Successful immunotherapy with IL-2/anti-CD40 induces the chemokine-mediated mitigation of an immunosuppressive tumor microenvironment

Abstract

Treatment of mice bearing orthotopic, metastatic tumors with anti-CD40 antibody resulted in only partial, transient anti-tumor effects whereas combined treatment with IL-2/anti-CD40, induced tumor regression. The mechanisms for these divergent anti-tumor responses were examined by profiling tumor-infiltrating leukocyte subsets and chemokine expression within the tumor microenvironment after immunotherapy. IL-2/anti-CD40, but not anti-CD40 alone, induced significant infiltration of established tumors by NK and CD8⁺ T cells. To further define the role of chemokines in leukocyte recruitment into tumors, we evaluated anti-tumor responses in mice lacking the chemokine receptor, CCR2. The anti-tumor effects and leukocyte recruitment mediated by anti-CD40 alone, were completely abolished in CCR2^−/− mice. In contrast, IL-2/anti-CD40-mediated leukocyte recruitment and reductions in primary tumors and metastases were maintained in CCR2^−/− mice. Treatment of mice with IL-2/anti-CD40, but not anti-CD40 alone, also caused an IFN-γ-dependent increase in the expression of multiple Th1 chemokines within the tumor microenvironment. Interestingly, although IL-2/anti-CD40 treatment increased Tregs in the spleen, it also caused a coincident IFN-γ-dependent reduction in CD4⁺/FoxP3⁺ Tregs, myeloid-derived suppressor cells and Th2 chemokine expression specifically within the tumor microenvironment that was not observed after treatment with anti-CD40 alone. Similar effects were observed using IL-15 in combination with anti-CD40. Taken together, our data demonstrate that IL-2/anti-CD40, but not anti-CD40 alone, can preferentially reduce the overall immunosuppressive milieu within the tumor microenvironment. These results suggest that the use of anti-CD40 in combination with IL-2 or IL-15 may hold substantially more promise for clinical cancer treatment than anti-CD40 alone.

Many strategies for cancer treatment use combinations of immunotherapeutic agents for enhanced anti-tumor responses. However, these approaches are often complicated by a need to overcome tumor-induced immune suppression in the tumor microenvironment. In this regard, T regulatory (Treg) cells and myeloid-derived suppressor cells (MDSC) have been identified as functional suppressor cells within tumors (1, 2). The most effective immunotherapeutic regimens are likely to consist of agents that restructure, within the tumor microenvironment, the composition of tumor-infiltrating leukocytes away from these inhibitory elements in favor of effector cells, such as NK cells and CD8⁺ T cells.

Chemokine expression can regulate the polarization of immune responses (3). For example, CXCR3 and CCR5 are preferentially expressed on Th1 T cells and M1 macrophages and their respective ligands are associated with enhanced cell-mediated immune responses (3–5) and favorable prognosis in human RCC (5, 6). Another chemokine, monocyte chemoattractant protein (MCP)-1 activates macrophages for enhanced anti-tumor activities (7), however MCP-1 expression is also associated with the recruitment of mononuclear cells capable of producing tumor promoting factors (8, 9), as well as MDSC that contribute to tumor progression through the inhibition of effector cell functions (8, 10).

We reported previously that IL-2 and agonistic antibody to CD40 (αCD40) synergize for the regression of metastatic tumors in mice (11). Although we identified CD8⁺ T cells and host IFNγ expression as critical components of this therapeutic approach (11), the specific mechanisms underlying the IL-2/αCD40 synergistic anti-tumor responses within the microenvironment remain unclear. We demonstrate in a murine model of metastatic renal cancer that αCD40 may be limited by its dependency upon MCP-1 and an inability to remove Tregs and MDSC specifically from within the tumor microenvironment, allowing for eventual tumor progression. In contrast, synergistic anti-tumor responses and protection achieved by IL-2/αCD40 are associated with the expression of Th1 chemokines that are associated with favorable prognosis in RCC (5, 6), an augmentation of effector leukocytes and concomitant removal of suppressive cells specifically within the tumor microenvironment.

Results

CCR2 Expression Is Required for αCD40, but Not IL-2/αCD40 Mediated Anti-Tumor Responses.

Our previous study showed that IL-2/αCD40 exhibited strong synergy for treatment of established metastatic tumors in mice, as compared to IL-2 or αCD40 as single agents (11). Furthermore, we found that αCD40 treatment of Renca-bearing mice induced significant reduction in tumors in association with high levels of systemic MCP-1 levels, suggesting a possible role for MCP-1 in leukocyte recruitment into tumors and CD40-dependent anti-tumor effects (12). To determine the relative contribution of MCP-1 to the αCD40- and IL-2/αCD40-mediated anti-tumor responses, we compared tumor outcomes in treated WT and mice deficient in CCR2, the receptor for MCP-1 (13). Treatment of WT mice with αCD40 significantly reduced primary tumor areas, but a substantially greater reduction was observed after IL-2/αCD40 treatment (Fig. 1A). Interestingly, the effects of αCD40 were abrogated in CCR2^−/− mice, whereas the efficacy of IL-2/αCD40 treatment was maintained in CCR2^−/− mice.

Fig. 1.

CCR2 is required for αCD40, but not IL-2/αCD40-mediated anti-tumor responses. (A) WT and CCR2^−/− tumor-bearing mice were treated as indicated. On day 22, mice were euthanized, the primary tumor was dissected and measured. (B) Lungs from these treated mice were collected on the same day and fixed in Bouin's solution. The number of lung metastases was counted under a dissecting microscope (*, P < 0.05 and **, P < 0.005 as compared to control-treated mice). (C) For studies of tumor progression, mice underwent surgical removal of the tumor-bearing kidney followed by treatment with IL-2 and/or αCD40. Survival analysis was plotted according to the Kaplan-Meier method and statistical differences determined using the log-rank test (*, P < 0.0001). For all tumor studies, the results from one experiment consisting of 10 mice/treatment group are shown and results are representative of three similar experiments.

Since the orthotopic tumor model that we used forms spontaneous tumor metastases in the lungs, we next examined the effect of αCD40 and IL-2/αCD40 treatment on lung metastases. Similar to primary tumors, αCD40 alone significantly reduced the number of metastases in WT, but not CCR2^−/− mice (Fig. 1B). In contrast, the reduction in metastases achieved by IL-2/αCD40 was maintained in CCR2^−/− mice.

To evaluate the efficacy of the different treatments on survival of mice bearing metastatic disease, we performed a unilateral nephrectomy of the tumor-bearing kidney, followed by treatment of residual metastatic disease. IL-2/αCD40-treated WT and CCR2^−/− mice had significantly prolonged survival, with >75% of the mice remaining disease-free for >80 days (Fig. 1C). Surviving mice were rechallenged s.c. with Renca on day 92 and these mice demonstrated complete resistance to tumors (Fig. S1). Although αCD40 mediated significant reduction in primary tumors (Fig. 1A), neither αCD40 nor IL-2 treatments alone prolonged survival (Fig. 1C). Thus, αCD40 mediates CCR2-dependent anti-tumor responses, whereas the more substantial, durable responses induced by IL-2/αCD40 are CCR2 independent.

CCR2 Is Required for αCD40, but Not IL-2/αCD40-Mediated Recruitment of Leukocytes into Tumors.

Next, we characterized the profile of tumor-infiltrating leukocytes in response to IL-2, αCD40, and IL-2/αCD40 immunotherapy. In contrast to IL-2 or αCD40 alone, the tumors from IL-2/αCD40-treated mice contained significant increases in the numbers of infiltrating CD8⁺ T cells, NK, and B cells (Fig. 2A). In contrast, CD4⁺ T cells did not increase after any treatment. Treatment of tumor-bearing mice with either αCD40 or IL-2/αCD40 also induced significant macrophage recruitment into tumors. These cellular changes were still evident when they were normalized for tumor area (Fig. S2). Thus, as compared to IL-2 or αCD40 alone, IL-2/αCD40 maximally enhances leukocyte recruitment into the tumor microenvironment.

Fig. 2.

αCD40 induces CCR2-dependent leukocyte recruitment into tumors, whereas IL-2/αCD40 maximally induces CCR2-independent increases in tumor-infiltrating leukocytes. (A) Wild-type and (B) CCR2^−/− tumor-bearing mice were treated, as indicated. On day 22, mice were euthanized and the primary tumor was dissected. Total leukocytes were counted and further identified by cell surface staining with the indicated antibodies and flow cytometry. The total number of each cell subset was calculated by multiplying the total number of leukocytes by the percent expressing each surface marker (*, P < 0.05; NS = not significant). Data are derived from two tumors per treatment group in each of three separate experiments.

To determine the relative contribution of MCP-1 to the αCD40- and IL-2/αCD40-mediated leukocyte recruitment into tumors, we analyzed the profile of leukocytes in treated CCR2^−/− mice (Fig. 2B). The IL-2/αCD40 mediated increases in CD8⁺ T cells, B cells and macrophages were maintained in CCR2^−/− mice. In contrast, the αCD40-mediated increase in macrophages was lost in CCR2^−/− mice (Fig. 2B). We also found that NK cell recruitment in response to IL-2/αCD40 was abolished in CCR2^−/− mice.

The results above were further confirmed by immunohistochemical analyses on tumors from treated WT or CCR2^−/− mice (Fig. S3). Whereas IL-2/αCD40 indued macrophage and CD8⁺ T cell recruitment in WT and CCR2^−/− mice, the αCD40-induced recruitment of macrophages was lost in CCR2^−/− mice (Fig. S3A). By computer-assisted quantitation of the immunoreactive areas, the IL-2/αCD40 mediated increase in F4/80⁺ macrophages and CD8⁺ T cell recruitment was significant (Fig. S3 A and B). These results indicate that CCR2 is required for the αCD40, but not IL-2/αCD40-mediated recruitment of leukocytes into tumors.

IL-2/αCD40 Increases the Expression of RANTES, MIP-1γ, MIG, and IP-10.

Our data indicated that leukocyte recruitment into the tumor induced by the αCD40 treatment was dependent upon CCR2-mediated responses, whereas recruitment caused by IL-2/αCD40 was CCR2-independent. To determine whether this was due to the increased expression of additional chemoattractants induced by IL-2/αCD40, we analyzed tumors for chemokine gene expression by qPCR. IL-2/αCD40 significantly increased CXCL9/MIG, CXCL10/IP-10, CCL5/RANTES, and CCL9/MIP-1γ expression within tumors (Fig. 3). Furthermore, the IL-2/αCD40 mediated increase in chemokine expression was completely lost in GKO mice (Fig. 3). Interestingly, CXCR3, the receptor for MIG and IP-10, and CCR5, a RANTES receptor, were also significantly upregulated (Fig. S4). Taken together, these data suggest that IL-2/αCD40 induces the IFNγ-dependent upregulation of several chemokines within tumors that may facilitate leukocyte recruitment.

Fig. 3.

IL-2/αCD40 induces the IFNγ-dependent increase in chemokine expression within tumors. WT and GKO tumor-bearing mice were treated, as indicated. On day 22, mice were euthanized and the primary tumor was dissected and placed in RNALater. Total RNA was extracted from tumors and analyzed by qPCR for (A) CXCL9, (B) CXCL10, (C) CCL5, and (D) CCL9 chemokine gene expression. For each gene, the results from saline control-treated samples were normalized to 1. Each closed circle represents results from one experiment using wild-type mice (n = 5) and the p values on each graph were derived from the comparison of αCD40 to IL-2/αCD40 treated, wild-type mice. No statistical differences were observed using GKO mice (open triangles and dotted mean lines; n = 3).

IFN Gamma Is Required for the IL-2/αCD40-Mediated Regulation of Tumor-Infiltrating CD4⁺ and CD8⁺ T Cells.

Since IFNγ expression was required for chemokine induction within the tumor microenvironment, we next examined its role in IL-2/αCD40-induced leukocyte recruitment. IL-2/αCD40 treatment of WT mice significantly increased the number of tumor-associated CD8⁺ T cells (Fig. S5A). This response was markedly reduced in GKO mice, although it was still significant compared to control mice. IL-2 and αCD40 as single agents also increased the number of CD8⁺ T cells in WT, but not GKO mice. IL-2/αCD40 also reduced the number of CD4⁺ T cells within tumors in WT, but not GKO mice (Fig. S5A). These cellular changes were still evident when they were normalized for tumor area (Fig. S5B). Taken together, IL-2/αCD40 significantly increased the CD8:CD4 ratio of tumor-infiltrating T cells and this was completely lost in GKO mice (Fig. S5C).

IL-2/αCD40 Selectively Reduces the Number of Tumor-Infiltrating Regulatory T Cells and Treg-Associated Chemokines Within Tumors.

Since the studies above demonstrated an overall reduction in CD4⁺ T cells by IL-2/αCD40, we hypothesized that part of the anti-tumor effect could be due to reduction in CD4⁺ Tregs. We therefore compared the ability of IL-2, αCD40, and IL-2/αCD40 treatments to regulate the numbers of CD4⁺/CD25⁺/FoxP3⁺ Tregs in tumors and spleens. Consistent with our previous finding (14), IL-2/αCD40 increased the number of Tregs within the spleen (Fig. 4A). However, we also found that the same treatment selectively reduced the numbers of tumor-associated Tregs (Fig. 4B). IL-2 and αCD40, as single agents, had no significant effect upon the number of Tregs in either the spleen or tumor. Furthermore, the IL-2/αCD40-mediated reduction in Treg numbers was lost in GKO mice. IL-15/αCD40, another combination immunotherapy recently shown to induce anti-tumor responses (15), also reduced tumor-associated Tregs (Fig. 4B). However, unlike IL-2, IL-15 by itself did not increase Treg numbers in tumors.

Fig. 4.

IL-2/αCD40 treatment selectively reduces the number of CD4⁺/FoxP3⁺ cells and Treg-associated chemokines within tumors. WT and GKO tumor-bearing mice were treated as indicated. On day 22, mice were euthanized and the spleens and tumors harvested. The total number of Tregs in each microenvironment was determined by multiplying the total number of (A) splenocytes or (B) tumor-associated leukocytes by the percentage of CD4⁺/FoxP3⁺ cells in each sample (*, P < 0.03 for both spleen and tumor). Data are derived from two tumors per treatment group in each of three separate experiments. (C) CCL17 and (D) CCL22 gene expression in tumors was analyzed by qPCR. The results from saline control-treated samples were normalized to 1. Each closed circle represents results from one experiment using wild-type mice (n = 3) and p-values on each graph were derived from the comparison of αCD40 to IL-2/αCD40 treated, wild-type mice. No statistical differences were observed using GKO mice (open triangles and dotted mean lines; n = 3). (E) CCL17 and CCL22 protein expression in tumors was analyzed by ELISA of five individual tumor lysates (*, P < 0.05 and **, P < 0.02 as compared PBS treated groups; NS = not significant). (F) Tumor-bearing mice were treated beginning day 12 with PC61 alone or in combination with IL-2 or αCD40. On day 22, mice were killed, the primary tumor was dissected and measured (*, P < 0.03; **, P < 0.002 as compared to PBS alone). Each treatment group had nine mice.

We hypothesized that the reduced numbers of tumor-associated Tregs could be due in part to a reduction in chemoattractant signals for these cells. Therefore, we analyzed tumor lysates for the expression of CCL17 and CCL22, two chemokines implicated in the association of Tregs with human (1, 16, 17) and murine (18) tumors. IL-2/αCD40 treatment significantly reduced the gene expression of both chemokines (Fig. 4 C and D); whereas either IL-2 or αCD40 alone had no effect. The IL-2/αCD40-mediated reduction of both chemokines was abolished in GKO mice. Next, we analyzed tumor lysates for CCL17 and CCL22 protein expression by ELISA. Consistent with our gene expression studies, IL-2/αCD40 significantly decreased the protein expression of both chemokines (Fig. 4E). Interestingly, in GKO mice, CCL17 protein expression was significantly increased in response to αCD40 or IL-2/αCD40 treatments (Fig. 4E). To determine whether the increases in splenic Tregs could be attributed to alterations in CCL17 or CCL22 gene expression, we analyzed the expression of each chemokines gene in spleen cDNA isolated from each treatment group. However, no significant change in the expression of either chemokine gene was identified for any treatment group (Fig. S6 A and B). Overall, these results demonstrate that IL-2/αCD40 specifically reduces the number of tumor-associated Tregs and that this correlated with the IFNγ-dependent downregulation of chemokines that may control their recruitment to the tumor site.

Finally, we sought to demonstrate a tumor-supporting role of Tregs in Renca-bearing mice. We treated mice with αCD25 depleting antibody (PC61) alone or in combination with either IL-2 or αCD40. On the one hand, treatment of tumor-bearing mice with PC61 alone increased the primary tumor area (Fig. 4F). However, when PC61 was used in combination with either IL-2 or αCD40 where substantial infiltration of Tregs into tumors was detected, a significant reduction in primary tumor area was observed. Furthermore, CD4⁺/CD25⁺ Tregs isolated from Renca tumors suppress T cell proliferation even more efficiently than those isolated from the periphery (Fig. S7), which indicates a tumor-promoting role for Tregs, in our model.

IL-2/αCD40 Treatment Selectively Reduces the Number of Myeloid-Derived Suppressor Cells, Arginase Expression, and Associated Chemokines Within Tumors.

Since IL-2/αCD40 increased the number of tumor-associated macrophages, we hypothesized that it might also alter macrophage subsets present in the tumor microenvironment. Thus, we compared the ability of IL-2, αCD40, or IL-2/αCD40 treatment to regulate the numbers of myeloid-derived suppressor cells (MDSC) in tumors and spleens. MDSC were identified as CD45⁺, CD11b⁺, Gr1^Lo, CD124⁺ cells (2). IL-2/αCD40 significantly increased the number of MDSC within spleens (Fig. 5A) but significantly reduced them within tumors (Fig. 5B). In contrast, IL-2 and αCD40, as single agents, had no effect on MDSC in either site. The ability of IL-2/αCD40 to reduce the number of tumor-infiltrating MDSC was lost in GKO mice. IL-15/αCD40 treatment similarly mediated a significant reduction in tumor-derived MDSC (Fig. 5B).

Fig. 5.

IL-2/αCD40 treatment selectively reduces the number of myeloid-derived suppressor cells, arginase expression, and associated chemokines within tumors. Wild-type and GKO tumor-bearing mice were treated as indicated. On day 22, mice were euthanized and the spleens and tumors harvested. The total number of MDSC in each microenvironment was determined by multiplying the total number of (A) splenocytes or (B) tumor-associated leukocytes by the percentage of CD11b⁺/Gr1^Lo/CD124⁺ cells in each sample (*, P < 0.05 for spleen and P < 0.01 for tumor). Data are derived from two tumors per treatment group in each of three separate experiments. (C) Arginase expression in three separate tumors/treatment group was analyzed as described in Materials and Methods (*, P < 0.02 as compared to both PBS and αCD40-treated wild-type groups). (D) CXCL5 gene expression in tumors was analyzed by qPCR. The results from saline control-treated samples were normalized to 1. Each closed circle represents results from one experiment using wild-type mice (n = 3) and p-values were derived from the comparison of αCD40 to IL-2/αCD40 treated, wild-type mice. No statistical differences were observed using GKO mice (open triangles and dotted mean lines; n = 3). (E) CXCL5 protein expression in tumors was analyzed by ELISA of five individual tumor lysates (**, P < 0.03 as compared to PBS treated groups; NS = not significant).

To functionally characterize the effects of IL-2/αCD40 treatment on MDSC, we analyzed arginase expression within tumor lysates. IL-2/αCD40 reduced tumor-associated arginase expression in WT, but not GKO, mice (Fig. 5C). We also quantitated changes in arginase expression by sorting F4/80⁺ macrophages from the tumors of treated mice and found that those cells isolated from the tumors of IL-2/αCD40 treated mice had reduced arginase expression on a per cell basis (Fig. S8). These results indicate that the reduction in MDSC achieved by IL-2/αCD40 therapy also results in functional differences in monocyte populations within the tumor microenvironment.

Since IL-2/αCD40 mediated a reduction in tumor-associated MDSC, we next analyzed tumor lysates for the expression of CXCL5/ENA-78, a chemokine involved in the recruitment of these cells (19). IL-2/αCD40 treatment significantly reduced CXCL5 expression in tumors (Fig. 5D), whereas IL-2 or αCD40 had no effect. The IL-2/αCD40-mediated reduction of CXCL5 expression was lost in GKO mice (Fig. 5D). ELISA analysis of tumor lysates revealed that IL-2/αCD40 treatment reduced CXCL5 protein expression in tumors isolated from wild-type, but not GKO mice (Fig. 5E). Although the number of MDSC increased in the spleens of IL-2/αCD40-treated mice, we found no significant differences in CXCL5 expression by qPCR analysis of spleen cDNA (Fig. S6C). Taken together, these results demonstrate that IL-2/αCD40 therapy results in the IFNγ-dependent downregulation of CXCL5 expression that may mediate the recruitment of MDSC into the tumor microenvironment.

Discussion

Our results demonstrate clear differences in immune and anti-tumor responses achieved by IL-2/αCD40, as compared to those achieved by either IL-2 or αCD40 alone. Elevated Tregs and MDSC have been described recently in human RCC and clinical approaches aimed at removing these immunosuppressive cells will likely be an important component of improved immunotherapies (20). Although αCD40 alone can mediate significant leukocyte recruitment into primary tumors, it fails to remove Tregs and MDSC within the tumor microenvironment, which may allow for eventual tumor outgrowth. Consequently, αCD40 only marginally improves primary tumor burden and has no significant impact upon lung metastases or long-term survival of treated mice. The complete dependency of αCD40 upon CCR2, a receptor associated with the recruitment of potentially tumor-promoting leukocytes (8, 10), and the inability of αCD40 to remove tumor-infiltrating suppressor cells, was in dramatic contrast to the local immune responses achieved by IL-2/αCD40 in which the net result appears to be a conversion of the overall tumor microenvironment from a potentially suppressive milieu to a predominantly anti-tumor, Th1 environment.

Another important distinction derived from our study was the divergent cellular responses to IL-2/αCD40 therapy in the spleen, as compared to that of the tumor microenvironment. Consistent with our previous observations, IL-2/αCD40 mediated a significant increase in splenic Tregs (14). Our ongoing studies suggest that this increase may be due to expansion, rather than recruitment of Tregs to the spleen. Since peripheral increases in Tregs and MDSC did not correlate with reduced anti-tumor responses, we suggest that specific cellular changes within the tumor microenvironment are more predictive of successful immunotherapeutic outcomes. Consistently, Treg accumulation within other tumors correlated with poor survival (17). We further demonstrate a tumor-supporting role of Tregs in Renca-bearing mice. First, CD25⁺ Tregs isolated from tumors suppressed T cell proliferation even better than Tregs isolated from the periphery. The reduction in primary tumor area using IL-2 in combination with PC61 antibody was particularly interesting, since IL-2 alone consistently failed to have any appreciable effect on primary tumor size. However, anti-CD25 depletion by itself caused an increase in tumor burden, presumably due to the removal of CD25⁺ effector cells. Nevertheless, anti-tumor responses achieved by IL-2/αCD40 remained superior to either IL-2 or αCD40 in combination with anti-CD25, presumably due to the ability of IL-2/αCD40 to mediate other effects, such as the removal of MDSC, effector cell recruitment, and synergistic IFNγ induction that are critical components of IL-2/αCD40 antitumor therapy (11). We also show that IL-15/αCD40, another combination immunotherapy recently shown to induce anti-tumor responses (15), similarly decreased tumor-associated Tregs and MDSC. In this regard, IL-15 may have certain advantages over IL-2, since it did not increase tumor-associated Tregs by itself as IL-2 did. The expression profile of chemokines within tumors clearly plays an important role in the redistribution of suppressor cells and represents an attractive target for immunotherapeutic regimens that seek to alter the balance of the tumor microenvironment toward a beneficial host immune response.

We also demonstrate a central role for IFNγ in regulating the composition of tumor-infiltrating leukocytes after therapy. Previously, we showed that the anti-tumor effect of IL-2/αCD40 was dependent on CD8⁺ T cells and IFNγ (11). We, and others, have shown that the IFNγ-dependent recruitment of effector NK and CD8⁺ T cells is mediated by Th1 chemokines such as RANTES, Mig and IP-10 that have been associated with favorable anti-tumor responses in patients (5, 6, 21, 22). Second, and perhaps more interesting, was the requirement for IFNγ in the reduction in Tregs and MDSC within the tumor microenvironment. This selective reduction was closely associated with the downregulation of chemokines implicated in recruiting these regulatory cells to tumors (1, 16, 18, 19). It is interesting to note that αCD40 treatment increased CCL17 expression in GKO, but not WT mice. These findings are consistent with reports that CCL17 expression can be upregulated by cytokines such as TNFα (itself induced potently by CD40 ligation) and downregulated by IFNγ (23, 24), which has emerged as a master regulatory cytokine in our model. Although the regulation of these chemokines by IFNγ has been established (23–26), we extend this observation to demonstrate the feasibility of preferentially redistributing effector and regulatory cells specifically within the tumor microenvironment by combination immunotherapy that depends on IFNγ. We further demonstrate the IFNγ-dependent reduction in arginase expression within tumor-derived leukocytes. Functional arginase production by MDSC has been demonstrated in RCC patients and may result in impaired T cell signaling and tumor-induced tolerance (27). Arginase production by macrophages is also impaired by IFNγ and has been suggested to define a population alternatively activated by Th2 cytokines (28). Finally, tumor- infiltrating leukocytes isolated from IL-2/αCD40-treated mice can produce more IFNγ upon stimulation (11), illustrating the potential for a feedback loop within the tumor microenvironment, whereby the ongoing recruitment of beneficial effector cells and the coordinated reduction of immunosuppressive Tregs and MDSC may be rapidly amplified.

We also investigated the relative contribution of MCP-1 to the recruitment of leukocytes into tumors. MCP-1 expression is elevated in many cancers (29, 30) and has been associated with the recruitment of M2 polarized, or alternatively activated macrophages that may promote tumor progression (8, 30). Our study contrasts divergent roles for CCR2 interactions in different immunotherapeutic approaches for treating solid tumors. Although CD40-activated macrophages mediate potent anti-tumor effects in vitro (31), our data suggest that the efficacy of αCD40 in vivo might be limited by a dependency upon MCP-1, the potential for recruiting M2 macrophages and an inability to overcome suppressive pathways within the tumor microenvironment. In contrast, the IL-2/αCD40-mediated shift away from a dependency on MCP-1/CCR2 interactions is desirable, in that it also leads to the induction of multiple Th1 chemokines more closely associated with Th1 effector cell recruitment and favorable anti-tumor responses in RCC (5, 6).

Overall, we suggest that IL-2/αCD40 and other approaches that simultaneously increase effector responses and decrease counterregulatory responses within the tumor microenvironment may generate more effective anti-tumor responses. IL-2 has been used in patients with metastatic melanoma and RCC although durable responses have only been observed in a minority of patients (32). Similarly, objective responses were observed in only a minority of patients with advanced solid tumors after treatment with αCD40 (33). It is tempting to speculate that the limited efficacy of αCD40 is due to an inability to overcome local regulatory mechanisms within the tumor microenvironment that ultimately allow the tumor to escape immune control. Nevertheless, CD40 is an attractive therapeutic target because, in addition to its ability to stimulate immune responses, its ligation by CD40⁺ tumors induces cytokine production (12) and enhances Fas-mediated tumor apoptosis (34). While ongoing trials involving agonistic CD40 antibodies continue to provide important insights into the cellular responses to developing tumors in vivo, we believe that our results support the development of IL-2/αCD40 or IL-15/αCD40 combination therapy for the improved treatment of solid tumors, including RCC.

Materials and Methods

Mice.

BALB/c wild-type mice were obtained from the Animal Production Area of the National Cancer Institute (NCI) Frederick Cancer Research and Development Center. BALB/c CCR2^−/− mice were provided by Dr. Cara Mack (University of Colorado Health Sciences Center) (13). BALB/c IFNγ^−/− (GKO) mice were obtained from Jackson Laboratories. Mutant alleles were confirmed by PCR genotyping. Mice (8–10 weeks of age) were used in accordance with an approved NCI Frederick Institutional Animal Care and Use protocol.

Cells and Reagents.

The renal adenocarcinoma of BALB/c origin (Renca) was passaged i.p. as described (35). Recombinant human IL-2 was obtained from the NCI. Recombinant human IL-15 was from Peprotech, Inc. Agonist rat anti-mouse CD40 (clone FGK115B3) was purified from ascites, as described (12). Endotoxin was <1 EU/mg antibody, as determined by chromogenic Limulus Amebocyte Lysate kit (Cambrex). Purified rat IgG was purchased from Jackson ImmunoResearch Laboratories. Antibody against IL-2Rα (anti-CD25; clone PC61) used for depletion was purified from ascites.

In Vivo Tumor Model.

Renca cells (1 × 10⁵) were injected under the kidney capsule of mice on day 0. Mice treated with IL-2 received 300,000 IU i.p. twice a day on days 11, 15, 18, and 21 post tumor injection. Mice treated with anti-CD40 received 100 μg i.p. on days 11–15 and 18–21 post tumor injection. Mice treated with IL-15 received 1 μg i.p. on days 11–15 and 18–21 post tumor injection. In some experiments, mice received 200 μL of PC61 (400 μg/mL) antibody or saline control i.p. on days 11, 15, and 18. On day 22, mice were euthanized and primary tumors were collected. Tumor length and width was measured using calipers. Lungs were fixed in Bouin's solution and lung metastases were counted under a dissecting microscope. In some studies, mice received a unilateral nephrectomy of the tumor-bearing kidney on day 11, followed by treatment with IL-2 and/or αCD40 and were monitored for tumor progression. For tumor rechallenge experiments, long-term survivors or control naïve mice were injected with 7.5 × 10⁴ RENCA s.c. and tumors were measured.

Isolation of Leukocytes from Spleen.

Spleens were harvested on day 22, placed in HBSS and filtered through a two-chamber sterile Filtra-Bag (Fisher Scientific). Spleens were gently pressed and the resulting single cell suspension was collected from the other side of the bag. Splenocytes were counted using a Sysmex KX-21 (Roche Diagnostics).

Isolation of Tumor-Infiltrating Leukocytes.

Tumors were dissected on day 22, filtered through a two-chamber sterile Filtra-Bag (Fisher Scientific) and digested in RPMI containing 5% FCS, 250 U/mL type IV collagenase (Invitrogen), 100 μg/mL DNase I (Roche Molecular Biochemicals) and 1 mM EDTA (pH 8.0), at 37 °C for 45 min. Then, the homogenate was processed in a tissue stomacher-80 (Seward) for 30 s, washed with HBSS (BioWhittaker), and resuspended in 40% Percoll (Amersham Pharmacia) in DMEM (BioWhittaker). The suspension was underlaid with 80% Percoll and centrifuged for 25 min at 1,000 × g. Leukocytes were collected from the interphase, washed, and counted.

Flow Cytometric Analysis.

Cells were analyzed by flow cytometry as detailed in SI Methods.

Treg Suppression Assay.

Treg suppression of T-cell proliferation was analyzed as detailed in SI Methods.

Arginase Assay.

Leukocytes were isolated from tumors, counted and lysed in 10 mM Tris-HCl (pH 7.4) containing 0.4% Triton X-100 and Halt protease inhibitor (Thermo Scientific). Lysates were spun briefly and arginase activity contained within the supernatants quantified using a commercially available kit (BioAssay Systems). In some experiments, CD45⁺, F4/80⁺ tumor-associated leukocytes were sorted and then lysed for arginase activity, as above.

ELISA.

Tumors were homogenized in RIPA lysis buffer (Thermo Scientific). Chemokine protein expression was determined using a commercially available ELISA kit following the manufacturer's instructions (R&D Systems). The amount of each chemokines was normalized against total protein, as determined using a BCA protein assay kit (Thermo Scientific).

Quantitative PCR (qPCR).

Tumor and spleen samples were analyzed by qPCR as detailed in SI Methods.

Immunohistochemistry.

Tumors were analyzed by immunohistochemistry as detailed in SI Methods.

Statistical Analysis.

Statistical differences were analyzed using a Mann-Whitney U test (GraphPad Prism, GraphPad Software, Inc.). Significance was indicated by P < 0.05 values.