Search

Fig. S1 shows characterization of GD2-specific CAR-T cells. Fig. S2 shows characterization of human MDSCs. Fig. S3 shows characterization of TME xenograft model. Fig. S4 shows GD2.CAR-T cell homing and infiltration into MDSC-containing tumors after NK cell infusion. Fig. S5 shows infiltration of NK cell and GD2.CAR-T cells by IHC.

Solid tumors are refractory to cellular immunotherapies in part because they contain suppressive immune effectors such as myeloid-derived suppressor cells (MDSCs) that inhibit cytotoxic lymphocytes. Strategies to reverse the suppressive tumor microenvironment (TME) should also attract and activate immune effectors with antitumor activity. To address this need, we developed gene-modified natural killer (NK) cells bearing a chimeric receptor in which the activating receptor NKG2D is fused to the cytotoxic ζ-chain of the T-cell receptor (NKG2D.ζ). NKG2D.ζ–NK cells target MDSCs, which overexpress NKG2D ligands within the TME. We examined the ability of NKG2D.ζ–NK cells to eliminate MDSCs in a xenograft TME model and improve the antitumor function of tumor-directed chimeric antigen receptor (CAR)–modified T cells. We show that NKG2D.ζ–NK cells are cytotoxic against MDSCs, but spare NKG2D ligand–expressing normal tissues. NKG2D.ζ–NK cells, but not unmodified NK cells, secrete proinflammatory cytokines and chemokines in response to MDSCs at the tumor site and improve infiltration and antitumor activity of subsequently infused CAR-T cells, even in tumors for which an immunosuppressive TME is an impediment to treatment. Unlike endogenous NKG2D, NKG2D.ζ is not susceptible to TME-mediated downmodulation and thus maintains its function even within suppressive microenvironments. As clinical confirmation, NKG2D.ζ–NK cells generated from patients with neuroblastoma killed autologous intratumoral MDSCs capable of suppressing CAR-T function. A combination therapy for solid tumors that includes both NKG2D.ζ–NK cells and CAR-T cells may improve responses over therapies based on CAR-T cells alone.

Supplementary Figure 1: CD34-IL7R* (C7R) is stably expressed in T-cells and enhances constitutive STAT5 activation relative to IL7R*.

Supplementary Figure 3: C7R signaling does not change phenotypic composition of GD2-CAR T-cells during culture.

Supplementary Figure 6: Efficient retroviral transduction of T-cells with Î"34, C7R, GD2-CAR, and GD2-CAR.C7R constructs.

Supplementary Figure 5: C7R does not significantly increase intracranial EphA2-CAR T-cell expansion against U373 tumors

Supplemental Figure S1. Contribution of NK and T cells to IFN-gamma production. Supplemental Figure S2. MICA expression on monocytes. Supplemental Figure S3. NKG2D Ligand Expression on C1R and C1R-MICA Cell Lines. Supplemental Figure S4. Monocyte MICA expression does not correlate with NK cell IFN-gamma production. Supplemental Figure S5. NKG2D and MICA expression levels prior to and following co-culture.

Supplementary Figure 4: C7R prolongs survival of GD2-CAR T-cells but does not support autonomous cell expansion.

Natural killer (NK) cells are large granular lymphocytes that promote the antitumor response via communication with other cell types in the tumor microenvironment. Previously, we have shown that NK cells secrete a profile of immune stimulatory factors (e.g., IFNγ, MIP-1α, and TNFα) in response to dual stimulation with the combination of antibody (Ab)-coated tumor cells and cytokines, such as IL12. We now demonstrate that this response is enhanced in the presence of autologous monocytes. Monocyte enhancement of NK cell activity was dependent on cell-to-cell contact as determined by a Transwell assay. It was hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of MICA on monocytes to NK cell NKG2D receptors. Strategies to block MICA–NKG2D interactions resulted in reductions in IFNγ production. Depletion of monocytes in vivo resulted in decreased IFNγ production by murine NK cells upon exposure to Ab-coated tumor cells. In mice receiving trastuzumab and IL12 therapy, monocyte depletion resulted in significantly greater tumor growth in comparison to mock-depleted controls (P < 0.05). These data suggest that NK cell–monocyte interactions enhance NK cell antitumor activity in the setting of monoclonal Ab therapy for cancer. Cancer Immunol Res; 5(9); 778–89. ©2017 AACR.

Supplemental Table 1: Differential gene expression in GD2-CAR.C7R T-cells compared to GD2-CAR T-cells.

Supplementary Figure 2: Efficient retroviral transduction of C7R and Î"34 in both CD4 and CD8 selected T-cells.

High expression levels of human epidermal growth factor receptor 2 (HER2) have been associated with poor prognosis in patients with pancreatic adenocarcinoma (PDAC). However, HER2-targeting immunotherapies have been unsuccessful to date. Here we increase the breadth, potency, and duration of anti-PDAC HER2-specific CAR T-cell (HER2.CART) activity with an oncolytic adeno-immunotherapy that produces cytokine, immune checkpoint blockade, and a safety switch (CAdTrio). Combination treatment with CAdTrio and HER2.CARTs cured tumors in two PDAC xenograft models and produced durable tumor responses in humanized mice. Modifications to the tumor immune microenvironment contributed to the antitumor activity of our combination immunotherapy, as intratumoral CAdTrio treatment induced chemotaxis to enable HER2.CART migration to the tumor site. Using an advanced PDAC model in humanized mice, we found that local CAdTrio treatment of primary tumor stimulated systemic host immune responses that repolarized distant tumor microenvironments, improving HER2.CART anti-tumor activity. Overall, our data demonstrate that CAdTrio and HER2.CARTs provide complementary activities to eradicate metastatic PDAC and may represent a promising co-operative therapy for PDAC patients., Rosewell Shaw et al. show that a previously developed immunotherapy strategy, coupling oncolytic adenoviral immunotherapy with clinically tested HER2-specific CAR T-cells, is effective against pancreatic ductal adenocarcinoma (PDAC). This combination therapy produces a curative response in both PDAC xenografts and humanized mouse models.

Pediatric and young adult (YA) patients with acute myeloid leukemia (AML) who relapse after allogeneic hematopoietic cell transplantation (HCT) have an extremely poor prognosis. Standard salvage chemotherapy and donor lymphocyte infusions (DLIs) have little curative potential. Previous studies showed that natural killer (NK) cells can be stimulated ex vivo with interleukin-12 (IL-12), -15, and -18 to generate memory-like (ML) NK cells with enhanced antileukemia responses. We treated 9 pediatric/YA patients with post-HCT relapsed AML with donor ML NK cells in a phase 1 trial. Patients received fludarabine, cytarabine, and filgrastim followed 2 weeks later by infusion of donor lymphocytes and ML NK cells from the original HCT donor. ML NK cells were successfully generated from haploidentical and matched-related and -unrelated donors. After infusion, donor-derived ML NK cells expanded and maintained an ML multidimensional mass cytometry phenotype for >3 months. Furthermore, ML NK cells exhibited persistent functional responses as evidenced by leukemia-triggered interferon-γ production. After DLI and ML NK cell adoptive transfer, 4 of 8 evaluable patients achieved complete remission at day 28. Two patients maintained a durable remission for >3 months, with 1 patient in remission for >2 years. No significant toxicity was experienced. This study demonstrates that, in a compatible post-HCT immune environment, donor ML NK cells robustly expand and persist with potent antileukemic activity in the absence of exogenous cytokines. ML NK cells in combination with DLI present a novel immunotherapy platform for AML that has relapsed after allogeneic HCT. This trial was registered at https://clinicaltrials.gov as #NCT03068819.

Solid tumors are refractory to cellular immunotherapies in part because they contain suppressive immune effectors such as myeloid-derived suppressor cells (MDSCs) that inhibit cytotoxic lymphocytes. Strategies to reverse the suppressive tumor microenvironment (TME) should also attract and activate immune effectors with antitumor activity. To address this need, we developed gene-modified natural killer (NK) cells bearing a chimeric receptor in which the activating receptor NKG2D is fused to the cytotoxic ζ-chain of the T-cell receptor (NKG2D.ζ). NKG2D.ζ–NK cells target MDSCs, which overexpress NKG2D ligands within the TME. We examined the ability of NKG2D.ζ–NK cells to eliminate MDSCs in a xenograft TME model and improve the antitumor function of tumor-directed chimeric antigen receptor (CAR)–modified T cells. We show that NKG2D.ζ–NK cells are cytotoxic against MDSCs, but spare NKG2D ligand–expressing normal tissues. NKG2D.ζ–NK cells, but not unmodified NK cells, secrete proinflammatory cytokines and chemokines in response to MDSCs at the tumor site and improve infiltration and antitumor activity of subsequently infused CAR-T cells, even in tumors for which an immunosuppressive TME is an impediment to treatment. Unlike endogenous NKG2D, NKG2D.ζ is not susceptible to TME-mediated downmodulation and thus maintains its function even within suppressive microenvironments. As clinical confirmation, NKG2D.ζ–NK cells generated from patients with neuroblastoma killed autologous intratumoral MDSCs capable of suppressing CAR-T function. A combination therapy for solid tumors that includes both NKG2D.ζ–NK cells and CAR-T cells may improve responses over therapies based on CAR-T cells alone.

Data mining of nanoparticle contrast–enhanced 3D images reveals phenotypes indicative of tumor response to cellular immunotherapy., Immunotherapies, including cell-based therapies, targeting the tumor microenvironment (TME) result in variable and delayed responses. Thus, it has been difficult to gauge the efficacy of TME-directed therapies early after administration. We investigated a nano-radiomics approach (quantitative analysis of nanoparticle contrast–enhanced three-dimensional images) for detection of tumor response to cellular immunotherapy directed against myeloid-derived suppressor cells (MDSCs), a key component of TME. Animals bearing human MDSC-containing solid tumor xenografts received treatment with MDSC-targeting human natural killer (NK) cells and underwent nanoparticle contrast–enhanced computed tomography (CT) imaging. Whereas conventional CT-derived tumor metrics were unable to differentiate NK cell immunotherapy tumors from untreated tumors, nano-radiomics revealed texture-based features capable of differentiating treatment groups. Our study shows that TME-directed cellular immunotherapy causes subtle changes not effectively gauged by conventional imaging metrics but revealed by nano-radiomics. Our work provides a method for noninvasive assessment of TME-directed immunotherapy potentially applicable to numerous solid tumors.

Myeloid-derived suppressor cells (MDSCs) represent a heterogenous population of immature myeloid cells capable of modulating immune responses. In the context of cancer, MDSCs are abnormally produced and recruited to the tumor microenvironment (TME) to aid in the establishment of an immunosuppressive TME that facilitates tumor escape. Additionally, MDSCs contribute to non-immunologic aspects of tumor biology, including tumor angiogenesis and metastasis. The clinical significance of MDSCs has recently been appreciated as numerous studies have suggested a correlation between circulating and intratumoral MDSC frequencies and tumor stage, progression, and treatment resistance. In this chapter, we review MDSC characterization, development, expansion, and mechanisms that facilitate immunosuppression and tumor progression. Furthermore, we highlight studies demonstrating the clinical significance of MDSCs in various disease states in addition to strategies that modulate various aspects of MDSC biology for therapeutic gain.

1032 Background: Successful targeting of solid tumors such as breast cancer (BC) using CAR T cells (CARTs) has proven challenging, largely due to the immune suppressive tumor microenvironment (TME). Myeloid derived suppressor cells (MDSCs) inhibit CART’s function and persistence within the breast TME. We generated CAR T cells targeting tumor-expressed mucin 1 (MUC1) (Bajgain P et al, 2018) for BC. To potentiate expansion and persistence of MUC1 CARTs and modulate the suppressive TME, we developed a novel chimeric co-stimulatory receptor, TR2.4-1BB, encoding a ScFv derived from a TNF-related apoptosis-inducing ligand receptor 2 (TR2) mAb followed by a 4-1BB endodomain. We hypothesize that engagement with TR2 expressed on TME-resident MDSCs, will lead to both MDSC apoptosis and CART co-stimulation, promoting T cell persistence and expansion at tumor site. Methods: Function of the novel TR2.4-1BB receptor, was assessed by exposing non-transduced (NT) and TR2.4-1BB transduced T cells to recombinant TR2 and nuclear translocation of NFκB was measured by ELISA. Functionality of in vitro generated MDSCs was determined by the suppression assay. In vitro CART/costimulatory receptor T cell function was measured by cytotoxicity assays using MUC1+ tumor targets in presence or absence of MDSCs. In vivo anti-tumor activity was assessed using MDSC enriched tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. Results: Nuclear translocation of NFκB was detected only in TR2.4-1BB T cells. MDSCs significantly attenuated T cell proliferation by 50±5% and IFNγ production by half compared with T cells cultured alone. Additionally, presence of MDSCs, diminished cytotoxic potential of MUC1 CARTs against MUC1+ BC cell lines by 25%. However, TR2.4-1BB expression on CAR.MUC1 T cells induced MDSC apoptosis thereby restoring the cytotoxic activity of CAR.MUC1 against MUC1+ BC lines in presence of TR2.4-1BB (67±8.5%). There was an approximate two-fold increase in tumor growth due enhanced angiogenesis and fibroblast accumulation in mice receiving tumors + MDSCs compared to tumors alone. Treatment of these MDSC-enriched tumors with MUC1.TR2.4-1BB CARTs led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared to CAR.MUC1 (469.79.9±81.46mm3) or TR2.4-1BB (434.86±64.25 mm3) T cells alone (Day 28 after T cell injection). The treatment also improved T cell proliferation and persistence at the tumor site. Thereby, leading to negligible metastasis demonstrating ability of CARTs to eliminate tumor and prevent dissemination. We observed similar results using HER2.TR2.4-1BB CARTs in a HER2+ BC model. Conclusions: Our findings demonstrate that CARTs co-expressing our novel TR2.4-1BB receptor have higher anti-tumor potential against BC tumors and infiltrating MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.

Natural killer (NK) cells are large granular lymphocytes that promote the antitumor response via communication with other cell types in the tumor microenvironment. Previously, we have shown that NK cells secrete a profile of immune stimulatory factors (e.g., IFNγ, MIP-1α, and TNFα) in response to dual stimulation with the combination of antibody (Ab)-coated tumor cells and cytokines, such as IL12. We now demonstrate that this response is enhanced in the presence of autologous monocytes. Monocyte enhancement of NK cell activity was dependent on cell-to-cell contact as determined by a Transwell assay. It was hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of MICA on monocytes to NK cell NKG2D receptors. Strategies to block MICA–NKG2D interactions resulted in reductions in IFNγ production. Depletion of monocytes in vivo resulted in decreased IFNγ production by murine NK cells upon exposure to Ab-coated tumor cells. In mice receiving trastuzumab and IL12 therapy, monocyte depletion resulted in significantly greater tumor growth in comparison to mock-depleted controls (P < 0.05). These data suggest that NK cell–monocyte interactions enhance NK cell antitumor activity in the setting of monoclonal Ab therapy for cancer. Cancer Immunol Res; 5(9); 778–89. ©2017 AACR.

Successful adoptive T-cell immunotherapy of solid tumors will require improved expansion and cytotoxicity of tumor-directed T cells within tumors. Providing recombinant or transgenic cytokines may produce the desired benefits but is associated with significant toxicities, constraining clinical use. To circumvent this limitation, we constructed a constitutively signaling cytokine receptor, C7R, which potently triggers the IL7 signaling axis but is unresponsive to extracellular cytokine. This strategy augments modified T-cell function following antigen exposure, but avoids stimulating bystander lymphocytes. Coexpressing the C7R with a tumor-directed chimeric antigen receptor (CAR) increased T-cell proliferation, survival, and antitumor activity during repeated exposure to tumor cells, without T-cell dysfunction or autonomous T-cell growth. Furthermore, C7R-coexpressing CAR T cells were active against metastatic neuroblastoma and orthotopic glioblastoma xenograft models even at cell doses that had been ineffective without C7R support. C7R may thus be able to enhance antigen-specific T-cell therapies against cancer. Significance: The constitutively signaling C7R system developed here delivers potent IL7 stimulation to CAR T cells, increasing their persistence and antitumor activity against multiple preclinical tumor models, supporting its clinical development. Cancer Discov; 7(11); 1238–47. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1201

Chimeric antigen receptors (CARs) combine T cell activation with antibody-mediated tumor antigen specificity, bypassing the need for T cell receptor (TCR) ligation. A limitation of CAR technology is on-target off-tumor toxicity caused by target antigen expression on normal cells. Using GD2 as a model cancer antigen, we hypothesized that this could be minimized by using T cells expressing Vγ9Vδ2 TCR, which recognizes transformed cells in a major histocompatibility complex (MHC)-unrestricted manner, in combination with a co-stimulatory CAR that would function independently of the TCR. An anti-GD2 CAR containing a solitary endodomain derived from the NKG2D adaptor DAP10 was expressed in Vγ9Vδ2+ T cells. Differential ligation of the CAR and/or TCR using antibody-coated beads showed that pro-inflammatory cytokine response depended on activation of both receptors. Moreover, in killing assays, GD2-expressing neuroblastoma cells that engaged the Vγ9Vδ2 TCR were efficiently lysed, whereas cells that expressed GD2 equivalently but did not engage the Vγ9Vδ2 TCR were untouched. Differentiation between X-on tumor and X-off tumor offers potential for safer immunotherapy and broader target selection.

Recent advances in methods for the ex vivo expansion of human natural killer (NK) cells have facilitated the use of these powerful immune cells in clinical protocols. Further, the ability to genetically modify primary human NK cells following rapid expansion allows targeting and enhancement of their immune function. We have successfully adapted an expansion method for primary NK cells from peripheral blood mononuclear cells or from apheresis products in gas permeable rapid expansion devices (G-Rexes). Here, we describe an optimized protocol for rapid and robust NK cell expansion as well as a method for highly efficient retroviral transduction of these ex vivo expanded cells. These methodologies are good manufacturing practice (GMP) compliant and could be used for clinical-grade product manufacturing.

Immunotherapy with tumor-directed immune cells, such as chimeric antigen receptor-bearing T (CAR-T) cells, have shown great promise. Indeed, CAR-T cells directed against leukemia antigens such as CD19 have produced impressive complete responses even in relapsed disease patients. In contrast, CAR-T cells directed against solid tumor have produced very few durable clinical responses. This may be, in part, due to the suppressive immune microenvironment of solid tumors that inhibits the anti-tumor activity of endogenous and CAR-T cells. Myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and inhibitory macrophages (M2s) are recruited by tumor-derived factors, and contribute to the immunosuppressive tumor microenvironment (TME) by secreting suppressive cytokines, expressing inhibitory ligands, and promoting tumor neovascularization. However, the contribution of each of these components to suppressing engineered cellular therapies has not been well defined. In order to design more effective cellular therapies for solid tumors, it is imperative that we understand TME-mediated immune suppression of human CAR-T cells. Therefore, the objective of this study was to evaluate the individual contribution of MDSCs, Tregs, and M2s to the suppressive TME and their ability to modulate CAR-T function. We hypothesized that MDSCs, Tregs, and M2s would inhibit GD2.CAR-T homing and proliferation at tumor sites, leading to decreased activity and tumor progression. To understand the individual contributions of these suppressive cells to inhibition of CAR-T cells, we developed a unique TME model in which human neuroblastoma tumor cells were co-inoculated subcutaneously with MDSCs, Tregs, or M2s in immunocompromised mice and then treated with GD2 neuroblastoma antigen-specific CAR-T cells. We found that while all three cell types decreased GD2.CAR-T cell-mediated tumor control in vivo, only MDSCs and M2s inhibited CAR-T homing and expansion at tumor sites. To investigate cross-talk between these suppressive cells, we assessed the phenotype and suppressive activity of MDSCs, Tregs, and M2 on autologous CAR-T cells after exposure to the other suppressive cell types. We found that the suppressive function of M2s increased by 30% after exposure to Tregs. Similarly, Treg suppressive function increased by 26% after exposure to M2s. Both M2 and Treg suppressive functions were enhanced in the presence of MDSCs. Our results suggest a potential cross-talk between suppressive cells of the TME and distinct roles in important aspects of CAR-T cell function, including homing and killing. Future studies in our lab aim to further understand the differential roles of these cells in regulating CAR-T cell function within the TME that will inform new approaches to engineer effective adoptive T-cell therapies. Citation Format: Charlotte H. Rivas, Anna Cole, Cliona M. Rooney, Robin Parihar. Suppressive myeloid cells of the solid tumor microenvironment enhance regulatory T cell function and differentially affect CAR-T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4734.

Natural killer (NK) cells express an activating receptor for the Fc portion of IgG (FcγRIIIa) that mediates interferon (IFN)–γ production in response to antibody (Ab)–coated targets. We have previously demonstrated that NK cells activated with interleukin-12 (IL-12) in the presence of immobilized IgG secrete 10-fold or more higher levels of IFN-γ as compared with stimulation with either agent alone. We examined the intracellular signaling pathways responsible for this synergistic IFN-γ production. NK cells costimulated via the FcR and the IL-12 receptor (IL-12R) exhibited enhanced levels of activated STAT4 and Syk as compared with NK cells stimulated through either receptor alone. Extracellular signal–regulated kinase (ERK) was also synergistically activated under these conditions. Studies with specific chemical inhibitors revealed that the activation of ERK was dependent on the activation of PI3-K, whose activation was dependent on Syk, and that sequential activation of these molecules was required for NK cell IFN-γ production in response to FcR and IL-12 stimulation. Retroviral transfection of ERK1 into primary human NK cells substantially increased IFN-γ production in response to immobilized IgG and IL-12, while transfection of human NK cells with a dominant-negative ERK1 abrogated IFN-γ production. Confocal microscopy and cellular fractionation experiments revealed that FcγRIIIa and the IL-12R colocalized to areas of lipid raft microdomains in response to costimulation with IgG and IL-12. Chemical disruption of lipid rafts inhibited ERK signaling in response to costimulation and significantly inhibited IFN-γ production. These data suggest that dual recruitment of FcγRIIIa and the IL-12R to lipid raft microdomains allows for enhanced activation of downstream signaling events that lead to IFN-γ production.

Recent advances in methods for the ex vivo expansion of human natural killer (NK) cells have facilitated the use of these powerful immune cells in clinical protocols. Further, the ability to genetically modify primary human NK cells following rapid expansion allows targeting and enhancement of their immune function. We have successfully adapted an expansion method for primary NK cells from peripheral blood mononuclear cells or from apheresis products in gas permeable rapid expansion devices (G-Rexes). Here, we describe an optimized protocol for rapid and robust NK cell expansion as well as a method for highly efficient retroviral transduction of these ex vivo expanded cells. These methodologies are good manufacturing practice (GMP) compliant and could be used for clinical-grade product manufacturing.

Background Traditionally, the CD56dimCD16+ subset of Natural Killer (NK) cells has been thought to mediate cellular cytotoxicity with modest cytokine secretion capacity. However, studies have suggested that this subset may exert a more diverse array of immunological functions. There exists a lack of well-developed functional models to describe the behavior of activated NK cells, and the interactions between signaling pathways that facilitate effector functions are not well understood. In the present study, a combination of genome-wide microarray analyses and systems-level bioinformatics approaches were utilized to elucidate the transcriptional landscape of NK cells activated via interactions with antibody-coated targets in the presence of interleukin-12 (IL-12). Methods We conducted differential gene expression analysis of CD56dimCD16+ NK cells following FcR stimulation in the presence or absence of IL-12. Next, we functionally characterized gene sets according to patterns of gene expression and validated representative genes using RT-PCR. IPA was utilized for biological pathway analysis, and an enriched network of interacting genes was generated using GeneMANIA. Furthermore, PAJEK and the HITS algorithm were employed to identify important genes in the network according to betweeness centrality, hub, and authority node metrics. Results Analyses revealed that CD56dimCD16+ NK cells co-stimulated via the Fc receptor (FcR) and IL-12R led to the expression of a unique set of genes, including genes encoding cytotoxicity receptors, apoptotic proteins, intracellular signaling molecules, and cytokines that may mediate enhanced cytotoxicity and interactions with other immune cells within inflammatory tissues. Network analyses identified a novel set of connected key players, BATF, IRF4, TBX21, and IFNG, within an integrated network composed of differentially expressed genes in NK cells stimulated by various conditions (immobilized IgG, IL-12, or the combination of IgG and IL-12). Conclusions These results are the first to address the global mechanisms by which NK cells mediate their biological functions when encountering antibody-coated targets within inflammatory sites. Moreover, this study has identified a set of high-priority targets for subsequent investigation into strategies to combat cancer by enhancing the anti-tumor activity of CD56dimCD16+ NK cells. Electronic supplementary material The online version of this article (doi:10.1186/s12920-015-0142-9) contains supplementary material, which is available to authorized users.

NK cells express an activating FcR (FcγRIIIa) that mediates Ab-dependent cellular cytotoxicity and the production of immune modulatory cytokines in response to Ab-coated targets. IL-21 has antitumor activity in murine models that depends in part on its ability to promote NK cell cytotoxicity and IFN-γ secretion. We hypothesized that the NK cell response to FcR stimulation would be enhanced by the administration of IL-21. Human NK cells cultured with IL-21 and immobilized IgG or human breast cancer cells coated with a therapeutic mAb (trastuzumab) secreted large amounts of IFN-γ. Increased secretion of TNF-α and the chemokines IL-8, MIP-1α, and RANTES was also observed under these conditions. NK cell IFN-γ production was dependent on distinct signals mediated by the IL-21R and the FcR and was abrogated in STAT1-deficient NK cells. Supernatants derived from NK cells that had been stimulated with IL-21 and mAb-coated breast cancer cells were able to drive the migration of naive and activated T cells in an in vitro chemotaxis assay. IL-21 also enhanced NK cell lytic activity against Ab-coated tumor cells. Coadministration of IL-21 and Ab-coated tumor cells to immunocompetent mice led to synergistic production of IFN-γ by NK cells. Furthermore, the administration of IL-21 augmented the effects of an anti-HER2/neu mAb in a murine tumor model, an effect that required IFN-γ. These findings demonstrate that IL-21 significantly enhances the NK cell response to Ab-coated targets and suggest that IL-21 would be an effective adjuvant to administer in combination with therapeutic mAbs.

In the current report, we have examined the ability of natural killer (NK) cells to produce T cell–recruiting chemokines following dual stimulation with interleukin (IL)-2 or IL-12 and human breast cancer cells coated with an antitumor antibody (trastuzumab). NK cells stimulated in this manner secreted an array of T cell–recruiting chemotactic factors, including IL-8, macrophage-derived chemokine, macrophage inflammatory protein 1α (MIP-1α), monocyte chemoattractant protein 1, and regulated on activation, normal T-cell expressed and secreted (RANTES), whereas stimulation of NK cells with either agent alone had minimal effect. Furthermore, these factors were functional for T-cell chemotaxis as culture supernatants derived from costimulated NK cells induced migration of both naïve and activated T cells in an in vitro chemotaxis assay. T-cell migration was significantly reduced when neutralizing antibodies to IL-8, MIP-1α, or RANTES were added to culture supernatants before their use in the chemotaxis assay. In addition, coadministration of trastuzumab-coated tumor cells and IL-12 to mice led to enhanced serum MIP-1α. As a clinical correlate, we examined the chemokine content of serum samples from breast cancer patients enrolled on a phase I trial of trastuzumab and IL-12, and found elevated levels of IL-8, RANTES, IFN-γ inducible protein 10, monokine induced by IFN-γ, and MIP-1α, specifically in those patients that experienced a clinical benefit. Sera from these patients exhibited the ability to direct T-cell migration in a chemotaxis assay, and neutralization of chemokines abrogated this effect. These data are the first to show chemokine production by NK cells, specifically in response to stimulation with antibody-coated tumor cells, and suggest a potential role for NK cell–derived chemokines in patients receiving therapeutic monoclonal antibodies. (Cancer Res 2006; 66(1): 517-26)

We have previously shown that natural killer (NK) cells secrete a distinct profile of immunomodulatory cytokines in response to dual stimulation with antibody-coated tumor cells and interleukin-12 (IL-12). This NK cell cytokine response is dependent on synergistic signals mediated by the activating receptor for the Fc portion of IgG (FcγRIIIa) and the IL-12 receptor (IL-12R), both constitutively expressed on NK cells. The phosphatase Src homology 2–containing inositol 5′-phosphatase 1 (SHIP1) is known to exert inhibitory effects on Fc receptor (FcR) signaling via its enzymatic activity on phosphatidylinositol 3-kinase (PI3-K) products within many cells of the immune system, most notably mast cells, B cells, and monocytes. However, its activity in the context of FcR activation on NK cells has not been fully explored. The current study focused on the regulation of FcγRIIIa-induced NK cell cytokine production by SHIP1. Inhibitor studies showed that NK cell IFN-γ production following FcR stimulation in the presence of IL-12 depended, in part, on the downstream products of PI3-K. Overexpression of wild-type (WT) SHIP1, but not a catalytic-deficient mutant, via retroviral transfection of primary human NK cells, resulted in a >70% reduction of NK cell IFN-γ production in response to costimulation. In addition, NK cells from SHIP1−/− mice produced 10-fold greater amounts of IFN-γ following culture with antibody-coated tumor cells plus IL-12 compared with NK cells from WT mice. Further, activation of the mitogen-activated protein kinase (MAPK) family member extracellular signal-regulated kinase (Erk; a downstream target of PI3-K) was significantly enhanced within SHIP1−/− NK cells compared with WT NK cells following costimulation. Pharmacologic inhibition of Erk activity, but not Jnk MAPK activity, led to significantly decreased IFN-γ production from both SHIP1−/− and WT NK cells under these conditions. These results are the first to show a physiologic role for SHIP1 in the regulation of NK cell cytokine production and implicate PI3-K in the induction of MAPK signal transduction following costimulation of NK cells via the FcR and the IL-12R.

Bacterial DNA contains a high frequency of unmethylated CpG motifs that stimulate immune cells via TLR9. NK cells express a low-affinity activating receptor for the Fc portion of IgG (FcγRIIIa), but were not thought to express TLR9 protein. The direct response of NK cells to CpG oligodeoxynucleotides (ODN) in the presence of FcR stimulation was investigated. Human NK cells cultured in the presence of CpG ODN plus immobilized IgG or Ab-coated tumor cells secreted large amounts of IFN-γ (>2000 pg/ml), whereas cells stimulated with Ab alone, CpG ODN alone, or Ab and control ODN produced negligible amounts. Enhanced secretion of IL-8, macrophage-derived chemokine, and MIP-1α was also observed after costimulation. NK cell cytokine production was not the result of interactions with APCs or their cytokine products. Flow cytometric analysis revealed that 36 ± 3.5% of human NK cells expressed basal levels of TLR9. TLR9 expression in human NK cells was confirmed by immunoblot analysis. Only TLR9-expressing NK cells responded to CpG ODN and Ab, because cytokine production was not observed in NK cells from TLR9-deficient mice. Mice receiving CpG ODN and HER2/neu-positive tumor cells treated with an anti-HER2 Ab exhibited enhanced systemic levels of IFN-γ compared with mice receiving either agent alone. TLR9−/− animals reconstituted with TLR9+/+ NK cells secreted IFN-γ in response to CpG ODN and Ab-coated tumor cells. These findings indicate that CpG ODN can directly enhance the NK cell cytokine response to Ab-coated targets via activation of TLR9.

Purpose: On the basis of preclinical studies, we hypothesized that interleukin (IL)12 would potentiate the antitumor actions of an antihuman epidermal growth factor receptor-2 (HER2) monoclonal antibody (trastuzumab). We conducted a Phase I trial to determine the safety and optimal biological dose of IL-12 when given in combination with trastuzumab.Patients and Methods: Patients with metastatic HER2-positive malignancies received trastuzumab on day 1 of each weekly cycle. Beginning in week 3, patients also received intravenous injections of IL-12 on days 2 and 5. The IL-12 component was dose-escalated within cohorts of 3 patients (30, 100, 300, or 500 ng/kg). Correlative assays were conducted using serum samples and peripheral blood cells obtained during the course of therapy.Results: Fifteen patients were treated, including 12 with HER2 2+ or 3+ breast cancer. The regimen was well tolerated with IL-12-induced grade 1 nausea and grade 2 fatigue predominating. Evaluation of dose-limiting toxicity and biological end points suggested that the 300 ng/kg dose was both the maximally tolerated dose and the optimal biological dose of IL-12 for use in combination with trastuzumab. Two patients with HER2 3+ breast cancer within the 500 ng/kg dose level experienced grade 1 asymptomatic decreases in left ventricular ejection fraction of 12% and 19% after 3 and 10 months of therapy, respectively. There was one complete response in a patient with HER2 3+ breast cancer metastatic to the axillary, mediastinal, and supraclavicular nodes, and 2 patients with stabilization of bone disease lasting 10 months and >12 months, respectively. Correlative assays showed sustained production of interferon (IFN)γ by natural killer cells only in those patients experiencing a clinical response or stabilization of disease. Elevated serum levels of macrophage inflammatory protein-1α, tumor necrosis factor-α, and the antiangiogenic factors IFN-γ inducible protein-10 and monokine induced by γ were also observed in these patients. Patient genotyping suggested that a specific IFN-γ gene polymorphism might have been associated with increased IFN-γ production. The ability of patient peripheral blood cells to conduct antibody-dependent cellular cytotoxicity against tumor targets in vitro did not correlate with clinical response or dose of IL-12.Conclusions: The addition of IL-12 to trastuzumab therapy did not appear to enhance the efficacy of this antibody treatment. Sustained production of IFN-γ and other cytokines were observed in three patients: One who exhibited a complete response and two others who had stabilization of disease lasting over 6 months. Given the small sample size and heterogeneity of the patient population, the effects of IL-12 on the innate immune response to trastuzumab therapy should be further explored in the context of a larger clinical trial.

Background. Cooperative interactions between natural killer (NK) cells and macrophages occur normally during the course of the early immune response to bacterial, protozoal, and viral pathogens, with each cellular compartment providing the other with critical stimulatory factors. We conducted the present study to determine whether NK cells contribute to the dysregulated immune response that accompanies septic shock. Methods. An analysis of the lethality of Escherichia coli CP9 was conducted in mice that had been depleted of NK cells via the injection of an anti-asialo GM1 antibody and in CD ϵ transgenic mice that are deficient in both NK cells and T cells. The 2 groups of mice were analyzed for serum levels of interferon-γ, tumor necrosis factor-α, and interleukin-1β as well as activation of NFκB and STAT1, 2 proinflammatory transcription factors. Results. NK cell-depleted and NK cell-deficient mice exhibited 80% survival in the face of an intraperitoneal bacterial challenge, whereas control mice all died within 12 hours. Serum levels of proinflammatory cytokines were markedly reduced in NK-depleted mice. NF κB and STAT1 activation were also reduced. NK-depleted mice exhibited less inflammation within multiple organs on histologic analysis. Conclusions. These results show that NK cells may contribute to the lethality of bacterial infections via effects on cytokine production. (Surgery 2002;132:205-12.)

Activation of macrophages by microbes results in the rapid production of monokines (e.g., interleukin-12 (IL-12), IL-15, and IL-18), which induce production of interferon-gamma (IFN-gamma) by natural killer (NK) cells. We examined the effects of administering IL-15 in combination with IL-12 in a murine toxicity model to determine how these two cytokines might contribute to the inflammatory state that accompanies infectious processes. The daily, simultaneous administration of IL-15 (3 x 10(5)U) and IL-12 (1 microg) to normal mice resulted in shock and 100% mortality within 3-7 days, whereas minimal toxicity was observed following the administration of IL-15 or IL-12 alone. Mice treated with IL-15 plus IL-12 exhibited lesions of the gastrointestinal tract, elevated serum levels of acute phase reactants and pro-inflammatory cytokines, and NK cell apoptosis. Neutralization of IFN-gamma, TNF-alpha, and IL-1beta was not protective in cytokine-treated mice, however, toxicity and death could be completely abrogated by depletion of NK cells. Mice deficient in the STAT4 transcription factor also exhibited complete protection while mice deficient in IFN-gamma or its downstream mediator, STAT1, did not. These findings suggest that cytokine- stimulated NK cells are able to promote systemic inflammation via the induction of STAT4-responsive genes other than IFN-gamma or TNF-alpha.

CD19-CAR T cells have shown antitumor activity for B-cell derived malignancies, but they also have shown limitations regarding their toxicity and development of CD19- escape variants. Given their short lifespan and potent cytolytic function NK cells are attractive candidate effectors to express CARs. The goal of this project was to devise a strategy to genetically modify NKs that express a CAR to target CD22 and secrete a CD19-specific T-cell engager (CD19-ENG) to redirect T cells to B-cell leukemias. NKs were transduced with a retroviral vector encoding a CD22-CAR and/or CD19-ENG molecules. To mimic immune escape, CD19 or CD22 knockout (ko) leukemia cells (BV173) were generated. We performed coculture and cytotoxicity assays using CD22 and/or CD19+ targets with NKs +/− T cells. In the absence of T cells, CD22-CAR +/− CD19-ENG NK cells recognized tumor cells in an antigen-dependent manner as judged by cytokine production and tumor killing. Moreover, CD22 CAR +/− NK cells secreting engager molecules efficiently redirected T cells in the presence of CD19+ targets. In vivo, CD22-CAR/CD19-ENG NK cells recognized tumor cells in an antigen-dependent manner, redirected T cells to tumor cells, and induced significant regression of leukemia in xenograft models in comparison to mice treated with CD22-CAR NK cells or NK cells targeting irrelevant antigens. We have generated for the first time NK cells that kill B-cell malignancies through a CAR and simultaneously redirect bystander T cells to a 2nd B-cell antigen to enhance antitumor effects and prevent immune escape. Genetic modification of NK cells to enhance their antitumor activity and redirect bystander T cells may represent a promising addition to current cell therapies for B-cell malignancies.

Natural Killer (NK) cells are large granular lymphocytes that are uniquely equipped to promote the anti-tumor response via communication with other immune cell types in the tumor microenvironment. We have shown that NK cells secrete a distinct profile of immune stimulatory factors e.g., interferon-gamma (IFN-γ) with potent anti-tumor activity in response to dual stimulation with antibody (Ab)-coated tumor cells and cytokines, such as interleukin (IL)-12 and that this response is significantly enhanced 10 fold in the presence of autologous monocytes (p < 0.05). Similar results were obtained when NK cell secretion of TNF-alpha (a) and MIP1-a were examined. This enhancement of the NK cell cytokine response by monocytes held true for every known stimulator of NK cell IFN-γ production. Further, autologous monocytes significantly increased the ability of IL-12 activated NK cells to lyse Trastuzumab-coated breast cancer cells in an ADCC assay (p < 0.05). Monocyte enhancement of NK cell activity was shown to be dependent on direct cell-cell contact as determined by a transwell assay. We hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of the stimulatory ligand MICA on monocytes to the NKG2D receptor on NK cells. Activation of monocytes with bacterial components (e.g., LPS) or IFN-a led to increased surface expression of the NKG2D ligand MICA and further enhanced the ability of monocytes to act as stimulators of NK cell cytokine secretion. The stimulatory effects of MICA-positive monocytes were duplicated via use of a MICA over-expressing cell line (C1R-MICA) (inducing a 4 fold increase in IFN-γ production) but not the parental MICA-negative cell line. Down-modulation of MICA via siRNA or incubation with a MICA neutralizing FAb fragment prior to co-culture of monocytes with NK cells led to a significant reduction in NK cell IFN-γ secretion (p < 0.05). Blockade of NKG2D on NK cells with a neutralizing Ab also reduced IFN-γ production in this co-culture system. In vivo, depletion of monocytes from Balb/c mice resulted in decreased IFN-γ production by murine NK cells upon exposure to Trastuzumab-coated tumor cells and IL-12. Extended depletion of monocytes in vivo abrogated the immune response to combination therapy with Trastuzumab and IL-12, resulting in significantly larger tumors in monocyte-depleted mice in comparison to mock-depleted controls (p < 0.02). These data suggest that direct cell-cell interaction between NK cells and monocytes may constitute an important mechanism for enhancement of the NK cell anti-tumor response in the setting of monoclonal Ab therapy for cancer.

The Her2 / neu (c-erbB-2) oncogene encodes a 185-kDa protein tyrosine kinase which is overexpressed in 20 % of breast adenocarcinomas and is recognized by a humanized anti-Her2 / neu monoclonal antibody (mAb) (rhu4D5 or Herceptin). Natural killer (NK) cells are capable of mediating antibody-dependent cell cytotoxicity (ADCC) against antibody-coated targets via their expression of a low-affinity receptor for IgG (FcγRIII or CD16). NK cells can be expanded in cancer patients via the administration of low-dose interleukin-2 (IL-2) and become potent cytotoxic effectors following exposure to high doses of IL-2. We tested IL-2-activated NK cells against Her2 / neu+ (MCF-7Her2 / neu) and Her2 / neu– (MDA-468) breast cancer cell lines in a 4-h 51Cr-release cytotoxicity assay in the presence or absence of rhu4D5 mAb (effector : target ratio = 10 : 1). Specific lysis of rhu4D5-coated MCF-7Her2 / neu and MDA-468 target cells by IL-2-activated NK cells was 35 % and 3 %, respectively (p

Background: T-cell therapy with CD19-CAR T cells has been very successful for the treatment of B-cell derived malignancies in humans. However, cytokine release syndrome, neurotoxicity, and development of CD19- escape variants have emerged as potential limitations. Developing CAR NK-cell based therapies might overcome some of these side effects since NK cells do not rapidly expand or persist long-term after adoptive transfer. However, CAR NK-cell therapies are less effective than CAR T-cell therapies in preclinical models. To overcome these limitations we have devised a strategy to genetically modify NK cells with CD22-CARs and CD19/CD3-bispecific T-cell engager (CD19-ENG) molecules. These NK cells should not only kill CD22+ B cells directly, but also redirect bystander T cells to malignant CD19+ B cells, enhancing antitumor effects and preventing immune escape. Methods: NK cells were generated using K562s expressing 41BBL and membrane bound IL15, and genetically modified with a retroviral vector encoding a CD22-CAR with a 41BB.ζ endodomain and/or a retroviral vector encoding CD19-ENG and mOrange separated by an IRES. To mimic immune escape, CD19 or CD22 knockout (ko) Ph+ leukemia cells (BV173) were generated by CRISPR/cas9. The effector function of genetically modified NK cells was evaluated using standard immunological assays. Results: After transduction 70-80% of NK cells expressed CD22-CARs, and ~50% expressed CD22-CARs and CD19-ENGs as judged by FACS analysis. We performed coculture and cytotoxicity assays using non-transduced (NT), CD22-CAR, CD19-ENG, and CD22-CAR/CD19-ENG NK cells as effectors and BV173 (CD19+/CD22+), BV173.koCD19, BV173.koCD22, Daudi (CD19+/CD22+), and KG1a (CD19-,CD22-) as targets. Cocultures were preformed +/- T cells and after 24 hours IFNγ and IL2 was determined by ELISA. In the absence of T cells, CD22-CAR and CD22-CAR/CD19-ENG NK cells only recognized CD22+ targets as judged by IFNγ production. Moreover, CD22-CAR/CD19-ENG and CD19-ENG NK cells efficiently redirected T cells to secrete IFNγ in the presence of CD19+/CD22- targets. No NK-cell population produced IL2, however CD22-CAR/CD19-ENG and CD19-ENG NK cells induced IL2 production of T cells in the presence of CD19+ targets. No significant cytokine production was observed in the absence of antigen (media, KG1a). Specificity of generated NK cells was confirmed in cytotoxicity assays. In vivo studies to confirm our in vitro findings are in progress. Conclusions: We have generated for the first time NK cells that kill B-cell malignancies through a CAR (CD22) and simultaneously redirect bystander T cells to a 2nd B-cell antigen (CD19) to enhance antitumor effects and prevent immune escape. Genetic modification of NK cells to enhance their antitumor activity and redirect bystander T cells may present a promising addition to current cell therapies for B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26HER2/neu) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ–deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4+ or CD8+ T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26HER2/neu tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.

Background: Myeloid derived suppressor cells or MDSC are a heterogeneous population of bone marrow-derived cells that consist of myeloid progenitor and immature. Cancer-induced pro-inflammatory signals recruit MDSC from the bone marrow and maintain them in an undifferentiated state. MDSCs inhibit T cell proliferation via up regulation of iNOS and ROS thereby suppressing antitumor immune responses. MDSC have also been shown to promote tumor growth by stimulating angiogenesis and other mechanisms. TGF-b1 is a pleiotropic cytokine abundantly expressed in the tumor microenvironment with diverse effects on myeloid, lymphoid and tumor cells. The aim of this study is to determine the effect of TGF-b1 in the generation and function of MDSC, including its effects on T cell proliferation and tumor growth. Methods: Ex vivo MDSC generation: Bone marrow progenitor cells were derived from WT C57bl/6 mice and co-cultured with MTEC (mouse tonsil epithelial cells transformed with HPV16 E6+E7 oncogenes and H-Ras ) tumor supernatants in the presence or absence of TGF-β1 for 5 days at 370C. Cells were then harvested, processed into single cell suspensions, and stained for MDSC surface markers, DAF-DA (to determine nitric oxide levels),iNOS and other functional markers analysis was performed by flow cytometry (FACS). MDSC functional assay: A) T cell proliferation assay MDSCs were generated in the presence or absence of TGF-β1 with supernatants from MTEC cells and co-cultured with CFSE labeled T cells activated with anti CD3 and anti CD28 antibodies. T cell proliferation was measured by using CFSE dilution, which was analyzed by flow cytometry. B) Effect of MDSC on tumor growth Control and TGF-β1 conditioned MDSC were co-cultured with MTEC tumor (grown as spheroids) for 72 hrs at the end of which histological sections of the spheroids were prepared and analyzed for tumor proliferation by Ki-67 staining. Sorted MDSCs were also co-cultured with T-hep3 cells grown in a monolayer (human oral cancer line) and tumor growth was determined by flow cytometry. Results: While control MDSC suppressed T cell proliferation in a dose-dependent fashion, we observed that TGF-β1 primed MDSCs lost the ability to inhibit T cell proliferation. Further, TGF-β1 primed MDSC inhibited tumor growth in an ex vivo co-culture system. Histological sections of tumor spheroid / MDSC co-cultures revealed diminshed ki-67 expression in spheroids cultured with TGF-B1 conditioned MDSC compared to control. Upon further examining the cellular mechanism, it was seen that TGF-β1 treated MDSCs down regulate iNOS expression and produced decreased amounts of nitric oxide compared to their control counterparts, without altering the expression of other MDSC functional markers like arginase, PD-1 and PD-L1. Conclusions: We conclude that TGF-β1 reprograms MDSC via an iNOS/NO dependent mechanism to a) T cell suppressive capacity and b) inhibit tumor cell growth. These observations have a direct translational implication wherein the inherent pro-tumor nature of MDSCs could potentially be reprogrammed with TGF-β1 and directed toward the tumor thereby suppressing tumor growth. Citation Format: Padmini Jayaraman, Falguni Parikh, Rosemarie Krupar, Robin Parihar, Indu Varier, Andrew Sikora. TGF-beta1 primed myeloid derived suppressor cells decrease tumor growth and lose their ability to inhibit T cell proliferation via iNOS downregulation. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr PR005.