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The Journal of Immunology, 2007, 179, 1669 -1680
Copyright © 2007 by The American Association of Immunologists, Inc.
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IFN-{gamma} and Fas Ligand Are Required for Graft-versus-Tumor Activity against Renal Cell Carcinoma in the Absence of Lethal Graft-versus-Host Disease1

Teresa Ramirez-Montagut*,{dagger}, Andrew Chow*, Adam A. Kochman*, Odette M. Smith*, David Suh*, Hamad Sindhi*, Sydney Lu*, Chiara Borsotti*, Jeremy Grubin*, Neel Patel*, Theis H. Terwey*, Theo D. Kim*, Glenn Heller{ddagger}, George F. Murphy§, Chen Liu, Onder Alpdogan* and Marcel R. M. van den Brink2,*

* Department of Immunology and Department of Medicine, Laboratory of the Immunology of Bone Marrow Transplantation, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; {dagger} Howard Hughes Medical Institute, Laboratory of Neuro-oncology, The Rockefeller University, New York, NY 10021; {ddagger} Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; § Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115; and Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32608


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To determine the mechanisms of graft-versus-tumor (GVT) activity in the absence of graft-versus-host disease (GVHD) against a solid tumor, we established two allogeneic bone marrow transplantation models with a murine renal cell carcinoma (RENCA). The addition of 0.3 x 106 donor CD8+ T cells to the allograft increased the survival of tumor-bearing mice without causing GVHD. The analysis of CD8+ T cells deficient in cytotoxic molecules demonstrated that anti-RENCA activity is dependent on IFN-{gamma} and Fas ligand (FasL), but does not require soluble or membrane-bound TNF-{alpha}, perforin, or TRAIL. Recipients of IFN-{gamma}–/– CD8+ T cells are unable to reject RENCA compared with recipients of wild-type CD8+ T cells and, importantly, neither group develops severe GVHD. IFN-{gamma}–/– CD8+ T cells derived from transplanted mice are less able to kill RENCA cells in vitro, while pretreatment of RENCA cells with IFN-{gamma} enhances class I and FasL expression and rescues the lytic capacity of IFN-{gamma}–/– CD8+ T cells. These results demonstrate that the addition of low numbers of selected donor CD8+ T cells to the allograft can mediate GVT activity without lethal GVHD against murine renal cell carcinoma, and this GVT activity is dependent on IFN-{gamma} and FasL.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Allogeneic bone marrow transplantation (allo-BMT)3 is an effective therapeutic strategy for hematological malignancies. Its therapeutic efficacy could be enhanced by facilitating the capacity of donor T cells to react against tumor cells, i.e., graft-versus-leukemia (GVL) effect, while avoiding graft-versus-host disease (GVHD), the primary complication of this therapy (1). We and others have determined that donor T cells use different cytolytic pathways to mediate GVHD and GVL effects. The effector mechanisms that have been studied include the Fas/Fas ligand (FasL), perforin/granzyme, and TRAIL/DR5 pathways and several cytokines, including TNF-{alpha} and IFN-{gamma} (2). Aside from differences in the results of several experimental models, most studies indicate an important role for Fas/FasL in systemic and liver GVHD (3, 4, 5), while TNF-{alpha} seems to be involved in intestinal GVHD (6, 7, 8). Conversely, different pathways have been described in GVL activity. Although perforin/granzyme is the dominant pathway used by alloreactive CD8+ T during GVL activity (9, 10, 11, 12, 13), recent studies indicate a role for the TRAIL/DR5 pathway during antileukemic responses (14). However, in models of graft-versus-lymphoma, both Fas/FasL and perforin/granzyme are dominant and required for optimal GVL activity (15). Therefore, the pathways used in optimal GVL reactions may depend on the tumor type.

Although allo-BMT has been proposed as a therapy for solid tumors, only few clinical trials, and even fewer experimental models, exist (16, 17). Therefore, in contrast to GVL activity, little is known regarding the mechanisms involved in graft-versus-tumor (GVT) activity. Recent studies have demonstrated the efficacy of non-myeloablative allo-BMT in patients with renal cell carcinoma (18, 19). In this study, we used two novel experimental allo-BMT models for the study of GVT activity against renal cell cancer to analyze and optimize GVT activity in the absence of lethal GVHD. The sensitivity of these models was further enhanced by the use of novel bioluminescence techniques, which allow sequential noninvasive monitoring of tumor growth in vivo in individual mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines

Renal cell carcinoma (RENCA; H-2d) was cultured in RPMI 1640, 10% heat-inactivated FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate at 1.5 g/L sodium bicarbonate, and 4.5 g/L glucose. RENCA was retrovirally transduced to express a triple-fusion protein consisting of herpes simplex virus thymidine kinase, enhanced GFP, and firefly luciferase (TGL: plasmid was provided by V. Ponomarev, Memorial Sloan-Kettering Cancer Center, New York, NY) (20). This line was used to challenge all transplanted mice, whereas untransduced RENCA was used for in vitro cytotoxicity experiments.

Mice, bone marrow transplantation (BMT), and tumor induction

Female C57BL/6 (H-2b), BALB/c (H-2d), and B10.D2 (H-2d) mice were obtained from The Jackson Laboratory. Mice were used between 8 and 12 wk old. Bone marrow (BM) cells were isolated by flushing femurs and tibias. Donor BM was T cell depleted by incubating with Low-TOX-M rabbit complement (Cedarlane Laboratories) for 40 min at 37°C. The donor T cell inoculum consisted of whole T cells or CD8+ T cells selected from the spleens of wild-type (WT), IFN-{gamma}–/–, TNF-{alpha}–/–, pfp–/–, gld, TRAIL–/–, or membrane-bound form of TNF-{alpha} (memTNF) mice with anti-CD8 magnetic microbeads (Miltenyi Biotec). Cells were transplanted by tail vein injection into lethally irradiated BALB/c recipients (900 cGy) from a 137Cs source as a split dose with a 3-h interval between doses. For GVT experiments, animals received 1 x 105 RENCA cells i.v. in a separate injection on day 0. BMT protocols were approved by the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee. Mice were housed in sterilized microisolator cages and received normal chow and autoclaved hyperchlorinated drinking water (pH 3.0).

Assessment of GVT by bioluminescence imaging (BLI)

For quantitative assessment of RENCA tumor burden by BLI, mice received an i.p. injection of 3 mg of D-Luciferin per mouse (Xenogen). Fifteen minutes later, mice were anesthetized and placed into the IVIS bioluminescence imaging system (Xenogen), and bioluminescent signal intensity was recorded for up to 5 min. In response to the saturation of images, recording time was brought down to 30 s or 1 min. Pseudocolor images showing the whole-body distribution of bioluminescent signal intensity were superimposed on conventional grayscale photographs, and total flux (photons/sec) was determined for individual mice.

Assessment of GVHD

The severity of GVHD was assessed with a clinical GVHD scoring system, as previously described (21). In brief, mice were individually scored every week for five clinical parameters on a scale from 0 to 2 as follows: weight loss, posture, activity, fur, and skin. A clinical GVHD index was generated by the summation of the five criteria scores (0–10). Survival was monitored daily. Animals with scores >5 were considered moribund and were sacrificed.

GVHD histopathologic analysis

GVHD target organ pathology for the small and large bowel and liver was assessed in a blinded fashion. Formalin-preserved organs were paraffin embedded, sectioned, H&E stained, and scored with a semiquantitative scoring system. Bowel and liver were scored for 18–22 different parameters associated with GVHD, as previously described (22), and skin was evaluated for the number of apoptotic cells per millimeter of epidermis, as previously described (23).

Flow cytometric analysis

Splenocytes were washed in FACS buffer (PBS with 2% FBS and 0.1% sodium azide) and incubated for 15 min at 4°C with anti-CD16/CD32 FcR block. Subsequently, cells were incubated for 30 min at 4°C with Abs and washed twice with FACS buffer. Stained cells were resuspended in FACS buffer and analyzed on a FACSCalibur flow cytometer (BD Biosciences) with CellQuest or FlowJo software (Tree Star). For detection of cell surface molecules, RENCA cells were treated with 5, 10, and 50 ng/ml murine rIFN-{gamma} (BD Pharmingen) for 12, 24, 36, and 48 h. RENCA cells were trypsinized (0.1% trypsin, 0.02% EDTA in HBSS without Ca2+ and Mg2+), washed twice in FACS buffer, and incubated for 15 min at 4°C with anti-CD16/CD32 FcR block. Subsequently, cells were washed and incubated for 30 min at 4°C with Ab, washed twice with FACS buffer, and analyzed. The following fluorochrome-labeled anti-murine Ab were used: CD16/CD32 FcR block (clone 2.4G2), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD45R/B220 (RA3-6B2), Gr-1 (RB6-8C5), Mac-1 (CD11b clone M1/70), NK1.1 (PK136), H-2Kb (AF6-88.5), Fas (Jo2), DR5 (MD5-1), I-Ad (39-10-8), and H-2Dd (34-2-12) (all from BD Pharmingen). For annexin V analysis, RENCA cells were treated with 50 ng/ml murine rIFN-{gamma} (BD Pharmingen) for 24 h. RENCA cells were plated in 24-well plates (Corning Glass) previously coated with sodium azide-free/low endotoxin anti-murine Fas (Jo2 Ab) or sodium azide-free/low endotoxin anti-trinitrophenyl isotype control hamster IgG Gr.1{kappa} (A119-3) (10 µg/ml in PBS at 4°C overnight). FasL-sensitive LK35.2 or RENCA cells were plated in 24-well plates at a concentration of 1 x 105 cells in 1 ml of medium. After 8 h at 37°C, the cells were harvested and resuspended in 100 µl of annexin V-binding buffer and 5 µl of annexin V (BD Pharmingen). The cells were incubated in the dark at room temperature for 20 min, and 300 µl of annexin V-binding buffer was added before FACS analysis.

Allogeneic CD8+ T cell cytotoxicity

Mice were transplanted with WT or IFN-{gamma}–/– CD8+ T cells, challenged with RENCA cells, and sacrificed 21 days after transplantation. CD8+ T cells were purified with magnetic beads (~90% purity; Miltenyi Biotec) and placed in coculture with untreated RENCA cells or pretreated with 50 ng/ml IFN-{gamma} for 24 h (Pre-IFN). The coculture consisted of 1 x 104 CD8+ T cells with 1 x 104 RENCA cells. After 8 h of coculture, all cells were harvested and stained for annexin V staining. For positive control, IFN-{gamma}-treated RENCA cells were cultured onto Jo2-precoated wells.

Histopathology

Lungs were preserved in formalin following death and then embedded in paraffin, sectioned, and stained with H&E. The immunohistochemical detection of CD3+ T cells was performed with a Discovery XT System (Ventana Medical Systems). The protocol was established at the Molecular Cytology Core Facility at Memorial Sloan-Kettering Cancer Center. In brief, sections were blocked for 30 min using 10% normal goat serum (Vector Laboratories) and 2% BSA, followed by avidin and biotin incubation for 4 min each. Incubation with 0.6 µg/ml CD3 Ab (DakoCytomation) was carried for 3 h at room temperature followed by a 60-min incubation with biotinylated anti-rabbit Ab at a 1/200 dilution (Vectastain ABC; Vector Laboratories). A diaminobenzidine (DAB) detection kit containing blocker D, copper D, inhibitor D, streptavidin-HRP D, DAB H2O2 D, and DAB D (Ventana Medical Systems) was used according to the manufacturer’s instructions.

Statistics

Kaplan-Meier curves were analyzed with log rank. Mouse studies were performed to assess changes in BLI over time. The mice were assigned to treatment groups, and the area under the curve (AUC) was used to summarize the BLI trajectory of each mouse under study. Not all of the mice were followed for the full length of the study. The primary reason for censoring was death or sacrifice, and ignoring this type of informative censoring may result in a biased treatment comparison. To eliminate this bias, a statistic test to compare two groups was formed using the information up to the minimum follow-up time for each cross-treatment mouse pair. By eliminating the uneven censorship between mouse pairs in different groups, an AUC-based statistic test can be constructed that has a mean of 0 when the growth rates in the two groups are equal. The statistic is based on the average difference in the censored AUC curves between treatment groups (24). The p values derived from this statistic were generated from a permutation test, because the number of animal per group in these studies was small. Analysis of CD8+ T cell cytotoxicity was done using a two-tailed Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The addition of selected donor CD8+ T cells to the BM allograft induces strong GVT activity without lethal GVHD in a MHC-disparate BMT model

We studied GVHD and GVT activity of unfractionated T cells in a MHC-disparate model. Lethally irradiated BALB/c mice were transplanted with C57BL/6 T cell-depleted bone marrow (TCD-BM) accompanied by 1 x 106 C57BL/6 whole T cells, and mice were challenged with 1 x 105 RENCA cells. We found that most mice reject RENCA (86%), but a high proportion (57%) succumb to lethal GVHD (data not shown). We then tested the possibility of uncoupling GVHD from GVT by excluding CD4+ T cells from the graft and infusing only purified CD8+ T cells. The dose of CD8+ T cells was titrated down until most of the mice survived up to 150 days after BMT (data not shown). Once the dose of 0.3 x 106 C57BL/6 CD8+ T cells was determined, we tested whether in the absence of lethal GVHD, the same number of CD8+ T cells would be sufficient to reject a tumor challenge (Fig. 1). Mice transplanted with TCD-BM and challenged with RENCA die rapidly and fail to reject the tumor, while 40% of the mice that receive CD8+ T cells rejected RENCA and survive >150 days (Fig. 1, A and B) with no significant weight loss due to GVHD (Fig. 1C). Importantly, all control mice that received only CD8+ T cells and no tumor challenge survived (Fig. 1A) and exhibited minimal GVHD as determined by weight loss (Fig. 1C).


Figure 1
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  		FIGURE 1. Allo-BMT with CD8+ T cells results in GVT activity without lethal GVHD. Lethally irradiated BALB/c mice were transplanted with 5 x 106 TCD-BM and challenged with 1 x 105 RENCA cells by tail vein injection. Mice received TCD-BM and RENCA cells (BM + RENCA), TCD-BM with 0.3 x 106 C57BL/6 CD8+ T cells and RENCA cells (BM + RENCA + CD8), or TCD-BM with 0.3 x 106 C57BL/6 CD8+ T cells without tumor challenge (BM + CD8). A, The Kaplan-Meier survival curve indicates percentage of mice that survived the transplant. The graphic includes data of two independent experiments (for BM + RENCA n = 4–5 mice/group; for BM + RENCA ± CD8 n = 10 mice/group). B, Tumor burden graphic represents average bioluminescence per group and shows data of one representative experiment from a total of two. C, Percent weight loss represents clinical GVHD. The graphic represents one experiment from a total of two. D, Lethally irradiated BALB/c mice were transplanted with 5 x 106 TCD-BM and challenged with different doses of RENCA tumor (BM + 5 x 104, 1 x 105, and 5 x 105 RENCA cells) or were transplanted with TCD-BM in addition to 0.3 x 106 C57BL/6 CD8+ T cells and different doses of RENCA cells (BM + 5 x 104, 1 x 105 RENCA cells, or 5 x 105 RENCA cells + CD8). Control mice were transplanted with TCD-BM in addition to 0.3 x 106 C57BL/6 CD8+ T cells without tumor challenge (BM + CD8). The Kaplan-Meier survival curve represents one experiment. E, Tumor burden graphic represents average bioluminescence per group and shows data from one experiment. *, p < 0.05.

 
To track the progression of tumor growth, we established a high sensitivity and high throughput bioluminescence imaging system (20, 25) that allows us to noninvasively detect the progress of the tumor without sacrificing animals. Bioluminescence of the RENCA tumor in vivo indicated a significant delay in tumor growth in the recipients of allogeneic CD8+ T cells compared with the TCD-BM group (Fig. 1B). These results indicate that in the absence of CD8+ T cells, transplanted mice die rapidly of tumor burden, while mice that receive CD8+ T cells with the allograft show a significant increase in survival and a delay in tumor growth without the detrimental effect of GVHD.

Next, we examined whether the same dose of CD8 cells could continue to reject different doses of RENCA cells (Fig. 1, D and E). Lethally irradiated BALB/c mice were transplanted with C57BL/6 TCD-BM accompanied by 0.3 x 106 C57BL/6 CD8+ T cells, and mice were challenged with 5 x 104, 1 x 105, and 5 x 105 RENCA cells. The results indicate that, in every case, the presence of CD8+ T cells increased survival (Fig. 1D) and the delay of tumor take (Fig. 1E).

Although the dose of CD8+ T cells is not inducing severe or lethal GVHD (100% survival in groups infused with CD8+ T cells only), we assessed subclinical GVHD in target organs (Fig. 2). Lethally irradiated BALB/c mice transplanted with C57BL/6 TCD-BM alone or accompanied by 0.3 x 106 C57BL/6 CD8+ T cells were sacrificed 20 days later, and GVHD target organs were analyzed by semiquantitative histopathology and flow cytometry. The results indicate no significant differences in some GVHD target organs, including skin (Fig. 2, table), liver, large bowel (Fig. 2A), and thymus (Fig. 2B), while mild GVHD was detected in the small bowel (Fig. 2A).


Figure 2
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  		FIGURE 2. Subclinical GVHD in mice transplanted with allo-BMT and CD8+ T cells. Lethally irradiated BALB/c mice were transplanted with 5 x 106 TCD-BM (BM) or TCD-BM with 0.3 x 106 C57BL/6 CD8+ T cells (BM + CD8) and target organ GVHD was analyzed in tissues harvested 20 days after transplantation. Results from skin GVHD are presented in the table. A, Semiquantitative histopathological analysis for GVHD in liver, small bowel, and large bowel. B, Absolute numbers of thymocytes (Thy) and double-positive cells (DP) were analyzed by flow cytometry. The data represent a single experiment with n = 10 mice/group; *, p < 0.05.

 
Overall, these results indicate that a dose of 0.3 x 106 CD8+ T cells infused in the allo-BMT does not induce severe or lethal GVHD, but rather a mild subclinical reaction that allows for 100% survival of the hosts. Importantly, this allogeneic CD8+ T cell dose provides anti-RENCA activity at different tumor doses.

GVT activity by selected donor CD8+ T cells is dependent on FasL and IFN-{gamma}

Previous studies have demonstrated that RENCA cells are sensitive to the lytic effects of perforin and Fas/FasL, insensitive to TNF-{alpha}, and have been inconclusive regarding the contribution of TRAIL (26, 27, 28). We tested the mechanism of tumor rejection by comparing WT donor CD8+ T cells against CD8+ T cells derived from mice deficient in different cytotoxic molecules. A first set of experiments compared WT CD8+ T cells to perforin-deficient (pfp–/–) CD8+ T cells (Fig. 3). Lethally irradiated BALB/c mice transplanted with TCD-BM and challenged with RENCA died from tumor burden (Fig. 3), while the majority of mice that receive either WT CD8+ T cells, or pfp–/– CD8+ T cells demonstrated an increase in survival (Fig. 3A) and delay in tumor growth (Fig. 3, B and C). Thus, perforin-deficient CD8+ T cells are as efficient as WT in rejecting RENCA cells, indicating that perforin is not required for RENCA protection. Importantly, all of the control mice that received TCD-BM and WT CD8+ T cells without tumor challenge survived, indicating an absence of lethal GVHD, and further demonstrating that the deaths in the groups that were challenged with RENCA were due to tumor burden.


Figure 3
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  		FIGURE 3. Perforin is not required for GVT activity against RENCA. Lethally irradiated BALB/c mice were transplanted with 5 x 106 TCD-BM and challenged with 1 x 105 RENCA cells by tail vein injection. Mice received TCD-BM and RENCA cells (BM + RENCA), TCD-BM with 0.3 x 106 WT CD8+ T cells and RENCA cells (BM + RENCA + WT CD8), TCD-BM with 0.3 x 106 pfp–/– CD8+ T cells and RENCA cells (BM + RENCA + pfp–/– CD8), or TCD-BM with 0.3 x 106 WT CD8+ T cells without tumor challenge (BM + CD8). A, The Kaplan-Meier survival curve indicates percentage of mice that survived the transplant. The graphic includes data from two independent experiments (for BM and BM + RENCA n = 4–5 mice/group; for all other groups n = 10 mice/group). *, p < 0.01. B, Tumor burden graphic represents average bioluminescence per group and shows data of one representative experiment from a total of two. C, Bioluminescence of RENCA cells per individual mice. The images show data of one representative experiment from a total of two.

 
Next, we tested whether FasL expression on allogeneic CD8+ T cells is required for tumor rejection (Fig. 4). Lethally irradiated BALB/c mice transplanted with TCD-BM and challenged with RENCA died from tumor burden (Fig. 4), while the majority of mice that received WT CD8+ T cells survive (Fig. 4A) and displayed a delay in tumor growth (Fig. 4, B and C). Conversely, although mice that received CD8+ T cells deficient in FasL (gld CD8+ T cells) show increased survival and a delay in tumor growth compared with the control group that received no CD8+ T cells, most mice are not able to reject RENCA and ultimately die with a detectable tumor present (Fig. 4C). These results indicate a requirement for FasL in the CD8-mediated rejection of RENCA.


Figure 4
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  		FIGURE 4. FasL is required for GVT activity against RENCA. Lethally irradiated BALB/c mice were transplanted with 5 x 106 TCD-BM and challenged with 1 x 105 RENCA cells by tail vein injection. Mice received TCD-BM (BM), TCD-BM and RENCA cells (BM + RENCA), TCD-BM with 0.3 x 106 WT CD8+ T cells and RENCA cells (BM + RENCA + WT CD8), or TCD-BM with 0.3 x 106 gld CD8+ T cells and RENCA cells (BM + RENCA + gld CD8). A, The Kaplan-Meier survival curve indicates percentage of mice that survived the transplant. The graphic includes data from two independent experiments (for BM and BM + RENCA n = 4–5 mice/group; for all other groups n = 8–10 mice/group). *, p < 0.01. B, Tumor burden graphic represents average bioluminescence per group and shows data of one representative experiment from a total of two. C, Bioluminescence of RENCA cells per individual mice. The images show data from one representative experiment from a total of two.

 
To test the role of other members of the TNF family during RENCA rejection, WT CD8+ T cells were compared with CD8+ T cells from mice deficient in TRAIL or from mice expressing a memTNF, a mutant form of TNF-{alpha} deficient in the TNF-{alpha} converting enzyme (TACE) cleavage site (29) (Fig. 5). Survival (Fig. 5A) and tumor rejection (Fig. 5, B and C) were comparable between mice that received WT CD8+ T cells and those that received TRAIL–/– or memTNF-{alpha} T cells, indicating that CD8-mediated GVT activity against RENCA does not require TRAIL or soluble TNF.


Figure 5
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  		FIGURE 5. TRAIL and memTNF are not required for GVT activity against RENCA. Lethally irradiated BALB/c mice were transplanted with 5 x 106 TCD-BM and challenged with 1 x 105 RENCA cells by tail injection. Mice received TCD-BM ({lambda}), TCD-BM, and RENCA cells ({nu}), TCD-BM with 0.3 x 106 WT CD8+ T cells and RENCA cells ({varsigma}), TCD-BM with 0.3 x 106 memTNF CD8+ T cells and RENCA cells ({upsilon}), or TCD-BM with 0.3 x 106 TRAIL–/– CD8+ T cells and RENCA cells ({varsigma}). A, Kaplan-Meier survival curve indicates percentage of mice that survived the transplant. The graphic includes data of two independent experiments (for {lambda} and {nu}, n = 4–5 mice/group; for {varsigma}, {varsigma}, and {upsilon}, n = 8–10 mice/group), *, p < 0.01. B, Tumor burden graphic represents average bioluminescence per group and shows data of one representative experiment from a total of two. C, Bioluminescence of RENCA cells per individual mice. The images show data of one representative experiment from a total of two.

 
Next, we compared WT CD8+ T cells against IFN-{gamma}–/– and TNF-{alpha}–/– CD8+ T cells to test the role of these cytotoxic cytokines during RENCA rejection (Fig. 6). Mice that received TNF-{alpha}–/– CD8+ T cells were able to protect the host from RENCA challenge to the same extent as WT CD8+ T cells. This further demonstrates that TNF-{alpha}, either in its soluble or membrane-bound form, is not required for tumor rejection. Mice that received CD8+ T cells deficient in IFN-{gamma} (IFN-{gamma}–/–) succumbed rapidly (Fig. 6A) due to tumor burden (Fig. 6B), indicating that IFN-{gamma} expression by donor CD8+ T cells is required for tumor rejection.


Figure 6
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  		FIGURE 6. IFN-{gamma} but not TNF-{alpha} is required for GVT activity against RENCA. Lethally irradiated BALB/c mice were transplanted with 5 x 106 TCD-BM and challenged with 1 x 105 RENCA cells by tail injection. Mice received TCD-BM (BM), TCD-BM and RENCA cells (BM + RENCA), TCD-BM with 0.3 x 106 WT CD8+ T cells and RENCA cells (BM + RENCA + CD8), TCD-BM with 0.3 x 106 TNF-{alpha}–/– CD8+ T cells and RENCA cells (BM + RENCA + TNF–/– CD8), or TCD-BM with 0.3 x 106 IFN-{gamma}–/– CD8+ T cells and RENCA cells (BM + RENCA + IFN–/– CD8). A, The Kaplan-Meier survival curve indicates percentage of mice that survived the transplant. The graphic represents one of three independent experiments (for BM and BM + RENCA n = 4–5 mice/group; for all other groups n = 8–10 mice/group). *, p < 0.01. B, Bioluminescence of RENCA cells per individual mice. The images show data of one representative experiment from a total of three. C, IFN{gamma}–/– CD8+ T cells do not exacerbate GVHD. Lethally irradiated BALB/c mice were transplanted with 5 x 106 C57BL/6 TCD-BM (BM), TCD-BM and 0.3 x 106 WT CD8+ T cells (BM + WT CD8), or TCD-BM with 0.3 x 106 IFN{gamma}–/– CD8+ T cells (BM + IFN–/– CD8) (for BM n = 5 mice; for all other groups n = 10 mice/group). Top panel, Kaplan-Meier survival curve indicates percentage of mice that survived the transplant. Middle panel, Clinical GVHD score based on five clinical parameters measured once a week on a scale from 0 to 2: weight loss, posture, activity, fur, and skin. Bottom panel, Percent weight loss represents one parameter of clinical GVHD.

 
Donor IFN-{gamma}–/– CD8+ T cells do not induce lethal GVHD

Several groups have suggested that IFN-{gamma} is necessary for tolerance induction and, thus, the absence of IFN-{gamma} in donor T cells may enhance GVHD (30, 31, 32, 33, 34, 35, 36, 37). Therefore, it is possible that the infusion of IFN-{gamma}–/– CD8+ T cells enhances GVHD and abrogates any GVT activity present in these cells. To address whether IFN-{gamma}–/– CD8+ T cells could enhance GVHD, mice were transplanted with TCD-BM accompanied with 0.3 x 106 WT CD8+ T cells or the same number of IFN-{gamma}–/– CD8+ T cells (Fig. 6C). We found that comparable numbers of mice receiving either WT or IFN-{gamma}–/– CD8+ T cells survived (Fig. 6C, top). GVHD clinical scores (Fig. 6C, middle) and weight loss (Fig. 6C, bottom) indicated no differences between the two groups, although mild GVHD was detected. Overall, these results demonstrate that IFN-{gamma}–/– CD8+ T cells do not exacerbate GVHD in our model.

IFN-{gamma}–/– CD8+ T cells efficiently infiltrate RENCA tumors

Previous studies have shown that IL-12-mediated regression of RENCA tumor is partly mediated by IFN-{gamma}-inducible chemokines, including IFN-{gamma}-inducible protein 10 (IP-10) and monokine induced by IFN-{gamma} (Mig), which aid in the recruitment of CXCR3+-activated T cells to the tumor bed (38, 39). To address whether IFN-{gamma}–/– CD8+ T cells failed to reject RENCA lung metastasis due to a defect in homing, lungs were harvested at necropsy and analyzed by histopathology for CD3+ staining. We found no significant differences in T cell infiltration of IFN-{gamma}–/– CD8+ T cells compared with WT CD8+ T cells (data not shown), indicating that IFN-{gamma}–/– CD8+ T cells are not impaired in their capacity to infiltrate RENCA tumors. Because IP-10 has been shown to inhibit angiogenesis (40, 41, 42), it remains to be determined the extent of tumor rejection contributed by a IFN-{gamma}-mediated antiangiogenic effect.

IFN-{gamma} enhances Fas expression on RENCA cells and increases killing by alloreactive CD8+ T cells

Previous studies have demonstrated that IFN-{gamma} up-regulates the cell surface expression of Fas on RENCA cells, rendering them more susceptible to cell death through members of the TNF superfamily (26, 27, 43). Because both IFN-{gamma} and FasL pathways are involved in GVT activity against RENCA, we hypothesized that IFN-{gamma} may modulate Fas expression on RENCA cells, leading to its in vivo rejection. RENCA cells treated in vitro with 10 ng/ml IFN-{gamma} for up to 2 days show no difference in the survival determined by annexin V staining (data not shown), indicating that IFN-{gamma} has no direct cytotoxic effect on RENCA cells. Under the same conditions, IFN-{gamma} enhances the cell surface expression of MHC class I (H-2Dd) and Fas, while it down-regulates the expression of DR5, the TRAIL receptor (Fig. 7A). Moreover, incubation with 20 ng/ml IFN-{gamma} for up to 2 days further down-regulated DR5 expression on RENCA cells. Class II molecules, as expected, are not present on the cell surface of RENCA cells.


Figure 7
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  		FIGURE 7. IFN-{gamma} up-regulates Fas on RENCA cells and enhances CD8+ lysis. A, Flow cytometric analysis of cell surface expression of Fas enhanced by IFN-{gamma} treatment. 1 x 105 RENCA cells were plated and treated with 10 and 20 ng/ml IFN-{gamma} for 24 and 48 h. Data represent cell surface expression on untreated cells (blue histogram) and 10 (red histogram) or 20 ng/ml (green histogram) of IFN-{gamma} at 24 and 48 h. Grey histogram represents the isotype control. B, IFN{gamma} pretreatment of RENCA cells is sufficient for Fas-mediated apoptosis. RENCA cells were cultured in 50 ng/ml IFN-{gamma} for 24 h and subsequently plated onto Jo2 or isotype control precoated wells. Adherent and floating cells were harvested and stained for annexin V. The data represent one of two independent experiments. C, Allogeneic CD8+ T cell-killing of RENCA cells is enhanced with IFN-{gamma} pretreatment. Lethally irradiated BALB/c mice were transplanted with 5 x 106 TCD-BM and challenged with 1 x 105 RENCA cells by tail vein injection. Mice received either 0.3 x 106 WT CD8+ T cells (n = 5) or 0.3 x 106 IFN{gamma}–/– CD8+ T cells (n = 5) and were sacrificed 21 days after transplantation. CD8+ T cells were purified and cocultured with RENCA cells for 8 h at a 1:1 ratio (1 x 104 CD8+ T cells vs 1 x 104 RENCA cells). Prior to coculture, RENCA cells were either left untreated or were pretreated for 24 h with 10 ng/ml IFN-{gamma} (pre-IFN). The data represents percentage of RENCA cells positive for annexin V. Results from individual mice are represented by closed circles. RENCA cells pretreated with IFN-{gamma} and cultured on Jo2-coated plates are used as positive controls and are represented by closed squares (pre-IFN Jo2). *, p < 0.05.

 
Next, we tested whether the incubation of RENCA cells with IFN-{gamma} and treatment with an activating anti-Fas Ab (clone Jo2) could induce direct lysis of RENCA cells (Fig. 7B). Less than ~20% of RENCA cells plated onto Jo2-treated wells for 8 h were positive for annexin V staining. When RENCA cells were preincubated with IFN-{gamma} for 18 h, washed, and then plated onto Jo2-coated wells for 8 h, annexin V staining increased 2-fold. These results indicate that IFN-{gamma} up-regulation of Fas followed by Fas stimulation is sufficient for the induction of apoptosis in RENCA cells.

To further elucidate the role of IFN-{gamma} during GVT activity, alloreactive WT and IFN-{gamma}–/– CD8+ T cells isolated from tumor-bearing transplanted hosts were added to RENCA cells (Fig. 7C). WT CD8+ T cells were able to induce apoptosis of RENCA cells (34.3 ± 2.2% SEM), while preincubation of RENCA cells with IFN-{gamma} significantly increased the percentage of apoptotic cells (41.6 ± 1.9% SEM). By contrast, IFN-{gamma}–/– CD8+ T cells induced significantly less RENCA lysis (26.3 ± 2.7% SEM), while preincubation of RENCA cells with IFN-{gamma} demonstrated similar levels of apoptosis as WT CD8+ T cells (36 ± 1.7% SEM), showing that the defect in lysis can be rescued by preincubation of the target with IFN-{gamma}. These results indicate that treatment of RENCA cells with IFN-{gamma} enhances Fas expression and correlates with an increase in alloreactive CD8+ T cell killing.

Addition of donor CD8+ T cells to the BM allograft maintains GVT activity without lethal GVHD in a clinically relevant MHC-matched allo-BMT model

To determine whether the infusion of CD8+ T cells maintains a strong GVT activity in a more clinically relevant model, we used a MHC-matched model of B10.D2 into BALB/c (Fig. 8). This model allows for tumor challenge with the same RENCA cell line from BALB/c origin. First, the role of whole T cells in GVT and GVHD was determined (Fig. 8A). Lethally irradiated BALB/c mice were transplanted with TCD-BM accompanied by 3 x 106 whole T cells, and challenged with 1 x 105 RENCA cells. We found an increase in the survival of mice that received RENCA and T cells compared with control mice that received no T cells in the allograft, although no mice survived the transplant. Control mice that received only T cells and no tumor challenge developed lethal GVHD and died at a comparable rate as those challenged with tumor. Tumor burden measured by bioluminescence indicated that, even when GVT activity delayed tumor growth (data not shown), some mice that were able to reject RENCA succumbed to acute GVHD, indicating that even in the presence of strong GVHD, optimal GVT activity was not achieved.


Figure 8
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  		FIGURE 8. Complete MHC-matched allo-BMT with CD8+ T cells results in significant GVT activity without lethal GVHD. Lethally irradiated BALB/c mice (H-2d) were transplanted with 5 x 106 TCD-BM from B10.D2 mice (H-2d) and challenged with 1 x 105 RENCA cells by tail vein injection. A, Mice received TCD-BM ({lambda}), TCD-BM and RENCA cells ({nu}), TCD-BM with 5 x 106 whole B10.D2 T cells and RENCA cells ({sigma}), or TCD-BM with 1 x 106 whole B10.D2 T cells without tumor challenge ({sigma}). B and C, Allo-BMT with CD8+ T cells results in GVT activity without GVHD. Lethally irradiated BALB/c mice (H-2d) were transplanted with 5 x 106 TCD-BM from B10.D2 mice (H-2d) and challenged with 1 x 105 RENCA cells by tail vein injection. Mice received TCD-BM and RENCA cells ({nu}), TCD-BM with 2 x 106 CD8+ T cells and RENCA cells ({sigma}), or TCD-BM with 2 x 106 CD8+ T cells without tumor challenge ({sigma}). B, Kaplan-Meier survival curve indicates percentage of mice that survived the transplant. The graphic includes data of two independent experiments (for {nu}, n = 4–5 mice/group; for {sigma} and {sigma}, n = 10 mice/group). C, Percent weight loss represents clinical GVHD. The graphic includes data of one representative experiment from a total of two. D, Tumor burden graphic represents average bioluminescence per group and shows data of one representative experiment from a total of two. E, Bioluminescence of RENCA cells per individual mice. The images show data of one representative experiment from a total of two.

 
Next, we tested whether GVT activity could be detected in the absence of lethal GVHD by infusion of 2 x 106 B10.D2 CD8+ T cells instead of unfractionated T cells (Fig. 8, B–E). Lethally irradiated BALB/c mice transplanted with TCD-BM and challenged with RENCA died rapidly (median survival of 27 days), while a significant increase in survival was observed in mice that received CD8+ T cells (median survival of 47 days). Importantly, all mice in the control group that received CD8+ T cells and no tumor challenge survived the transplant, indicating an absence of lethal GVHD (Fig. 8B). Bioluminescence of tumor cells in vivo indicated a delay in tumor growth in mice that received CD8+ T cells compared with BM-only control mice (Fig. 8, D and E). Importantly, the unchallenged CD8+ T cell control group did not demonstrate clinical GVHD or significant weight loss (Fig. 8C).

Taken together, these results demonstrate in two different allo-BMT models that separation of GVT activity against a RENCA from GVHD is possible when purified CD8+ T cells are added to the allograft. Allogeneic CD8+ T cells can induce a potent GVT activity, resulting in a tumor delay or even tumor rejection in the absence of lethal GVHD.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We demonstrate in two independent allo-BMT models, including a MHC-disparate and a MHC-matched model, that the addition of low numbers of CD8+ T cells to the allograft confers significant GVT activity against RENCA in the absence of lethal GVHD. We determined the mechanism of tumor rejection and demonstrated a dependence on IFN-{gamma} and FasL. In vitro studies indicate that upon IFN-{gamma} treatment, Fas is up-regulated and activation of this cell surface molecule can induce RENCA apoptosis. Overall, these results suggest a model where alloreactive CD8+ T cells produce IFN-{gamma}, which up-regulates Fas on RENCA cells and induces their elimination from the host via FasL expressed on alloactivated CD8+ T cells. Consistent with our model, in studies of acute and chronic GVHD IFN-{gamma} was shown to regulate the expression of both Fas/FasL on alloreactive T cells (4). Our data do not exclude the possibility that an additional antiantiogenic effect of IFN-{gamma} may be involved in RENCA rejection.

Also consistent with our findings are several reports on the susceptibility of RENCA FasL-induced apoptosis (26, 27, 43). In particular, Seki et al. (26) demonstrated that CD8+ CTL responses against RENCA are different in vitro vs in vivo. Whereas perforin and FasL mediate in vitro CTL activity against RENCA cells, in vivo tumor regression is perforin independent. These results are consistent with our findings demonstrating that perforin is not required for in vivo rejection of RENCA. A more confounding result in our BMT model is the absence of TRAIL-mediated tumor rejection, because this pathway seems crucial for anti-RENCA responses both in vitro (27) and in vivo (26, 28, 44, 45). In vivo experiments (44, 45) determined that tumor rejection was dependent on TRAIL expression by NK cells induced by IFN-{gamma}. In our GVT model, the role of NK cells is negligible because the only effector cells in the graft are purified CD8+ T cells. A second explanation for the lack of TRAIL-dependent RENCA lysis could be attributed to the differences in the local environment of the metastatic nodules. An isolated clone of RENCA (clone R331), which is more sensitive to FasL- and TRAIL-induced cell death, displayed differences in the ability to form liver and lung metastases (28). R331 readily metastasizes to the lung, while few liver metastases develop. Assuming that TRAIL-mediated killing of RENCA cells occurs preferentially in the liver compartment, then it is not surprising that GVT activity against lung metastases is TRAIL independent. Furthermore, our experiments show a mild down-regulation of DR5, the TRAIL receptor, on the cell surface of RENCA cells after IFN-{gamma} treatment.

Although IFN-{gamma} plays an important role in the cytokine cascade during the pathogenesis of GVHD, as demonstrated by the laboratories of Ferrara and Sykes (46, 47, 48), lethal acute GVHD can be induced in the absence of IFN-{gamma} (31). Moreover, several groups propose a role for IFN-{gamma} as an inhibitor of acute GVHD (30, 32, 33, 34, 35, 36, 37). For example, in a MHC-disparate model (30), injection of 7.5–10 x 106 BALB/c CD4-depleted splenocytes from IFN-{gamma}–/– mice leads to increased mortality of C57BL/6 mice, while injection of WT cells results in long-term survival of most recipients. Furthermore, injection of 12 x 106 transgenic H-2b CD8+ T cells (CD8 plus 2C TCR) specific against H-2Ld (e.g., BALB/c) does not induce lethal GVHD, unless these cells are derived from IFN-{gamma}–/– mice. The authors contend that because IFN-{gamma} contributes to the death phase of activated CD8+ T cells, IFN-{gamma}–/– donors will expand and augmentation of GVHD will follow. Importantly, these studies are coinciding in the requirement of IFN-{gamma} for adequate GVT activity. Our results indicate that IFN-{gamma} is necessary for CD8+ T cell-mediated rejection of RENCA cells and infusion of IFN-{gamma}–/– CD8+ T cells does not exacerbate GVHD. Our model predicts that small numbers of allogeneic CD8+ T cells are sufficient for local production of IFN-{gamma} that render RENCA cells susceptible for Fas-mediated death while GVHD may require more numbers of CD8+ T cells or the presence of alloreactive CD4+ T cells.

Since the seminal clinical study by Childs et al. (18), renal cell cancer has been a target for allo-BMT therapy because of its sensitivity to GVT after a non-myeloablative BMT and resistance to chemotherapy or other conventional treatments. To our knowledge, only two studies in mice address the role of allo-BMT for the rejection of tumor challenge with RENCA cells (49, 50). Both use a non-myeloablative conditioning regimen and determine that this regimen by itself does not suffice for tumor rejection. RENCA rejection is only achieved when donor leukocyte infusion (DLI) is added to the regimen. In the study by Harano et al. (50), mice are injected with allogeneic splenocytes and BM, and 2 days later tolerance was induced by treatment with cyclophosphamide. These cyclophosphamide-induced tolerant mice have low chimerism and fail to reject RENCA. The same treatment followed by DLI on day 3 increased mixed chimerism and rejects s.c. RENCA. In a second study by Zoller et al. (49), BALB/c hosts received on day 7 DLI (containing 5 x 106 lymph node cells from 129SVEV donors (H-2b) previously immunized with RENCA cells) simultaneously with a tumor lysate-pulsed dendritic cell vaccine. Under these conditions, GVHD is still observed and only when donor T cells are "host-tolerant" does the vaccination strategy become advantageous. To generate "host-tolerant" T cells, BALB/c mice were lethally irradiated, reconstituted, and vaccinated with RENCA cells. Although both studies provide evidence for GVT activity, the conditioning, early DLI, and/or vaccination regimens used are difficult to translate to clinical trials. Importantly, both models require a "tolerization" strategy of donor T cells that may impair long-term antitumor responses and do not address the immunological mechanism required for RENCA rejection. In contrast, our results are directly clinically relevant for the development of novel clinical trials. For example, after non-myeloablative stem cell transplantation and infusion of HLA-matched CD34+ cells in patients with renal cell carcinoma, the expansion of CD8+ T cells that produce IFN-{gamma} is associated with clinical responses (51). Moreover, in a Phase I clinical trial, donor-derived CD8+ CTL clones specific for minor H Ags have also been successfully used to mediate GVT activity (52).

In conclusion, we have demonstrated in two independent allo-BMT models that GVT activity against a renal cell carcinoma can be uncoupled from GVHD and is dependent on IFN-{gamma} and Fas/FasL. We believe that these data are important for the understanding of GVT activity and for the development of new treatments for renal cell carcinoma and the treatment of solid tumors.


   Acknowledgments
 
T. Ramirez-Montagut thanks Robert B. Darnell for support during the elaboration of this manuscript.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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.

1 This work was supported by Grants HL69929, HL72412, and CA33049 from the National Institutes of Health and awards from the Leukemia and Lymphoma Society, Emerald Foundation and the Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center funded by William H. Goodwin and Alice Goodwin and the Commonwealth Foundation for Cancer Research. Back

2 Address correspondence and reprint requests to Dr. Marcel M. R. van den Brink, Laboratory of Allogeneic Bone Marrow Transplantation, Memorial Sloan-Kettering Cancer Center, Z1404, Box 111, 1275 York Avenue, New York, NY 10021. E-mail address: vandenbm{at}mskcc.org Back

3 Abbreviations used in this paper: allo-BMT, allogeneic bone marrow transplantation; GVL, graft-versus-leukemia; GVHD, graft-versus-host disease; FasL, Fas ligand; GVT, graft-versus-tumor; RENCA, renal cell carcinoma; BMT, bone marrow transplantation; BM, bone marrow; BLI, bioluminescence imaging; AUC, area under the curve; TCD-BM, T cell-depleted BM; WT, wild type; memTNF, membrane-bound form of TNF-{alpha}; DLI, donor leukocyte infusion; DAB, diaminobenzidine. Back

Received for publication September 7, 2006. Accepted for publication May 1, 2007.


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 Introduction
 Materials and Methods
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 Discussion
 Disclosures
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K. Ishiyama, A. Takami, S. Suzuki, H. Konaka, M. Namiki, A. Ooi, and S. Nakao
Relationship Between Tumor-infiltrating T Lymphocytes and Clinical Response After Reduced-intensity Allogeneic Hematopoietic Stem Cell Transplantation for Advanced Renal Cell Carcinoma: A Single Center Prospective Study
Jpn. J. Clin. Oncol., September 20, 2009; (2009) hyp104v1.
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