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Laboratory and Animal Investigations |

Double Signal Stimulation Was Required for Full Recovery of the Autologous Tumor-Killing Effect of Effusion-Associated Lymphocytes* FREE TO VIEW

Yuh-Min Chen, MD, PhD, FCCP; Chun-Ming Tsai, MD; Jacqueline Whang-Peng, MD; Reury-Perng Perng, MD, PhD, FCCP
Author and Funding Information

*From the Chest Department (Drs. Chen, Tsai, and Perng), Taipei Veterans General Hospital, and School of Medicine, National Yang-Ming University; and Division of Cancer Research (Dr. Whang-Peng), National Health Research Institute, Taipei 112, Taiwan, ROC.

Correspondence to: Yuh-Min Chen, MD, PhD, FCCP, Chest Department, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Rd, Taipei, Taiwan, ROC; e-mail: ymchen@vghtpe.gov.tw



Chest. 2002;122(4):1421-1427. doi:10.1378/chest.122.4.1421
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Study objectives: To determine the different effects of interleukin (IL)-2, IL-4, IL-7, IL-10, IL-12, and/or T-cell receptor (TCR)-CD3 engagement in recovering the functions of cytotoxic T lymphocytes (CTL) from malignant effusion.

Setting: National teaching hospital.

Materials and methods: Effusion-associated lymphocytes (EAL) were isolated from 35 malignant pleural effusions. Interferon (IFN)-γ production, proliferative response, and cytolytic activity of the cultured EAL against autologous tumors and K-562 cells were measured.

Results: It was found that EAL had a significantly depressed function. Stimulation with two signals, including IL-2 plus IL-7, IL-2 plus IL-12, or IL-2 plus TCR-CD3 engagement, could fully restore the functions of EAL, including IFN-γ production, proliferative response, and a specific increase in cytolytic activity against autologous tumor cells. IL-4 and IL-10, whether or not in combination with IL-2, did not augment the function of EAL, and even depressed it in some cases. The lymphocyte-depletion test showed that most of the recovered functions were from CD8+ CTL.

Conclusion: The depressed cellular function of EAL could be reversed with double signal stimulation, including IL-2 plus IL-7, IL-2 plus IL-12, or IL-2 plus TCR-CD3 engagement. These recovered cellular functions were mainly from CD8+ CTL.

Figures in this Article

Effusion-associated lymphocytes (EAL) from the malignant pleural effusion of cancer patients exist in a hyporesponsive state.13 The usage of EAL as a model to study the activation of cytotoxic T lymphocytes (CTL) by various cytokines or monoclonal antibodies (MoAbs) is a convenient way to investigate tumor-induced immunosuppression and to explore the possibility of using EAL as a source of adoptive immunotherapy.24

We previously found that interleukin (IL)-2 in combination with IL-12 or T-cell receptor (TCR)-CD3 engagement could restore the cytolytic activity of immunosuppressed EAL against autologous tumors.23 Whether or not other cytokines that had been found to be useful in the augmentation of antitumor activity, such as IL-4, IL-7, and IL-10,57 especially IL-4 and IL-10, which had been noted to have controversial effects on antitumor immunity,12,69 were also effective in situations such as that of malignant pleural effusion is unknown. There is also no report comparing the ability of these cytokines in the recovery of the functioning of immunosuppressed EAL from cancer patients.

In the present study, we have used EAL to study the different effects of IL-2, IL-4, IL-7, IL-10, IL-12, and/or αCD3 (for TCR-CD3 engagement) in the recovery of cellular functions, including interferon (IFN)-γ production, cell proliferation, and cytolytic activity against tumor cells, of immunosuppressed lymphocytes.

Patient Population

Pleural effusions were collected from 35 patients with newly diagnosed cancer with malignant pleural effusion. All patients had a positive effusion cytology finding for malignant cells, and included 28 patients with pulmonary adenocarcinoma, 4 patients with poorly differentiated carcinoma of the lung, and 3 patients with adenocarcinoma of the breast. None of the patients had received anticancer treatment, corticosteroid, or other nonsteroid anti-inflammatory drugs within 1 month before sample collections.

Preparation of Effusions

Pleural effusion specimens were collected in acid citrate dextrose (solution C) bottles to prevent clotting. The effusion was immediately centrifuged, and the cell pellet was resuspended in RPMI 1640 medium (Whittaker Bioproducts; Walkersville, MD), and then layered on Ficoll-Hypaque cushions. After centrifugation, the tumor cells and mononuclear cells were collected from the interface and washed twice in RPMI 1640 medium. Tumor cells were then separated from mononuclear cells by centrifugation on a discontinuous Percoll density gradient (Amersham Pharmacia Biotech AB; Uppsala, Sweden).

Separation of EAL From Tumor Cells

The lymphocyte culture medium (LCM) contained RPMI 1640 medium supplemented with 6% fetal bovine serum (GIBCO; Grand Island, NY), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (Flow Laboratories; McLean, VA), 5 × 10-5 mol/L 2-mercaptoethanol, 2 mM glutamine (GIBCO), 50 μg/mL of streptomycin, and 500 U/mL of penicillin. Percoll medium was adjusted to 20% and 40% in the LCM. Cells from pleural effusion were resuspended in 20% Percoll medium and layered on 40% Percoll medium, and then centrifuged at 312g for 60 min at 25°. After centrifugation, three layers of cells were noted. Cells from each band were then collected, washed, and resuspended in LCM. Cells from the upper band of the effusions were 90 to 98% tumor cells; they were washed and subsequently cultured for use as autologous tumor target cells. Cells from the middle band of the effusions were mainly mesothelial cells, with < 10 to 15% mononuclear cells and/or tumor cells each. Cells from the bottom band consisted of > 98% mononuclear cells with < 1 to 2% tumor cells or mesothelial cells, and some red cells. Cells from the bottom band were washed with LCM twice and served as EAL for culturing in different conditions. The cell viability of each fraction was > 95%, as determined by trypan blue dye exclusion.

Cytokines and MoAbs

Purified human IL-2, IL-4, IL-7, IL-10, and IL-12 was purchased from R&D Systems (Minneapolis, MN). The αCD3 antibody was produced in hybridoma OKT3, and the culture supernatant was used.10

Immunophenotyping of EAL

The phenotype of different lymphocyte subpopulations in various conditions was determined by flow cytometry. Fluorescein isothiocynate or phycoerythrin-conjugated MoAb (anti-IgG1, anti-IgG2a, anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16, anti-CD19, anti-CD45, anti-CD56) was purchased from Becton Dickinson (Mountain View, CA), and was used for detecting the corresponding surface antigens according to the recommended procedure of the supplier. Briefly, the EAL were incubated with the relevant MoAb at 4°C for 30 min. After processing with sodium azide and paraformaldehyde, the cells were analyzed in a FAC Star Plus Cytofluorometer (Becton Dickinson).

MoAb Incubation and Magnetic Beads Depletion of Subpopulations of EAL

EAL, 2 × 107/mL, were washed once with a bead-separation medium (consisting of Hank’s balanced salt solution with bovine serum albumin, glutamine, and antibiotics), and incubated with specific mouse antihuman MoAb at a final dilution titer of 1:1000 for 1 h at 4°C. The specific mouse antihuman MoAb used included αCD19 (from hybridoma FMC63), αCD16 (from hybridoma 3G8), αCD4 (from hybridoma OKT4), and αCD8 (from hybridoma B9.8) for B cells, natural-killer (NK) cells, helper T (CD4+) cells, and cytotoxic T (CD8+) cells, respectively. Then, BioMag (PerSeptive Biosystems; Framingham, MA), containing goat antimouse IgG conjugated with magnetic beads, was used at 2.5 × 107/mL for lymphocyte depletion. BioMag depletion was done twice in order to ensure > 99% depletion of the desired population of lymphocytes.

Activation of Effectors

EAL were incubated at 1 to 2 × 106 cells/mL in LCM alone or with the different concentrations of cytokines mentioned above and/or αCD3. A cell proliferation assay was performed after 3 days of culture at 37°C in a carbon dioxide incubator. Cell-mediated cytotoxicity and IFN-γ production by the EAL were determined after 6 days of culture at 37°C in a carbon dioxide incubator.

Enzyme-Linked Immunosorbent Assay Analysis of IFN-γ Levels

The 6-day cultured media of the various groups were centrifuged at 4°C, and supernatants were collected and stored at − 70°C in aliquots of 0.25 mL in microcentrifuge tubes. IFN-γ was determined in duplicate by employing the enzyme-linked immunosorbent assay (ELISA) kit (Quantikine; R&D Systems) for a solid-phase ELISA method that employed the quantitative “sandwich” enzyme immunoassay technique. Positive and negative controls were included in the assay.

Cell Proliferation Assay

The effects of different treatments on the proliferation of activated lymphocytes were determined by 3H-thymidine incorporation assay. In brief, 0.1 mL of cultured EAL was dispensed into a 96-well flat-bottom microplate in triplicate. 3H-thymidine at 2 μCi/mL was added to each well, and the cells were incubated at 37°C in a carbon dioxide incubator overnight. Incorporation of 3H was determined by a β-plate counter, with results expressed as counts per minute (cpm) ± SEM.

Cell-Mediated Cytotoxicity Assay

The overnight 51Cr release assay was used to detect the cell-mediated cytotoxicity. The results were expressed as total percentage of lysis and net percentage of lysis, according to the following formulas:

where percentage of lysis = (total percentage of lysis of the test effectors) − (total percentage of lysis of the medium control). One lytic unit (LU) was defined as the number of effectors required to cause a 30% lysis (specific or net percentage of lysis) of 5 × 103 target cells.

Statistical Analysis

The Wilcoxon signed-ranks test was used to determine whether or not there existed a significant difference in IFN-γ levels, proliferative activity, and cytolytic activity among the different culture conditions.

Single Signal Stimulation Is Enough for EAL To Produce IFN-γ

Twelve of 35 EAL cases underwent cytokine analysis. It was found that IL-2 and IL-12 alone could enhance IFN-γ production by EAL, but not IL-4, IL-7, or IL-10 alone. αCD3 gave an even stronger stimulation that enhanced IFN-γ production. The study cytokine in combination with αCD3 did not further augment IFN-γ production, and even depressed production when αCD3 was combined with IL-10 (Fig 1 ). IL-12 in combination with IL-2 also had a synergistic effect on IFN-γ production, at a degree similar to αCD3 stimulation (Fig 1). MoAb incubation and the BioMag depletion of lymphocyte subpopulation test were then performed on another two freshly isolated EAL samples, and the results showed that CD8+ T cells were responsible for IFN-γ production (Fig 2 ).

Profile of EAL Subpopulation Distribution

To determine whether there was any change in the proportion of the lymphocyte subpopulation, flow cytometry analysis was performed on the EAL after a culture of 6 days. Only IL-2, IL-7, IL-12, and αCD3 were used in this experiment. Samples from three patients were tested, and all showed similar results. It was found that the proportion of CD8+ T cells could be increased even with only a single cytokine treatment. The effect was highest when EAL was treated with IL-2, IL-7, and IL-12 together. The NK cell population did not change markedly after cytokine treatment (Table 1 ).

Both IL-2 and IL-7 Alone Were Able To Induce Proliferative Responses in the EAL, and Their Effect Was Enhanced Markedly by TCR-CD3 Engagement

Twenty-three freshly isolated EAL samples from the 35 patients with malignant pleural effusion received a proliferative examination on day 3 of culturing. The proliferation elicited by IL-2, IL-4, IL-7, IL-10, IL-12, and αCD3 on freshly isolated EAL is depicted in Figure 3 . A significantly higher proliferative rate was achieved with IL-2, IL-7, and αCD3. Next, the combined effect of two or more cytokines on the EAL was examined; a significant additive or synergistic effect was found when EAL was treated with IL-2 plus IL-7, IL-2 plus IL-7 and IL-12, IL-2 plus αCD3, IL-7 plus αCD3, IL-2 plus αCD3 and IL-7, and IL-2 plus αCD3 and IL-12 (Fig 4 ). IL-4 and IL-10 could not significantly enhance the proliferation of EAL no matter whether they were used alone, with IL-2, or in combination with αCD3 stimulation. After obtaining the above findings, MoAb incubation and the BioMag depletion of the lymphocyte subpopulation test were performed on four freshly isolated EAL cases with the culture condition of IL-2 plus IL-7, IL-12, or αCD3. It was found that the cells responsible for the proliferative response were the CD8+ T cells (Table 2 ).

IL-7, IL-12, or a Combined TCR-CD3 Engagement Could Enhance the Cytolytic Activity of EAL Against Autologous Tumor Cells in the Presence of IL-2

Thirteen freshly isolated EAL samples received a cytotoxic test after culturing under different conditions. Cytolytic activity against autologous tumors and K-562 cells was depressed in the EAL. The addition of IL-2, 300 IU/mL, slightly but significantly increased EAL cytolytic activity against both autologous tumors and K-562 cells (Table 3 ). The cytolytic activity against autologous tumors was decreased when IL-4 (p = 0.155) or IL-10 (p = 0.003) was added to IL-2, while it was markedly increased when αCD3 (p = 0.021), IL-7 (p = 0.037), or IL-12 (p = 0.131) was added, compared to IL-2 alone (Table 3). The further addition of IL-12 to IL-7 plus IL-2 did not increase its cytolytic activity against autologous tumors (p = 0.109). IL-2 plus αCD3 had a significantly better antiautologous tumor activity compared to IL-2 plus IL-4 (p = 0.003) and IL-2 plus IL-10 (p = 0.002). However there was no significant difference between IL-2 plus αCD3 and the following: IL-2 plus IL-7 (p = 0.075), IL-2 plus IL-12 (p = 0.173), or IL-2 plus IL-7 plus IL-12 (p = 0.328).

Among these stimulations, IL-2 plus IL-7 (p = 0.006), IL-2 plus IL-12 (p = 0.009), and IL-2 plus αCD3 (p = 0.002) had significantly better antiautologous tumor activity than K-562 killing activity, suggesting specific killing effects on autologous tumor cells (Table 3). In contrast, L-2 plus IL-4 had the highest K-562 killing activity among all conditioned EAL samples, and this effect was greater than that of killing autologous tumor cells (p = 0.087), suggesting a nonspecific killing effect.

MoAb incubation and the BioMag depletion test on cytotoxicity were performed with another five freshly isolated EAL samples. It was found that the cells responsible for the cytolytic activity of autologous tumor cells were CD8+ T cells (Table 4 ).

The use of immunostimulatory cytokines has become an increasingly promising approach in cancer immunotherapy. The major goal of immunotherapy is the activation of tumor-specific T lymphocytes capable of rejecting tumor cells in cancer patients. IL-2, IL-4, IL-7, IL-10, and IL-12 have been used to promote the generation of lymphokine-activated killer cells, to stimulate CTL development, or to induce tumor rejection or reduce metastases.23,57,1114 The data regarding IL-2, IL-7, and IL-12 were more consistently in favor of lymphokine-activated killer cell or CTL development, or driving the T-helper pathway to the T-helper 1 pathway. The data regarding IL-4 and IL-10 were more controversial.12,69,15 We tried to define the role of these cytokines on the function of EAL in the present study.

Our studies revealed that EAL from cancer patients were in an immunosuppressed status that could be reversed with cytokine stimulation. However, this full recovery of the cellular functioning (including cytokine production, cell proliferation, and cytolytic activity) of EAL required at least a double signal stimulation, and could be achieved with IL-2 plus IL-7, IL-2 plus IL-12, or IL-2 plus TCR-CD3 engagement. In addition, there was no significant difference in the functional performance of the activated EAL of various cancer subtypes.

There were still some minor differences among the EAL samples that were reactivated with IL-2 plus IL-7, IL-2 plus IL-12, and IL-2 plus αCD3 stimulation. IL-2 plus IL-12-stimulated EAL had a higher IFN-γ production than IL-2 plus IL-7, and IL-2 plus αCD3-stimulated EAL (Fig 1). The proliferative response was higher in IL-2 plus αCD3-stimulated EAL, followed by IL-2 plus IL-7-stimulated EAL, and IL-2 plus IL-12 stimulated EAL (Fig 4). For cytolytic activity against autologous tumors, IL-2 plus αCD3-stimulated EAL had higher, but insignificant, activity than IL-2 plus IL-12-stimulated EAL, followed by IL-2 plus IL-7. In contrast, cytolytic activity against K562 cells indicating a nonspecific killing activity was very low under these three conditions. Regardless of these differences, the effector cells reactivated by these stimulations were mainly CD8+ T cells. Based on the above findings, the CD8+ T cellular functions recovered through different cytokine treatment methods all belonged to CTL activities.

We found that it was not practical to add three cytokines or stimulations together. Based on the present study, IL-2 plus IL-7 and IL-12-derived EAL did not yield a higher cellular activity than IL-2-based double signal stimulation. In addition, IL-2 was required for TCR-CD3 engagement to be useful in the full restoration of the cellular functioning of immunosuppressed EAL, but not IL-4, IL-7, IL-10, or IL-12. Except for the situation of the IL-4 plus IL-2-stimulated EAL samples, which had a relatively high nonspecific killing effect (against K562 cells), IL-4, and especially IL-10, had a negative effect on the cellular functioning of EAL when IL-2 stimulation and/or TCR-CD3 engagement was performed.

It was found that different kinds of cellular functions (cytokine production, proliferation, cytotoxicity) did not correlate with each other, even within the same cellular function category; for example, the amounts of IFN-γ produced by stimulated EAL did not correlate with each other. Further, TCR-CD3 engagement alone could produce a rather high IFN-γ level. It was also found that the degree of cytokine production (IFN-γ) did not correlate with cytolytic activity, nor did proliferative response correlate with cytolytic activity.

Activated EAL after double signal stimulation may be an ideal source of effectors for use in adoptive immunotherapy. It can be obtained in large quantities and can be activated to give high levels of cytolytic activity against the autologous tumor. In addition, a small dose of IL-2 is sufficient to activate the EAL when another stimulation is also used, thus reducing the risk of the toxicity induced by the high-dose IL-2 that is usually used clinically. Furthermore, the pleural cavity is within easy reach, and thus treatment can be directly performed in situ after adequate drainage of the malignant pleural effusion. Therefore, the use of EAL or the direct manipulation of EAL in the pleural cavity may prove to be an alternative method to treat patients with malignant effusion. These methods may include the development of biological response modifier programs with combinations of ILs derived from the present study, and/or the management of malignant pleural effusions that may respond to regional infusions of combinations of these cytokines.

In summary, IL-4 and IL-10 had a negative effect on the recovery of the cellular functioning of EAL. However, TCR-CD3 engagement, IL-7, or IL-12 stimulation can restore the cellular functioning of immunosuppressed EAL when in the presence of IL-2. The recovered cellular function was mainly from CD8+ T cells.

Abbreviations: cpm = counts per minute; CTL = cytotoxic T lymphocytes; EAL = effusion-associated lymphocytes; ELISA = enzyme-linked immunosorbent assay; IFN = interferon; IL = interleukin; LCM = lymphocyte culture medium; LU = lytic unit; MoAb = monoclonal antibody; NK = natural-killer; TCR = T-cell receptor

Figure Jump LinkFigure 1. IFN-γ levels (picograms per milliliter) of 12 tested EAL cases. Freshly isolated EAL were cultured alone or with IL-2 300 IU/mL or 1200 IU/mL, IL-4 10 ng/mL or 30 ng/mL, IL-7 10 ng/mL or 30 ng/mL, IL-10 10 ng/mL or 30 ng/mL, IL-12 10 ng/mL or 30 ng/mL, and αCD3 at 1/100 dilution, or in combination, for 6 days. The culture supernatant was collected, and then IFN-γ levels were checked with ELISA. The results showed that single signal stimulation is enough for IFN-γ production by EAL.Grahic Jump Location
Figure Jump LinkFigure 2. The MoAb incubation and BioMag depletion test for the study of the lymphocyte subpopulation responsible for IFN-γ production. MoAb incubation and BioMag depletion were done with the EAL immediately after their separation from the tumor cells on day 0; each subgroup of cells was then cultured with IL-2, 300 IU/mL, with/without IL-12, 10 ng/mL, or αCD3 1/100 dilution for 6 days. The culture supernatant was collected, and then IFN-γ levels were checked with ELISA. The results showed that it was the CD8+ T cells that were responsible for IFN-γ production.Grahic Jump Location
Table Graphic Jump Location
Table 1. Mean Value of the Lymphocyte Subpopulation (%) of Cultured EAL*
* 

αCD3, 1/100 dilution; IL-2, 300 IU/mL; IL-4, IL-7, IL-10, IL-12, 10 ng/mL.

Figure Jump LinkFigure 3. Proliferation elicited by IL-2, IL-4, IL-7, IL-10, IL-12, or αCD3 on freshly isolated EAL. Freshly isolated EAL (2 × 106/mL) were cultured with IL-2, 300 IU/mL; IL-2, 1,200 IU/mL; IL-4, 10 ng/mL; IL-4, 30 ng/mL; IL-7, 10 ng/mL; IL-7, 30 ng/mL; IL-10, 10 ng/mL; IL-10, 30 ng/mL; IL-12, 10 ng/mL; IL-12, 30 ng/mL; or αCD3 1/100 dilution. A proliferation assay was performed on day 3. A significantly higher proliferative response was achieved with IL-2, IL-7, and αCD3 (*p < 0.01, compared with EAL alone). cpm = counts per minute.Grahic Jump Location
Figure Jump LinkFigure 4. Proliferative response of EAL treated with cytokines in combination. Freshly isolated EAL (2 × 106/mL) were cultured with IL-2, 300 IU/mL, alone, or αCD3 at 1/100 dilution alone, or in combination with IL-4, 10 ng/mL; IL-7, 10 ng/mL; IL-10, 10 ng/mL; or IL-12, 10 ng/mL. A proliferation assay was performed on day 3. A significant additive or synergistic effect was found when EAL were treated with IL-2 plus IL-7, IL-2 plus IL-7 and IL-12, IL-2 plus αCD3, IL-7 plus αCD3, IL-2 plus αCD3 and IL-7, and IL-2 plus αCD3 and IL-12. **p < 0.001, compared with IL-2 alone; *p < 0.01, compared with IL-2 alone. See Figure 3 legend for expansion of abbreviation.Grahic Jump Location
Table Graphic Jump Location
Table 2. The Effect of the MoAb Incubation and BioMag Depletion of the EAL Subpopulation on Proliferation*
* 

Data are presented as mean ± SEM. αCD3, 1/100 dilution; IL-2, 300 IU/mL; IL-7, IL-12, 10 ng/mL.

Table Graphic Jump Location
Table 3. Cytotoxicity Against Autologous Tumor Cells and K562 Cells*
* 

αCD3, 1/100 dilution; IL-2, 300 IU/mL; IL-4, IL-7, IL-10, IL-12, 10 ng/mL.

 

Wilcoxon signed ranks test (two-related samples), compared with EAL alone.

Table Graphic Jump Location
Table 4. The Effect of the MoAb Incubation and BioMag Depletion of the EAL Subpopulation on Cytotoxicity in Five EAL Samples*
* 

Data are presented as mean ± SEM. αCD3; 1/100 dilution; IL-2, 300 IU/mL; IL-7, IL-12, 10 ng/mL.

Chen, YM, Yang, WK, Whang-Peng, J, et al (1996) Elevation of interleukin-10 levels in malignant pleural effusion.Chest110,433-436. [PubMed] [CrossRef]
 
Chen, YM, Yang, WK, Ting, CC, et al Cross regulation by IL-10 and IL-2/IL-12 of the helper T cells and the cytolytic activity of lymphocytes from malignant effusion of lung cancer patients.Chest1997;112,960-966. [PubMed]
 
Chen, YM, Yang, WK, Whang-Peng, J, et al Restoration of the immunocompetence by IL-2 activation and TCR-CD3 engagement ofin vivoanergized tumor specific CTL from lung cancer patients.J Immunother1997;20,354-364. [PubMed]
 
Chen, YM, Ting, CC, Whang-Peng, J, et al Restoration of cytotoxic T lymphocytes function in malignant pleural effusion: interleukin-15 vs interleukin-2.J Interferon Cytokine Res2000;20,31-39. [PubMed]
 
Mackensen, A, Lindemann, A, Mertelsmann, R Immunostimulatory cytokines in somatic cells and gene therapy of cancer.Cytokine Growth Factor Rev1997;8,119-128. [PubMed]
 
Tsunoda, T, Tanimura, H, Yamaue, H, et al The promotive effect of interleukin 4 with interleukin 2 in the proliferation of tumor-infiltrating lymphocytes from patients with malignant tumor.Biotherapy1992;4,9-15. [PubMed]
 
Zheng, LM, Ojcius, DM, Garaud, F, et al Interleukin-10 inhibits tumor metastasis through an NK cell-dependent mechanism.J Exp Med1996;184,579-584. [PubMed]
 
Salazar-Onfray, F Interleukin-10: a cytokine used by tumors to escape immunosurveillance.Med Oncol1999;16,86-94. [PubMed]
 
Nabioullin, R, Sone, S, Mizuno, K, et al Interleukin-10 is a potent inhibitor of tumor cytotoxicity by human monocytes and alveolar macrophages.J Leukoc Biol1994;55,437-442. [PubMed]
 
Kung, PC, Goldstein, G, Reinherz, EL, et al Monoclonal antibodies defining distinctive human T cell surface antigens.Science1979;206,347-349. [PubMed]
 
Trinchieri, G Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes.Blood1994;84,4008-4027. [PubMed]
 
Wit, DD, Flemming, CL, Harris, JD, et al IL-12 stimulation but not B7 expression increases melanoma killing by patient cytotoxic T lymphocytes (CTL).Clin Exp Immunol1996;105,353-359. [PubMed]
 
Jicha, DL, Mule, JJ, Rosenberg, SA Interleukin 7 generates antitumor cytotoxic T lymphocytes against murine sarcomas with efficacy in cellular adoptive immunotherapy.J Exp Med1991;174,1511-1515. [PubMed]
 
Alderson, MR, Sassenfeld, HM, Widmer, MB Interleukin 7 enhances cytolytic T lymphocyte generation and induces lymphokine-activated killer cells from human peripheral blood.J Exp Med1990;172,577-587. [PubMed]
 
Terres, G, Coffman, RL The role of IL-4 and IL-10 cytokines in controlling an anti-tumor responsein vivo.Int Immunol1998;10,823-832. [PubMed]
 

Figures

Figure Jump LinkFigure 1. IFN-γ levels (picograms per milliliter) of 12 tested EAL cases. Freshly isolated EAL were cultured alone or with IL-2 300 IU/mL or 1200 IU/mL, IL-4 10 ng/mL or 30 ng/mL, IL-7 10 ng/mL or 30 ng/mL, IL-10 10 ng/mL or 30 ng/mL, IL-12 10 ng/mL or 30 ng/mL, and αCD3 at 1/100 dilution, or in combination, for 6 days. The culture supernatant was collected, and then IFN-γ levels were checked with ELISA. The results showed that single signal stimulation is enough for IFN-γ production by EAL.Grahic Jump Location
Figure Jump LinkFigure 2. The MoAb incubation and BioMag depletion test for the study of the lymphocyte subpopulation responsible for IFN-γ production. MoAb incubation and BioMag depletion were done with the EAL immediately after their separation from the tumor cells on day 0; each subgroup of cells was then cultured with IL-2, 300 IU/mL, with/without IL-12, 10 ng/mL, or αCD3 1/100 dilution for 6 days. The culture supernatant was collected, and then IFN-γ levels were checked with ELISA. The results showed that it was the CD8+ T cells that were responsible for IFN-γ production.Grahic Jump Location
Figure Jump LinkFigure 3. Proliferation elicited by IL-2, IL-4, IL-7, IL-10, IL-12, or αCD3 on freshly isolated EAL. Freshly isolated EAL (2 × 106/mL) were cultured with IL-2, 300 IU/mL; IL-2, 1,200 IU/mL; IL-4, 10 ng/mL; IL-4, 30 ng/mL; IL-7, 10 ng/mL; IL-7, 30 ng/mL; IL-10, 10 ng/mL; IL-10, 30 ng/mL; IL-12, 10 ng/mL; IL-12, 30 ng/mL; or αCD3 1/100 dilution. A proliferation assay was performed on day 3. A significantly higher proliferative response was achieved with IL-2, IL-7, and αCD3 (*p < 0.01, compared with EAL alone). cpm = counts per minute.Grahic Jump Location
Figure Jump LinkFigure 4. Proliferative response of EAL treated with cytokines in combination. Freshly isolated EAL (2 × 106/mL) were cultured with IL-2, 300 IU/mL, alone, or αCD3 at 1/100 dilution alone, or in combination with IL-4, 10 ng/mL; IL-7, 10 ng/mL; IL-10, 10 ng/mL; or IL-12, 10 ng/mL. A proliferation assay was performed on day 3. A significant additive or synergistic effect was found when EAL were treated with IL-2 plus IL-7, IL-2 plus IL-7 and IL-12, IL-2 plus αCD3, IL-7 plus αCD3, IL-2 plus αCD3 and IL-7, and IL-2 plus αCD3 and IL-12. **p < 0.001, compared with IL-2 alone; *p < 0.01, compared with IL-2 alone. See Figure 3 legend for expansion of abbreviation.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1. Mean Value of the Lymphocyte Subpopulation (%) of Cultured EAL*
* 

αCD3, 1/100 dilution; IL-2, 300 IU/mL; IL-4, IL-7, IL-10, IL-12, 10 ng/mL.

Table Graphic Jump Location
Table 2. The Effect of the MoAb Incubation and BioMag Depletion of the EAL Subpopulation on Proliferation*
* 

Data are presented as mean ± SEM. αCD3, 1/100 dilution; IL-2, 300 IU/mL; IL-7, IL-12, 10 ng/mL.

Table Graphic Jump Location
Table 3. Cytotoxicity Against Autologous Tumor Cells and K562 Cells*
* 

αCD3, 1/100 dilution; IL-2, 300 IU/mL; IL-4, IL-7, IL-10, IL-12, 10 ng/mL.

 

Wilcoxon signed ranks test (two-related samples), compared with EAL alone.

Table Graphic Jump Location
Table 4. The Effect of the MoAb Incubation and BioMag Depletion of the EAL Subpopulation on Cytotoxicity in Five EAL Samples*
* 

Data are presented as mean ± SEM. αCD3; 1/100 dilution; IL-2, 300 IU/mL; IL-7, IL-12, 10 ng/mL.

References

Chen, YM, Yang, WK, Whang-Peng, J, et al (1996) Elevation of interleukin-10 levels in malignant pleural effusion.Chest110,433-436. [PubMed] [CrossRef]
 
Chen, YM, Yang, WK, Ting, CC, et al Cross regulation by IL-10 and IL-2/IL-12 of the helper T cells and the cytolytic activity of lymphocytes from malignant effusion of lung cancer patients.Chest1997;112,960-966. [PubMed]
 
Chen, YM, Yang, WK, Whang-Peng, J, et al Restoration of the immunocompetence by IL-2 activation and TCR-CD3 engagement ofin vivoanergized tumor specific CTL from lung cancer patients.J Immunother1997;20,354-364. [PubMed]
 
Chen, YM, Ting, CC, Whang-Peng, J, et al Restoration of cytotoxic T lymphocytes function in malignant pleural effusion: interleukin-15 vs interleukin-2.J Interferon Cytokine Res2000;20,31-39. [PubMed]
 
Mackensen, A, Lindemann, A, Mertelsmann, R Immunostimulatory cytokines in somatic cells and gene therapy of cancer.Cytokine Growth Factor Rev1997;8,119-128. [PubMed]
 
Tsunoda, T, Tanimura, H, Yamaue, H, et al The promotive effect of interleukin 4 with interleukin 2 in the proliferation of tumor-infiltrating lymphocytes from patients with malignant tumor.Biotherapy1992;4,9-15. [PubMed]
 
Zheng, LM, Ojcius, DM, Garaud, F, et al Interleukin-10 inhibits tumor metastasis through an NK cell-dependent mechanism.J Exp Med1996;184,579-584. [PubMed]
 
Salazar-Onfray, F Interleukin-10: a cytokine used by tumors to escape immunosurveillance.Med Oncol1999;16,86-94. [PubMed]
 
Nabioullin, R, Sone, S, Mizuno, K, et al Interleukin-10 is a potent inhibitor of tumor cytotoxicity by human monocytes and alveolar macrophages.J Leukoc Biol1994;55,437-442. [PubMed]
 
Kung, PC, Goldstein, G, Reinherz, EL, et al Monoclonal antibodies defining distinctive human T cell surface antigens.Science1979;206,347-349. [PubMed]
 
Trinchieri, G Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes.Blood1994;84,4008-4027. [PubMed]
 
Wit, DD, Flemming, CL, Harris, JD, et al IL-12 stimulation but not B7 expression increases melanoma killing by patient cytotoxic T lymphocytes (CTL).Clin Exp Immunol1996;105,353-359. [PubMed]
 
Jicha, DL, Mule, JJ, Rosenberg, SA Interleukin 7 generates antitumor cytotoxic T lymphocytes against murine sarcomas with efficacy in cellular adoptive immunotherapy.J Exp Med1991;174,1511-1515. [PubMed]
 
Alderson, MR, Sassenfeld, HM, Widmer, MB Interleukin 7 enhances cytolytic T lymphocyte generation and induces lymphokine-activated killer cells from human peripheral blood.J Exp Med1990;172,577-587. [PubMed]
 
Terres, G, Coffman, RL The role of IL-4 and IL-10 cytokines in controlling an anti-tumor responsein vivo.Int Immunol1998;10,823-832. [PubMed]
 
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