, 16, 17), although these cells in some instances did not express cytotoxic factors and required Ag exposure for functional reactivation.

However, despite this evidence, it remains to be elucidated, at the population level, whether T cell differentiation is promoted at the tumor site as a result of the natural evolution of immunity to the disease compared with a tumor-free tissue. The surgical treatment of American Joint Committee on Cancer (AJCC) (18) stage III melanoma patients can provide ideal test and control tissue samples to address the question of T cell differentiation at the tumor site. In fact, in several patients both tumor-invaded (TILN) and tumor-free (TFLN) lymph nodes are isolated from the same nodal basin. Moreover, the issue of T cell differentiation at the tumor site is relevant not only for a better understanding of the natural evolution of tumor immunity, but to designing improved protocols of immunotherapy. This goal is being pursued through Ag-specific vaccination, an approach that may promote activation of T cells directed to a few different melanoma Ags (1). Nevertheless, the identification of efficient means to promote Ag-independent differentiation of CD8⁺ T cells would further expand the repertoire of tumor-specific T lymphocytes that could be turned into effector cells.

These issues were addressed by investigating the differentiation profile and the response to cytokines of CD8⁺ T cells in a large panel of TILN and TFLN from stage III melanoma patients. Here we show that a considerable fraction of TILN display a CD8⁺ T cell phenotypic profile characterized by increasing content of lymphocytes at the CCR7⁻ cytotoxic factor⁺ stage, a phenotype not found in CD8⁺ T cells from most TFLN. Furthermore, in patients whose invaded lymph nodes contained a predominant fraction of CD8⁺ T cells in early maturation stages (i.e., CCR7⁺ CD45RA^+/− cytotoxic factor⁻), we show that cytokines such as IL-2 and IL-15 can promote Ag-independent differentiation of a fraction of CD8⁺ T cells associated with the acquisition of tumor Ag-specific effector functions.

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

Different combinations of the following mouse anti-human mAbs were used: 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 PharMingen), followed by biotin-conjugated rat anti-mouse IgM and then by CyChrome-conjugated streptavidin (BD PharMingen). To detect intracellular perforin or granzyme B, cells were permeabilized with Cytofix/Cytoperm (BD PharMingen) and then stained with FITC anti-perforin (BD PharMingen) or PE-anti-granzyme B (CLB, Amsterdam, The Netherlands) in the presence of Perm/Wash solution (BD PharMingen). In some experiments CCR7⁺ lymphocytes from TILN and PBL were purified by cell sorting after staining with anti-CCR7 mAb. Sorting, acquisition, and analysis by four-color flow cytometry were conducted by a dual-laser FACSCalibur cytofluorometer (BD Biosciences, Mountain View, CA) using CellQuest software. PE-labeled tetramers of HLA-A*0201-containing peptides from Melan-A/Mart-1_26–35 (21, 22), gp100_209–217 (23), MAGE-3_271–279 (24), NY-ESO-1_157–165 (25), and influenza matrix_58–66 peptides (26) were purchased from ProImmune (Oxford, U.K.). Tetramers were titrated against peptide-specific T cell lines to minimize background staining while preserving the mean fluorescence intensity of positive cells and then were used at a final dilution of 1/200 of the stock solution. Cells (2 × 10⁶) were stained for 15 min with PE-labeled tetramers at 37°C and then stained for 30 min on ice with other cell surface Abs. Negative controls for tetramer staining included PBL from HLA-A*0201⁻ healthy controls.

Lymphocytes from peripheral blood, TILN, or short term T cell cultures were stained with PE-tetramers for 15 min at 37°C and then stimulated in the presence of autologous PBMC loaded with 2 μM Melan-A/Mart-1_27–35 or gp100_209–217 peptides for 4–6 h. After the first hour, GolgiStop (BD PharMingen) was added. Cell surface staining was then conducted with PerCP-anti-CD8 and/or CyChrome-anti-CCR7, followed by cell permeabilization with Cytofix/Cytoperm and staining with allophycocyanin-anti-IFN-γ mAb in the presence of Perm/Wash solution. The expression of IFN-γ was then analyzed by flow cytometry after imposing a double gating for CD8⁺ and tetramer⁺ T cells (7).

Lymphocytes from peripheral blood or TILN were cultured with 20 ng/ml PMA (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 (BD PharMingen) and analyzed by flow cytometry. Alternatively, PMA- plus ionomycin-stimulated lymphocytes were analyzed for intracellular IFN-γ production by staining with PE-anti-CD8, CyChrome-anti-CCR7, FITC-anti-CD45RA, and allophycocyanin-anti-IFN-γ Abs after permeabilization.

Lymphocytes from TILN were cultured at 1 × 10⁶/ml in RPMI 1640 containing 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, cells were stained with 2 μM CFSE (Molecular Probes, Eugene, OR) for 10 min at 37°C, followed by three washes with cold RPMI 1640. CFSE-stained lymphocytes were then cultured in the presence of cytokines, or on plates precoated with anti-CD3 mAb (10 ng/ml) through cross-linking mediated by 10 μg/ml goat anti-mouse IgG (Sigma-Aldrich).

Lymphocytes from TILN were cultured at 1 × 10⁶/ml in RPMI 1640 containing 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 cytotoxic activity of these cultures was tested on Melan-A/Mart-1⁺, gp100⁺ HLA-A*0201⁺ melanomas, preincubated or not with anti-HLA-A2 mAb CR11.351 (27), and on T2 cells loaded with Melan-A/Mart-1_27–35 or gp100_209–217 peptides. Lysis of targets was evaluated by a 4-h ⁵¹Cr release assay at E:T cell ratios from 20:1 to 2.5:1, as described previously (19).

Comparison of TILN and TFLN phenotypes for the proportion of CD3⁺ CD8⁺ T cells expressing each of the eight phenotypes defined by CCR7 vs CD45RA and by CCR7 vs perforin was evaluated by ANOVA, followed by Student-Newman-Keuls multiple comparison test. Hierarchical cluster analysis (28) of the CD3⁺ CD8⁺ T cell differentiation phenotype was conducted using J-Express Pro software (www.Molmine.com). Data were hierarchically clustered by tissue (TILN or TFLN) and phenotype using a complete linkage as clustering method and the Pearson correlation as the similarity measure.

CD3⁺ CD8⁺ T cells from TILN of 142 AJCC stage III melanoma patients were assessed for the expression of markers of differentiation. In addition, from 42 of such patients, a tissue sample of a TFLN, removed from the same nodal basin as the TILN, was obtained and analyzed. Hierarchical cluster analysis was performed to obtain a comprehensive classification of the CD8⁺ T cell phenotypic profiles of TILN and TFLN on the basis of the eight subsets defined by CCR7 vs CD45RA, and by CCR7 vs perforin. Four major CD8⁺ T cell phenotypic clusters were identified (Fig. 1). CD8⁺ T cells from all TFLN and 56% of the TILN samples fell into cluster 1, characterized by frequent CCR7⁺ CD45RA⁺ (T_N) and CCR7⁺ CD45RA⁻ (T_CM) phenotypes or even CCR7⁻ CD45RA⁻ (T_EM) cells, but lacking perforin in most instances or expressing it at very low levels and only in TILN samples, not in TFLN (Fig. 1). Increasing expression of perforin in the CCR7⁻ subset characterized CD8⁺ T cells in clusters 2, 3, and 4, although not all CCR7⁻ cells were perforin⁺, at either the T_EM or T_EMRA stage (see CCR7⁻/perforin⁻ column in Fig. 1). These three clusters contained only T cells from TILN and showed a progressive increase in the proportion of CD8⁺ T cells at the CCR7⁻ CD45RA⁻ (T_EM) and CCR7⁻ CD45RA⁺ (T_EMRA, or terminally differentiated) (7, 11) stages (Fig. 1). Significant differences in the differentiation profile between TILN and TFLN were confirmed by the Student-Newman-Keuls multiple comparison test for seven of eight phenotypic subsets defined by CCR7 vs CD45RA and CCR7 vs perforin (see Fig. 1). In agreement with these results, analysis of matched TILN and TFLN samples from the same patient indicated, in several instances, a shift of CD8⁺ T cells from TILN toward the CCR7⁻ CD45RA⁻ T_EM stage, with expression of perforin in the CCR7⁻ fraction, compared with the matched TFLN (representative results from seven patients shown in Fig. 2). Such differences between matched TILN and TFLN pairs were less pronounced when the TILN were isolated from patients in cluster 1, as in this instance both TILN and TFLN showed the early stages of differentiation (data not shown). Further evidence for differentiation of CD3⁺ CD8⁺ T cells, from a fraction of TILN, to cytotoxic factor⁺ stage was obtained by comparing CCR7 vs perforin and CCR7 vs granzyme B phenotypes. This analysis indicated that both cytotoxic factors were expressed in the CD8⁺ CCR7⁻ T cells of TILN from 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 cell differentiation phenotype was performed in several patients belonging to the maturation clusters 1–4, but no relationship was found between PBL and TILN phenotypes (data not shown). Taken together these results indicate accumulation of CD8⁺ T cells at CCR7⁻ cytotoxic factor⁺ stages in a large fraction of TILN, but not in TFLN, from metastatic melanoma patients.

Lymphocytes from TILN in the panel of stage III patients were evaluated for CD69 up-regulation and cytokine secretion in response to PMA and ionomycin. All four CCR7/CD45RA subsets of CD8⁺ T lymphocytes from TILN (Fig. 4,A) responded similarly in terms of CD69 up-regulation (Fig. 4B). In contrast, the CCR7⁺CD45RA⁺ T_N subset did not produce IFN-γ in response to PMA plus ionomycin (Fig. 4C), while this cytokine was mainly produced by CD8⁺ cells at the T_CM and T_EM stages (CCR7⁺CD45RA⁻ and CCR7⁻ CD45RA⁻) and to a lesser extent by T_EMRA cells (CCR7⁻ CD45RA⁺ subset; Fig. 4,C). Furthermore, the proliferative response of CD8⁺ T cells from TILN to immobilized anti-CD3 mAb, as evaluated by CFSE staining, was found mainly in the CCR7⁺ CD45RA⁺ subset and to a lesser extent in the CCR7⁺ CD45RA⁻ subset (Fig. 4 D). These data indicate that the TILN CD8⁺ T cell differentiation subsets defined by CCR7 vs CD45RA expression are associated with distinct functional stages.

The CCR7 vs CD45RA profile of melanoma-Ag-specific T cells recognized by HLA-A*0201 tetramers was investigated in HLA-A*0201 patients found in the panel of 142 TILN from stage III patients. The differentiation phenotype of tetramer⁺ T cells directed to melanocyte lineage Ags (Melan-A/Mart-1_26–35 and gp100_209–217), tumor-restricted epitopes (Mage-3_271–279 and NY-ESO-1_157–165), or influenza matrix_58–66 peptide, from TILN in cluster 1 (data from two representative patients in cluster 1 are shown in the upper two rows of panels in Fig. 5) indicated a predominant CCR7⁺ CD45RA⁺, T_N phenotype, in agreement with the frequent profile of the bulk CD8⁺ T cell population. Evidence for progressive differentiation of tetramer⁺ T cells was obtained in TILN samples from patients in cluster 2, 3, and 4 (lower three rows of panels in Fig. 5). This was indicated by the increasing proportion of tetramer⁺ T cells expressing a T_CM phenotype (as in the representative patient from cluster 2, Fig. 5) or expressing the T_EM or T_EMRA phenotype (see representative patients from clusters 3 and 4, respectively; Fig. 5). Interestingly, the pattern of differentiation of tetramer⁺ T cells directed to influenza matrix peptide was similar in each phenotype cluster to the phenotype of T cells directed to the four tumor Ags (Fig. 5). Analysis of TFLN samples from HLA-A*0201⁺ patients for the CCR7 vs CD45RA phenotype of CD8⁺ T cells directed to melanoma and influenza epitopes indicated a predominant T_N or T_CM phenotype, without expression of perforin (data not shown). Taken together, these data suggest that melanoma-specific and viral Ag-specific T cells from the same TILN or TFLN tissue sample may share a similar differentiation profile, in agreement with analysis at the bulk CD8⁺ T cell level.

The CD8⁺ T cell maturation profile of a large fraction of TILN samples in cluster 1 indicated a prevalence of CCR7⁺ CD45RA⁺, T_N, or CCR7⁺ CD45RA⁻, T_CM cells, lacking cytotoxic factors. To evaluate whether CD8⁺ T cells from cluster 1 could be induced through the differentiation process, CCR7⁺ T lymphocytes were sorted from TILN, labeled with CFSE, and then cultured with γc cytokines that may play a role in T cell differentiation (10, 11, 29). IL-2 and IL-15 induced a proliferative response in the sorted CCR7⁺ CD8⁺ T cells, while the response to IL-7 was minimal (Fig. 6,A). Furthermore, while nonproliferating cells remained CCR7⁺, this marker was down-modulated in most CD8⁺ T lymphocytes that could proliferate to IL-2 and IL-15 (Fig. 6,A). Sorted CCR7⁺ CD8⁺ T cells from PBL of healthy donors showed a similar response (Fig. 6,B). In contrast to IL-2 and IL-15, the sorted CCR7⁺ CD8⁺ T cells from TILN did not down-modulate CCR7 when proliferating to immobilized anti-CD3 mAb (Fig. 6,A). Analysis of CD45RA expression of the sorted CCR7⁺ CD8⁺ T cells from TILN confirmed the presence of two subsets, CD45RA⁺ (T_N) and CD45RA⁻ (T_CM; Fig. 6,C). In the CD8⁺ subset, proliferation to IL-2 or IL-15 was observed mainly in the CD45RA⁻ fraction and to a lesser extent in the CD45RA⁺ fraction (Fig. 6,C). By contrast, in the CD4⁺ subset the response to γc cytokines was mainly found in the CD45RA⁻ subset (Fig. 6,C). By gating for CCR7 and CD45RA on CFSE-labeled lymphocytes, the CD8⁺ T cells that proliferated to γc cytokines expressed, at the end of culture (day 7), either a CCR7⁻ CD45RA⁻ (T_EM) or a CCR7⁻CD45RA⁺ (T_EMRA) phenotype (data not shown). Further evidence for cytokine-induced CD8⁺ T cell differentiation was obtained in TILN from cluster 1 by looking at the expression of cytotoxic factors. Freshly isolated CD8⁺ T cells were mostly CCR7⁺ perforin⁻ (Fig. 7). In contrast, after culture with IL-2, IL-15, or IL-2 plus IL-15, the CD3⁺ CD8⁺ lymphocytes expressed a predominant CCR7⁻ perforin⁺ phenotype (Fig. 7). Culture with IL-7 led to up-regulation of perforin in a fraction of cells, but most of them remained CCR7⁺ (Fig. 7). Taken together these data indicate that differentiation of CCR7⁺ CD8⁺ T cells from TILN can be promoted in vitro by γc cytokines such as IL-2 and IL-15. This process involves mainly the proliferation of CCR7⁺ CD45RA⁻ T_CM cells and, to a lesser extent, of CCR7⁺ CD45RA⁺ T_N cells, and leads to differentiation to CCR7⁻ perforin⁺ stage.

The impact of γc cytokines on T cell phenotype and function was evaluated in Melan-A/Mart-1- or gp100-specific T cells in TILN from HLA-A*0201⁺ patients in cluster 1. In these patients, freshly isolated tetramer⁺ T cells expressed mostly a CCR7⁺ CD45RA⁺ phenotype (Fig. 8), a phenotype that did not change by culture in medium without cytokines (data not shown). In contrast, after culture 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% of the cells (Fig. 8) or even a predominant CCR7⁻ CD45RA⁻ phenotype in some patients (data not shown).

At the functional level, intracellular expression of IFN-γ in response to peptide-loaded APCs was observed by Melan-A/Mart-1-specific T cells after culture of lymphocytes from TILN with IL-2 and IL-15, but not with IL-7 or in freshly isolated cells (Fig. 9,A). Similar results were obtained with gp100-specific T cells from TILN (see Fig. 9,B for results on IL-2-cultured lymphocytes). However, while most of the tetramer⁺ T cells produced IFN-γ when stimulated with PMA plus ionomycin (Fig. 9,B), only a fraction of the cytokine-cultured tetramer⁺ T cells became positive for intracellular IFN-γ in response to peptide-loaded APCs (Fig. 9, A and B). This was observed even in instances where most of the Ag-specific T lymphocytes were at the CCR7⁻ stage after cytokine culture (Fig. 9 B), suggesting heterogeneity for Ag-specific IFN-γ production. Similar results, indicating production of IFN-γ in response to peptide-loaded APCs after cytokine culture, were obtained even with flu matrix-specific T cells from TILN (data not shown).

Lymphocytes from TILN in cluster 1, after culture for 7 days with γc cytokines, were assessed for Ag-specific cytotoxicity. After culture with IL-2, IL-15, or IL-2 plus IL-15, the T cell cultures from TILN exhibited HLA-A2-restricted lysis of HLA-A*0201⁺, Melan-A/Mart-1⁺, gp100⁺ autologous melanoma (Fig. 10,A) and lysed APCs loaded with Melan-A/Mart-1 peptide and, to a lesser extent, APCs loaded with gp100_209–217 peptide (Fig. 10, C–E). By contrast, no lytic activity on melanoma cells (Fig. 10,A), or on peptide-loaded APCs (Fig. 10 B) was exerted by freshly isolated T cells from TILN. Taken together these results suggest that γc cytokines, such as IL-2 and IL-15, can promote in-vitro Ag-independent differentiation of melanoma-specific T cells from TILN.

Comparison of TILN and TFLN from melanoma patients by hierarchical cluster analysis provided evidence for a marked shift of CD8⁺ T cell differentiation toward CCR7⁻ cytotoxic factor⁺ stages at the tumor site in a relevant fraction of tissue samples. In fact, in three of four T cell phenotype clusters, representing ∼44% of the TILN samples, we found that the differentiation profile of the CD8⁺ T cell fraction was gradually shifted toward an increasing content of CCR7⁻ CD45RA^−/+ cells containing the cytotoxic factors perforin and granzyme B. Thus, in these clusters, the CD8⁺ T cell differentiation was consistent with a progressive shift toward the T_EM and T_EMRA/terminally differentiated stages. The remaining TILN samples and all TFLN defined a single cluster in which CD8⁺ T cell differentiation was mainly characterized by CCR7⁺ CD45RA⁺, T_N and CCR7⁺ CD45RA⁻, T_CM stages. Moreover, most TILN and TFLN in this cluster lacked perforin or expressed it at very low levels and only in some TILN samples. The distinct CD8⁺ T cell subsets found in TILN and defined by intracellular and extracellular markers reflected distinct functional stages, as indicated by CFSE analysis for proliferative response to immobilized anti-CD3 mAb and by IFN-γ release in response to PMA plus ionomycin. Taken together these results suggest that tumor-invaded, but not tumor-free, lymph nodes of a relevant fraction of stage III melanoma patients are involved in a process of CD8⁺ T cell differentiation beyond T_N, T_CM, and cytotoxic factor⁻ stages.

Analysis of T cell maturation stages in melanoma-specific T cells from TILN indicated that the process of T cell differentiation could involve even tumor-specific T lymphocytes directed to melanocyte-lineage Ags or to tumor-restricted epitopes, as indicated by the phenotype of tetramer⁺ T cells from TILN of clusters 2–4. This may indicate a role of anti-tumor immunity in shaping the CD8⁺ T cell differentiation profile at the tumor site. However, interestingly, maturation along the T_CM→T_EM→T_EMRA pathway was documented in the same TILN even for T cells directed to a viral epitope (influenza matrix). Such an effect may be promoted through Ag-independent bystander mechanisms (30, 31), and tumor-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 melanoma (32). Moreover, other factors, such as IL-6 and IL-10, are frequently expressed by melanoma cells (33, 34). These factors could exert an indirect effect on T cell differentiation through their activity on the expression of IL-2/IL-15Rβ and γ-chains on T cells (10, 11), thus promoting the response of CD8⁺ T lymphocytes to soluble regulators of differentiation.

Culture of CCR7⁺ CD8⁺ lymphocytes from TILN of phenotypic cluster 1 in the presence of γc cytokines IL-2 and/or IL-15 promoted differentiation of a fraction of lymphocytes to the CCR7⁻ perforin⁺ stage. This was associated with acquisition of Ag-specific effector functions, such as cytokine release and cytotoxicity. This cytokine-induced differentiation was restricted to a fraction of CD8⁺ lymphocytes that could proliferate in response to IL-2 and IL-15, suggesting differential expression of functional cytokine receptors in CCR7⁺ CD8⁺ T cells. Indeed, recent evidence indicates that IL-15Rα and IL-2/IL-15Rβ-chain expression is low, although detectable, in CCR7⁺ CD45RA⁺ T_N cells and increases progressively along the T_N→T_CM→T_EM differentiation pathway (10, 11), while γc is similarly expressed in the three stages (11). In agreement, analysis of the expression of differentiation markers indicated that the response of CCR7⁺ CD8⁺ T cells to γc cytokines was due mainly to proliferation of CD45RA⁻ cells (i.e., T_CM), with some response even in the CD45RA⁺ fraction (i.e., T_N), and led to down-modulation of CCR7. Down-modulation of CCR7 after culture with γc cytokines is in agreement with the findings in the CD4⁺ subset (10), where the proliferative response has been shown to lead T_CM (i.e., CCR7⁺ CD45RA⁻) cells to down-modulate CCR7 and acquire effector functions, such as cytokine release, associated with the CCR7⁻ CD45RA⁻ T_EM stage. In the CD8⁺ subset, Geginat et al. (11) found that upon culture of PBL with IL-7 plus IL-15, T_N cells maintained their phenotype, while the T_CM fraction gave rise to cells expressing CCR7 and CD45RA in all possible combinations, including T_EM and T_EMRA. These latter phenotypes were the two profiles that we found expressed at the end of culture by CD8⁺ T cells that proliferated to IL-2 or IL-15, used as single cytokines. In addition, a direct comparison of IL-15 alone vs IL-7 plus IL-15 has not been performed, but it remains possible that CD8⁺ T cell maturation may be affected differently by culture with single γc cytokines, performed in our study, vs the combination of γc cytokines. Furthermore, we observed down-modulation of CCR7 in the proliferative response of CD8⁺ T cells to γc cytokines, but not in the response to immobilized anti-CD3 mAb. Interestingly, down-modulation of CCR7 has been described in T_N (naive) cells upon stimulation with immobilized anti-CD3 plus anti-CD28 (10), suggesting that T cell proliferation can be coupled, or not, to differentiation depending on the specificity of signals (i.e., TCR triggering vs TCR triggering plus costimulation).

The Ag-independent maturation of a fraction of CD8⁺ T cells from TILN suggests that γc cytokines, such as IL-2 and IL-15, may be exploited for promoting the development of antitumor effectors in cancer patients for immunotherapy purposes (35). This goal appears relevant in the light of the frequent identification of anti-tumor CD8⁺ T cells at early stages of differentiation at the tumor site. Thus, the available evidence suggests that a large fraction of melanoma patients may benefit from strategies aimed at promoting functional maturation of their antitumor T cells. In principle, Ag-independent T cell differentiation to the effector stage could reduce the need for targeting specific tumor Ags, thus avoiding the risk of tumor resistance due to Ag loss variants. In addition, T cells from TILN may respond to γc cytokines mainly on the basis of their differentiation stage, which controls the expression of functional cytokine receptors, and independently from their Ag specificity. If antitumor T cells can be found at the tumor site, although in early stages of maturation, then cytokine-mediated differentiation would provide a way of activating T cells directed against a wide spectrum of tumor Ags. Thus, cytokine-induced differentiation of TILN populations, enriched for antitumor T cells, may represent a potential application for improving protocols of immunotherapy for cancer.

We are grateful to the melanoma patients for their generous participation in this study.