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Immunology |
Immunotherapy and Gene Therapy Unit, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy [G. G., C. C., E. G., M. P. C.]; Department of Experimental Medicine and Pathology, 2nd Chair of Pathology, University of Rome "La Sapienza," 00161 Rome, Italy [A. S.]; and Department of Immunology and Bacteriology, University of Glasgow, Glasgow G11 6NT, United Kingdom [F. Y. L.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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knockout mice (GKO) rejected C26 colon carcinoma cells transduced to secrete interleukin(IL)-12 but do not reject similarly transduced TSA mammary adenocarcinoma (C26/12 and TSA/12 cells, respectively). To determine whether such difference could be because of a different tumor response to IFN-, we injected BALB/c mice with TSA, C26, and their IL-12-transduced counterparts rendered unresponsive to IFN- by stable transduction of a dominant negative (DN), truncated IFN- receptor 
chain. TSA/DN and C26/DN showed the same in vivo growth kinetics as parental cells, whereas coexpression of IL-12 induced rejection independent of tumor-cell responsiveness to IFN-. This suggests that the role of IFN- is primarily in activating the host immune response, which appears to depend on the intrinsic immunogenicity of the target tumor. C26 and TSA share a common tumor-associated antigen, yet C26 cells are more immunogenic than TSA. C26/12 expressed 10-fold higher levels of class I MHC molecules and induced higher CTL activity compared with TSA/12 cells in GKO mice. Moreover, whereas in GKO mice the TSA/12 tumor was associated with a greater number of infiltrating T cells, only those infiltrating C26/12 tumor expressed the activation marker OX40. The search for cytokine(s) that might contribute in determining the different T-cell response to these IL-12-transduced tumors in GKO mice revealed a role of IL-15. In situ hybridization showed IL-15 expression in C26/12 but not in TSA/12 tumors. In addition, injection of GKO mice with soluble IL-15 receptor- to block IL-15 expression prevented rejection of C26/12 cells. Together, the results suggest that in the absence of IFN-, IL-12 can exert antitumor activity through alternative mechanisms, depending on the tumor cell type and the availability of cytokines that can replace IFN- in sustaining T-cell functions. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production by T and NK cells (6)
. IL-12-induced IFN- has a direct antiproliferative effect on several tumor cell types and up-regulates the expression of class I MHC, thus enhancing immune recognition of TAAs (7)
. IFN- also stimulates the production of nitric oxide by tumor and/or host cells, which inhibits tumor growth (8
, 9)
. Furthermore, IFN- is involved in the production of chemokines such as IFN--inducible protein-10 and a monokine induced by IFN- by a variety of cell types, including mononuclear cells, fibroblasts, keratinocytes, endothelial cells, T cells, and some tumor cells. These chemokines act as chemoattractants for lymphocytes, and inhibit the differentiation and proliferation of endothelial cells (10, 11, 12, 13, 14, 15, 16)
. IFN- mediates its effects through a receptor that is widely expressed on normal and malignant cells (17)
. The IFN-R consists of and ß chains, which dimerize on ligand binding. Dimerization triggers receptor transphosphorylation and activation of the associated JAK1 and JAK2 tyrosine kinases (18
, 19)
. Activated JAK kinases then phosphorylate downstream STAT1, causing its dimerization and nuclear translocation (20)
. Nuclear STAT1 binds GAS in IFN--responsive genes, thereby modulating their transcription (21)
.
Although IFN- is the main effector of IL-12 antitumor activities, systemic injection of exogenous IFN- cannot completely substitute for IL-12 for activity (7)
. This finding might reflect differences in the half-life of the two proteins, but more likely, the differing distribution of their receptors; IFN-R is ubiquitously expressed, whereas the IL-12R ß2-chain is expressed only on NK cells and activated lymphocytes (22)
. We demonstrated previously that GKO mice still partially reject growth of an IL-12-transduced colon carcinoma cell line (C26/12) through a mechanism that requires the presence of CD4+ T cells (23)
, which produce GM-CSF associated with the reduction of tumor blood vessels through the activity of polymorphonuclear leukocytes and CD8+ T cells (24)
. These findings suggest that the antitumor effect of IL-12 can be mediated via an IFN--independent pathway at least in the C26 model.
Here, we show that, unlike C26/12 tumor cells, mammary adenocarcinoma TSA cells transduced with IL-12 genes (TSA/12) are fully tumorigenic in GKO mice. Recently, the ability of the immune system to control tumor development in carcinogen-treated or tumor-prone mice, as well as the rejection of transplantable tumor, have been correlated with the direct effect of IFN- on tumor cells (25, 26, 27)
. Using IL-12-transduced or nontransduced TSA and C26 tumor cells rendered unresponsive to IFN- by stable transduction and overexpression of a IFN-RDN, we tested whether the differential rejection of C26/12 and TSA/12 cells observed in GKO mice might rest in different tumor sensitivity to IFN-. The results indicate that tumor cell type and intrinsic immunogenicity are the determinants of the need for IFN- in tumor rejection. In our experimental setting, IFN- acts on cells of the immune response rather than directly on tumor cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment with sIL-15R.
A soluble fragment of the murine IL-15R (31)
and the functionally mutated form of sIL-15R (M4), containing an amino acid substitution at the Sushi domain necessary for binding to IL-15 were used to block endogenous IL-15 bioactivity on GKO mice (32)
. sIL-15R (40 µg/mouse) was injected i.p. daily for 20 days starting 2 days before C26/12 tumor inoculation. Untreated- or M4-treated mice were used as controls.
Vector Construction and Retroviral Infection.
An 860-bp fragment containing the extracellular and transmembrane domains of the IFN- receptor, together with the FLAG sequence, derived from pEF2-FlmugR-DNM (a kind gift of Sidney Pestka, Johnson Medical School, Piscatway, NJ), was excised with XbaI and KpnI, blunt-ended, and cloned into the HpaI site of the LXSH retroviral vector (33)
to obtain the vector IFN-RDNLSH. This retroviral vector was transfected into the gp+E86 packaging cell line (34)
by standard calcium phosphate coprecipitation, and the 48-h culture supernatant was used to infect the amphotropic PA317 packaging cell line (35)
. Infected PA317 cells were selected with hygromycin and used to generate helper-free, virus-containing supernatants. TSA, TSA/12 (36)
, C26, and C26/12 (37)
target cells were infected by four cycles of exposure to undiluted supernatant for 2 h in the presence of Polybrene (8 mg/ml). At 48 h after infection, cells were diluted and selected in hygromycin (500 µg/ml). Bulk cultures and single resistant colonies were expanded and screened by FACS analysis for IFN-R and FLAG expression, and by ELISA for IL-12 production (106 cells/ml; 48 h at 37°C). Limiting dilution cloning was used to obtain optimal levels of IFN-RDN expression.
Flow Cytometry.
Expression of IFN-RDN in transduced cell lines was assayed by flow cytometry after conventional staining with biotin-conjugated anti-IFN-R (CD119, clone GR20) followed by streptavidin-phycoerytrin (PharMingen, San Diego, CA) or with mouse anti-FLAG M2 (Kodak, New Haven, CT) followed by fluorescence-conjugated antimouse immunoglobulin (Biosource, Camarillo, CA). For assay of class I MHC expression, biotin-conjugated anti-H-2Ld/H-2Db clone 28–14-8 (PharMingen) was used. Hybridoma 35/299, which expresses a rat IgG2a mAb specific for gp70 was a gift from Drew Pardoll (Johns Hopkins University, Baltimore, MD). Isotype-matched mAbs of unrelated specificity were used as controls. Analysis was performed on a FACScan (Becton Dickinson, Franklin Lakes, NJ). Data were collected on 5,000–10,000 viable cells and analyzed using winMDI 2.8 software.
EMSA.
Nuclear extracts were isolated from control, and transduced C26 or TSA cells (2 x 106/ml) either unstimulated or stimulated with the indicated amount of mrIFN- (PharMingen) according to Schreiber et al. (38)
. End-labeled DNA probes (50,000 cpm/sample) were mixed with 4 µg of crude nuclear extract and incubated at room temperature for 20–30 min in the presence of 1 µg of poly(dI·dC; Boehringer Mannheim, Roche Diagnostics, Monza, Italy) in 10 µl of buffer C [20 mM HEPES (pH 7.9), 10% glycerol, 0.4 M NaCl, 0.1 mM EDTA, 0.1 M EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin A]. Samples were resolved on a 6% polyacrylamide gel in running buffer (0.5 x Tris-borate EDTA) for 1 h at 200 V. The gel was dried and exposed in a PhosphorImager storage screen and visualized using PhosphorImager 445S1 (Molecular Dynamics, Sunnyvale, CA). Probe competition experiments were carried out using an unlabeled GAS probe (5' AGC-TTG-TAT-TTC-CCA-GAA-AAG-GGA-TC; Ref. 39
). Supershift experiments were performed by preincubating nuclear extracts with 1–2 µg of Abs for 30 min at 4°C before the probe was added. All of the polyclonal Abs recognizing STAT family protein and used in supershift experiments were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
NK Cell Assay.
To measure NK cell activity, fresh splenocytes from SCID mice (44% NK-positive cells) were incubated for 2 days in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% FCS and 500 units/ml of human IL-2 (Chiron-Italia, Milan, Italy) in the presence or absence of murine rIL-12 (2.5 ng/ml; kindly provided by M. Gately, Roche, Nutley, NJ) and tested for cytotoxic activity against 51Cr-labeled YAC, TSA, or C26 target cells.
Cell-mediated Cytotoxicity Assay.
Naïve BALB/c or GKO mice were inoculated into the footpad with -irradiated (15,000 rad) C26/12 or TSA/12 cells at a dose of 5 x 106 irradiated cells/mouse. After 5 days, popliteal lymph nodes were removed, and lymphoid cells were suspended to 5 x 105 cells/ml in RPMI 1640 supplemented with 10% FCS and restimulated in vitro for 5 days at 37°C in 5% CO2 in the presence of the common tumor antigenic peptide AH1 (SPSYVYHQF) at a final concentration of 1 µg/ml (synthesized by Primm S.r.l., Milan, Italy). Effector cells were used in cell-mediated cytotoxicity assays, with AH1-pulsed BALB/c blasts. In selected experiments restimulation in vitro of effector cells was performed for 5 days in the presence of 5 x 104/ml irradiated (15,000 rad) C26 or TSA cells, and in these experiments C26 and TSA cells were used as specific targets.
Immunocytochemistry and in Situ Hybridization.
Tumor fragments, tumor-draining lymph nodes, and spleens were collected 1, 3, and 7 days after tumor inoculation, embedded in OCT compound (Miles Laboratories, Inc., Elkhart, IN), snap-frozen in liquid nitrogen, and stored at -80°C. Immunocytochemical analysis using the biotinylated antirat IgG/streptavidin-horseradish peroxidase method was performed as follows: 5-µm cryostat sections were fixed in acetone and immunostained with rat antimouse mAb against TCR Vß6 (clone RR4-7; PharMingen), Vß8.3 (clone 1B3.3; PharMingen), CD8 (53.6.72 hybridoma, Lyt2; American Type Culture Collection), CD4 (GK1.5 hybridoma, T200; American Type Culture Collection), OX40 (OX86 hybridoma; European Collection of Animal Cell Cultures, Salisbury, United Kingdom), and NK (pan-NK, clone DX5; PharMingen). Sections were sequentially blocked using a tissue biotin blocking kit (DAKO) and incubated with optimal dilutions of primary Abs, biotynilated-antirat IgG (DAKO), and streptavidin-horseradish peroxidase (DAKO). Each incubation step was for 30 min and was followed by a 10-min wash in PBS. Sections were then incubated with 0.03% H2O2 and 0.06% 3,3'-diaminobenzidine (BDH Chemicals, Poole, United Kingdom) for 2–5 min, washed in tap water, and counterstained with hematoxylin. The number of immunostained cells was determined by light microscopy (magnification x400) in 5 fields on a 1-mm2 grid and given as cells/mm2 (mean ± SD).
The presence of IL-15 mRNA was investigated by in situ hybridization using IL-15 cDNA obtained by cutting the pVKLIL-15IRES1neo plasmid (kindly provided by Silvano Ferrini, National Cancer Institute, Genova, Italy) at EcoRI and BamHI sites. After digestion, the 675-bp fragment was run on 1% agarose gel, purified using the Qiaex II gel extraction kit (Qiagen, Hiden, Germany) and biotin-labeled using a random primer biotin labeling kit (NEL804; ENZO Diagnostics, Farmingdale, NY). Cryostat sections were harvested on RNA-grade slides, air-dried, and fixed in 4% buffered paraformaldehyde for 10 min, dehydrated in ethanol, sequentially washed in PBS/50 mM MgCl2 and 200 mM Tris-HCl-glycine, acetylated in 2x SSC, 0.1 M triethanolamine, and 0.5% acetic anhydride (pH 8.0), washed in 2x SSC, and finally dehydrated in ethanol. Slides were prehybridized for 10 min at 42°C in 2x SSC, 50% formamide, and 500 µg/ml salmon sperm DNA, and hybridized overnight at 42°C with biotin-labeled-specific IL-15 cDNA probe (20 ng/ml), 2x SSC, 500 µg/ml salmon sperm DNA, 5x Denhardt’s solution, 10 mM dithiothreitiol, and 10% dextran sulfate. Unbound and nonspecifically bound probes were removed by sequential washes in 2x SSC, 50% formamide, and 1x SSC, 50% formamide at 50°C, and in 0.1x SSC at room temperature. Slides were air-dried and the biotnylated signal was detected using a TSA biotin system kit (NEN700; ENZO Diagnostics). Slides were counterstained with hematoxylin. RNA-specific binding was controlled by previous digestion with 100 µg/ml RNase A and 10 units/ml RNase T (Sigma Chemical Co., Poole, United Kingdom). PstI-digested pUC9 plasmid fragments were used as negative controls.
Statistical analysis was performed using the Student t test (Microsoft Office).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, indicating that the IL-12 effect is strictly IFN--dependent. In contrast, C26/12 tumor growth was rejected in 70% of both BALB/c and GKO mice (Fig. 1B)
. These contradictory results cannot be explained by a disparity in IL-12 production, because both transduced cell lines were selected to release similar amounts of IL-12 (6 ± 0.5 ng/106 cells/48 h). No differences between TSA and C26 cells in the inhibition of cell proliferation in response to rIFN- even at high doses of rIFN- (2000 units/ml) was observed in vitro (data not shown). Thus, a direct antiproliferative effect of IFN- on tumor cells is unlikely.
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RDN-transduced TSA and C26 Cells. acts on host immune cells or directly on tumor cells, C26, C26/12, TSA, and TSA/12 tumor cells were rendered IFN--unresponsive by stable transduction with an IFN-RDN chain construct (27)
. Transduced clones were selected for IFN-R chain overexpression and for FLAG reactivity to distinguish the transgene encoded protein (data not shown). Gel EMSA for inhibition of nuclear STAT1 was used to confirm the block in IFN- signaling in these clones. Murine rIFN--treated TSA, TSA/12, C26, and C26/12 cells revealed a nuclear complex (Fig. 2, A and B)
that was inhibited by anti-STAT1 Ab and by cold GAS probe but not by an anti-STAT4 Ab (Fig. 2C)
, whereas no STAT1 induction was observed in rIFN--treated nuclear extracts from IFN-RDN-transfected cells. Involvement of a recently described novel STAT1-independent IFN- signaling pathway through c-myc and c-jun induction in the absence of STAT1 phosphorylation (40)
seems unlikely because this signal requires an intact IFN-R chain (41)
missing in the DN constructs (27)
.
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RDN-transduced TSA and C26 cells no longer up-regulated class I MHC in response to rIFN-, and this loss of responsiveness was not because of a general defect in the class I MHC induction pathway, because both cell lines responded normally to murine IFN- (Fig. 3
; data not shown).
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RDN-transduced TSA and C26 Cells.RDN-transduced TSA and C26 cells formed rapidly progressing tumors (Fig. 4, A and B)
. Identical results were obtained using other IFN-RDN-transduced TSA and C26 cell clones (data not shown). Reverse transcription-PCR and immunostaining of C26/DN and TSA/DN tumors collected 15 days after injection demonstrated intact expression of the DN receptors (data not shown).
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-unresponsive TSA/12 and C26/12 in the Presence or Absence of Endogenous IFN-. responsiveness of TSA and C26 cells is important for local IL-12-induced rejection of these tumors, BALB/c mice were injected with TSA/12/DN and C26/12/DN cells and tumor growth was evaluated (Fig. 4, C and D)
.
TSA/12, TSA/12/DN, C26/12, and C26/12/DN tumors showed equally impaired tumor take, with 
80% of mice remaining tumor-free at 70 days after inoculation. These results strongly suggests that the antitumor effect of IL-12-induced IFN- is most likely not directed to tumor cells but instead to host cells. Mice that rejected C26/12/DN cells did not reject a challenge with BALB/c fibrosarcoma cells either transduced with the IFN-RDN vector or nontransduced (data not shown), ruling out the possibility that construct- (e.g., the FLAG epitope present in the extracellular portion of the DN chain) or vector-derived antigens increase the immunogenicity of DN-transduced cells.
Additional proof that the status of IFN--responsiveness of TSA and C26 cells does not determine the take or rejection of the tumors came from analysis of GKO mice injected with TSA/12/DN or C26/12/DN cells (Fig. 5, A and B)
. As expected, all of these mice developed TSA/12/DN tumors, whereas after initial take, 80% of mice rejected C26/12/DN tumors. Thus, IL-12-induced rejection of TSA tumors requires the presence of endogenous IFN-, but rejection of C26 cells does not.
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. C26, C26/12, TSA, and TSA/12 were analyzed for expression of the shared TAA gp70, the envelope protein of an endogenous murine leukemia virus. The immunodominant epitope recognized within gp70 is AH1, a nonamer peptide presented in the context of class I MHC Ld (42)
. Immunofluorescence and FACS analysis revealed higher gp70 (Fig. 6A)
but lower class I MHC Ld (Fig. 6B)
expression in both TSA and TSA/12 as compared with C26 and C26/12 cells. In 51Cr release assays, splenocytes derived from SCID mice enriched in NK cells and activated in vitro with IL-2 or IL-2 + IL-12 killed TSA target cells more efficiently than C26 targets (Fig. 6C)
, indicating that innate immunity is more effective against the tumor cells expressing fewer class I MHC molecules on their surface. We then compared C26/12 and TSA/12 cells for the ability to induce CTLs in both BALB/c and GKO mice. Irradiated tumor cells were injected into the footpad, and lymphocytes from draining popliteal nodes were restimulated in vitro with the AH1 peptide (Fig. 6D)
, or irradiated TSA or C26 tumor cells accordingly to the tumor used for priming (Fig. 6, E and F)
. Whereas C26/12 cells primed CTL induction in both strains, TSA/12 cells induced weak or undetectable CTL activity against AH1-pulsed blast or TSA cells (Fig. 6, D–F)
.
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The site of injection was excised 1, 3, and 7 days after tumor cell inoculation. As shown in Table 1
, TSA/12 tumors had more infiltrating T than C26/12. A more in depth analysis of the two major TCR types, Vß6 and Vß8.3, involved in recognition of the AH1 immunodominant peptide was performed (42
, 43)
. Vß6 was mainly present in C26/12 tumors, and in GKO mice these Vß6 T lymphocytes accounted for almost all of the CD8+ tumor-associated lymphocytes. In contrast, Vß8.3 was present in TSA/12 tumors regardless of whether the tumors were collected from BALB/c or GKO mice. This apparently controversial result was resolved by evaluating a functional marker of T cell activation, OX40 (44)
. Indeed, almost all of the T cells derived from the C26/12 tumor, either from BALB/c or GKO mice, stained positively for OX40 (Table 1
; Fig. 7, B and D
). In contrast, TSA/12 tumors from BALB/c mice, although richer in infiltrating T cells, had fewer OX40-positive cells (Table 1
; Fig. 7A
). An almost complete depletion of OX40 expression was observed in TSA/12 tumors from GKO mice (Table 1
; Fig. 7C
). In accordance with the analysis of this functional marker, TSA/12 is rejected in BALB/c but not in GKO mice.
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in Mediating IL-12 Antitumor Activity.
; Table 1
) and, to a slightly lesser extents, in GKO mice injected with C26/12 cells (Fig. 7H)
, whereas rare IL-15-positive cells with macrophage morphology were present in TSA/12-injected BALB/c mice (Fig. 7E)
, and no IL-15 was detected in TSA/12-injected GKO mice (Fig. 7G)
. To prove that IL-15 produced during the rejection of C26/12 in GKO mice is functionally needed, mice were treated with a sIL-15R (31)
. None of the treated GKO mice were able to reject C26/12, whereas mice treated with M4, a functionally mutated sIL-15R unable to bind IL-15, showed 50% of rejection as did untreated mice (Fig. 8)
. This observation provides new data implicating IL-15 in IL-12-mediated antitumor activity at least in the absence of endogenous IFN-.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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for rejection by the host. In fact, rIL-12 treatment of s.c.-injected C26 cells was shown to be ineffective, whereas treatment of C26 transduced with IL-2, which changes the pattern and number of infiltrating cells without affecting tumor take, resulted in rejection by 80% of the mice (50)
. In a different approach in which the effect of rIL-12 was tested against C26 cells injected intrasplenically, systemic rIL-12 completely inhibited liver metastases because of the abundant presence of NK T cells (51)
. Unlike C26, TSA cells injected s.c. are highly susceptible to rIL-12 treatment, leading to T lymphocyte and macrophages infiltration associated with necrosis (36)
.
In our studies, both IL-12-transduced-C26 and -TSA cells selected to release the same amount of cytokine were rejected in BALB/c mice, but only the C26/12 tumor was rejected in the absence of IFN- (Fig. 1
; Ref. 23
). The role of IFN- and the nature of IFN- target cells in IL-12-induced tumor rejection have remained unclear because of contrasting results on the possible direct effect of IFN- on tumor cells. This issue has been addressed by overexpressing a mutant form of the IFN-R chain in transplantable tumors (26
, 27
, 52
, 53)
or by using mice knocked out for this molecule (53
, 54)
. We have stably transduced the DN truncated IFN-R chain into TSA and C26 cells alone or together with IL-12 genes, and injected them into BALB/c and GKO mice. In BALB/c mice, tumor take and growth kinetics of TSA/DN and C26/DN were similar to that of their parental counterparts, and both tumors were rejected in the presence of locally released IL-12 (TSA/12/DN and C26/12/DN; Fig. 4
). These results contrast with those of Coughlin et al. (52)
, who reported that IFN--unresponsive tumor cells were more tumorigenic and less susceptible to rIL-12 therapy in vivo. This discrepancy likely rests in the use of different tumor models, and rejection of the K1735 melanoma and the SCK mammary carcinoma used in that study was through IFN--inducible protein-10-mediated inhibition of angiogenesis rather than by leukocyte killing. In contrast, rejection of both C26/12 and TSA/12 tumors requires leukocyte infiltration and killing. Moreover, we used tumor cells that express IFN-RDN and IL-12 simultaneously, thus colocalizing IL-12 induction of IFN- and the block in its effect on tumor cells at the injection site.
Our results are consistent with those of Mumberg et al. (53)
, who also reported that tumor rejection was independent of tumor sensitivity to IFN-. In the same context, Qin et al. (54)
, using a carcinogen-induced tumor from IFN-R knockout mice, observed that tumor IFN-R expression on nonhematopoietic host cells but not on tumor cells was required for CD4+ T-cell mediated tumor rejection. Thus, cell type characteristics of the target tumor represent a key factor in determining tumor responsiveness to IFN-. Indeed, IFN- was shown recently to promote tumor escape of CT26 colon carcinoma cells, which are closely related to the C26 tumor, by down-regulating surface expression of the endogenous TAA gp70 (55)
. Our C26 and TSA tumor cells express the gp70 antigen (Fig. 6)
, but down-modulation of this molecule by IFN- was slight in C26 cells and undetectable in TSA cells (data not shown).
Expression of gp70 on TSA and C26 cells did not correlate with their immunogenicity. The gp70 immunodominant epitope AH1 is presented in the context of class I MHC Ld (42)
, which was expressed at higher levels on C26 and C26/12 cells than on TSA and TSA/12 cells (Fig. 6B)
. Although such MHC differences were between 8- and 10-fold, they exceeded the 5-fold difference that dictates the selection of effector cells mediating immunocytokine therapy of the CT26 colon carcinoma (56)
. This difference in class I MHC expression might explain why NK cells killed TSA cells more efficiently than C26 target cells, and why C26/12 but not TSA/12 cells were able to prime CTLs in the absence of IFN- (Fig. 6, C–F)
. An attempt to correlate these in vitro data with the in vivo tumor infiltration of T lymphocytes bearing TCR Vß used for AH1 recognition (42
, 43)
gave apparently contradictory results. Vß6 were mainly found in C26/12 tumors and accounted for almost all CD8+ T cells in GKO mice. The alternative TCR type, Vß8.3 was dominant in TSA/12 tumors either from BALB/c or GKO mice (Table 1)
. Although the number of Vß6+ T lymphocytes in C26/12 appears to correlate with tumor rejection, the number of Vß8.3+ T lymphocytes does not, as TSA/12 is rejected in BALB/c only. A possible explanation of these results may be the activation status of infiltrating T cells. Analysis of OX40, an activation marker for CD4+ and CD8+ T cells (44)
, indeed revealed that all of the tumors, except TSA/12 from GKO mice, were positive for OX40, correlating with tumor rejection.
Our preliminary efforts to identify a cytokine(s) that might plays a role in determining the differential TCR in these tumors has thus far eliminated GM-CSF, which is involved in C26/12 tumor rejection in GKO mice but is similarly induced in TSA/12-injected GKO mice (data not shown). By contrast, TSA cells transduced to express IL-15, a cytokine that plays a role in activation of NK and T-cell effector function (reviewed in Ref. 57
), are rejected in BALB/c mice but not in GKO mice (4)
. In situ hybridization analysis of C26/12- and TSA/12-injected GKO mice revealed IL-15 expression at the C26/12 but not the TSA/12 tumor site (Fig. 8
; Table 1
). Moreover, block of endogenous IL-15 resulted in complete loss of protection from C26/12 tumor development in GKO mice (Fig. 8)
. This data complemented well the demonstration that TSA double transduced with IL-12 and IL-15 is rejected in GKO mice through CD8 T cells (4)
.
Together, the results suggest that IL-12 can exert antitumor activity throughout alternative mechanisms independent from IFN- but related to the intrinsic tumor immunogenicity and the availability of cytokines, such as IL-15 and GM-CSF, that can replace IFN- in sustaining T-cell functions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Associazione Italiana Ricerca sul Cancro, ISS-ITA-USA n. T00.A15, Consiglio Nazionale delle Ricerche Finalized Project on Biotechnology (PF49). G. G. was supported by an Italian Foundation for Cancer Research fellowship. 
2 To whom requests for reprints should be addressed, at Immunotherapy and Gene Therapy Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. Fax: 39-02-23902630; E-mail: mcolombo{at}istitutotumori.mi.it
3 The abbreviations used are: IL, interleukin; NK, natural killer; TAA, tumor-associated antigen; IFN-R, functional IFN- receptor; FACS, fluorescence-activated cell sorter; IFN-RDN, dominant-negative truncated IFN- receptor; Ab, antibody; sIL-15R, soluble fragment of the murine IL-15R; GAS, -activated sites; r, recombinant; GKO, IFN- knockout mice; GM-CSF, granulocyte-macrophage colony-stimulating factor; EMSA, electrophoretic mobility shift assay; SCID, severe combined immunodeficiency; JAK, Janus-activated kinase; STAT, signal transducers and activators of transcription; mAb, monoclonal antibody; DN, dominant-negative; TCR, T-cell receptor.
4 A. Comes, E. Di Carlo, P. Musiani, O. Rosso, R. Meazza, C. Chiodoni, M. P. Colombo, and S. Ferrini. IFN- independent synergistic effect of IL-12 and IL-15 induces antitumor responses in syngeneic mice, submitted for publication, 2001.
Received 12/13/01. Accepted 6/ 3/02.
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) or GKO (
) mice. Groups of 18 mice were injected with 105 tumor cells. TSA/12 and C26/12 cells produced comparable amounts of IL-12 (6 ± 0.5 ng/106 cells/ml) after 48 h of in vitro culture.
) target cells. D–F, CTL activity after in vivo priming of BALB/c (filled symbols) and GKO mice (open symbols) with irradiated TSA/12 (
) or C26/12 cells (
). D, target cells were AH1-pulsed BALB/c blasts. E and F, target cells were C26 (
