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[Cancer Research 64, 406-414, January 1, 2004]
© 2004 American Association for Cancer Research

Immunology

Effector Cell-Derived Lymphotoxin {alpha} and Fas Ligand, but not Perforin, Promote Tc1 and Tc2 Effector Cell-Mediated Tumor Therapy in Established Pulmonary Metastases

Mark J. Dobrzanski, Joyce B. Reome, Joseph A. Hollenbaugh, James C. Hylind and Richard W. Dutton

Trudeau Institute, Saranac Lake, New York


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytolytic CD8+ effector cells fall into two subpopulations based on cytokine secretion. Type 1 CD8+ T cells (Tc1) secrete IFN-{gamma}, whereas type 2 CD8+ T cells (Tc2) secrete interleukin (IL)-4 and IL-5. Although both effector cell subpopulations display Fas ligand (FasL) and tumor necrosis factor (TNF), tumor lysis is predominantly perforin dependent in vitro. Using an ovalbumin-transfected B16 lung metastasis model, we show that heightened numbers of adoptively transferred ovalbumin-specific Tc1 and Tc2 cells accumulated at the tumor site by day 2 after therapy and induced tumor regression that enhanced survival in mice with pulmonary metastases. Transfer of either TNF-{alpha}- or perforin-deficient Tc1 or Tc2 effector cells generated from specified gene-deficient mice showed no differences in therapeutic efficiency when compared with corresponding wild-type cells. In contrast, both Tc1 and Tc2 cells, derived from either FasL or TNF-{alpha}/lymphotoxin (LT) {alpha} double knockout mice, showed that therapeutic effects were dependent, in part, on effector cell-derived FasL or LT{alpha}. Six days after effector cell therapy, elevated levels of activated endogenous CD8/CD44High and CD4/CD44High T cells localized and persisted at sites of tumor growth, whereas donor cell numbers concomitantly decreased. Both Tc1 and Tc2 effector cell subpopulations induced endogenous antitumor responses that were dependent, in part, on recipient-derived IFN-{gamma} and TNF-{alpha}. However, neither effector cell-mediated therapy was dependent on recipient-derived perforin, IL-4, IL-5, or nitric oxide. Collectively, tumor antigen-specific Tc1 and Tc2 effector cell-mediated therapy is initially dependent, in part, on effector cell-derived FasL or LT{alpha} that may subsequently potentiate endogenous recipient-derived type 1 antitumor responses dependent on TNF-{alpha} and IFN-{gamma}.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adoptive T-cell immunotherapy has been shown to be effective for the treatment of patients with certain metastatic cancers. Recent clinical studies have shown that selection, expansion, and infusion of high-avidity melanoma-reactive CD8 T-cell populations derived from either peripheral blood or tumors of patients with late-stage disease can induce either partial or complete tumor regression (1, 2, 3, 4) . Such studies have shown that infused tumor peptide-specific CD8 T cells can persist in cancer patients but may be ineffective and/or unresponsive to specific tumor antigen (Ag) in vivo (1 , 3, 4, 5, 6, 7, 8, 9, 10) . This may be due in part to alterations in T-cell signal transduction (11 , 12) , inhibition through natural killer-like cell receptors on CD8 T cells (13 , 14) , or the presence of immunosuppressive cytokines and regulatory T cells (9 , 15) . Alternatively, others have shown that endogenous CD4 helper T cells and/or exogenous cytokines, such as interleukin (IL)-2 and IFN-{gamma}, can augment tumor eradication by enhancing cell survival, persistence, and therapeutic function of adoptively transferred tumor Ag-specific CD8 T cells (2 , 3 , 9 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) . These observations provide a further impetus to characterize endogenous effector cell subpopulations and their antitumor responses that arise as a result of adoptively transferred tumor-reactive CD8 T cells associated with tumor regression in vivo.

For the most part, studies in experimental animals have shown that CD8 T cells recognize tumor-associated Ag in the context of MHC class I molecules and play a major role in immune surveillance and tumor immunity. The mechanisms of CD8 T-cell-mediated cytotoxicity have been described extensively and include direct cognate interactions with tumor target cells and/or release of various cytokines that aid in tumor eradication (28 , 29) . The predominant effector molecules involved are perforin, which forms pores in the membrane of target cells, and either membrane-bound Fas ligand (FasL) or tumor necrosis factor (TNF), which induce apoptosis in susceptible cells (28, 29, 30) . Among these mechanisms, granule exocytosis mediated by perforin/granzyme is thought to be the principal mechanism of CD8-mediated tumor Ag-specific cytotoxicity (28, 29, 30, 31) .

As in the case of CD4 T cells, CD8 T lymphocytes can be further classified into distinct effector cell types based on their cytokine-secreting profiles after tumor Ag encounter (32, 33, 34) . Type 2 CD8 T cells (Tc2) preferentially secrete IL-4, IL-5, and IL-10 and kill predominantly by the perforin pathway, whereas, type 1 CD8 T cells (Tc1) predominantly secrete IFN-{gamma} and kill by either perforin- or Fas-mediated mechanisms in vitro (33 , 34) . Although both effector cell subpopulations have been identified in human peripheral blood and in patients with various clinical disorders (35, 36, 37, 38) , their role and effects on endogenous antitumor immune responses and tumor eradication of established malignancy remain relatively undefined. Using an ovalbumin (OVA)-transfected B16 lung metastasis/immunotherapy model, we have shown previously that not only can adoptively transferred Tc1 or Tc2 effector cells induce tumor regression through potentially different cytokine-mediated mechanisms, but, in some cases, they can also promote long-term survival among mice with established malignancy (39 , 40) . In the current study, we extended our observations to assess the potential direct killing mechanisms involved in effective tumor Ag-specific Tc1/Tc2 effector cell therapy and subsequent "indirect" mechanisms mediated by endogenous effector cell responses in mice treated for established pulmonary malignancy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Female C57BL/6 mice, 6–10 weeks of age, were obtained from the Animal Breeding Facility at the Trudeau Institute. The OT-I mouse strain (Thy 1.2), on a C57BL/6 background (H-2b), was originally obtained from Dr. Michael Bevan (University of Washington, Seattle, WA). These mice express a transgenic T-cell receptor V{alpha}2 and Vß5 specific for the SIINFEKL peptide of OVA in the context of MHC class I, H2-Kb (41) . Perforin-/-, gld-/-, IL-4-/-, IL-5-/-, IFN-{gamma}-/-, TNF-{alpha}-/-, and B6.PL/Thy 1.1 mice, on a B6 background, were purchased from The Jackson Laboratory (Bar Harbor, ME). TNF-{alpha}/lymphotoxin (LT) {alpha}-/- double knockout mice, on a C57BL/6 background (H-2b), were originally obtained from Dr. Jonathon Sedgwick (DNAX Research Institute, Palo Alto, CA). Homozygous perforin-/- (OT-I.PKO), gld-/- (OT-I.FasL), TNF-{alpha}-/- (OT-I.TNF-{alpha}), and TNF-{alpha}/LT{alpha}-/- (OT-I.TNF-{alpha}/LT{alpha}) knockout mice expressing the T-cell receptor V{alpha}2 and Vß5 transgenes were generated by back-crossing OT-I mice onto specified syngeneic knockout mice (H-2b) until they were homozygous for the gene deletion. Animals were maintained and treated according to animal care committee guidelines of the NIH and Trudeau Institute.

Tumor Cells.
The weakly immunogenic OVA-transfected B16 tumor cell line (B16-OVA) that is syngeneic to the C57BL/6 background was kindly provided by Drs. Edith Lord and John Frelinger (Rochester, NY). EL4 and the derivative OVA-expressing EG.7-OVA cell lines were obtained from the American Type Culture Collection (Manassas, VA).

Spleen and Lung Cell Preparation.
Spleens were collected from mice, and single cell suspensions were prepared, washed twice in HBSS, and resuspended in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 2 mM pyruvate, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, and 10% heat-inactivated FCS (Gibco). CD8-enriched T cells were obtained by treating cells with anti-CD4 (RL172.4), anti-heat stable Ag (J11D), and anti-MHC class II (D3.137, M5114, CA4) monoclonal antibodies for 30 min at 4°C. Cells were washed and incubated with rabbit (Pel Freeze Inc., Rogers, AK) and guinea pig (Harlan Inc., Indianapolis, IN) complement for 30 min at 37°C (40) . For preparation of single cell suspensions from lung parenchyma, lungs were flushed in situ with HBSS via cannulation of the heart to remove residual intravascular blood pools. Minced lung tissues were incubated for 1 h at 37°C on a rocker platform in 1.5 ml RPMI 1640/lung, supplemented with DNase I (50 units/ml; Sigma, St. Louis, MO.), collagenase A (100 mg/ml; Roche Diagnostics), and 5% FCS. After incubation, digested lung tissues were mechanically dispersed through nylon mesh screens in RPMI 1640/5% FCS. After three washes in RPMI 1640/5% FCS, lymphoid cells were resuspended in RPMI 1640/10% FCS to attain a cell concentration of 1 x 107 viable cells/ml. Cytospin preparations of cells from lung homogenates were fixed with methanol and stained with eosin and methylene blue (Fisher, Pittsburgh, PA). Cell differential counts were performed on a total of 200–300 cells on coded slides.

Generation of OVA-Specific Tc1 and Tc2 Effector Cells.
CD8 cells were obtained from spleen and lymph nodes of designated OT-I mice and cultured in RPMI 1640 (Irvine Scientific, Santa Ana, CA) supplemented with penicillin, streptomycin, glutamine, 2-Mercaptoethanol (2-ME), and 10% FCS (Hyclone Laboratories, Logan, UT). C57BL/6 B-cell blasts, stimulated with lipopolysaccharide for 3 days, were used as Ag-presenting cells and loaded with the OVA peptide (10 µM) at 37°C for 30 min, treated with mitomycin C (100 µg/ml; Sigma) at 37°C for 40 min, and washed three times before use. CD8 effector T cells were prepared from OT-I transgenic mice by 4-day culture under polarizing conditions as described previously (40) . On day 4 of culture, effector cells were 95–99% CD8+ V{alpha}2+ cells.

CTL Assays.
Tc1 and Tc2 effector cell cytolytic activity was determined in a standard 51Cr release assay (40) .

Adoptive Immunotherapy Model.
Syngeneic B6 or B6.PL/Thy 1.1 mice received i.v. injection with 2 or 5 x 105 B16-OVA melanoma cells to establish pulmonary metastases. Seven days after tumor challenge, mice were treated i.v. with 2 x 106 Tc1 or Tc2 OVA-specific effector T cells, and survival times were monitored daily (39) . Control groups of mice received no treatment. Metastases on freshly isolated lungs appeared as discrete black pigmented foci that were easily distinguishable from normal lung tissue. The number of pulmonary metastases in treated and untreated control groups was counted in a blind fashion. Metastatic foci too numerous to count were assigned an arbitrary value of >250. Alternatively, cytospin preparations of cells from lung homogenates were fixed with methanol and stained with eosin and methylene blue (Fisher). Although tumor cells appeared heterogeneous in size, they were easily differentiated as predominately larger cells with an elevated nuclear:cytoplasm ratio and, in some cases, contained black pigment. Counts were performed on a total of 200–300 cells on coded slides.

Flow Cytometric Analysis.
Single cell suspensions of processed murine lung were washed three times in a fluorescent antibody buffer consisting of 1% BSA and 0.02% sodium azide in 0.01 M PBS (pH 7.2). Recipient immune cell populations were phenotyped by their expression of surface markers using direct immunofluorescence staining techniques. Lymphocytes (106), pretreated with FcR block, were incubated for 20 min on ice with 100 µl of fluorescent antibody buffer containing 1 µg of various monoclonal antibodies conjugated to either biotin, phycoerythrin, FITC, CyChrome, or Tricolor. For biotinylated monoclonal antibodies, streptavidin Allophycocyanin (APC) or streptavidin CyChrome was used as a second-step reagent. The monoclonal antibodies used include anti-CD90.1 (Thy 1.1; clone HIS51; PharMingen, San Diego, CA), anti-CD90.2 (Thy 1.2; clone 53-2.1; PharMingen), anti-CD8 (Caltag Laboratories, Burlingame, CA), anti-CD4 (PharMingen), anti-CD44 (clone IM7; PharMingen), anti-Ly6c (PharMingen), anti-CD25 (PharMingen), and anti-FasL (PharMingen). Stained cell preparations were than washed three times in fluorescent antibody buffer and analyzed by multiparameter flow cytometry using a Becton Dickinson FACSCalibur (San Jose, CA). Ten thousand cells were analyzed per sample, and dead cells were excluded on the basis of forward light scatter. Surface marker analysis was performed using Cell Quest Software, and the percentage of positively stained cells and absolute cell numbers were determined.

RNase Protection assays.
Pelleted cells were resuspended in TRIzol reagent (Gibco). Total RNA was isolated by chloroform extraction and ethanol precipitation and analyzed using the RiboQuant Multiprobe RNase Protection Assay system (BD PharMingen) with the mAPO-3 "death gene" mRNA detection multiprobe set. Bands were detected using Molecular Imager FX with the Quantity One Software analysis program (Bio-Rad, Hercules, CA) and normalized against the L32 housekeeping gene as relative units.

Statistical Analysis.
For statistical analysis, two-tailed Student’s t test or nonparametric Mann-Whitney rank-sum test was used.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro-Generated OVA-Specific Tc1 and Tc2 Effector T Cells Express FasL, TNF-{alpha}, and LT{alpha}.
CD8+ Tc1 or Tc2 effector T cells were generated in vitro from OVA-specific T-cell receptor transgenic OT-I mice as described in "Materials and Methods." As shown in earlier studies, both Tc1 and Tc2 effector cell populations were highly polarized for type 1 (IFN-{gamma}) and type 2 (IL-4 and IL-5) cytokines, respectively, and demonstrated potent tumor Ag-specific cytolytic activity to OVA Ag-expressing tumor cells (EG.7-OVA) in vitro (40) . Moreover, effector cell populations were CD8+CD4-, Ly6C-, and expressed up-regulated levels of both CD44 and CD25 and down-regulated levels of CD62L. In the current study, we extended our phenotypic characterization of both Tc1 and Tc2 effector cells by assessing effector cell-derived FasL and TNF. Effector cells were generated in vitro and harvested, and either cell surface or message expression of FasL, TNF-{alpha}, and LT{alpha} was determined by either flow cytometry or standard RNase protection assays as described in "Materials and Methods." As shown in Fig. 1Citation , flow cytometric analysis showed that large proportions of either Tc1 or Tc2 effector cell subpopulations expressed low, yet detectable, levels of surface FasL when compared with that of naïve OT-I CD8 T cells. Because detection and expression of surface FasL among cultured lymphocytes may be somewhat transient or difficult to detect due to surface Ag cleaving and other cell dynamics [i.e., "cryptically" found in granules of some T-cell populations (42) ], we also assessed message expression using mRNA protection assays. mRNA expression for FasL among naïve OT-I CD8 cells was negligible, whereas both Tc1 and Tc2 effector cell subpopulations expressed similarly elevated levels (Fig. 1B)Citation . In parallel studies, we show that Tc1 and Tc2, but not naïve cells, express substantial message for the pro-inflammatory cytokines TNF-{alpha} and LT{alpha} (Fig. 1C)Citation . Effector cell populations derived from either perforin, FasL, or TNF knockout mice were phenotypically comparable with those of corresponding wild-type effector cell populations, with the exception of function associated with the deleted genes (data not shown).



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  		Fig. 1. Expression of Fas ligand (FasL) and tumor necrosis factor by Tc1 and Tc2 effector cells in vitro. Ovalbumin-specific Tc1 and Tc2 effector cells were generated in vitro from naïve CD8 T cells from OT-I T-cell receptor transgenic mice as described in "Materials and Methods." A, cells were dual-labeled with FITC-conjugated anti-CD8 and phycoerythrin-conjugated FasL monoclonal antibodies. Lymphocytes were distinguished by their forward light scatter (FSC)/side light scatter (SCC) profiles, and CD8 populations were gated and analyzed for expression of surface FasL. In parallel studies, effector cells were harvested, and FasL (B) and tumor necrosis factor {alpha} or lymphotoxin {alpha} (C) mRNA expression was detected by RNase protection assay using the mAPO-3 and mCK-3 multiprobe template sets, respectively. Optical density values were determined as described in "Materials and Methods, normalized against the L32 housekeeping gene, and expressed as relative units. Results are representative of two similar experiments.

 
In other studies, effector cell subpopulations were evaluated for tumor Ag-specific cytolytic potentials in standard 4-h chromium release assays. As shown in Fig. 2Citation , both Tc1 and Tc2 effector cells derived from either wild-type, TNF-, or FasL-deficient OT-I mice demonstrated comparable tumor antigen-specific cytolytic activity to OVA-expressing tumor cell targets. Effector cells derived from perforin knockout OT-I mice showed markedly lower levels of tumor cell lysis at all E:T cell ratios tested (Fig. 2, D and H)Citation . All effector cell subpopulations showed negligible lytic activity to non-OVA-expressing EL4 tumor cell targets. These results confirm that killing by all four Tc1 or Tc2 effector cell populations was highly Ag specific and occurred, with the exception of perforin-deficient effectors, at comparable levels in vitro.



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  		Fig. 2. Comparison of ovalbumin-specific cytolytic activity by select functionally deficient Tc1 and Tc2 effector cells in vitro. Tc1 and Tc2 effector cell populations were generated from either wild-type (A and E), tumor necrosis factor {alpha}/lymphotoxin {alpha} (B and F), Fas ligand (C and G), or perforin (D and H) knockout OT-I mice as described in "Materials and Methods." Cytolytic activity was assessed in a standard 4-h 51Cr release assay against ovalbumin-expressing EG.7 or parent EL4 tumor cell lines at various E:T ratios. Spontaneous release in all assays were <15%. Data are expressed as the mean ± SE of three independent experiments.

 
Decreased Therapeutic Efficiency by FasL-Deficient but not Perforin-Deficient Tc1 and Tc2 Effector Cell Subpopulations in Mice with Established Pulmonary Tumors.
Because the therapeutic efficacy of adoptive immunotherapy is proportional to the number of transferred immune cells, we quantitatively analyzed the antitumor effects of Tc1 and Tc2 effector cells by transfer of different effector cell concentrations and monitoring survival times in mice with established pulmonary tumors. As shown in Fig. 3, A and BCitation , mice receiving doses of 4–20 x 105 Tc1 effector cells, derived from either wild-type or perforin knockout OT-I mice, showed a similar increase in survival time when compared with untreated control tumor-bearing animals. Moreover, transfer of 5-fold fewer Tc1 effector cells, at numbers as low as 0.8 x 105, resulted in a similarly effective prolongation in survival times among mice bearing 7 day established tumors. In contrast, groups of mice receiving similar doses of FasL-deficient Tc1 effector cells showed greatly diminished therapeutic effects at all doses tested when compared with that of corresponding cell numbers from either wild-type or perforin-deficient Tc1 effector cells. All mice receiving no treatment succumbed to progressively growing tumor within 30–40 days after tumor challenge (Fig. 3)Citation . These results suggest that, on a per-cell basis, FasL-deficient Tc1 effector cells were at least 25–125-fold less effective than either wild-type or perforin-deficient Tc1 effector cells because the former demonstrated only modest therapeutic effects at cell numbers as high as 20 x 105 when transferred into tumor-bearing mice. Concomitantly, we assessed the therapeutic efficiency of Tc2 effector cells derived from corresponding OT-I mice. As shown in Fig. 3Citation , D-F, only groups of mice receiving 20 x 105 wild-type Tc2 effector cells showed comparative increases in survival times when compared with that of corresponding groups of Tc1 effector cell-treated mice. Although therapeutic effects on a per-cell basis by the former were nearly 25-fold less than that of the latter, Tc2 effector cells derived from perforin-deficient, but not FasL-deficient, OT-I mice were similar to wild-type Tc2 effector cells (Fig. 3Citation , D-F). Collectively, this suggests that effector cell-derived FasL may play a substantial role in either Tc1 or Tc2 effector cell-mediated therapeutic responses in vivo.



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  		Fig. 3. Therapeutic efficacy of select functionally deficient ovalbumin (OVA) antigen-specific Tc1 and Tc2 effector cells in mice bearing established lung tumors. Syngeneic mice (n = 6–8 mice/group) received i.v. injection with 2 x 105 B16-OVA tumor cells. Seven days later, various doses of wild-type (A and D), perforin (B and E), or Fas ligand deficient (C and F) OVA antigen-specific Tc1 (left panel) or Tc2 (right panel) effector cells were adoptively transferred into tumor-bearing mice, and survival was monitored. Results are representative of two similar experiments.

 
Decreased Therapeutic Efficiency by LT{alpha}-Deficient Tc1 and Tc2 Effector Cell Populations in Mice with Established Pulmonary Tumor.
Because Tc1 and Tc2 effector cell subpopulations were found to generate substantial levels of both TNF-{alpha} and LT{alpha}, we assessed the role of effector cell-derived TNF in both Tc1 and Tc2 effector-mediated antitumor responses. OVA Ag-specific Tc2 effector cell subpopulations, generated from either wild-type OT-I or OT-I.TNF-{alpha}-/- mice, were transferred into syngeneic C57BL/6 tumor-bearing mice, and survival times were monitored as described previously. As shown in Fig. 4Citation , survival among tumor-bearing mice receiving TNF-{alpha}-deficient Tc2 effector cell therapy did not differ significantly from that of corresponding groups of tumor-bearing mice receiving wild-type Tc2 effector cell therapy (P = 0.69).



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  		Fig. 4. Role of effector cell-derived tumor necrosis factor (TNF)-{alpha} and lymphotoxin {alpha} on effector cell therapy of mice with established B16-OVA lung tumor. Syngeneic mice (n = 6–8 mice/group) received i.v. injection with 2 x 105 B16-OVA tumor cells. Seven days later, 2 x 106 OVA antigen-specific Tc2 effector cells, generated from either OT-I.TNF-{alpha} (A) or OT-I.TNF-{alpha}/LT{alpha} (B) double knockout mice, were adoptively transferred into tumor-bearing mice, and survival times were monitored. Groups of untreated and corresponding wild-type effector cell-treated tumor-bearing mice served as controls. Results are representative of two similar experiments.

 
Because TNF-{alpha} and LT{alpha} (TNF-ß) display a similar spectra of biological activities and share the same CD120a (TNFR-p55) and CD120b (TNFR-p75) receptors (43) , we assessed the role of LT{alpha} in effector cell-mediated therapy. Using TNF-{alpha} and LT{alpha} double knockout mice, we show that survival times among mice receiving TNF-{alpha}/LT{alpha}-deficient Tc2 effector cell therapy were substantially prolonged when compared with that of untreated tumor-bearing mice (Fig. 4B)Citation . However, the former was significantly (P < 0.001) less effective than that of corresponding groups of mice receiving wild-type Tc2 effector cell therapy. This suggests that effector cell-derived LT{alpha}, and not TNF-{alpha}, may play a substantial role in Tc2 effector cell-mediated therapeutic responses in vivo. Similar results were obtained using corresponding Tc1 effector cell subpopulations (data not shown).

Endogenous T-Cell Localization Kinetics after Adoptively Transferred OVA Ag-Specific Effector Cell Treatment in Mice with Established Pulmonary Tumors.
Using Thy 1.1 congenic mice, donor (Thy 1.2) and tumor-bearing recipient (Thy 1.1) T-cell populations from either lung or spleen of effector cell-treated mice were enumerated by multicolor flow cytometric analysis. As shown in Fig. 5ACitation , donor Thy 1.2/CD8 Tc1 effector cells were detectable within lungs of tumor-bearing mice as early as 1–2 days, with peak levels at days 2–4 after therapy. However, cell numbers and frequencies decreased and appeared to remain at consistent levels with time. Similar cell kinetic profiles were observed in spleens from these same animals (Fig. 5ACitation , inset). Concomitantly, we evaluated the accumulation and kinetics of recipient-derived CD8 and CD4 T-cell populations coexpressing CD44High at sites of tumor growth. As shown in Fig. 5BCitation , activated recipient CD8 T-cell numbers (Thy 1.1/CD8/CD44High) were markedly elevated by day 7 after effector cell therapy. Similarly, activated recipient CD4 T-cell numbers (Thy 1.1/CD4/CD44High) accumulated at similar kinetics but at higher cell numbers and frequencies than recipient-derived CD8 T cells after effector cell therapy (Fig. 5C)Citation . In contrast, recipient-derived T-cell population numbers from lungs of untreated tumor-bearing control mice were not comparatively different from those of corresponding T-cell populations from lungs of normal age-matched mice (Fig. 5, B and C)Citation . Collectively, this suggests that although local donor effector cell numbers substantially decreased within 1 week after therapy, accumulation of activated recipient-derived CD8/CD44High and CD4/CD44High T-cell subpopulations at sites of tumor cell growth was dependent, in part, on donor effector cell treatment. Similar cell kinetics were obtained using corresponding Tc2 effector cell subpopulations (data not shown).



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  		Fig. 5. Early T-cell localization kinetics after Tc1 effector cell therapy in mice with established lung metastases. Tumor-bearing mice (Thy 1.1) were treated with 2 x 106 Tc1 effector cells (Thy 1.2) as described in the Fig. 3Citation legend. Lungs were harvested from groups of either untreated, effector cell-treated, or normal age-matched mice at specified time points after tumor challenge (n = 2–3 mice/group/time point). Lung cell suspensions were labeled with either anti-Thy 1.1, anti-Thy 1.2, anti-CD8, anti-CD4, or anti-CD44 monoclonal antibody and assessed by multicolor flow cytometry as described in "Materials and Methods." Gates were set on either donor Thy 1.2/CD8+ (A), recipient Thy 1.1/CD8+ (B), or Thy 1.1/CD4+ (C) T cells coexpressing elevated levels of CD44. Data are expressed as absolute cell numbers and were calculated as the percentage of positively stained cells x the total number of monocytes/tissue. Results are representative of two independent experiments.

 
Role of Endogenous Perforin in Tumor-Bearing Mice Treated with Either Tc1 or Tc2 Effector Cell Therapy.
Because effector cell therapy induced a profound accumulation and localization of activated recipient-derived T-cell populations at the site of tumor growth, we next assessed the role of potential recipient-derived antitumor mechanisms among tumor-bearing mice treated with Ag-specific effector cell therapy. Because we have shown previously that effector cell-derived perforin does not play a potential role in either Tc1 or Tc2 effector cell therapy, we extended our current studies to assess the role of recipient-derived perforin in Tc1 or Tc2 effector cell-mediated tumor regression. OVA-Ag-specific Tc1 or Tc2 effector cell subpopulations were generated as described previously and transferred into either wild-type or syngeneic perforin-/- knockout C57BL/6 mice bearing 7 day established B16-OVA lung tumors. As shown in Fig. 6ACitation , survival times among groups of Tc1 effector cell-treated tumor-bearing wild-type or perforin-/- knockout recipients were significantly (P < 0.001) prolonged when compared with corresponding groups of untreated control mice. However, effector cell-treated tumor-bearing perforin-/- knockout recipients showed no significant (P = 0.86) differences in therapeutic effects when compared with similarly treated tumor-bearing wild-type recipients. Similar results were obtained in tumor-bearing animals receiving Tc2 effector cell therapy (Fig. 6B)Citation .



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  		Fig. 6. Role of recipient-derived perforin in either Tc1 or Tc2 effector cell-mediated therapy. Wild-type or perforin-deficient mice (n = 6–10 mice/group) received injection with 2 x 105 B16-OVA tumor cells. Seven days later, 2 x 106 OVA antigen-specific Tc1 (A) or Tc2 (B) effector cells were adoptively transferred into specified groups of mice bearing established lung metastases, and survival times were monitored. Corresponding groups of untreated tumor-bearing mice served as controls. Results are representative of two independent experiments.

 
Role of Recipient-Derived IL-4 or IL-5 in Tumor-Bearing Mice Treated with Tc2 Effector Cell Therapy.
Because we have shown previously that effective Tc2 effector cell therapy aids in the recruitment of activated recipient-derived T cells to the site of tumor growth and is dependent, in part, on effector cell-derived IL-4 and IL-5 (44) , we next extended our current studies to assess the roles of such type 2-like cytokines derived from tumor-bearing recipient mice after Tc2 therapy. OVA-Ag-specific Tc2 effector cell subpopulations were generated as described previously and transferred into either wild-type or syngeneic IL-4-/- or IL-5-/- knockout C57BL/6 mice bearing 7 day established B16-OVA lung tumors. As shown in Fig. 7Citation , adoptive transfer of Tc2 effector cells into either IL-4 or IL-5 knockout animals showed no differences in therapeutic effects (P > 0.05) when compared with similarly treated wild-type mice. This suggests that effective Tc2 effector cell-mediated therapy was not markedly dependent on either recipient-derived IL-4 or IL-5 type 2-related cytokine-mediated antitumor responses.



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  		Fig. 7. Role of recipient-derived interleukin (IL)-4 and IL-5 in Tc2 effector cell-mediated therapy. Wild-type and either IL-4 (A) or IL-5 (B) cytokine-deficient mice (n = 6–10 mice/group) received injection with 2 x 105 B16-OVA tumor cells. Seven days later, 2 x 106 OVA antigen-specific Tc2 effector cells were adoptively transferred into specified groups of mice bearing established lung metastases, and survival times were monitored. Groups of untreated wild-type or corresponding cytokine-deficient tumor-bearing mice served as controls. Results are representative of two independent experiments.

 
Role of Recipient-Derived TNF-{alpha} and IFN-{gamma} in Tumor-Bearing Mice Treated with Either Tc1 or Tc2 Effector Cell Therapy.
Because we have shown previously that either Tc1 or Tc2 effector cell treatment resulted in the accumulation of activated recipient-derived T cells that predominantly secreted cytokines associated with a type 1-like immune response (44 , 45) , we next investigated the role of recipient-derived IFN-{gamma} and TNF-{alpha} in Tc1 or Tc2 effector cell-mediated tumor regression. OVA Ag-specific Tc1 or Tc2 effector cell populations were generated as described previously and transferred into either wild-type or syngeneic cytokine gene knockout C57/BL/6 mice bearing 7 day established B16-OVA lung tumors. As shown in Fig. 8ACitation , survival times among both Tc1 effector cell-treated wild-type and TNF-{alpha}-/- knockout tumor-bearing recipient mice were significantly (P < 0.001) prolonged when compared with those of corresponding groups of untreated control mice. However, Tc1 effector cell therapy was significantly (P < 0.001) less efficacious in prolonging survival times in tumor-bearing TNF-{alpha}-/- knockout recipient mice than in tumor-bearing wild-type mice receiving similar doses of Tc1 effector cell therapy. In parallel studies, transfer of these same effector cell populations into tumor-bearing IFN-{gamma}-/- knockout recipients showed a similarly marginal therapeutic effect when compared with corresponding groups of untreated tumor-bearing control knockout mice (Fig. 8B)Citation . Similar therapeutic effects were seen with corresponding groups of tumor-bearing mice treated with OVA Ag-specific Tc2 effector cells (Fig. 8, C and D)Citation . Control groups of untreated tumor-bearing wild-type or cytokine knockout mice similarly died within 40 days after tumor challenge (Fig. 8)Citation . Collectively, this suggested that both Tc1 and Tc2 effector cell-mediated tumor regression in vivo were markedly dependent, in part, on recipient-derived IFN-{gamma} and TNF-{alpha} type 1-like cytokine-mediated antitumor responses.



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  		Fig. 8. Role of recipient-derived tumor necrosis factor {alpha} and IFN-{gamma} in Tc1 or Tc2 effector cell-mediated therapies. Wild-type, tumor necrosis factor {alpha}- or IFN-{gamma}-deficient mice (n = 6–10 mice/group) received i.v. injection with 2 x 105 B16-OVA tumor cells. Seven days later, 2 x 106 OVA antigen-specific Tc1 (A and B) or Tc2 (C and D) effector cells were adoptively transferred into specified groups of mice bearing established lung metastases, and survival times were monitored. Groups of untreated wild-type or corresponding cytokine-deficient tumor-bearing mice served as controls. Results are representative of two independent experiments.

 
Role of Endogenous Nitric Oxide in Tumor-Bearing Mice Treated with Either Tc1 or Tc2 Effector Cell Therapy.
Previously, we have shown that over time (>day 14 after therapy), endogenous myeloid cell populations infiltrate sites of tumor growth after effector cell-mediated therapy (44 , 45) . Because it has been shown that induction and release of nitric oxide from tumor-associated macrophages is dependent on a number of cytokines, including IFN-{gamma} and TNF-{alpha} (46 , 47) , we next assessed the role of nitric oxide in Tc1- or Tc2-mediated therapy. OVA-Ag-specific Tc1 or Tc2 effector cell subpopulations were generated as described previously and transferred into either wild-type or syngeneic inducible NO synthase-/- knockout C57BL/6 mice bearing 7 day established B16-OVA lung tumors. As shown in Fig. 9ACitation , survival times among groups of Tc1 effector cell-treated tumor-bearing wild-type or inducible NO synthase knockout recipients were significantly (P < 0.001) prolonged when compared with that of corresponding groups of untreated control mice. However, effector cell-treated tumor-bearing inducible NO synthase knockout recipients showed no significant (P = 0.80) differences in therapeutic effects when compared with effector cell-treated tumor-bearing wild-type recipients. Similar results were obtained in tumor-bearing animals receiving Tc2 effector cell therapy (Fig. 9B)Citation .



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  		Fig. 9. Role of endogenous nitric oxide in effector cell-mediated therapy of mice with established lung tumor. Wild-type or inducible NO synthase-deficient mice (n = 6–10 mice/group) received injection with 2 x 105 B16-OVA tumor cells. Seven days later, 2 x 106 OVA antigen-specific Tc1 (A) or Tc2 (B) effector cells were adoptively transferred into specified groups of mice bearing established lung metastases, and survival times were monitored. Groups of untreated wild-type or knockout tumor-bearing mice served as controls. Results are representative of two independent experiments.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown previously that protection and tumor regression by Tc2 and Tc1 effector cells were dependent, in part, on potentially different mechanisms involving effector cell-derived IL-4, IL-5, and IFN-{gamma}, respectively (39 , 40 , 44 , 45) . In the current study, we extended our observations to assess the potential direct and indirect cell contact-dependent mechanisms involved with Tc1 and Tc2 effector cell-mediated tumor regression and their effects on potential endogenous antitumor response mechanisms in vivo. We show that transfer of either TNF-{alpha}- or perforin-deficient Tc1 or Tc2 effector cells, generated from specified gene-deficient mice, showed no differences in therapeutic efficiency when compared with tumor-bearing mice treated with corresponding wild-type effector cells, whereas both Tc1 and Tc2 cells, derived from either OT-I.gld or OT-I.TNF-{alpha}/LT{alpha} double knockout mice, showed that therapeutic effects were dependent, in part, on effector cell-derived FasL or LT{alpha}. Moreover, both effector cell therapies induced endogenous antitumor responses that were dependent, in part, on recipient-derived IFN-{gamma} and TNF-{alpha}, but not recipient-derived perforin, IL-4, IL-5, or nitric oxide.

Generally, effector T cells induce tumor cell cytotoxicity by two principle contact-dependent mechanisms that involve perforin-dependent granule exocytosis and Fas-mediated lysis (28, 29, 30, 31) . Although both Tc1 and Tc2 CD8 effector T-cell subpopulations display FasL, we have shown that tumor lysis is predominantly perforin dependent in vitro (33 , 40) . Interestingly, perforin derived from either polarized Tc1 or Tc2 effector cell subpopulations had little or no effect on enhancing survival among tumor-bearing mice in vivo. This is in agreement with the reports of others showing that perforin from nonpolarized CD8 T-cell populations not only played little, if any, role in T cell-mediated antitumor responses (48, 49, 50) but also had the potential to regulate various CD4 and CD8 T-cell-mediated immune responses in vivo (51) . However, our results show that in the absence of effector cell-derived FasL, disease onset and survival among tumor-bearing mice receiving either Tc1 or Tc2 effector cell therapy are significantly diminished. This suggests that both Tc1 and Tc2 effector cells contribute to the early stages of the effector phase of the antitumor response, in part, by "contact-dependent" mechanisms involving either membrane-bound or soluble effector cell-derived FasL. Investigations on the role and effects of the FasL/CD95 network on the functional status of Tc1 and Tc2 effector cells and subsequent impact on tumor-effector cell interactions are currently under way.

Similarly, both soluble and membrane-bound TNF have been shown to exhibit potent cytotoxic and tumorcidal activities in vivo (52, 53, 54) . In our current study, we also show that both Tc1 and Tc2 effector cells produced comparable levels of both TNF-{alpha} and LT{alpha}. Because LT{alpha} can engage the same cell surface receptors as TNF-{alpha} and therefore possesses similar biological activities (43) , we next independently assessed the potential roles of LT{alpha} and TNF-{alpha} in Tc1 and Tc2 effector cell-mediated therapies. Both Tc1 and Tc2 effector cells derived from TNF-{alpha} knockout mice had similar therapeutic effects on tumor-bearing mice when compared with that of corresponding wild-type effector cell-treated animals. However, when Tc1 and Tc2 effector cells were generated from TNF-{alpha}/LT{alpha} double knockout mice, both therapeutic efficiency and survival were significantly impaired among treated tumor-bearing mice. Interestingly, this suggested that both Tc1 and Tc2 effector cell-mediated therapies were dependent, in part, on effector cell-derived LT{alpha} and not TNF-{alpha} or that LT{alpha} and TNF-{alpha} were interchangeable. One possible explanation for such functional discrepancies may be that, despite sharing the same CD120a (TNFR-p55) and CD120b (TNFR-p75) receptors, LT{alpha} and TNF-{alpha} may directly engage a variety of distinct, yet overlapping, signaling pathways that result in either enhanced cell survival/activation and proliferation or apoptosis. Consequently, this would appear to be highly dependent on cell lineage, metabolic state, and/or kinetics of ligand/receptor binding at the sites of tumor growth. Alternatively, others have suggested that functional disparities among TNF-{alpha} and LT{alpha} may be attributed to their differential effects on tumor-associated stromal cells that produce select chemokines that indirectly influence effector cell recruitment to sites of tumor growth and inflammation (55 , 56) . Additional studies investigating tumor stroma and select chemokine-mediated T-cell migration kinetics are currently in progress.

With reference to T-cell recruitment kinetics to sites of tumor growth, our studies show that peak levels of both donor Tc1 and Tc2 effector cells accumulated at the tumor site 2 days after adoptive therapy. This led to the emergence and persistence of substantial levels of activated endogenous CD4/CD44High and CD8/CD44High T cells to the site of tumor growth as early as 6 days after effector cell therapy. With the local emergence of recipient-derived effector cells, we next assessed the potential role of various endogenous antitumor immune response mechanisms after Tc1 or Tc2 effector cell therapy. Because perforin is the dominant pathway in tumor cell contact-dependent killing by CTLs (28, 29, 30, 31) , we next assessed the role of recipient-derived perforin among tumor-bearing mice treated with either Tc1 or Tc2 effector cell therapies. Interestingly, similar to the results obtained for donor effector cell-derived perforin, endogenous perforin played little, if any, role in either effector cell-mediated therapy. Similarly, corresponding studies using effector cell-treated inducible NO synthase knockout tumor-bearing recipients showed no differences in therapeutic efficiency when compared with that of similarly treated wild-type tumor-bearing mice. This suggested that neither recipient-derived Ag-specific perforin nor nonspecific nitric oxide cytolytic mechanisms affected Tc1 or Tc2 effector cell-mediated therapies.

Because we and others have demonstrated favorable contributions by type 2-like cytokines in tumor rejection (39 , 40 , 44 , 57, 58, 59, 60) , we next assessed the roles of recipient-derived IL-4 and IL-5 in effector cell-mediated immunotherapy. In contrast to our previous findings that effector cell-derived IL-4 and IL-5 played a substantial role in Tc2-mediated therapy, the absence of endogenous recipient-derived IL-4 and IL-5 had no therapeutic effect on effector cell-treated tumor-bearing animals. Conceivably, differences between donor and recipient-derived IL-4 and IL-5 may be related to differences in spatial and/or temporal patterns of cytokine expression during tumor-effector cell interaction and/or local responding cell population differences during endogenous antitumor responses.

Alternatively, we have previously shown that B16-OVA tumor cells are both sensitive and responsive to the type 1-associated cytokines IFN-{gamma} and TNF-{alpha} in vitro (45) .1 Addition of IFN-{gamma} up-regulated both CD95 and CD120a receptor gene expression among tumor cells in vitro. Moreover, addition of TNF-{alpha} in the presence of IFN-{gamma} not only showed a marked decrease in tumor cell growth but also provided an increase in the proportion of tumor cells undergoing apoptosis. This suggests that such direct synergistic cytokine-mediated effects can promote potential TNF family-related death mechanisms that are highly dependent on IFN-{gamma}. Moreover, these findings were substantiated in vivo when tumor regression was markedly dependent on recipient-derived IFN-{gamma} and TNF-{alpha} in studies using effector cell-treated tumor-bearing knockout recipients. Aside from their previously described immunoenhancing effects on local immune responses (9 , 20 , 26) , the data suggest that endogenous IFN-{gamma} and TNF-{alpha} directly contribute, in part, to enhancing tumor cell susceptibility to either Tc1 or Tc2 effector cell-derived FasL- or LT{alpha}-mediated death mechanisms.

These observations, in conjunction with our previous studies, suggest that either Tc1 or Tc2 effector cell-mediated immunotherapy is dependent on sequential events involving initial tumor recognition by effector cell-derived FasL and/or TNF/LT{alpha}. Such interactions may lead, in part, to Tc2 and Tc1 release of effector cell-derived IL-4, IL-5, and IFN-{gamma}, respectively. Through potentially different cytokine-mediated mechanisms, Tc1 and Tc2 cellular therapies may aid in the recruitment and potentiation of recipient type 1-like antitumor responses that are markedly dependent on recipient-derived IFN-{gamma} and TNF-{alpha}. With the capacity to clinically isolate tumor Ag-specific T-cell populations from cancer patients, ex vivo generation, propagation, and reinfusion of polarized effector cell subpopulations may offer a new strategy for successful tumor immunotherapy in cancer patients with select primary and metastatic disease.


   ACKNOWLEDGMENTS
 
We thank David Niederbuhl for excellent assistance with the generation of both wild-type and knockout OVA T-cell receptor transgene-positive mice. We are particularly grateful to Drs. Edith Lord and John Frelinger for providing the B16-OVA cell line.


   FOOTNOTES
 
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.

Grant support: NIH Grant CA71833.

Requests for reprints: Mark J. Dobrzanski, Trudeau Institute, Algonquin Avenue, Saranac Lake, New York 12983. E-mail: mdobrzanski{at}trudeauinstitute.org

1 M. J. Dobranski, J. B. Reome, J. A. Hollenbaugh, and R. W. Dutton. Tc1 and Tc2 effector cell therapy elicit long-term immunity by contrasting mechanisms that result in complementary endogenous type 1 antitumor responses, manuscript in preparation. Back

Received 8/20/03. Revised 10/10/03. Accepted 10/21/03.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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