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[Cancer Research 66, 4913-4921, May 1, 2006]
© 2006 American Association for Cancer Research

Immunology

Ex vivo Culture with Interleukin (IL)-12 Improves CD8+ T-Cell Adoptive Immunotherapy for Murine Leukemia Independent of IL-18 or IFN-{gamma} but Requires Perforin

Jennifer N. MacGregor1,3, Qiao Li2, Alfred E. Chang2,3, Thomas M. Braun4, Dennis P.M. Hughes5 and Kevin T. McDonagh6

Departments of 1 Microbiology and Immunology, 2 Surgery, and 3 Graduate Program in Immunology, and 4 School of Public Health, University of Michigan Medical School, Ann Arbor, Michigan; 5 Department of Pediatrics, M.D. Anderson Cancer Center, Houston, Texas; and 6 Division of Hematology, Oncology, and Blood and Marrow Transplantation, Department of Internal Medicine, University of Kentucky College of Medicine, Lexington, Kentucky

Requests for reprints: Jennifer N. MacGregor, Department of Microbiology and Immunology, University of Michigan Medical School, 1150 West Medical Center Drive, 6606 Medical Science Building II, Ann Arbor, MI 48109-0620. Phone: 734-615-9014; E-mail: jmacgreg{at}umich.edu.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In animal models and clinical trials, adoptive transfer of activated, antigen-specific CD8+ T cells mediates tumor regression in a cell dose-dependent manner. The cytokine interleukin (IL)-12 promotes CD8+ T-cell cytotoxicity and, with IL-18, synergistically up-regulates IFN-{gamma} release. We have shown that culturing CD8+ T cells ex vivo with IL-12 and IL-18 enhanced antitumor responses in vivo and in vitro using a model of C1498/ovalbumin, a murine acute myeloid leukemia cell line expressing the antigen ovalbumin. Activated ovalbumin-specific CD8+ T cells cultured with IL-12, IL-18, both, or neither were assayed for antigen-specific cytokine production and cytolytic activity and adoptively transferred to C57BL/6 mice with established tumors. Maximal IFN-{gamma} release occurred after T-cell culture with IL-12 and IL-18. Tumor-specific in vitro cytotoxicity was enhanced by IL-12, unaffected by addition of IL-18, and abrogated in perforin-deficient T cells irrespective of cytokine exposure. T cells cultured with IL-12 more effectively eliminated tumors, and addition of IL-18 did not further augment responses. IFN-{gamma}-deficient CD8+ T cells showed effective antitumor activity that was enhanced by IL-12 with or without IL-18. Perforin-deficient CD8+ T cells were poor mediators of antitumor activity, though, and showed no improvement after culture with IL-12 and/or IL-18. Thus, ex vivo culture with IL-12 was sufficient to augment antigen-specific in vitro cytotoxicity and antitumor activity in vivo in an IFN-{gamma}-independent but perforin-dependent manner. Ex vivo culture with IL-12 may improve CD8+ T-cell immunotherapy of cancer in the absence of donor cell–derived IFN-{gamma} via perforin-mediated cytolysis. (Cancer Res 2006; 66(9): 4913-21)


   Introduction
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Materials and Methods
 Results
 Discussion
 References
 
Advances in immunology and an appreciation for the role of T cells as anticancer effectors have led to identification of populations of differentiated, tumor-reactive T cells as candidates for cellular immunotherapy, including tumor-draining lymph node (TDLN) cells and tumor-infiltrating lymphocytes, which can be recovered from cancer patients' lymph nodes and tumor biopsies for ex vivo expansion and reinfusion (1–4). Another application of cellular adoptive immunotherapy is a donor lymphocyte infusion following bone marrow transplantation for hematopoietic malignancies, which promotes graft-versus-leukemia antitumor activity. Numerous technical innovations have made it possible to rapidly expand these cells ex vivo to generate many enriched, tumor-specific T cells that respond robustly to in vitro tumor stimulation and mediate long-lasting tumor immunity in vivo (5, 6).

Tumor-specific IFN-{gamma} production after in vitro stimulation is a common clinical end point for cancer immunotherapy trials (7–9). The frequency of IFN-{gamma}-producing cells, as measured by flow cytometry and enzyme-linked immunospot, has become an accepted method of analyzing antitumor responses by effector T cells.

T-cell cytolytic function is another effector mechanism that has been correlated with successful cellular antitumor activity in many cancer models and clinical trials. There is no direct test for T-cell cytotoxicity in vivo, but tumor-reactive T cells are often capable of lysing primary tumor targets from patients in vitro. T cells may lyse tumor targets via Fas/Fas ligand interactions, release of directly toxic cytokines, such as tumor necrosis factor-{alpha} (TNF-{alpha}), or through perforin-mediated cell lysis (10–13).

To improve both T-cell-mediated IFN-{gamma} production and cytolysis, cytokine adjuvants have been used in immunotherapies. Interleukin (IL)-12 promotes polarization of T cells to an IFN-{gamma}-producing population and, for CD8+ T cells, up-regulates cytolytic activity (14–16). IL-18 alone has little effect on IFN-{gamma} production but acts in synergy with IL-12 to dramatically enhance IFN-{gamma} production in the absence of T-cell receptor (TCR) stimulation and may augment cytotoxicity (17–19). These cytokines have been used together systemically with significant therapeutic benefit (20–22). Unfortunately, the toxicity of the therapy has limited its utility in several model systems (23–26). Studies have been undertaken to address this problem, which remains unresolved (27–29). We developed a model in which we cultured antigen-specific effector CD8+ T cells ex vivo with IL-12 and IL-18 and then adoptively transferred the cells to tumor-bearing hosts, after which we observed significant enhancement of tumor regression. In vitro analysis of T-cell function revealed that exposure of these cells to IL-12 and IL-18 showed synergy for IFN-{gamma} release, in amount and cell frequency, and that cells cultured with IL-12 or the combination of IL-12 and IL-18 had greater tumor-specific cytolytic function.

In this report, we show that ex vivo exposure to IL-12 is sufficient to enhance in vivo antitumor responses on adoptive transfer without IL-18. We further show that in vitro and in vivo cytolytic function of IL-12-cultured T cells is dependent on perforin. IFN-{gamma} release was not required in our model to achieve efficacious tumor suppression, as IFN-{gamma}-deficient CD8+ T cells mediated antitumor activity as well as wild-type (WT) cells. These data suggest that the effect of IL-12 on T-cell-mediated antitumor activity in this model is IFN-{gamma}-independent, perforin-mediated cytotoxicity.


   Materials and Methods
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 Materials and Methods
Results
 Discussion
 References
 
Mice. C57BL/6 and BALB/c mice were purchased from the National Cancer Institute (Frederick, MD), and IL-18 knockout mice were from The Jackson Laboratory (Bar Harbor, ME). OT-1 mice were a gift from Michael Bevan (University of Washington, Seattle, WA) and express transgenic, H2b-restricted TCRs specific for an ovalbumin-derived peptide (SIINFEKLL), providing a uniform source of antigen-specific CD8+ T cells. OT-1 mice deficient in IFN-{gamma} production (IFN-{gamma}–/–) or perforin (Pfp–/–) production were gifts from Mark Dobrzanski (Trudeau Institute, Inc., Saranac Lake, NY). Sex- and age-matched C57BL/6 mice were tumor-bearing recipients for in vivo studies. Animals were maintained in specific pathogen-free facilities in the Unit for Animal Laboratory Medicine (University of Michigan Medical School, Ann Arbor, MI). Animal protocols were approved and in compliance with guidelines from the University of Michigan Committee on the Use and Care of Animals.

Cell lines. C1498 is a H2b-positive murine acute myeloid leukemia (AML) line provided by Daniel Valera (University of Minnesota, Minneapolis, MN). MT901 is a H2d-positive murine sarcoma line. C1498/ovalbumin and MT901/HER-2 were generated by retroviral transduction with a bicistronic Moloney murine leukemia viral vector encoding either the full-length ovalbumin or HER-2/neu gene and a puromycin resistance gene (adapted from G. Nolan; available at http://www.stanford.edu/group/nolan). Transduced cells were selected by culture with puromycin. Cell cultures were maintained in complete medium (RPMI 1640 with 10% heat-inactivated FCS, 100 units/mL penicillin, 100 µg/mL streptomycin, and 1 mmol/L L-glutamine).

Antibodies and cytokines. Purified monoclonal antibodies (mAb) specific for murine CD3-{varepsilon}, CD28, IL-10, TNF-{alpha}, and IFN-{gamma} and biotinylated anti-IL-10, anti-TNF-{alpha}, and anti-IFN-{gamma} mAbs were purchased from BD PharMingen (San Diego, CA). Recombinant purified murine IFN-{gamma}, TNF-{alpha}, IL-10, IL-12, and IL-18 were purchased from R&D Systems (Minneapolis, MN). Recombinant human IL-2 was purchased from Chiron Therapeutics (Emeryville, CA).

T-cell activation. Spleens from naive mice were processed into single-cell suspensions, passed through 70-µm pore nylon mesh filters to remove debris, and depleted of RBC with ACK lysis buffer (pH 7.2). Cells were maintained in T-cell medium (complete medium plus 1 mmol/L 2-mercaptoethanol and 1 mmol/L sodium pyruvate). Cell suspensions were enriched for T cells by nonadherence to nylon wool and cultured in T-cell medium with 2 µg/mL plate-bound anti-CD3-{varepsilon} mAb and 2 µg/mL anti-CD28 mAb for 2 days to activate. Activated T cells were then expanded for 2 days in T-cell medium with 60 IU/mL IL-2. Parallel cultures were supplemented with 10 ng/mL IL-12 and/or 100 ng/mL IL-18. Where indicated, HER-2/neu-specific BALB/c T cells were generated by retroviral transduction (adapted from G. Nolan) after activation and resulted in ~30% transgene efficiency (data not shown). After expansion, T cells were washed in PBS for in vitro assays or adoptively transferred to tumor-bearing mice for in vivo studies.

In vitro quantification of IFN-{gamma}-producing cells. Activated, expanded T cells were resuspended in T-cell medium without cytokines and cocultured with antigen-positive and parental tumor at an E:T of 5:1 in the presence of 10 µg/mL brefeldin A. After 4 hours, cells were washed in PBS, stained with an anti-CD8 mAb, washed again in PBS, and fixed with 4% formaldehyde. Cells were then washed in permeabilization buffer (0.1% saponin in PBS with 0.1% NaN3 and 1% bovine serum albumin), stained with a phycoerythrin-conjugated IFN-{gamma} mAb or isotype control, and then washed again in permeabilization buffer. After resuspension in cold PBS, cells were sorted on an Epics XL flow cytometer with System II software (Coulter, Miami, FL) and data were subsequently analyzed with WinList software (Verity Software House, Inc., Topsham, ME). Data are averages ± SD of duplicate samples for each condition and represent four independent experiments with OT-1 T cells and two independent experiments with HER-2/neu-specific BALB/c T cells.

In vitro detection of antigen-specific cytokine secretion. Activated, expanded T cells were resuspended in T-cell medium without cytokines and stimulated with antigen-positive and parental tumor at an E:T of 5:1. Cell-free supernatants were analyzed by ELISA for cytokine release 16 hours later. A standard protocol from BD PharMingen was followed. Known concentrations of purified cytokines were titrated to establish a standard curve on each assay plate. A405 was measured on a Vmax microplate reader and analyzed with SoftMax Pro software (Molecular Devices, Sunnyvale, CA). Data are reported as averages of triplicate wells for each sample ± SD and are representative of six independent assays to measure IFN-{gamma} from WT OT-1 T cells, two independent assays to quantify IL-10 and TNF-{alpha}, and two independent assays of HER-2/neu-specific IFN-{gamma} production from BALB/c T cells.

Cellular cytotoxicity assay. C1498 and C1498/ovalbumin were labeled with 100 nmol/L carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) in PBS for 10 minutes at 37°C and then washed in PBS and resuspended in T-cell medium with 1 µg/mL propidium iodide (PI). Activated, expanded OT-1 T cells were resuspended in T-cell medium and added in 100 µL volumes to 96-well V-bottomed plates in duplicate, diluted 2-fold to achieve E:T of 20:1 and 5:1 with tumor targets present in 100 µL volumes of 5 x 104 per well. CFSE-stained and PI-counterstained tumor cells plated without T cells served as "spontaneous lysis" controls. Plates were centrifuged briefly to pellet effectors and targets, incubated for 4 hours at 37°C with 5% CO2, and then kept on ice during analysis by flow cytometry (as described). Percentage lysis was determined by dividing the number of CFSE and PI–double-positive cells by the total number of CFSE-labeled cells for each sample less the percentage of CFSE and PI-double positive target cells in spontaneous lysis control wells. Specific lysis was calculated by subtracting the percentage of control C1498 cells lysed at the same E:T ratio. Data are reported as averages ± SD of duplicate wells for each condition and are representative of four independent assays with WT OT-1 T cells and two independent experiments with perforin-deficient OT-1 T cells.

In vivo AML induction. To establish s.c. tumors, 1 x 106 C1498/ovalbumin cells were injected in 0.2 mL PBS under the skin of naive C57BL/6 mice. Mice were randomized (n = 5) to receive i.v. adoptive transfer of T cells at the indicated cell doses 3 days later. Serial measurements of tumor areas were taken with Vernier calipers and are reported as mean tumor areas for each cohort. For survival studies, 1 x 106 tumor cells were injected into the peritoneum (i.p.) of randomized cohorts of mice (n = 5-6) 8 days before the adoptive transfer of 1 x 107 T cells that were activated and cultured as described. Mice were monitored >60 days for survival. Data are representative of four independent in vivo experiments with OT-1 T cells and two independent studies with IFN-{gamma}- and perforin-deficient OT-1 cells. Survival curves are representative of three independent experiments.

Statistical analysis. Data were analyzed with GraphPad Prism software (San Diego, CA) to determine statistical differences between groups using the Student's unpaired t test. P < 0.05 was considered statistically significant.


   Results
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 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-12 and IL-18 synergistically enhance the amount and frequency of antigen-specific CD8+ T-cell IFN-{gamma} release. It has been widely reported that IL-12 and IL-18 together synergize to increase antigen-specific and nonspecific IFN-{gamma} secretion from activated T cells as confirmed in Fig. 1 (30–34). For this report, we closely examined the effect of IL-12 and IL-18 individually as well as in combination. We observed an increase in the frequency of IFN-{gamma}-producing cells in IL-12 and IL-18 cultured T cells following intracellular cytokine staining and flow cytometry (Fig. 1A). A modest increase in the number of cytokine-secreting cells was also evident in T cells that have been cultured with only IL-12 but not other controls. In contrast, only the combination of IL-12 and IL-18 enhanced the in vitro secretion of antigen-specific IFN-{gamma} compared with all controls (Fig. 1B). To be sure that these results were not unique to ovalbumin-specific transgenic TCR-expressing T cells, we also examined the effect of IL-12 and IL-18 on T-cell responses in a sarcoma model, in which the target tumor antigen was HER-2/neu. T cells expressing a HER-2/neu-specific TCR showed an increase in IFN-{gamma} production both in terms of frequency and amount after culture with IL-12, but this effect was dramatically enhanced by previous culture with both IL-12 and IL-18 (Fig. 1C and D). T cells expressing a control gene, green fluorescent protein, had minimal IFN-{gamma} responses toward antigen-positive or parental tumor (Fig. 1D; data not shown). These results suggest that IL-12 is sufficient to increase the fraction of CD8+ T cells capable of producing IFN-{gamma} in response to antigen but that the combination of IL-12 and IL-18 is necessary to enhance the amount of IFN-{gamma} secreted.


Figure 1
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  		Figure 1. IL-12 and IL-18 increase frequency and amount of antigen-specific IFN-{gamma} production by activated CD8+ T cells. Activated T cells were cultured with IL-12 and/or IL-18 and then unstimulated or restimulated with antigen-positive or parental tumor for analysis of IFN-{gamma} production. A, OT-1 T cells cultured with IL-12 or the combination of IL-12 and IL-18 contained significantly greater numbers of IFN-{gamma}-producing cells in response to ovalbumin antigen. IL-12 stimulated a slight increase in the number of IFN-{gamma}-producing cells compared with IL-18 and no cytokine culture (P < 0.004). Culture with IL-12 plus IL-18 generated the greatest number of IFN-{gamma}-producing cells (P < 0.0013, compared with all other culture conditions). Inset, mean fluorescence intensity (MFI) of IFN-{gamma} staining was greatest in response to antigen-specific stimulation with C1498/ovalbumin (C1498/OVA; P < 0.05). B, activated OT-1 T cells cultured with IL-12, IL-18, both, or neither were unstimulated or cocultured with C1498 or C1498/ovalbumin overnight, and supernatants were tested by ELISA for IFN-{gamma} secretion. All cell populations produced antigen-specific IFN-{gamma} on the stimulation, but only T cells cultured with IL-12 and IL-18 produced an enhanced amount (P < 0.00005). C, frequency of IFN-{gamma}-producing HER-2/neu-specific CD8+ T cells was increased modestly by IL-12 alone and dramatically by IL-12 plus IL-18 compared with controls (no cytokine and IL-18). D, the amount of HER-2/neu-specific IFN-{gamma} released by T cells was increased slightly by IL-12 (P < 0.0000003, compared with controls) but synergistically increased by culture with IL-12 and IL-18 (P < 0.003). Control T cells lacking HER-2/neu specificity did not release IFN-{gamma} in response to MT901 irrespective of HER-2/neu expression.

 
IL-12 enhances perforin-dependent CD8+ T-cell cellular cytotoxicity. Both IL-12 and IL-18 have been shown to improve T-cell cellular cytotoxicity (15, 17, 35–37). We further investigated the mechanism of cytolytic function, hypothesizing that perforin was an important effector molecule based on published reports of IL-12-mediated up-regulation of its expression. WT ovalbumin-specific CD8+ T cells from OT-1 mice were assayed in parallel with perforin-deficient CD8+ OT-1 T cells to determine their cytotoxicity against C1498 and C1498/ovalbumin leukemia targets in vitro. Before the assay, T cells were activated and expanded with IL-2 plus IL-12, IL-18, both, or neither. Figure 2 summarizes the results. Among WT cells, all cytokine culture conditions supported T-cell cytotoxicity against antigen-positive targets, but the greatest activity was observed when T cells have been cultured with IL-12. Perforin-deficient CD8+ T cells showed severely impaired antigen-specific cytotoxicity irrespective of cytokine culture conditions before the assay. Thus, not only was perforin required for antigen-specific cytolysis of the leukemia targets, but there were no perforin-independent cytotoxic functions up-regulated by IL-12 or IL-18.


Figure 2
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  		Figure 2. IL-12 enhances perforin-dependent, antigen-specific cellular cytotoxicity of activated CD8+ T cells. Activated WT or perforin-deficient (Pfp–/–), ovalbumin-specific T cells were cultured at the indicated E:T ratios for 4 hours with CFSE-labeled tumor in the presence of the vital dye PI, and the fraction of killed tumor target cells was determined by flow cytometry. After culture with IL-12 alone, OT-1 cells showed enhanced specific lysis at all E:T ratios (P < 0.02), whereas the combination of IL-12 and IL-18 resulted in improved lysis only at the highest E:T ratio (P < 0.01) compared with WT controls. Perforin-deficient T-cell lysis of tumor targets was severely impaired irrespective of cytokine culture conditions before the assay (P < 0.01, compared with all WT populations).

 
Adoptive transfer of antigen-specific CD8+ T cells cultured with IL-12 improves tumor suppression independent of IL-18. We next evaluated ex vivo cytokine adjuvants IL-12 and IL-18 in a CD8+ T-cell-dependent adoptive immunotherapy model to determine if in vitro T-cell responses correlated with enhanced antitumor activity in vivo. We adoptively transferred three doses of ovalbumin-specific CD8+ T cells to mice bearing s.c. AML nodules or C1498/ovalbumin. These doses were high (1 x 107), intermediate (5 x 106), or low (1 x 106) and have been established previously in our laboratory as a range of effective to moderately effective to ineffective doses.7 Transferred T cells were cultured with IL-12, IL-18, both, or neither before the adoptive transfer. We found that antitumor activity was enhanced after culture of antigen-specific T cells with IL-12 alone as with the combination of IL-12 and IL-18 (Fig. 3A-D ). The intermediate dose of T cells given was sufficient to suppress tumor growth if culture with IL-12 alone or IL-12 plus IL-18 preceded adoptive transfer, whereas culture with IL-18 mediated modest but statistically insignificant tumor suppression only at the highest dose of T cells transferred. Cohorts receiving T cells cultured without IL-12 or IL-18 (no cytokine) had tumor growth similar to no treatment (no adoptive transfer) controls. These data are summarized in Table 1 . WT C57BL/6 T cells cultured with or without IL-12 and/or IL-18 did not mediate tumor regression of C1498/ovalbumin (data not shown), and OT-1 T cells did not mediate suppression of parental C1498 tumor (even with cytokine exposure; data not shown).


Figure 3
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  		Figure 3. Adoptive transfer of tumor-specific CD8+ T cells cultured with IL-12 mediates enhanced tumor suppression independent of IL-18. Activated OT-1 T cells cultured with IL-12, IL-18, both, or neither were adoptively transferred at a high (1 x 107), intermediate (5 x 106), or low (1 x 106) dose to mice bearing C1498/ovalbumin tumors. One cohort received no treatment (no adoptive transfer; No AT), and the average tumor growth curve for this group is represented in all similar panels for comparison. A, transfer of T cells cultured with no polarizing cytokine failed to mediate tumor suppression at any cell dose. B, tumor suppression was enhanced modestly by transfer of the highest dose of T cells cultured with IL-18, but by day 21 the effect was indistinguishable from untreated (no adoptive transfer) control tumor growth (P > 0.1). C, IL-12-cultured T cells enhanced tumor suppression at both high (1 x 107) and intermediate (5 x 106) cell doses compared with the highest dose of cells transferred after culture with no cytokine or IL-18 (P < 0.0007). D, culture with IL-12 and IL-18 was equally effective as culture with IL-12 alone (P > 0.7 at all cell doses on day 21). E, overall survival with i.p. tumor was similarly enhanced by adoptive transfer of 1 x 107 IL-12-cultured T cells or T cells cultured with IL-12 plus IL-18 (P < 0.05, compared with no cytokine and IL-18).

 

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  		Table 1. Ex vivo culture with IL-12 enhances adoptive tumor immunotherapy

 
To evaluate the in vivo effect of IL-12 and IL-18 as ex vivo adjuvants in a more stringent tumor model, we adoptively transferred cytokine-cultured or control OT-1 T cells to mice bearing 8-day i.p. C1498/ovalbumin and monitored cohorts for survival. Similar to results from 3-day s.c. tumor studies, IL-12 alone was sufficient and necessary to improve survival to 100%, whereas IL-18 neither helped nor hindered disease survival rates. These data show that varying the location of tumor (i.p. versus s.c.) and increasing the tumor burden (8-day versus 3-day) does not impair the benefit of IL-12 alone as an adjuvant for CD8+ T-cell adoptive immunotherapy.

We considered the possibility that IL-12 was as effective as the combination of IL-12 with IL-18 on adoptive transfer due to recipient-derived IL-18. To rule this out, we did adoptive transfer of OT-1 T cells cultured with IL-12 alone or the combination of IL-12 plus IL-18 to s.c. tumor-bearing mice genetically deficient in IL-18 production. The two tumor growth curves remained identical, showing that recipient-derived IL-18 had no effect on the effector function of IL-12-cultured cells and their enhancement of antitumor activity (data not shown). Conversely, to determine if the modest effect of IL-18-cultured T cells on tumor suppression was due to recipient-derived IL-12, we transferred the highest dose of IL-18-cultured and IL-12 plus IL-18–cultured OT-1 T cells to tumor-bearing mice deficient in production of IL-12. IL-18-cultured T cells did not mediate any tumor suppression, similar to no adoptive transfer, showing that the slight tumor suppression observed after transfer of a high dose of IL-18-cultured T cells requires recipient-derived IL-12 (data not shown).

IFN-{gamma}-deficient CD8+ T cells mediate antigen-specific tumor suppression that is enhanced by ex vivo culture with IL-12. We began our studies in this model with the hypothesis that the synergy between IL-12 and IL-18 for effector CD8+ T cell production of IFN-{gamma} was important to potentiate an increase in antitumor activity on adoptive transfer, but our in vivo results suggested that the amount of IFN-{gamma} produced was not critical to the success of the therapy. It remained possible that an increase in the number of T cells producing IFN-{gamma}, which was affected by IL-12 alone or in combination with IL-18, was still important. To determine the requirement for IFN-{gamma} in our clinical responses, we adoptively transferred IFN-{gamma}-deficient OT-1 T cells cultured with IL-12, IL-18, both, or neither at a single intermediate dose of 5 x 106 to mice bearing 3-day s.c. tumors. T cells cultured with IL-12 alone or the combination of IL-12 and IL-18 had enhanced antitumor activity relative to controls cultured with IL-18 alone or no cytokine (Fig. 4A ). The latter yielded tumor curves similar to the untreated cohort, which received no adoptive transfer.


Figure 4
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  		Figure 4. Tumor-specific, IFN-{gamma}-deficient CD8+ T cells mediate tumor regression that is enhanced by IL-12. A, activated OT-1 T cells (WT; open symbols) or deficient in IFN-{gamma} production (IFN-{gamma}–/–; closed symbols) were adoptively transferred at a single, intermediate cell dose (5 x 106) to mice bearing 3-day s.c. tumors, and tumors were measured twice weekly. An untreated cohort (no adoptive transfer) was maintained as a control. Tumor-specific T cells deficient in IFN-{gamma} production were capable of mediating tumor suppression after culture with IL-12 or the combination of IL-12 and IL-18 (P < 0.05 for both on day 12 after transfer) similar to WT cells under the same conditions (P < 0.04). T cells cultured with no polarizing cytokine or with IL-18 before the transfer were as ineffective as no adoptive transfer (P > 0.4 at all time points). Inhibition of tumor growth for groups receiving T cells cultured with IL-12 or IL-12 plus IL-18 was statistically indistinguishable irrespective of donor cell ability to produce IFN-{gamma} (P > 0.1). B, TNF-{alpha} secretion was increased after culture with the combination of IL-12 and IL-18 (P < 0.00002, compared with other conditions). IFN-{gamma}-deficient T cells showed the same pattern of expression, with enhancement of TNF-{alpha} secretion occurring only in the population of T cells cultured previously with IL-12 and IL-18 (P < 0.00006). C, antigen-specific IL-10 production was stimulated by T-cell culture with IL-12 and greater than that stimulated by IL-18 or no cytokine culture (P < 0.0006). IL-12 plus IL-18 stimulated ovalbumin-specific IL-10 similar to IL-12 culture (P > 0.4, compared with IL-12; P < 0.0001, compared with other controls). Loss of IFN-{gamma} production did not alter IL-10 expression.

 
These data supported two important conclusions about our tumor model. First, IFN-{gamma}-independent effector functions were enhanced by IL-12 alone and were sufficient to improve CD8+ T-cell-mediated antitumor activity. Second, IFN-{gamma} itself was not required; thus, the synergy between IL-12 and IL-18 for CD8+ donor T-cell production of IFN-{gamma} was not necessary to achieve enhanced tumor suppression.

We considered that rejection of tumors in our IFN-{gamma}-deficient T-cell adoptive transfer studies might be affected by compensatory overexpression of other cytokines. IL-10, a type 2 and immunoregulatory cytokine known to be up-regulated by IL-12, was of particular interest (38, 39). We cultured OT-1 T cells, either WT or IFN-{gamma} deficient, alone or with C1498 or C1498/ovalbumin. T cells had been activated and cultured with IL-12, IL-18, both, or neither before the assay. Supernatants from the cocultures were tested for IL-10 secretion by ELISA. Figure 4B shows that IFN-{gamma}-deficient T cells have the same pattern of IL-10 secretion as the WT cells following cytokine exposure, suggesting no alteration in IL-10 production by CD8+ T-cell effector function as a result of genetic loss of IFN-{gamma} expression. As expected, IL-12 stimulated antigen-specific IL-10 secretion, and the combination of IL-12 and IL-18 supported the same level of production. No significant secreted IL-4 was detectable with or without stimulation of T cells, irrespective of cytokine exposure, and whether endogenous IFN-{gamma} could be produced or not (data not shown). Thus, IFN-{gamma}-deficient OT-1 T cells did not default to a type 2 phenotype.

We also measured secretion of type 1 cytokine TNF-{alpha}. Supernatants from cocultures of WT OT-1 or IFN-{gamma}-deficient OT-1 T cells with tumor were tested by ELISA for TNF-{alpha} secretion (Fig. 4C). Interestingly, we found that IL-12 plus IL-18 promoted synergy for antigen-specific TNF-{alpha} production, but no other cytokine culture conditions enhanced secretion. An important role for enhanced TNF-{alpha} under WT conditions was ruled out because IL-12-cultured T cells did not up-regulate TNF-{alpha} production and yet effectively improved antileukemia responses in vivo in s.c. tumor and leukemia survival studies. IFN-{gamma}-deficient T cells showed an identical production profile as WT T cells, so there was no compensation for loss of one cytokine with the other.

Perforin-deficient CD8+ T cells do not mediate antigen-specific tumor suppression and are not enhanced by culture with IL-12 or IL-18. The other major CD8+ T-cell effector function we found to be affected by our ex vivo cytokine culture regimen was antigen-specific, perforin-dependent cellular cytotoxicity enhanced by IL-12 but not IL-18. In vitro, genetic loss of perforin expression resulted in ablation of cytotoxicity whether T cells were cultured with any cytokine before the assay. To confirm translation of this finding to in vivo antitumor activity, we adoptively transferred perforin-deficient OT-1 T cells at an intermediate dose of 5 x 106 per s.c. tumor-bearing mouse. Activated T cells were cultured with IL-12, IL-18, both, or neither before the transfer, and one cohort of untreated animals was monitored in parallel (no adoptive transfer). In support of our in vitro cytotoxicity assay, perforin-deficient T cells did not mediate tumor regression irrespective of cytokine exposure, suggesting an absolute requirement for perforin-dependent lysis to eliminate established s.c. tumors (Fig. 5 ).


Figure 5
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  		Figure 5. Tumor-specific, perforin-deficient CD8+ T cells do not effectively control tumor growth. Activated T cells from WT and perforin-deficient OT-1 donor mice were cultured with IL-12, IL-18, both, or neither and then adoptively transferred at a single, intermediate cell dose (5 x 106) to animals bearing 3-day s.c. tumors. Tumors were measured twice weekly. Perforin-deficient OT-1 T cells were incapable of controlling tumor growth irrespective of cytokine culture conditions before the transfer, resulting in average tumor growth curves similar to untreated mice (no adoptive transfer; P > 0.1 for all time points and cohort comparisons). WT OT-1 donor cells treated with IL-12 or IL-12 plus IL-18 mediated enhanced tumor suppression compared with WT cells cultured with IL-18 or no cytokine (P < 0.05 on day 21) and compared with all perforin-deficient OT-1 recipients (P < 0.005).

 
Our data collectively suggest that CD8+ T-cell antitumor activity was enhanced by IL-12 and required perforin-dependent cytolysis but was not dependent on an increase in the number of IFN-{gamma}-producing T cells or amount of IFN-{gamma} secreted. Thus, IL-12-mediated up-regulation of CD8+ T-cell cytotoxicity was the primary effector of enhanced antitumor responses independent of the effects of IL-18 on T cells and in the absence of donor T-cell IFN-{gamma} production.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We showed that culturing effector CD8+ T cells with IL-12 before the adoptive transfer mediated enhanced immunotherapy of murine tumors equivalent to culture with both IL-12 and IL-18, which suggests that IFN-{gamma} production is not a central mechanism of antitumor activity in our model as we had originally hypothesized. This was confirmed in vivo by transferring IFN-{gamma}-deficient, antigen-specific CD8+ T cells cultured with IL-12 or the combination of IL-12 and IL-18 to tumor-bearing mice. Both IFN-{gamma}+/+ and IFN-{gamma}–/– donor T cells had equivalent antitumor activity under each culture condition. Tumor regression induced by IFN-{gamma}–/– OT-1 T cells was not simply due to compensatory up-regulation of other antitumor type 1 cytokines or default to the type 2 phenotype, as production of TNF-{alpha}, IL-4, and IL-10 was not exaggerated in IFN-{gamma}-deficient T cells after in vitro stimulation.

IFN-{gamma}-independent mechanisms of IL-12- and IL-18-mediated CD8+ T-cell effector function are seldom explored, as the synergy for IFN-{gamma} production has overshadowed other potential benefits of the cytokine effects on T cells (37, 40–43). In this report, we showed that, in the absence of donor T-cell production of IFN-{gamma}, another mechanism was proven critical to both enhancement of CD8+ T-cell adoptive immunotherapy and its effectiveness in general. This mechanism was perforin-dependent cytolysis, which we showed was required for in vitro cytotoxicity against murine leukemia targets and for in vivo antileukemia activity.

Our data support the conclusions of other reports of ex vivo IL-12 and IL-18 T-cell adjuvant use for adoptive immunotherapy and show several significant differences. First, a recent report published by Li et al. showed the effectiveness of ex vivo IL-12 and IL-18 as an adjuvant for T-cell immunotherapy of cancer. Their tumor model was a murine sarcoma micrometastasis model. The adoptively transferred cells, although activated and cultured ex vivo similarly to ours, were derived from TDLN cells of donor mice and consisted of both CD4+ and CD8+ T cells. They showed that IL-12 plus IL-18 synergistically increased IFN-{gamma} and decreased IL-10 production in vitro. In our model, we confirmed the increase in IFN-{gamma} but did not observe a decrease in IL-10. This difference in cytokine production is likely due to the inclusion of CD4+ T cells in the sarcoma immunotherapy model and the mixed milieu of cells present in TDLN. Further, the previously published study showed that neither IL-12 nor IL-18 alone provided the same benefit as the combination of cytokines, whereas our data suggested that IL-12 alone might be sufficient and necessary to improve T-cell adoptive immunotherapy. We attribute this key difference again to the source of adoptively transferred cells because the effect of IL-12 on cytotoxic CD8+ T cells is expected to be profoundly different than its effect on mixed populations of CD4+, CD8+, and other TDLN-resident cells with varied polarization and effector status. The report by Li et al. did not evaluate IFN-{gamma}-independent T-cell effector functions nor did it address cytolytic function or production of TNF-{alpha} or IL-4 (44).

We also tested the effectiveness of IL-12 and IL-18 as ex vivo adjuvants in another tumor model, a sarcoma micrometastasis model, in which HER-2/neu was the target tumor antigen. In the sarcoma model, both CD4+ and CD8+ T cells were retrovirally transduced with a TCR specific for HER-2/neu and then adoptively transferred to mice bearing HER-2/neu+ lung micrometastases. As reported here, IL-12 was sufficient to increase the frequency of antigen-specific CD8+ IFN-{gamma}-producing T cells, but IL-12 and IL-18 together were required to synergistically increase IFN-{gamma} production in vitro. Preliminary data suggest that both IL-12 and IL-18 are required to improve antitumor activity in this model in vivo. Our laboratory has established previously that this tumor model requires only CD8+ T cells for tumor clearance and that antitumor activity depends on both cellular cytotoxicity and tumor-specific IFN-{gamma}.7 In consideration of these and published data from Li et al., we further refine our criteria for the use of IL-12 alone versus IL-12 plus IL-18 to conclude that, in tumor models that do not require CD4+ T-cell help but are sensitive to donor cell–derived IFN-{gamma}, the combination of IL-12 and IL-18 may be necessary to improve T-cell adoptive immunotherapy. Conversely, when IFN-{gamma}-independent mechanisms, such as cellular cytotoxicity, are the main requirement for tumor clearance (as in the major model presented in this report), IL-12 alone may be sufficient. Of note, our data indicate that in vitro analysis of frequency of IFN-{gamma}-producing cells correlates well with in vivo results, as both C1498 and MT901 models show increased numbers of IFN-{gamma}-producing cells after tumor-specific T cells are cultured with IL-12 and IL-12 plus IL-18, and these populations of cells are the best antitumor effectors in their respective tumor models. Thus, we show that even for an IFN-{gamma}-insensitive tumor model the ability of effector T cells to increase the frequency of tumor-specific IFN-{gamma}-secreting cells in vitro is a reliable predictor of clinical response.

One other similar report has been published using only ex vivo IL-12 as adjuvant for another solid tumor model of adoptive immunotherapy. The authors found that mixed effector T cells became skewed to the type 1 phenotype and had improved cellular cytotoxicity and in vivo antitumor activity. The killing mechanism, and addition of IL-18, was not addressed (45). Our data bridge the gap between these studies and provide further insight into effector mechanisms affected by, and sometimes required for, the observed increase in antitumor activity mediated by ex vivo cytokine adjuvant.

Cytotoxicity has been identified as an important effector function in the elimination of many tumors, including leukemias (46, 47). Our studies reveal a murine model of leukemia, in which perforin-mediated T-cell cytotoxicity seems to be the major effector mechanism required for tumor suppression. A variety of leukemia-associated antigens have been identified, including a Wilms' tumor antigen, proteinase 3, and minor histocompatability antigens, which serve as targets of antigen-specific cytotoxicity in vitro and are eliminated by tumor-specific T-cell populations in vivo (9, 48). A precedent thus exists for cytotoxicity serving as the key mechanism and a valid predictor of clinical responses in cancer therapy.

Collectively, we have shown that ex vivo cytokines can be used as adjuvant for CD8+ T-cell adoptive tumor immunotherapy, and predictive tests of in vivo effector T-cell antitumor activity, such as IFN-{gamma} production, may agree with but do not necessarily translate into antitumor responses. Whereas tumor-specific IFN-{gamma} production correlated with in vivo antitumor activity, IFN-{gamma} production by donor T cells was not required for therapeutic benefit. We identified a key function, perforin-mediated lysis, which was enhanced by IL-12 culture in vitro and required in vivo to eliminate tumors. These data suggest a preclinical model of CD8+ T-cell adoptive immunotherapy for IFN-{gamma}-independent antitumor activity elicited by an ex vivo cytokine adjuvant, IL-12, while providing supporting in vitro correlates to successful antitumor activity.


   Acknowledgments
 
Grant support: NIH grant R01CA92488 and Leukemia and Lymphoma Society Award (K.T. McDonagh) and University of Michigan Miller Fund Award (J.N. MacGregor).

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.

We thank Frank A. Urban and Srilakshmi Yalavarthi for expert technical support; Michael S. Khodadoust, Shengping Li, and Jianmin Yang for assistance with experimental design and data analysis; and Mark Dobrzanski and David Niederbuhl for generously assisting with procurement of mice described in this report.


   Footnotes
 
7 Unpublished data. Back

Received 9/30/05. Revised 2/14/06. Accepted 3/ 8/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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