HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nagoshi, M. Articles by Eberlein, T. J. Search for Related Content PubMed PubMed Citation Articles by Nagoshi, M. Articles by Eberlein, T. J.

Successful Adoptive Cellular Immunotherapy Is Dependent on Induction of a Host Immune Response Triggered by Cytokine (IFN- and Granulocyte/Macrophage Colony-Stimulating Factor) Producing Donor Tumor-Infiltrating Lymphocytes¹

Laboratory of Biologic Cancer Therapy, Department of Surgery, Division of Surgical Oncology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and systemic low dose rIL-2 effectively eradicates pulmonary metastases of the murine MCA-105 sarcoma. We described earlier that host CD8⁺ T cells are critical for tumor eradication and that successful treatment is associated with production of high levels of IFN- and granulocyte/macrophage (GM)-CSF by donor TIL in vitro. Here, we propose the mechanism through which adoptively transferred Thy-1.1⁺ TIL induce a host antitumor response in congenic Thy-1.2⁺ tumor-bearing mice. Donor Thy-1.1⁺ TIL were detected at the tumor site 12 h after transfer. These Thy-1.1⁺ cells produced IFN- and GM-CSF in situ. The percentage of Thy-1.1⁺ TIL at the tumor site increased up to 16.4 ± 4.9% 24 h after transfer but decreased to undetectable levels thereafter. In contrast, the percentages of host cells producing IFN- and GM-CSF continued to increase at the tumor site. These increases were significantly higher in TIL + rIL-2-treated mice compared with untreated mice and rIL-2-treated mice 48 h after TIL transfer. The appearance of IFN-⁺ and GM-CSF⁺ cells was followed by a large influx of host CD4⁺, CD8⁺, and Thy-1.2⁺ TIL and eventually by tumor eradication. This response was tumor specific since TIL obtained from MCA-205 did not induce high levels of IFN- and GM-CSF and did not induce tumor eradication of MCA-105 tumor. Coinjection of Thy-1.1⁺ TIL and anti-IFN- or anti-GM-CSF mAb significantly inhibited antitumor efficacy of the TIL + rIL-2 treatment. We conclude that successful adoptive immunotherapy in this model is mediated through cytokine production by adoptively transferred TIL that induce a host T cell-dependent antitumor response.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adoptive cellular immunotherapy (AIT)⁵ refers to a procedure that transfers ex vivo-prepared cells with antitumor activity to a tumor-bearing host. Examples of ex vivo-prepared cells used for this purpose include lymphokine-activated killer (LAK) cells, tumor-infiltrating lymphocytes (TIL) and tumor-draining lymph node cells (1, 2, 3, 4, 5, 6, 7, 8, 9). In animal experiments, especially in murine pulmonary micrometastasis models, adoptive immunotherapy with TIL and rIL-2 demonstrated a 50 to 100 times stronger antitumor efficacy than comparable therapy with LAK cells (10, 11). Early reports in animal models found the correlation between in vitro cytotoxic activities of T cells and in vivo antitumor efficacy (12, 13). This correlation suggested that the mechanism of tumor eradication by TIL was direct cytolysis. On the contrary, noncytolytic TIL were reported to show in vivo antitumor efficacy (14, 15, 16). Additionally, Barth et al. demonstrated that the effectiveness of TIL in vivo correlated better with their ability to specifically secrete lymphokines, such as IFN- and TNF-, than with their cytotoxicity in vitro (14). Tumor-specific secretion of granulocyte/macrophage colony-stimulating factor (GM-CSF) by lymph node lymphocytes was also found to be associated with in vivo therapeutic efficacy (17). In addition, we have shown not only that the CD3-activated TIL used in these AIT experiments produced high levels of IFN- and GM-CSF in vitro but also that the level of cytokines was associated with the in vivo efficacy of TIL (18). The controversy whether TIL induce tumor eradication by direct cytolysis, cytokine secretion, or both is still ongoing. It is also unknown to what extent a host immune response is required for tumor eradication during TIL + rIL-2 therapy and therefore how adoptively transferred TIL contribute to tumor eradication. In some reports, suppression of host immune cells by sublethal dose irradiation of the host or administration of anti-Thy-1.2 Ab before the treatment efficacy did not abrogate the efficacy of the AIT (14, 16). On the other hand, our group has shown that host CD8⁺ T cells are critical in this type of AIT by CD8⁺ T cell depletion and normal splenocyte recruitment study (19). To evaluate the role of adoptively transferred TIL in inducing a host immune response during TIL + rIL-2 treatment, we explored the changes in T cell subset concentrations of both donor and host at the tumor site using FACS analysis and immunohistochemistry.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Female 6- to 8-wk-old C57BL/6 (Thy-1.2⁺) and B6PL-Thy-1^a (Thy-1.1⁺) mice were obtained from The Jackson Laboratory, Bar Harbor, ME. C57BL/6 mice were used as recipients for all AIT experiments, and female B6PL-Thy-1^a mice were used for TIL preparation. SCID mice (C57BL/6J-prkdc^SCID/SzJ) were also used for recipient in some experiments. IFN- gene knockout mice (C57BL/6ifng^tmlTs) were used for IFN- gene knockout (gko) TIL preparation. The animals were housed in the Dana-Farber Animal Facility under National Institutes of Health/Harvard Medical School-approved animal subject conditions. All mice received animal laboratory chow and water ad libitum.

Tumor

The weakly immunogenic syngeneic methylcholanthrene (MCA)-induced fibrosarcomas MCA-105 and MCA-205 were kindly provided by Drs. J. Yang and S. A. Rosenberg (National Cancer Institute, Bethesda, MD). They were maintained in vivo by s.c. passage of 5 x 10⁵ cryopreserved or fresh tumor cells in C57BL/6 syngeneic mice or cultured in vitro (20, 21). MCA-105 and MCA-205 tumors were also used for generating TIL 2 to 3 wk after s.c. injection when tumor had reached a diameter of 1 to 2 cm.

Digestion of tumor and murine lungs

s.c. tumor and murine lungs were dissected, minced, and digested by mechanical stirring in a solution of 0.1% collagenase type IV, 0.01% hyaluronidase type V, and 0.002% DNase type I (Sigma Chemical Co., St. Louis, MO) in 40 ml of HBSS (BioWhittaker, Walkersville, MD) for 2 h at room temperature. The resulting single-cell suspension was filtrated through a sterile Nitex mesh (Lawshe Instrument Co., Rockville, MD) and washed twice in HBSS.

Preparation of TIL

The resulting single-cell suspension recovered from s.c. MCA-105 or MCA-205 tumors of B6PL-Thy-1^a (Thy-1.1⁺) or C57/6ifng^tm1Ts (IFN- gko) mice was resuspended at a density of 5 x 10⁵ cells/ml in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 IU/ml penicillin, 100 µg/ml streptomycin (all BioWhittaker), 5 x 10^-5 M 2-ME (Sigma), and 100 IU/ml rIL-2 (AMGEN, Thousand Oaks, CA). Then it was placed onto solid phase anti-CD3 mAb (supernatant from 145-2C11 hybridoma; American Type Culture Collection, Rockville, MD)-coated flasks (4). After 48 h, the cells were collected with cell scrapers (Costar, Cambridge, MA). The cells were washed and resuspended in culture medium as described above. Every 3 to 4 days, cultures were split by cell redistribution into new flasks with addition of new culture medium. TIL were expanded for 3 to 4 wk before they were used in in vivo experiments.

Cytokine release assay

T cells were placed in rIL-2-free medium for 24 h before experiments. For cytokine release, T cells (10⁶/ml) were placed in culture medium with or without anti-CD3 mAb coating. T cells were incubated at 37°C for 24 h; then the supernatant was collected, centrifuged to remove any cells, and frozen at -20°C. Aliquots were thawed and tested by ELISA for the presence of IFN- and GM-CSF.

Adoptive immunotherapy model

Thy-1.2⁺ mice were injected i.v. with 5 x 10⁵ MCA-105 or MCA-205 tumor cells in 1 ml of HBSS on day 0. Thy-1.1⁺ or IFN- gko TIL from s.c. MCA-105 or MCA-205 tumor were transferred i.v. at 5 x 10⁶ cells on day 3 after tumor injection. rIL-2 was given i.p. twice daily at 30,000 IU/ml from day 3 to day 7. Control groups received no treatment or rIL-2 only. Animals were killed on day 14 (MCA-205 tumor-injected animals) or day 21 (MCA-105 tumor-injected animals). Murine lungs were insufflated using a 15% solution of India ink (Farber-Castell Co., Lewisburg, TN), washed in water, and bleached in Fekete’s solution (22). The number of pulmonary metastases in treatment and control groups was counted in a blinded fashion and reported as the mean ± SEM. Each group consisted of 5 to 10 animals. Murine lungs were dissected and prepared for phenotyping and immunohistochemistry at different intervals following TIL transfer up to day 21.

Flow cytometric analysis

Single-cell suspensions of processed murine lungs were aliquoted at a concentration of 5 x 10⁵ lymphocytes/ml and stained by rat anti-mouse CD4/PE, CD8/FITC (both Becton Dickinson Immunocytometry Systems, Mountain View, CA), mouse anti-mouse Thy-1.1/PE (PharMingen, San Diego, CA), biotinylated rat anti-mouse Thy-1.2 (Becton Dickinson), rat anti-mouse Mac-3/FITC, mouse-anti-mouse NK-1.1/PE, rat anti-mouse IFN-, and GM-CSF (all PharMingen) mAb or control murine IgG For staining of intracellular cytokines (IFN- and GM-CSF); cells were prefixed with 4% paraformaldehyde (Sigma) for 10 min at 4°C and washed with staining buffer (BSS with 0.1% sodium azide, 0.1% saponin, and 5% FCS) twice (23, 24). For indirect staining, cells were incubated with primary mAb in staining medium (HBSS with 0.1% sodium azide and 5% FCS) for 30 min at 4°C. After two washings with staining buffer, cells were stained with FITC-conjugated goat anti-rat IgG (mouse adsorbed, Life Technologies, Gaithersburg, MD) or FITC-conjugated avidin (Becton Dickinson) for 30 min at 4°C. The stained cells were fixed with 1% paraformaldehyde at 4°C. Flow cytometric analysis of the stained lymphocytes was performed on a Coulter Epics C cytometer (Coulter Electronics, Hialeah, FL). Percentages of positive cells were calculated of the total lymphocyte population. Multiple samples were compared using the same gate settings for lymphocytes. Five to seven mice were analyzed in each group on each day. Each experiment was repeated at least three times using newly prepared TIL that showed efficacy in eradication of pulmonary metastatic tumors in vivo.

Immunohistochemistry

Specimens were embedded in OCT compound (Miles, Elkhart, IN) and snap frozen in liquid nitrogen (-170°C). Frozen blocks were stored at -80°C until used. Frozen serial sections were cut 5 mm thick on a microtome-cryostat. Sections were fixed in cold acetone for 5 min, preincubated with rabbit serum at room temperature for 15 min, and subsequently incubated with primary Abs (rat anti-mouse CD8, CD4, Thy-1.2, IFN-, GM-CSF, and biotinylated mouse anti-mouse Thy-1.1) at 4°C overnight, biotinylated rabbit anti-rat IgG (mouse adsorbed, Vector Laboratories Inc., Burlingame, CA) at room temperature for 30 min, and avidin-peroxidase conjugate at room temperature for 30 min. Then sections were incubated with 0.03% H₂O₂ and 0.06% diaminobenzidine (Vector).

Cytokine depletion

On day 3 at the same time of TIL injection, some mice were injected with 1.0 ml of HBSS containing 100 µg of rat anti-mouse IFN- IgG1 (R4-6A2 hybridoma) obtained from American Type Culture Collection (ATCC, Rockville, MD) (25). Previous experiments showed depletion of IFN- lasting for at least 48 h (14, 16, 25). Because of the timing, this depletion inhibited the secretion of IFN- by the adoptively transferred Thy-1.1⁺ cells as well as host Thy-1.2⁺ cells. Similarly, mice were injected on day 3 with 1.0 ml of HBSS containing 100 µg of rat anti-mouse GM-CSF IgG2a (clone: MP1-22E9, PharMingen) (26). Injection of control rat IgG1 (PharMingen) did not affect the number of pulmonary metastases in TIL + rIL-2 treatment.

Statistical analysis

The statistical significance between treatment and control groups was determined by the nonparametric Wilcoxon rank sum test and was considered significant when the p value was <0.05. All p values are two-sided.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vivo efficacy of Thy-1.1⁺ donor TIL

To test the efficacy of tumor eradication using Thy-1.1⁺ donor TIL in vivo, the numbers of pulmonary metastases were counted on day 21 after tumor injection in MCA-105 tumor-bearing mice (Fig. 1A). Each treatment group consisted of 5 to 10 mice. Only the TIL-105 + rIL-2 treatment significantly reduced the numbers of pulmonary metastases compared with control untreated mice (p < 0.01; Fig. 1A). The number of metastases in rIL-2-treated mice in the absence of TIL or TIL-105-treated mice in the absence of rIL-2 was similar to those in control animals. To determine the specificity of TIL-mediated tumor eradication, TIL obtained from MCA-205 tumor was used as control. TIL-205 + rIL-2 did not eradicate MCA-105 tumor. However, in MCA-205 tumor-bearing mice, the TIL-205 + rIL-2 treatment significantly reduced the numbers of pulmonary metastases compared with control untreated mice, IL-2 only-treated mice, or TIL-205 only-treated mice (Fig. 1B). However, TIL-105 + rIL-2 had no effect in MCA-205 tumor-bearing mice.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Treatment of MCA-105 (A) and MCA-205 (B) tumor-bearing mice. Data are expressed as the numbers of pulmonary metastases counted on day 21 (MCA-105) and day 14 (MCA-205) in untreated control, rIL-2-treated, TIL-treated, and TIL + rIL-2-treated groups of tumor-bearing mice. Only when TIL and tumor were from the same origin did TIL + rIL-2 treatment significantly reduce the numbers of pulmonary metastases compared with other groups (* and **, p < 0.01, p < 0.05, respectively).

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Treatment of MCA-105 tumor-bearing w.t. C57BL/6 or C57BL/6SCID mice. Data are expressed as the numbers of pulmonary metastases in rIL-2-treated control and TIL + rIL-2-treated groups of either w.t. or SCID tumor-bearing mice on day 21 after tumor injection. TIL + rIL-2 treatment significantly reduced the numbers of pulmonary metastases in w.t. mice, but not in SCID mice (p < 0.01).

Utilizing the MCA-105 model, we further investigated the mechanisms of AIT with TIL + rIL-2. Donor Thy-1.1⁺ TIL-105 reproducibly contained >95% CD8⁺ T cells and 0 to 1% CD4⁺ T cells at the time of AIT. The changes in T cell subsets at the tumor sites were followed by flow cytometric analysis. Percentages of positive cells of the total lymphocyte population were calculated. Multiple samples were compared using the same gate settings for lymphocytes. In vivo, donor Thy-1.1⁺ cells were detectable in the lungs of TIL-105 + rIL-2-treated mice for only 72 h. The percentage of donor Thy-1.1⁺ cells in the lungs of TIL-105 + rIL-2-treated mice increased up to 16.4% 24 h after TIL transfer and then gradually decreased. On day 3 after transfer (day 7 after tumor injection), donor Thy-1.1⁺ cells were undetectable (Fig. 3A). In contrast, the percentage of host Thy-1.2⁺ cells rapidly increased in TIL-105 + rIL-2-treated but not in untreated mice after the disappearance of donor Thy-1.1⁺ cells. These Thy-1.2⁺ cells consisted of almost equal percentages of CD8⁺ and CD4⁺ T cells. On day 7 after tumor injection, the percentages of CD8⁺, CD4⁺, and Thy-1.2⁺ cells in TIL-105 + rIL-2-treated mice had increased to significantly higher levels compared with those of the control mice (Thy-1.2⁺ cells: 83.9% vs 34%, p < 0.05; CD8⁺ cells: 16.2% vs 10.1%, p < 0.05; CD4⁺ cells: 15.9% vs 8.1%, p < 0.05, respectively). The high percentages of host Thy-1.2⁺, CD8⁺, and CD4⁺ T cells were also seen in lungs of rIL-2-treated mice as well as TIL-205 + rIL-2-treated mice (Fig. 3, B and C, respectively), suggesting that the composition of lymphocytic infiltrates were similar for all three treatments. After day 7, high levels of CD8⁺, CD4⁺, and Thy-1.2⁺ T cells were sustained in TIL-105 + rIL-2-treated mice but were not always significantly different from those of control mice.

View larger version (33K): [in this window] [in a new window]   FIGURE 3. The percentages of Thy-1.1+ donor cells, Thy-1.2+ host cells, and CD8+ and CD4+ T cells in lungs of untreated (, A, B), TIL-105 + rIL-2-treated (, A, C), IL-2-treated (, B), and TIL-205 +rIL-2-treated mice (, C) bearing MCA-105 tumor. The percentage of Thy-1.1+ cells peaked 24 h after TIL transfer and decreased thereafter, and Thy-1.1+ cells were undetectable after 3 days as determined by FACS analysis (A). The percentages of Thy-1.2+, CD8+, and CD4+ T cells in TIL + rIL-2-treated mice increased to significantly higher levels by day 7 compared with untreated control mice (p < 0.05, respectively (A)). The increases in CD8, CD4, and Thy-1.2+ cells in IL-2 alone- or TIL-205-treated mice were considerably albeit not statistically significant. In all the FACS analysis experiments, percentages of positive cells were calculated of the total lymphocyte population. Five to seven mice were analyzed in each group on each day. Each experiment was repeated at least three times using newly prepared TIL that showed efficacy in eradication of pulmonary metastatic tumors in vivo.

View larger version (118K): [in this window] [in a new window]   FIGURE 4. Microscopic findings in the lungs of control (A, x50, original magnification), rIL-2-treated (B, x50), and TIL-105 + rIL-2-treated mice on day 7 after tumor injection in the MCA-105 model (C, x50). Control untreated lungs showed viable metastatic foci (between arrows) with minimal lymphocytes outside the tumor. rIL-2-treated lungs showed viable metastatic foci (surrounded by arrowheads) with scattered (B) lymphocytes surrounding the tumor. In contrast, TIL-105 + rIL-2-treated lungs showed exudate and a large amount of lymphocytic infiltration (C).

View larger version (114K): [in this window] [in a new window]   FIGURE 5. Microscopic findings of mice treated with TIL-105 + rIL-2 using consecutive sections of the lungs in MCA-105 model. Hematoxylin and eosin staining (A, x200, original magnification) and immunostainings for CD8 (B, x200) and CD4 (C, x200) were performed on day 7. Both CD8+ and CD4+ T cells were found in tumor foci without obvious difference in number.

View larger version (147K): [in this window] [in a new window]   FIGURE 6. Microscopic findings in the lungs in MCA-105 tumor-bearing mice treated with TIL-105 + rIL-2. Immunostainings for Thy-1.2 (A, x200; inset, x400, original magnification) and Thy-1.1 (B, x200) were performed on day 7. Thy-1.2+ host cells infiltrate into tumor foci (A). Thy-1.1+ donor cells were undetectable in tumor foci (B).

Earlier, we showed that anti-CD3-activated TIL-105 produced cytokines among which IFN- and GM-CSF were the most prominent (18). In addition, phenotype analysis showed that in vitro-activated Thy-1.1⁺ TIL-105 consisted of 68.5 ± 10.5% IFN-⁺ cells and 11.5 ± 1.4% GM-CSF⁺ cells (average ± SE; data not shown). As shown in Figure 3A, the percentage of Thy-1.1⁺ cells in lungs of TIL-105 + rIL-2-treated mice increased up to 16.4 ± 5.0% after 24 h and decreased to undetectable levels thereafter (Fig. 7). The rapid increase in the percentages of IFN-⁺ cells and GM-CSF⁺ cells followed the appearance of donor Thy-1.1⁺ cells. The sustained levels of IFN-⁺ cells and GM-CSF⁺ cells from day 5 to day 10 could not be due to donor Thy-1.1⁺ cells (Fig. 7). As shown in Figure 8, the percentages of IFN-⁺ and GM-CSF⁺ cells in lungs of TIL-105 + rIL-2-treated mice were higher than those in lungs of rIL-2-treated or untreated control mice, reaching significance on day 5 after tumor injection (IFN-⁺ cells: 24.7% vs 5.7% and 7.0%, p < 0.01; GM-CSF⁺ cells: 38.9% vs 17.4% and 21.1%, p < 0.05, Fig. 8A). Likewise, the increase in percentage of IFN-⁺ and GM-CSF⁺ cells was significantly higher in MCA-105 tumor treated with TIL-105 than in MCA-105 tumor treated with TIL-205 on day 5 (IFN-⁺ cells: 24.7% vs 7.7%; GM-CSF⁺ cells: 38.9% vs 25.9%, p < 0.05, respectively; Fig. 8B). Similarly, the increases in IFN-⁺ and GM-CSF⁺ cells in MCA-205 tumor treated with TIL-205 were significantly higher than those treated with TIL-105 on day 5 (IFN-⁺ cells: 29.1% vs 12.9%, p < 0.05; GM-CSF⁺ cells: 35.1% vs 18.8%, p < 0.05, respectively; Fig. 8C).

View larger version (15K): [in this window] [in a new window]   FIGURE 7. The percentages of Thy-1.1+, IFN-+, and GM-CSF+ cells in lungs of TIL-105 + rIL-2-treated mice as determined by FACS analysis in the MCA-105 model. After the appearance of Thy-1.1+ donor cells on day 4, IFN-+, and GM-CSF+ cells rapidly increased on day 5 and gradually decreased thereafter.

View larger version (29K): [in this window] [in a new window]   FIGURE 8. Cytokine production detected by FACS analysis. A, Percentages of IFN-+ cells, GM-CSF+ cells in lungs of untreated (), rIL-2-treated (), and TIL-105 + rIL-2-treated mice in MCA-105 tumor-bearing mice (). The percentages of IFN-+ and GM-CSF+ cells were significantly higher in TIL + rIL-2-treated mice than in untreated control mice on day 5 (*, **; p < 0.01, p < 0.05, respectively). B, Percentages of IFN-+ and GM-CSF+ cells in lungs of MCA-105 tumor-bearing mice treated with MCA-105 or MCA-205-derived TIL. The increases in IFN-+ and GM-CSF+ cells in MCA-105 tumor treated with TIL-105 were significantly higher than those in tumors treated with TIL-205 on day 5 (p < 0.05). C, Percentages of IFN-+ and GM-CSF+ cells in lungs of MCA-205-bearing mice treated with MCA-105 or MCA-205-derived TIL. The increases in IFN-+ and GM-CSF+ cells in MCA-205 tumor treated with TIL-205 were significantly higher than those in tumors treated with TIL-105 on day 5 (p < 0.05). In all the FACS analysis experiments, percentages of positive cells were calculated of the total lymphocyte population.

View larger version (33K): [in this window] [in a new window]   FIGURE 9. Double staining and FACS analysis on lungs of TIL-105 + rIL-2-treated mice in the MCA-105 model at 12 h (A) and 96 h (B) after TIL injection. The percentages of IFN-+ and GM-CSF+ cells are expressed on the x-axis, and the percentage of Thy-1.1+ cells is expressed on the y-axis. Twelve hours after TIL transfer, Thy-1.1+ IFN-+ donor cells, and Thy-1.1+ GM-CSF+ donor cells were detected at the tumor site (A). On the contrary, 96 h after TIL transfer, Thy-1.1+ cells disappeared but Thy-1.1- IFN-+ host cells and Thy-1.1- GM-CSF+ host cells were detected at the tumor sites (B).

View larger version (176K): [in this window] [in a new window]   FIGURE 10. Immunostainings of consecutive sections for Thy-1.1 (A, x200, original magnification), IFN- (B, x200), GM-CSF (C, x200), and Thy-1.2 (D, x200) in TIL-105 + rIL-2-treated MCA-105 tumor-bearing mice 24 h after treatment. Thy-1.1+ donor TIL (arrows), IFN-+ cells (small arrowheads), and GM-CSF+ cells (large arrowheads) were located at the periphery of the tumor foci. The distributions of IFN-+ cells, GM-CSF+ cells, and Thy-1.1+ cells were almost identical, at the periphery of the tumor foci. However, in contrast, Thy-1.2+ cells were detected inside the metastatic foci (large arrows).

View larger version (26K): [in this window] [in a new window]   FIGURE 11. Appearance of macrophages and NK cells detected by FACS analysis. A, Percentages of Mac-3+ and NK-1.1+ cells in lungs of untreated (), rIL-2-treated (), and TIL-105 + rIL-2-treated mice () in MCA-105 tumor-bearing mice. The percentages of Mac-3+ and NK-1.1+ cells were significantly higher in TIL + rIL-2 than in untreated control mice on day 5 (*, **; p < 0.01, p < 0.05, respectively). B, Percentages of Mac-3+ and NK-1.1+ cells in lungs of MCA-105 tumor-bearing mice treated with MCA-105 or MCA-205-derived TIL as determined by FACS analysis. The increases in Mac-3+ and NK-1.1+ cells in MCA-105 tumor treated with TIL-105 were significantly higher compared with those in tumors treated with TIL-205 on day 7 (Mac-3+ cells: p < 0.05; NK-1.1+ cells: p < 0.01, respectively). C, Percentages of Mac-3+ and NK-1.1+ cells in lungs of MCA-205-bearing mice treated with MCA-105 or MCA-205-derived TIL as determined by FACS analysis. The increases in Mac-3+ and NK-1.1+ cells in MCA-205 tumor treated with TIL-205 were significantly higher than those in tumors treated with TIL-105 on day 7 (p < 0.05). In all the FACS analysis experiments, percentages of positive cells of the total lymphocyte population were calculated.

IFN- and GM-CSF depletions

To confirm the importance of IFN- and GM-CSF in TIL + rIL-2 treatment, MCA-105 tumor-bearing mice were injected i.p. with anti-mouse IFN- or anti-mouse GM-CSF mAbs at the same time as TIL injection. The numbers of pulmonary metastases in anti-IFN- mAb injected animals were similar to those of control untreated animals (172 metastases), whereas administration of anti-GM-CSF mAb resulted in 86 metastases (Table I). Thus, antitumor efficacy of TIL + rIL-2 treatment was completely inhibited by depletion of IFN- and partially inhibited by depletion of GM-CSF. Because of its timing (day 3, immediately following adoptive transfer of Thy-1.1⁺ cells), it should be noted that mAbs depleted host- as well as donor-produced IFN- and GM-CSF.

View larger version (24K): [in this window] [in a new window]   FIGURE 12. Treatment of MCA-105 tumor-bearing mice with rIL-2 alone, with TIL from w.t. C57BL/6 mice +rIL-2, or TIL from IFN- gko C57BL/6 mice (gko-TIL) + rIL-2. Data are expressed as the numbers of pulmonary metastases. Only TIL from w.t. C57BL/6 combined with rIL-2 significantly reduced the numbers of pulmonary metastases compared with other groups (* and **, p < 0.01 and p < 0.05, respectively).

View this table: [in this window] [in a new window]   Table II. Cytokine-producing ability of conventional and IFN- gko-TILa in vitro

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We detected Thy-1.1⁺ donor TIL at the tumor site immediately following adoptive transfer. The percentage of Thy-1.1⁺ cells actually increased over time. After 96 h, however, no donor Thy-1.1⁺ cells could be detected in the lungs by immunohistochemistry or FACS analysis (Figs. 3, 6, and 9). These findings are consistent with those of Matsumura et al. in the MCA-106 and MCA-205 model (5): Cloned MCA-106 TIL transferred into MCA-106 tumor-bearing mice accumulated at the tumor sites. This accumulation was apparent over a 24-h period after transfer and was associated with tumor eradication. Apparent from this study was also that the accumulation and subsequent tumor eradication was tumor specific. No accumulation of MCA-106 TIL was detected in MCA-205 tumor-bearing animals. Likewise, in our study, TIL-105 used in AIT induced eradication of MCA-105 tumor but TIL-205 did not (Fig. 1). Therefore, tumor eradication induced by adoptively transferred TIL in vivo seems to be dependent on a transient accumulation of tumor-specific donor TIL at the tumor site.

The hypothesis that donor TIL induce tumor eradication through cytokine secretion was based on the observation that noncytolytic TIL could eradicate murine pulmonary micrometastases in vivo (14, 15, 16, 17). Some investigators also demonstrated that anti-cytokine Abs inhibited the antitumor efficacy of TIL in vivo (14, 16). Similarly, our study showed that Thy-1.1⁺ TIL cultured in low dose rIL-2 (100 IU/ml) induced in vivo antitumor efficacy in Thy-1.2⁺ hosts (Fig. 1), yet these TIL showed little or no tumor-specific cytotoxic activity in vitro in 4-h or 6-h chromium release assays (data not shown) and are detectable in the lungs of the host for only 72 h. This would imply that by itself the donor T cell-mediated cytolysis of tumor is insufficient to yield the results observed on day 21 (MCA-105) or day 14 (MCA-205 (Fig. 1)).

We previously reported that depletion of host CD8⁺ T cells but not CD4⁺ T cells before TIL transfer abrogated antitumor efficacy. Additionally, adding back normal splenocytes after CD8⁺ cell depletion reconstituted antitumor efficacy (19). In the current study, we demonstrated that successful AIT with TIL + rIL-2 was dependent on an immunocompetent host (Fig. 2). A significant infiltration of host Thy-1.2⁺, CD8⁺, and CD4⁺ T cells at tumor sites was observed in TIL-105 + rIL-2-treated mice as determined by FACS analysis and histology (Figs. 3A and 4C). Although increased percentages of Thy-1.2⁺, CD8⁺, and CD4⁺ T cells were also found in rIL-2-treated and TIL-205 + rIL-2-treated animals (Fig. 3, B and C),, histologic evaluation showed a much larger infiltrate in TIL-105 + rIL-2-treated animals compared with all other groups (Fig. 4). This suggests that the infiltrate of host T cells is more a reflection of IL-2 than anything else. However, only treatment with TIL + rIL-2 caused a massive infiltration of host CD8⁺ and CD4⁺ cells into metastatic foci and tumor lysis on day 7 (Figs. 4C, 5, and 6). TIL derived from MCA-205 tumor caused no tumor lysis or lymphocytic infiltration even in combination with rIL-2 (data not shown). Perhaps with higher doses of rIL-2, a larger percentage of CD4⁺ and CD8⁺ T cells would infiltrate into the tumor, consistent with prior reports that rIL-2 treatment can be effective at very high doses, but occurs at the expense of host toxicity. We are utilizing relatively low doses of rIL-2. Thus, recruitment of host immune cells and infiltration into the tumor in response to adoptive transfer of T cells in a crucial event in successful eradication.

How does recruitment of host cells occur? The evaluation of cytokines (IFN- and GM-CSF) using FACS analysis and immunohistochemistry over time showed the appearance of cytokine-producing donor TIL at the periphery of the tumor site within 24 h (Figs. 9 and 10). Shortly after the increase in Thy-1.1⁺ T cells, there was an increase in IFN-⁺ and GM-CSF⁺ Thy-1.1-negative host cells (Figs. 7 and 9B). Only the combination of tumor-specific TIL and IL-2 induced the significant increase of IFN-⁺ and/or GM-CSF⁺ host cells on day 2 after TIL transfer (Figs. 7 and 8). Depletion of these cytokines completely (IFN-) or partially (GM-CSF) inhibited tumor eradication (Table I). In addition, TIL deficient in production of IFN- (gko-TIL) failed to induce an effective antitumor response (Fig. 12). These data suggest that cytokines produced by the transferred TIL trigger the initiation of a cascade of events in the host resulting in recruitment of host immune cells. This may partially explain why previous LAK cell and TIL therapies in humans had unpredictable results. Some patients with large amounts of disease responded completely, and some patients with very little metastatic disease had no response. It may also explain the observation during these trials of a "triggering" response; i.e., once switched on, the patient had a rapid and dramatic antitumor response.

It is controversial to what extent a host immune response is required for tumor eradication in AIT. There have been some reports supporting that AIT is independent from host immune response. Sublethal dose irradiation of host before treatment has been reported not to affect the efficacy of TIL+ rIL-2 therapy (14, 16). However, irradiation-resistant host cells such as macrophages may participate in the tumor eradication. Tuttle et al. reported that IFN- produced by adoptively transferred T cells plays a key role in AIT, but depletion of Thy-1.2⁺ cells by mAb 24 h before treatment did not abrogate the effectiveness of AIT in a 14-day protocol (16). These findings are not necessarily in contrast with our findings if the depletion of Thy-1.2⁺ cells lasted for only 24 to 48 h. In our earlier studies (19), we found that depletion of either CD4 or CD8 T cells by mAbs lasted for only 48 h. In addition, our experiment in C57BL6/SCID mice suggests that an immunocompetent host is required for successful AIT (Fig. 2).

It is possible that cytokines other than IFN- and GM-CSF are produced locally at the tumor site in this model such as TNF- (14, 16, 30). Preliminary data show that the percentage of TNF-⁺ cells in the lungs of TIL + rIL-2-treated mice is significantly higher than in those in untreated and rIL-2-treated mice in MCA-105 and MCA-205 models (data not shown). IFN- and TNF- are cytokines that may directly exert a cytotoxic effect on tumor cells. Recombinant murine IFN- had a significant antiproliferative effect against MCA-105 tumor cells when tested in a [³H]TdR uptake assay (16). Nonspecific inflammatory responses can be mediated by IL-2, IFN-, GM-CSF, and TNF- which can further activate macrophages, dendritic cells, NK cells, and granulocytes that may produce cytokines such as IL-1, IL-1ß, IL-2, IL-6, and IL-12 (14, 25, 31, 32, 33, 34, 35, 36). Macrophages activated by IFN- had increased antitumor cytotoxicity in vitro (37, 38, 39). Mule et al. reported that host macrophages seemed to play an important role in AIT using splenocytes from tumor-bearing mice (40). Therefore, host immune cells, such as macrophages and CD8⁺ T cells, are likely to play an important role in eradication of tumor. APC such as dendritic cells and macrophages activated by GM-CSF may participate in the establishment of an antitumor immune response (17, 41, 42, 43, 44, 45). Our data demonstrated an enhanced infiltration of macrophages, NK cells, and dendritic cells at the tumor sites in TIL + rIL-2-treated mice compared with other treatment groups (Fig. 11 and data not shown).

In spite of the nonspecific infiltration, tumor eradication by TIL + rIL-2 was a tumor-specific reaction. Through restimulation by specific tumor-associated Ags (TAA) (one of which was recently identified by Itoh et al. (46)) at the tumor site, adoptively transferred TIL may express cytotoxicity against tumor cells and release cytokines. The importance of this was indicated by AIT experiments in which TIL-105 had little efficacy against MCA-205 tumor and TIL-205 had little efficacy against MCA-105 tumor (Fig. 1). However, highly significant tumor eradication and increased levels of IFN- and GM-CSF production at the tumor sites were observed when TIL-105 or TIL-205 were transferred into MCA-105 and MCA-205 tumor-bearing animals, respectively (Fig. 8, B and C). Thus, local reactivation of adoptively transferred TIL by TAA may initiate the cytokine-mediated recruitment of host cells. Fig. 13 summarizes our proposed mechanism of AIT. Immediately after TIL transfer, adoptively transferred T cells migrate to the tumor sites and release cytokines. This local cytokine production initiates the influx of host immune cells such as NK cells, macrophages, dendritic cells, CD8⁺ T cells, and CD4⁺ T cells to the tumor sites. The availability of APC may promote Ag presentation, which may induce T cells to proliferate and exhibit tumor eradication.

View larger version (18K): [in this window] [in a new window]   FIGURE 13. The proposed mechanism of AIT using TIL + rIL-2 in MCA-105 tumor-bearing mice. Adoptively transferred CD8+ T cells migrate to the periphery of the tumor and are restimulated by specific TAA (initial phase). Adoptively transferred TIL may express cytotoxicity against tumor cells and release cytokines (IFN-, GM-CSF, and TNF-) that induce the influx of host immune cells such as macrophages, NK cells, granulocytes, dendritic cells, CD8+ T cells, and CD4+ T cells into tumor (host phase). This event further promotes the availability of APC to present Ag, enhance cytokine production at the tumor sites, and induce T cell proliferation and a subsequent host immune response against tumor (tumor destruction phase).

Our findings confirm the hypothesis that the initiation of a cytokine cascade at the tumor site is triggered by cytokine-producing adoptively transferred TIL. We conclude that successful AIT is dependent on the production of cytokines such as IFN- and GM-CSF at the tumor site by donor TIL, followed by the infiltration of host CD8⁺ and CD4⁺ T cells into tumor with further production of these cytokines. The result is specific tumor eradication.

	Footnotes

 
¹ This work is supported by National Institute of Health R01 Grants CA45854 and CA606662, and by an American Cancer Society Faculty Research Award, FRA407.

² Both authors contributed equally to this work.

³ Current address: Surgical University Hospital, University of Heidelberg, INF 110, 69120 Heidelberg, Germany.

⁴ Address correspondence and reprint requests to Dr. Timothy J. Eberlein, Laboratory of Biologic Cancer Therapy, Department of Surgery, Division of Surgical Oncology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115. E-mail address:

⁵ Abbreviations used in this paper: AIT, adoptive immunotherapy; LAK cells, lymphokine-activated killer cells; TIL, tumor-infiltrating lymphocytes; GM-CSF, granulocyte/macrophage colony-stimulating factor; gko, -interferon gene knockout; w.t., wild-type; TAA, tumor-associated antigens; MCA, methylcholanthrene.

Received for publication May 27, 1997. Accepted for publication September 19, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Eberlein, T. J., M. Rosenstein, S. A. Rosenberg. 1982. Regression of a disseminated syngeneic solid tumor by systemic transfer of lymphoid cells expanded in interleukin 2. J. Exp. Med. 156:385.[Abstract/Free Full Text]
2. Geiger, J. D., P. D. Wagner, M. J. Cameron, S. Shu, A. E. Chang. 1993. Generation of T-cells reactive to the poorly immunogenic B16-BL6 melanoma with efficacy in the treatment of spontaneous metastases. J. Immunother. 13:153.
3. Lafreniere, R., S. A. Rosenberg. 1985. Successful immunotherapy of murine experimental hepatic metastases with lymphokine-activated killer cells and recombinant interleukin 2. Cancer Res. 45:3735.[Abstract/Free Full Text]
4. Massaro, A. F., D. D. Schoof, A. Rubinstein, M. Zuber, V. F. Leonard, T. J. Eberlein. 1990. Solid-phase anti-CD3 antibody activation of murine tumor-infiltrating lymphocytes. Cancer Res. 50:2587.[Abstract/Free Full Text]
5. Matsumura, T., J. J. Sussman, R. A. Krinock, A. E. Chang, S. Shu. 1994. Characteristics and in vivo homing of long-term T-cell lines and clones derived from tumor-draining lymph nodes. Cancer Res. 54:2744.[Abstract/Free Full Text]
6. Rosenstein, M., I. Yron, Y. Kaufmann, S. A. Rosenberg. 1984. Lymphokine-activated killer cells: lysis of fresh syngeneic natural killer-resistant murine tumor cells by lymphocytes cultured in interleukin 2. Cancer Res. 44:1946.[Abstract/Free Full Text]
7. Tuttle, T. M., T. H. Inge, K. P. Bethke, C. W. McCrady, G. R. Pettit, H. D. Bear. 1992. Activation and growth of murine tumor-specific T-cells which have in vivo activity with bryostatin 1. Cancer Res. 52:548.[Abstract/Free Full Text]
8. Wallace, P. K., L. D. Palmer, L. D. Perry, E. S. Bolton, R. B. Alexander, P. K. Horan, J. C. Yang, K. A. Muirhead. 1993. Mechanisms of adoptive immunotherapy: improved methods for in vivo tracking of tumor-infiltrating lymphocytes and lymphokine-activated killer cells. Cancer Res. 53:2358.[Abstract/Free Full Text]
9. Yoshizawa, H., A. E. Chang, S. Shu. 1991. Specific adoptive immunotherapy mediated by tumor-draining lymph node cells sequentially activated with anti-CD3 and IL-2. J. Immunol. 147:729.[Abstract]
10. Rosenberg, S. A., P. Spiess, R. Lafreniere. 1986. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 233:1318.[Abstract/Free Full Text]
11. Spiess, P. J., J. C. Yang, S. A. Rosenberg. 1987. In vivo antitumor activity of tumor-infiltrating lymphocytes expanded in recombinant interleukin-2. J. Natl. Cancer Inst. 79:1067.
12. Burton, R. C., N. L. Warner. 1977. In vitro induction of tumor-specific immunity. IV. Specific adoptive immunotherapy with cytotoxic T cells induced in vitro to plasmacytoma antigens. Cancer Immunol. Immunother. 2:91.
13. Kedar, E., M. Schwartzbach, Z. Raanan, S. Hefetz. 1977. In vitro induction of cell-mediated immunity to murine leukemia cells. II. Cytotoxic activity in vitro and tumor-neutralizing capacity in vivo of anti-leukemia cytotoxic lymphocytes generated in macrocultures. J. Immunol. Methods 16:39.[Medline]
14. Barth, R. J. J., J. J. Mule, P. J. Spiess, S. A. Rosenberg. 1991. Interferon gamma and tumor necrosis factor have a role in tumor regressions mediated by murine CD8⁺ tumor-infiltrating lymphocytes. J. Exp. Med. 173:647.[Abstract/Free Full Text]
15. Shu, S., R. A. Krinock, T. Matsumura, J. J. Sussman, B. A. Fox, A. E. Chang, D. S. Terman. 1994. Stimulation of tumor-draining lymph node cells with superantigenic staphylococcal toxins leads to the generation of tumor-specific effector T cells. J. Immunol. 152:1277.[Abstract]
16. Tuttle, T. M., C. W. McCrady, T. H. Inge, M. Salour, H. D. Bear. 1993. Gamma-interferon plays a key role in T-cell-induced tumor regression. Cancer Res. 53:833.[Abstract/Free Full Text]
17. Aruga, A., S. Shu, A. E. Chang. 1995. Tumor-specific granulocyte/macrophage colony-stimulating factor and interferon gamma secretion is associated with in vivo therapeutic efficacy of activated tumor-draining lymph node cells. Cancer Immunol. Immunother. 41:317.[Medline]
18. Goedegebuure, P. S., M. Zuber, V. D. Leonard, U. L. Burger, J. J. Cusack, M. P. Chang, L. M. Douville, T. J. Eberlein. 1994. Reactivation of murine tumour-infiltrating lymphocytes with solid-phase anti-CD3 antibody: in vitro cytokine production is associated with in vivo efficacy. Surg. Oncol. 3:79.[Medline]
19. Burger, U. L., M. P. Chang, P. S. Goedegebuure, T. J. Eberlein. 1995. Recruitment of host CD8+ T cells by tumor-infiltrating lymphocytes and recombinant interleukin-2 during adoptive immunotherapy of cancer. Surgery 117:325.[Medline]
20. Shu, S., S. A. Rosenberg. 1985. Adoptive immunotherapy of a newly induced sarcoma: immunologic characteristics of effector cells. J. Immunol. 135:2895.[Abstract]
21. Parker, G. A., S. A. Rosenberg. 1977. Serologic identification of multiple tumor-associated antigens on murine sarcomas. J. Natl. Cancer Inst. 58:1303.
22. Fekete, E.. 1938. A comparative morphological study of the mammary gland in a high and low tumor strain of mice. Am. J. Pathol. 14:557.
23. Sander, B., J. Andersson, U. Andersson. 1991. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol. Rev. 119:65.[Medline]
24. Sander, B., I. Hoiden, U. Andersson, E. Moller, J. Abrams. 1993. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and sera. J. Immunol. Methods 166:201.[Medline]
25. Stoppacciaro, A., C. Melani, M. Parenza, A. Mastracchio, C. Bassi, C. Baroni, G. Parmiani, M. P. Colombo. 1993. Regression of an established tumor genetically modified to release granulocyte colony-stimulating factor requires granulocyte-T cell cooperation and T cell-produced interferon gamma. J. Exp. Med. 178:151.[Abstract/Free Full Text]
26. Suda, T., A. O’Garra, I. MacNeil, M. Fischer, M. W. Bond, A. Zlotnik. 1990. Identification of a novel thymocyte growth-promoting factor derived from B cell lymphomas. Cell. Immunol. 129:228.[Medline]
27. Aebersold, P., C. Hyatt, S. Johnson, K. Hines, L. Korcak, M. Sanders, M. Lotze, S. Topalian, J. Yang, S. A. Rosenberg. 1991. Lysis of autologous melanoma cells by tumor-infiltrating lymphocytes: association with clinical response. J. Natl. Cancer Inst. 83:932.[Abstract/Free Full Text]
28. Alexander, R. B., S. A. Rosenberg. 1990. Long-term survival of adoptively transferred tumor-infiltrating lymphocytes in mice. J. Immunol. 145:1615.[Abstract]
29. Alexander, R. B., S. A. Rosenberg. 1991. Adoptively transferred tumor-infiltrating lymphocytes can cure established metastatic tumor in mice and persist long-term in vivo as functional memory T lymphocytes. J. Immunother. 10:389.
30. Kurt, R. A., J. A. Park, M. C. Panelli, S. F. Schluter, J. J. Marchalonis, B. Carolus, E. T. Akporiaye. 1995. T lymphocytes infiltrating sites of tumor rejection and progression display identical V beta usage but different cytotoxic activities. J. Immunol. 154:3969.[Abstract]
31. Bannerji, R., C. D. Arroyo, C. C. Cordon, E. Gilboa. 1994. The role of IL-2 secreted from genetically modified tumor cells in the establishment of antitumor immunity. J. Immunol. 152:2324.[Abstract]
32. Dranoff, G., E. Jaffee, A. Lazenby, P. Golumbek, H. Levitsky, K. Brose, V. Jackson, H. Hamada, D. Pardoll, R. C. Mulligan. 1993. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc. Natl. Acad. Sci. USA 90:3539.[Abstract/Free Full Text]
33. Evans, R., T. M. Duffy, S. J. Kamdar. 1991. Differential in situ expansion and gene expression of CD4⁺ and CD8⁺ tumor-infiltrating lymphocytes following adoptive immunotherapy in a murine tumor model system. Eur. J. Immunol. 21:1815.[Medline]
34. Evans, R., S. J. Kamdar, T. Duffy, L. Edison. 1993. Intratumor gene expression after adoptive immunotherapy in a murine tumor model: regulation of messenger RNA levels associated with the differential expansion of tumor-infiltrating lymphocytes. J. Immunol. 150:177.[Abstract]
35. Mule, J. J., M. C. Custer, W. D. Travis, S. A. Rosenberg. 1992. Cellular mechanisms of the antitumor activity of recombinant IL-6 in mice. J. Immunol. 148:2622.[Abstract]
36. Vagliani, M., M. Rodolfo, F. Cavallo, M. Parenza, C. Melani, G. Parmiani, G. Forni, M. P. Colombo. 1996. Interleukin 12 potentiates the curative effect of a vaccine based on interleukin 2-transduced tumor cells. Cancer Res. 56:467.[Abstract/Free Full Text]
37. Meltzer, M. S.. 1981. Macrophage activation for tumor cytotoxicity: characterization of priming and trigger signals during lymphokine activation. J. Immunol. 127:179.[Medline]
38. Boraschi, D., D. Soldateschi, A. Tagliabue. 1982. Macrophage activation by interferon: dissociation between tumoricidal capacity and suppressive activity. Eur. J. Immunol. 12:320.[Medline]
39. Pace, J. L., S. W. Russell, B. A. Torres, H. M. Johnson, P. W. Gray. 1983. Recombinant mouse gamma interferon induces the priming step in macrophage activation for tumor cell killing. J. Immunol. 130:2011.[Abstract]
40. Mule, J. J., M. Rosenstein, S. Shu, S. A. Rosenberg. 1985. Eradication of a disseminated syngeneic mouse lymphoma by systemic adoptive transfer of immune lymphocytes and its dependence upon a host component(s). Cancer Res. 45:526.[Abstract/Free Full Text]
41. Sallusto, F., A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179:1109.[Abstract/Free Full Text]
42. Paglia, P., C. Chiodoni, M. Rodolfo, M. P. Colombo. 1996. Murine dendritic cells loaded in vitro with soluble protein prime cytotoxic T lymphocytes against tumor antigen in vivo (see comments). J. Exp. Med. 183:317.[Abstract/Free Full Text]
43. Heufler, C., F. Koch, G. Schuler. 1988. Granulocyte/macrophage colony-stimulating factor and interleukin 1 mediate the maturation of murine epidermal Langerhans cells into potent immunostimulatory dendritic cells. J. Exp. Med. 167:700.[Abstract/Free Full Text]
44. Armstrong, C. A., R. Botella, T. H. Galloway, N. Murray, J. M. Kramp, I. S. Song, J. C. Ansel. 1996. Antitumor effects of granulocyte-macrophage colony-stimulating factor production by melanoma cells. Cancer Res. 56:2191.[Abstract/Free Full Text]
45. Zitvogel, L., J. I. Mayordomo, T. Tjandrawan, A. B. DeLeo, M. R. Clarke, M. T. Lotze, W. J. Storkus. 1996. Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines. J. Exp. Med. 183:87.[Abstract/Free Full Text]
46. Itoh, T., W. J. Storkus, E. Gorelik, M. T. Lotze. 1994. Partial purification of murine tumor-associated peptide epitopes common to histologically distinct tumors, melanoma and sarcoma, that are presented by H-2Kb molecules and recognized by CD8⁺ tumor-infiltrating lymphocytes. J. Immunol. 153:1202.[Abstract]
47. Rosenberg, S. A., B. S. Packard, P. M. Aebersold, D. Solomon, S. L. Topalian, S. T. Toy, P. Simon, M. T. Lotze, J. C. Yang, C. A. Seipp, C. Simpson, C. Carter. 1988. Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma: a preliminary report. N. Engl. J. Med. 319:1676.[Abstract]
48. Rosenberg, S. A., P. Aebersold, K. Cornetta, A. Kasid, R. A. Morgan, R. Moen, E. M. Karson, M. T. Lotze, J. C. Yang, S. L. Topalian, M. J. Merino, K. Culver. 1990. Gene transfer into humans: immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N. Engl. J. Med. 323:570.[Abstract]
49. Eberlein, T. J., M. L. Rodrick, A. F. Massaro, S. E. Jung, J. A. Mannick, D. D. Schoof. 1989. Immunomodulatory effect