The mechanisms by which tumor-associated neutrophils (TANs) affect tumor growth are to a large extent unknown. Regulatory T-cells (T-regs) are functionally immune-suppressive subsets of T-cells. Depletion or inhibition of T-regs can enhance antitumor immunity. We demonstrated both by RT-PCR and by ELISA that murine TANs secrete significant amounts of the T-regs chemoattractant, CCL17, much more than circulating or splenic neutrophils, and at a level progressively increasing during tumor development. Migration assays, both in vitro and in vivo, showed recruitment of T-regs by TANs, which was inhibited with anti-CCL17 monoclonal antibodies. Systemic neutrophil depletion in tumor-bearing mice using anti-Ly6G monoclonal antibodies reduced the migration of T-regs into the tumors. We further showed, using flow cytometry, that CCL17 secretion by TANs is not limited to mouse models of cancer but is also relevant to human TANs. Our results suggest a new indirect mechanism by which TANs may inhibit antitumor immune activity, thus promoting tumor growth. We further describe, for the first time, a clear link between TANs and T-regs acting together to impair antitumor immunity.Neutrophils make up a significant portion of the inflammatory cell infiltrate in many models of cancer. It is becoming increasingly clear that tumor-associated neutrophils (TANs) play a major role in cancer biology. 1,2 Despite some recent advancement in appreciating the role of neutrophils in cancer, the exact mechanisms by which TANs affect tumor growth are to a large extent unknown. In untreated tumors, neutrophils have been reported to suppress the antitumor immune response. 3 Depletion of these TANs inhibits tumor growth 4-6 and reduces the level of immunosuppression in the tumor microenvironment. 4 As mentioned above, we have recently demonstrated that in untreated tumors, neutrophils assume a protumorigenic state, which by analogy to tumor-associated macrophages is termed the "N2" phenotype. We, however, also noted that under certain conditions (e.g., after Transforming Growth Factor beta (TGF-b) blockade), TANs can take on an "N1" phenotype, which is proinflammatory and anti-immunogenic. 4 Gregory and Houghton raised the interesting question of whether the differences between N1 and N2 TANs were due to two unique transcriptional programs or instead represent two states of activation. 7 Although our work suggests a unique transcriptional program, we were, thus far, unable to identify clear markers for N1 or N2 TANs, somewhat limiting the separation between these neutrophil subsets, and their functions.The role of chemokines in the pathogenesis of lung cancer, as well as other cancers, is being increasingly appreciated. 8 One example is the involvement of CCL5 (Rantes) in tumor invasiveness. 9 Another example that we have investigated is the role of CCL2 (MCP-1), which was found to have protumorigenic function in lung cancer by reducing the recruitment and/ or polarization of M2 macrophages. 10 We have previously found that TANs overexpress many ...

Adjuvant arthritis (AA) is an experimental model of autoimmune arthritis that can be induced in susceptible strains of rats such as inbred Lewis upon immunization with CFA. AA cannot be induced in resistant strains like Brown-Norway or in Lewis rats after recovery from arthritis. We have previously shown that resistance to AA is due to the presence of natural as well as acquired anti-heat shock protein (HSP) Abs. In this work we have studied the fine specificity of the protective anti-HSP Abs by analysis of their interaction with a panel of overlapping peptides covering the whole HSP molecule. We found that arthritis-susceptible rats lack Abs to a small number of defined epitopes of the mycobacterial HSP65. These Abs are found naturally in resistant strains and are acquired by Lewis rats after recovery from the disease. Active vaccination of Lewis rats with the protective epitopes as well as passive vaccination with these Abs induced suppression of arthritis. Incubation of murine and human mononuclear cells with the protective Abs induced secretion of IL-10. Analysis of the primary and tertiary structure of the whole Mycobacterium tuberculosis HSP65 molecule indicated that the protective epitopes are B cell epitopes with nonconserved amino acid sequences found on the outer surface of the molecule. We conclude that HSP, the Ag that contains the pathogenic T cell epitopes in AA, also contains protective B cell epitopes exposed on its surface, and that natural and acquired resistance to AA is associated with the ability to respond to these epitopes.

The role of neutrophils in tumor progression has become in recent years a subject of growing interest. Tumor-associated neutrophils (TANs), which constitute an important portion of the tumor microenvironment, promote immunosuppression in advanced tumors by modulating the proliferation, activation and recruitment of a variety of immune cell types. Studies which investigated the consequences of manipulating TAN polarization suggest that the impact of these neutrophils on tumor progression is considerably mediated by and dependent on the presence of CD8 T-cells. It has been previously shown that granulocytic myeloid regulatory cells, i.e. TANs and granulocytic myeloid-derived suppressor cells (G-MDSCs) are capable of suppressing CD8 T-cell proliferation and affect their activation. In the current study, we find that in addition, TANs isolated from different models of murine cancer promote immunosuppression by strongly inducing CD8 T-cell apoptosis. We demonstrate that the TNFα pathway in TANs is critical for the induction of apoptosis, and that the mechanism through which apoptosis is induced involves the production of NO, but not ROS. In the absence of pre-activation, TANs are capable of activating CD8 T-cells, but specifically induce the apoptosis of non-activated CD8CD69 cells. Despite this contradictive effect on T-cell function, we show that TANs suppress the anti-tumor effect of CD8 T-cells and abolish their ability to delay tumor growth. Our results add another important layer on the understanding of the possible mechanisms by which TANs regulate the anti-tumor immune response mediated by CD8 T-cells, therefore promoting a tumor-supportive environment.

Vesicular stomatitis virus (VSV) has shown promise as an oncolytic agent, although unmodified VSV can be neurotoxic. To avoid toxicity, a vector was created by introducing the interferon-b (IFN-b) gene (VSV.IFN-b). We conducted this study to determine the ability of VSV.IFN-b to lyse human cancer (mesothelioma) cells and to evaluate the potential of this recombinant virus for clinical translation. Four normal human mesothelial and 12 mesothelioma cell lines were tested for their susceptibility to VSV vectors in vitro. VSV.hIFN-b did not cause cytotoxicity in any normal lines. Only 4 of 12 lines were effectively lysed by VSV.hIFN-b. In the eight resistant lines, pretreatment with IFN-b prevented lysis of cells by VSV.GFP, and VSV infection or addition of IFN-b protein resulted in the upregulation of double-stranded RNA-dependent protein kinase (PKR), myxovirus resistance A (MxA), and 2 0 ,5 0 -oligo-adenylate-synthetase (2 0 5 0 -OAS) mRNA. In the susceptible lines, there was no protection by pretreatment with IFN-b protein and no IFN-or VSV-induced changes in PKR, MxA, and 2 0 5 0 -OAS mRNA. This complete lack of IFN responsiveness could be explained by marked downregulation of interferon alpha receptors (IFNARs), p48, and PKR in both the mesothelioma cell lines and primary tumor biopsies screened. Presence of p48 in three tumor samples predicted responsiveness to IFN. Our data indicate that many mesothelioma tumors have partially intact IFN pathways that may affect the efficacy of oncolytic virotherapy. However, it may be feasible to prescreen individual susceptibility to VSV.IFN-b by immunostaining for the presence of p48 protein.

Locally produced transforming growth factor-B (TGF-B) promotes tumor-induced immunosuppression and contributes to resistance to immunotherapy. This article explores the potential for increased efficacy when combining immunotherapies with TGF-B suppression using the TGF-B type I receptor kinase inhibitor SM16. Adenovirus expressing IFN-B (Ad.IFN-B) was injected intratumorally once in established s.c. AB12 (mesothelioma) and LKR (lung cancer) tumors or intratracheally in a Kras orthotopic lung tumor model. Mice bearing TC1 (lung cancer) tumors were vaccinated with two injections of adenovirus expressing human papillomavirus-E7 (HPV-E7; Ad.E7). SM16 was administered orally in formulated chow. Tumor growth was assessed and cytokine expression and cell populations were measured in tumors and spleens by real-time PCR and flow cytometry. SM16 potentiated the efficacy of both immunotherapies in each of the models and caused changes in the tumor microenvironment. The combination of SM16 and Ad.IFN-B increased the number of intratumoral leukocytes (including macrophages, natural killer cells, and CD8 + cells) and increased the percentage of T cells expressing the activation marker CD25. SM16 also augmented the antitumor effects of Ad.E7 in the TC1 flank tumor model. The combination did not increase HPV-E7 tetramer-positive CD8 + T cells in the spleens but did induce a marked increase in the tumors. Tumors from SM16-treated mice showed increased mRNA and protein for immunostimulatory cytokines and chemokines, as well as endothelial adhesion molecules, suggesting a mechanism for the increased intratumoral leukocyte trafficking. Blockade of the TGF-B signaling pathway augments the antitumor effects of Ad.IFN-B immune-activating or Ad.E7 vaccination therapy. The addition of TGF-B blocking agents in clinical trials of immunotherapies may increase efficacy.

Background:Successful immunotherapy will require alteration of the tumour microenvironment and/or decreased immune suppression. Tumour-associated macrophages (TAMs) are one major factor affecting tumour microenvironment. We hypothesised that altering TAM phenotype would augment the efficacy of immunotherapy.Methods:We and others have reported that 5,6-Dimethylxanthenone-4-acetic-acid (DMXAA, Vadimezan) has the ability to change TAM phenotypes, inducing a tumour microenvironment conducive to antitumour immune responses. We therefore combined DMXAA with active immunotherapies, and evaluated anti-tumour efficacy, immune cell phenotypes (flow cytometry), and tumour microenvironment (RT–PCR).Results:In several different murine models of immunotherapy for lung cancer, DMXAA-induced macrophage activation significantly augmented the therapeutic effects of immunotherapy. By increasing influx of neutrophils and anti-tumour (M1) macrophages to the tumour, DMXAA altered myeloid cell phenotypes, thus changing the intratumoural M2/non-M2 TAM immunoinhibitory ratio. It also altered the tumour microenvironment to be more pro-inflammatory. Modulating macrophages during immunotherapy resulted in increased numbers, activity, and antigen-specificity of intratumoural CD8+ T cells. Macrophage depletion reduced the effect of combining immunotherapy with macrophage activation, supporting the importance of TAMs in the combined effect.Conclusion:Modulating intratumoural macrophages dramatically augmented the effect of immunotherapy. Our observations suggest that addition of agents that activate TAMs to immunotherapy should be considered in future trials.

Since previous work using a nonreplicating adenovirus-expressing mouse interferon-β (Ad.mIFNβ) showed promising preclinical activity, we postulated that a vector-expressing IFNβ at high levels that could also replicate would be even more beneficial. Accordingly a replication competent, recombinant vaccinia viral vector-expressing mIFNβ (VV.mIFNβ) was tested. VV.mIFNβ-induced antitumor responses in two syngeneic mouse flank models of lung cancer. Although VV.mIFNβ had equivalent in vivo efficacy in both murine tumor models, the mechanisms of tumor killing were completely different. In LKRM2 tumors, viral replication was minimal and the tumor killing mechanism was due to activation of immune responses through induction of a local inflammatory response and production of antitumor CD8 T-cells. In contrast, in TC-1 tumors, the vector replicated well, induced an innate immune response, but antitumor activity was primarily due to a direct oncolytic effect. However, the VV.mIFNβ vector was able to augment the efficacy of an antitumor vaccine in the TC-1 tumor model in association with increased numbers of infiltrating CD8 T-cells. These data show the complex relationships between oncolytic viruses and the immune system which, if understood and harnessed correctly, could potentially be used to enhance the efficacy of immunotherapy.

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