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Agonistic αCD40 therapy has been shown to inhibit cancer progression in only a fraction of patients. Understanding the cancer cell–intrinsic and microenvironmental determinants of αCD40 therapy response is therefore crucial to identify responsive patient populations and to design efficient combinatorial treatments. Here, we show that the therapeutic efficacy of αCD40 in subcutaneous melanoma relies on preexisting, type 1 classical dendritic cell (cDC1)–primed CD8+ T cells. However, after administration of αCD40, cDC1s were dispensable for antitumor efficacy. Instead, the abundance of activated cDCs, potentially derived from cDC2 cells, increased and further activated antitumor CD8+ T cells. Hence, distinct cDC subsets contributed to the induction of αCD40 responses. In contrast, lung carcinomas, characterized by a high abundance of macrophages, were resistant to αCD40 therapy. Combining αCD40 therapy with macrophage depletion led to tumor growth inhibition only in the presence of strong neoantigens. Accordingly, treatment with immunogenic cell death–inducing chemotherapy sensitized lung tumors to αCD40 therapy in subcutaneous and orthotopic settings. These insights into the microenvironmental regulators of response to αCD40 suggest that different tumor types would benefit from different combinations of therapies to optimize the clinical application of CD40 agonists.Significance:This work highlights the temporal roles of different dendritic cell subsets in promoting CD8+ T-cell–driven responses to CD40 agonist therapy in cancer.

Supplementary Figure from Efficacy of CD40 Agonists Is Mediated by Distinct cDC Subsets and Subverted by Suppressive Macrophages

Supplementary Data from Efficacy of CD40 Agonists Is Mediated by Distinct cDC Subsets and Subverted by Suppressive Macrophages

PDF file - 702K, RIG-I expression in human pancreatic carcinoma specimens

PDF file - 57K, Sequences of DNA templates for in vitro transcription of ppp-RNAs and sequences of siRNAs

PDF file - 60K, CXCL10 production and lymphocyte activation induced by ppp TGF-β treatment is independent of TRIF or TLR7 signaling

PDF file - 42K, Effect of RNA treatment on splenic MDSC populations in tumor-bearing mice

PDF file - 45K, Toxicological assessment of systemic RNA treatment

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Deregulated TGF-β signaling in pancreatic cancer promotes tumor growth, invasion, metastasis, and a potent immunosuppressive network. A strategy for disrupting this tumor-promoting pathway is silencing TGF-β by siRNA. By introducing a triphosphate group at the 5′ end of siRNA (ppp-siRNA), gene silencing can be combined with immune activation via the cytosolic helicase retinoic acid-inducible gene I (RIG-I), a ubiquitously expressed receptor recognizing viral RNA. We validated RIG-I as a therapeutic target by showing that activation of RIG-I in pancreatic carcinoma cells induced IRF-3 phosphorylation, production of type I IFN, the chemokine CXCL10, as well as caspase-9–mediated tumor cell apoptosis. Next, we generated a bifunctional ppp-siRNA that combines RIG-I activation with gene silencing of TGF-β1 (ppp-TGF-β) and studied its therapeutic efficacy in the orthotopic Panc02 mouse model of pancreatic cancer. Intravenous injection of ppp-TGF-β reduced systemic and tumor-associated TGF-β levels. In addition, it induced high levels of type I IFN and CXCL10 in serum and tumor tissue, systemic immune cell activation, and profound tumor cell apoptosis in vivo. Treatment of mice with established tumors with ppp-TGF-β significantly prolonged survival as compared with ppp-RNA or TGF-β siRNA alone. Furthermore, we observed the recruitment of activated CD8+ T cells to the tumor and a reduced frequency of CD11b+ Gr-1+ myeloid cells. Therapeutic efficacy was dependent on CD8+ T cells, whereas natural killer cells were dispensable. In conclusion, combing TGF-β gene silencing with RIG-I signaling confers potent antitumor efficacy against pancreatic cancer by breaking tumor-induced CD8+ T cell suppression. Cancer Res; 73(6); 1709–20. ©2013 AACR.

PDF file - 46K, Bi-functional ppp-siRNA directed against TGF-β1 combines gene silencing with RIG-I signaling in human pancreatic cancer cells

Background: It is hypothesized that TLR7 agonists reprogram myeloid cells in the TME, which may lead to activation of antitumor CD8 T effector cells and subsequently tumor cell death. The orally available TLR7 agonist prodrug RO7119929 is mainly converted into its active form in the liver. Here we report preclinical data informing the clinical proof of mechanism (PoM) strategy for RO7119929, as well as confirmatory clinical data from the first-in-human, Phase I study (NCT04338685). Methods: Prior to the Phase 1 study, bulk RNA sequencing of tumor and liver tissue was performed in a RO7119929 -treated HEP55.1c mouse model and RO7119929-treated cynomolgus monkeys. Single cell and single nucleus RNA-sequencing (snRNA-seq) was also performed in RO7119929 -treated human peripheral blood mononuclear cells (PBMCs). The Phase 1 study subsequently enrolled 55 pts with advanced primary liver cancers or other solid tumors with predominant liver involvement to receive weekly RO7119929. Paired pre- and post-treatment tumor liver biopsies were assessed. Pharmacodynamic (PD) monitoring of proximal and distal TLR7-related PD markers was performed using snRNA-seq, immunohistochemistry (IHC) and flow cytometry. Results: Preclinical mouse, cynomolgus monkey as well as human derived data showed dose-dependent, TLR7 mode of action-related, immune stimulatory reprogramming of PBMCs, the TME and liver tissue via myeloid cell activation. These findings were translated into a Phase I expansion cohort to demonstrate PoM using predefined PD gating criteria. By implementing snRNA-seq of paired liver biopsies from 11 pts, clinical PoM of RO7119929 could be demonstrated on a cellular and cytokine/chemokine level. Macrophages were the predominant immune infiltrate in liver tumor tissue and showed a relative increase in numbers and a trend towards an M1-immune stimulatory phenotype reprogramming on treatment. The local induction of proximal and distal cytokines and chemokines further confirmed a general shift towards a proinflammatory TME. In addition, peripheral CD8 T cell expansion was observed in most pts analyzed. An increase in CD8 tumor-infiltrating lymphocytes (TILs) occurred in 4/9 pts (all presented with stable disease) while most pts with no TIL increase had disease progression as best response. However, a general change towards a predominant CD8 inflamed immune phenotype was not detected. There was no trend for TLR7-induced increase in PD-L1 or MHC class I expression. Conclusions: Single-agent TLR7 agonist treatment of advanced primary or metastatic liver cancers induces local inflammation of the TME by reprogramming myeloid cells. Combination therapies may be needed to increase the relocation of antitumor CD8 T effector cells to the TME and unlock the full potential of the local immune stimulatory effect. Citation Format: Carles Fabregat-Franco, Changhoon Yoo, Bruno Sangro, Camilla Qvortrup, Hyung-Don Kim, Teresa Macarulla, Mariano Ponz-Sarvisé, Chia-Chi Lin, Do-Youn Oh, Thomas Yau, Juliana Bessa, Petra C. Schwalie, Steffen Dettling, Ramona Schlenker, Natascha Riede, Elia Hall, Felix Lichtenegger, Nicole Kratochwil, Thomas Pöschinger, Zhiwen Jiang, Tianyi Jiang, Christina Godfried Sie, Audrey Yeo Te-Ying, Christina Schiff, Sabine Hoves, Michael Cannarile. RO7119929, a TLR7 agonist prodrug, induces local inflammation of the tumor microenvironment (TME) by reprogramming myeloid cells in patients (pts) with advanced primary or metastatic liver cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2342.

Agonistic αCD40 therapy has been shown to inhibit cancer progression in only a fraction of patients. Understanding the cancer cell–intrinsic and microenvironmental determinants of αCD40 therapy response is therefore crucial to identify responsive patient populations and to design efficient combinatorial treatments. Here, we show that the therapeutic efficacy of αCD40 in subcutaneous melanoma relies on preexisting, type 1 classical dendritic cell (cDC1)–primed CD8+ T cells. However, after administration of αCD40, cDC1s were dispensable for antitumor efficacy. Instead, the abundance of activated cDCs, potentially derived from cDC2 cells, increased and further activated antitumor CD8+ T cells. Hence, distinct cDC subsets contributed to the induction of αCD40 responses. In contrast, lung carcinomas, characterized by a high abundance of macrophages, were resistant to αCD40 therapy. Combining αCD40 therapy with macrophage depletion led to tumor growth inhibition only in the presence of strong neoantigens. Accordingly, treatment with immunogenic cell death–inducing chemotherapy sensitized lung tumors to αCD40 therapy in subcutaneous and orthotopic settings. These insights into the microenvironmental regulators of response to αCD40 suggest that different tumor types would benefit from different combinations of therapies to optimize the clinical application of CD40 agonists. Significance: This work highlights the temporal roles of different dendritic cell subsets in promoting CD8+ T-cell–driven responses to CD40 agonist therapy in cancer.

Antiangiogenic and cytotoxic effects are considered the principal mechanisms of action of sorafenib, a multitarget kinase inhibitor approved for the treatment of hepatocellular carcinoma (HCC). We report that sorafenib also acts through direct immune modulation, indispensable for its antitumor activity. In vivo cell depletion experiments in two orthotopic HCC mouse models as well as in vitro analysis identified macrophages (MΦ) as the key mediators of the antitumoral effect and demonstrate a strong interdependency of MΦ and natural killer (NK) cells for efficient tumor cell killing. Caspase 1 analysis in sorafenib-treated MΦ revealed an induction of pyroptosis. As a result, cytotoxic NK cells become activated when cocultured with sorafenib-treated MΦ, leading to tumor cell death. In addition, sorafenib was found to down-regulate major histocompatibility complex class I expression of tumor cells, which may reduce the tumor responsiveness to immune checkpoint therapies and favor NK-cell response. In vivo cytokine blocking revealed that sorafenib efficacy is abrogated after inhibition of interleukins 1B and 18. Conclusion: We report an immunomodulatory mechanism of sorafenib involving MΦ pyroptosis and unleashing of an NK-cell response that sets it apart from other spectrum kinase inhibitors as a promising immunotherapy combination partner for the treatment of HCC.

Colony-stimulating factor 1 receptor (CSF1R) blockade abates tumor-associated macrophage (TAM) infiltrates and provides marked clinical benefits in diffuse-type tenosynovial giant cell tumors. However, facial edema is a common adverse event associated with TAM elimination in patients. In this study, we examined molecular and cellular events associated with edema formation in mice and human patients with cancer treated with a CSF1R blocking antibody. Extended antibody treatment of mice caused marked body weight gain, an indicator of enhanced body fluid retention. This was associated with an increase of extracellular matrix–remodeling metalloproteinases (MMPs), namely MMP2 and MMP3, and enhanced deposition of hyaluronan (HA) and proteoglycans, leading to skin thickening. Discontinuation of anti-CSF1R treatment or blockade of MMP activity restored unaltered body weight and normal skin morphology in the mice. In patients, edema developed at doses well below the established optimal biological dose for emactuzumab, a CSF1R dimerization inhibitor. Patients who developed edema in response to emactuzumab had elevated HA in peripheral blood. Our findings indicate that an early increase of peripheral HA can serve as a pharmacodynamic marker for edema development and suggest potential interventions based on MMP inhibition for relieving periorbital edema in patients treated with CSF1R inhibitors.

Significance Cancer immunotherapy demonstrates clinical efficacy in selected cancer types. Yet, as the majority of patients do not respond to the most effective immunotherapeutics, novel immunotherapy combinations are extensively investigated. There is increasing clinical interest in combining cancer immunotherapies with antiangiogenic agents, particularly VEGFA pathway inhibitors. Here we show that anti-CD40 immunotherapy increases CD8+ T cell infiltration in the tumor, yet tumor regression is achieved more robustly when anti-CD40 is combined with dual Ang2 and VEGFA blockade, but not VEGFA inhibition alone. Tumor regression was associated with proinflammatory skewing of the tumor microenvironment and intratumoral redistribution of CD8+ T cells. These data emphasize the rationale for blocking Ang2 as a vascular-modulatory strategy in combination with T cell-targeting immunotherapies., Cancer immunotherapies are increasingly combined with targeted therapies to improve therapeutic outcomes. We show that combination of agonistic anti-CD40 with antiangiogenic antibodies targeting 2 proangiogenic factors, vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (Ang2/ANGPT2), induces pleiotropic immune mechanisms that facilitate tumor rejection in several tumor models. On the one hand, VEGFA/Ang2 blockade induced regression of the tumor microvasculature while decreasing the proportion of nonperfused vessels and reducing leakiness of the remaining vessels. On the other hand, both anti-VEGFA/Ang2 and anti-CD40 independently promoted proinflammatory macrophage skewing and increased dendritic cell activation in the tumor microenvironment, which were further amplified upon combination of the 2 treatments. Finally, combined therapy provoked brisk infiltration and intratumoral redistribution of cytotoxic CD8+ T cells in the tumors, which was mainly driven by Ang2 blockade. Overall, these nonredundant synergistic mechanisms endowed T cells with improved effector functions that were conducive to more efficient tumor control, underscoring the therapeutic potential of antiangiogenic immunotherapy in cancer.

Immunotherapy with checkpoint inhibitors has proved to be highly effective, with durable responses in a subset of patients. Given their encouraging clinical activity, checkpoint inhibitors are increasingly being tested in clinical trials in combination with chemotherapy. In many instances, there is little understanding of how chemotherapy might influence the quality of the immune response generated by checkpoint inhibitors. In this study, we evaluated the impact of chemotherapy alone or in combination with anti–PD-L1 in a responsive syngeneic tumor model. Although multiple classes of chemotherapy treatment reduced immune cell numbers and activity in peripheral tissues, chemotherapy did not antagonize but in many cases augmented the antitumor activity mediated by anti–PD-L1. This dichotomy between the detrimental effects in peripheral tissues and enhanced antitumor activity was largely explained by the reduced dependence on incoming cells for antitumor efficacy in already established tumors. The effects of the various chemotherapies were also agent specific, and synergy with anti–PD-L1 was achieved by different mechanisms that ultimately helped establish a new threshold for response. These results rationalize the combination of chemotherapy with immunotherapy and suggest that, despite the negative systemic effects of chemotherapy, effective combinations can be obtained through distinct mechanisms acting within the tumor.

Despite decades of research, cancer remains a devastating disease and new treatment options are needed. Today cancer is acknowledged as a multifactorial disease not only comprising of aberrant tumor cells but also the associated stroma including tumor vasculature, fibrotic plaques, and immune cells that interact in a complex heterotypic interplay. Myeloid cells represent one of the most abundant immune cell population within the tumor stroma and are equipped with a broad functional repertoire that promotes tumor growth by suppressing cytotoxic T cell activity, stimulating neoangiogenesis and tissue remodeling. Therefore, myeloid cells have become an attractive target for pharmacological intervention. In this review, we summarize the pharmacological approaches to therapeutically target tumor-associated myeloid cells with a focus on advanced programs that are clinically evaluated. In addition, for each therapeutic strategy, the preclinical rationale as well as advantages and challenges from a drug development perspective are discussed.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and has a high mortality rate due to limited treatment options. Hence, the response of HCC to different cancer immunotherapies is being intensively investigated in clinical trials. Immune checkpoint blockers (ICB) show promising results, albeit for a minority of HCC patients. Mouse models are commonly used to evaluate new therapeutic agents or regimens. However, to make clinical translation more successful, better characterized preclinical models are required. We therefore extensively investigated two immune-competent orthotopic HCC mouse models, namely transplanted Hep-55.1c and transgenic iAST, with respect to morphological, immunological and genetic traits and evaluated both models’ responsiveness to immunotherapies. Hep-55.1c tumors were characterized by rich fibrous stroma, high mutational load and pronounced immune cell infiltrates, all of which are features of immune-responsive tumors. These characteristics were less distinct in iAST tumors, though these were highly vascularized. Cell depletion revealed that CD8+ T cells from iAST mice do not affect tumor growth and are tumor tolerant. This corresponds to the failure of single and combined ICB targeting PD-1 and CTLA-4. In contrast, combining anti-PD-1 and anti-CTLA-4 showed significant antitumor efficacy in the Hep-55.1c mouse model. Collectively, our data comprehensively characterize two immune-competent HCC mouse models representing ICB responsive and refractory characteristics. Our characterization confirms these models to be suitable for preclinical investigation of novel cancer immunotherapy approaches that aim to either deepen preexisting immune responses or generate de novo immunity against the tumor.

Tumour-associated macrophages (TAMs) largely express an alternatively activated (or M2) phenotype, which entails immunosuppressive and tumour-promoting capabilities. Reprogramming TAMs towards a classically activated (M1) phenotype may thwart tumour-associated immunosuppression and unleash anti-tumour immunity. Here we show that conditional deletion of the microRNA (miRNA)-processing enzyme DICER in macrophages prompts M1-like TAM programming, characterized by hyperactive IFN-γ/STAT1 signalling. This rewiring abated the immunosuppressive capacity of TAMs and fostered the recruitment of activated cytotoxic T lymphocytes (CTLs) to the tumours. CTL-derived IFN-γ exacerbated M1 polarization of Dicer1-deficient TAMs and inhibited tumour growth. Remarkably, DICER deficiency in TAMs negated the anti-tumoral effects of macrophage depletion by anti-CSF1R antibodies, and enabled complete tumour eradication by PD1 checkpoint blockade or CD40 agonistic antibodies. Finally, genetic rescue of Let-7 miRNA activity in Dicer1-deficient TAMs partly restored their M2-like phenotype and decreased tumour-infiltrating CTLs. These findings suggest that DICER/Let-7 activity opposes IFN-γ-induced, immunostimulatory M1-like TAM activation, with potential therapeutic implications.

BackgroundThis phase Ib study evaluated the safety, clinical activity, pharmacokinetics, and pharmacodynamics (PD) of emactuzumab (anti-colony stimulating factor 1 receptor monoclonal antibody (mAb)) in combination with selicrelumab (agonistic cluster of differentiation 40 mAb) in patients with advanced solid tumors.MethodsBoth emactuzumab and selicrelumab were administered intravenously every 3 weeks and doses were concomitantly escalated (emactuzumab: 500 to 1000 mg flat; selicrelumab: 2 to 16 mg flat). Dose escalation was conducted using the product of independent beta probabilities dose-escalation design. PD analyzes were performed on peripheral blood samples and tumor/skin biopsies at baseline and on treatment. Clinical activity was evaluated using investigator-based and Response Evaluation Criteria In Solid Tumors V.1.1-based tumor assessments.ResultsThree dose-limiting toxicities (all infusion-related reactions (IRRs)) were observed at 8, 12 and 16 mg of selicrelumab together with 1000 mg of emactuzumab. The maximum tolerated dose was not reached at the predefined top doses of emactuzumab (1000 mg) and selicrelumab (16 mg). The most common adverse events were IRRs (75.7%), fatigue (54.1%), facial edema (37.8%), and increase in aspartate aminotransferase and creatinine phosphokinase (35.1% both). PD analyzes demonstrated an increase of Ki67+-activated CD8+ T cells accompanied by a decrease of B cells and the reduction of CD14Dim CD16bright monocytes in peripheral blood. The best objective clinical response was stable disease in 40.5% of patients.ConclusionEmactuzumab in combination with selicrelumab demonstrated a manageable safety profile and evidence of PD activity but did not translate into objective clinical responses.Trialregistration numberNCT02760797.

Macrophage infiltration has been identified as an independent poor prognostic factor for several cancer entities. In mouse tumor models macrophages orchestrate various tumor-promoting processes. This observation sparked an interest to therapeutically target these plastic innate immune cells. To date, blockade of colony stimulating factor-1 or its receptor represents the only truly selective approach to manipulate macrophages in cancer patients. Here, we discuss the currently available information on efficacy and safety of various CSF-1/CSF-1R inhibitors in cancer patients and highlight potential combination partners emerging from preclinical studies while considering the differences between mouse and human macrophage biology.

Deregulated TGF-β signaling in pancreatic cancer promotes tumor growth, invasion, metastasis, and a potent immunosuppressive network. A strategy for disrupting this tumor-promoting pathway is silencing TGF-β by siRNA. By introducing a triphosphate group at the 5′ end of siRNA (ppp-siRNA), gene silencing can be combined with immune activation via the cytosolic helicase retinoic acid-inducible gene I (RIG-I), a ubiquitously expressed receptor recognizing viral RNA. We validated RIG-I as a therapeutic target by showing that activation of RIG-I in pancreatic carcinoma cells induced IRF-3 phosphorylation, production of type I IFN, the chemokine CXCL10, as well as caspase-9–mediated tumor cell apoptosis. Next, we generated a bifunctional ppp-siRNA that combines RIG-I activation with gene silencing of TGF-β1 (ppp-TGF-β) and studied its therapeutic efficacy in the orthotopic Panc02 mouse model of pancreatic cancer. Intravenous injection of ppp-TGF-β reduced systemic and tumor-associated TGF-β levels. In addition, it induced high levels of type I IFN and CXCL10 in serum and tumor tissue, systemic immune cell activation, and profound tumor cell apoptosis in vivo. Treatment of mice with established tumors with ppp-TGF-β significantly prolonged survival as compared with ppp-RNA or TGF-β siRNA alone. Furthermore, we observed the recruitment of activated CD8+ T cells to the tumor and a reduced frequency of CD11b+ Gr-1+ myeloid cells. Therapeutic efficacy was dependent on CD8+ T cells, whereas natural killer cells were dispensable. In conclusion, combing TGF-β gene silencing with RIG-I signaling confers potent antitumor efficacy against pancreatic cancer by breaking tumor-induced CD8+ T cell suppression. Cancer Res; 73(6); 1709–20. ©2013 AACR.

Summary Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8 + cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1 + myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies.

Granzymes A and B (GrAB) are known principally for their role in mediating perforin-dependent death of virus-infected or malignant cells targeted by CTL. In this study, we show that granzymes also play a critical role as inducers of Ag cross-presentation by dendritic cells (DC). This was demonstrated by the markedly reduced priming of naive CD8+ T cells specific for the model Ag OVA both in vitro and in vivo in response to tumor cells killed in the absence of granzymes. Reduced cross-priming was due to impairment of phagocytosis of tumor cell corpses by CD8α+ DC but not CD8α− DC, demonstrating the importance of granzymes in inducing the exposure of prophagocytic “eat-me” signals on the dying target cell. Our data reveal a critical and previously unsuspected role for granzymes A and B in dictating immunogenicity by influencing the mode of tumor cell death and indicate that granzymes contribute to the efficient generation of immune effector pathways in addition to their well-known role in apoptosis induction.

Numerous studies have been performed in vitro and in various animal models to modulate the interaction of dendritic cells (DC) and T cells by Fas (CD95/Apo-1) signalling to delete activated T cells via induction of activation-induced cell death (AICD). Previously, we could demonstrate that Fas ligand (FasL/CD95L)-expressing 'killer-antigen-presenting cells' can be generated from human monocyte-derived mature DC (mDC) using adenoviral gene transfer. To evaluate whether these FasL-expressing mDC (mDC-FasL) could eliminate alloreactive primary human T cells in vitro, co-culture experiments were performed. Proliferation of human T cells was markedly reduced in primary co-cultures with allogeneic mDC-FasL, whereas a strong proliferative T-cell response could be observed in co-cultures with enhanced green fluorescent protein-transduced mDC. Inhibition of T-cell proliferation was related to the transduction efficiency, and the numbers of mDC-FasL present in co-cultures. In addition, proliferation of pre-activated alloreactive CD4(+) and CD8(+) T cells could be almost completely inhibited in secondary co-cultures using mDC-FasL as stimulatory cells, which was the result of induction of apoptosis in the majority of preactivated T cells. The specific deletion of alloreactive T cells by mDC-FasL was confirmed by an unaffected proliferative response of surviving T cells towards allogeneic 'third-party' peripheral blood mononuclear cells in a third stimulation, or upon unspecific stimulation with anti-CD3/CD28 beads. The results of this study demonstrate that allospecifically activated T cells are efficiently eliminated by mDC-FasL, supporting further investigations to apply FasL-expressing 'killer-DC' as a novel strategy for the treatment of allograft rejection.

The pore-forming protein perforin is synthesized as an inactive precursor in natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), and becomes active when a short C-terminal peptide is cleaved within acidic lysosome-like cytotoxic granules. Although it was shown more than a decade ago that this cleavage is pH dependent and can be inhibited by the generic cysteine cathepsin inhibitor E-64d, no protease capable of processing the perforin C terminus has been identified. Neither is it known whether a single protease is responsible or the processing has inbuilt redundancy. Here, we show that incubation of human NK cells and primary antigen-restricted mouse CTLs with the cathepsin L (CatL) inhibitor L1 resulted in a marked inhibition of perforin-dependent target cell death and reduced perforin processing. In vitro, CatL preferentially cleaved a site on full-length recombinant perforin close to its C terminus. The NK cells of mice deficient in CatL showed a reduction but not a complete absence of processed perforin, indicating that cysteine proteases other than CatL are also able to process perforin. We conclude that granule-bound cathepsins are essential for processing perforin to its active form, and that CatL is an important, but not exclusive, participant in this process.

Introduction Immunotherapy is a promising treatment strategy for hepatocellular carcinoma (HCC). A phase III trial for advanced HCC shows favorable results for nivolumab compared to sorafenib. In order to evaluate the outcome of different therapeutic strategies, we compared two HCC mouse models. The orthotopic transplanted HCC fragment Hep-55.1c model and the inducible HCC mouse model (iAST) were extensively characterized ex vivo and in vivo in response to immunotherapies, and were compared to the clinical situation of HCC patients. Experimental Procedures Fragments of mouse Hep-55.1c tumors were implanted in the left lateral liver lobe of C57/Bl6 mice and tumor growth kinetic was monitored by µ-CT. HCC tumor growth in iAST mice was induced via intravenous injection of the adenovirus expressing Cre recombinase. iAST mice express a loxP flanked stop cassette and the SV40 large T-antigen under control of a hepatocyte-specific albumin promoter. Both tumor models were characterized with respect to immune infiltration, cytokine release, somatic mutational load and histopathological characteristics. Mice were treated using different immunotherapeutic agents in monotherapy or in combination such as anti-PD-1, anti-CTLA-4 or the TLR7/8 agonist R848. Human HCC samples were analyzed for mutational load by sequencing, while tumor architecture and immune infiltrate were analyzed by histology. Results Hep-55.1c tumors showed high stroma content accompanied by a low and disorganized vasculature whereas multinodular iAST tumors were highly vascularized lacking stroma content. Furthermore, a high mutational load and a strong immune cell infiltrate including cytotoxic T cells, NK cells and myeloid cells were found in Hep-55.1c tumors. The iAST tumors were characterized by a relatively low immune infiltrate and a small number of mutations. Comparison of these baseline results with data obtained from immune-histopathological analysis and sequencing of human HCC samples confirmed this differentiated picture in the clinics. Following treatment, iAST mice showed no response to immune checkpoint monotherapies and only a marginal number of reactive T cells was found within the tumor. In contrast, tyrosine kinase inhibitor sorafenib led to tumor growth inhibition in iAST model. Treatment of mice bearing Hep-55.1c tumors with immunotherapy e.g. anti-PD-1 showed good response. Combination of anti-PD-1 with R848 increased therapeutic efficacy compared to monotherapies. Conclusions In this study, we have established two orthotopic HCC mouse models that reflect the diverse clinical situation of human HCC. Our findings demonstrate that the composition of the tumor microenvironment has a tremendous influence on the outcome of therapeutic strategies for HCC. Hence, thorough characterization of tumor patients is indispensable in order to predict response to immunotherapy. Citation Format: Carina Hage, Sabine Hoves, Mailin Ashoff, Leanne Strauss, Mario Perro, Frank Herting, Fabian Kiessling, Thomas Pöschinger. Identifying characteristics of orthotopic HCC mouse models to predict response to immunotherapy [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 4084.

The use of dendritic cells (DCs) for the generation of anti-tumour immunity has been the focus of a vast array of scientific and clinical studies. The ability of DCs to present protein tumour antigens (T-Ags) to CD4(+) and CD8(+) T cells is pivotal to the success of therapeutic cancer vaccines. DC's specialised capacity to cross-present exogenous Ags onto major histocompatibility (MHC) class I molecules for the generation of T-Ag-specific cytotoxic T lymphocytes (CTLs) has made these cells the focal point of vaccine-based immunotherapy of cancer. However, although DC-based strategies can induce T cell responses in cancer patients, recent reviews of clinical studies demonstrate that DC-based approaches have essentially failed to meet their clinical end points. These findings highlight the need to re-evaluate the DC-based vaccine strategies and incorporate recent advancements in DC biology and tumour immunology. The current review considers the issues related to how best to target the Ag-processing pathway of DCs, the role of adjuvants, the appropriate conditioning of the DCs and strategies to overcome tumour-mediated immune escape.

Novel immunosuppressive strategies are targeting for an antigen-specific deletion of T cells responsible for organ damage in autoimmunity and allograft rejection. Here, we present a new flow cytometry-based assay that allows the reliable and efficient detection of T cells that were eliminated in an antigen-specific fashion. A stable cell-labelling technique utilizing the two membrane dyes PKH26 and PKH67 has been combined with annexin V and 7-aminoactinomycin (7-AAD) staining to detect apoptotic cells. A differential gating strategy enabled us to determine the viability/apoptosis for each PKH-stained T cell subpopulation independently. The capability to simultaneously analyze apoptosis within T cell mixtures of different antigen specificities establishes this assay as a superior tool for the further development of novel antigen-specific immunosuppressive approaches.

Altered expression of major histocompatibility complex (MHC) class I molecules can be caused by defects in genes of the antigen-processing machinery (APM), and is often correlated to progression in solid tumours. However, little is known about expression of the APM components in blasts from patients with acute myeloid leukaemia (AML). In this study, we investigated the expression of the APM components large multifunctional peptidases (LMP) 2 and 7, transporter-associated with antigen processing (TAP) 1 and 2, beta-2-microglobulin (beta2m) and MHC class I heavy chain in situ by tissue microarray from bone marrow biopsies of 30 AML patients. APM components were heterogeneously expressed in all AML samples tested, but no significant correlation with the AML subtype according to the French-American-British classification was found. Depending on the APM component tested, up to 90% of the trephines showed no or weak expression, whereas the LMP7 protein was detected in 66% of all samples. By following disease progression in individual AML patients, we found severe downregulation of APM components in two out of four patients from initial diagnosis to relapse. We conclude that downregulation of APM components may play a role in the failure of immuno-surveillance and may therefore contribute to relapse in acute leukaemia.

Activation of alloreactive T cells by APCs such as dendritic cells (DC) has been implicated as crucial step in transplant rejection. In contrast, it has been proposed that macrophages (Mφ) maintain tolerance toward alloantigens. It was therefore the aim of this study to further analyze the T cell-stimulatory capacity of mature DC and Mφ in vitro using the model of allogeneic MLR. There was a strong proliferative response in T cells cocultured with DC, which was further increased upon restimulation in a secondary MLR. In contrast, T cells did not proliferate in cocultures with Mφ despite costimulation with anti-CD28 and IL-2. Cytokine analysis revealed considerable levels of IL-10 in cocultures of T cells with Mφ, whereas high amounts of IL-2 and IFN-γ were present in cocultures with DC. There was only minimal T cell proliferation in a secondary MLR when T cells were rescued from primary MLR with Mφ and restimulated with DC of the same donor, or DC of an unrelated donor (third party), whereas a strong primary proliferative response was observed in resting T cells, demonstrating induction of T cell anergy by Mφ. Functional analysis of T cells rescued from cocultures with Mφ demonstrated that anergy was at least partly mediated by IL-10-producing regulatory T cells induced by Mφ. These results demonstrate that Mφ drive the differentiation of regulatory T cells and mediate anergy in allogeneic T cells, supporting the concept that Mφ maintain peripheral tolerance in vivo.

Social stress in mammals has repeatedly been shown to cause substantial alterations in the distribution pattern of immune cells in the peripheral blood. The studies described here investigated the role of adrenergic mechanisms in mediating stressor-induced changes in blood leukocyte numbers using a social confrontation procedure in the rat. Experimental manipulations were carried out to eliminate the stress-associated release of adrenal hormones or to block the binding of endogenous catecholamines to alpha- and beta-adrenergic receptors. Adrenalectomy completely abolished the stressor-induced decreases in circulating numbers of T helper cells (CD4+), cytotoxic T cells (CD8+) and B cells but was ineffective in preventing neutrophil granulocytosis, monocytosis and an increase in natural killer (NK) cells. Treatment with the alpha-adrenergic antagonist phentolamine (PHE) was highly effective in preventing granulocytosis and partially blocked lymphopenia, but failed to abolish monocytosis and an increase in NK cells. Treatment with the beta-adrenergic antagonists propranolol (PROP) or nadolol (NAD) entirely blocked the increases in monocytes and NK cells. In addition, beta-adrenergic blockade also significantly reduced neutrophilia, with PROP being more effective than NAD. The results presented here provide evidence that catecholamines play an important role in the redistribution of blood leukocytes during social stress. In particular, the mobilization of cells of the innate immune response seems to be regulated by adrenergic mechanisms.

Summary Dendritic cells (DC) genetically engineered to express high levels of Fas ligand (FasL/CD95L) have been demonstrated to delete T cells in an antigen specific manner in several different animal models in vivo. However, the immunomodulatory capacity of primary human FasL-expressing Killer-DC has not been determined. Therefore, human Killer-DC were generated from mature monocyte-derived DC using the inducible CRE/LoxP adenoviral vector system, and the immunoregulatory capacity of these cells was analyzed in cocultures with primary human T cells in vitro. Combined transductions of DC by AdloxPFasL and AxCANCre resulted in FasL expression in >70% of DC without affecting the mature phenotype. Proliferation of activated primary human T cells was inhibited up to 80% in cocultures with FasL-expressing DC but not EGFP-transduced DC, which was due to induction of apoptosis in activated but not resting CD4+ and CD8+ T cells. Apoptosis induced by Killer-DC could be blocked by an anti-FasL-antibody in a dose dependent fashion. The present results demonstrate that FasL-expressing Killer-DC eliminate activated but not resting primary human CD4+ and CD8+ T cells by induction of Fas-mediated apoptosis supporting the concept to apply Killer-DC as a novel strategy for the treatment of T cell-dependent autoimmune disease and allograft rejection in humans.

The cytotoxic properties of granzymes are well established, though recent publications suggest additional roles for granzymes in immunity. We demonstrated that granzymes can act as regulators of cross-presentation by dendritic cells by inducing critical “eat-me” signals on the dying tumor cell, resulting in efficient phagocytosis of cell-associated tumor antigen.

Macrophage infiltration has been identified as an independent, poor prognostic factor for patients afflicted with various cancer entities. However, the characterization of tumor-associated macrophages (TAMs) prior to and following cancer patient treatment has been limited. Our study analyzed tumor biopsies before and after anti-CSF-1R antibody treatment unraveling the nature of TAMs and providing novel insights into their phenotypic and functional characteristics in cancer.

A sensitive, selective, and comprehensive method for the quantitative determination of tryptophan and 18 of its key metabolites in serum, urine, and cell culture supernatants was developed. The analytes were separated on a C18 silica column by reversed-phase liquid chromatography and detected by electrospray ionization tandem mass spectrometry in positive ion multiple reaction monitoring (MRM) mode, except for indoxyl sulfate which was measured in negative ion MRM mode in a separate run. The limits of detection and lower limits of quantification were in the range of 0.1-50 and 0.5-100 nM, respectively. Fully (13)C isotope-labeled and deuterated internal standards were used to achieve accurate quantification. The applicability of the method to analyze serum, urine, and cell culture supernatants was demonstrated by recovery experiments and the evaluation of matrix effects. Precision for the analysis of serum, urine, and cell culture supernatants ranged between 1.3% and 16.0%, 1.5% and 13.5%, and 1.0% and 17.4%, respectively. The method was applied to analyze changes in tryptophan metabolism in cell culture supernatants from IFN-gamma-treated monocytes and immature or mature dendritic cells.

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-gamma production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8 alpha(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8 alpha(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8 alpha(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8 alpha(+) DCs in vivo for efficient induction of CTL responses. The Journal of Immunology, 2011, 187: 55-63.

The review discusses the controversies in the field of cytotoxic lymphocyte secretory granule death pathways. A pore-forming protein, PRF, and serine proteases, Grz, are key effector molecules of CL. These toxins are stored within secretory granules, which exocytose their contents in response to immune synapse formation between the CL and virus-infected or transformed target cell. There, PRF and Grz synergize to induce various apoptotic death pathways and to maintain immune homeostasis. Mechanistic aspects of the synergy and apoptotic mechanisms are still not fully understood, and the current review will address some of the hotly debated controversies in the field.

A characteristic feature of tumors is high production of lactic acid due to enhanced glycolysis. Here, we show a positive correlation between lactate serum levels and tumor burden in cancer patients and examine the influence of lactic acid on immune functions in vitro. Lactic acid suppressed the proliferation and cytokine production of human cytotoxic T lymphocytes (CTLs) up to 95% and led to a 50% decrease in cytotoxic activity. A 24-hour recovery period in lactic acid–free medium restored CTL function. CTLs infiltrating lactic acid–producing multicellular tumor spheroids showed a reduced cytokine production. Pretreatment of tumor spheroids with an inhibitor of lactic acid production prevented this effect. Activated T cells themselves use glycolysis and rely on the efficient secretion of lactic acid, as its intracellular accumulation disturbs their metabolism. Export by monocarboxylate transporter-1 (MCT-1) depends on a gradient between cytoplasmic and extracellular lactic acid concentrations and consequently, blockade of MCT-1 resulted in impaired CTL function. We conclude that high lactic acid concentrations in the tumor environment block lactic acid export in T cells, thereby disturbing their metabolism and function. These findings suggest that targeting this metabolic pathway in tumors is a promising strategy to enhance tumor immunogenicity.

Myeloid cells represent the most abundant immune cell type within the tumor microenvironment of certain tumor entities, including tumor associated macrophages (TAMs). Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for TAMs is macrophage colony stimulating factor 1 (CSF1). We generated a monoclonal antibody (RG7155) that binds to the secondary dimerization interface of CSF1 receptor (CSF1R) as a specific and potent allosteric inhibitor. In vitro, RG7155 treatment results in cell death of CSF1-differentiated macrophages. In animal models, CSF1R inhibition reduced the F4/80+ TAMs infiltrate by 90% and was accompanied by an increase of the CD8+/CD4+ T cell ratio. The ability of RG7155 to reduce TAMs is currently evaluated in a first-in-man phase I clinical study in patients suffering either from pigmented villonodular synovitis (PVNS), a neoplastic disorder characterized by CSF1 overexpression, or other tumor entities. The associated biomarker program involves mandatory paired pre- and on-treatment biopsies of tumor and surrogate skin tissue as well as pharmacodynamic marker assessment in circulating blood. In patients treated with RG7155 an increase of CSF1 associated with a sustained decrease of CD14+CD16+ alternatively activated monocytes in peripheral blood was detected. In PVNS patients administration of RG7155 led to striking reductions of CSF1R+ and CD163+ macrophages in tumor tissue resulting in objective clinical responses according to RECIST (Response Evaluation Criteria in Solid Tumors) in 5 out of 6 patients. All six evaluable PVNS patients showed partial metabolic response in FDG-PET imaging and significant symptomatic improvement as early as 4 weeks after treatment initiation. Furthermore, TAM reduction was also observed in paired tumor samples of patients with various advanced solid malignancies, suggesting broad applicability of this therapeutic approach. This abstract is also presented as Poster A50. Citation Format: Michael Cannarile, Sabine Hoves, Ann-Marie Broeske, Joerg Benz, Katharina Wartha, Valeria Runza, Flora Rey-Giraud, Leon P. Pradel, Friedrich Feuerhake, Irina Klaman, Tobin Jones, Ute Jucknischke, Stefan Scheiblich, Ingo H. Gorr, Antje Walz, Keelara Abiraj, Philippe Cassier, Antonio Sica, Carlos Gomez-Roca, Christophe Le Tourneau, Jean-Pierre Delord, Antoine Italiano, Hyam Levitsky, Jean-Yves Blay, Dominik Ruettinger, Carola H. Ries. Targeting tumor-asoociated macrophages with a novel anti-CSF1R antibody in cancer patients. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR04. doi:10.1158/1538-7445.CHTME14-PR04

Apoptosis induced by interaction of members of the TNF-/TNF-receptor superfamily has been considered as a major mechanism of cell-mediated cytotoxicity. For functional analysis, the 51Cr release assay has been widely used, which requires loss of membrane integrity in the apoptotic target cell. However, loss of membrane integrity is a late event during apoptosis and therefore only late apoptotic cells will be detected by this method. In contrast, the JAM-assay first described by Polly Matzinger has been demonstrated to be more sensitive than the 51Cr release assay, since this method is dependent on DNA-fragmentation which precedes loss of membrane integrity in most apoptotic cells. The JAM-assay is easier to perform, less expansive, and safer than the current standard (51)Cr release assay. Therefore, this article will focus on optimized conditions of the JAM-assay to detect and quantitate Fas (CD95/Apo-1)-induced apoptosis as an example of death-receptor-mediated cytotoxicity.