Migration of cytotoxic T lymphocytes toward melanoma cells in three-dimensional organotypic culture is dependent on CCL2 and CCR4

Authors


Abstract

Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor-positive T lymphocytes into the tumor area. However, in cancer patients, the role of chemokines in T lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the migration of a melanoma patient's CTL toward autologous tumor cells has been studied in a novel three-dimensional organotypic melanoma culture. In this model, CTL migrated toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by the CC chemokine receptor (CCR)4 expressed by the CTL and the CC chemokine ligand (CCL)2 secreted by the tumor cells, as evidenced by blockage of CTL migration by CCL2 or antibodies to CCL2 or CCR4. These results were confirmed in a Transwell migration assay in which the CTL actively migrated toward isolated CCL2 and migration was inhibited by anti-CCR4 antibody. These studies, together with previous studies in mice indicating regression of CCL2-transduced tumor cells, suggest that CCL2 may be useful as an immunotherapeutic agent for cancer patients.

Abbreviations:
CCL:

CC chemokine ligand

CCR:

CC chemokine receptor

CXCL:

CXC chemokine ligand

CXCR:

CXC chemokine receptor

LAK:

lymphokine-activated killer

MLTC:

mixed lymphocyte tumor cell culture

PHA:

phytohemagglutinin

Introduction

Chemokines produced by tumor cells have been demonstrated to attract chemokine receptor-positive T lymphocytes into the tumor area, potentially leading to tumor growth inhibition in vitro and in vivo13. These studies have been largely confined to mouse tumor systems, and the role of chemokines in T lymphocyte trafficking toward tumor cells derived from patients is relatively unexplored. In mouse systems, ex vivo transduction of chemokines into tumor cells has provided potent tumor vaccines inducing tumor rejection, which was mediated by infiltrating T cells at the vaccine site 1. Chemokines may be fused to tumor Ag-specific Ab to attract adoptively transferred or endogenous T cells to the tumor site, or incorporated into Ag vaccines to attract T cells to the vaccine site, which has been shown to lead to tumor destruction in mouse models 4, 5.

To develop immunotherapeutic strategies for cancer patients based on chemokines and their receptors, similar to the approaches already successfully used in mice (see above), we must identify chemokines and their receptors involved in T cell migration toward tumor cells derived from patients. Such information, to our knowledge, is not available.

Migration of T cells is usually studied in a chemotaxis assay using Transwell plates. In this chemotaxis system, T cells migrate from the upper culture chamber through a polycarbonate or nitrocellulose membrane to the lower chamber containing the chemoattractant. This culture system has several disadvantages: (i) tumor cells are missing from the culture system; (ii) artificial high chemokine concentrations are often used that do not reflect the concentrations produced by tumor cells; (iii) the stromal components important for T cell migration and activation are missing; (iv) T cells migrate only a short distance (approximately 10 μm) through non-physiological membranes (nitrocellulose or polycarbonate); and (v) study of the migration process is limited to a period of only a few hours.

We have developed a novel three-dimensional organotypic melanoma culture system (referred to hereafter as reconstruct), which allows the study of chemokines and their receptors involved in T cell migration under in vivo-like conditions. This model consists of a bottom layer of collagen type I with fibroblasts, superimposed by a tumor cell layer, collagen/fibroblast separating layer and finally a top layer of collagen, fibroblasts and T cells, and has the following advantages over the Transwell plate cultures: (i) the cultures include growing tumor cells; (ii) chemokines are produced by the growing tumor cells in physiological concentrations; (iii) collagen and fibroblasts provide stromal components important for T cell migration and activation 6; (iv) the model allows determination of T cell migration over 6–9 days; and (v) T cells migrate through a distance of at least 500 μm. Thus, the reconstruct has many advantages over the traditionally used Transwell culture system as it mimics the in vivo conditions that play a role in T cell migration.

We show here that CTL migrate toward autologous melanoma cells in the reconstruct, resulting in tumor cell apoptosis, and that migration is mediated by the CC chemokine ligand (CCL)2 produced by the tumor cells and the CC chemokine receptor (CCR)4 expressed by the T cells.

Results

Functional characteristics of CTL MLTC35 in mixed lymphocyte tumor cell culture (MLTC)

CTL MLTC35 lysed autologous WM35 melanoma cells in a 6-h 51Cr-release assay and lysis was dependent on E/T cell ratio. K562 NK target cells and Daudi lymphokine-activated killer (LAK) target cells were not lysed in the same assay (Fig. 1A). The CTL were predominantly of CD8 phenotype (Fig. 1B) and lysis of melanoma cells by the CD8+ CTL may have been mediated by granzyme A (Fig. 1C). Melanoma cell lysis was inhibited by mAb to HLA class I, as well as an mAb with dual binding specificity for HLA-A2/B17 (57/58) (Table 1). Since patient 35 was HLA-A2 positive, but HLA-B17 negative, CTL were most likely HLA-A2 restricted. Proliferation of CTL MLTC35 was dependent on the presence of tumor cells (Fig. 1D), and proliferating lymphocytes secreted both IFN-γ and GM-CSF (Fig. 1E), but not TNF-α and IL-4.

Figure 1.

Immunological functions of CTL MLTC35. CTL MLTC35 was generated as described in the Materials and methods. (A) CTL responses. CTL MLTC35 were stimulated for 3–4 days with irradiated autologous WM35 melanoma cells and IL-2. CTL lysis of 51Cr-labeled autologous melanoma cells, and Daudi and K562 control target cells was determined in 6-h 51Cr-release assay at various E/T ratios. Data represent means ± SD of specific lysis derived from three independent experiments. Each experiment included three wells per data point. Expression of CD8 (B) and granzyme (C) by CTL. CTL MLTC35 were stained with PE-conjugated mouse anti-human CD8 mAb (solid line) or mouse IgG1 isotype control (dotted line) (B), or FITC-conjugated mouse anti-human granzyme A mAb (solid line) or mouse IgG1 isotype control (dotted line) (C) and analyzed by flow cytometry. (D) Proliferative CTL responses. CTL MLTC35 were incubated with or without irradiated WM35 melanoma cells for 4 days in the presence of IL-2. Proliferative responses of CTL were determined in [3H]thymidine incorporation assay. One of four experiments is presented in (D). Among the four experiments the ratio of proliferation in cultures with CTL MLTC35 + WM35 compared to WM35 ranged from 2.7 to 29, and compared to CTL MLTC35 ranged from 1.8 to 5.2; all comparisons were statistically significant (p<0.01). (E) Cytokine production. IFN-γ (left panel) and GM-CSF (right panel) in supernatants obtained from cultured CTL after 2 days were measured in ELISA. Values (D and E) are means ± SD (error bars) of triplicate determinations. Differences between experimental values (CTL MLTC35 + WM35) and control values (CTL MLTC35, WM35) were analyzed for significance by Student's two-sample t-test. One of two experiments is presented in (E). Among the two experiments the ratio of proliferation in cultures with CTL MLTC35 + WM35 compared to WM35 ranged from 14.7 to 52.1, and compared to CTL MLTC35 ranged from 2.0 to 22.4; all comparisons were statistically significant (p<0.05).

Table 1. Blocking of cytotoxicity of CTL MLTC35 against WM35 melanoma cells by mAb
Antibodya)
  1. a) Tumor targets were incubated with Ab (20–50 μg/mL) for 1 h at room temperature and excess Ab was removed before addition of effector cells in a 6-h 51Cr-release assay.

  2. b) Means of triplicate wells at E/T ratio of 5.

  3. c) NA = not applicable.

  4. d) Values are significantly (p<0.05 or less, Student's two-sample t-test) different from the values obtained with cultures that received control mouse IgG. Experiment was repeated once with similar results using a different E/T ratio.

DesignationSpecificityIsotype% Lysisb)% Inhibition
Mouse IgGNAc)IgG17.2 ± 1.8-
W6/32HLA class IIgG2a7.7 ± 2.5d)57.2
MA2.1HLA-A2/B17IgG17.8 ± 2.8d)65.2
B33.1HLA class IIIgG2a14.2 ± 0.517.3

Functional characteristics of CTL MLTC35 in the reconstruct

A schematic presentation of the four reconstruct layers, each consisting of cells embedded in collagen is shown in Fig. 2A. CTL MLTC35 labeled with carboxyl-fluorescein diacetate-Green migrated from the top layer of collagen and fibroblasts through a separating layer (about 500 μm) of collagen and fibroblasts toward WM35 tumor cells labeled with 7-amino-4-chloromethylcoumarin-Blue (Fig. 2Bi), whereas no T cells were found in control cultures with tumor cells only (Fig. 2Bii). The CTL induced massive apoptosis in the autologous melanoma cells (Fig. 2 Ci), as compared to reconstructs with tumor cells and phytohemagglutinin (PHA) blasts (Fig. 2Cii) or tumor cells alone (Fig. 2Ciii). CTL values were significantly (p<0.0001, two-sample t-test) higher than either of the two control values (Table 2).

Figure 2.

CTL MLTC35 migration toward autologous WM35 melanoma cells in reconstruct. (A) Reconstruct schema. (B) CTL MLTC35 migration. Reconstructs contained WM35 melanoma cells stained with CellTracker Blue 7-amino-4-chloromethylcoumarin, and carboxyl-fluorescein diacetate Green-labeled CTL MLTC35 (i). Control wells contained no lymphocytes (ii). Reconstructs were harvested on day 4, fixed in buffered formalin and embedded in paraffin. Sections were photographed in the Nikon fluorescence microscope using appropriate filters. The number of green-labeled T cells that migrated toward tumor cells is indicated in the lower left corner in (i) and (ii). These values represent means ± SD per field (ten fields were counted). The value in (i) is significantly (p<0.001) higher than the value in (ii). (C) Induction of apoptosis in WM35 cells by CTL MLTC35. Reconstructs were harvested on day 6, fixed in buffered formalin, embedded in paraffin and stained by hematoxylin and eosin (HE). Magnification ×400. The percentage of apoptotic tumor cells was determined by counting apoptotic nuclei and intact tumor cells in 10 fields of sections stained with HE.

Table 2. CTL MLTC35 induce apoptosis of melanoma cells in reconstructa)
LymphocytesTotal number of tumor cellsmean ± SD/field(10 fields from duplicate wells)Number of apoptotic tumor cellsmean ± SD/field(10 fields from duplicate wells)Percentage of apoptotic tumor cellsmean ± SD/field(10 fields from duplicate wells)
  1. a) For generation of reconstructs, see Fig. 2 legend.

  2. b), c) Values with the same symbol differ significantly (p<0.0001, Student's two-sample t-test) from each other. Experiment was repeated twice with similar results (see Table 5 and 6).

CTL MLTC3541.9 ± 10.219.3 ± 6.047.4 ± 12.0b), c)
PHA blast 3538.1 ± 14.23.0 ± 1.96.3 ± 2.7b)
None47.8 ± 17.81.7 ± 1.03.9 ± 2.0c)

Phenotypic characteristics of CTL MLTC35 and WM35 melanoma cells

CTL MLTC35 and WM35 melanoma cells were phenotyped with special emphasis on molecules that might be involved in the interactions of these cells with each other and components of the reconstruct (Table 3). CTL MLTC35 (90.0% CD8+ positive; Fig. 1B and Table 3) expressed α2 and β1 integrins (Table 3), which are important for T cell activation by collagen in the reconstruct 7, 8. LFA-1a, ICAM-1, and CD44 expressed by the CTL (Table 3) facilitate interaction of the lymphocytes with fibroblasts in the reconstruct 9.

Table 3. Phenotypic and functional markers of CTL MLTC35 and WM35 melanoma cells
Parameter investigateda)WM35CTL MLTC35
  1. a) All markers were determined by FACS analysis.

  2. b) MFI: mean fluorescence intensity.

  3. c) NA: not applicable.

% cells positiveMFIb)% cells positiveMFI
HLA class I99.015.598.054.2
HLA class II75.05.995.317.5
CD4NAc)NA6.89.15
CD8NANA90.031.9
CD25NANA48.94.2
CD401.03.111.73.7
CD40L8.03.39.73.9
CD44100.0368.097.417.5
CD95 (FAS)4.03.163.72.0
CD95L (FASL)4.211.29.40.5
CD80 (B7–1)2.03.480.616.9
CD86 (B7–2)1.04.222.93.6
CD54 (ICAM-1)100.077.437.14.8
CD11a (LFA-1a)4.03.389.56.7
CD49a (α1 integrin)70.13.854.59.8
CD49b (α2 integrin)97.519.259.38.8
CD29 (β1 integrin)87.09.364.64.4
CD61 (β3 integrin)89.04.621.53.6

WM35 melanoma cells expressed both HLA class I and II molecules, ICAM-1, and various integrins. The cells expressed very low amounts of FAS, FAS ligand, B7–1 and B7–2 (Table 3). Expression of α2 and β1 integrins by the melanoma cells (Table 3) facilitates migration of the cells through collagen 10. ICAM-1 on the melanoma cells interacts with LFA-1a on the CTL, resulting in T cell stimulation 11.

Chemokine and chemokine receptor involved in CTL MLTC35 migration toward WM35 cells

We evaluated a possible role of CCR4 expressed by CTL MLTC35 cells and CCL2, CCL3, CCL5 and CCL17 produced by WM35 cells (Table 4; Fig. 3) in the migration of the T cells toward melanoma cells in the reconstruct. The concentrations of the various chemokines in supernatant of WM35 melanoma cells were 2,396 pg/mL (CCL2), 55.2 pg/mL (CCL3), 71.0 pg/mL (CCL5), and less than 10.0 pg/mL (CCL17). T cell migration was measured as a function of tumor cell apoptosis. Mouse anti-CCL2 mAb, rabbit anti-CCR4 Ab and CCL2 significantly (p<0.001) inhibited tumor cell apoptosis (Table 5, 6). Thus, CTL migration toward tumor cells is induced by tumor-derived CCL2 chemokine, which binds to CCR4 on the CTL.

Table 4. Chemokine receptors expressed by CTL MLTC35, and chemokines produced by WM35
Chemokine receptors expressed by CTL MLTC35a)Chemokines known to bind to receptorChemokine expressed by WM35b)
  1. a) Chemokine receptor was detected by FACS. The following receptors were not expressed by CTL MLTC35: CCR1, CCR2, CCR3, CCR5, CCR6, CCR8, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR5, CXCR6 and CX3CR1.

  2. b) Chemokine expression was detected by RT-PCR (CCL17, CCL19, CCL22, CCL25 and CXCL12) or ELISA (CCL2, CCL3, CCL5, CCL21 and CXCL12).

CCR4

CCL2 (MCP-1)

CCL3 (MIP-1α)

CCL5 (RANTES)

CCL17 (TARC)

CCL22 (MDC)

+

+

+

+

CCR7

CCL21 (6Ckine)

CCL19 (MIP-3β)

CCR9CCL25 (TECK)
CXCR4CXCL12 (SDF-1α)
Figure 3.

Fig. 3. Autologous WM35 melanoma cells produce CCL2 and CTL MLTC35 express the corresponding receptor CCR4. (A) CCL2 in supernatants obtained from various cells (WM35 melanoma cells, FF2441 fibroblasts, and CTL MLTC35) was measured in ELISA. Values are means ± SD of triplicate wells. (B) Expression of CCR4. Cultured cells were incubated with saturating concentration (5 μg/mL) of rabbit anti-CCR4 Ab (solid line) or rabbit IgG control (dotted line) in RPMI 1640 with 5% human AB serum for 1 h at 4°C. After washing, PE-labeled monkey anti-rabbit IgG was added. Expression of CCR4 was detected by flow cytometry.

Table 5. Blocking of CTL MLTC35 migration by anti-chemokine receptor Ab in reconstruct
Treatmenta)

Total number of tumor cells

Mean ± SD/field

(10 fields from duplicate wells)

Number of apoptotic tumor cells

Mean ± SD/field

(10 fields from duplicate wells)

Percent apoptotic tumor cells

Mean ± SD/field

(10 fields from duplicate wells)

  1. a) Reconstructs consisted of a bottom layer of collagen and fibroblasts, followed by a tumor cell layer and a separating layer of collagen and fibroblasts. Anti-chemokine, anti-chemokine receptor or control Abs were added at 10 μg/mL, followed by a top layer containing CTL mixed with fibroblasts and collagen (E/T=1:1). Percentage of apoptotic tumor cells in 4-day cultures was determined as described in Materials and methods.

  2. b) Data from similar experiments are presented in Table 2 and 6.

  3. c), d) Values with the same letter differ significantly from each other (p<0.001, Student's two-sample t-test).

WM35b)49.9 ± 5.44.8 ± 1.79.5 ± 2.7
WM35 + CTL MLTC35b)39.3 ± 9.38.5 ± 2.221.9 ± 4.6
WM35 + CTL MLTC35 + control rabbit IgG40.4 ± 7.08.5 ± 2.121.1 ± 4.4c)
WM35 + CTL MLTC35 + anti-CCR4 Ab (rabbit)45.6 ± 10.54.3 ± 2.19.2 ± 3.2c)
WM35 + CTL MLTC35 + control mouse IgG 35.9 ± 6.47.9 ± 2.421.9 ± 5.1d)
WM35 + CTL MLTC35 + anti-CCL2 mAb (mouse)47.6 ± 7.34.9 ± 1.110.3 ± 1.9d)
WM35 + CTL MLTC35 + anti-CCL3 mAb (mouse)43.9 ± 6.58.3 ± 3.218.7 ± 5.7
WM35 + CTL MLTC35 + anti-CCL17 mAb (mouse)39.8 ± 4.87.7 ± 1.819.3 ± 3.5
WM35 + CTL MLTC35 + anti-CCL5 mAb (mouse)41.6 ± 6.68.0 ± 1.619.4 ± 3.9
Table 6. Blocking of CTL MLTC35 migration by chemokine in reconstruct
Treatmenta)

Total number of tumor cells

Mean ± SD/field

(10 fields from duplicate wells)

Number of apoptotic tumor cells

Mean ± SD/field

(10 fields from duplicate wells)

Percent apoptotic tumor cells

Mean ± SD/field

(10 fields from duplicate wells)

  1. a) Reconstructs consisted of a bottom layer of collagen and fibroblasts, followed by a tumor cell layer and a separating layer of collagen and fibroblasts, followed by a top layer containing CTL mixed with fibroblasts and collagen (E/T=1:1). Separate cultures received CCL2 (250 ng/mL) on top of the T cell layer. Percentage of apoptotic tumor cells in 4-day cultures was determined as described in Materials and methods.

  2. b) Data from similar experiments are presented in Table 2 and 5.

  3. c) Values with the same letter differ significantly from each other (p<0.001, Student's two-sample t-test).

WM35b)39.0 ± 9.09.5 ± 2.524.5 ± 3.0
WM35 + CTL MLTC35b)41.8 ± 7.623.5 ± 4.756.3 ± 4.9c)
WM35 + CTL MLTC35 + CCL239.5 ± 5.413.3 ± 3.433.3 ± 5.3c)

To evaluate whether fibroblasts, through CCL2 production (Fig. 3A), play a role in CTL MLTC35 migration toward WM35 melanoma cells, migration of the T cells and subsequent tumor cell apoptosis was tested in the absence of fibroblasts. CTL MLTC35 induced significant (p<0.001) apoptosis in WM35 tumor cells in the absence of fibroblasts (Table 7). Thus, the CTL migration toward tumor cells is induced by tumor-derived CCL2 chemokine and not by fibroblast-derived chemokine.

Table 7. CTL MLTC35 induce apoptosis of WM35 melanoma cells in reconstruct without FF2441 fibroblastsa)
Cells

Total number of tumor cells

Mean ± SD/field

(10 fields from duplicate wells)

Number of apoptotic tumor cells

Mean ± SD/field

(10 fields from duplicate wells)

Percent apoptotic tumor cells

Mean ± SD/field

(10 fields from duplicate wells)

  1. a) Reconstructs consisted of a bottom layer of collagen, followed by a tumor cell layer and a separating layer of collagen. The top layer contained CTL mixed with collagen (E/T=1:1). Percentage of apoptotic tumor cells in 4-day cultures was determined as described in Materials and methods.

  2. b) Values with same letter differ significantly from each other (p<0.001, Student's two-sample t-test).

WM3528.3 ± 3.71.2 ± 0.94.1 ± 2.7b)
WM35 + CTL MLTC3525.2 ± 3.29.8 ± 2.638.7 ± 8.5b)

Induction of CCR4-positive CTL MLTC35 migration by recombinant CCL2 in Transwell

Recombinant CCL2 and supernatant from WM35 melanoma cell culture induced a dose-dependent migration of T cells in the Transwell migration assay (Fig. 4A). Treatment of T cells with anti-CCR4 mAb significantly (p<0.05) inhibited the migration of CTL MLTC35 toward recombinant CCL2, whereas anti-CCR7 mAb treatment had no effect (Fig. 4B). We can conclude from these studies that CTL migration toward tumor cells (Fig. 2, Table 5) is induced by tumor-derived CCL2 chemokine. CCL2 also induced CTL MLTC35 migration toward tumor cells in the reconstruct (see Table 6).

Figure 4.

CTL MLTC35 migrate toward CCL2 in Transwell. (A) CTL MLTC35 suspension was placed in the top chamber of the Transwell. CCL2, prepared at the indicated concentrations in T cell medium, or supernatant obtained from WM35 cell culture were added to the bottom of the Transwell (duplicate wells). After 90 min of culture, the number of migrated cells in the bottom chamber was counted under the microscope. (B) As in (A), but T cells were incubated with rabbit anti-CCR4 Ab or anti-CCR7 mAb (Ab control). Results shown in (A) and (B) were obtained in two different experiments.

Discussion

We have shown here that CTL migrate through a 500-μm collagen/fibroblast separating layer toward tumor cells, resulting in tumor cell apoptosis. We have also shown that migration is dependent on CCL2 produced by tumor cells and CCR4 expressed by CTL. To our knowledge, this is the first demonstration of chemokine dependency of human CTL migration toward tumor cells.

The reconstruct is a novel three-dimensional culture system in which the migration of leukocytes toward tumor cells and the factors that influence leukocyte migration can be studied under in vivo-like conditions. In the reconstruct, human melanoma is recapitulated in vitro using a mixture of collagen and fibroblasts (lattices or matrices). Ag-elicited T cells are stimulated by collagen, most likely through the interaction of α2 and β1 integrins on T cells with collagen 8. Since activated fibroblasts play an important role in the activation of T lymphocytes, they were included in the reconstruct. T lymphocytes bind to fibroblasts via LFA-1a, ICAM-1 and CD44. The adhesive interaction stimulates fibroblasts to secrete inflammatory cytokines such as IL-1, IL-6 and IL-7 9, 12, and fibronectin 13. The main biological role of IL-1 is the stimulation of T cells to express IL-2 receptor and secrete IL-2 14. IL-6 and IL-7 are T cell survival factors 15, and fibronectin stimulates predominantly resting lymphocytes 13. However, in our studies, induction of tumor cell apoptosis by CTL was similar in the absence and presence of fibroblasts, suggesting that fibroblasts do not play a significant role in T cell activation and migration. This finding was surprising as fibroblast factors were expected to substitute for the absence of exogenous cytokines in the cultures. Thus, CTL MLTC35 was IL-2 dependent in its growth outside the reconstruct, whereas the CTL migrated toward tumor cells and lysed these cells in the absence of exogenous IL-2 in the reconstruct.

Other investigators have used collagen matrices to study interaction of leukocytes with tumor cells, but they have not demonstrated CTL migration resulting in tumor cell apoptosis in a culture system similar to the reconstruct shown here 1618. In the present study, CCL2 produced by melanoma cells attracted CTL through binding to CCR4 on the T cells. This was demonstrated by blocking CTL migration toward tumor cells with CCL2, anti-CCR4 Ab, or anti-CCL2 Ab. Each of the compounds inhibited CTL migration, and inhibition by the Ab was most likely due to blocking of CCR4 on the CTL.

In preliminary studies, we have delineated the role of chemokine receptor/chemokine in the migration of four additional melanoma- and one colon carcinoma-specific T cell lines or clones toward tumor cells. For three of these T cell lines, different receptor/ligand pairs were involved in T cell migration and induction of tumor apoptosis (CCR2/CCL2; CXCR3/CXCL10; CXCR3/CXCL11; unpublished data). Two T cell lines used CXCR4 to migrate toward CXCL12 produced by melanoma cells 19. These studies suggest that in general, in different patients, different chemokine receptors/chemokines are involved in T cell migration toward tumor cells, followed by induction of tumor cell apoptosis by T cells.

Most melanomas from primary and metastatic lesions produce CCL2 20, 21. CCL2 was identified as a monocyte-specific chemoattractant 22, 23 that was later shown to attract T lymphocytes and NK cells 24, 25.

CCR4, a seven-transmembrane G-protein-coupled receptor, is expressed on monocytes, T cells, B cells, basophils, NK cells, immature DC and adult T cell leukemia 2628. CCR4 binds CCL2, –3, –5, –17 and –22 26, 28. However, other reports have shown that CCL17 and –22, but not CCL2, –3 and –5 are specific functional ligands for CCR4 2931. Thus, CCL2 binding to CCR4, as indicated by our results obtained in the reconstruct (Table 5) and Transwell (Fig. 4) is a controversial issue.

Previous studies have indicated that high levels of CCL2 lead to massive monocyte/macrophage accumulation and tumor destruction 21, and CCL2-transduced tumor cells provide effective vaccines in experimental animals 32. These studies and our studies presented here, suggesting that CCL2 attracts CTL into the tumor area resulting in tumor cell apoptosis, emphasize the potential usefulness of this chemokine in immunotherapeutic treatment of cancer patients, using CCL2-transduced tumor cells, tumor-associated Ag fused to CCL2, or anti-tumor Ab/CCL2 fusion protein 1, 4, 5. However, patients will have to be selected for expression of CCR4 by tumor-reactive T cells. Thus, patients’ treatments may have to be individualized.

In addition to therapeutic implications, the results of our study have prognostic potential. Infiltration of melanoma lesions with T lymphocytes is correlated with a favorable prognosis 33. Expression of CXCR3 and CCR4 on CD8+ T cells correlated with a statistically significant survival advantage in melanoma patients with stage III disease 34, suggesting that expression of these receptors may serve as a biomarker of potential clinical responses to immunotherapy.

Materials and methods

Patient

Melanoma patient 35 and her primary lesion have been described 35.

Cell lines

Melanoma cell line WM35 was maintained in MCDB153-L15 medium (Sigma, St. Louis, MO) supplemented with 2% FBS 35. HLA types of WM35 cells were A2, B18, B51, C2, C1203, DR7, DR1602 and DQb1. Fetal foreskin fibroblast cell line FF2441 was maintained in DMEM (GIBCO-Invitrogen, Carlsbad, CA) supplemented with 10% FBS. NK cell target K562 (human erythroleukemia cell line) and LAK cell target Daudi (human lymphoblastoid cell line) were obtained from American Type Culture Collection (ATCC, Rockville, MD). All lymphoid cell lines were maintained in RPMI 1640 medium (GIBCO-Invitrogen) supplemented with 10% FBS.

Reagents

The following mAb and polyclonal Ab were used: HLA class I-specific mAb W6/32 and HLA class II-specific mAb B33.1 and D1.B6 (obtained from Dr. B. Perussia, Thomas Jefferson University, and Dr. G. Trinchieri, The Wistar Institute); mAb MA2.1 to HLA-A2 and -B17 (57/58) (ATCC); mAb Nok-1 to Fas ligand (PharMingen, Los Angeles, CA); mAb CH-11 to CD95 and anti-CD11a mAb (Immunotech, Westbrook, ME); fluorescein- or phycoerythrin-labeled anti-CD4, –8, –25, –29, –40, –40L, –44, –49a, –49b, –54, –61, –80, and –86, mAb (PharMingen); anti-CCL2, –3, –5 and –17 mAb; anti-human CCR1, –2, –3, –5, –6, –7, –9, and CXCR1, –2, -3, –4, –5 and –6 mAb (R&D Systems, Minneapolis, MN); anti-human CCR4, –8, and –10 mAb (Imgenex, San Diego, CA); anti-CCR11 and -CX3CR1 polyclonal Ab (Abcam, Cambridge, MA); fluorescein-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR); phycoerythrin-labeled monkey anti-rabbit IgG (Immunotech); recombinant human CCL2 (R&D Systems).

Generation of anti-melanoma CTL line

CTL were generated from PBMC in MLTC as described previously 36. Cultures were stimulated weekly with irradiated autologous WM35 melanoma tumor cells in T cell medium 36 containing partially purified IL-2 (20 U/mL; ABI, Columbia, MA).

T lymphocyte proliferation assay

T lymphocyte proliferation assay was performed as described previously 36. Briefly, lymphocytes were stimulated with irradiated WM35 cells and 20 U recombinant IL-2 (gift from Biological Resources Branch, NCI-Frederick Cancer Research and Development Center, Frederick, MD) for 4 days. Proliferative responses of the lymphocytes were determined by [3H]thymidine incorporation assay. All determinations were performed in triplicate.

Chemokine determination by RT-PCR or ELISA

mRNA was extracted from WM35 melanoma cells (3 × 106) using Dynabeads mRNA DIRECTTM kit (DYNAL, Lake Success, NY). PCR was performed using published primers (CCL17, CCL22, CXCL12, CCL9; 3739), or primer 5′-TGT AGG GCG ACG GTT TTA-3′ and 5′-TCC ACC ACA ACA TGC AG-3′ for CCL25. PCR reactions were performed for 35 cycles (94°C, 1 min; 60°C for CCL17, CCL19 and CCL22, 57°C for CXCL12, 55°C for CCL25, 1 min; 72°C, 1 min) using the SuperScript One Step RT-PCR kit (Invitrogen, Carlsbad, CA). All PCR products were analyzed on 10% Novex-TBE gel (Invitrogen).

Supernatants obtained from melanoma cells WM35 on day 4 of culture were tested for the presence of CCL2, –3 and –5 using ELISA Quantikine kit (R&D Systems).

Cytokine measurements

Cytokines produced by CTL MLTC35 were determined using ELISA kits (Endogen, Rockford, IL) 35, 36.

Cytotoxicity assay

Cytotoxicity 51Cr-release assay was performed as described previously 36, 40. To determine HLA restriction of the CTL, tumor targets were incubated with various amounts of anti-HLA class I mAb W6/32 (IgG2a; 60 μg/mL), anti-HLA class II mAb B33.1 or D1.B6 (IgG2a; 60 μg/mL), or anti-HLA-B17/A2 mAb MA2.1 (IgG1; 20 μg/mL). Isotype-matched control mAb were used at similar concentrations. All incubations were performed for 1 h at room temperature. Excess blocking mAb was removed and cytotoxicity assay performed. The percentage of CTL lysis inhibition was determined using the following formula:

% lysis inhibition = 100 – [(% lysis with anti-HLA mAb / % lysis with control mAb) × 100].

Phenotyping of tumor cells and T cells

Cultured T cells were incubated with saturating concentrations (5 μg/mL) of fluorescein- or phycoerythrin-labeled mAb detecting human lymphocyte markers (see Table 3) in RPMI 1640 medium supplemented with 5% human AB serum for 1 h at 4°C. Binding of the mAb was analyzed by FACS. All values given in the Results section are corrected for irrelevant, isotype-matched control Ab binding.

T cell migration in the organotypic melanoma culture system (reconstruct)

Cultures were initiated by mixing 4.5 × 104 human fetal fibroblasts FF2441 with collagen matrix [1.6 mL 10× Eagle's minimum essential medium EMEM), 0.16 mL L-glutamine, 1.82 mL heat-inactivated human AB serum, 0.52 mL NaHCO3 and 14.8 mL bovine collagen type I], and plating 450 μL of the mixture into wells of a 24-well plate. After 1 h, WM35 melanoma cells (1 × 105) were seeded on top of the collagen matrix. After 2 days, melanoma cells were stained with CellTracker Blue 7-amino-4-chloromethylcoumarin (Invitrogen-Molecular Probes, Carlsbad, CA) and a separating layer of fibroblasts in collagen gel (100 μL, 500 μm) was added on top of the melanoma cells. Fibroblast-collagen overlay containing pre-stained (carboxyl-fluorescein diacetate-Green, Invitrogen-Molecular Probes) CTL was prepared by mixing 3 × 104 fibroblasts FF2441 and 1 × 105 CTL with collagen matrix, and adding 250 μL per well. For control reconstruct, PHA blasts prepared from PBMC of the same patient were used. Reconstructs were incubated in medium (50% DMEM, 50% MCDB153-L15 medium supplemented with 2% FBS). Four days after the addition of T cells, reconstructs were fixed in 10% buffered formalin for 4 h at room temperature, and processed for histological evaluation. The percentage of apoptotic tumor cells was determined by counting apoptotic nuclei and intact tumor cells in sections stained with hematoxylin and eosin. This method has been superior to other methods used for staining of apoptotic cells, such as caspase-3, TUNEL and cytokeratin 18 staining methods 41. In T cell migration blocking studies, anti-chemokine or chemokine receptor mAb or isotype-matched control Ab (10 μg/mL) were added on top of the separating layer, and chemokine (250 ng/mL) was added on top of the T cell layer. The percentage of apoptotic tumor cells in the presence and absence of inhibitor was determined.

Chemotaxis assay

CTL migration was evaluated using a 24-well, 5.0-μm pore size Transwell plate (Costar, Cambridge, MA). T cells were washed once with T cell medium 36 and adjusted to 5 × 105 cells/mL in the same medium. An aliquot (100 μL) of the cell suspension containing 5 × 104 T cells was placed in the top chamber of the Transwell. Chemokine, prepared at the indicated concentration in T cell medium (500 μL total volume), was added to the bottom chamber of the Transwell. After 90 min incubation at 37°C in a 5% CO2 atmosphere, the top chamber was removed, and the number of T cells that had migrated into the bottom chamber was counted under the microscope.

Statistical analyses

Differences between experimental and control values were analyzed for significance by 2-sample Student's t-test.

Acknowledgements

This work was supported by SPORE grant P50 CA93372, grants CA25874 and CA10815 from the National Institutes of Health, and by the Commonwealth Universal Research Enhancement Program, Pennsylvania, Department of Health. We thank James Hayden for his advice in microscopy imaging and Jeffrey S. Faust for assistance in flow cytometry analyses. We also thank Elsa Aglow for providing assistance in histotechnology and Marion Sacks for editorial assistance.

Footnotes

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