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Blood, 1 March 2007, Vol. 109, No. 5, pp. 2234-2242. Prepublished online as a Blood First Edition Paper on November 2, 2006; DOI 10.1182/blood-2006-07-037473.
TRANSPLANTATION A GMCSF and IL-15 fusokine leads to paradoxical immunosuppression in vivo via asymmetrical JAK/STAT signaling through the IL-15 receptor complex1 The Montreal Centre for Experimental Therapeutics in Cancer, Jewish General Hospital, McGill University, Montreal, QC, 2 Molecular Oncology Laboratory, McGill University, Montreal, QC; 3 Department of Chemistry, University of Quebec at Montreal (UQAM), Montreal, QC; and 4 Division of Hematology/Oncology, Jewish General Hospital, McGill University, Montreal, QC
We hypothesized that a granulocyte macrophage colony-stimulating factor (GMCSF) and interleukin 15 (IL-15) fusokine (GIFT15) would possess greater immune-stimulatory properties than their combined use. Unexpectedly, tumor cells engineered to secrete GIFT15 protein led to suppression of natural killer (NK) and NKT-cell recruitment in vivo, suggesting an unanticipated immune-suppressive effect. We found GIFT15 to have pleiotropic effects on an array of immune-competent cells. Among these, macrophages treated with GIFT15 secrete de novo the tissue inhibitor of metalloproteinase-2 (TIMP-2); activated matrix metalloproteinase-2 (MMP-2); transforming growth factor-ß (TGF-ß); as well as vascular endothelial growth factor (VEGF). We show that the GIFT15 fusokine has increased affinity for the  chain component of the IL-15R, leading to aberrant signaling through the ß chain manifested by the hyperphosphorylation of STAT3 both in macrophages and splenocytes. Suppression of common  chain–mediated STAT5 phosphorylation and blockade of the IL-15–dependent IFN- response in mouse splenocytes were also observed. We tested GIFT15 as an immunosuppressor and demonstrated that it allowed engraftment of allogeneic B16F0 and human xenograft U87GM glioma cells in immunocompetent mice. Thus, GIFT15 defines a new class of fusokine that mediates proangiogenic and immunosuppressive effects via aberrant signaling by the IL-15R in lymphomyeloid cells.
Immune-stimulatory cytokines can be exploited to treat human ailments including cancer. Among the cytokines identified for such use, GMCSF has been under much scrutiny due to its direct action on the adaptive immune system through the enhancement of antigen presentation as well as costimulation.1,2 Furthermore, second-generation strategies linking innate and adaptive immunity using GMCSF delivered as a fusion cytokine (fusokine) with other immune-stimulatory proteins such as IL-2 are currently being developed.3,4 The utility of GMCSF-containing fusokines having been established in animal models of cancer immunotherapy begets the testing of novel combinatorial fusokines, especially since the biochemical behavior of such chimeras can lead to unprecedented biopharmaceutical properties as we have previously reported.4 IL-15 possesses overlapping activities with IL-2 such as the activation of T cells and the stimulation of natural killing5 as well as additive stimulatory effects on the immune system distinct from IL-2.6,7 These features make IL-15 an attractive companion to GMCSF as part of an immunotherapeutic fusokine. In fact, it was previously reported that cotreatment of dendritic cell (DC) precursors with GMCSF and IL-15 as separate entities can generate a powerful T-helper 1 (Th1) immune response both in vitro and in vivo.8 Thus, we hypothesized that the generation of a GMCSF and IL-15 fusion transgene (hereafter GIFT15) would lead to immunostimulatory synergy in the setting of cancer immunotherapy. Unexpectedly, we found that the GIFT15 fusokine behaved in a manner opposite to what was anticipated and possessed profoundly immune-suppressive properties as well as robust proangiogenic features in vivo. These unanticipated features were found to arise from asymmetrical JAK/STAT signaling through the IL-15 receptor complex in responsive lymphomyeloid cells. This novel pharmaceutical effect was further characterized in the setting of allogeneic and xenogeneic somatic cell transplantation. Here we describe our findings that support the potential use of GIFT15 as a novel immunosuppressive compound.
Animals, cell lines, recombinant proteins, antibodies, and ELISA kits
All experimental mice were females aged 6 to 8 weeks (Jackson Laboratory, Bar Harbor, ME). The C57Bl/6-derived B16F0 and human U87GM cell lines were cultured in DMEM (Wisent Technologies, Rocklin, CA) supplemented with 10% FBS (Wisent Technologies) and 50 U/mL Pen/Strep (Wisent Technologies). The cell lines JAWSII and CTLL2 (American Type Culture Collections [ATCC], Manassas, VA) were grown according to ATCC's recommendations. Biochemical reagents used were as follows: recombinant proteins (rIL-15/rIL-15R Vector construct and protein modeling
The mouse IL-15 cDNA (Invivogen, San Diego, CA) was modified to remove the 3' nucleotides encoding the STOP codon and subsequently cloned in frame with the cDNA encoding mouse GMCSF cDNA to generate the cDNA for GIFT15 fusokine. The GIFT15 cDNA was incorporated into a bicistronic retrovector allowing the expression of both the fusokine and GFP reporter.4 To build a structural model of GIFT15 by homology modeling, crystal structures of human GMCSF (accession code 2gmf) and human IL-2 (D chain, accession 1erj) were used as templates for mouse GMCSF and mouse IL-15, respectively. The structural template for the region connecting GMCSF and IL-15 (accession code 1orc) was identified by fold recognition methods, using software PROSPECT v2 (Oak Ridge National Laboratory, Oak Ridge, TN). Based on the templates identified, 50 structural models of GIFT15 were generated using software MODELLER v6 (University of California at San Francisco). A structural model of GIFT15 in complex with cytokine receptor was generated based on the crystal structure of the IL-2 signaling complex (accession 1erj), which is the trimeric assembly of IL-2R Fusokine expression and functional assays Infectious retroparticles encoding GIFT15 were generated with 293-GP2 packaging cell (Clontech, Mountain View, CA) and concentrated retroparticles were used to gene modify B16F0 melanoma and U87 glioma cells. To test the bioactivity of GIFT15, the IL-2–responsive CTLL-2 or GMCSF-responsive JAWSII cell lines were plated at a density of 105 cells/well in a 96-well plate and treated with increasing concentrations of cytokines for 72 hours. Cell proliferation was assessed with a 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Murine B16F0 tumor implantation in syngeneic C57Bl/6 mice and immune infiltrate analysis One million cytokine-secreting B16F0 cells were injected subcutaneously in C57bl/6 mice, and tumor growth was monitored over time. All implanted B16F0 polyclonal populations produced comparable molar quantities of cytokines (0.6 ± 0.1 pmol/106 cells/24 hours). For immune infiltrate analysis, one million cytokine-secreting B16F0 cells were mixed with 500 µL Matrigel (BD Biosciences) at 4°C and injected subcutaneously in C57Bl/6 mice. Implants were surgically removed 2 weeks after transplantation and digested as reported previously.4 After incubation with anti–FcR III/II mAb for 1 hour, cells were incubated for 1 hour at 4°C with appropriate antibodies and analyzed by flow cytometry using a Becton Dickinson FACScan (San Jose, CA). Murine B16F0 tumor implantation in NOD-SCID mice One million GIFT15-secreting or GFP-expressing B16F0 cells were injected subcutaneously in immunocompromised nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice, and tumor growth was monitored over time. For VWF immunostaining, animals were killed and tumors retrieved for paraffin embedment before being cut and probed with anti-VWF as reported elsewhere.9 Total blood vessels were counted and divided by the total surface area calculated using Scion image software (Scion, Frederick, MD) to obtain blood vessel density. Macrophage migration assays, intracellular signaling, and cytokine secretion Peritoneal macrophages were isolated from C57Bl/6 mice by peritoneal lavage and used for all macrophage-based in vitro assays. For migration assays, 105 macrophages per well were plated in the top chambers of a Transwell plate and the lower chambers were filled, in triplicate with 500 µL serum-free RPMI to which conditioned media (CM) derived from cytokine-expressing B16 cells were added to achieve a final cytokine concentration of 0.1 or 1 nM. After 18 hours of incubation at 37°C, the cells on the bottom filter of each well were counted at 10 high-power fields (x 400). For signaling analysis, GIFT15 was purified using an immunoaffinity column packed using CNBr-sepharose (Amersham, Piscataway, NJ) according to the manufacturer's instructions and 30 pmol purified cytokines were added to 106 macrophages for 15 minutes before being lysed and probed by Western blot (WB) with rabbit antiphosphorylated STAT3 or STAT5. Total STAT3 or STAT5 proteins were used as loading controls. For analysis of macrophage secretome, macrophages were cultured with 30 pmol cytokines for 72 hours at 37°C, and supernatants screened using angiogenic protein arrays and ELISA according to the manufacturer's instructions. Surface plasmon resonance (SPR)
The binding interaction between GIFT15 and the IL-15 receptor GIFT15-mediated biochemical responses in splenocytes
Splenocytes were collected from normal C57Bl/6 mice. Media containing 30 pmol cytokines were used to stimulate 106 splenocytes for 15 minutes and cell lysates generated for Western blot analysis with antiphosphorylated STAT3, STAT5, and Bcl-XL. For apoptosis assays, 106 splenocytes were cultured using the same conditions for 36 hours and then stained with propidium iodine (PI) and for annexin-V. For splenocyte proliferation assays, 105 splenocytes were cultured with increasing concentrations of cytokines for 72 hours at 37°C and compared by MTT assay. To assess IFN- Allogeneic B16F0 and xenogenic U87GM tumor implantation Allogeneic tumor cell implantation was performed by injecting 107 live B16-GFP or B16-GIFT15 in BALB/c mice and tumor growth followed over time. For spleen analysis, animals with GIFT15 tumors exceeding 1000 mm3 or with the largest B16-GFP tumors were killed and the spleen was removed and weighed. Paraffin-embedded slides were also prepared for hematoxylin and eosin (H&E) staining and visualized using a Zeiss Axiovert 25 microscope (Carl Zeiss, Toronto, ON, Canada) with a 40x/0.55 NA objective. Pictures were taken using a Sony VX-DSC-W5 Mpegmovie digital camera (Sony, Tokyo, Japan). For flow cytometry analysis, splenocytes were stained and analyzed by fluorescence-activated cell sorting (FACS). For xenogenic tumor cell implantation, 107 live human U87-GFP or U87-GIFT15 transduced tumor glioma cells were implanted subcutaneously. Tumor growth and graft survival were monitored over time in wild-type C57Bl/6, CD4–/–, CD8–/–, or beige (natural killer [NK] deficient) mice. Statistical analysis P values were calculated using the paired Student t test.
Design and characterization of murine GIFT15 The fusokine was created by cloning a modified GMCSF cDNA missing the nucleotides coding for the last 11 carboxy-terminal amino acids (aa's), in frame with the 5' end of the mouse IL-15 cDNA, including its long signal peptide.6,7 The final fusokine GIFT15 cDNA encodes for a single polypeptide chain of 299 aa's (Figure 1A). Computer-based analysis of the 3-dimensional structure revealed that the 7-aa peptidic bridge and the uncleaved IL-15 long signal peptide sequence form an intercytokine bridge of 55 aa's in length with a 3 alpha helix configuration (Figure 1B). Denaturing immunoblotting performed on the supernatant from B16F0 cells retrovirally transduced to express GIFT15 showed that the chimeric protein was efficiently secreted into the extracellular space and migrated at a molecular weight of 55 kDa (Figure 1C). The bioactivity of both cytokine subunits within GIFT15 was confirmed by the proliferation of the GMCSF-dependent JAWSII and IL-15–dependent CTLL2 cell lines, respectively (Figure 1D).
GIFT15 promotes tumor growth by blocking the recruitment of NK/NKT cells and by inducing angiogenesis To assess the ability of GIFT15 to induce an immune modulatory effect, polyclonal populations of B16F0 cells engineered to secrete equimolar levels of cytokines were injected subcutaneously in syngeneic immune-competent C57Bl/6 mice. We observed that B16F0 cells secreting GIFT15 (hereafter B16-GIFT15) had acquired aggressive growth properties with an average tumor size 3-fold larger than that of control groups in the weeks following implantation (Figure 2A). To determine whether this phenomenon was linked to an atypical immune response, we analyzed tumor infiltration by immune cells 2 weeks after implantation of Matrigel matrix embedded cells. We found that NK and NKT cells were virtually absent in GIFT15-secreting tumors when compared with B16-GMCSF or B16–IL-15 control groups, while the number of other CD3+ T-cell subsets was similar to controls (Figure 2B). The observed absence in NK/NKT-cell recruitment by B16-GIFT15 cells contradicted what we predicted would occur in vivo, especially since IL-15 has been shown by others to directly stimulate the development, expansion, recruitment, and activation of NK and NKT cells.10–13 B16-GIFT15 cells implanted in immunocompromised NOD-SCID mice also showed significantly enhanced tumorigenicity where we would have predicted a similar tumor growth rate to controls if immunosuppression was solely at play (Figure 2C). Histologic analysis of explanted tumors by immunostaining against VWF revealed a 3-fold increase in blood vessel density (P < .05) in B16-GIFT15 tumors compared with the control (Figure 2D) as well as a robust 4-fold increase of infiltration by tumor-associated macrophages (TAMs) (data not shown).
GIFT15 effect on macrophages
TAMs play an important role in immunosuppression and angiogenesis.14–16 The GIFT15-secreting tumors showed a 4-fold increase in macrophage content when compared with control B16 tumors (data not shown), suggesting a powerful macrophage chemotactic property for the fusokine in vivo. Macrophages are known to express the IL-15 receptor (IL-15R) where intracellular signaling occurs through the ß chain (JAK1/STAT3) and the
GIFT15 leads to hyperphosphorylation of STAT3 and blockade of STAT5 activation in lymphoid cells
CD3+ and NK/NKT lymphoid cells express the full IL-15R composed of the
GIFT15 protects splenocytes from apoptosis, stimulates proliferation, and blocks IFN- secretion
STAT3 activation has been linked to a variety of biochemical and cellular events such as survival, proliferation, angiogenesis, and immunosuppression.23–26 Since we have shown that splenocytes hyperphosphorylate STAT3 in response to GIFT15, we tested whether GIFT15 enhances survival of splenocytes in vitro using serum-free media as a proapoptotic stimulus. Splenocytes stained for PI and annexin-V revealed that 83% of cells treated with GIFT15 survived compared with 33% with rGMCSF, 43% using rIL-15, or 41% with both molecules (Figure 5A). In addition, cell lysate immunoblotting against the antiapoptotic molecule Bcl-XL (Figure 5A) provided evidence that GIFT15 rescues splenocytes from cell death through an increase in Bcl-XL level, a process known to occur when STAT3 is dominantly activated.23,24 Of interest, splenocyte proliferation does not seem to be affected by the relative decrease in STAT5 phosphorylation (Figure 5B; P < .05) despite the fact that the latter is associated with mitogenic activities.27–29 Based on the asymmetrical signaling mediated by GIFT15 through the IL-15R, we tested for expression levels of known target genes of STAT5 in immune-competent cells—such as IFN
GIFT15 can block lymphocyte activation arising from a mixed lymphocyte reaction and allows engraftment of GIFT15-expressing allogeneic somatic cells in immune-competent recipients
Since GIFT15 can signal aberrantly through the IL-15R by hyperphosphorylating STAT3 without leading to IFN-
GIFT15 allows for xenotransplantation of the human glioma U87GM in immunocompetent mice We tested the limits of GIFT15-mediated tolerance in the context of xenotransplantation using the human glioma cell line U87GM transduced to secrete the GIFT15 fusokine. GIFT15-secreting tumors were accepted in all mice until day 224, at which time half of the group rejected the graft. All control mice had rejected the U87-GFP graft 12 days after transplantation (Figure 7A-B). However, wild-type C57Bl/6 mice allowed the GIFT15-secreting tumor graft to survive for up to 2 months, starting from the day the control tumors were rejected (Figure 7C). Of interest, implanted CD8–/– mice reproduced the same graft survival curve obtained by the wild-type C57Bl/6 strain, indicating that CD8 T cells are not implicated in the rejection of xenogenic cells. In contrast, NK deficiency is permissive to xenografting since 80% of beige mice accepted the transplant for a period longer than 100 days after transplantation (Figure 7C). In addition, the rejection profile was faster in CD4–/– mice when compared with wild-type or CD8–/– mice, implying that CD4 lymphocytes are necessary for GIFT15-mediated graft tolerance (Figure 7C).
We have previously demonstrated that the merging of 2 distinct immune-stimulatory cytokines—GMCSF and IL-2—can lead to a fusokine with synergistic proinflammatory properties in the setting of cancer immunotherapy.4 Operating within the same developmental mind frame, we sought to improve this antitumor biopharmaceutical by generating a GMCSF and IL-15 fusokine (aka, GIFT15). We observed that B16 melanoma cells expressing GIFT15 displayed markedly enhanced tumor growth properties in vivo. This observation was contradictory to what our working hypothesis had predicted. In an attempt to decipher this totally unexpected result, we first observed that tumor-associated angiogenesis was markedly enhanced in association with a robust recruitment of TAMs, and we further noted that tumor-associated lymphoid cells, in particular NK/NKT cells, were significantly reduced in number. These 2 seminal observations led us to revise our hypothesis on the putative biochemical effect of GIFT15: GIFT15 behaves as a proangiogenic and immunosuppressive compound. These interesting and unanticipated properties suggest that GIFT15 may be useful as a compound for treatment of those medical ailments in which pharmaceutical immunosuppression and/or angiogenesis are desirable. To best understand the cellular physiology and biochemistry of GIFT15, we chose to tackle the investigation of angiogenesis and immunosuppression distinctly. Our initial observation that B16-GIFT15 cells led to massive recruitment of TAMs gave us the insight that host-derived macrophages may be playing a significant role in the observed enhanced angiogenesis. It has been previously shown that GMCSF and IL-15 can induce migration of macrophages both in vitro and in vivo.4–7 Since macrophages express both the GMCSFR and the IL-15R, it follows that GIFT15 would lead to selective recruitment of these cells. Not surprisingly, we found GIFT15 to be a potent chemotactic agent for macrophages in vitro. There is much precedent in the field of cancer angiogenesis of TAMs playing an important role in releasing a wide array of proangiogenic factors as part of a maladaptive injury repair response to a "wound that does not heal."14–16 We demonstrate that primary peritoneal macrophages stimulated with GIFT15 protein preferentially phosphorylate STAT3 via the IL-2/IL-15 ß chain and subsequently adopt an unprecedented phenotype where they secrete TIMP-2 de novo. Indeed, there is virtually no published precedent describing TIMP-2 production by monocyte/macrophages, suggesting a completely novel property of macrophages. TIMP-2 is known to act either as an inhibitor of MMP-2 if a soluble complex is formed or as an activator if it is bound to MT1-MMP on cell surfaces, which can lead to matrix remodeling and angiogenesis.17,18 Moreover, STAT3 phosphorylation has been shown to control the level of MMP-2 secretion by directly binding to its promoter and activating gene expression,30 suggesting that both TIMP-2 and STAT3 hyperphosphorylation can lead to MMP2 secretion and activation. Indeed, enzymatically active MMP-2 was found to be produced by GIFT15-treated macrophages. MMP-2 possesses a variety of functions, such as anti-inflammation, increasing the levels of active TGF-ß, as well as the activation of VEGF and of other growth factors.31–37 In fact, significantly increased levels of active TGF-ß and VEGF levels were documented in GIFT15-treated macrophages. Although the qualitative interaction of GIFT15 with the GMCSFR appears identical to that of monomeric GMCSF, it must be noted that GMCSF's half-life in vivo is more than 240 minutes,38,39 whereas IL-15 has a much shorter plasma half-life of less than 1 minute.40 Therefore, it is possible that the in vivo half-life of GIFT15 is closer to that of native GMCSF and may lead to prolonged interaction with the IL-15R. In sum, GIFT15 appears to act as a chemotactic agent for macrophages in vivo and also modulates their phenotype in a manner rendering them profoundly proangiogenic.
A second seminal observation is the extensive cell-mediated immune suppression by GIFT15 that tolerizes to both allogeneic and xenogenic somatic cell implantation in otherwise immunologically intact recipient mice. On a molecular basis, we have found that GIFT15 has increased affinity for the IL-15R The B16 melanoma cell line is syngeneic to C57Bl/6 mice, and will lead to tumor growth when implanted subcutaneously in immune-competent C57Bl/6 mice. Indeed, B16 tumors fail to induce an effective immune response in immunologically naive C57Bl/6 mice and are incapable of generating tumors in MHC-mismatched BALB/c mice. In light of the remarkable immunosuppressive effects of GIFT15, we tested whether its expression could protect allogeneic tumor cells from rejection in immune-competent MHC-mismatched recipient animals. As proof of concept, B16-GIFT15 (H-2Kb) cells were grafted in MHC-mismatched BALB/c (H-2Kd) mice, and we observed that tumors secreting GIFT15 were accepted in all recipient mice, while controls were tumor free. We observed that B16 tumor cells are associated with a CD3+ lymphoid cell infiltrate and that the composition of this infiltrate is characterized by depletion of NK/NKT cells in the presence of GIFT15. Since tumor growth is accelerated in this setting, it suggests that NK/NKT cells may play a role in delaying syngeneic tumor growth in immunologically naive mice. We further investigated the utility of GIFT15 immunosuppression in the context of xenotransplantation. In this case, a transduced polyclonal population of the human glioma cell line U87GM was transplanted subcutaneously in BALB/c mice. All mice accepted the GIFT15 xenograft for up to 7 months, whereas the control U87-GFP xenograft was rejected 12 days after injection. Since different mouse strains generate variable immune responses,41 we pursued our studies by performing xenotransplantation on C57Bl/6 mice, which are known to possess a biased Th1 immune response.42 Even though both control and GIFT15-expressing xenografts were rejected in these mice, there was a 2-month delay for the complete regression of the U87-GIFT15 transplants in comparison with the U87-GFP group. In contrast, in the allogeneic tumor implant model, xenogenic human tumor cells cannot lead to a physiological immune synapse in murine hosts due to the interspecies discrepancy between human MHC and murine TCR, thereby limiting cellular rejection to MHC-independent cytotoxic cells such as NK. Indeed, NK cells appear to be important effectors in xenotissue rejection.43 Our model system behaves similarly, since we observed that NK-deficient mice are permissive to human xenograft tumor growth and that CD4–/– and CD8–/– mice robustly reject xenografts. The observation that GIFT15 allows human xenograft tumor growth in normal mice strongly supports the hypothesis that its suppressive effect—in the xenogenic setting—is targeted toward NK cells. Furthermore, this anti-NK effect of GIFT15 is dependent upon interplay with CD4 cells. We speculate that GIFT15 modulates a subset of CD4 cells that may interact with NK directly. Therefore, in the absence of a species-paired immunologic synapse between effector and target cell, GIFT15 likely tolerizes to xenografts by promoting a suppressive cross talk between host-derived CD4 and NK cells.44
Taken together, our observations and experiments support the hypothesis that GIFT15 possesses novel biochemical properties leading to altered affinities to components of the IL-15R and asymmetrical downstream signaling via its 2 dependent STAT/JAK pathways in lymphomyeloid cells. As a result, cellular proliferation, reduced apoptosis, and blunting of the IFN-
Contribution: M.R. designed research, performed research, analyzed data, and wrote the paper; J.H.W. designed and performed research; B.A., L.L., and M.F. performed research; and J.G. designed research, analyzed data, and wrote the paper. Conflict of interest disclosure: The authors declare no competing financial interests. Correspondence: Jacques Galipeau, Jewish General Hospital, 3755 Cote Ste-Catherine Road, Montreal, QC, Canada, H3T1E2; e-mail: jacques.galipeau{at}mcgill.ca.
This study was supported by a Canadian Institute for Health Research (CIHR) operating grant (MOP-15017). Sheldon Biotechnology Centre at McGill University is supported by a Multi-User Maintenance Grant from CIHR. M.R. is a recipient of a Fonds de recherches en Santé du Québec (FRSQ) Scholarship, and J.G. is an FRSQ chercheur boursier sénior. We thank Dr M. Hancock (Sheldon Biotechnology Center) for the BIAcore experiments. We thank Drs N. Eliopoulos, I. Copland, and J. Stagg for technical advice and materials.
Submitted July 25, 2006; accepted October 17, 2006.
Prepublished online as Blood First Edition Paper, November 2, 2006
DOI: 10.1182/blood-2006-07-037473
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
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Abstract
Introduction
], rIL-15 + rGMCSF [X], GIFT15 [
], and rGMCSF [
], or GFP CM [
]). (C) Splenocyte activation and secretion of IFN
) subcutaneously, and tumor volume as well as percentage survival were monitored over 8 months (P < .05 between the GIFT15 and GFP group). (C) Xenotransplantation of U87-GFP/GIFT15 in WT C57BL/6 or KO mice. Live U87-GFP or GIFT15 cells (107) were transplanted subcutaneously in WT C57BL/6 (n = 6/group), CD8–/– (n = 10/group), CD4–/– (n = 10/group), or beige (n = 10/group) mice, and graft survival was monitored over time (P < .05 between U87-GIFT15 and GFP). Results are shown as mean tumor volume ± SED.