Tumor-Infiltrating Lymphocytes

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CD40-Ligated Dendritic Cells Effectively Expand Melanoma-Specific CD8+ CTLs and CD4+ IFN-γ-Producing T Cells from Tumor-Infiltrating Lymphocytes Patrick Terheyden, Per thor Straten, Eva-B. Bröcker, Eckhart Kämpgen and Jürgen C. Becker J Immunol 2000; 164:6633-6639; ; http://www.jimmunol.org/content/164/12/6633

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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References

CD40-Ligated Dendritic Cells Effectively Expand Melanoma-Specific CD8ⴙ CTLs and CD4ⴙ IFN-␥-Producing T Cells from Tumor-Infiltrating Lymphocytes1 Patrick Terheyden,* Per thor Straten,† Eva-B. Bro¨cker,* Eckhart Ka¨mpgen,* and Ju¨rgen C. Becker2*

S

ubsequent to Ag uptake, immature dendritic cells (DC)3 leave peripheral tissues and migrate to lymphoid organs for presentation of these Ags in the context of MHC molecules to T cells (1). This contact is initiated via interaction of the TCR with MHC-peptide complexes; however, costimulatory signals such as CD80 or CD86 are essential to ensure T cell activation. These molecules are rapidly induced on differentiation and/or activation on DC (2). Notably, DC contact with CD4⫹CD154⫹ T lymphocytes is a potent inducer for CD80/86 expression (3–5) and IL-12 production (6 – 8). This stimulation allows DC to prime CD8⫹ T cells (9 –12). This two-step model of DC-mediated T cell activation explains the need for CD4⫹ T cell help to induce CTLs in vitro. Furthermore, there is increasing evidence that CD40/ CD154 interactions are essential for the Th1/Th2 differentiation (6, 8). The direction of Th cell differentiation involves a variety of factors including MHC genotype, Ag dose, the nature of costimulation, the route of immunization, and the cytokine production by both APC and T cells (8, 13–15). DC subsets with differential *Department of Dermatology, School of Medicine, University of Wu¨rzburg, Wu¨rzburg, Germany; and †Department of Tumor Cell Biology, Danish Cancer Society, Copenhagen, Denmark Received for publication September 17, 1999. Accepted for publication March 15, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from the Interdisziplina¨res Zentrum fu¨r klinische Forschung, Bundesministerium fu¨r Forschung und Technik (Wu¨rzburg B-4). 2

Address correspondence and reprint requests to Dr. Ju¨rgen C. Becker, University of Wu¨rzburg, Josef-Schneider Strasse 2, D-97080 Wu¨rzburg, Germany. E-mail address: [email protected] Abbreviations used in this paper: DC, dendritic cell; BV, ␤-variable; FCM, flow cytometry; LCM, lymphocyte-conditioned medium; RT-PCR/DGGE, reverse transcriptase PCR/denaturing gradient gel electrophoresis; TCC, T cell clone; TIL, tumor infiltrating lymphocytes. 3

Copyright © 2000 by The American Association of Immunologists

potential to direct Th cell responses have been defined in mice (16) and humans (17). Several cytokines secreted by these DC possess a pivotal role in the polarization of Th cell responses. In particular, IL-12 is important for the development of Th1, whereas IL-4 and IL-10 favor Th2 responses (13). DC activation via CD154 results in an increased IL-12 production (6) leading to enhanced IFN-␥ production of T cells in autologous and allogeneic MLRs (18, 19). The cloning of T cells has proved to be an important tool in tumor immunology, because it allows the study of cell-cell interactions on the level of a single cell (20). The generation of TCC established in classical cloning procedures enabled the identification of tumor Ags that are now used in clinical protocols for specific cancer immunotherapy (21). This procedure may also serve as an ideal model to study cellular interaction during T cell priming and expansion, because it allows single interactions of either a CD4⫹ or a CD8⫹ T cell with APC. Hence, we developed a human model to scrutinize DC-T cell interaction, based on the induction of anti-melanoma immune responses. For this purpose, a cloning procedure was established in which TIL were cocultured with autologous DC that were pulsed with autologous tumor material. Using this model, we provide evidence that CD40 maturation signals are critical for the direction of cellular immune responses. Mature DC that had not received a CD40 signal predominantly induced IL-4-producing Th2 cells, and the expansion of CD8⫹ T cells was not observed. In contrast, CD40 stimulation of DC enables a shift to IFN-␥-producing Th1 cells as well as the expansion of melanoma-reactive CD8⫹ CTL clones.

Patients and Methods Patients and cells Patients involved in this study suffered from American Joint Committee on Cancer stage IV melanoma. Informal consent was given. Tumor biopsies were used as sources for both TIL and melanoma cells. Metastatic lesions were localized either in the skin (three patients) or lymph node (four patients). Small pieces of tumor biopsies were snap frozen in liquid nitrogen 0022-1767/00/$02.00


Professional APC, notably dendritic cells (DC), are necessary for stimulation and expansion of naive T cells. By means of murine models, the interaction between CD40 on DC and its ligand CD154 has been recognized as an important element for conditioning of DC to prime and expand CTL. We translated these findings into the human system, scrutinizing the ability of DC to initiate clonal expansion of single T cells. DC generated under completely autologous conditions from peripheral blood monocytes were cocultured at a rate of 0.3 cell/well with melanoma-infiltrating T cells; this procedure guaranteed that either a CD4ⴙ or a CD8ⴙ cell interacted with the DC, thus avoiding the contact of more than one T cell to the DC. In the absence of further stimulation, this cloning protocol yielded almost exclusively CD4ⴙ T cell clones that predominantly exhibited a Th2 phenotype. However, cross-linking of CD40 on DC resulted in the induction of IFN-␥-producing Th1 CD4ⴙ T cell clones. In addition, CD40-activated DC were capable of expanding CD8ⴙ CTL clones. The ratio of CD4 to CD8 T cell clones corresponded to the ratio present in the initial tumor-infiltrating lymphocyte preparation. The CTL clones efficiently lysed autologous tumor cells whereas autologous fibroblasts or MHC-mismatched melanoma cells were not killed. Our findings support the critical role of CD40/CD154 interactions for the induction of cellular immune responses. The Journal of Immunology, 2000, 164: 6633– 6639.

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DIFFERENTIAL EXPANSION OF T CELL SUBSETS BY CD40 SIGNALS ON DC TCR BV clonotype mapping RNA was extracted from tumor biopsies and established TCC using the Purescript isolation kit (Gentra Systems, Minneapolis MN). cDNA synthesis and quantitation of TCR cDNA in each sample was conducted as described (25). Using equal amounts of TCR template in all reactions, cDNA was amplified using specific primer panels. Amplifications were conducted in a total volume of 25 ␮l containing 1⫻ PCR buffer (50 mM KCl, 20 mM Tris (pH 8.4), 2.0 mM cresol red, 12% sucrose, 0.005% (w/v) BSA (Boehringer Mannheim)), 2.5 pmol of each primer, 40 mM dNTPs (Pharmacia LKB, Uppsala, Sweden), and 1.25 U AmpliTaq polymerase (Perkin-Elmer Cetus, Emeryville, CA). Parameters used for the amplification were 94°C for 60 s, 60°C for 60 s, and 72°C for 60 s for 40 cycles. Taq polymerase and dNTPs were added to the reaction tube at a 80°C step between the denaturation and the annealing steps of the first cycle. For denaturing gradient gel electrophoresis analysis, 10-␮l aliquots were loaded onto a denaturing gradient gel containing 6% polyacrylamide and a gradient of urea and formamide from 20% to 80%. Gel electrophoresis was conducted at 160 V for 4.5 h in 1⫻ TA buffer (0.04 M Tris-acetate, 0.001 M EDTA) kept at a constant temperature of 58°C. After electrophoresis, the gel was stained with ethidium bromide and photographed under UV transillumination.

Assay for Ag specificity of CD4⫹ TCC Specificity of CD4⫹ T cells was established by their proliferative response to DC presenting relevant or irrelevant Ags. For this purpose, 1 ⫻ 104 cloned T cells were starved for at least 24 h in cytokine-free medium, then cultured in round-bottom 96-well plates (Falcon) with either 2.5 ⫻ 104 irradiated (50 Gy) mature melanoma oncolysate-pulsed DC, 2.5 ⫻ 104 irradiated tetanus toxoid-pulsed (Behring Chiron, Marburg, Germany), or nonpulsed DC, respectively, at a final volume of 200 ␮l RPMI 1640 containing 10% AB-plasma and 1000 U/ml IL-2 (Aldesleukin, Chiron). After 48 h, 0.5 ␮Ci [6-3H]thymidine (Amersham, Braunschweig, Germany) was added, and the cells were incubated for another 18 h. Cells were harvested onto glass fiber filters. After drying, the filters were placed in scintillation fluid (Packard, Groningen, The Netherlands) and counted in a Wallac scintillation spectrometer (Wallac, Munich, Germany).

Cytotoxicity assay for Ag specificity of CD8⫹ TCC Specific MHC-restricted lysis of autologous melanoma cells by TCC was analyzed using the microcytotoxicity assay modified according to the method of Bro¨cker et al. (26). One hundred target cells per well were plated on Terasaki plates in 10 ␮l medium and incubated overnight. After removal of medium by rinsing the plates twice with PBS, effector T cells were cocultured with targets at E:T ratios of 50:1, 25:1, and 12:1 overnight. TCC were starved for at least 24 h in cytokine-free culture medium. To remove lysed cells, plates were rinsed once with warm 10% FCS and twice with PBS. Before washing, the plates were placed upside down for 20 min to facilitate cellular detachment. Subsequently, plates were fixed and stained according to the method of Pappenheim. Data were quantified by determination of target cell reduction (control ⫺ probe) expressed in percent of control count. Only cells with obvious nuclei were counted. Autologous and allogeneic melanoma cell lines as well as HeLa cells (ATCC) grew in 10% FCS in RPMI, whereas SCC cells (ATCC) were cultured in 10% FCS in DMEM (high glucose) (PAA Laboratories, Linz, Austria).

Flow cytometry For flow cytometry, the following anti-human mAbs were used (PharMingen, if not noted otherwise): anti-CD3 (mouse IgG1, clone UCHT1); anti-CD4 (mouse IgG1, clone RPA-T4); anti-CD8 (mouse IgG1, clone HIT8a); anti-HLA-DR (mouse IgG1, clone HB-55, American Type Culture Collection (ATCC), Manassas, VA); anti-CD14 (mouse IgG2a, clone UCHM1, Sigma); anti-CD25 (mouse IgG1, clone ACT-1, DAKO, Hamburg, Germany); anti-CD80 (mouse IgG1, clone L-307.4); anti-CD83 (mouse IgG1, clone HB15A, Immunotech, Marseille, France); anti-CD86 (mouse IgG2b, clone IT2.2); anti-CD115 (rat IgG2a, clone GR12, Calbiochem, Bad Soden, Germany). Cells were washed in PBS, resuspended with 0.1% BSA in PBS at 1 ⫻ 104–1 ⫻ 105/100 ␮l, incubated with mouse or rat IgG for 30 min at 4°C, washed with 0.1% BSA in PBS, and treated with FITC-labeled either goat anti-mouse or anti-rat IgG F(ab⬘)2 fragments (Dianova) for 30 min. Isotype-matched mouse or rat IgG served as controls (Sigma). For CD40 cross-linked DC, cells were preincubated with 2% irrelevant mouse serum; thereafter, biotinylated Ab preparations were used for staining together with R-PE-conjugated streptavidin (DAKO). After washing, fluorescence was analyzed with a FACScan (Becton Dickinson). Dead cells, e.g., irradiated DC, were identified by propidium iodide staining (5 ␮g/ml).

Cytokine release assays Cytokine production of 1 ⫻ 103 cloned T cells was measured in culture supernatants 72 h after stimulation with irradiated DC pulsed with oncolysate at an initial DC:T cell ratio of 2:1. The culture medium was identical with that used for T cell cloning. IFN-␥ (detection range, 0.4 –25 IU/ml) and IL-4 (detection range, 16 –1000 pg/ml) were assayed by ELISA (ICN, Costa Mesa, CA) according to the manufacturer’s instructions. Results are corrected for cytokine content present in supernatants of irradiated tumor lysate-pulsed DC cultured under identical conditions that contained 15 IU/ml IFN-␥, but no IL-4.

Results

DC induce the growth of Ag-specific CD4⫹ TCC of Th2 phenotype Cloning of T cells infiltrating melanoma metastases was performed by means of autologous DC. DC were prepared from peripheral blood by enrichment for CD14⫹ cells and cultured in the presence of IL-4 and GM-CSF under strictly autologous conditions. At day


for in situ analysis of the TCR ␤-variable (BV) repertoire. The remaining native material was stored in blocks 0.5 cm in diameter in 10% DMSO in FCS at ⫺80°C, which allowed repeated isolation of vital cells and oncolysates. For the generation of autologous DC from peripheral blood, a procedure like that of Romani et al. (22) was used. Briefly, PBMC of melanoma patients were isolated by Lymphoprep density gradient centrifugation (Nycomed, Oslo, Norway). CD2⫹ and CD19⫹ cells were depleted by using the corresponding Ab-loaded magnetic beads (Dynal, Oslo, Norway). The remaining cells were cultured in RPMI 1640 (Life Technologies, Eggenstein, Germany) supplemented with 1% autologous plasma, 500 U/ml IL-4 (Strahtmann Biotech, Hannover, Germany), and 800 U/ml GMCSF (Molgramostim, Sandoz, Nuremberg, Germany). At day 5, the cells were pulsed with oncolysate. Whole tumor cell lysates were obtained according to the method of Grabbe et al. (23). Subsequent to two washes, 1 ⫻ 105 autologous tumor cells were resuspended in 1 ml ice-cold water. After 10 min on ice, cells were vortexed vigorously and subsequently centrifuged at 10,000 ⫻ g for 15 min at 4°C. Supernatant was discarded. After two additional cycles, the pellet was dissolved in 400 ␮l PBS and added to the DC at a final dilution of 1:100. In one of the patients, no tumor cells grew in vitro; thus, we repeatedly used frozen pieces of the tumor for the preparation of oncolysates. At day 7, a cocktail of cytokines containing IL-1, IL-6, TNF-␣ (Strahtmann Biotech), and PGE2 (Sigma, Deisenhofen, Germany) was added to induce final differentiation of DC (24). CD40 maturation signals were provided at day 9; DC were preincubated for 10 min in 3% autologous plasma/PBS at 1 ⫻ 106 cells/ml, followed by a 20-min incubation step in the presence of an anti-CD40 Ab (5 ␮g/ml, clone 5C3, mouse IgG1; PharMingen, San Diego, CA). Subsequent to two washes, 10 ␮g/ml rabbit anti-mouse IgG Ab (Dianova, Hamburg, Germany) was added, and cells were incubated overnight. Maturation was documented by flow cytometry analysis by labeling with anti-CD14, anti-CD25, antiCD80, anti-C83, anti-CD86, anti-CD115, and anti-HLA-DR Abs. At day 10, DC were harvested, irradiated with 50 Gy, and used as feeders for T cell cloning. Loss of proliferative capacity was confirmed by [3H]thymidine uptake (data not shown). Melanoma-infiltrating T cells for T cell cloning were obtained by mincing tumor biopsies into small pieces after careful removal of adjacent nonmalignant tissue. Tumor pieces were digested for 2.5 h in RPMI 1640 supplemented with 0.25% collagenase D (Boehringer Mannheim, Mannheim, Germany) and 100 ␮g/ml DNase type I (Sigma) followed by a passage through a steel sieve. After additional washes, cells were initially expanded in bulk cultures at 1 ⫻ 105 cells/ml in RPMI 1640, supplemented with 10% heat-inactivated human AB plasma (Transfusion Medicine Center, University of Wu¨rzburg, Wu¨rzburg, Germany), 1% penicillin-streptomycin, 1% sodium pyruvate (Life Technologies), 2 mM L[]r-glutamine, 1000 IU/ml IL-2 (Aldesleukin, Chiron, Ratingen, Germany), and 10% LCM. LCM was generated as previously described (20). For T cell cloning, 0.3 cell were placed into 96-well, V-bottom microtiter plates (Falcon, Becton Dickinson, Heidelberg, Germany) with 2000 irradiated DC at a final volume of 150 ␮l/well. The culture medium was identical with that used in bulk cultures. One-half of the culture medium was replaced after 7 days followed by additional changes in 3- to 4-day intervals. Wells showing cell growth were subcloned at 1000 cells/well; this was normally done after 10 to 14 days.

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5, immature DC were pulsed with tumor Ags derived from autologous oncolysates. When nonautologous material or no antigenic material at all was used to pulse DC, no lesional T cells could be expanded. This was tested several times for those instances when in vitro expansion of autologous tumor cells was not successful. In these cases, mixtures of three melanoma cell lines, i.e., Bro P9, SK-Mel 28, Mel-2a, were used. Final maturation of DC was initiated at day 7 of culture by addition of TNF-␣, IL-1, IL-6, and PGE2. Phenotyping of the mature DC, harvested on day 10, demonstrated high expression of CD25, CD80, CD83, CD86, and HLA-DR as well as low expression of CD115 (Fig. 1, A–F). Light microscopy confirmed that these cells displayed the characteristic morphological appearance of mature DC with veiled edges and multiple processes. TIL were obtained from either skin or lymph node metastases of melanoma patients. T cells, i.e., original dilution 0.3 cell/well, were expanded by Ag-driven restimulation with oncolysate-pulsed DC every 2 wk. When a cell pellet became visible after 10 –14 days, a fraction of the cells was removed for further analysis. Phenotypic analysis of the TCC obtained under these conditions revealed the almost exclusive growth of CD4⫹ T cells. The results of a representative experiment are given in Fig. 2, and summarized data are presented in Table I. Initial characterization of TIL preparations revealed that these contained both CD4⫹ and CD8⫹ T cells (Table I). For the experiment depicted in Fig. 2, this ratio was 2.7 (57.8% CD4⫹, 21.2% CD8⫹). The ratio, however, varied from patient to patient. Clonality of these T cell lines was confirmed by RT-PCR/DGGE-based clonotype mapping, demonstrating that each TCC expressed only one individual TCR V␤-chain (Fig. 3). Ag-specific reactivity was tested by the proliferative response of CD4⫹ T cells to autologous DC that were pulsed with oncolysate, irrelevant Ag, i.e., tetanus toxoid, or not pulsed at all. This analysis confirmed that the majority of TCC showed a specific Ag-dependent proliferation. However, as illustrated in Fig. 4, ⬃25% of TCC possess a nonspecific reactivity that was uncovered by a strong proliferative response to nonpulsed DC. The cytokine production pattern of the CD4⫹ TCC was characterized. To delineate Th1- or Th2-like T cells, IFN-␥ and IL-4 were measured by immunochemical detection in supernatants of coculture experiments with oncolysate-pulsed DC. Representative data are given in Fig. 5, in which IL-4 production is plotted against that of IFN-␥. The majority of clones produced either high (⬎1000

pg/ml) or intermediate (between 100 and 1000 pg/ml) amounts of IL-4. Only a few clones produced IFN-␥ in addition to IL-4 (3 of 15) and just 1 displayed an exclusive production of this Th1 cytokine. Thus, using the culture conditions described above, we obtained predominantly TCC with a Th2-like cytokine expression. Cross-ligation of CD40 on DC induces Th1 CD4⫹ TCC The potential of murine DC to induce IFN-␥ producing T cells after CD40 ligation was demonstrated previously (6). The following experiments were designed to translate these observations into

FIGURE 2. Exclusive growth of CD4⫹ TCC in the presence of oncolysate-pulsed DC. Five representative clones from patient 7 (0.3 T cell/ well, of ⬃600 wells); 1 ⫻ 104 T cells were stained with murine mAb against CD4 (B) or CD8 (C). Isotype-matched murine IgG served as control (A). Dead cells, e.g., irradiated DC, were identified by propidium iodine staining (5 ␮g/ml). The x-axis represents log10 fluorescence intensity.


FIGURE 1. Phenotype of DC before (A–F) and after (G–L) CD40 cross-ligation. Representative experiment is shown. No significant changes for surface Ag expression; however, cellular morphology is more uniform after treatment (A and G, cells in the cytograms). Signal for specific Ab shown in black lines (B and H, CD25; C and I, CD80; D and J, CD83; E and K, CD86; F and J, HLA-DR); gray lines are the corresponding isotype control, cells were gated as shown in A and G, respectively. x-axis represents counted events per channel (y-axis).

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DIFFERENTIAL EXPANSION OF T CELL SUBSETS BY CD40 SIGNALS ON DC

Table I. Number of TCC obtained via CD40 or non-CD40-ligated DC Cloning Experiments Non-CD40-ligated DC

Patient

1 2 3 4 5 6 7 a b

In situa

CD4

CD8

3.6 8.0 0.52 ND 0.24 ND 2.72

16 17 1 4 0 7 12

1 0 0 1 0 0 0

CD4/CD8

16.0 4.0

CD40-ligated DC Cloning efficiacyb

CD4

CD8

1.21 (14) 2.8 (6) 0.33 (3) 1.7 (3) 0 (4) 1.2 (6) 1.2 (10)

22 19 0 13 0 3 ND

3 4 1 9 0 2 ND

CD4/ CD8

Cloning efficiacy

7.33 4.75

1.79 (14) 2.3 (10) 0.33 (3) 3.7 (6) 0 (4) 0.8 (6) ND

1.44 1.5 ND

Ratio of number of CD4⫹ T cells to number of CD8⫹ T cells in the tumor lesion. Rate of clones obtained among 100 wells (number of 96-well plates used in this experiment is in parentheses).

FIGURE 3. TCR clonotype mapping. RNA was extracted from tumor biopsies (top) and established TCC (bottom) and subjected to RT-PCR/ DGGE clonotype mapping using specific primers covering the TCR BV regions 1–24. One representative TCC is shown.

IFN-␥ and IL-4 production. Only two IL-4-producing Th2 clones were expanded under these conditions. Cross-ligation of CD40 on DC induces the expansion of both CD4⫹ and CD8⫹ TCC When DC lacked CD40 signals, their use as stimulator cells yielded almost exclusively CD4⫹ TCC (Table I). We next tested the hypothesis that CD40-mediated maturation signals for DC would enable CTL expansion. Indeed, CD40-treated DC allowed the clonal expansion of CD8⫹ T cells ( p ⬍ 0.001, ␹2 test). The mean of the CD4:CD8 ratio was 3.7 (range, 1.44 –7.3) (Table I). In total, we obtained 45 CD4⫹ TCC with nonstimulated DC and 57 CD4⫹ TCC with CD40 stimulated DC. Only 2 CD8⫹ TCC were gained with nonstimulated DC, whereas 17 CD8⫹ TCC grew in the CD40 group. The ratio of CD4⫹ to CD8⫹ TCC obtained using CD40-treated DC corresponded to the ratio present in the initial preparation of TIL, suggesting that CD40-conditioned DC are equally effective in stimulating growth of both T cell subsets (Table I). Clonality of the obtained T cell lines was confirmed by

FIGURE 4. Oncolysate-pulsed DC induce proliferation of specific cloned CD4⫹ T cells. Specificity of CD4⫹ T cells was established by their proliferative response to DC presenting relevant, i.e., autologous oncolysate (f) or irrelevant Ag (tetanus toxoid (u)) or to nonpulsed DC (䡺). Cloned T cells (1 ⫻ 104), starved for 24 h in cytokine-free medium, were cultured in round-bottom 96-well plates with 2.5 ⫻ 104 irradiated (50 Gy) mature DC in a final volume of 200 ␮l RPMI 1640 containing 10% AB plasma and 1000 U/ml IL-2. After 48 h, 0.5 ␮Ci [6-3H]thymidine was added, and the cells were incubated for another 18 h. Data are given as stimulation indices of [3H]thymidine uptake of T cells in response to nonpulsed DC.


the human system. DC were cultured as described above, but at day 9 an additional maturation signal was delivered by cross-ligation of CD40. This was achieved by a specific mAb followed by a secondary cross-linking Ab. After overnight culture, DC were harvested and used for cloning of melanoma-infiltrating T cells. In general, the phenotype of CD40 cross-linked DC was not significantly altered to their nonstimulated controls (Fig. 1). The use of this CD40-ligated DC yielded a cloning efficacy, i.e., the number of positive clones of 100 wells, of 1.49% (range, 0 –3.7%), which was not significantly higher than that obtained with the DC preparation described above which was 1.21% (range, 0 –2.8%) (Table I). Analysis of cytokine expression of the obtained CD4⫹ TCC, however, revealed a significant difference that clones expanded in the presence of nonligated DC ( p ⬍ 0.001, ␹2 test); the majority of the CD4⫹ T cell lines induced by CD40-ligated DC expressed IFN-␥ as shown in Fig. 6. Of 18 TCC, 5 produced exclusively IFN-␥, and 11 additional clones showed a mixture of

The Journal of Immunology

FIGURE 5. Predominant production of IL-4 by DC-cloned T cells. Cytokine production of 1 ⫻ 103 cloned T cells was measured in culture supernatants 72 h after stimulation with irradiated oncolysate-pulsed DC at an initial DC:T cell ratio of 2:1. IFN-␥ and IL-4 were assayed by ELISA. The results are corrected for cytokine content present in supernatants of irradiated oncolysate-pulsed DC, i.e., 15 IU/ml IFN-␥ and 0 pg/ml IL-4, respectively. IFN-␥ is present in LCM and is not produced by irradiated DC which was controlled several times (data not shown).

FIGURE 7. Specific MHC-restricted lysis of autologous melanoma cells by TCC. Cytotoxicity against autologous melanoma (A–C), autologous fibroblasts (D–F), and allogeneic HLA-mismatched melanoma (HLA-A1/24, -B8/44 and HLA-A2, -B35, -Cw3, respectively) (G–I) was analyzed using the microcytotoxicity assay. Effector T cells and targets at E:T ratios of 50:1, 25:1, and 12:1 were cocultured overnight. Target cells without additional T cells served as controls (A, D, and G). Data were quantified by determination of target cell reduction (control ⫺ probe) expressed in percent of control count (C, F, and I). The x-axis shows different E:T ratio and the y-axis represents target cell reduction in percent.

FIGURE 6. Cytokine expression of CD4⫹ TCC that were expanded in the presence of CD40 cross-linked, oncolysate-pulsed DC. The assay was performed as described in the legend of Fig. 5. Such DC predominantly induce IFN-␥ producing T cells (Th clones obtained from two patients).

neic melanoma were killed. Because the tumor cell line established from patient 2 served as allogeneic control for CD8⫹ TCC obtained from patient 1 and vice versa, the possibility that the control cell lines were not lysed because lack of sufficient amounts of MHC class I molecules could be excluded. Additional allogeneic melanoma targets were also not attacked if a HLA mismatch was given (patient 6 (HLA-A3/26, B35/49, Cw4/w6) did not react against patient’s 4 melanoma (HLA A1/11, B51/53, Cw4/w5)). Furthermore, CD8⫹ TCC did not attack nonmelanoma targets. Neither HeLa nor SCC cells were killed by the obtained TCC (Table II).

Discussion Numerous murine studies provided evidence for the critical role of CD40 maturation signals offered by its ligand. CD154 is up-regulated on Th cells in response to the initial contact with the DC. The importance of this interaction is shown by the fact that


RT-PCR/DGGE-based clonotypic mapping. Fig. 3 depicts the high number of T cell clonotypes comprising most TCR BV regions present in situ in a tumor metastasis. In contrast, if cloned T cell lines derived from the same tumor were analyzed, only one TCR BV transcript was present for each line (Fig. 3 gives a representative example of a TCR BV14-expressing CD8⫹ T cell line). The clonality of these TCR was unequivocally shown by sequence analysis (data not shown). Specificity and cytolytic capacity of CTL clones was shown by microcytotoxicity assays against autologous melanoma cells (Fig. 7, A–C), autologous fibroblasts (Fig. 7, D–F), and MHC-mismatched allogeneic melanoma cells (Fig. 7, G–I). This analysis clearly demonstrated that the majority of TCC efficiently lyse the autologous tumor in a specific, MHC-restricted fashion, because neither the autologous fibroblasts nor the alloge-

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DIFFERENTIAL EXPANSION OF T CELL SUBSETS BY CD40 SIGNALS ON DC Table II. Autologous fibroblasts, allogeneic melanoma, SCC, and HeLa cells are not attacked by TCC (two CD8⫹ TCC of patient 6)a E:T Ratio

Autologous Melanoma

Autologous Fibroblasts

Allogenous Melanoma

SCC

HeLa

1

20:1 10:1 5:1

69 42 18

14 21 21

10 15 13

7 4 2

21 13 14

2

20:1 10:1 5:1

45 39 39

0 20 20

19 11 10

0 0 0

0 2 4

a Reduction of target cells. Data were quantified by determination of target cell reduction (control ⫺ probe) expressed in percent of control count.

regulated cytokine production are involved (6, 12). In the DC generated according to the given protocol, no up-regulation of either costimulatory molecules or MHC class II molecules could be detected. This may be explained by the fact that these molecules are already maximally expressed by mature DC. A nonspecific mechanism of induction of T cell proliferation could be binding and stimulation of the constitutively expressed CD40 molecule on the cloned T cell (32). In these experiments, we did not exclude this possibility, but likelihood that unbound anti-CD40 mAb and/or unbound cross-linking mAb carried over from the DC culture to the T cell culture is rather small because of multiple washing steps. However, the in vivo situation differs to most in vitro models in that multiple cells react with Ag-pulsed DC; thus, peptide-pulsed and tumor lysate-pulsed DC without CD40 cross-linking were reported to induce CTL-mediated regressions in murine B16 melanoma (33, 34). In a recent clinical trial, objective tumor remissions could be induced by vaccination with autologous DC, pulsed with either tumor lysates or a cocktail of MHC class I-restricted peptides together with keyhole limpet hemocyanin. The objective of adding the latter was to ensure that DC are not interacting solely with CD8⫹ but also with CD4⫹ T cells (35). CD4⫹ T cells can differ in their cytokine profile. The development of Th1- and Th2-like cells is defined by the microenvironmental milieu, e.g., IL-12 fosters Th1 responses, whereas IL-4 and IL-10 favor Th2 responses. This milieu is critically influenced by the type of APC and the character of the APC/T cell interaction. DC are a heterogeneous population of cells and their phenotype may depend on local factors. There is increasing evidence that different DC subsets can be generated in vitro depending on the progenitor cells and/or culture conditions. Some recent reports demonstrated that DC subsets can be obtained with different potential to induce Th cell subsets (16, 17). In alloreactions that allow multiple intercellular reactions, DC generated under autologous conditions in the presence of TNF-␣, IL-1, IL-6, and PGE2, as done herein, were reported to direct naive T cells to IFN-␥ production (24). In melanoma, it is still contentious whether the cross-regulation between Th1 and Th2 subsets is beneficial for mounting an efficient antitumor response. In general, Th1-like responses are thought to be associated with tumor regression, whereas Th2-like responses are correlated with increased suppressor activity (36, 37). Nevertheless, murine Th2 TCC augment CTL responses and induce tumor regression when transferred into tumor-bearing mice (38). Furthermore, Th2 TCC may represent natural T cells. Natural T cells are a special type of Th2 cells with a very restricted TCR V␣ and V␤ repertoire that are thought to be essential in the initial phase of cellular immune responses; they are characterized by their production of high amounts of IL-4 on binding to the nonclassical


CD154-deficient mice are incapable of generating CD4⫹ T cell responses to various Ags (27) and have deficits in antiviral immunity and impaired memory CTL responses (10, 28). CD40-deficient hosts cannot provide help to Ag-specific T cells on adoptive transfer (29). Cross-ligation of CD40 on DC induces an enhanced IL-12 production directing naive CD4⫹ T cells to Th1 cytokineproducing effector cells (6, 8). Moreover, CD154 engagement is required for protective cell-mediated immunity to Leishmania major infection (7). Except from some viruses directly infecting the APC, CD4⫹ T cell help is required for CD8⫹ CTL responses. Recently, it has been demonstrated that for the induction of CTL responses DC and Th cells do not need to meet simultaneously but that the Th cell can induce the DC to autonomously trigger CTL responses (9). This “licensing” model, first proposed by Matzinger, has been substantiated in further animal studies (9 –12). The licensing is, in part, dependent on the interaction between CD40 and CD154 and can be mimicked by artificial ligation of CD40 by mAb. Although several reports suggest that the licensing model is relevant for the human immune system, this had yet to be confirmed (3, 5). CD40-stimulated DC guide T cells to Th1 effector cell types due to enhanced costimulation and IL-12 expression (18). However, to the best of our knowledge, there is no functional study demonstrating that CD40 maturation signals direct DC help to CTL responses. In the present report, we demonstrate that CD40 maturation signals are critical for the direction of cellular immune responses in human tumor immunity: the interaction of a mixture of melanoma-reactive T cells with Ag-bearing DC under strictly autologous conditions exclusively yielded IL-4-producing Th2 cells; whereas after CD40 cross-linking, CD8⫹ CTL clones as well as IFN-␥-producing Th1 cells were expanded. Our model system described within this report allows only a single interaction of either a CD4⫹ or a CD8⫹ T cell with the DC and supports the licensing model in that non-CD40-treated DC were incapable of directly activating CD8⫹ T cells. The exclusive expansion of CD4⫹ TCC in the absence of CD40 maturation signals appears to result from the interaction of a single T cell, thus avoiding DC licensing. Consequently, our data provide convincing evidence for the essential role of CD4⫹ T cell help during the induction of CTL responses. This hypothesis is illustrated by several observations: the generation of Melan-A/MART-1 specific CTL from naive precursors is possible only in the presence of CD4⫹ T cells (30), and CTL could be induced in vivo only by coimmunization with a CD4 helper-epitope (31). Neither the precise mechanism(s) by which CD4⫹ Th cells deliver help for CTL priming nor the changes brought about in the APC during such activation are fully understood. However, most likely a combination of improved Ag processing, increased expression of costimulatory and adhesion molecules, as well as up-

The Journal of Immunology MHC class I protein, CD1, which is up-regulated at inflammatory sites and binds nonpeptide Ags (39). Cloning of T cells obtained from melanoma patients facilitated the identification of tumor-associated Ags (21). In the present study, we report a cloning protocol for TIL based on autologous, tumor-pulsed DC and results in the growth of CD4⫹ and/or CD8⫹ T cells depending on the status of the APC. Thus, we were able to establish the influence of CD40-CD154 interactions present on Th cells and DC, respectively, on the licensing of DC for the human immune system as deduced from murine studies. Although we thus far did not characterize the fine specificity of the TCC, this model allows their further characterization. The reproducible high efficacy together with the possibility to generate CD4⫹ or CD8⫹ TCC opens the opportunity to generate those from different sources, i.e., TIL, PBMC, or lymphocytes derived from sentinel lymph node biopsies, or different parts of the primary tumors. The latter is of particular interest in melanoma because the analysis of TIL in regressive areas of primary tumors may reveal more potent tumor regression Ags than currently are known (25, 40).

We thank Alexander D. McLellan for the critical discussions during preparation of the manuscript.

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Acknowledgments

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