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Inadequate persistence of tumor-infiltrating natural killer (NK) cells is associated with poor prognosis in cancer patients. The solid tumor microenvironment is characterized by the presence of immunosuppressive factors, including prostaglandin E2 (PGE2), that limit NK cell persistence. Here, we investigate if the modulation of the cytokine environment in lung cancer with IL-2 or IL-15 renders NK cells resistant to suppression by PGE2. Analyzing Cancer Genome Atlas (TCGA) data, we found that high NK cell gene signatures correlate with significantly improved overall survival in patients with high levels of the prostaglandin E synthase (PTGES). In vitro, IL-15, in contrast to IL-2, enriches for CD25(+)/CD54(+) NK cells with superior mTOR activity and increased expression of the cAMP hydrolyzing enzyme phosphodiesterase 4A (PDE4A). Consequently, this distinct population of NK cells maintains their function in the presence of PGE2 and shows an increased ability to infiltrate lung adenocarcinoma tumors in vitro and in vivo. Thus, strategies to enrich CD25(+)/CD54(+) NK cells for adoptive cell therapy should be considered.

Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor postinfusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK-cell functions following cytokine withdrawal to model postinfusion performance. In contrast to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided cell function advantages. Genome-wide analysis of cytosolic and polysome-associated messenger RNA (mRNA) revealed not only cytokine-dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mammalian target of rapamycin (mTOR) signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15-induced cell function advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK-cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15-stimulated NK cells was also observed using a clinically applicable protocol for NK-cell expansion in vitro and in vivo. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B-cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR-regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK-cell cancer therapy.

Dendritic cell (DC) vaccines induce T-cell responses in cancer patients. However, there is a paucity of data regarding the role of DC vaccines in shaping natural killer (NK) cell responses. Here, we observe that NK cells are less activated following DC vaccination. In vitro, DC-mediated inhibition of NK cells did not require cell-to-cell contact, but required increased Signal transducer and activator of transcription 3 (STAT3) phosphorylation (pSTAT3) in DCs. When phosphorylation of STAT3 was inhibited in DCs, we found that DCs did not suppress NK cells, and observed an increase in the production of lymphotoxin-alpha (LTα) and interleukin-12 (IL-12) as well as reduced release of transforming growth factor beta (TGF-β). The addition of recombinant LTα or IL-12 to the DC-NK-cell cocultures restored NK-cell activity, and neutralization of TGF-β resulted in elevated production of LTα and IL-12 from DCs. Compared with LPS, DCs matured with a cocktail of R848, poly I:C, and IFN-γ showed reduced levels of pSTAT3 and higher levels of LTα and IL-12 and did not inhibit NK-cell activity. These results show that LTα, IL-12, and TGF-β are involved in the cross-talk between NK cells and DCs. Our findings have important implications for the development of DC-based vaccination strategies to potentiate NK-cell responses in patients with cancer.

Purpose: Increased frequencies of myeloid-derived suppressor cells (MDSC) correlate with poor prognosis in patients with cancers. Tumor-derived prostaglandin-E2 (PGE2) plays an important role in inducing MDSCs. However, the detailed mechanisms of this induction remain unknown. To develop targeted therapies for MDSCs, we sought to investigate the molecular basis of PGE2-regulated accumulation of MDSCs and their functional consequence on natural killer (NK) cell activity. Experimental Design: The effects of PGE2 in inducing phenotypic, signaling, and functional alternations on monocytes were analyzed in vitro. Suppression of NK-cell activity by PGE2-treated monocytes was compared with that of freshly isolated CD14+HLA-DRlow/− monocytic MDSCs (moMDSC) from patients with melanoma. In addition, to explore the in vivo relevance of targeting PGE2 to reduce MDSC-mediated suppression of NK cells, we established a murine model, where tumor cells were disabled from cyclooxygenase-2 (COX-2) production. Results: Patient-derived moMDSCs inhibited NK-cell activity through the production of TGFβ. In vitro, binding of PGE2 to EP2 and EP4 receptors on monocytes activated the p38MAPK/ERK pathway and resulted in elevated secretion of TGFβ. Similar to moMDSCs, PGE2-treated monocytes potently suppressed NK-cell activity through production of TGFβ. Furthermore, silencing COX-2 in murine 4T1 tumor cells reduced the accumulation of CD11b+Gr1+ MDSCs in the spleen, resulting in concomitant improved in vivo clearance of NK-cell sensitive YAC-1 cells. Conclusions: Our results reveal an indispensable role of tumor-derived PGE2 in inducing MDSCs and suggest a favorable outcome of combining COX-2–targeted therapy and adoptive NK-cell transfer in patients with cancer. Clin Cancer Res; 20(15); 4096–106. ©2014 AACR.

Tumour-induced immune dysfunction is a serious challenge to immunotherapy for cancer, and intact adaptive and innate cellular immunity is key to its success. Myelomonocytic cells have a central role in this immune suppression, and tumour-associated macrophages, eosinophils, neutrophils and myeloid-derived suppressor cells have all been shown to be of major importance. These myelomonocytic cells secrete a broad repertoire of inflammatory mediators providing them with powerful tools to inhibit tumour-reactive T cells and natural killer cells; free oxygen radicals including reactive oxygen species and NO, arginase, indoleamine 2,3-dioxygenase, prostaglandins, the pro-inflammatory heterodimer S100A8/9 and cytokines, such as granulocyte-macrophage colony-stimulating factor and transforming growth factor-β, have proven particularly potent in suppressing antitumour cellular immunity. Determining which of these factors prevail in individual cancer patients and designing methods aimed at neutralization or inhibition of their effects on target tissues have the potential to greatly enhance the clinical efficacy of immunotherapy.

Blocking the immune checkpoint molecule CTL antigen-4 (CTLA-4) with ipilimumab has proven to induce long-lasting clinical responses in patients with metastatic melanoma. To study the early response that takes place after CTLA-4 blockade, peripheral blood immune monitoring was conducted in five patients undergoing ipilimumab treatment at baseline, three and nine weeks after administration of the first dose. Along with T-cell population analysis, this work was primarily focused on an in-depth study of the myeloid-derived suppressor cell (MDSC) populations. Ipilimumab treatment resulted in lower frequencies of regulatory T cells along with reduced expression levels of PD-1 at the nine-week time point. Three weeks after the initial ipilimumab dose, the frequency of granulocytic MDSCs was significantly reduced and was followed by a reduction in the frequency of arginase1-producing CD3− cells, indicating an indirect in trans effect that should be taken into account for future evaluations of ipilimumab mechanisms of action. Cancer Immunol Res; 1(3); 158–62. ©2013 AACR.

Tumors can suppress the host immune system by employing a variety of cellular immune modulators, such as regulatory T cells, tumor-associated macrophages, and myeloid-derived suppressor cells (MDSC). In the peripheral blood of patients with advanced stage melanoma, there is an accumulation of CD14+HLA-DRlo/− MDSC that suppress autologous T cells ex vivo in a STAT-3–dependent manner. However, a precise mechanistic basis underlying this effect is unclear, particularly with regard to whether the MDSC induction mechanism relies on cell–cell contact of melanoma cells with CD14+ cells. Here, we show that early-passage human melanoma cells induce phenotypic changes in CD14+ monocytes, leading them to resemble MDSCs characterized in patients with advanced stage melanoma. These MDSC-like cells potently suppress autologous T-cell proliferation and IFN-γ production. Notably, induction of myeloid-suppressive functions requires contact or close proximity between monocytes and tumor cells. Further, this induction is largely dependent on production of cyclooxygenase-2 (COX-2) because its inhibition in these MDSC-like cells limits their ability to suppress T-cell function. We confirmed our findings with CD14+ cells isolated from patients with advanced stage melanoma, which inhibited autologous T cells in a manner relying up prostaglandin E2 (PGE2), STAT-3, and superoxide. Indeed, PGE2 was sufficient to confer to monocytes the ability to suppress proliferation and IFN-γ production by autologous T cells ex vivo. In summary, our results reveal how immune suppression by MDSC can be initiated in the tumor microenvironment of human melanoma. Cancer Res; 73(13); 3877–87. ©2013 AACR.

Myeloid-derived suppressor cells (MDSC) are important regulators of the immune system and key players in tumor-induced suppression of T-cell responses. CD14+HLA-DR-/low MDSC have been detected in a great number of malignancies, including melanoma. MDSC are known to be impaired in their ability to differentiate along the myeloid lineage, e.g., into dendritic cells (DC). This is a concern for utilization of monocyte-derived DC for vaccination of patients with melanoma or other cancers exhibiting accumulation of CD14+ MDSC. When producing DC according to standard operating procedures of two currently ongoing clinical trials, we found that MDSC co-purified with monocytes isolated by elutriation. MDSC frequencies did not affect yield or viability of the produced DC, but induced a dose-dependent decrease in DC maturation, ability to take up antigen, migrate and induce T-cell IFNγ production. Changes in DC characteristics were most notable when 'pathological' frequencies of >50% CD14+HLA-DR- cells were present in the starting culture. The impaired DC quality could not be explained by altered cytokine production or increased oxidative stress in the cultures. Tracking of HLA-DR- cells throughout the culture period revealed that the observed changes were partially due to the impaired maturation and functionality of the originally HLA-DR- population, but also to their negative effects on HLA-DR+ cells. In conclusion, MDSC could be induced to differentiate into DC but, due to the impairment of overall DC vaccine quality when >50% HLA-DR- cells were present in the starting culture, their removal could be advisable.

Meeting abstracts Cancer progression is associated with a severe impairment of anti-tumor immune responses. The accumulation of peripheral blood CD14+HLA-DRlo/- monocytic MDSCs has been identified as a prognostic factor in advanced stage melanoma patients. These cells suppress T cell functions ex

Oncogenes have been traditionally viewed as molecular drivers for tumor growth and survival. Recent evidence indicates that oncogenes may facilitate the escape of malignant cells from immune recognition and elimination. In this article, we discuss the implications of the overexpression of epidermal growth factor receptor (EGFR) family members on immune escape of tumors and immunotherapy.

Overexpression of the receptor tyrosine kinases HER2 and HER3 is associated with a poor prognosis in several types of cancer. Presently, HER2- as well as HER3-targeted therapies are in clinical practice or evaluated within clinical trials, including treatment with mAbs mediating growth inhibition and/or activation of Ab-induced innate or adaptive cellular immunity. A better understanding of how HER2/HER3 signaling in tumors influences cellular immune mechanisms is therefore warranted. In this study, we demonstrate that HER2/HER3 signaling regulates the expression of MHC class I-related chain A and B (MICA and MICB) in breast cancer cell lines. The MICA and MICB (MICA/B) molecules act as key ligands for the activating receptor NK group 2, member D (NKG2D) and promote NK cell-mediated recognition and cytolysis. Genetic silencing of HER3 but not HER2 downregulated the expression of MICA/B, and HER3 overexpression significantly enhanced MICA expression. Among the major pathways activated by HER2/HER3 signaling, the PI3K/AKT pathway was shown to predominantly regulate MICA/B expression. Treatment with the HER3-specific ligand neuregulin 1β promoted the expression in a process that was antagonized by pharmacological and genetic interference with HER3 but not by the ataxia-telangiectasia–mutated (ATM) and ATM and Rad3-related protein kinases inhibitor caffeine. These observations further emphasize that HER2/HER3 signaling directly, and not via genotoxic stress, regulates MICA/B expression. As anticipated, stimulating HER2/HER3 enhanced the NKG2D-MICA/B–dependent NK cell-mediated cytotoxicity. Taken together, we conclude that signaling via the HER2/HER3 pathway in breast carcinoma cell lines may lead to enhanced NKG2D-MICA/B recognition by NK cells and T cells.

Suppressive factors produced by tumors or tumor-associated cells represent a major obstacle to immune-mediated tumor eradication and immunotherapy. We studied tumor-dependent changes in expression of the ζ-chain, a key molecule for the transduction of stimulatory signals through the T-cell receptor and activating receptors on natural killer (NK) cells, in patients with early (stages 1 and 2) breast cancer. Ex-vivo levels of ζ-chain expression, proliferation and cytokine expression in lymphocyte subpopulations were measured in breast tumors, their draining lymph nodes and in pre- and postoperative blood samples of cancer patients and healthy controls by multi-parametric flow cytometry. We found that T-cell ζ-chain expression in peripheral blood of breast cancer patients (n = 29) was down-regulated compared with healthy controls (n = 10) (p ≤ 0.033), which was most pronounced in stage 2 (n = 15, p ≤ 0.004). T- and NK-cell ζ-chain loss was most distinct in the tumor and decreased with increasing distance (p ≤ 0.015). After surgical tumor resection, peripheral blood ζ-chain levels normalized to those observed in healthy controls. ζ-chain expression in peripheral blood T-cells correlated with lymphocytic proliferative activity (p ≤ 0.035), and with the expression of proinflammatory cytokines in CD8+ T-cells (p ≤ 0.035). Breast cancer patients had lower numbers of circulating early memory CD8+ T-cells than healthy donors (p ≤ 0.000), correlating with lower T-cell ζ-chain levels (p ≤ 0.011). Our findings suggest that phenotypic and functional alterations in lymphocytes can be detected even in early stage breast cancer patients. The observed immunosuppression appears to be systemic and tumor dependent, as it is strongest in the tumor, correlates with tumor stage and normalizes after surgery.

Dendritic cells (DC) induce T-cell responses in cancer patients. However, less is known about the role of DC-vaccines in shaping natural killer (NK) cell responses. In this study, we sought to investigate the activity of NK cells following vaccination with DC. Patients with chronic lymphocytic leukemia (CLL) and melanoma were vaccinated with tumor-antigen loaded monocytes-derived DC and NK cell activity was analyzed before and two weeks after vaccination. Compared with pre-vaccination samples, NK cells expressed reduced levels of several activation markers and produced less IFNγ (p=0.03). To further explore the mechanism of the reduced NK cell responses, DC were differentiated from monocytes in GM-CSF, IL-4 and LPS and thereafter co-cultured with autologous NK cells in vitro. In these experiments, DC-mediated inhibition of NK cells was independent of cell-contact but dependent on the phosphorylation status of STAT3 in DC. Compared with untreated DC, STAT3-inhibited-DC produced higher amounts of lymphotoxin-alpha (LTα, 5.5±5.0 vs. 38.1±25.9 pg/ml; p=0.005) and IL-12 (29.6±39.0 vs. 176.8±96.8 pg/ml; p=0.005) and reduced amounts of tumor growth factor-beta (TGFβ, 495.0±138.7 vs. 300.4±141.8 pg/ml; p=0.03) and did not suppress NK cell proliferation (p=0.03), cytotoxicity (p=0.0003), and IFNγ production (p=0.02). Neutralization of TGFβ resulted in elevated production of LTα (2.25±4.5 vs. 251.5±12.0 pg/ml; p=0.03) and IL-12 (0.25±0.5 vs. 15.0±1.82 pg/ml; p=0.02) by DC and addition of recombinant LTα or IL-12 in co-cultures of DC and NK cells was indispensable to maintain NK cell activity. Compared with LPS, DC matured with a cocktail of R848, poly I:C, and IFNγ showed reduced levels of pSTAT3 and produced higher levels of LTα and IL-12 and did not inhibit NK cell activity. These results show that LTα, IL-12 and TGFβ are involved in the cross-talk between NK cells and DC. Our findings have important implications for the development of DC-based vaccination strategies to potentiate NK-cell responses in patients with cancer. Disclosures Österborg: Celgene: DMC memeber of Celgene-initiated phase 3 trials Other.

Cyclooxygenase-2 and its product PGE2 play essential roles in inducing and maintaining the suppressive functions of MDSCs in cancer. Blockade of the COX-2/PGE2 pathway abolishes the induction of MDSCs in response to tumor-derived factors and thus may provide new opportunities for targeting MDSCs in cancer patients.

Tumors can suppress the host immune system through a variety of cellular mechanisms, such as regulatory T cells, tumor-associated macrophages and most recently discovered, myeloid-derived suppressor cells (MDSCs). We have previously demonstrated that CD14+HLA-DRlo/- cells accumulate in the peripheral blood of advanced melanoma patients and can suppress autologous T cells ex vivo in a STAT-3-dependent manner. However, the role of MDSCs in the melanoma tumor microenvironment remains to be elucidated. In the current study, early-passage human melanoma cell lines induce significant phenotypic changes on CD14+ monocytes resembling MDSCs in advanced stage melanoma patients. Moreover, such melanoma-educated monocytes strongly impair both autologous T cell proliferation and interferon-γ production. Of note, the induction of the suppressive functions in healthy donor-derived monocytes appears to require tumor contact and/or close proximity to the tumor cells, and is largely dependent on cyclooxygenase-2 (Cox-2) production and activation of STAT-3 signaling pathway. Intriguingly, we have also demonstrated that these two mechanisms are directly involved in the T cell suppression induced by these MDSC-like cells. Last but not least, CD14+ cells isolated ex vivo from advanced stage melanoma patient PBMCs (n=5) exhibited inhibition of autologous T cell functions in a dose dependent manner. Blockade of STAT-3 signaling (p=0.0358, n=4) and superoxide production (p=0.0195, n=4) significantly alleviated the T cell suppression. In conclusion, our data demonstrated the importance of STAT-3 and Cox-2 activity in inducing CD14+ monocytes to exhibit MDSC-like properties, which contributes to our knowledge of how immune suppression is initiated in the human melanoma tumor microenvironment. Citation Format: Yumeng Mao, Isabel Poschke, Yago Pico de Coaña, Erik Wennerberg, Andreas Lundqvist, Rolf Kiessling. Melanoma-educated CD14+ monocytes become myeloid-derived suppressor cell-like and are potent inhibitors of autologous T cells through Cox-2 production and STAT-3 signaling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A63.

The aim of this project was to generate cytotoxic T lymphocytes (CTL) specific for the weakly immunogenic MAGE-A4 antigen, by an alloreactive approach. CTL may recognize and eliminate tumor cells. However, to avoid generation of autoreactive CTL, most CTL specific for tumor-associated antigens (TAA) are eliminated by negative selection during their development in the thymus. Thus, compared to virus-specific CTL, TAA-specific CTL precursors are often rare and more challenging to expand from peripheral blood. One TAA that is difficult to raise specific CTL against is the MAGE-A4 antigen, a cancer-testis antigen expressed by many tumors but not normal tissues. A MAGE-A4 230-239 peptide is processed (Duffour et al. Eur J Immunol 1999;29;3329–37) and presented by the HLA-A2 complex (Hilling et al. J Mol Biol 2001;310:1167-76). Still, rarely have MAGE-A4 230-239 specific CTL been found after in vitro stimulation or in vivo vaccination. We speculated that the reason was elimination of basically all precursors by negative selection. To get around this, we transfected HLA-A2 into mature dendritic cells (moDC) generated from HLA-A2 negative donor monocytes. In contrast to previous studies using this approach for other TAA (Stronen et al. Scand J Immunol 2009;69:319-28), we used the HLA-A2 gene in a plasmid instead of the HLA-A2 mRNA for transfection. This approach excludes in vitro transcription and avoids RNA stability issues. The transfection efficiency with our method was between 25 to 50% after 24 hours. We pulsed the HLA-A2 transfected moDC with peptide and co-cultured them with autologous CD8 T cells. As these CD8 T cells have not encountered HLA-A2 during development, they should contain higher frequencies of precursors specific for peptides presented by this HLA allele. Thus, the setting is allogeneic only for peptides presented by HLA-A2, and will from hereon be referred to as the HLA-A2 allogenic setting. As a control, we used the optimized MART-1 26-35 (A27L) peptide, known to generate potent in vitro responses even in CD8 T cells from healthy HLA-A2 positive donors using an autologous setting. We managed to expand MART-1 specific CTL in vitro from 50% of healthy HLA-A2 positive donors (2/4), when using PBMC and in every donor when using moDC (3/3) as antigen-presenting cells (APC). Thus it was not surprising that we could also generate MART-1 specific cells from all donors with the HLA-A2 allogeneic setting (3/3). In the case of MAGE-A4, we have never succeeded to generate specific CTL from HLA-A2 positive donors, neither with peptide-pulsed PBMC (0/8) nor with moDC (0/3) as APC. Therefore, it is very promising that we were able to generate MAGE-A4 specific cells from almost half of the donors (2/5) with the HLA-A2 allogenic setting. More donors will be included to determine the efficiency of the method. The MAGE-A4 and MART-1 specific cells generated by the HLA-A2 allogeneic approach were cloned and functionally tested. The clones for both specificities were similarly capable of killing peptide-pulsed target cells, for some at a high efficiency (80% lysis). MART-1 clones generally required less peptide for lysis (picomolars) than MAGE-A4 clones (nanomolars). This may be due to superior binding of the optimized MART-1 26-35 (A27L) peptide to the HLA-A2. The clones will be further phenotyped and tested for other effector functions such as cytokine production and lysis of MAGE-A4 expressing tumor-cells. We conclude that functional MAGE-A4 230-239 specific CTL can be generated by the HLA-A2 allogeneic method. Citation Format: Tanja Lövgren, Yumeng Mao, Maxi-Lu Böschen, Eva B. Allerbring, Adnane Achour, Isabel Poschke, Rolf Kiessling. Generation of MAGE-A4 230-239 peptide-specific cytotoxic T lymphocytes using an alloreactive approach. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B70.

We report that antibodies can be spontaneously loaded in functionalized mesoporous silica (FMS) with superhigh density (0.4−0.8 mg of antibody/mg of FMS) due to their comprehensive noncovalent interaction. The superhigh loading density and noncovalent interaction between FMS and antibodies allow long-lasting local release of the immunoregulatory molecules from FMS under physiological conditions. Preliminary data indicate that FMS-anti-CTLA4 antibody injected directly into a mouse melanoma induces much greater and extended inhibition of tumor growth than the antibody given systemically. Our findings open up a novel approach for local delivery of therapeutically active proteins to tumors and, potentially, other diseases.