In recent years, immunotherapy strategies have been aggressively pursued to enhance anti-tumor immune responses and many phase II and III clinical trials have been conducted (22
). Although a variety of immunotherapeutic approaches have been shown to generate active cytotoxic T-lymphocytes (CTLs), success in patients has been limited. It has become increasingly clear that the generation of CTL is necessary, but not sufficient for an effective response (22
). There may be a number of reasons for this. First, in addition to inducing immune stimulation, cancer immunotherapies also appear to trigger counter-regulatory immune-suppressive mechanisms such as upregulation of inhibitory surface molecules on T-cells (like CTLA-4 or PD1) or production of T-regulatory cells (24
). Second, tumors are known to produce inhibitory cytokines and chemokines (26
), as well as induce populations of suppressor cells (28
). Thus, it is becoming increasingly apparent that in addition to the generation of CTL, successful immunotherapy will also require “inhibiting the inhibitors”. The studies presented here, using specific anti-murine CCL2 and CCL12 mAb in three different models of immunotherapy, suggest that MCPs may be additional unrecognized key proximal cytokines able to block the immune responses elicited by immunotherapy.
Human CCL2 has two murine orthologues – CCL2 (MCP-1) and CCL12 (MCP-5). Both bind to the CCR2 receptor, although CCL2 is a better agonist of murine CCR2 (29
). Most functions described for CCL12 are similar to those found for CCL2 (13
). We found that each of these mAbs had some effect on tumor growth, but saw significantly more growth inhibition when the two mAb were combined by themselves () or in combination with immunotherapy (Supplemental Fig. 1
). To most accurately model potential effects in humans, we therefore used a mixture of both mAbs for all of our experiments.
There has been some controversy in the literature about the role of CCL2 in tumor development. CCL2, originally identified as a potent chemoattractant for monocytes (7
), can also function as a T-cell chemoattractant and induce T-cell tumor tropism, including memory T-cells (30
). It seemed reasonable that CCL2 would thus function to inhibit tumor growth. Indeed, early work showed that transfection of tumor cells that secreted high levels of CCL2 resulted in massive monocyte/macrophage infiltration into the tumor mass, leading to its destruction (33
). However, in patients, CCL2 has been found at high levels in multiple tumor types, including NSCLC (7
) and high levels usually correlate with poor clinical outcome (36
). Studies, such as those by Loberg et al., showed that systemic administration of anti-CCL2 neutralizing antibodies significantly retarded tumor growth (8
). The use of α-CCL2 mAb in mice has been recently shown to reduce tumorigenesis and metastasis in prostate cancer xenograft models (37
These observations support mounting evidence suggesting that most of the effects of CCL2 in non-transduced tumors are actually pro-tumorigenic (7
). First, it is now recognized that most monocytes recruited into tumors do not kill tumor cells, but are subverted to an M2 phenotype where they actually support tumor growth (38
). Second, CCL2 appears to directly augment the growth and invasiveness of certain tumor cells, that express the CCR2 receptor (7
). Third, CCR2 is expressed by endothelial cells and CCL2 appears to promote angiogenesis (40
). Fourth, it has been observed that CCL2 can also serve as a chemoattractant for T-regs (41
). Finally, it is now recognized that CCL2 also has direct immunoinhibitory (pro-tumorigenic) effects on T-cell function (10
), such as inhibiting T cell effector functions and switching T cell differentiation towards Th2-like cells (12
We noted that vaccines induced an influx of macrophages, compared to control, that was not prevented with CCL2 blockade. It was somewhat surprising that, neutralization of a major chemokine attracting monocytes to the tumor did not reduce the total number of monocytes. We have no definitive answer to this question, but we speculate that in tumors there are many other agents (including CCL5, CCL7, CCL8, CXCL8, CXCL12, CXCL1, M-CSF, and VEGF) that can replace MCP-1 in terms of chemoattraction of monocytes (38
). Another possibility is that inducing CD8+
activity and reducing T-regs changed the total balance of chemokines in tumor microenvironment, allowing for the influx of monocytes to continue based on other chemokines.
Our mechanistic studies in the TC1 model show that CCL2 blockade in combination with the Ad.E7 vaccine clearly resulted in increased numbers, activity, and antigen-specificity of CD8+
T cells, and in the percentage of activated CD4+
T cells in the spleens (), and even more importantly, intra-tumorally (). These data are consistent with those of Peng et al. in adoptive transfer studies who used neutralizing α-CCL2 antibodies and showed that this led to the generation of T-cells that were substantially more active and more vigorous at eliminating tumor suggesting increased tumor specificity (10
), and later studies, showing that prevention of tumor-secretion of CCL2 had a positive effect on CTLs that were more active in both spleen and draining lymph nodes (43
It is likely that some of these activating effects on CD8+ T-cells were due to blockade of the direct effects of CCL2 on the T-cells; however, we also explored other factors that might be involved. First, we did not note any changes in the levels of CD11b+/GR1+ cells, generally accepted as MDSC. In our studies, we did find some effects of α-CCL2 mAb alone on TAM phenotype (manuscript in preparation), however, we could not implicate these changes in explaining the augmented effects we saw in combination therapy, since there were no obvious differences in macrophages phenotype populations when we compared Ad.E7 treatment alone with combination treatment ().
In contrast, we found significant reductions in T-regs in the spleens () and tumors of mice treated with the combination therapy versus vaccine alone (), suggesting an important possible mechanism for reduction of immune suppression. The idea that T-regs are important inhibitors of anti-tumor immune responses is well established (45
) and their presence correlates with poor prognosis of cancer patients (46
). The finding that CCL2 blockade could inhibit T-reg recruitment is consistent with previous studies showing that: 1) CD4+
T-regs selectively over-express the CCL2/CCL12 receptors CCR2 (47
), and CCR4 (48
), 2) CCL2 has been shown to specifically chemoattract T-regs in vitro
), and 3) blocking CCR2 in vivo
reduced the influx of T-regs to disease sites in a model of arthritis (47
). Interestingly, in the TC1 tumor model, depletion of CD4 cells using a specific mAb leads to slower growth (data not shown), suggesting that these tumors do induce T-regs that then augment their growth.
Given their strong immuno-inhibitory properties, reduction of T-regs has been a goal of many groups. To date, most attempts to reduce T-regs have used non-specific agents such as low dose cyclophosphamide or antibodies/antibody-toxins directed toward the IL2-receptor (CD25). Targeting CD25 may have disadvantages, however, since it is also expressed on activated CD8+
). Our data suggest that a novel, and possibly safer way to prevent the influx of T-regs into the tumor microenvironment may be via CCL2 blockade. This may be particularly important when the strong immune reaction induced by vaccines is also accompanied by a strong induction of T-regs.
Finally, we also found that the tumor microenvironment was altered in the combination-treated tumors with increased mRNA levels of Th1 type mediators such as TNF-α, IFNγ, CXCL10, and ICAM-1 (), and protein levels of TNF-α. It is currently uncertain if this is a direct result of CCL2 blockade leading to enhanced T-cell activation or whether increased numbers of activated CD8+ T cells results in a more immunostimulatory microenvironment.
In summary, we demonstrated here that blocking CCL2 dramatically augmented the effect of immunotherapy for NSCLC and mesothelioma in a multifactorial immunologic mechanism. Our observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials.