Restricting Glycolysis Preserves T Cell Effector Functions and Augments Checkpoint Therapy

Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials

Untersuchungen zur transkriptionellen Regulation der Immunevasion maligner Melanome durch ICER

In den letzten drei Jahrzehnten ist die Zahl der Neuerkrankungen an malignen Melanomen dramatisch angestiegen, wohingegen es hinsichtlich der Sterberate aufgrund ausbleibender therapeutischer Erfolge zu keiner Verbesserung kam.173 Zur Entwicklung neuer innovativer therapeutischer Strategien ist das Verständnis der Moleküle und Signalwege, die an der Bildung eines malignen Melanoms und einer effizienten anti-Melanom-Immunantwort beteiligt sind, essentiell. Der intrazelluläre cAMP-Gehalt von Tumorzellen korreliert mit deren metastasierenden Potential, wobei der genaue Mechanismus und dessen Verbindung zum Immunsystem bisher noch nicht aufgeklärt werden konnte.85 In der vorliegenden Dissertation zeigte eine Datenbankanalyse, dass in humanen Melanombiopsien insbesondere der transkriptionelle Repressor ICER, dessen Expression durch cAMP induziert wird, stark exprimiert wird. Um einen Zusammenhang zwischen einer erhöhten ICER-Expression und dem beobachteten Tumorwachstum in vivo aufzudecken, wurde die Entwicklung unterschiedlicher Tumorzelllinien in Icer-defizienten Mäusen analysiert. Hierbei wurde gefunden, dass sowohl Icer-defiziente Mäuse als auch Mäuse mit einer konditionellen Defizienz für Icer in Makrophagen, Tumorzellen mit einem hohen cAMP-Gehalt abstoßen. Mithilfe von Metabolismus-, Transkriptom- und durchflusszytometrischen Analysen konnte gezeigt werden, dass Tumor-infiltrierende Makrophagen aus Icer-defizienten Mäusen einen Phänotyp des Subtyps 1 (M1) besitzen. Pro-inflammatorische M1 Makrophagen fördern effiziente anti-Tumor-Immunantworten, während anti-inflammatorische M2 Makrophagen diese eher inhibieren und folglich das Tumorwachstum indirekt fördern.179 Der M1 Phänotyp Icer-defizienter Mäuse ist vor allem im Vergleich zu wildtypischen Makrophagen durch eine starke Produktion des pro-inflammatorischen Cytokins TNF-α gekennzeichnet. Weiterhin wurde gezeigt, dass ICER durch Interaktion mit dem Tnf-Promotor dessen Aktivität inhibiert, so dass Icer-kompetente Makrophagen unter dem Einfluss von cAMP nur sehr wenig TNF-α produzieren. Schließlich wurde gezeigt, dass Icer-/Tnf-doppel-defiziente Mäuse nicht mehr in der Lage sind Tumorzellen abzustoßen. Um die Möglichkeit zu testen, inwieweit eine Blockade der cAMP-Bildung in der Behandlung eines malignen Melanoms eingesetzt werden könnte, wurde der therapeutische Einsatz des Adenylatzyklase-Inhibitors MDL-12 getestet. Durch peritumorale MDL-12-Injektionen und der damit verbundenen Inhibition der cAMP-Synthese konnte das Wachstum von B16F10 Melanomzellen in C57BL/6J Mäusen inhibiert werden. In der vorliegenden Dissertation wurde ein cAMP-abhängiger Mechanismus von Melanomzellen aufgedeckt, durch den dieser Tumortyp mittels Inhibition einer effizienten Makrophagen-abhängigen Immunantwort dem Immunsystem entkommen kann.During the last three decades the incidence of malignant melanoma has rapidly increased whereas the mortality rate has not changed because of less success in therapeutic treatment. To develop new innovative therapeutic strategies a detailed understanding of molecules and signaling pathways involved in malignant melanoma formation and efficient anti-melanoma immune responses is essential. The intracellular cAMP concentration of tumor cells is correlated to their metastatic potential whereas the mechanism and the connection to the immune system is still unclear.85 The expression of the transcriptional repressor ICER is induced by cyclic AMP (cAMP). In this dissertation it could be shown that the expression of ICER is upregulated in human melanoma. To link ICER and tumor growth in vivo the development of different tumor cell lines was analyzed in Icer deficient mice. Herein it could be shown that Icer-deficient mice as well as mice with a conditional deletion for ICER in macrophages reject tumor cells with high cAMP concentrations as a common feature. Using metabolism, transcriptome and flow cytometry analyses it could be shown that tumor-infiltrating macrophages of Icer deficient mice have a macrophage phenotype of the M1 subtype. Proinflammatory M1 macrophages promote efficient anti-tumor immune responses whereas M2 macrophages inhibit those anti-tumor immune responses but promote tumor development.179 Icer deficient macrophages are producing high amounts of the proinflammatory cytokine TNF-α in comparison to macrophages of WT mice. In addition it could be shown that ICER interacts with the Tnf promoter. Due to this interaction the Tnf promoter activity is downregulated and Icer competent macrophages do not produce high amounts of TNF-α. Furthermore, it could be shown that double deficient mice for Icer and Tnf are not able to control tumor cell growth anymore. To test the possibility of cAMP signaling pathway modulation in melanoma treatment the therapeutic application of the adenylate cyclase inhibitor MDL-12 was tested. Inhibition of de novo cAMP synthesis by peritumoral injection of MDL-12 lead to a rejection of B16F10 melanoma cells in WT mice. Taken together, a yet unknown cAMP-dependent escape mechanism of melanoma cells leading to inhibition of efficient macrophage-dependent anti-melanoma immune responses is demonstrated

Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram Range

Efficient and reproducible sample preparation is a prerequisite for any robust and sensitive quantitative bottom-up proteomics workflow. Here, we performed an independent comparison between single-pot solid-phase-enhanced sample preparation (SP3), filter-aided sample preparation (FASP), and a commercial kit based on the in-StageTip (iST) method. We assessed their performance for the processing of proteomic samples in the low μg range using varying amounts of HeLa cell lysate (1–20 μg of total protein). All three workflows showed similar performances for 20 μg of starting material. When handling sample sizes below 10 μg, the number of identified proteins and peptides as well as the quantitative reproducibility and precision drastically dropped in case of FASP. In contrast, SP3 and iST provided high proteome coverage even in the low μg range. Even when digesting 1 μg of starting material, both methods still enabled the identification of over 3000 proteins and between 25 000 and 30 000 peptides. On average, the quantitative reproducibility between experimental replicates was slightly higher in case of SP3 (<i>R</i>² = 0.97 (SP3); <i>R</i>² = 0.93 (iST)). Applying SP3 toward the characterization of the proteome of FACS-sorted tumor-associated macrophages in the B16 tumor model enabled the quantification of 2965 proteins and revealed a “mixed” M1/M2 phenotype

Tick Salivary Sialostatin L Represses the Initiation of Immune Responses by Targeting IRF4-Dependent Transcription in Murine Mast Cells

Co-evolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the parasite. Herein, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells while degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1β and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1 receptor-deficient mast cells exhibit strong impairment in IL-9 production demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9 suggesting that sialostatin L suppresses mast cell-derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell-specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction of asthma symptoms demonstrating the immunosuppressive potency of tick-derived molecules

Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages

Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein–coupled receptor–dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system

Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages

Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein-coupled receptor-dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system