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International Immunology Advance Access originally published online on September 29, 2008
International Immunology 2008 20(11):1467-1479; doi:10.1093/intimm/dxn104
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© The Japanese Society for Immunology. 2008. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Deliberately provoking local inflammation drives tumors to become their own protective vaccine site

Connie Jackaman1, Andrew M. Lew2, Yifan Zhan2, Jane E. Allan1, Biljana Koloska3, Peter T. Graham3, Bruce W. S. Robinson1 and Delia J. Nelson1,3

1 School of Biomedical Sciences, Curtin University, Kent St Bentley, Perth, Western Australia 6102, Australia
2 School of Medicine and Pharmacology, University of Western Australia, Perth, Western Australia 6009, Australia
3 Walter and Eliza Hall Institute, Melbourne, Victoria 3050, Australia

Correspondence to: D. Nelson; E-mail: delianelson{at}curtin.edu.au


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Anti-cancer immunotherapies aim to generate resolution of all existing tumors, including inaccessible ones, and provide long-term protection against recurrence. This is rarely achieved. Thus, we aimed to determine if the tumor microenvironment could be turned into a potent ‘self’-vaccine site. Our target was to eradicate larger tumor burdens. Our models respond to single-agent immunotherapies; however, they fail at a precisely defined ‘cut-off’ tumor burden. Thus, this system was used to define the immune mechanisms required to mediate regression of larger tumors that are resistant to mono-immunotherapies. We report that direct injection of IL-2 with agonist anti-CD40 antibody into the tumor bed resulted in permanent resolution of treated and untreated distal tumors. Tumor-infiltrating CD8+ T cells and neutrophils collaborated to eradicate treated tumors, IFN{gamma} was not critical and protective memory was preserved. This approach relied only on tumor antigens expressed within the tumor microenvironment. It also avoided systemic toxicities, did not require chemotherapy or surgery and is clinically useful because only one tumor site has to be accessible for treatment. We conclude that provoking intra-tumoral inflammation skews the tumor microenvironment from tumorigenic to immunogenic, resulting in the resolution of treated and untreated distal tumors, as well long-term protective memory.

Keywords: agonist anti-CD40 antibody, anti-tumor immunity, IL-2, T cells, neutrophils


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Intravenous administration of cytokines (1–5) or agonist anti-CD40 antibody (6) can enhance pre-existing anti-tumor immunity. However, as monotherapies, they are rarely curative unless tumor burden is sufficiently small (7, 8). This may be because they do not adequately access the larger tumor microenvironment. Furthermore, their toxicities may be problematic (9–11). Combining systemic anti-CD40 antibody with surgery (12), chemotherapy (13) or cytokines, such as IL-2 (14) improves anti-tumor activity, but can worsen toxicity and may destroy immunological memory (15). None of these approaches targets the tumor such that it becomes it own vaccine site, i.e. its own source of tumor antigens, and as a result, do not simultaneously achieve the systemic release of CD8+ T cells that eradicate distal tumors, as well the long-term protection expected from a vaccine.

An alternative strategy is targeting these agents directly into the tumor microenvironment by intra-tumoral (i.t.) injection, catheterization or other approaches (16, 17). Our clinical studies in malignant mesothelioma (MM) patients found that this approach avoided toxic side effects and provoked local immune responses that, in some cases, were associated with objective responses (18, 19). These data suggest that the tumor can be its own source of tumor antigens. We have used our pre-clinical models of mesothelioma to extend these observations and have shown that i.t. IL-2 generated T cell-mediated curative regression of small tumors without the deleterious side effects seen with systemic IL-2 administration (20). However, as for humans, i.t. IL-2 failed when challenged with larger tumor burdens (21). Similar studies have demonstrated that i.t. anti-CD40 antibody retains its anti-tumor activity without serious side effects (10).

Importantly, our murine studies revealed a clearly defined ‘cut-off’ tumor size when 100% of mice become resistant to i.t. IL-2 (20) or i.t. anti-CD40 antibody (shown herein). The precise delineation of size-related resistance to the two monotherapies provides an ideal model to study immune mechanisms required to mediate regression of larger, monotherapy-resistant tumors. Based on our previous studies, we chose to combine IL-2 with anti-CD40 antibody. Both agents are involved in immune activation, target numerous cell types and used together may enhance the anti-tumor immune response. Their combined systemic use generates potent anti-tumor immunity (14, 22); however, it also induces IFN{gamma}-dependent apoptosis of CD4+ memory T cells (15).

In this study, we targeted anti-CD40 antibody and/or IL-2 directly into tumors and compared the consequent anti-tumor activity in small, monotherapy-susceptible versus larger, monotherapy-resistant tumors. We not only focussed on MM but also looked at other cancer models. MM is generally resistant to conventional therapies and desperately requires a more effective alternative treatment strategy. Importantly, MM is accessible for i.t. therapy. Furthermore, as MM patients rarely die from metastases treating the primary tumor may result in significant palliative and survival benefit. The MM model was established in our laboratory by inoculating mice with the relevant human carcinogen, i.e. asbestos fibers (20). Re-inoculation of cloned tumor cells into syngeneic mice results in progressing tumors confirmed by histopathology to be representative of human MM. These features make this model highly relevant to the human disease which remains rare in pre-clinical models of solid tumors.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 Funding
 References
 
Mice
Female C57BL/6J (H-2b), BALB/c (H-2d) and nude (BALB/c—Foxn1nu/nu) aged 6–8 weeks were obtained from the Animal Resources Center (Perth, Australia). GK mice are transgenic for the depleting anti-CD4 Ab, GK1.5, and so have no peripheral CD4 T cells; GK and GK/perforin–/– mice (23) were bred and housed at the Walter and Eliza Hall Institute (WEHI) where the relevant experiments were performed. Animals were maintained under SPF conditions in QEII Medical Centre and WEHI animal holding areas. This project was approved by the Animal Experimentation Ethics Committees of the University of Western Australia and WEHI with the condition that tumors did not grow larger than 100 mm2. Mice were injected subcutaneously (s.c.) with varying numbers of tumor cells in 100 µl PBS and tumor growth monitored using microcallipers.

Cell lines
AE17 is a MM cell line derived from the peritoneal cavity of C57BL/6J mice injected with asbestos fibers and has been previously described (20). AB1 is another MM cell line on the H-2d BALB/c background (24). The Lewis lung carcinoma cell line (LL) developed in C57BL/6J mice was obtained from the American Tissue Culture Collection (Manassas, VA, USA). Tumor and hybridoma cell lines were maintained in RPMI 1640 (GIBCO BRL, NY, USA) supplemented with 10 or 5% FCS (CSL Ltd, Parkville, Victoria, Australia), 48 mg l–1 gentamicin (Pharmacia and Upjohn, Western Australia, Australia), 60 mg l–1 benzylpenicillin (CSL Ltd) and 0.05 mM 2-mercaptoethanol (Merck, BDH).

Depletion using mAbs
For depletion of CD4+ or CD8+ cells, two doses (150 µg per dose) of either YTS-191 or YTS-169 (hybridomas obtained from the European Collection of Animal Cell Cultures, Salisbury, UK), respectively, were injected intra-peritoneal (i.p.) 1 day before treatment and three doses per week (100–150 µg per dose) continued until the end of treatment. Mice were sacrificed and spleens tested (by FACS analysis); CD8+ depletions were 95–99% effective, while CD4+ depletions were 90–95% effective (data not shown).

Anti-asialo GM1 (Wako Chemicals, Neuss, Germany) was used according to the manufacturer's instructions to deplete NK cells. Animals were given two doses i.p. before treatment (20 µl per dose) and then a dose every 4 to 5 days, continued throughout treatment. Depletion of NK cells was checked at the start and end of the experiment by double staining for NK1.1 and {alpha}β TCR and was 80–90% effective.

For depletion of granulocytes the 1A8 mAb (isotypeIgG2a; kindly donated by T. Malek) was used. 1A8 is specific for Ly-6G+ granulocytes, does not bind to Ly-6C and is not expressed on activated T or B cells (25). The 1A8 depleting protocol involved two doses (50 µg per dose) injected i.p. 2 days before treatment and continued every second day; this protocol was determined to 90% effective.

IFN{gamma} was blocked using two doses of 250 µg per dose of the XMG1.2 clone (PharMingen) injected i.p. before treatment and continued (three doses per week) until the end of the treatment.

In vivo treatments—IL-2 and activating anti-CD40 antibody
Lyophilized Proleukin (recombinant IL-2; Cetus Corporation, Emeryville, CA, USA) was reconstituted in sterile PBS (Sigma). Purified and LPS-free agonist anti-CD40 antibody, FGK45 (0.9–1.4 mg ml–1) was obtained from the WEHI Monoclonal Antibody Facility and diluted in PBS for injection into mice.

FACS analysis
Samples were prepared as a single-cell suspension and stained for FACS analysis using antibodies obtained from PharMingen (anti-IL-2R{alpha} or CD25, clone 3C7; anti-GR-1, clone RB6.8C5; B220, clone RA3-6B2; anti-CD11c, clone HL3; anti-CD4, clone RM4-5 and anti-CD8, clone 53-6.7), from eBiosciences (anti-CD40, clone 1C10) and from Caltag (anti-F4/80). Analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA, USA) using Cell Quest software.

Histochemistry
Hematoxylin and eosin staining and immunohistochemistry were performed as described previously (20). For immunohistochemistry, primary antibodies directed against murine CD4 and CD8 T cells (clones RM4-5 and 53-6.7, respectively; PharMingen), macrophages (F4/80), CD11c dendritic cells (DCs) (N418; both antibodies supplied by A. McWilliam, Curtin University, Perth, Australia), B cells (B220) and granulocytes (GR-1; clone RB6.8C5) were used with the appropriate isotype controls.

Statistical analysis
Statistical significance was calculated using GraphPad PRISM. Student's t-test and Mann–Whitney U-test were used to determine differences between two populations. One-way analysis of variance was used to determine differences between more than two populations.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 Funding
 References
 
Systemic IL-2/anti-CD40 combination only generates weak anti-MM immunity
The AE17 MM tumor cell line does not express either IL-2R{alpha} (CD25) or anti-CD40 (Fig. 1A). It is also not directly killed by IL-2 and/or anti-CD40 antibody in vitro (data not shown). Therefore, IL-2 and anti-CD40 antibody treatment cannot target tumor cells directly.


Figure 1
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  		Fig. 1. Systemic IL-2 or anti-CD40 antibody offer minimal survival benefits to CD40– MM tumors. A total of 100 µl of PBS, IL-2 (20 µg per dose) or anti-CD40 antibody (40 µg per dose) on their own or in combination were injected via the tail vein every third day for 2 weeks into tumor-bearing mice. Tumor growth showing individual mice (A) and survival were monitored (B). Two of four mice of mice died due to toxicity from the combination regimen. Data are from one representative of two experiments with four mice group.

 
The next series of experiments addressed the effect systemic administration of different doses of IL-2 and anti-CD40 antibody alone, or in combination, has on MM tumor growth. To do this, AE17 tumors ranging from 25 to 50 mm2 were given three i.p. injections per week for 2 weeks with PBS (the diluent control), IL-2 or anti-CD40 antibody as monotherapies, as well the IL-2/anti-CD40 combination regimen. High (2000 µg) doses of IL-2 caused visible distress such as ruffled fur, weight loss, inappetance and reduced mobility resulting in a number of animals having to be culled (data not shown), while a lower dose (20 µg) was well tolerated and offered slight anti-tumor activity (Fig. 1A and B). Similarly, high (100 µg) doses of anti-CD40 antibody were associated with significant toxicity leading to 50% fatality [(10) and data not shown]. A lower dose (40 µg) generated significant survival benefits with 33% of mice demonstrating complete tumor resolution without obvious side effects (Fig. 1A and B). Combining the lower doses of IL-2 (20 µg) with anti-CD40 antibody (40 µg) resulted in a high death rate with 50% of deaths related to toxicity, and unexpectedly, no tumors resolved (Fig. 1A and B).

I.t. IL-2 and anti-CD40 antibody alone induce regression of small MM tumors
We then used the i.t. route for the next series of experiments. The i.t. regimen consisted of three doses per week for 2 weeks of i.t. PBS, anti-CD40 antibody (40 µg per dose), the IgG2a isotype control (40 µg per dose), IL-2 (20 µg per dose) or combined IL-2/anti-CD40 (20 µg per dose IL-2 and 40 µg per dose anti-CD40 antibody) in 100 µl.

Based on our previous studies, we first focussed on the responses of small tumors that are highly susceptible to i.t. IL-2 [i.e. tumor must be <25 mm2 (20)]. Thus, s.c. injected AE17 tumor cells were allowed to develop into small, ‘early-stage tumors’. These tumors weighed 0.15–0.3 g (representing ~1% of total body) and were well enough established to contain their own blood supply (data not shown).

The IL-2 or anti-CD40 monotherapies and the IL-2/anti-CD40 combination demonstrated significant anti-tumor activity against a small tumor burden. Intra-tumoral anti-CD40 inhibited tumor growth (Fig. 2A) resulting in a significant survival advantage (Fig. 2B) with 69% of animals exhibiting complete tumor regression for >1 year. As previously reported (20), i.t. IL-2 alone was effective against small tumors, leading to complete tumor regression in 83% of mice with retarded tumor growth and prolonged survival seen in the remaining mice (Fig. 2A and B).


Figure 2
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  		Fig. 2. Small established AE17 tumors respond well to i.t. immunotherapeutic monotherapies. C57BL/6J mice inoculated with 5 x 105 AE17 tumor cells were left to develop into small tumors (range 4–24 mm2) and the i.t. regimens commenced (arrow). Tumor growth (A) and survival (B) were monitored. Data are pooled from three experiments with 15 mice per group; total 75 mice. The role of T cells in these regimens was addressed using nude mice (10 per group; C) and by depleting C57BL/6J mice bearing small tumors of CD8+ cells (D), CD4+ cells (E) or both (F) using mAbs throughout the treatment period. Data are pooled from three experiments for CD4+ depletion or CD8+ depletion (15 mice per group; total 180 mice) and two experiments for CD4+/CD8+ depletion (10 mice per group; total 80 mice). Data shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = not significant, comparing treatment groups to PBS-treated controls.

 
A significant survival advantage was seen in mice treated with combined IL-2/anti-CD40 as complete tumor regression was seen in 100% of mice with small AE17 tumors. When treatment first commenced tumor growth continued, and in some cases grew faster, before regression. Note that pooled data from three representative experiments is shown. However, these experiments have been repeated at least 30 times, involving >300 mice; all experiments produced >80% response rate (complete resolution of tumor). All cured mice remained tumor free for the duration of their natural lives (i.e. >18 months). Importantly, i.t. treatment with IL-2 and/or anti-CD40 did not lead to any obvious side effects.

I.t. IL-2 and anti-CD40 antibody as monotherapies or in combination use different effector mechanisms to mediate regression of small tumors
Initially, the role of T cells in these regimens was explored in nude mice that lack mature T cells. Once established, untreated AE17 tumors in nude mice grew at a faster rate (Fig. 2C) than in C57BL/6 mice. Nude mice bearing small tumors did not respond to treatment with the IL-2. Anti-CD40 antibody-driven responses were impaired but not completely abrogated (Fig. 2C). However, the combination of IL-2/anti-CD40 maintained significant anti-tumor immunity compared with PBS-treated controls, although most tumors eventually progressed suggesting a long-term role for T cells, as the tumors progressed sometime after treatment cessation.

The lack of mature T cells in nude mice does not address the role of endogenous tumor-specific T cells generated during normal tumor progression and prior to treatment. Thus, we also used depleting antibodies to remove CD4+ and/or CD8+ T cells during the therapeutic window. This approach ensures that the normal endogenous anti-tumor T cell response (26) is not interfered with until treatment and addresses the role of CD4+ and/or CD8+ T cells during therapy. In these experiments, PBS-treated control tumors from both the non-depleted and depleted groups grew at similar rates (Fig. 2A and D–F).

When CD8+ T cells were depleted, IL-2 treatment was rendered completely ineffective (Fig. 2D). Surprisingly, the absence of CD8+ T cells during therapy only impaired rather than prevented the ability of anti-CD40 to exert anti-tumor activity (Fig. 2D) and long-term survival was slightly reduced (data not shown). Note that CD8+ depletion was >95% effective. The IL-2/anti-CD40 combination therapy remained effective despite removal of CD8+ T cells; most of these CD8+-depleted mice subsequently died over 1 year later of non-tumor-related causes.

Depletion of CD4+ cells during treatment rendered i.t. IL-2 completely ineffective (Fig. 2E). Loss of CD4+ cells diminished, but did not ablate, anti-CD40-driven tumor regression and the majority of anti-CD40-treated tumors eventually completely resolved. The combination therapy maintained full anti-tumor activity when CD4+ cells were depleted during the treatment period. Similar results were seen using anti-CD4 Ab transgenic mice (GK mice) in which the anti-CD4 Ab is produced mainly from the pancreata thereby preventing the presence of peripheral CD4+ T cells for the duration of their natural lives (data not shown).

The removal of both CD4+ and CD8+ cells together during therapy completely ablated IL-2 anti-tumor activity confirming that both CD4+ and CD8+ T cells are required for i.t. IL-2 to be effective against small tumors (Fig. 2F). Animals depleted of both CD4+ and CD8+ cells demonstrated a diminished ability to retard tumor growth during anti-CD40 treatment; nonetheless, there was significant (relative to PBS controls) anti-tumor activity. These data suggest that i.t. anti-CD40-induced regression of small tumors involves CD8+ T cells as well as other unidentified mechanisms. The IL-2/anti-CD40 combination therapy remained effective in doubly CD4+- and CD8+-depleted mice bearing small tumors; i.e. tumors regressed (Fig. 2F) and >75% of mice survived beyond 60 days (data not shown). These data clearly show that T cells are not required for eradicating small tumors using IL-2/anti-CD40 and suggest that other effector mechanisms may be involved. However, the return of T cells was associated with the resolution of the few tumors that emerged after treatment cessation, once again implying a long-term role for T cells. Thus, while T cells are not initially required for tumor eradication, our data suggest that T cells are required for long-term eradication.

Only the IL-2/anti-CD40 combination induces regression of large tumors and generates tumor-specific protective memory
The next series of experiments used the same therapies in larger tumors; i.e. treatment commenced when tumors were >25 mm2. These tumors were monotherapy resistant as neither i.t. IL-2 nor anti-CD40 alone altered tumor growth, and no animals were cured (Fig. 3A and B). In contrast, a significant survival advantage was seen in mice treated with combined IL-2/anti-CD40 as complete tumor regression was seen in 87% of mice with large AE17 tumors. These mice lived well past 1 year when at various time points they were re-challenged with AE17 cells. No mice developed tumors and they subsequently started to die of age and other non-tumor-related factors ≥18 months later (Fig. 3B).


Figure 3
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  		Fig. 3. Only the IL-2/anti-CD40 combination induces regression of large MM tumors. Tumor growth (A) and survival (B) were monitored in four experiments (20 mice per group) with tumor sizes ranging from 25–45 mm2 before treatment began with PBS, IL-2 or anti-CD40 or both. Mice that experienced complete tumor resolution and remained tumor free for longer than a year were re-challenged with 5 x 105 AE17 tumor cells. The role of T cells during the combination therapy was addressed in nude mice (10 per group; C) and C57BL/6 mice bearing large tumors depleted of CD8+ cells (D), CD4+ cells (E) or both (F). Data are pooled from three experiments for CD4+ depletion or CD8+ depletion (15 mice per group) and two experiments for CD4+/CD8+ depletion (10 mice per group). All data shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = not significant, comparing treatment groups to PBS-treated controls.

 
The role of CD4+ and CD8+ T cells was also examined in large tumor-bearing mice treated with the IL-2/anti-CD40 combination therapy. At first, anti-CD4 Ab transgenic GK mice with perforin deficiency were used. The efficacy of the combination regimen in GK/perforin-deficient mice with large tumors was lost in 50% of mice and partially ablated in the remainder (data not shown), although all mice eventually succumbed to tumors; i.e. no mice were cured. Thus, perforin-dependent mechanisms appear critical to eradicate large tumors upon IL-2/anti-CD40 treatment. Nonetheless, the 50% of mice that transiently maintained anti-tumor activity suggest that another mechanism is also operating.

Depleting antibodies to remove CD4+ and/or CD8+ T cells during the therapeutic window were also used. Treatment of large tumors with IL-2/anti-CD40 remained effective in CD4+-depleted animals (Fig. 3C) with the majority of mice completely cured. In contrast, IL-2/anti-CD40 treatment into large tumors in 70% of animals depleted of CD8+ or CD8+ and CD4+ T cells no longer restrained tumor growth (Fig. 3D and E). Although 30% of these mice demonstrated transient responses, all mice eventually succumbed to tumor burden (data not shown). Taken together, the data from Figs 2 and 3 show that CD8+ cells play a critical role in eradicating large, but not small, tumors during IL-2/anti-CD40 treatment.

Only the IL-2/anti-CD40 combination provokes an inflammatory response within the tumor microenvironment
Tumors collected after three doses showed a minimal cellular infiltrate after PBS treatment (Supplementary figure 1 is available at International Immunology Online). The only cell type that proportionally increased in anti-CD40-treated mice was B cells: FACS analysis (data not shown) demonstrated that B cells increased from 14 ± 3% in PBS-treated mice to 30 ± 5% in anti-CD40-treated mice. IL-2 recruited primarily CD8+ T cells and no other cell types (20); changing from 3.7 ± 0.4 to 8.2 ± 2% in PBS versus IL-2-treated mice, respectively, measured by FACS (data not shown). Only mice treated with the combination demonstrated an inflammatory infiltrate consisting primarily of neutrophils (increasing from 5.2 ± 1.3 in PBS mice to 53 ± 5% in IL-2/anti-CD40 mice) and CD8+ cells (increasing from 3.7 ± 0.4 to 8.2 ± 2.4%; data not shown).

NK cell are not critical effectors in IL-2/anti-CD40 antibody anti-tumor immunity
The role of NK cells during IL-2 with or without anti-CD40 was assessed using a depleting antibody. The loss of NK cells reduced the IL-2-mediated survival rate from 80 to 40% suggesting that NK cells do play a role in IL-2 induced immunity (Fig. 4A–D). The IL-2/anti-CD40 combination treatment into small tumors retained significant anti-tumor efficacy in the absence of NK cells. The cure rate of IL-2/anti-CD40 combination into large tumors decreased from 90 to 65% when NK cells were removed, but this was not statistically significant, and both groups maintained statistical difference to their PBS-treated counterparts. Similar results were seen using NK-deficient Beige (bg/bg) mice (data not shown). Thus, while there is a hint that NK cells may play a role in IL-2/anti-CD40-mediated immunity, their role appears to be a minor one.


Figure 4
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  		Fig. 4. IL-2/anti-CD40-driven tumor resolution is independent of NK cells and INF{gamma}. Mice bearing small or large tumors were depleted of NK cells throughout IL-2 ± anti-CD40 treatment and tumor growth (C) and survival (D) rates measured for comparison to no depletion controls (A and B). Data are pooled from two experiments (6–10 mice per group). Similarly, IFN{gamma} was neutralized throughout treatment with IL-2 ± anti-CD40 with tumor growth (E) and survival monitored (F): data from one experiment with four mice per group given anti-IFN{gamma} mAb and no anti-IFN{gamma} controls consisting of 10 mice per group. Arrow indicates time treatment commenced. Data represented as mean ± SEM. **P < 0.01, ***P < 0.001 comparing treated groups to PBS-treated controls.

 
IFN{gamma} is not essential for i.t. IL-2/anti-CD40-mediated anti-tumor activity
A recent report demonstrated that systemic administration of IL-2/anti-CD40 provoked an IFN{gamma} response that was crucial for anti-tumor activity, but this IFN{gamma} was also responsible for the apoptosis of memory CD4+ T cells (15). While delivery of the same agents directly into tumors preserves tumor-specific memory (Fig. 3B), we were still interested in the role of IFN{gamma} during therapy. Thus, IFN{gamma} was neutralized with an IFN{gamma}-specific mAb during therapy. Once again, this was performed to avoid compromising endogenous effector T cells generated during normal tumor progression prior to treatment (26). Only IL-2 on its own into small tumors and IL-2/anti-CD40 into small versus large tumors were examined. The loss of IFN{gamma} during therapy interfered with the ability of IL-2 to restrain small tumors (Fig. 4E and F) with all mice eventually demonstrating tumor progression (Fig. 4F). In contrast, regardless of tumor size when treatment began, the combination regimen maintained its potent anti-tumor activity with >80% of mice permanently cured.

Simultaneous recruitment of polymorphonuclear neutrophils and T cells is a key event for IL-2/anti-CD40-driven eradication of large tumors
The data above suggest that there may be number of effector mechanisms that play an important anti-tumor role in response to the anti-CD40 monotherapy and to the IL-2/anti-CD40 combination in small tumors. Thus, there must be another mechanism involved in the combination therapy that is capable of, at least transiently, restraining tumor growth. C57BL/6 mice (Fig. 5A) and nude mice (Fig. 5D) responding to the combination therapy developed necrosis and had a significant polymorphonuclear neutrophil (PMN) infiltrate (Fig. 5C and E) suggesting that IL-2/anti-CD40-driven PMN may play a role in restraining small tumors. To test the role of PMN, AE17 tumor-bearing mice were depleted of PMN using the 1A8 mAb, which detects Ly-6G and specifically targets PMN, during treatment with IL-2/anti-CD40. Complete abrogation of anti-tumor immunity was seen in the absence of PMN (Fig. 5F). The return of PMN coincided with renewed anti-tumor activity and the development of tumor necrosis. However, only partial control of tumor growth was achieved, as after transient regression, tumor growth appeared static. Forty percent of these tumors eventually progressed, while the remaining 60% of tumors subsequently resolved (Fig. 5G). These data suggest that PMNs play a critical role in eradicating large tumors responding to the combination IL-2/anti-CD40 treatment. Note that the data in Figs 2 and 3 show that CD8+ T cells were also critically required to eradicate IL-2/anti-CD40-treated large, but not small, tumors. Taken together, these results imply that CD8+ T cells and PMNs need to co-exist for optimal tumor destruction.


Figure 5
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  		Fig. 5. PMNs are a critical effector cell type that mediates IL-2/anti-CD40 driven eradication of small and large tumors. C57BL/6 mice (A) and nude mice (D) treated with IL-2/anti-CD40 develop tumor necrosis. Representative photographs of ex vivo AE17 tumors during treatment with PBS (B and D) or IL-2/anti-CD40 (C and E) collected from C57BL/6 (B and C) or nude mice (D and E) and stained with hematoxylin and eosin (magnification x630). Mice treated with PBS or the IL-2/anti-CD40 combination were depleted of PMN using the 1A8 mAb (anti-Ly-6G) throughout treatment. Tumor growth (F) and survival (G) were monitored. Tumor size at commencement of treatment was 28 ± 10 mm2. Data are from one representative experiment of two experiments (five mice per group; total 40 mice).

 
IL-2/anti-CD40 eradicates large, fast-growing, untreated distal site tumors
To determine the effect of these treatments on an untreated, distal tumor (representing metastatic tumors), C57BL/6J mice were s.c. inoculated with tumor cells into the left and right flanks and treatment given into one tumor. Tumor cell inocula were adjusted to compare the effectiveness of these treatments on fast- versus slow-growing distal tumors. Treated tumors were also divided into small and large tumors. Small treated tumors completely regressed in response to the monotherapies as well as the combination (Fig. 6A and C). Eighty percent of large treated tumors responded to the combination IL-2/anti-CD40 regimen (Fig. 6A and C).


Figure 6
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  		Fig. 6. The IL-2/anti-CD40 combination is effective against untreated distal tumors. C57BL/6J mice were injected s.c. with 5 x 105 AE17 tumor cells into the left flank and either 1 x 105 AE17 cells (slow-growing distal tumors; C and D) or 5 x 105 AE17 cells (fast-growing distal tumors; A and B) into the right flank on day 0. The i.t. treatments were delivered into one tumor indicated by arrows (treated tumor; A, C and E) while the second (distal) tumor was left untreated (B, D and F). In a further experiment, mice were given 5 x 105 cells at both sites on day 0 and left to develop into large tumors before treatment began into one tumor (E and F). Data are from one experiment (five or six mice per group; total 68 mice) as mean ± SEM. Treated mice were compared with PBS-treated control mice; *P < 0.05, **P < 0.01, ***P < 0.001.

 
IL-2 had no impact on untreated, distal site tumors. In contrast, the anti-CD40 monotherapy induced regression of untreated distal tumors in 100 and 60% of animals with slow-growing and fast-growing distal tumors, respectively (Fig. 6B and D). However, the most effective treatment was the IL-2/anti-CD40 combination as fast- and slow-growing distal tumors were eradicated in 100% animals that had small treated tumors and in 80% of animals bearing large treated tumors (Fig. 6B and D). Note that all PBS-treated tumors progressed and that only small tumor responses to PBS are shown in Fig. 6(A–D).

As we have shown that large tumors are more resistant to treatment, we also assessed the responses of untreated, large, distal tumors in mice bearing large AE17 tumors treated with IL-2/anti-CD40 (Fig. 6E and F). Eighty-three percent of distal (untreated) large tumors exhibited complete tumor regression (Fig. 6F).

I.t. IL-2/anti-CD40 is effective in other tumor models
To ensure that the anti-tumor response was not restricted to a single genetic (C57BL/6J) background or to one tumor type (i.e. MM), BALB/c mice were inoculated s.c. with AB1 MM tumor cells, while C57BL/6J mice were inoculated s.c. with LL lung carcinoma tumor cells. The IL-2 and anti-CD40 monotherapies were only tested on animals bearing small AB1 and LL tumors, while the IL-2/anti-CD40 combination was tested on small and large tumors.

I.t. IL-2 treatment had a minimal effect on the growth of small AB1 MM tumors (Fig. 7A and C). I.t. anti-CD40 inhibited the growth of small AB1 tumors with 66% of animals exhibiting complete tumor regression. The IL-2/anti-CD40 combination eradicated small and large AB1 tumors in 83% of animals (Fig. 7A and C) and induced tumor necrosis (data not shown). All animals that achieved complete tumor eradication remained tumor free for >1 year.


Figure 7
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  		Fig. 7. The i.t. IL-2/anti-CD40 antibody combination is effective in other tumor models. BALB/c mice were injected s.c. with 106 AB1 tumor cells (A and C) and C57BL/6J mice were s.c. injected with 5 x 105 LL tumor cells (B, D and E). Mice bearing small AB1 and LL tumors were given the same i.t regimen of PBS, IL-2 or anti-CD40 antibody alone, while those bearing large tumors were only given combined IL-2/anti-CD40. Survival (A and B) and tumor growth rates (C, D and E) were measured and shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. For AB1 tumors, one experiment with six mice per group (total 36 mice) and for LL tumors, three experiments with 10 mice per group (total 180 mice). In a separate experiment (five mice per group; total 15 mice), LL small or large tumor-bearing mice were given an extended period of therapy covering 27 days (E).

 
I.t. IL-2 alone or anti-CD40 alone into small LL tumors did not prevent tumor growth (Fig. 7B and D) and exhibited no survival advantage. The IL-2/anti-CD40 combination was effective against small and large LL tumors, with 70% of animals in each group exhibiting tumor regression. Twenty-nine percent of animals were permanently cured; however, cessation of treatment resulted in tumor recurrence in the remaining mice. Extending the treatment period by a further four injections increased the number of permanent cures to 40% (Fig. 7E).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 Funding
 References
 
Here, we have taken advantage of our previous studies in which we identified a tumor size-restricted response to specific single-agent immunotherapies; i.e. small tumors which are susceptible to IL-2 or anti-CD40 versus larger tumors that are 100% resistant to the same agents. The latter provide a model with which we can study the immune mechanisms required to mediate regression of larger tumors that are already resistant to mono-immunotherapies. We show that T cells require assistance before they can destroy a larger tumor burden; this assistance comes in the form of large numbers of tumor-infiltrating PMN. Furthermore, we show these PMNs can eradicate a small tumor on their own, in the absence of T cells.

The PMN/T cell response was only achieved when we targeted, via injection, a combination of the cytokine IL-2 and an agonistic anti-CD40 antibody directly into the tumor bed. This provoked a potent local anti-tumor inflammatory response that was curative not only for the injected tumor but also for untreated distal tumors; i.e. only one tumor has to be treated for a profound systemic effect. While this therapy was highly effective in genetically different MM models, it also mediated regression of a lung carcinoma. Importantly, this local delivery approach overcame the ablation of immune memory seen when the same reagents are used systemically (15). Permanently cured mice subsequently died (up to 2 years later) of old age, despite tumor re-challenge 12 months after tumor resolution. Furthermore, this treatment was effective in large tumor burdens; the same percent (~5%) of body weight in humans would generally be non-responsive to current treatment modalities. This response is rare for current or novel therapies, including immunotherapies; it also reproduces the effect a potent vaccine has in infectious systems. Thus, we have successfully skewed the tumor microenvironment from an immunosuppressive one to an acute pro-inflammatory state that enables the tumor to function as its own vaccine site. As result, potent tumor-specific T cells are systemically released such that they patrol the body and access untreated distal tumors. Simultaneously, and importantly, potent memory T cells are generated.

When used intravenously the IL-2 or anti-CD40 monotherapies were barely effective, unless higher doses were used, while the combination produced distressing and potentially fatal side effects in association with disappointing anti-tumor activity. Targeting the tumor microenvironment by i.t. injections enabled lower doses to produce highly effective anti-tumor immunity without serious side effects during treatment. Hence, a real bonus (in terms of side effects and costs) is the ability to use much lower doses. This i.t. approach is immediately applicable for accessible tumors and has been pioneered by our group in MM clinical trials (18, 21). However, the data also support the use of more technically challenging methods that could deliver these agents into inaccessible tumors via e.g. gene therapy (21, 27), tumor cell–anti-CD40 bispecific mAbs or tumor vascular targeting peptides (28).

Interestingly, each treatment induced a different tumor infiltrate profile that coincided with different, size-related, critical effector mechanisms. For example, IL-2-induced regression of small tumors was mediated by an enhanced tumor-specific CD8+ CTL response that required CD4+ help (20), yet the infiltrate was primarily CD8+ T cells. However, IL-2 did not prevent growth of a distal tumor despite complete regression of the treated tumor and the generation of protective immunity. This response was not reproduced in mice with a large tumor burden.

Anti-CD40 therapy was also only effective in small tumors and, unlike its systemic delivery into MM tumor-bearing mice which only induced a temporal response during treatment (6), i.t. administration offered responding mice a permanent cure. This i.t. therapy induced regression of distal tumors and offered long-term protection suggesting an important role for CD8+ T cells and depleting CD8+ T cells during the therapy diminished the long-term, but not the short-term, anti-tumor response (data not shown). Unlike its systemic delivery, i.t. anti-CD40 did not recruit CD8+ T cells into MM tumors (6) and CD8 depletion only partially abrogated anti-tumor efficacy. In agreement with others, CD4+ T cells were not required for eradication of tumors and this may be because DC licensing at the tumor site by i.t. anti-CD40 antibody bypasses the need for CD4+ T cell help (29). However, it was also clear that other effector mechanisms played a significant role in i.t. anti-CD40-induced tumor regression, although PMNs were not involved.

An added degree of complexity was identified with the IL-2/anti-CD40 combination therapy as tumor burden appeared to require different effector mechanisms. The combination treatment provoked a massive inflammatory infiltrate and necrosis in small and large tumors that preceded tumor regression and led to a complete cure in >80% of mice. CD4+ and CD8+ T cells, as well as PMNs were recruited into these tumors. The latter are reported to posses the ability to directly kill tumor cells (30, 31) and our data suggest that PMNs are able to eradicate small tumors on their own. Thus, it is possible that at least two different anti-tumor effector cell types were recruited into the tumor microenvironment (i.e. CD8+ CTL and PMN). Small tumors treated with IL-2/anti-CD40 could be still eradicated when one of these populations was removed. Therefore, the presence of multiple effector cells may mean that the removal of one cell population does not jeopardize the destruction of a small tumor by the remaining effector cells. In contrast, large tumors treated with IL-2/anti-CD40 required CD8+ (but not CD4+) T cells and PMNs together for tumor eradication. Note that removal of NK cells slightly perturbed IL-2/anti-CD40-driven tumor eradication regardless of burden. Similarly, blocking IFN{gamma} only impacted upon the IL-2 monotherapy and did not impair the potency of the combination therapy. However, it is very difficult to assess blocking efficiency in the face of potentially overwhelming IFN{gamma} response. Nonetheless, the retention of memory suggests that either IFN{gamma} does not deleteriously affect memory CD4+ T cells or local administration does not generate the IFN{gamma} cytokine storm seen after systemic administration (15).

Our data suggest that anti-tumor CD8+ T cells on their own are not enough to eradicate firmly established, advanced, tumors and support the notion of generating acute inflammation within a tumor. Indeed, our data show that the local inflammatory response must consist of both CD8+ T cells and PMNs such that they ‘co-operate’ with each other to destroy large tumors. We have not yet identified the nature of this T cell:PMN collaboration. It is possible that PMNs recruit, promote or maintain CD8+ T effectors (32–34) within tumors. A recent report has shown that heat-killed Mycobacterium induces the development of two distinct GR-1+ myeloid lineages which interact via their respective free radical products to modulate T cell expansion (35). Thus, it is also possible that PMN sub-populations are skewed away from NO production and toward superoxide (Formula) which promotes expansion of activated T cells in mice.

Tumor cells (particularly those in large tumors) have the opportunity to employ multiple escape mechanisms such as MHC class I down-regulation and antigen loss and avoid killing by T cells, but not by PMNs. Thus, killing large tumor burdens may in fact require two different effector cell types; T cells that attack MHC class I+ tumor cells expressing the appropriate antigen and PMNs that kill MHC class Ilow and/or tumor antigen loss variants. Furthermore, the release of increased levels of tumor antigen in an inflammatory context may promote, or even spread, T cell responses to other tumor antigens and up-regulate MHC class I on target cells guaranteeing tumor destruction.

Combining IL-2 with anti-CD40 also cured untreated distal tumors suggestive of the dissemination of effectors that reach and destroy distal tumors. This is potentially promising for the treatment of metastases, although it is recognized that this may be an over-interpretation of the two tumor model. It is, however, particularly important for MM patients as the cause of death is usually dispersed primary tumor, only some of which is accessible and treatable; thus, the model reassures us that untreated primary tumors can respond.

In summary, we report that the i.t. IL-2 and anti-CD40 antibody monotherapies were only effective against small tumors. However, their combination provoked impressive local inflammation which enabled the tumor to function as its own vaccine site resulting in the regression of large MM and LL tumors in at least 80% of mice, without deleterious side effects. This response was permanently curative of treated tumors and untreated distal tumors. Blocking INF{gamma} did not ablate anti-tumor activity and protective memory was retained. Each treatment (IL-2 or anti-CD40 antibody alone versus their combination) relied on different effector cells for complete tumor eradication. In particular, the IL-2/anti-CD40 combination could only permanently eradicate a large tumor if both CD8+ cells and PMNs were present. These results give new insights into the immune mechanisms required for rejection of both small and large tumors, thereby allowing the development of improved treatments for cancer patients.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
Funding
 References
 
Supplementary figure 1 is available at International Immunology Online.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
References
 
National Health and Medical Research Council of Australia (211999); Cancer Council of Western Australia; RAINE Medical Research Foundation; Australian Lung Foundation; Mesothelioma Applied Research Foundation.


   Acknowledgements
 
We thank Kathy Heel and the Centre for Microscopy, Characterization and Analysis for help with the FACScan, as well as the staff at UWA animal holding areas in QEII hospital. The authors would also like to thank the National Health and Medical Research Council, the Mesothelioma Applied Research Foundation, the Australian Lung Foundation and the Western Australian Cancer Council for their funding contributions to these studies. The three latter funding bodies involve the efforts of dedicated people including a large number of volunteers, many of whom have experienced tragic circumstances. A special note of thanks goes to the Canberra-based Asbestos Research Group who raised funds in an effort to honor the memory of their relatives, Gerald Willey and Peter Thurbon, both of whom died after a courageous struggle against mesothelioma. Their award was given to our group based upon the theories and data of one of our PhD students (B.K.) who is now engaged in her own battle against cancer.


   Abbreviations
 
DC, dendritic cell
i.p., intra-peritoneal
i.t., intra-tumoral
MM, malignant mesothelioma
PMN, polymorphonuclear neutrophil

   Notes
 
Transmitting editor: A. Kelso

Received 20 June 2008, accepted 26 August 2008.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 

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