High hypoxia status in pancreatic cancer is associated with multiple hallmarks of an immunosuppressive tumor microenvironment.

Introduction: Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is a particularly lethal disease that is often diagnosed late and is refractory to most forms of treatment. Tumour hypoxia is a key hallmark of PDAC and is purported to contribute to multiple facets of disease progression such as treatment resistance, increased invasiveness, metabolic reprogramming, and immunosuppression. Methods: We used the Buffa gene signature as a hypoxia score to profile transcriptomics datasets from PDAC cases. We performed cell-type deconvolution and gene expression profiling approaches to compare the immunological phenotypes of cases with low and high hypoxia scores. We further supported our findings by qPCR analyses in PDAC cell lines cultured in hypoxic conditions. Results: First, we demonstrated that this hypoxia score is associated with increased tumour grade and reduced survival suggesting that this score is correlated to disease progression. Subsequently, we compared the immune phenotypes of cases with high versus low hypoxia score expression (HypoxiaHI vs. HypoxiaLOW) to show that high hypoxia is associated with reduced levels of T cells, NK cells and dendritic cells (DC), including the crucial cDC1 subset. Concomitantly, immune-related gene expression profiling revealed that compared to HypoxiaLOW tumours, mRNA levels for multiple immunosuppressive molecules were notably elevated in HypoxiaHI cases. Using a Random Forest machine learning approach for variable selection, we identified LGALS3 (Galectin-3) as the top gene associated with high hypoxia status and confirmed its expression in hypoxic PDAC cell lines. Discussion: In summary, we demonstrated novel associations between hypoxia and multiple immunosuppressive mediators in PDAC, highlighting avenues for improving PDAC immunotherapy by targeting these immune molecules in combination with hypoxia-targeted drugs.

SHAP value-based ERP analysis (SHERPA): Increasing the sensitivity of EEG signals with explainable AI methods.

Conventionally, event-related potential (ERP) analysis relies on the researcher to identify the sensors and time points where an effect is expected. However, this approach is prone to bias and may limit the ability to detect unexpected effects or to investigate the full range of the electroencephalography (EEG) signal. Data-driven approaches circumvent this limitation, however, the multiple comparison problem and the statistical correction thereof affect both the sensitivity and specificity of the analysis. In this study, we present SHERPA - a novel approach based on explainable artificial intelligence (XAI) designed to provide the researcher with a straightforward and objective method to find relevant latency ranges and electrodes. SHERPA is comprised of a convolutional neural network (CNN) for classifying the conditions of the experiment and SHapley Additive exPlanations (SHAP) as a post hoc explainer to identify the important temporal and spatial features. A classical EEG face perception experiment is employed to validate the approach by comparing it to the established researcher- and data-driven approaches. Likewise, SHERPA identified an occipital cluster close to the temporal coordinates for the N170 effect expected. Most importantly, SHERPA allows quantifying the relevance of an ERP for a psychological mechanism by calculating an "importance score". Hence, SHERPA suggests the presence of a negative selection process at the early and later stages of processing. In conclusion, our new method not only offers an analysis approach suitable in situations with limited prior knowledge of the effect in question but also an increased sensitivity capable of distinguishing neural processes with high precision.

Electrophysiological correlates of face and object perception: A comparative analysis of 2D laboratory and virtual reality conditions.

Human face perception is a specialized visual process with inherent social significance. The neural mechanisms reflecting this intricate cognitive process have evolved in spatially complex and emotionally rich environments. Previous research using VR to transfer an established face perception paradigm to realistic conditions has shown that the functional properties of face-sensitive neural correlates typically observed in the laboratory are attenuated outside the original modality. The present study builds on these results by comparing the perception of persons and objects under conventional laboratory (PC) and realistic conditions in VR. Adhering to established paradigms, the PC- and VR modalities both featured images of persons and cars alongside standard control images. To investigate the individual stages of realistic face processing, response times, the typical face-sensitive N170 component, and relevant subsequent components (L1, L2; pre-, post-response) were analyzed within and between modalities. The between-modality comparison of response times and component latencies revealed generally faster processing under realistic conditions. However, the obtained N170 latency and amplitude differences showed reduced discriminative capacity under realistic conditions during this early stage. These findings suggest that the effects commonly observed in the lab are specific to monitor-based presentations. Analyses of later and response-locked components showed specific neural mechanisms for identification and evaluation are employed when perceiving the stimuli under realistic conditions, reflected in discernible amplitude differences in response to faces and objects beyond the basic perceptual features. Conversely, the results do not provide evidence for comparable stimulus-specific perceptual processing pathways when viewing pictures of the stimuli under conventional laboratory conditions.

Induced oscillatory brain responses under virtual reality conditions in the context of repetition priming.

In the human electroencephalogram (EEG), induced oscillatory responses in various frequency bands are regarded as valuable indices to examine the neural mechanisms underlying human memory. While the advent of virtual reality (VR) drives the investigation of mnemonic processing under more lifelike settings, the joint application of VR and EEG methods is still in its infancy (e.g., due to technical limitations impeding the signal acquisition). The objective of the present EEG study was twofold. First, we examined whether the investigation of induced oscillations under VR conditions yields equivalent results compared to standard paradigms. Second, we aimed at obtaining further insights into basic memory-related brain mechanisms in VR. To these ends, we relied on a standard implicit memory design, namely repetition priming, for which the to-be-expected effects are well-documented for conventional studies. Congruently, we replicated a suppression of the evoked potential after stimulus onset. Regarding the induced responses, we observed a modulation of induced alphaband in response to a repeated stimulus. Importantly, our results revealed a repetition-related suppression of the high-frequency induced gammaband response (>30 Hz), indicating the sharpening of a cortical object representation fostering behavioral priming effects. Noteworthy, the analysis of the induced gammaband responses required a number of measures to minimize the influence of external and internal sources of artefacts (i.e., the electrical shielding of the technical equipment and the control for miniature eye movements). In conclusion, joint VR-EEG studies with a particular focus on induced oscillatory responses offer a promising advanced understanding of mnemonic processing under lifelike conditions.

Low-Cycle Fatigue Behavior of Wire and Arc Additively Manufactured Ti-6Al-4V Material.

Additive manufacturing (AM) techniques, such as wire arc additive manufacturing (WAAM), offer unique advantages in producing large, complex structures with reduced lead time and material waste. However, their application in fatigue-critical applications requires a thorough understanding of the material properties and behavior. Due to the layered nature of the manufacturing process, WAAM structures have different microstructures and mechanical properties compared to their substrate counterparts. This study investigated the mechanical behavior and fatigue performance of Ti-6Al-4V fabricated using WAAM compared to the substrate material. Tensile and low-cycle fatigue (LCF) tests were conducted on both materials, and the microstructure was analyzed using optical microscopy and scanning electron microscopy (SEM). The results showed that the WAAM material has a coarser and more heterogeneous grain structure, an increased amount of defects, and lower ultimate tensile strength and smaller elongation at fracture. Furthermore, strain-controlled LCF tests revealed a lower fatigue strength of the WAAM material compared to the substrate, with crack initiation occurring at pores in the specimen rather than microstructural features. Experimental data were used to fit the Ramberg-Osgood model for cyclic deformation behavior and the Manson-Coffin-Basquin model for strain-life curves. The fitted models were subsequently used to compare the two material conditions with other AM processes. In general, the quasi-static properties of WAAM material were found to be lower than those of powder-based processes like selective laser melting or electron beam melting due to smaller cooling rates within the WAAM process. Finally, two simplified estimation models for the strain-life relationship were compared to the experimentally fitted Manson-Coffin-Basquin parameters. The results showed that the simple "universal material law" is applicable and can be used for a quick and simple estimation of the material behavior in cyclic loading conditions. Overall, this study highlights the importance of understanding the mechanical behavior and fatigue performance of WAAM structures compared to their substrate counterparts, as well as the need for further research to improve the understanding of the effects of WAAM process parameters on the mechanical properties and fatigue performance of the fabricated structures.

Regulator of G-protein signaling 1 critically supports CD8+ TRM cell-mediated intestinal immunity.

Members of the Regulator of G-protein signaling (Rgs) family regulate the extent and timing of G protein signaling by increasing the GTPase activity of Gα protein subunits. The Rgs family member Rgs1 is one of the most up-regulated genes in tissue-resident memory (TRM) T cells when compared to their circulating T cell counterparts. Functionally, Rgs1 preferentially deactivates Gαq, and Gαi protein subunits and can therefore also attenuate chemokine receptor-mediated immune cell trafficking. The impact of Rgs1 expression on tissue-resident T cell generation, their maintenance, and the immunosurveillance of barrier tissues, however, is only incompletely understood. Here we report that Rgs1 expression is readily induced in naïve OT-I T cells in vivo following intestinal infection with Listeria monocytogenes-OVA. In bone marrow chimeras, Rgs1 -/- and Rgs1 +/+ T cells were generally present in comparable frequencies in distinct T cell subsets of the intestinal mucosa, mesenteric lymph nodes, and spleen. After intestinal infection with Listeria monocytogenes-OVA, however, OT-I Rgs1 +/+ T cells outnumbered the co-transferred OT-I Rgs1- /- T cells in the small intestinal mucosa already early after infection. The underrepresentation of the OT-I Rgs1 -/- T cells persisted to become even more pronounced during the memory phase (d30 post-infection). Remarkably, upon intestinal reinfection, mice with intestinal OT-I Rgs1 +/+ TRM cells were able to prevent the systemic dissemination of the pathogen more efficiently than those with OT-I Rgs1 -/- TRM cells. While the underlying mechanisms are not fully elucidated yet, these data thus identify Rgs1 as a critical regulator for the generation and maintenance of tissue-resident CD8+ T cells as a prerequisite for efficient local immunosurveillance in barrier tissues in case of reinfections with potential pathogens.

Antigen recognition detains CD8+ T cells at the blood-brain barrier and contributes to its breakdown.

Blood-brain barrier (BBB) breakdown and immune cell infiltration into the central nervous system (CNS) are early hallmarks of multiple sclerosis (MS). High numbers of CD8+ T cells are found in MS lesions, and antigen (Ag) presentation at the BBB has been proposed to promote CD8+ T cell entry into the CNS. Here, we show that brain endothelial cells process and cross-present Ag, leading to effector CD8+ T cell differentiation. Under physiological flow in vitro, endothelial Ag presentation prevented CD8+ T cell crawling and diapedesis resulting in brain endothelial cell apoptosis and BBB breakdown. Brain endothelial Ag presentation in vivo was limited due to Ag uptake by CNS-resident macrophages but still reduced motility of Ag-specific CD8+ T cells within CNS microvessels. MHC class I-restricted Ag presentation at the BBB during neuroinflammation thus prohibits CD8+ T cell entry into the CNS and triggers CD8+ T cell-mediated focal BBB breakdown.

Real-life relevant face perception is not captured by the N170 but reflected in later potentials: A comparison of 2D and virtual reality stimuli.

The perception of faces is one of the most specialized visual processes in the human brain and has been investigated by means of the early event-related potential component N170. However, face perception has mostly been studied in the conventional laboratory, i.e., monitor setups, offering rather distal presentation of faces as planar 2D-images. Increasing spatial proximity through Virtual Reality (VR) allows to present 3D, real-life-sized persons at personal distance to participants, thus creating a feeling of social involvement and adding a self-relevant value to the presented faces. The present study compared the perception of persons under conventional laboratory conditions (PC) with realistic conditions in VR. Paralleling standard designs, pictures of unknown persons and standard control images were presented in a PC- and a VR-modality. To investigate how the mechanisms of face perception differ under realistic conditions from those under conventional laboratory conditions, the typical face-specific N170 and subsequent components were analyzed in both modalities. Consistent with previous laboratory research, the N170 lost discriminatory power when translated to realistic conditions, as it only discriminated faces and controls under laboratory conditions. Most interestingly, analysis of the later component [230-420 ms] revealed more differentiated face-specific processing in VR, as indicated by distinctive, stimulus-specific topographies. Complemented by source analysis, the results on later latencies show that face-specific neural mechanisms are applied only under realistic conditions (A video abstract is available in the Supplementary material and via YouTube: https://youtu.be/TF8wiPUrpSY).

The reality of virtual reality.

Virtual reality (VR) has become a popular tool for investigating human behavior and brain functions. Nevertheless, it is unclear whether VR constitutes an actual form of reality or is more like an advanced simulation. Determining the nature of VR has been mostly achieved by self-reported presence measurements, defined as the feeling of being submerged in the experience. However, subjective measurements might be prone to bias and, most importantly, do not allow for a comparison with real-life experiences. Here, we show that real-life and VR height exposures using 3D-360° videos are mostly indistinguishable on a psychophysiological level (EEG and HRV), while both differ from a conventional 2D laboratory setting. Using a fire truck, three groups of participants experienced a real-life (N = 25), a virtual (N = 24), or a 2D laboratory (N = 25) height exposure. Behavioral and psychophysiological results suggest that identical exogenous and endogenous cognitive as well as emotional mechanisms are deployed to process the real-life and virtual experience. Specifically, alpha- and theta-band oscillations in line with heart rate variability, indexing vigilance, and anxiety were barely indistinguishable between those two conditions, while they differed significantly from the laboratory setup. Sensory processing, as reflected by beta-band oscillations, exhibits a different pattern for all conditions, indicating further room for improving VR on a haptic level. In conclusion, the study shows that contemporary photorealistic VR setups are technologically capable of mimicking reality, thus paving the way for the investigation of real-world cognitive and emotional processes under controlled laboratory conditions. For a video summary, see https://youtu.be/fPIrIajpfiA.

New is not always better: Virtual reality does not necessarily enhance mnemonic processing.

Integrating new technologies such as Virtual Reality (VR) can contribute to increasing efficiency in several areas relevant to society. VR can be applied in various contexts and has the potential to improve mnemonic processes and memory performance. However, the specific conditions under which VR is more beneficial than conventional learning methods remain unclear. To further investigate the value of VR for mnemonic processing, participants performed a memory task under three different conditions. For that task, they were presented with rules regarding the spatial arrangement of building blocks with a written text or a video in 2D on a screen or in 3D/360° with a head-mounted display. Following the learning session, memory performance was measured by a recognition test involving a multiple-choice questionnaire, in which participants had to mark the correct arrangement of building blocks, and a construction test, in which they had to arrange five different building blocks according to the rules learned. Additionally, participants had to arrange 38 building blocks according to the rules in a free recall test the following day. Surprisingly, results revealed no superiority effect for learning in VR. Instead, learning the rules with the text yielded the best memory performance results, indicating that prior experience with conventional learning methods facilitates declarative knowledge acquisition. Considering previous findings regarding cognitive processing in VR, our results suggest that in passive learning, processing the more salient and personally relevant virtual stimuli in the surrounding VR environment requires more attentional resources. Therefore, VR impairs focusing on the relevant declarative information and impedes the transfer of the learned knowledge to different contexts. When considering to implement VR, the value to the particular domain and specific learning task should be taken into consideration: For learning basic declarative information without actively involving the students, conventional learning methods seem sufficient and more efficient for mnemonic processing compared to new technologies.

Comparing encoding mechanisms in realistic virtual reality and conventional 2D laboratory settings: Event-related potentials in a repetition suppression paradigm.

Although the human brain is adapted to function within three-dimensional environments, conventional laboratory research commonly investigates cognitive mechanisms in a reductionist approach using two-dimensional stimuli. However, findings regarding mnemonic processes indicate that realistic experiences in Virtual Reality (VR) are stored in richer and more intertwined engrams than those obtained from the conventional laboratory. Our study aimed to further investigate the generalizability of laboratory findings and to differentiate whether the processes underlying memory formation differ between VR and the conventional laboratory already in early encoding stages. Therefore, we investigated the Repetition Suppression (RS) effect as a correlate of the earliest instance of mnemonic processes under conventional laboratory conditions and in a realistic virtual environment. Analyses of event-related potentials (ERPs) indicate that the ERP deflections at several electrode clusters were lower in VR compared to the PC condition. These results indicate an optimized distribution of cognitive resources in realistic contexts. The typical RS effect was replicated under both conditions at most electrode clusters for a late time window. Additionally, a specific RS effect was found in VR at anterior electrodes for a later time window, indicating more extensive encoding processes in VR compared to the laboratory. Specifically, electrotomographic results (VARETA) indicate multimodal integration involving a broad cortical network and higher cognitive processes during the encoding of realistic objects. Our data suggest that object perception under realistic conditions, in contrast to the conventional laboratory, requires multisensory integration involving an interconnected functional system, facilitating the formation of intertwined memory traces in realistic environments.

2D-dwell-time analysis with simulations of ion-channel gating using high-performance computing.

Single-channel patch-clamp recordings allow observing the action of a single protein complex in real time and hence the deduction of the underlying conformational changes in the ion-channel protein. Commonly, recordings are modeled using hidden Markov chains, connecting open and closed states in the experimental data with protein conformations. The rates between states denote transition probabilities that could be modified by membrane voltage or ligand binding. Modeling algorithms have to deal with limited recording bandwidth and a very noisy background. It was previously shown that the fit of two-dimensional (2D)-dwell-time histograms with simulations is very robust in that regard. Errors introduced by the low-pass filter or noise cancel out to a certain degree when comparing experimental and simulated data. In addition, the topology of models (that is, the chain of open and closed states) could be inferred from 2D-histograms. However, the 2D-fit was never applied to its full potential. A major reason may be the extremely time-consuming and often unreliable fitting process, due to the stochastic variability in the simulations. We have now solved these issues by introducing a message-passing interface (MPI) allowing massive parallel computing on a high-performance computing (HPC) cluster and obtaining ensemble solutions. With ensembles, we have demonstrated how important ranked solutions are for difficult tasks related to a noisy background, fast gating events beyond the corner frequency of the low-pass filter, and topology estimation of the underlying Markov model. Finally, we have shown that, by combining the objective function of the 2D-fit with the deviation of the current amplitude distributions, automatic determination of the current level of the conducting state is possible, even with an apparent current reduction due to low-pass filtering. Making use of an HPC cluster, the power of 2D-dwell-time analysis can be used to its fullest with minor input of the experimenter.

Rhythms of human attention and memory: An embedded process perspective.

It remains a dogma in cognitive neuroscience to separate human attention and memory into distinct modules and processes. Here we propose that brain rhythms reflect the embedded nature of these processes in the human brain, as evident from their shared neural signatures: gamma oscillations (30-90 Hz) reflect sensory information processing and activated neural representations (memory items). The theta rhythm (3-8 Hz) is a pacemaker of explicit control processes (central executive), structuring neural information processing, bit by bit, as reflected in the theta-gamma code. By representing memory items in a sequential and time-compressed manner the theta-gamma code is hypothesized to solve key problems of neural computation: (1) attentional sampling (integrating and segregating information processing), (2) mnemonic updating (implementing Hebbian learning), and (3) predictive coding (advancing information processing ahead of the real time to guide behavior). In this framework, reduced alpha oscillations (8-14 Hz) reflect activated semantic networks, involved in both explicit and implicit mnemonic processes. Linking recent theoretical accounts and empirical insights on neural rhythms to the embedded-process model advances our understanding of the integrated nature of attention and memory - as the bedrock of human cognition.

BCG hydrogel promotes CTSS-mediated antigen processing and presentation, thereby suppressing metastasis and prolonging survival in melanoma.

BACKGROUND: The use of intralesional Mycobacterium bovis BCG (intralesional live BCG) for the treatment of metastatic melanoma resulted in regression of directly injected, and occasionally of distal lesions. However, intralesional-BCG is less effective in patients with visceral metastases and did not significantly improve overall survival. METHODS: We generated a novel BCG lysate and developed it into a thermosensitive PLGA-PEG-PLGA hydrogel (BCG hydrogel), which was injected adjacent to the tumor to assess its antitumor effect in syngeneic tumor models (B16F10, MC38). The effect of BCG hydrogel treatment on contralateral tumors, lung metastases, and survival was assessed to evaluate systemic long-term efficacy. Gene expression profiles of tumor-infiltrating immune cells and of tumor-draining lymph nodes from BCG hydrogel-treated mice were analyzed by single-cell RNA sequencing (scRNA-seq) and CD8+ T cell receptor (TCR) repertoire diversity was assessed by TCR-sequencing. To confirm the mechanistic findings, RNA-seq data of biopsies obtained from in-transit cutaneous metastases of patients with melanoma who had received intralesional-BCG therapy were analyzed. RESULTS: Here, we show that BCG lysate exhibits enhanced antitumor efficacy compared to live mycobacteria and promotes a proinflammatory tumor microenvironment and M1 macrophage (MΦ) polarization in vivo. The underlying mechanisms of BCG lysate-mediated tumor immunity are dependent on MΦ and dendritic cells (DCs). BCG hydrogel treatment induced systemic immunity in melanoma-bearing mice with suppression of lung metastases and improved survival. Furthermore, BCG hydrogel promoted cathepsin S (CTSS) activity in MΦ and DCs, resulting in enhanced antigen processing and presentation of tumor-associated antigens. Finally, BCG hydrogel treatment was associated with increased frequencies of melanoma-reactive CD8+ T cells. In human patients with melanoma, intralesional-BCG treatment was associated with enhanced M1 MΦ, mature DC, antigen processing and presentation, as well as with increased CTSS expression which positively correlated with patient survival. CONCLUSIONS: These findings provide mechanistic insights as well as rationale for the clinical translation of BCG hydrogel as cancer immunotherapy to overcome the current limitations of immunotherapies for the treatment of patients with melanoma.

Addressing the role of PKD3 in the T cell compartment with knockout mice.

BACKGROUND: The Protein kinase D3 (PKD3) has been implicated in signal transduction downstream of the T cell receptor (TCR). However, its role for the activation of primary T lymphocytes has not been elucidated so far. METHODS: Expression of PKD isoforms in primary murine T cells was determined by RT-PCR and SDS-Page. A germline PKD3-knockout mouse line was analyzed for its immune response to OVA/alum intraperitoneal immunization. Phenotyping of the T cell compartment ex vivo as well as upon stimulation in vitro was performed by flow cytometry. Additionally, cytokine expression was assessed by flow cytometry, RT-PCR and Luminex technology. RESULTS: PKD expression in T cells is modulated by TCR stimulation, leading to a rapid down-regulation on mRNA and on protein level. PKD3-deficient mice respond to immunization with enhanced T follicular helper cell generation. Furthermore, peripheral PKD3-deficient CD4+ T cells express more interleukin-2 than wild type CD4+ T cells upon TCR stimulation ex vivo. However, purified naïve CD4+ T cells do not differ in their phenotype upon differentiation in vitro from wild type T cells. Moreover, we observed a shift towards an effector/memory phenotype of splenic T cells at steady state, which might explain the contradictory results obtained with pan-T cells ex vivo and naïve-sorted T cells. CONCLUSION: While PKD3-deficiency in vivo in mice leads to a skewing of the T cell compartment towards a more activated phenotype, this kinase seems to be dispensable for naïve CD4+ T cell differentiation in vitro. Video Abstract.

An organophotocatalytic late-stage N-CH3 oxidation of trialkylamines to N-formamides with O2 in continuous flow.

We report an organophotocatalytic, N-CH3-selective oxidation of trialkylamines in continuous flow. Based on the 9,10-dicyanoanthracene (DCA) core, a new catalyst (DCAS) was designed with solubilizing groups for flow processing. This allowed O2 to be harnessed as a sustainable oxidant for late-stage photocatalytic N-CH3 oxidations of complex natural products and active pharmaceutical ingredients bearing functional groups not tolerated by previous methods. The organophotocatalytic gas-liquid flow process affords cleaner reactions than in batch mode, in short residence times of 13.5 min and productivities of up to 0.65 g per day. Spectroscopic and computational mechanistic studies showed that catalyst derivatization not only enhanced solubility of the new catalyst compared to poorly-soluble DCA, but profoundly diverted the photocatalytic mechanism from singlet electron transfer (SET) reductive quenching with amines toward energy transfer (EnT) with O2.

Tumor rejection in Cblb-/- mice depends on IL-9 and Th9 cells.

BACKGROUND: Casitas B lymphoma-b (Cbl-b) is a central negative regulator of cytotoxic T and natural killer (NK) cells and functions as an intracellular checkpoint in cancer. In particular, Th9 cells support mast cell activation, promote dendritic cell recruitment, enhance the cytolytic function of cytotoxic T lymphocytes and NK cells, and directly kill tumor cells, thereby contributing to tumor immunity. However, the role of Cbl-b in the differentiation and antitumor function of Th9 cells is not sufficiently resolved. METHODS: Using Cblb-/- mice, we investigated the effect of knocking out Cblb on the differentiation process and function of different T helper cell subsets, focusing on regulatory T cell (Treg) and Th9 cells. We applied single-cell RNA (scRNA) sequencing of in vitro differentiated Th9 cells to understand how Cbl-b shapes the transcriptome and regulates the differentiation and function of Th9 cells. We transferred tumor-model antigen-specific Cblb-/- Th9 cells into melanoma-bearing mice and assessed tumor control in vivo. In addition, we blocked interleukin (IL)-9 in melanoma cell-exposed Cblb-/- mice to investigate the role of IL-9 in tumor immunity. RESULTS: Here, we provide experimental evidence that Cbl-b acts as a rheostat favoring Tregs at the expense of Th9 cell differentiation. Cblb-/- Th9 cells exert superior antitumor activity leading to improved melanoma control in vivo. Accordingly, blocking IL-9 in melanoma cell-exposed Cblb-/- mice reversed their tumor rejection phenotype. Furthermore, scRNA sequencing of in vitro differentiated Th9 cells from naïve T cells isolated from wildtype and Cblb-/- animals revealed a transcriptomic basis for increased Th9 cell differentiation. CONCLUSION: We established IL-9 and Th9 cells as key antitumor executers in Cblb-/- animals. This knowledge may be helpful for the future improvement of adoptive T cell therapies in cancer.

Cannabinoid Receptor Type-2 in B Cells Is Associated with Tumor Immunity in Melanoma.

Agents targeting the endocannabinoid system (ECS) have gained attention as potential cancer treatments. Given recent evidence that cannabinoid receptor 2 (CB2R) regulates lymphocyte development and inflammation, we performed studies on CB2R in the immune response against melanoma. Analysis of The Cancer Genome Atlas (TCGA) data revealed a strong positive correlation between CB2R expression and survival, as well as B cell infiltration in human melanoma. In a murine melanoma model, CB2R expression reduced the growth of melanoma as well as the B cell frequencies in the tumor microenvironment (TME), compared to CB2R-deficient mice. In depth analysis of tumor-infiltrating B cells using single-cell RNA sequencing suggested a less differentiated phenotype in tumors from Cb2r-/- mice. Thus, in this study, we demonstrate for the first time a protective, B cell-mediated role of CB2R in melanoma. This gained insight might assist in the development of novel, CB2R-targeted cancer therapies.

Pancreatic Cancers with High Grade Tumor Budding Exhibit Hallmarks of Diminished Anti-Tumor Immunity.

Tumor budding is associated with epithelial-mesenchymal transition and diminished survival in a number of cancer types including pancreatic ductal adenocarcinoma (PDAC). In this study, we dissect the immune landscapes of patients with high grade versus low grade tumor budding to determine the features associated with immune escape and disease progression in pancreatic cancer. We performed immunohistochemistry-based quantification of tumor-infiltrating leukocytes and tumor bud assessment in a cohort of n = 111 PDAC patients in a tissue microarray (TMA) format. Patients were divided based on the ITBCC categories of tumor budding as Low Grade (LG: categories 1 and 2) and High Grade (HG: category 3). Tumor budding numbers and tumor budding grade demonstrated a significant association with diminished overall survival (OS). HG cases exhibit notably reduced densities of stromal (S) and intratumoral (IT) T cells. HG cases also display lower M1 macrophages (S) and increased M2 macrophages (IT). These findings were validated using gene expression data from TCGA. A published tumor budding gene signature demonstrated a significant association with diminished survival in PDAC patients in TCGA. Immune-related gene expression revealed an immunosuppressive TME in PDAC cases with high expression of the budding signature. Our findings highlight a number of immune features that permit an improved understanding of disease progression and EMT in pancreatic cancer.