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Myeloid-derived suppressor cells (MDSC) include immature monocytic (M-MDSC) and granulocytic (PMN-MDSC) cells that share the ability to suppress adaptive immunity and to hinder the effectiveness of anticancer treatments. Of note, in response to IFNγ, M-MDSCs release the tumor-promoting and immunosuppressive molecule nitric oxide (NO), whereas macrophages largely express antitumor properties. Investigating these opposing activities, we found that tumor-derived prostaglandin E2 (PGE2) induces nuclear accumulation of p50 NF-κB in M-MDSCs, diverting their response to IFNγ toward NO-mediated immunosuppression and reducing TNFα expression. At the genome level, p50 NF-κB promoted binding of STAT1 to regulatory regions of selected IFNγ-dependent genes, including inducible nitric oxide synthase (Nos2). In agreement, ablation of p50 as well as pharmacologic inhibition of either the PGE2 receptor EP2 or NO production reprogrammed M-MDSCs toward a NOS2low/TNFαhigh phenotype, restoring the in vivo antitumor activity of IFNγ. Our results indicate that inhibition of the PGE2/p50/NO axis prevents MDSC-suppressive functions and restores the efficacy of anticancer immunotherapy. Significance: Tumor-derived PGE2-mediated induction of nuclear p50 NF-κB epigenetically reprograms the response of monocytic cells to IFNγ toward an immunosuppressive phenotype, thus retrieving the anticancer properties of IFNγ.

Cancer induces alteration of hematopoiesis to fuel disease progression. We report that in tumor-bearing mice the macrophage colony-stimulating factor elevates the myeloid cell levels of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD salvage pathway, which acts as negative regulator of the CXCR4 retention axis of hematopoietic cells in the bone marrow. NAMPT inhibits CXCR4 through a NAD/Sirtuin 1–mediated inactivation of HIF1α-driven CXCR4 gene transcription, leading to mobilization of immature myeloid-derived suppressor cells (MDSC) and enhancing their production of suppressive nitric oxide. Pharmacologic inhibition or myeloid-specific ablation of NAMPT prevented MDSC mobilization, reactivated specific antitumor immunity, and enhanced the antitumor activity of immune checkpoint inhibitors. Our findings identify NAMPT as a metabolic gate of MDSC precursor function, providing new opportunities to reverse tumor immunosuppression and to restore clinical efficacy of immunotherapy in patients with cancer. Significance: These findings identify NAMPT as a metabolic gate of MDSC precursor function, providing new opportunities to reverse tumor immunosuppression and to restore clinical efficacy of immunotherapy in cancer patients.

Telomerase (TERT) is overexpressed in 80% to 90% of primary tumors and contributes to sustaining the transformed phenotype. The identification of several TERT epitopes in tumor cells has elevated the status of TERT as a potential universal target for selective and broad adoptive immunotherapy. TERT-specific cytotoxic T lymphocytes (CTL) have been detected in the peripheral blood of B-cell chronic lymphocytic leukemia (B-CLL) patients, but display low functional avidity, which limits their clinical utility in adoptive cell transfer approaches. To overcome this key obstacle hindering effective immunotherapy, we isolated an HLA-A2–restricted T-cell receptor (TCR) with high avidity for human TERT from vaccinated HLA-A*0201 transgenic mice. Using several relevant humanized mouse models, we demonstrate that TCR-transduced T cells were able to control human B-CLL progression in vivo and limited tumor growth in several human, solid transplantable cancers. TERT-based adoptive immunotherapy selectively eliminated tumor cells, failed to trigger a self–MHC-restricted fratricide of T cells, and was associated with toxicity against mature granulocytes, but not toward human hematopoietic progenitors in humanized immune reconstituted mice. These data support the feasibility of TERT-based adoptive immunotherapy in clinical oncology, highlighting, for the first time, the possibility of utilizing a high-avidity TCR specific for human TERT. Cancer Res; 76(9); 2540–51. ©2016 AACR.

Myeloid-derived suppressor cells (MDSC) contribute significantly to the malignant characters conferred by hypoxic tumor microenvironments. However, selective biomarkers of MDSC function in this critical setting have not been defined. Here, we report that miR-210 expression is elevated by hypoxia-inducible factor-1α (HIF1α) in MDSC localized to tumors, compared with splenic MDSC from tumor-bearing mice. In tumor MDSC, we determined that HIF1α was bound directly to a transcriptionally active hypoxia-response element in the miR-210 proximal promoter. miR-210 overexpression was sufficient to enhance MDSC-mediated T-cell suppression under normoxic conditions, while targeting hypoxia-induced miR-210 was sufficient to decrease MDSC function against T cells. Mechanistic investigations revealed that miR-210 modulated MDSC function by increasing arginase activity and nitric oxide production, without affecting reactive oxygen species, IL6, or IL10 production or expression of PD-L1. In splenic MDSC, miR-210 regulated Arg1, Cxcl12, and IL16 at the levels of both mRNA and protein, the reversal of which under normoxic conditions decreased T-cell–suppressive effects and IFNγ production. Interestingly, miR-210 overexpression or targeting IL16 or CXCL12 enhanced the immunosuppressive activity of MDSC in vivo, resulting in increased tumor growth. Taken together, these results provide a preclinical rationale to explore miR-210 inhibitory oligonucleotides as adjuvants to boost immunotherapeutic responses in cancer patients. Cancer Res; 75(18); 3771–87. ©2015 AACR.

Tumor vaccines have shown promise in early clinical trials. Among them, tumor cells genetically engineered to secrete biologically active granulocyte-macrophage colony-stimulating factor (GM-CSF) can generate a systemic antitumor immune response. Although the minimal required GM-CSF dose produced by modified tumor cells to achieve a measurable antitumor effect is well known, no data examined whether an upper therapeutic limit may exist for this vaccination strategy. Because recent data demonstrate an immunosuppressive effect of GM-CSF produced by growing tumors, we thus sought to determine whether high GM-CSF doses administered in a vaccine formulation could impair antitumor immunity. Using a vaccine strategy involving a GM-CSF-producing bystander cell line (B78H1-GM) admixed with autologous tumor, we assessed the impact of varying doses of GM-CSF while maintaining a constant antigen dose. Our results defined a threshold above which a GM-CSF-based vaccine not only lost its efficacy, but more importantly for its clinical implications resulted in substantial immunosuppression in vivo. Above this threshold, GM-CSF induced Gr1+/CD11b+ myeloid suppressor cells that substantially impaired antigen-specific T-cell responses and adversely affected antitumor immune responses in vivo. The dual effects of GM-CSF are mediated by the systemic and not local concentration of this cytokine. Myeloid suppressor cell-induced immunosuppression is mediated by nitric oxide production via inducible nitric oxide synthase (iNOS) because the specific iNOS inhibitor, l-NMMA, restored antigen-specific T-cell responsiveness in vitro. Taken together, our data demonstrated the negative impact of supra-therapeutic vaccine doses of GM-CSF and underscored the importance of identifying these critical variables in an effort to increase the therapeutic efficacy of tumor vaccines.

Introduction: Depending on their polarization, myeloid cells can either promote or restrain tumor progression. By secreting myelopoietic factors tumours promote myeloid cells polarization toward a phenotype that provides immune protection and that stimulate neoplastic cells growth, invasiveness, and metastasis. Chemokines and their cognate receptors are thought to play a key role in myeloid cell trafficking, however, their study in vivo is hindered by their signal redundancy and integration. Methods: By taking advantage of a newly developed nanoplatform that allows the in vivo preferential transfection of tumor infiltrating myeloid cells (TIMC, aka Myeloid derived suppressor cells, MDSC, and tumor associated macrophages, TAM), we evaluated the effect of CCR-1, CCR-2, CCR-4, CCR-5 and/or CCR-7 silencing on TAMC trafficking, phenotype, and function. Results: While CCR-4 and -7 targeted silencing did not affect tumor progression, simultaneous silencing of CCR-1 and CCR-5 significantly restrained tumor growth and greatly modifies tumor micro-environment. Mechanistic in vivo and in vitro analysis surprisingly indicate that myeloid cell trafficking was not altered whereas myeloid cell polarization was. In particular, CCR-1 and CCR-5 blockade restrained MDSC differentiation and promote the accumulation of retinoblastoma positive, neutrophil-like cells with a strong tumoricidal activity. Discussion: Our data indicate that CCR-1 and CCR-5 engagement (most likely via CCL-3 and CCL-4) is necessary for the pro-tumoral polarization of myeloid cells in cancer. Our findings not only improve our understanding of myeloid cells differentiation in cancer but, also, strongly suggest that targeted inhibition of CCR1 and CCR5 on TIMC can be a new and effective anti-tumor immune-therapeutic strategy that convert pro-tumoral MDSCs into tumoricidal neutrophils. Citation Format: Serena Zilio, Jennifer Vella, Adriana De La Fuente, Vincenzo Bronte, Emilia Mazza, Silvio Bicciato, Paolo Serafini. In vivo targeted silencing of CCR1 and CCR5 repolarizes pro-tumoral myeloid cells in retinoblastoma positive neutrophils with a strong anti-tumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1449.

A major obstacle for the development of effective immunotherapy is the ability of tumors to escape the immune system. The possibility to kill tumor cells because they are recognized as infected rather than as malignant could help overcome immune escape mechanisms. Here we report a conceptually new approach of cancer immunotherapy based on in vivo infection of tumors and killing of infected tumor cells. Attenuated but still invasive, Salmonella typhimurium can be successfully exploited to invade melanoma cells that can present antigenic determinants of bacterial origin and become targets for anti-Salmonella–specific T cells. However, to fully appreciate the anticancer therapeutic properties of S. typhimurium, tumor-bearing mice need to be vaccinated against S. typhimurium before intratumoral Salmonella injection. Tumor infection when coupled to anti-Salmonella vaccination leads to 50% to 100% tumor-free mice with a better outcome on larger tumors. Invasive Salmonella also exert an indirect toxic effect on tumor cells through the recruitment of inflammatory cells and the cross-presentation of tumor antigens, which allow induction of tumor-specific immune response. This is effective in retarding the growth of untreated established distant tumors and in protecting the mice from subsequent tumor challenges.

Translational research and the clinical development of therapeutic cancer vaccines requires stronger scientific rationalization. Here we demonstrate how immune response markers as well as biomarkers defining the immune regulatory environment were utilized as guiding tools from discovery to advanced clinical trials of IMA901, a novel therapeutic vaccine for the treatment of renal cell carcinoma (RCC). IMA901 consists of multiple tumor-associated peptides (TUMAPs) confirmed to be naturally presented in human RCC tissue by mass spectrometry, selected using differential transcriptomics and preclinically validated by systematic analysis of immunogenicity with artificial antigen-presenting cells. Two consecutive independent clinical studies in a total of 96 HLA-A*02+ advanced/metastatic RCC patients were conducted. The phase I study revealed that T-cell responses to multiple IMA901 antigens were significantly associated with disease control and negatively associated with the presence of FoxP3+ regulatory T cells (Tregs). The subsequent randomized phase II study demonstrated that pre-treatment with a single low dose of cyclophosphamide (Cy) reduced Treg frequencies and prolonged overall survival (OS) in patients who mounted an immune response to the IMA901 vaccine. Additionally, T-cell responses to multiple IMA901 antigens were again associated with clinical benefit. Furthermore, a comprehensive prognostic and predictive biomarker program was conducted. Among cellular biomarkers, highly significantly elevated levels of myeloid-derived suppressor cells (MDSC), IL-17-/IL-10-secreting T cells and dysfunctional T cells in RCC patients vs. healthy individuals were found. Two MDSC populations (CD14+ HLA-DR- and CD14- CD11b+ CD15+) were significantly negatively associated with survival in vaccinated RCC patients. Interestingly, both MDSC populations were also found to be negatively associated with OS in an independent trial in colorectal cancer patients (N=79) implying a broader role for these MDSC species. Additionally, among over 300 serum biomarkers tested, apolipoprotein A-I (ApoA1) and the chemokine CCL17 were found to be predictive for both immune responses to IMA901 and survival of the RCC patients. The knowledge acquired in these trials was used to design a randomized phase III study. In this ongoing study, IMA901 is combined with the tyrosine kinase inhibitor sunitinib based on the findings that sunitinib downmodulates the two MDSC populations described above. Furthermore, in this phase III study, the relevance of ApoA1/CCL17 will be explored by prospectively defined subgroup analyses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5365. doi:1538-7445.AM2012-5365

IMA901 is a therapeutic cancer vaccine for the treatment of renal cell cancer patients based on the selection of naturally presented tumor-associated peptides. A previous phase I study (N=28 patients) showed significant correlations of clinical benefit with T-cell responses to multiple IMA901 peptides. Based on this experience, a randomized phase II study was designed to explore the biological and clinical efficacy of IMA901. Different regulatory cell populations were assessed in patients prior to vaccination, including 6 phenotypically defined populations of myeloid derived suppressor cells (MDSCs). A total of 68 HLA-A*02-positive RCC patients with documented progression during or after first-line therapy with cytokines or TKI were randomized to receive or not a single infusion of low-dose cyclophosphamide (CY; 300 mg/m2) three days prior to start repeated i.d. vaccinations with IMA901 in association with 75 µg GM-CSF i.d. Among them, 64 pts were eligible according to the pre-specified, per-protocol analysis. Patients receiving a single infusion of CY had a significant decrease of FoxP3-positive regulatory T cells (Tregs) after 3 days (p=0.014), particularly in Ki67+ (proliferating) Tregs (p=0.006). Patients receiving low-dose CY also showed a trend for improved overall survival (OS) as compared to the non-CY group (OS not reached after 25 months of follow-up vs. 16 months, respectively; p=0.086). Marginally significantly better OS rates were seen in immune responders compared to non-responders in the overall population (p=0.048), but highly significantly so in the subgroup of patients randomized to CY (p=0.006). 6 previously reported MDSC populations were analyzed from cryopreserved PBMCs by a single multi-color flow cytometry staining panel. A high overlap (>25%) was found for only 2 MDSC populations, so 6 populations represented five distinct MDSC subtypes. MDSC levels were significantly increased in patient samples as compared to matched healthy donors (p To our knowledge, this is the first study assessing the impact of MDSC levels on the survival of cancer patients by employing a novel method that can differentiate five distinct MDSC populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 302. doi:10.1158/1538-7445.AM2011-302

One of the major hurdles for cancer immunotherapy is that most tumors express self-antigens to which the immune system is unresponsive due to self-tolerance. Our lab and others have demonstrated that high-avidity tumor-specific CD8+ T cells are superior in anti-tumor activity to low avidity T cells. However, adoptive transfer of high-avidity, tumor-specific CTL populations may fail due to loss of CTL effector functions upon tumor infiltration. Therefore, strategies to maintain effector functions of high avidity CTL, or enhancement of low avidity CTL are desirable. To investigate whether provision of CD4+ T cell help will enhance the effectiveness of high-avidity CTLs in anti-tumor immunity as compared to lower avidity T cells, we utilized two transgenic mouse lines developed in our lab that bear different TCR transgenes specific for a common melanoma differentiation antigen, tyrosinase-related protein 2 (TRP-2(180-188)). T cells from clone 24 TCR Tg mice (TCRhi) show higher avidity than those from clone 37 (TCRlo) mice based on tetramer dissociation and in vitro effector function assays. Here we report that co-activation of CD4+ T cells (OVA323-339 specific, CD4-OVA) with TRP-2-specific CD8+ T cells can boost the in vitro function of TCRhi cells, including proliferation, IFN-γ secretion, Gr-B secretion as well as cell-mediated cytotoxicity, but had minimal effect on TCRlo cells. We observed that only adoptive co-transfer of CD4-OVA cells with TCRhi cells suppressed tumor growth of (5 day) established B16 melanoma tumors. Furthermore, those mice receiving co-transferred CD4-OVA cells with TCRhi cells developed autoimmune depigmentation at the sites of vaccination and tumor challenge. Our finding may have important implications for enhancement of cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 473. doi:10.1158/1538-7445.AM2011-473

We have generated myeloid-derived suppressor cells (MDSCs) from bone marrow precursors (BM-MDSCs) as a tool to investigate the molecular pathways involved in their immunosuppressive program. BM-MDSCs suppress in vitro both antigen-specific and unspecific CD8+ T cells. The tolerogenic properties of BM-MDSC were further confirmed in vivo following their adoptive transfer in a setting of allogeneic pancreatic insulae transplantation in pharmacologically-induced diabetic mice. The cytokines leading to generation of MDSCs are normally produced during an inflammatory response and act on a common transcription factor which is considered a master regulator of the emergency granulopoyesis. We investigated the effect of genetic knock out of this transcription factor in vivo by creating mice lacking it in myelomonocytic cells. In these mice tumor growth did not cause dysfunction in CD8+ T cells, suggesting that this transcription factor is crucial for their tolerogenic activity and a potential target for new approaches aimed at controlling tumor-induced tolerance. Citation Format: Vincenzo Bronte. Transcriptional control of tumor-induced myeloid-derived suppressor cells [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY13-02