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Immunology |
in Rejection of Established Tumors by IL-12
Department of Surgery, University of California, VA Medical Center, San Francisco, California 94121
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and T cells. The role of IFN- in IL-12-induced tumor rejection is unclear, because after IL-12 administration IFN- is produced by multiple cell types, and it acts on most cell types because of the ubiquitous expression of its receptor. Using a T-cell-adoptive transfer model, we show that after IL-12 treatment, tumor-specific T-cell production of IFN- is necessary and sufficient for rejection of established tumors. Furthermore, by testing tumors using IFN--unresponsive tumor cells, we show that tumor cell expression of MHC class I molecules in vivo is abrogated by blocking the response to IFN-. However, tumor response to IFN- is not essential for rejection of established small and large tumors by IL-12 and Cy+IL-12, respectively; neither is it essential for expression of tumor immunogenicity. Our results indicate that the rejection of established tumors by IL-12/Cy+IL-12 is dependent on the induction of a Th1 response producing IFN- that acts on host cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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14Vß8 TCR (2
, 3)
, conventional T cells are not required. Although IFN- is produced by NKT cells on IL-12 stimulation (4)
, it may not be essential for the antitumor effect of IL-12-activated NKT cells (2
, 5)
. The second type of IL-12-induced antitumor response is mediated by T cells and is associated with rejection of established palpable tumors (6
, 7)
. It is limited to only immunogenic tumors, because tumor rejection requires the presence of tumor-sensitized antigen-specific T cells (8)
. NK/NKT cells are not required as depletion of NK1.1+ cells with anti-NK1.1 antibody only delays, but does not affect, the dramatic tumor rejection induced by IL-12 treatment (9)
. The effector mechanism in the rejection of established tumors is not fully understood but does not involve perforin-dependent cell lysis, as tumor rejection is fully preserved in perforin KO mice.4
IFN- is required for rejection of established tumors (10, 11, 12, 13)
, but the role of the cytokine itself in tumor rejection seems to be necessary but not sufficient in that exogenous administration of IFN- does not produce the dramatic antitumor effect seen with IL-12 (14)
. We have described previously two models of IL-12-induced tumor regression in murine MCA207 tumors. In the established small tumor regression model, 7–10-day established s.c. palpable tumors with sizes of 4–8 mm in diameter are completely eradicated by treatment with recombinant murine IL-12 alone (15)
. In the established large tumor regression model, 3–4-week established large s.c. tumors with sizes of 15–20 mm in diameter are completely eradicated by treatment with a single dose of Cy followed by a short course of IL-12 (13)
. Again, depletion of NK cells with anti-NK1.1 antibody only delays but does not abolish the curative effect of Cy+IL-12 (16)
. A Th1 type of immune response has been found to be associated with tumor rejection in both models (13
, 15)
. Rejection of both small and large MCA207 tumors by IL-12 and Cy+IL-12 is abolished in IFN- KO mice (13)
and in normal mice depleted of IFN- by antibody treatment (10)
.
IFN- is produced by T cells, NK cells, as well as by macrophages and dendritic cells in response to IL-12 (17, 18, 19)
. On the other hand, IFN- also has pleiotropic effects on many cells because of the ubiquitous expression of its receptor on nearly all of the cells (20)
. Because of the multiple sources of IFN-production and its wide variety of cellular targets, a critical step in determining the role of IFN- in the IL-12-mediated antitumor response is to identify its source of production and its target cell during tumor rejection. In an earlier study, Brunda et al. (14)
observed that although the overall levels of IL-12-induced IFN- production were not reduced in T-cell-deficient nude mice, the antitumor effect of IL-12 in these mice was reduced significantly. This observation suggests that T-cell production of IFN-, possibly at the tumor site, may be critical for tumor rejection, as intratumoral IFN- production has been observed through immunohistochemical (15)
and PCR (21)
analysis. The target cell of IFN- during IL-12-induced tumor rejection has also been investigated in a recent study. Through the overexpression of a mutant form of the IFN- receptor subunit in tumor cells, Coughlin et al. (22)
showed that IL-12-mediated antitumor activity is diminished when tumor cells are made unresponsive to IFN-. They concluded that IFN- exerts its effect by inducing tumor-derived secondary chemokines (IP-10), which in turn inhibit angiogenesis. This approach of using IFN- unresponsive tumor cells made through dominant negative block of normal IFN-receptor function has been used by others as well, and resulted in mixed findings. In endotoxin-induced IFN--dependent tumor rejection, Dighe et al. (23)
showed that tumor rejection is abolished in mice bearing IFN--unresponsive MethA tumors. On the other hand, Mumberg et al. (24)
showed that the ability of adoptively transferred CD4+ T cells in T-cell-deficient mice to eliminate MHC class II-negative tumors was not affected by blocking tumor cell response to IFN-.
To determine the source of IFN- production that is most critical for IL-12-induced tumor rejection, we used an adoptive transfer model that we have developed in which tumor-sensitized, antigen-specific, but not naïve, T cells transferred into tumor-bearing TCR-ß KO mice are able to mediate tumor rejection after IL-12 treatment (8)
. To see whether production of IFN- by tumor-sensitized T cell is sufficient for tumor rejection, we transferred tumor-sensitized T cells into tumor-bearing T cell and IFN- double-deficient mice, and treated the reconstituted mice with IL-12. Conversely, we transferred tumor-sensitized T cells from IFN-KO mice into tumor-bearing T-cell-deficient mice to see whether IFN-production by tumor-sensitized T cells is necessary for tumor regression. To determine whether tumor cells are the targets of IFN-in our tumor regression model, we created IFN- unresponsive tumor cells through overexpression of a dominant-negative form of the IFN-receptor and tested these tumors for rejection by IL-12/Cy+IL-12 in vivo. Our results demonstrate that tumor-sensitized T cells and their production of IFN- are necessary and sufficient for IL-12-induced rejection of established tumor cells, and the target cell of IFN- during tumor rejection is likely to be host, but not tumor, cells. These findings help to explain IL-12-induced tumor rejection and the role of IFN- during this process.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-insensitive MCA207 transfectants were prepared by transfection with the plasmid pEF2-FlmugR-DNM (generously provided by Dr. William M. F. Lee, University of Pennsylvania, Philadelphia, PA) that encodes a COOH-terminal mutant form of the murine IFN- receptor subunit (25)
. Transfectants were first selected using G418-containing medium. Individual clones were established by limiting dilution. Clones were characterized by surface staining with biotinylated antibodies specific for the IFN- receptor (CD119;PharMingen) followed by flow cytometry analysis (Becton Dickinson FACScalibur). Clones that overexpressed the IFN- receptor >100-fold above that of parental MCA207 were then tested for resistance to IFN-. After incubation with IFN- (100 units/ml), transfected cells were tested for the ability of IFN- to enhance MHC class I protein expression using biotinylated anti-H-2kb antibody followed by flow cytometry analysis. Only clones that failed to up-regulate MHC I expression in the presence of IFN- were selected for in vivo experiments. For all of the subsequent in vitro and in vivo experiments, multiple clones were used, and representative results are shown. Controls were parental MCA207 and clones that did not overexpress the IFN- receptor. C57BL/6 normal mice were purchased from the Biological Testing Branch, National Cancer Institute, NIH (Frederick, MD). TCR-ß and IFN-double KO mice were created by first crossing single KO mice and selecting F1 progenies with homozygous double gene deletion followed by breeding between F1 progenies to obtain F2 mice. Female mice 8–12 weeks old were used in these experiments.
In Vivo Immunotherapy Treatment Models.
In both the small and large tumor regression models, mice were injected s.c. with 5 x 105 cells in 0.2-ml saline on the left flank. Tumor size was assessed using a caliper to measure tumor diameter. IL-12 treatment of small tumors (8–10 days established, 3–6 mm in diameter) consisted of three i.p. injections of 300 ng of recombinant murine IL-12 (5)
in 0.5 ml of 1% mouse serum in saline given once every other day for three doses. Cy+IL-12 treatment of large tumors consisted of a single i.p. treatment of 3 mg (120 mg/kg) of Cy (Sigma) in 0.5 ml of saline followed 3 days later by a course of IL-12 as described above. IFN- treatment consisted of daily i.p. injections of 20,000 units of murine recombinant IFN- (Peprotech, Rocky Hill, NJ) for 7 days. Cy+IFN- treatment consisted of a single i.p. injection of 300 mg of Cy followed by 7 daily i.p. injections of IFN- as described above.
Adoptive T-Cell Transfer.
Tumor-immune donor mice were generated by immunization of naïve mice twice with 0.5–1 x 106 irradiated (5000 rads) tumor cells s.c. The immunized mice were challenged with 5 x 105 live tumor cells 1 week after the second immunization. The spleens from the immunized mice were collected 10–30 days after tumor challenge. Spleen cells were prepared by removing RBCs from single cell suspension by osmolysis followed by extensive washing with saline. The donor T cells were isolated from a single cell suspension of spleen cells by anti-Thy1.2-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA). Five to 10 million (5–10 x 106) purified T cells (>93% CD3 positive) were used for each adoptive transfer. TCR-ß gene KO or TCR-ß/IFN- double KO mice were used as recipients of adoptively transferred spleen and T cells from various donor sources. TCR-ß KO mice were first inoculated with 5 x 105 MCA207 tumor cells s.c. Fourteen days after tumor establishment, when most tumors were 7–12 mm in diameter, tumor-bearing TCR-ß mice received an indicated numbers of spleen or purified T cells from indicated donor mice via i.v. tail vein injection. Two days after adoptive cell transfer, recipients were treated with IL-12 (300 ng once every other day for three doses).
Prophylactic and Concomitant Immunity Tests.
Concomitant immunity tests were carried out as described previously (6
, 7)
. Briefly, s.c. tumors were first established in naïve mice on one flank with 5 x 105 tumor cells. At the indicated time points after the first tumor inoculation, another inoculation of 5 x 105 tumor cells was given to naïve or tumor-bearing mice on the contralateral flank. The development of tumors from the second inoculation in both naïve and tumor-bearing mice was assessed. Protective immunity was tested by immunizing naïve mice with 1 x 106 irradiated (5000 rads) tumor cells s.c. once a week for 2 weeks followed by challenge with 5 x 105 live tumor cells on the opposite flank 1 week after the second immunization. Naïve mice were used as controls at the time of live tumor challenge.
Immunohistochemistry.
Tumors harvested for immunohistochemistry were immediately frozen in OTC medium for cryosection. Tumor sections at 6 µm were prepared and fixed in cold acetone as described previously (13)
. The sections were blocked with 2% goat serum and 1% BSA in PBS and stained with antibodies specific to IFN- receptor (CD119, 10 µm/ml; PharMingen) and MHC I (H2kb; 10 µm/ml;PharMingen). Microscopic images were photographed with a digital camera (Spot II; Diagnostic Instruments, Sterling Heights, MI). Images were assembled and enhanced using Adobe Photoshop (version 5) software.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, but not Th2 Cytokines IL-4 and IL-10, Is Essential for Tumor Rejection Induced by IL-12/Cy+IL-12., IL-4, and IL-10. We found that rejection of established small and large tumors by IL-12 alone and Cy+IL-12 is abolished in mice lacking IFN-, but not IL-4 or IL-10 (Table 1)
. These results demonstrate that the Th1 cytokine IFN-, but not Th2 cytokines IL-4 and IL-10, is essential for IL-12-induced rejection of established tumors.
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Is Necessary but not Sufficient for IL-12-mediated Tumor Regression. is necessary for tumor rejection, it is not sufficient. Thus, treatment of 10-day established palpable MCA207 tumors with IL-12 resulted in complete tumor eradication in normal mice, whereas only transient inhibition of tumor growth was observed with daily exogenous IFN- for 7 days and tumors progressed on termination of exogenous IFN- (Fig. 1A)
. Similarly, treatment of 4-week established large MCA207 tumors with Cy+IL-12 but not Cy alone cured all of the tumors in normal mice. Similar to what was observed previously, treating large tumors with Cy alone induced transient tumor regression, and then tumors resumed progressive growth by 2 weeks after Cy treatment (13)
. In contrast, treatment with Cy followed 3 days later by exogenous daily IFN- (Cy+IFN-) for 7 days in normal mice resulted in only transient tumor regression as seen with Cy treatment alone, and none of the tumors were completely eradicated (Fig. 1B)
.
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Are Necessary and Sufficient for IL-12-induced Tumor Rejection. is necessary but not sufficient for tumor rejection suggests that its antiproliferative activity is not the ultimate effector mechanism in the direct killing of tumor cells. This is supported by the lack of in vitro toxicity of IFN- to MCA207 cells. Culturing MCA207 cells in the presence of IFN- at 100 units/ml does not significantly inhibit tumor growth, and increasing the concentration of IFN- in the culture medium to 5000 units/ml results in reduction but not arrest of tumor cell proliferation. Because previous studies have shown that the antitumor effect of IL-12 is reduced significantly in T-cell-deficient nude mice despite 10-fold higher systemic IFN- release by other non-T cells (14)
, we thought that both T cells and their production of IFN- may be critical for tumor rejection. We investigated this possibility by taking advantage of our recent finding that tumor-sensitized, but not naïve, T cells transferred into tumor-bearing TCR-ß KO mice are able to mediate tumor rejection after IL-12 or Cy+IL-12 treatment in an antigen-specific manner (8)
. To see whether T-cell production of IFN- is able to mediate complete tumor rejection in the absence of IFN- production by other host cells, we created T-cell and IFN- double-deficient mice and transferred tumor-sensitized T cells from normal tumor-immune donors into these tumor-bearing double KO mice followed by IL-12 treatment. Conversely, to determine whether T-cell production of IFN- is essential for tumor rejection, we transferred tumor-sensitized T cells from IFN-KO mice to T-cell-deficient tumor-bearing mice, and treated with IL-12. In the latter case, the major source of IFN- production would be NK cells in the host. The results (Table 2)
show that transfer of tumor-sensitized T cells from normal mice into T-cell and IFN- double-deficient mice result in tumor rejection after IL-12 treatment. In contrast, transfer of tumor-sensitized T cells from IFN- KO mice into single T-cell-deficient tumor-bearing mice failed to cause tumor rejection by IL-12. The tumor rejection kinetics are similar to what was observed in our previous adoptive transfer experiments using single T-cell-deficient hosts (8)
. The results from these adoptive transfer experiments show that IFN- and its production by tumor-sensitized T cells, but not non-T cells, is necessary and sufficient for rejection of established tumors by IL-12.
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Unresponsive MCA207 Cells. during tumor rejection. For this, we constructed IFN-unresponsive MCA207 tumor lines through the blockade of normal IFN-receptor function in tumor cells by causing them to overexpress a mutant form of the IFN- receptor subunit. This mutant receptor is under the control of a strong promoter, which causes overexpression of the mutant receptor and formation of inactive dimers with endogenous wild-type receptor in a dominant negative fashion. This strategy has proven to be highly effective in previous studies (22
, 23)
. Using this approach, we selected three clones (clone 11, 16, and 29) of MCA207 tumor cells that showed >100-fold overexpression of IFN- receptor (CD119) on the cell surface. As controls, we selected one clone (clone 24) that does not demonstrate overexpression of IFN- receptor . These clones were used in subsequent in vitro and in vivo experiments. Fig. 2A
shows the overexpression of the dominant negative form of the IFN- receptor in one of the MCA207 clones, clone 11, compared with parental MCA207 cells. Clones 16 and 29 exhibit the same profile of IFN- receptor as clone 11, and clones 24 demonstrates the same profile as parental MCA207 (not shown).
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in vitro was confirmed by testing the ability of IFN- to up-regulate MHC class I expression in these cells. Whereas parental MCA207 tumor cells responded to IFN- (100 units/ml) with up-regulation of MHC class I molecule (H-2 Kb) expression, clones overexpressing the mutant form of IFN- receptor lost this response (Fig. 2B)
even under concentrations of IFN- as high as 5000 units/ml (Fig. 2C)
. Like parental MCA207, clone 24 showed clear up-regulation of H-2Kb expression in response to IFN- (not shown). Therefore, unlike parental MCA207 cells, IFN- unresponsive clones overexpress a mutant IFN- receptor and lose their response to IFN- in vitro.
Response of IFN- Unresponsive Tumors to Immunotherapy.
We have demonstrated previously that large (15–20 mm in diameter) established (3–4 weeks) s.c. MCA207 tumors are eradicated by Cy+IL-12 treatment and that tumor rejection is dependent on IFN- (Ref. 13
; Fig. 1B
; Table 1
). To determine whether the tumor is the target of IFN-, we tested tumor rejection using the IFN- unresponsive MCA207 clones. When implanted s.c. with 5 x 105 cells, each IFN-unresponsive clone showed a slightly faster tumor growth rate than parental MCA207, whereas the IFN- responsive control, clone 24, showed a significantly slower growth rate than parental MCA207 tumors. Despite this difference in growth rate, we found that like parental MCA207, all of the large (>15 mm in diameter) tumors formed from inoculation of mice with IFN--unresponsive clones were completely eradicated by treatment with Cy+IL-12 but not Cy alone (Table 3)
. Similar to our previous study (13)
and Fig. 1B
, treatment of tumors with Cy alone only induced a transient tumor regression. Between the various clones tested, clone 11 showed slower, whereas clones 16, 24, and 29 showed similar tumor regression as compared with parental MCA207 after Cy+IL-12 therapy (Fig. 1B)
. Furthermore, cured animals resisted rechallenge with both their respective clone and parental MCA207 cells.
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unresponsive tumors to therapy with IL-12 alone. We have shown previously that small (4–8 mm in diameter) 10-day established s.c. MCA207 tumors are eradicated by IL-12 treatment alone (15)
. To determine whether tumor responsiveness to IFN-is required in this setting, small (3–6 mm in diameter) IFN--unresponsive tumors and control parental MCA207 tumors were treated with IL-12 alone. Like parental MCA207 tumors, all of the tumors of IFN--unresponsive clones 29 were highly responsive to IL-12 treatment and demonstrated complete tumor regression (Fig. 3, A and B)
. Clone 11 tumors showed a partial response to IL-12 treatment, in that all of the tumors initially responded to IL-12 with regression compared with untreated tumors (Fig. 3C)
, but the majority of clone 11 tumors regrew, and 4 of 14 animals in three experiments were cured (Table 4)
. This partial response still forms a clear contrast to the total resistance of MCA207 tumors to IL-12 therapy in IFN- KO mice (Fig. 3D)
.
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-unresponsive tumors responded like parental MCA207 tumors to treatment with IL-12 and Cy+IL-12, it is possible that these clones may lose their ability to block IFN- action in vivo. To determine whether this is the case, we harvested several IFN--unresponsive tumors before and after treatment with IL-12 and Cy+IL-12, and examined the overexpression of mutant IFN- receptor and MHC class I expression by immunohistochemistry. Normal untreated MCA207 tumors showed low staining of IFN- receptor and moderate staining of H-2Kb (Fig. 4
, MCA207), indicating that tumor cells express abundant MHC class I molecules in vivo. In contrast, untreated IFN-unresponsive clone 11 showed a very strong staining of IFN- receptor (Fig. 4
, clone 11), consistent with the overexpression of CD119 found in vitro (Fig. 2A)
. Surprisingly, clone 11 tumor cells also showed a total lack of H-2Kb expression (Fig. 4
, clone 11), indicating that in vivo expression of MHC class I molecule by tumor cells is dependent on tumor cell response to IFN-. Furthermore, this pattern of overexpression of CD119 and total lack of expression of MHC class I molecule by tumor cells is preserved in clone 11 tumors after treatment with IL-12 and Cy+IL-12 (Fig. 4
, clone 11/IL-12 and clone 11/Cy+IL-12). It is noted that the peripheral of the Cy+IL-12-treated clone 11 tumors stained positive for H-2Kb. This is an area of intense immune infiltration including T cells and macrophages. Another example of clear contrast between tumor and normal tissues can be seen in Fig. 4
in which a lymph node adjacent to IL-12-treated clone 11 tumors shows the opposite staining pattern with the two antibodies (Fig. 4
, clone 11/IL-12). Other IFN- unresponsive clones showed similar characteristics to those of clone 11 in vivo.
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Unresponsive MCA207 Tumors Are Equally Immunogenic in Vivo. unresponsive clones to see whether the partial response of clone 11 to IL-12 alone is because of reduction in its immunogenicity. Specifically, we wanted to see whether the failure of IFN- unresponsive clones to up-regulate MHC I in vivo renders these tumors less immunogenic than parental MCA207. In a prophylactic setting, we immunized naïve normal mice with irradiated tumor cells from either MCA207 or its IFN- unresponsive clones. The immunized mice as well as naïve control mice were then challenged with live MCA207 tumor cells. In another setting of concomitant tumor bearing, we first established s.c. tumors with MCA207 or IFN- unresponsive clones. When tumors became palpable in 8 days, a second inoculation with the same tumor cells as the primary tumor was given to naïve control or tumor-bearing mice on the opposite flanks. By both tests of prophylactic and concomitant immunity, we observed that IFN- unresponsive clones 11 and 29 exhibit immunogenicity similar to that of parental MCA207, as immunization with both inactivated MCA207 and IFN- unresponsive clones was able to induce strong protection against subsequent challenge with live tumor cells (Fig. 5A)
, and mice bearing 8-day tumors of both parental MCA207 and IFN- unresponsive clones were able to reject a second tumor inoculation (Fig. 5B)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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has been found to be essential for IL-12- (10)
and Cy+IL-12-induced rejection of established MCA207 tumors (13)
. Previous studies in other tumor models of the role of IFN- in IL-12-induced tumor rejection have shown that IFN- is likely to be involved in multiple steps and mechanisms of tumor rejection. For example, IFN- has been found to be responsible for T-cell migration to the tumor bed (26, 27, 28)
, inhibition of tumor vascular development through induction of IP-10 (25
, 29)
, and activation of tumor and macrophage expression of iNOS (12
, 15)
. It is likely that other, as yet unidentified, properties of IFN- are involved as well. To elucidate the role of IFN- in IL-12- and Cy+IL-12-induced tumor rejection in the MCA207 models, the current study determines the source of IFN- production and its target cell.
First we have shown that IFN- per se is necessary but not sufficient for IL-12-mediated tumor rejection. This conclusion is consistent with a previous study in another tumor model (14)
. Because IFN- alone is not able to cause tumor regression in vivo, the direct cytotoxic and antiproliferative activity of IFN- is unlikely to be responsible for tumor rejection after IL-12 treatment. This is consistent with our in vitro observation regarding the lack of direct toxicity of IFN- to MCA207 tumor cells. Thus, the role of IL-12 in tumor rejection is not simply to induce IFN- production in vivo, and as a result, the antitumor mechanism of IL-12 in our tumor rejection models cannot be explained by mechanisms activated by IFN- alone.
Our previous study indicated that a Th1 antitumor response is associated with tumor rejection induced by IL-12 and Cy+IL-12 (13
, 15)
. Indeed, using cytokine gene KO mice, we show here that the Th1 cytokine, IFN-, but not Th2 cytokines, IL-4 and IL-10, is essential for tumor rejection induced by IL-12 and Cy+IL-12 treatment. This finding forms a clear contrast to the recently reported requirement of IL-4 during the priming phase of the Th1-mediated CTL memory antitumor immune response (30)
. Because we have shown in a recent study that rejection of established MCA207 tumors requires the priming of tumor-sensitized T cells before the start of treatment (8)
, the fact that established MCA207 tumors from IL-4 KO mice are fully rejected by IL-12/Cy+IL-12 treatment (Table 1)
suggests that IL-4 is not required for the generation of pre-existing immunity in our tumor rejection models. This finding led us to question whether the Th1 response associated with IL-12 treatment and tumor regression is responsible for tumor rejection, and, more specifically, if T cells and T-cell production of IFN- are necessary and sufficient for tumor rejection. In a recent study using adoptive transfer experiments, we have shown that tumor-sensitized, but not naïve, T cells are essential for IL-12-induced tumor rejection (8)
. We show here that rejection of large established tumors by IL-12 is accomplished in mice in which the only cells capable of producing IFN- are tumor-sensitized T cells. Whereas transfer of tumor-immune T cells into T-cell-deficient hosts confers some degree of antitumor activity as shown by many previous studies, the efficacy of such T-cell transfer alone is usually ineffective against well-established s.c. tumors as we have shown in a recent study (8)
. However, the transfer of normal T cells into T-cell and IFN- double-deficient hosts has not been reported previously. The partial tumor eradication (2 of 10; Table 2
) by transfer of normal tumor-sensitized T cells alone may be caused by an uncontrolled expansion of CTL response in the absence of IFN- (31)
. On the other hand, tumor rejection by IL-12 is abolished when the only cells that cannot produce IFN- are tumor-sensitized T cells from IFN- KO mice. Two recent studies have shown that T-cell sensitization by incipient tumors is not affected in IFN- KO mice. Nakajima et al. (27)
showed that immunization with inactivated tumor cells in IFN- KO mice resulted in T-cell sensitization. This can be demonstrated by the ability of these cells to suppress tumor formation when mixed with tumor cells and injected s.c. (Winn assay), and by their ability to produce IL-2 on tumor restimulation in vitro. Similarly, Winter et al. (32)
showed that T cells from the tumor-draining lymph nodes of normal and IFN- KO mice have similar profiles of activation markers on the cell surface, and are similar in their ability to suppress 5-day established experimental lung metastases when infused via tail vein injection. These results demonstrate that the critical source of IFN- production during IL-12-induced rejection of established MCA207 tumors is tumor-sensitized T cells. In other words, Th1 cells are responsible for IL-12-induced tumor rejection in the MCA207 tumor model.
What, then, is the function of the IFN- produced by these T cells? We approached this question by first asking whether the target cell of IFN- is the tumor itself or host-derived. Using IFN- unresponsive tumors in normal mice, we have shown that tumors are not likely to be the critical targets of IFN- during IL-12-induced tumor rejection. Our finding differs from a previous study (25)
. However, two differences between the studies may account for the different conclusions. First, the previous study used a nonestablished (nonpalpable) tumor model in which the antitumor response was likely mediated by NK/NKT cells, whereas our study used established palpable (large) tumors in which the antitumor response is T-cell-dependent (8)
. Second, whereas in the previous study the effect of IFN- was associated with collapse of tumor blood vessels because of its antiangiogenic induction of IP-10 on tumor cells, tumor cell expression of IP-10 is not a major event in our model (13)
, and we have observed increased rather than decreased numbers of blood vessels in regressing tumors after treatment with IL-12 and Cy+IL-12.4
Our result also differs from that of a previous study in which the tumor cell is identified as the target of IFN- during endotoxin-induced tumor rejection (23)
. Because endotoxin-induced IFN- production and shock have been found to link to IL-12 in vivo (33)
, it is possible that some or all of the antitumor effect of endotoxin is mediated through IL-12. Testing tumor rejection with the IFN- unresponsive MCA207 clones showed a split response in that 8-day established clone 29 is readily rejected by endotoxin treatment, whereas clone 11 resisted treatment.5
If the target cell of IFN- during tumor rejection by IL-12 and Cy+IL-12 is not the tumor itself, it is then likely to be host-derived. A recent study by Nakajima et al. (27)
found that lack of IFN- affected T-cell migration to tumor sites. In the MCA207 tumor models, slightly reduced but still significant immune cell infiltration into MCA207 tumors was found after IL-12 and Cy+IL-12 treatment in IFN- KO mice.5
This suggests that IFN- may also be required at steps after immune infiltration in the effector phase. One way that IFN- could cause tumor killing by targeting a host cell is through macrophage activation, as activated macrophages have been shown to be tumoricidal in vitro (34)
. By assessing iNOS expression, the presence of activated macrophages in IL-12/Cy+IL-12-treated regressing tumors has been observed in a number of tumor models by others (12)
and by us (13
, 15)
. Activation of macrophages after IL-12 treatment is dependent on IFN-, as in anti-IFN- antibody-treated (21)
and IFN- KO mice,5
and IL-12-induced macrophage expression of iNOS is abolished, indicating lack of macrophage activation. We have suggested the role of macrophages as effector cells in IL-12-induced rejection of established tumors in a recent previous study (15)
. This view is supported by findings from other studies, which showed that Th1 cell-directed antitumor response depends on macrophages in the effector phase (35, 36, 37, 38)
. The requirement for both IFN- produced by antigen-specific T cells and activation by these T cells (39)
would explain why IFN- is necessary but not sufficient, whereas T cells and their production of IFN- together are both necessary and sufficient for IL-12-induced tumor rejection.
One interesting finding from the current study is the lack of MHC expression of IFN- unresponsive tumors in vivo and its lack of effect on tumor immunogenicity (Fig. 4
, clone 11). This observation of IFN--dependent expression of MHC I by tumor cells in vivo has been sustained by a similar observation made on normal MCA207 tumors grown in IFN- KO mice.5
The response of the IFN- unresponsive tumor cell clones to other IFNs such as IFN- in terms of up-regulating MHC class I expression in vitro was not analyzed in the current study. The in vivo observation made in the current study argues that the expression of MHC class I molecules is more critically dependent on IFN-. There seems to be no compensatory mechanism in the absence of the IFN- signaling pathway. Whereas it is not clear what makes a tumor immunogenic, there has been a general belief that lack of MHC expression is one of the ways that tumor cells escape immune recognition. Thus, many nonimmunogenic and metastatic tumors have low levels of MHC class I expression. In some studies forced expression of MHC I molecules were found to increase tumor immunogenicity (40, 41, 42)
. However, it is not known whether the loss of MHC I expression by an immunogenic tumor would lead to a loss of tumor immunogenicity. Results from the current study indicate that this is not the case (Fig. 5)
, suggesting that expression of MHC I is not what makes certain tumors immunogenic. These observations can be explained by assigning the task of T-cell priming to host antigen-presenting cells. However, the low MHC I expression by tumor cells may affect tumor killing by cytotoxic T cells if such effector mechanism is involved in the expression of antitumor immunity. Because IFN-unresponsive tumors that express low or no MHC molecules are rejected by IL-12-based therapy in the current study, the role of classic CTL as effector thus seems unlikely. This is consistent with our recent study that suggests that macrophages are the effector cells in our tumor rejection models.4
In summary, this study demonstrates that IFN- is essential for the antitumor effects of IL-12 and Cy+IL-12. The role of IFN- is necessary, but not sufficient, for tumor rejection. Tumor-sensitized T cells are the source of IFN- production that is both necessary and sufficient for tumor rejection. The IFN- produced by Th1 cells does not work directly on tumor cells, but works rather through host cells for complete tumor eradication. Despite that MHC I expression by tumor cells in vivo is dependent on tumor response to IFN-, the loss of MHC I expression by tumor cells does not affect the immunogenicity of immunogenic tumors. Future experiments are needed to test whether macrophage response to IFN- is critical for tumor rejection by IL-12 and, if so, the mechanism by which activated macrophages eradicate tumor cells in these tumor regression models.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Merit Review Grants from VA Medical Research Services (to J. A. N. and K. T.). 
2 To whom requests for reprints should be addressed, at Surgical Services, San Francisco VA Medical Center, 4150 Clement Street, San Francisco, CA 94121. Phone: (415) 221-4810, extension 3708; Fax: (415) 750-2181; E-mail: kangla1{at}itsa.ucsf.edu
3 The abbreviations used are: IL, interleukin; NK, natural killer; Cy, cyclophosphamide; iNOS, inducible nitric oxide synthase; TCR, T-cell receptor; KO, knockout.
4 K. Tsung, J. P. Dolan, Y. L. Tsung, and J. A. Norton. Macrophages as effector cells in IL-12-induced T cell-dependent tumor rejection, Cancer Res., in press, 2002.
Received 10/31/01. Accepted 6/18/02.
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  M. Numasaki, M. Tagawa, F. Iwata, T. Suzuki, A. Nakamura, M. Okada, Y. Iwakura, S. Aiba, and M. Yamaya IL-28 Elicits Antitumor Responses against Murine Fibrosarcoma J. Immunol., April 15, 2007; 178(8): 5086 - 5098. [Abstract] [Full Text] [PDF]
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J. J. Sussman, R. Parihar, K. Winstead, and F. D. Finkelman Prolonged Culture of Vaccine-Primed Lymphocytes Results in Decreased Antitumor Killing and Change in Cytokine Secretion Cancer Res., December 15, 2004; 64(24): 9124 - 9130. [Abstract] [Full Text] [PDF]
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H.-L. Ma, M. J. Whitters, R. F. Konz, M. Senices, D. A. Young, M. J. Grusby, M. Collins, and K. Dunussi-Joannopoulos IL-21 Activates Both Innate and Adaptive Immunity to Generate Potent Antitumor Responses that Require Perforin but Are Independent of IFN-{gamma} J. Immunol., July 15, 2003; 171(2): 608 - 615. [Abstract] [Full Text] [PDF]
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K. Tsung, J. P. Dolan, Y. L. Tsung, and J. A. Norton Macrophages as Effector Cells in Interleukin 12-induced T Cell-dependent Tumor Rejection Cancer Res., September 1, 2002; 62(17): 5069 - 5075. [Abstract] [Full Text] [PDF]
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, naïve mice; 
, MCA207-immunized (A) or tumor-bearing (B) mice; 
, clone 11-immunized (A) or tumor-bearing (B) mice; 
, clone 29-immunized (A) or tumor-bearing (B) mice.