LFA-1 activation enriches tumor-specific T cells in a cold tumor model and synergizes with CTLA-4 blockade.

The inability of CD8+ effector T cells (Teffs) to reach tumor cells is an important aspect of tumor resistance to cancer immunotherapy. The recruitment of these cells to the tumor microenvironment (TME) is regulated by integrins, a family of adhesion molecules that are expressed on T cells. Here, we show that 7HP349, a small-molecule activator of lymphocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrin cell-adhesion receptors, facilitated the preferential localization of tumor-specific T cells to the tumor and improved antitumor response. 7HP349 monotherapy had modest effects on anti-programmed death 1-resistant (anti-PD-1-resistant) tumors, whereas combinatorial treatment with anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) increased CD8+ Teff intratumoral sequestration and synergized in cooperation with neutrophils in inducing cancer regression. 7HP349 intratumoral CD8+ Teff enrichment activity depended on CXCL12. We analyzed gene expression profiles using RNA from baseline and on treatment tumor samples of 14 melanoma patients. We identified baseline CXCL12 gene expression as possibly improving the likelihood or response to anti-CTLA-4 therapies. Our results provide a proof-of-principle demonstration that LFA-1 activation could convert a T cell-exclusionary TME to a T cell-enriched TME through mechanisms involving cooperation with innate immune cells.

Interleukin-6 blockade abrogates immunotherapy toxicity and promotes tumor immunity.

Immune checkpoint blockade (ICB) therapy frequently induces immune-related adverse events. To elucidate the underlying immunobiology, we performed a deep immune analysis of intestinal, colitis, and tumor tissue from ICB-treated patients with parallel studies in preclinical models. Expression of interleukin-6 (IL-6), neutrophil, and chemotactic markers was higher in colitis than in normal intestinal tissue; T helper 17 (Th17) cells were more prevalent in immune-related enterocolitis (irEC) than T helper 1 (Th1). Anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) induced stronger Th17 memory in colitis than anti-program death 1 (anti-PD-1). In murine models, IL-6 blockade associated with improved tumor control and a higher density of CD4+/CD8+ effector T cells, with reduced Th17, macrophages, and myeloid cells. In an experimental autoimmune encephalomyelitis (EAE) model with tumors, combined IL-6 blockade and ICB enhanced tumor rejection while simultaneously mitigating EAE symptoms versus ICB alone. IL-6 blockade with ICB could de-couple autoimmunity from antitumor immunity.

The Glutaminase Inhibitor CB-839 (Telaglenastat) Enhances the Antimelanoma Activity of T-Cell-Mediated Immunotherapies.

Immune-checkpoint inhibitors and adoptive tumor-infiltrating lymphocyte (TIL) therapies have profoundly improved the survival of patients with melanoma. However, a majority of patients do not respond to these agents, and many responders experience disease relapse. Although numerous innovative treatments are being explored to offset the limitations of these agents, novel therapeutic combinations with immunotherapies have the potential to improve patient responses. In this study, we evaluated the antimelanoma activity of immunotherapy combinations with Telaglenastat (CB-839), a potent glutaminase inhibitor (GLSi) that has favorable systemic tolerance. In in vitro TIL:tumor coculture studies, CB-839 treatment improved the cytotoxic activity of autologous TILs on patient-derived melanoma cells. CB-839 treatment decreased the conversion of glutamine to alpha-ketoglutarate (αKGA) more potently in tumor cells versus TILs in these cocultures. These results suggest that CB-839 may improve immune function in a tumor microenvironment by differentially altering tumor and immune cell metabolism. In vivo CB-839 treatment activated melanoma antigen-specific T cells and improved their tumor killing activity in an immune-competent mouse model of adoptive T-cell therapy. Additionally, the combination of CB-839 with anti-PD1 or anti-CTLA4 antibodies increased tumor infiltration by effector T cells and improved the antitumor activity of these checkpoint inhibitors in a high mutation burden mouse melanoma model. Responsiveness to these treatments was also accompanied by an increase of interferon gamma (IFNγ)-associated gene expression in the tumors. Together, these results provide a strong rationale for combining CB-839 with immune therapies to improve efficacy of these treatments against melanoma.

Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy.

High dose interleukin-2 (IL-2) is active against metastatic melanoma and renal cell carcinoma, but treatment-associated toxicity and expansion of suppressive regulatory T cells (Tregs) limit its use in patients with cancer. Bempegaldesleukin (NKTR-214) is an engineered IL-2 cytokine prodrug that provides sustained activation of the IL-2 pathway with a bias to the IL-2 receptor CD122 (IL-2Rß). Here we assess the therapeutic impact and mechanism of action of NKTR-214 in combination with anti-PD-1 and anti-CTLA-4 checkpoint blockade therapy or peptide-based vaccination in mice. NKTR-214 shows superior anti-tumor activity over native IL-2 and systemically expands anti-tumor CD8+ T cells while inducing Treg depletion in tumor tissue but not in the periphery. Similar trends of intratumoral Treg dynamics are observed in a small cohort of patients treated with NKTR-214. Mechanistically, intratumoral Treg depletion is mediated by CD8+ Teff-associated cytokines IFN-γ and TNF-α. These findings demonstrate that NKTR-214 synergizes with T cell-mediated anti-cancer therapies.

Vaccinating with Stem Cells to Stop Cancer.

In a recent study, Kooreman and colleagues identify a set of genes expressed in both induced pluripotent stem cells (iPSCs) and cancer cells. Vaccination of mice with iPSCs induces prophylactic and therapeutic anticancer immunity to shared antigens, opening a possible avenue towards rapid generation of iPSC-based, personalized cancer vaccines.

Peptide Vaccine Formulation Controls the Duration of Antigen Presentation and Magnitude of Tumor-Specific CD8+ T Cell Response.

Despite remarkable progresses in vaccinology, therapeutic cancer vaccines have not achieved their full potential. We previously showed that an excessively long duration of Ag presentation critically reduced the quantity and quality of vaccination-induced T cell responses and subsequent antitumor efficacy. In this study, using a murine model and tumor cell lines, we studied l-tyrosine amino acid-based microparticles as a peptide vaccine adjuvant with a short-term Ag depot function for the induction of tumor-specific T cells. l-Tyrosine microparticles did not induce dendritic cell maturation, and their adjuvant activity was not mediated by inflammasome activation. Instead, prolonged Ag presentation in vivo translated into increased numbers and antitumor activity of vaccination-induced CD8+ T cells. Indeed, prolonging Ag presentation by repeated injection of peptide in saline resulted in an increase in T cell numbers similar to that observed after vaccination with peptide/l-tyrosine microparticles. Our results show that the duration of Ag presentation is critical for optimal induction of antitumor T cells, and can be manipulated through vaccine formulation.

Protein targeting chimeric molecules specific for bromodomain and extra-terminal motif family proteins are active against pre-clinical models of multiple myeloma.

Bromodomain and extraterminal (BET) domain containing protein (BRD)-4 modulates the expression of oncogenes such as c-myc, and is a promising therapeutic target in diverse cancer types. We performed pre-clinical studies in myeloma models with bi-functional protein-targeting chimeric molecules (PROTACs) which target BRD4 and other BET family members for ubiquitination and proteasomal degradation. PROTACs potently reduced the viability of myeloma cell lines in a time-dependent and concentration-dependent manner associated with G0/G1 arrest, reduced levels of CDKs 4 and 6, increased p21 levels, and induction of apoptosis. These agents specifically decreased cellular levels of downstream BRD4 targets, including c-MYC and N-MYC, and a Cereblon-targeting PROTAC showed downstream effects similar to those of an immunomodulatory agent. Notably, PROTACs overcame bortezomib, dexamethasone, lenalidomide, and pomalidomide resistance, and their activity was maintained in otherwise isogenic myeloma cells with wild-type or deleted TP53. Combination studies showed synergistic interactions with dexamethasone, BH3 mimetics, and Akt pathway inhibitors. BET-specific PROTACs induced a rapid loss of viability of primary cells from myeloma patients, and delayed growth of MM1.S-based xenografts. Our data demonstrate that BET degraders have promising activity against pre-clinical models of multiple myeloma, and support their translation to the clinic for patients with relapsed and/or refractory disease.

Cancer vaccine formulation dictates synergy with CTLA-4 and PD-L1 checkpoint blockade therapy.

Anticancer vaccination is a promising approach to increase the efficacy of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death ligand 1 (PD-L1) checkpoint blockade therapies. However, the landmark FDA registration trial for anti-CTLA-4 therapy (ipilimumab) revealed a complete lack of benefit of adding vaccination with gp100 peptide formulated in incomplete Freund's adjuvant (IFA). Here, using a mouse model of melanoma, we found that gp100 vaccination induced gp100-specific effector T cells (Teffs), which dominantly forced trafficking of anti-CTLA-4-induced, non-gp100-specific Teffs away from the tumor, reducing tumor control. The inflamed vaccination site subsequently also sequestered and destroyed anti-CTLA-4-induced Teffs with specificities for tumor antigens other than gp100, reducing the antitumor efficacy of anti-CTLA-4 therapy. Mechanistically, Teffs at the vaccination site recruited inflammatory monocytes, which in turn attracted additional Teffs in a vicious cycle mediated by IFN-γ, CXCR3, ICAM-1, and CCL2, dependent on IFA formulation. In contrast, nonpersistent vaccine formulations based on dendritic cells, viral vectors, or water-soluble peptides potently synergized with checkpoint blockade of both CTLA-4 and PD-L1 and induced complete tumor regression, including in settings of primary resistance to dual checkpoint blockade. We conclude that cancer vaccine formulation can dominantly determine synergy, or lack thereof, with CTLA-4 and PD-L1 checkpoint blockade therapy for cancer.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

Intratumoral CD40 activation and checkpoint blockade induces T cell-mediated eradication of melanoma in the brain.

CD40 agonists bind the CD40 molecule on antigen-presenting cells and activate them to prime tumor-specific CD8+ T cell responses. Here, we study the antitumor activity and mechanism of action of a nonreplicating adenovirus encoding a chimeric, membrane-bound CD40 ligand (ISF35). Intratumoral administration of ISF35 in subcutaneous B16 melanomas generates tumor-specific, CD8+ T cells that express PD-1 and suppress tumor growth. Combination therapy of ISF35 with systemic anti-PD-1 generates greater antitumor activity than each respective monotherapy. Triple combination of ISF35, anti-PD-1, and anti-CTLA-4 results in complete eradication of injected and noninjected subcutaneous tumors, as well as melanoma tumors in the brain. Therapeutic efficacy is associated with increases in the systemic level of tumor-specific CD8+ T cells, and an increased ratio of intratumoral CD8+ T cells to CD4+ Tregs. These results provide a proof of concept of systemic antitumor activity after intratumoral CD40 triggering with ISF35 in combination with checkpoint blockade for multifocal cancer, including the brain.

Macrophages Facilitate Resistance to Anti-VEGF Therapy by Altered VEGFR Expression.

Purpose: VEGF-targeted therapies have modest efficacy in cancer patients, but acquired resistance is common. The mechanisms underlying such resistance are poorly understood.Experimental Design: To evaluate the potential role of immune cells in the development of resistance to VEGF blockade, we first established a preclinical model of adaptive resistance to anti-VEGF therapy. Additional in vitro and in vivo studies were carried out to characterize the role of macrophages in such resistance.Results: Using murine cancer models of adaptive resistance to anti-VEGF antibody (AVA), we found a previously unrecognized role of macrophages in such resistance. Macrophages were actively recruited to the tumor microenvironment and were responsible for the emergence of AVA resistance. Depletion of macrophages following emergence of resistance halted tumor growth and prolonged survival of tumor-bearing mice. In a macrophage-deficient mouse model, resistance to AVA failed to develop, but could be induced by injection of macrophages. Downregulation of macrophage VEGFR-1 and VEGFR-3 expression accompanied upregulation of alternative angiogenic pathways, facilitating escape from anti-VEGF therapy.Conclusions: These findings provide a new understanding of the mechanisms underlying the modest efficacy of current antiangiogenesis therapies and identify new opportunities for combination approaches for ovarian and other cancers. Clin Cancer Res; 23(22); 7034-46. ©2017 AACR.

Absence of Grail promotes CD8+ T cell anti-tumour activity.

T-cell tolerance is a major obstacle to successful cancer immunotherapy; thus, developing strategies to break immune tolerance is a high priority. Here we show that expression of the E3 ubiquitin ligase Grail is upregulated in CD8+ T cells that have infiltrated into transplanted lymphoma tumours, and Grail deficiency confers long-term tumour control. Importantly, therapeutic transfer of Grail-deficient CD8+ T cells is sufficient to repress established tumours. Mechanistically, loss of Grail enhances anti-tumour reactivity and functionality of CD8+ T cells. In addition, Grail-deficient CD8+ T cells have increased IL-21 receptor (IL-21R) expression and hyperresponsiveness to IL-21 signalling as Grail promotes IL-21R ubiquitination and degradation. Moreover, CD8+ T cells isolated from lymphoma patients express higher levels of Grail and lower levels of IL-21R, compared with CD8+ T cells from normal donors. Our data demonstrate that Grail is a crucial factor controlling CD8+ T-cell function and is a potential target to improve cytotoxic T-cell activity.Grail is an E3 ubiquitin ligase that inhibits T-cell receptor signalling in CD4+ T cells. Here the authors show Grail also limits IL-21 receptor expression and function in CD8+ T cells, is overactive in these cells in patients with lymphoma, and promotes tumour development in a lymphoma transplant mouse model.

Multifaceted Role of BTLA in the Control of CD8+ T-cell Fate after Antigen Encounter.

Purpose: Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown an overall clinical response rate 40%-50% in metastatic melanoma patients. BTLA (B-and-T lymphocyte associated) expression on transferred CD8+ TILs was associated with better clinical outcome. The suppressive function of the ITIM and ITSM motifs of BTLA is well described. Here, we sought to determine the functional characteristics of the CD8+BTLA+TIL subset and define the contribution of the Grb2 motif of BTLA in T-cell costimulation.Experimental Design: We determined the functional role and downstream signal of BTLA in both human CD8+ TILs and mouse CD8+ T cells. Functional assays were used including single-cell analysis, reverse-phase protein array (RPPA), antigen-specific vaccination models with adoptively transferred TCR-transgenic T cells as well as patient-derived xenograft (PDX) model using immunodeficient NOD-scid IL2Rgammanull (NSG) tumor-bearing mice treated with autologous TILs.Results: CD8+BTLA- TILs could not control tumor growth in vivo as well as their BTLA+ counterpart and antigen-specific CD8+BTLA- T cells had impaired recall response to a vaccine. However, CD8+BTLA+ TILs displayed improved survival following the killing of a tumor target and heightened "serial killing" capacity. Using mutants of BTLA signaling motifs, we uncovered a costimulatory function mediated by Grb2 through enhancing the secretion of IL-2 and the activation of Src after TCR stimulation.Conclusions: Our data portrays BTLA as a molecule with the singular ability to provide both costimulatory and coinhibitory signals to activated CD8+ T cells, resulting in extended survival, improved tumor control, and the development of a functional recall response. Clin Cancer Res; 23(20); 6151-64. ©2017 AACR.

Soluble interleukin-15 complexes are generated in vivo by type I interferon dependent and independent pathways.

Interleukin (IL)-15 associates with IL-15Rα on the cell surface where it can be cleaved into soluble cytokine/receptor complexes that have the potential to stimulate CD8 T cells and NK cells. Unfortunately, little is known about the in vivo production of soluble IL-15Rα/IL-15 complexes (sIL-15 complexes), particularly regarding the circumstances that induce them and the mechanisms responsible. The main objective of this study was to elucidate the signals leading to the generation of sIL-15 complexes. In this study, we show that sIL-15 complexes are increased in the serum of mice in response to Interferon (IFN)-α. In bone marrow derived dendritic cells (BMDC), IFN-α increased the activity of ADAM17, a metalloproteinase implicated in cleaving IL-15 complexes from the cell surface. Moreover, knocking out ADAM17 in BMDCs prevented the ability of IFN-α to induce sIL-15 complexes demonstrating ADAM17 as a critical protease mediating cleavage of IL-15 complexes in response to type I IFNs. Type I IFN signaling was required for generating sIL-15 complexes as in vivo induction of sIL-15 complexes by Poly I:C stimulation or total body irradiation (TBI) was impaired in IFNAR-/- mice. Interestingly, serum sIL-15 complexes were also induced in mice infected with Vesicular stomatitis virus (VSV) or mice treated with agonistic CD40 antibodies; however, sIL-15 complexes were still induced in IFNAR-/- mice after VSV infection or CD40 stimulation indicating pathways other than type I IFNs induce sIL-15 complexes. Overall, this study has shown that type I IFNs, VSV infection, and CD40 stimulation induce sIL-15 complexes suggesting the generation of sIL-15 complexes is a common event associated with immune activation. These findings reveal an unrealized mechanism for enhanced immune responses occurring during infection, vaccination, inflammation, and autoimmunity.

Mouse model for pre-clinical study of human cancer immunotherapy.

This unit describes protocols for developing tumors in mice, including subcutaneous growth, pulmonary metastases of B16 melanoma, and spontaneous melanoma in B-Raf V600E/PTEN deletion transgenic mouse models. Two immunization methods to prevent B16 tumor growth are described using B16.GM-CSF and recombinant vaccinia virus. A therapeutic approach is also included that uses adoptive transfer of tumor antigen-specific T cells. Methods including CTL induction, isolation, testing, and genetic modification of mouse T cells for adoptive transfer by using retrovirus-expressing genes of interest are provided. Additional sections, including growing B16 melanoma, enumerating pulmonary metastases, tumor imaging technique, and use of recombinant viruses for vaccination, are discussed together with safety concerns.

Generation of a new therapeutic peptide that depletes myeloid-derived suppressor cells in tumor-bearing mice.

Immune evasion is an emerging hallmark of cancer progression. However, functional studies to understand the role of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are limited by the lack of available specific cell surface markers. We adapted a competitive peptide phage display platform to identify candidate peptides binding MDSCs specifically and generated peptide-Fc fusion proteins (peptibodies). In multiple tumor models, intravenous peptibody injection completely depleted blood, splenic and intratumoral MDSCs in tumor-bearing mice without affecting proinflammatory immune cell types, such as dendritic cells. Whereas control Gr-1-specific antibody primarily depleted granulocytic MDSCs, peptibodies depleted both granulocytic and monocytic MDSC subsets. Peptibody treatment was associated with inhibition of tumor growth in vivo, which was superior to that achieved with Gr-1-specific antibody. Immunoprecipitation of MDSC membrane proteins identified S100 family proteins as candidate targets. Our strategy may be useful to identify new diagnostic and therapeutic surface targets on rare cell subtypes, including human MDSCs.

Cancer vaccines: Trafficking of tumor-specific T cells to tumor after therapeutic vaccination.

Cancer vaccines can induce robust activation of tumor-specific CD8(+) T cells that can destroy tumors. Understanding the mechanism by which cancer vaccines work is essential in designing next-generation vaccines with more potent therapeutic activity. We recently reported that short peptides emulsified in poorly biodegradable, Incomplete Freund's Adjuvant (IFA) primed CD8(+) T cells that did not localize to the tumor site but accumulated at the persisting, antigen-rich vaccination site. The vaccination site eventually became a T cell graveyard where T cells responded to chronically released gp100 peptide by releasing cytokines, including interferon-γ (IFN-γ), which in turn upregulated Fas ligand (FasL) on host cells, causing apoptosis of Fas(+) T cells. T cells that escaped apoptosis rapidly became exhausted, memory formation was poor, and therapeutic impact was minimal. Replacing the non-biodegradable IFA-based formulation with water-based, short-lived formulation in the presence of immunostimulatory molecules allowed T cells to traffic to tumors, causing their regression. In this review, we discuss recent advances in immunotherapeutic approaches that could enhance vaccine-primed immune cells fitness and render the tumor microenvironment more accessible for immune cell infiltration.

Peptide-based anticancer vaccines: The making and unmaking of a T-cell graveyard.

Poorly biodegradable, incomplete Freund's adjuvant (IFA)-based anticancer vaccines primed CD8+ T cells that did not localize to the tumor site but to the persisting, antigen-rich vaccination site, which became a T-cell graveyard. Short-lived, water-based formulations and the provision of immunostimulatory molecules overcame this issue, resulting in tumor suppression. Here, we discuss the implications of these findings for the development of therapeutic anticancer vaccines.

Persistent antigen at vaccination sites induces tumor-specific CD8⁺ T cell sequestration, dysfunction and deletion.

To understand why cancer vaccine-induced T cells often do not eradicate tumors, we studied immune responses in mice vaccinated with gp100 melanoma peptide in incomplete Freund's adjuvant (peptide/IFA), which is commonly used in clinical cancer vaccine trials. Peptide/IFA vaccination primed tumor-specific CD8(+) T cells, which accumulated not in tumors but rather at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, interferon-γ (IFN-γ)- and Fas ligand (FasL)-mediated apoptosis, resulting in hyporesponsiveness to subsequent vaccination. Provision of CD40-specific antibody, Toll-like receptor 7 (TLR7) agonist and interleukin-2 (IL-2) reduced T cell apoptosis but did not prevent vaccination-site sequestration. A nonpersisting vaccine formulation shifted T cell localization toward tumors, inducing superior antitumor activity while reducing systemic T cell dysfunction and promoting memory formation. These data show that persisting vaccine depots can induce specific T cell sequestration, dysfunction and deletion at vaccination sites; short-lived formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines.

Tumor-expressed inducible nitric oxide synthase controls induction of functional myeloid-derived suppressor cells through modulation of vascular endothelial growth factor release.

Inducible NO synthase (iNOS) is a hallmark of chronic inflammation that is also overexpressed in melanoma and other cancers. Whereas iNOS is a known effector of myeloid-derived suppressor cell (MDSC)-mediated immunosuppression, its pivotal position at the interface of inflammation and cancer also makes it an attractive candidate regulator of MDSC recruitment. We hypothesized that tumor-expressed iNOS controls MDSC accumulation and acquisition of suppressive activity in melanoma. CD11b(+)GR1(+) MDSC derived from mouse bone marrow cells cultured in the presence of MT-RET-1 mouse melanoma cells or conditioned supernatants expressed STAT3 and reactive oxygen species (ROS) and efficiently suppressed T cell proliferation. Inhibition of tumor-expressed iNOS with the small molecule inhibitor L-NIL blocked accumulation of STAT3/ROS-expressing MDSC, and abolished their suppressive function. Experiments with vascular endothelial growth factor (VEGF)-depleting Ab and recombinant VEGF identified a key role for VEGF in the iNOS-dependent induction of MDSC. These findings were further validated in mice bearing transplantable MT-RET-1 melanoma, in which L-NIL normalized elevated serum VEGF levels; downregulated activated STAT3 and ROS production in MDSC; and reversed tumor-mediated immunosuppression. These beneficial effects were not observed in iNOS knockout mice, suggesting L-NIL acts primarily on tumor- rather than host-expressed iNOS to regulate MDSC function. A significant decrease in tumor growth and a trend toward increased tumor-infiltrating CD8(+) T cells were also observed in MT-RET transgenic mice bearing spontaneous tumors. These data suggest a critical role for tumor-expressed iNOS in the recruitment and induction of functional MDSC by modulation of tumor VEGF secretion and upregulation of STAT3 and ROS in MDSC.