Immune Cell Interactions Research Articles

Abstract 2924: Organoid co-cultures with autologous T cells to assess toxicity and efficacy of bispecific antibodies

Abstract Recent advances in cancer immunotherapy had a positive impact on the life expectancy of patients for a large range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy since conventional 2D cancer models hold low clinical predictive value. HUB developed an innovative alternative, build on the discovery that adult stem cells proliferate and organise into three-dimensional organotypic structures when they are embedded into extracellular matrix. Patient specific organoids are generated from healthy and malignant tissues and stored as biobanks with high quality and reproducibility. HUB Organoids recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity, and morphological and functional traits. Since cancer progression and responses to immunotherapy are governed by immune cell interactions in the tumour microenvironment, we developed an assay in which various types of tumour organoids are co-cultured with T cells modified with chimeric antigen receptors (CAR T cells) to assess the cytotoxic potential. CAR-T cell therapy has been demonstrated to be efficacious against many hematological malignancies, however therapeutic application to treat solid cancers remains challenging. Our organoid-T cell co-culture assay offers a good platform to study the response of tumour organoids on the CAR T cells and will greatly contribute to our understanding of the critical factors that determine a successful CAR T cell therapy. In collaboration with GSK, HUB tested the cytotoxicity of GSK CAR T cells which target specific target antigen on human colorectal cancer (CRC) organoids. Six HUB CRC organoid models were selected based on differential expression levels of target antigen that was confirmed by FACS antibody staining. To evaluate GSK CAR T cell mediated organoid killing, caspase 3/7 expression was monitored at various timepoints and IFNγ secretion was measured by ELISA. For control, staurosporine and nonspecific-CAR T cells were included. Detected CRC-organoid killing demonstrated to be in line with antigen expression levels, indicating the specificity of the tested CAR T cells. Nevertheless, some CRC-organoid models demonstrated more resilient to CAR T cell mediated killing and further evaluation revealed upregulation of check point molecules such as PD-L1. This study reveals that tumour organoid-T cell co-culture assay hold value for preclinical development of CAR T-cell products and for evaluating tumour-evasion strategies. Moreover, this platform could potentially predict patient specific responses to CAR- T-cell therapy. Citation Format: Lorenz Jahn, Maxime Kempers, Roel Verker, Wies van Dooremalen, Linda Steinacher, Pleun Hombrink, Farzin Pourfarzad, Robert G. Vries, Lauriane Cabon, Sylvia F. Boj. Organoid co-cultures with autologous T cells to assess toxicity and efficacy of bispecific antibodies [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 2924.

Abstract 4599: Blood and lymphatic endothelial cell contractility-mediated vessel permeability regulates interstitial fluid pressure and T cell infiltration in the tumor microenvironment

Abstract Solid tumors such as breast and pancreatic cancer are characterized by poor prognostic outcomes with high interstitial fluid pressure (IFP), which promotes tumor progression by modulating angiogenesis, matrix metalloproteinase activity, and cancer cell motility and compromises immunotherapies by keeping immune cells (e.g., T cells) from infiltrating into the tumor. To reduce tumor IFP, researchers tried to decrease solid stress and normalize hyperpermeable blood vessels (BV) in tumors, but how lymphatic vessels (LV) are impaired in the tumor microenvironment is poorly understood. Several studies reported that vascular endothelial growth factor C (VEGFC) treatment defeated tumors by promoting lymphangiogenesis and boosting T cell recruitment, while it has also been recognized to promote lymph node metastasis. Given the controversial roles of VEGFC, the main goals of this study are to control LV permeability and promote drainage by identifying currently unappreciated roles of rho-associated protein kinase 1 and 2 (ROCK1/2) in regulating lymphatic endothelial cell (LEC) contractility, junctions, and drainage in tumors. We will have blood endothelial cells (BEC) as controls to understand how ROCK1/2 and cell contractility affect LEC/BEC junctions and permeability differently. Here, we created a three-dimensional (3D) bioengineered model of vascularized pancreatic and breast tumors to study vessel junctions, permeability, and IFP formation. The device consisted of a LEC or BEC lined, perfusable channel and a tumor-filled ECM chamber. We observed impaired barrier function of tumor BV/LV with increased permeability and loss of tight junctions compared to healthy vessels. To quantify the contractility of LEC/BEC, we employed traction force microscopy (TFM) by creating a 2D biomimicry of the endothelial monolayers using micropatterned polyacrylamide gel substrates with tunable stiffness. Using TFM, we showed that the combination of tumor-secreted factors and elevated matrix stiffness increased the traction of LEC/BEC, resulting in their loss of tight junctions. Using Rho kinase blockade to inhibit ROCK1/2 reduced the contractility of endothelial cells, reconstructed vessel structural integrity and functionality, and reduced intratumoral IFP in animal models. We expect that a decrease in the tumor IFP due to the restoration of lymphatic drainage would increase T cell infiltration, suppress tumor growth, and improve the survival of tumor-bearing mice combined with the anti-PD-1 or anti-PD-L1 therapy in our future studies. Together, our study provides new insights into understanding the physical interaction of endothelial, tumor, and immune cells, evidence of rho-kinases as a target for cancer immunotherapy, as well as a promising way to increase the efficacy of immunotherapies in cancer patients. Citation Format: Yansong Peng, Esak Lee. Blood and lymphatic endothelial cell contractility-mediated vessel permeability regulates interstitial fluid pressure and T cell infiltration in the tumor microenvironment. [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 4599.

Abstract 75: Impaired metastatic seeding underlies concomitant tumor resistance

Abstract Cancer metastasis, the spread of cancer cells from the primary tumor to distant organs, is responsible for poor overall survival of cancer patients even with advances in the development of targeted therapeutics. One of the reasons behind failure of current treatments is the continuous evolution of tumor cells and of their microenvironment at both the primary and distant sites, which leads to drug resistance. Primary tumor-derived signals such as secreted chemokines, cytokines, and growth factors can alter the host microenvironment at the metastatic site, leading to the recruitment of immune cells and to the formation of pre-metastatic microenvironments which promote metastasis. However, in some cases, primary tumor-derived signals have also been shown to have metastasis-suppressive effects, a phenomenon known as concomitant resistance. For example, unexpected increase in metastatic outgrowth upon surgical resection of a primary tumor has been reported for some breast cancer patients. Loss of concomitant resistance is a barrier to surgical intervention of primary tumor. This is currently being managed by chemotherapy and further identification of actionable mechanisms behind this phenomenon is necessary. The molecular mechanisms leading to concomitant resistance are poorly understood, and it remains poorly defined how this phenomenon evolves over time, during tumor progression. To investigate the effect of tumor progression on metastasis efficiency, non-tumor bearing (naïve) and tumor bearing mice were challenged with tail-vein injection of tumor cells and lung metastases were quantified. Our data indicate that primary tumors inhibit overt lung metastasis by impairing the ability of circulating tumor cells to seed in the lungs. Through time course studies, we found that early-stage (day 7 and 14) tumors impede early metastatic seeding, but later-stage tumors (day 28 and 35) do not. While adaptive immunity, involving T cells, can inhibit metastasis, results from depletion experiments suggest that the observed effect is T and B cell independent. Platelets can bind circulating tumor cells and recruit neutrophils at metastatic sites to facilitate seeding. We discovered that platelet activation and neutrophil recruitment to platelet-tumor cell clusters are impaired in tumor-bearing mice, suggesting that defects in these immune cell-tumor cell interactions might cause concomitant tumor resistance. Overall, our study uncovered that inefficient metastatic seeding mediates concomitant tumor resistance. Current and future work is directed towards identifying precise mechanisms by which platelets and neutrophils mediate concomitant tumor resistance, so that novel strategies could be developed to prevent metastasis. Citation Format: Akshita B. Bhatt, Manon Baures, Cecile Garcin, Myriam Labelle. Impaired metastatic seeding underlies concomitant tumor resistance [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 75.

Abstract 4559: Evaluation of the chemotherapy potential to improve the anti-cancer efficacy of PD-L1 inhibition using novel 3D microfluidic co-culture platform

Abstract Immunotherapy is now recognized as a powerful therapeutic approach to treat and cure cancer. However, the objective response rates for most solid tumors are low (below 30%). Accumulating evidence has shown that chemotherapy can increase the efficacy of immune checkpoint blockade and improve cancer outcomes. We have investigated if chemotherapy can modulate anti-PDL1 enhanced T cell cytotoxicity in breast cancer using novel in vitro 3D co-culture assay system. 3D co-culture of cancer and immune cells is a powerful platform for disease modeling and therapies testing because it can mimic the tumor micro-environment and complex cellular interactions. In this study we evaluated the immunomodulatory effect of chemotherapy on spheroid-T cells interactions in response to PD-L1 inhibition in triple-negative breast cancer. MCF7 and MDA231 cell lines with differential PD-L1 status were formed into spheroids and used as a tumor model. T cells were activated from PBMCs using ImmunoCult CD3/CD28 T cell Activator. Co-culture assays were performed over 72 hr in a Pu·MA System. The Pu·MA System is an automated microfluidic platform that enables phenotypic and functional assays using physiologically relevant 3D cell models. Cancer spheroids and T cells were cultured, manipulated, and measured in a single well of a microfluidic flowchip. The platform integrates 1) 3D cell model with delivery of immune cells, 2) drug delivery, 3) imaging of phenotypic features and 4) functional profiling for cytokine secretion and viability. T cells-spheroid interaction, invasion and cell viability was assessed using high-content fluorescence imaging CellVoyager CQ1 system. IL2, IFN-γ and TNF-α secretion was measured in collected supernatants using Lumit immunoassays. After viability determination, co-cultures were fixed and stained for cancer and T cell markers (E-Cadherin, F-Actin, CD3, CD8) using automated IF staining protocol and then imaged. Image analysis revealed colocalization and infiltration of the T cells into the spheroids. Disintegration of spheroid, loss of circular shape, and increased number of dead cancer cells indicated T cell mediated cancer cell death. To evaluate the immunomodulatory effect of chemotherapy on spheroid-T cells interactions in response to PD-L1 inhibition, co-cultures were exposed to PD-L1 inhibitor Atezolizumab in the presence or absence of Cisplatin/Pemetrexed. We have analyzed a shift in T cell-mediated killing activity and function in the presence or absence of chemotherapeutics. The proposed co-culture platform can be further extended to a more complex patient-derived 3D models using different cell types. Our functional immune-oncology 3D platform allows to study the crosstalk between immune, cancer and other cell interactions, evaluate new drug candidates and assess individual therapeutic approaches to advance precision medicine. Citation Format: Ekaterina Moroz Nikolov, Anthony Thai, Lila Cooper, Mahomi Suzuki, Arvonn Tully, Rashmi Rajendra, Evan F. Cromwell. Evaluation of the chemotherapy potential to improve the anti-cancer efficacy of PD-L1 inhibition using novel 3D microfluidic co-culture platform. [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 4559.

Abstract 4703: Applying spatial omics and computational integrative analyses to study drug responses and cancer immune cell interactions

Abstract Cancer responses to drug treatment are highly heterogeneous. We postulate that spatial determinants in the tumour play a critical role in cancer therapy outcomes. Here, we will present two spatial transcriptomics studies on spatial responses to immunotherapy and chemotherapy. Immune checkpoint inhibitors (ICI) are used to treat recurrent metastatic oropharyngeal squamous cell carcinomas (OPSCC). Unfortunately, less than 30% of patients benefit from this therapy. Thus, we performed spatial transcriptomics (ST) and in-situ multiprotein detection (PhenoCycler-Fusion) on tissue isolated from a patient diagnosed with metastatic OPSCC. The patient’s primary oral tumour responded to chemo-radio therapy, followed by nivolumab ICI. However, new soft pallet OPSCCs resurged. Subsequent pembrolizumab combined with lenvatinib (VEGFR inhibitor) treatment had an initial effect, butnew recurrent oral tumours re-emerged suggesting drug resistance. Using ST, we observed high expression of drug resistance genes such as SNAI2, SOX4 and NDRG1 consistent with the disease aggressive behaviour. Although, PD-1/PD-L1 expression was not observed, we identified 13 over-expressed druggable targets (i.e., EGFR, TF, VEGF) and >10 experimental targets. To rank each drug’s potential success, we measured the co-expression of each target ligand-receptor pair (L/R), reducing the candidates to 4 pathways (TF/TFRC> VEGFA/NRP1> PGF/NRP1> TGFB1/VASN>VEGFA/GPC1). Furthermore, TF/TFRC and VEGFA/NRP1 expected downstream genes were differentially over-expressed where positive LR signal was detected. Similarly, we used ST to define the cellular diversity within a sonic hedgehog (SHH) patient-derived model of Medulloblastoma (MB) and identified how cells specific to a transcriptional state or spatial location are pivotal in responses to treatment with the CDK4/6 inhibitor, Palbociclib. We distinguished neoplastic and non-neoplastic cells within tumours and from the surrounding cerebellar tissue, further refining pathological annotation. We identified a regional response to Palbociclib, with reduced proliferation and induced neuronal differentiation in both treated tumours. Additionally, we resolved at cellular resolution a distinct tumour “interface” where the tumour cells contacted neighbouring mouse brain tissue, consisting of abundant astrocytes and microglia, and continued to proliferate despite Palbociclib treatment. Our data highlight the power of a spatial multi-omics approach to characterise the response of a tumour to targeted therapy and provide further insights into the molecular and cellular basis underlying the response and resistance to cancer therapies. Citation Format: Xiao Tan, Andrew Causer, Tuan Quang Anh Vo, Ning Ma, Bassem Ben Cheikh, Laura Genovesi, Jazmina Gonzalez-Cruz, Quan Nguyen, Oliver Braubach. Applying spatial omics and computational integrative analyses to study drug responses and cancer immune cell interactions. [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 4703.

Macrophage–Neuroglia Interactions in Promoting Neuronal Regeneration in Zebrafish

The human nervous system exhibits limited regenerative capabilities following damage to the central nervous system (CNS), leading to a scarcity of effective treatments for nerve function recovery. In contrast, zebrafish demonstrate remarkable regenerative abilities, making them an ideal model for studying the modulation of inflammatory processes after injury. Such research holds significant translational potential to enhance our understanding of recovery from damage and disease. Macrophages play a crucial role in tissue repair and regeneration, with their subpopulations indirectly promoting axonal regeneration through developmental signals. The AP-1 signaling pathway, mediated by TNF/Tnfrsf1a, can elevate HDAC1 expression and facilitate regeneration. Furthermore, following spinal cord injury (SCI), pMN progenitors have been observed to switch between oligodendrocyte and motor neuron fates, with macrophage-secreted TNF-α potentially regulating the differentiation of ependymal-radial glia progenitors and oligodendrocytes. Radial glial cells (RGs) are also essential for CNS regeneration in zebrafish, as they perform neurogenesis and gliogenesis, with specific RG subpopulations potentially existing for the generation of neurons and oligodendrocytes. This review article underscores the critical role of macrophages and their subpopulations in tissue repair and regeneration, focusing on their secretion of TNF-α, which promotes axonal regeneration in zebrafish. We also offer insights into the molecular mechanisms underlying TNF-α's ability to facilitate axonal regeneration and explore the potential of pMN progenitor cells and RGs following SCI in zebrafish. The review concludes with a discussion of various unresolved questions in the field, and ideas are suggested for future research. Studying innate immune cell interactions with neuroglia following injury may lead to the development of novel strategies for treating the inflammatory processes associated with regenerative medicine, which are commonly observed in injury and disease.

The function of immunomodulation and biomaterials for scaffold in the process of bone defect repair: A review.

The process of bone regeneration involves the interaction of the skeletal, blood, and immune systems. Bone provides a solid barrier for the origin and development of immune cells in the bone marrow. At the same time, immune cells secrete related factors to feedback on the remodeling of the skeletal system. Pathological or traumatic injury of bone tissue involves changes in blood supply, cell behavior, and cytokine expression. Immune cells and their factors play an essential role in repairing foreign bodies in bone injury or implantation of biomaterials, the clearance of dead cells, and the regeneration of bone tissue. This article reviews the bone regeneration application of the bone tissue repair microenvironment in bone cells and immune cells in the bone marrow and the interaction of materials and immune cells.

Identification and Characterisation of Infiltrating Immune Cells in Malignant Pleural Mesothelioma Using Spatial Transcriptomics

Increasing evidence strongly supports the key role of the tumour microenvironment in response to systemic therapy, particularly immune checkpoint inhibitors (ICIs). The tumour microenvironment is a complex tapestry of immune cells, some of which can suppress T-cell immunity to negatively impact ICI therapy. The immune component of the tumour microenvironment, although poorly understood, has the potential to reveal novel insights that can impact the efficacy and safety of ICI therapy. Successful identification and validation of these factors using cutting-edge spatial and single-cell technologies may enable the development of broad acting adjunct therapies as well as personalised cancer immunotherapies in the near future. In this paper we describe a protocol built upon Visium (10x Genomics) spatial transcriptomics to map and characterise the tumour-infiltrating immune microenvironment in malignant pleural mesothelioma. Using ImSig tumour-specific immune cell gene signatures and BayesSpace Bayesian statistical methodology, we were able to significantly improve immune cell identification and spatial resolution, respectively, improving our ability to analyse immune cell interactions within the tumour microenvironment.

A Preliminary Volterra Lotka Model Analysing Cancer, Intracellular Parasite, and Immune Cells Interaction

A Preliminary Volterra Lotka Model Analysing Cancer, Intracellular Parasite, and Immune Cells Interaction

Dermatologic Manifestations of Noninflammasome-Mediated Autoinflammatory Diseases.

Autoinflammatory diseases (AIDs) arise from disturbances that alter interactions of immune cells and tissues. They give rise to prominent (auto)inflammation in the absence of aberrant autoantibodies and/or autoreactive T cells. AIDs that are predominantly caused by changes in the inflammasome pathways, such as the NLRP3- or pyrin-associated inflammasome, have gained substantial attention over the last years. However, AIDs resulting primarily from other changes in the defense system of the innate immune system are less well-studied. These noninflammasome-mediated AIDs relate to, for example, disturbance in the TNF or IFN signaling pathways or aberrations in genes affecting the IL-1RA. The spectrum of clinical signs and symptoms of these conditions is vast. Thus, recognizing early cutaneous signs constitutes an important step in differential diagnoses for dermatologists and other physicians. This review provides an overview of the pathogenesis, clinical presentation, and available treatment options highlighting dermatologic aspects of noninflammasome-mediated AIDs.

Abstract GS5-06: InteractPrint predicts clinically meaningful interactions between cancer epithelial cells and immune cells: Lessons from a single-cell breast cancer atlas

Abstract BACKGROUND: While immunotherapy has revolutionized the treatment of many solid tumors, the efficacy of immunotherapy regimens is comparatively lower in breast cancer. Immunotherapy efficacy is often negatively correlated with intratumor heterogeneity. Novel immunotherapy approaches in breast cancer should leverage how cancer epithelial cell heterogeneity affects immune cells in the tumor microenvironment. However, current definitions of cancer epithelial cell heterogeneity in breast cancer have limited resolution. Single cell RNA-seq (scRNA-seq) provides an unprecedented opportunity to further define cancer epithelial cell heterogeneity and identify how heterogeneity influences interactions with immune cells. METHODS: We generated a novel scRNA-seq dataset of 236,363 cells from 119 primary breast tumors biopsied from 88 patients taken from 8 publicly available datasets, currently the largest published scRNA-seq dataset in breast cancer. To define cancer epithelial cell heterogeneity, we performed unsupervised clustering and supervised clustering based on molecular subtype and expression of clinical target genes on all cancer epithelial cells. This identified 11 gene elements (GEs), which reflect key molecular features that vary between cancer epithelial cells. Receptor-ligand pairing analysis allowed us to determine how cells that highly express each GE interact with various immune cells. We developed InteractPrint, a score to predict the predominant tumor-interacting immune cells, based on the GE composition of an individual patient tumor. RESULTS: In our dataset, 17% of samples were HER2+, 41% were HR+, and 42% were TNBC. This dataset was statistically powered to characterize cancer epithelial cell heterogeneity. For each of the 11 GEs, we predicted interactions with immune cells. Experimentally, GEs with predicted NK cell interactions showed sensitivity to NK cell cytotoxicity. In a spatially resolved transcriptomics dataset, GEs with predicted T cell interactions demonstrated colocalization with CD8+ T cells, while those with limited predicted T cell interactions did not. To infer GE-immune interactions at the patient level (GEs define cell-level interactions), we developed InteractPrint. To validate InteractPrint, we assessed the accuracy of the T cell InteractPrint in predicting response to anti-PD-1 therapy. Across two trials and all breast cancer subtypes, T cell InteractPrint demonstrated significant improvement over PD-L1 in predicting response to anti-PD-1 therapy. In an scRNA-seq dataset of samples from patients treated with pembrolizumab, we observed AUC of 85% (p < 0.005) for T cell InteractPrint vs. 61% (p > 0.05) for PD-L1 in predicting response to anti-PD-1 therapy. In patients treated with paclitaxel + pembrolizumab in the I-SPY 2 trial, we observed AUC of 81% (p < 0.00001) for T cell InteractPrint versus 72% (p = 0.001) for PD-L1. CONCLUSIONS: Our results demonstrate considerable cancer epithelial cell heterogeneity across primary breast tumor samples and clinical subtypes. We defined this heterogeneity and leveraged it to predict immune cell interactions within a patient’s tumor. We developed T cell InteractPrint to capture heterogeneous interactions between cancer epithelial cells and CD8+ T cells. T cell InteractPrint is predictive of response to anti-PD-1 immune checkpoint inhibition at higher AUC than PD-L1. This provides a path forward for the interpretation of cancer epithelial cell heterogeneity in a clinically meaningful way. Citation Format: Lily Xu, Kaitlyn Saunders, Hildur Knutsdottir, Kenian Chen, Julia Maues, Christine Hodgdon, Evanthia T. Roussos Torres, Sangeetha Reddy, Lin Xu, Isaac Chan. InteractPrint predicts clinically meaningful interactions between cancer epithelial cells and immune cells: Lessons from a single-cell breast cancer atlas [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS5-06.

Single nuclei transcriptomics delineates complex immune and kidney cell interactions contributing to kidney allograft fibrosis

Chronic allograft dysfunction (CAD), characterized histologically by interstitial fibrosis and tubular atrophy, is the major cause of kidney allograft loss. Here, using single nuclei RNA sequencing and transcriptome analysis, we identified the origin, functional heterogeneity, and regulation of fibrosis-forming cells in kidney allografts with CAD. A robust technique was used to isolate individual nuclei from kidney allograft biopsies and successfully profiled 23,980 nuclei from five kidney transplant recipients with CAD and 17,913 nuclei from three patients with normal allograft function. Our analysis revealed two distinct states of fibrosis in CAD; low and high extracellular matrix (ECM) with distinct kidney cell subclusters, immune cell types, and transcriptional profiles. Imaging mass cytometry analysis confirmed increased ECM deposition at the protein level. Proximal tubular cells transitioned to an injured mixed tubular (MT1) phenotype comprised of activated fibroblasts and myofibroblast markers, generated provisional ECM which recruited inflammatory cells, and served as the main driver of fibrosis. MT1 cells in the high ECM state achieved replicative repair evidenced by dedifferentiation and nephrogenic transcriptional signatures. MT1 in the low ECM state showed decreased apoptosis, decreased cycling tubular cells, and severe metabolic dysfunction, limiting the potential for repair. Activated B, T and plasma cells were increased in the high ECM state, while macrophage subtypes were increased in the low ECM state. Intercellular communication between kidney parenchymal cells and donor-derived macrophages, detected several years post-transplantation, played a key role in injury propagation. Thus, our study identified novel molecular targets for interventions aimed to ameliorate or prevent allograft fibrogenesis in kidney transplant recipients.

Integrated Immune Cell-Cell Communication Networks Underpinning Rheumatoid Arthritis Pathogenesis

Rheumatoid Arthritis (RA) is a chronic, systemic auto-inflammatory disease that targets peripheral joints causing bone erosion, worsened mobility, and lower quality of life. RA is a complex autoimmune disease that develops through the combined actions of many cell types. In particular, monocytes, fibroblasts, and T cells play critical roles within the arthritic joint driving disease pathogenesis. These cells communicate with each other via ligand-receptor interactions orchestrating inflammatory responses ultimately leading to joint destruction. However, it remains unclear how the combined efforts of these different cells form an integrated network of immune responses which ultimately lead to disease development. Therefore, we hypothesized that an integrated communication network between the different immune cells found within RA joints functions to promote inflammation and drive RA disease pathogenesis. I utilized predictive assessment of cell-cell communication networks through single-cell RNA-sequencing to determine how immune cell interactions differ between autoimmune and non-autoimmune joint destruction. These studies identified a novel cell-cell communication network in which monocytes, fibroblasts and T cells cooperate to promote cell-cell interactions, immune activation, and induction of pro-inflammatory gene networks. Together, these results identify numerous potential therapeutic targets for the intervention of RA disease.

The benefits of adipocyte metabolism in bone health and regeneration.

Patients suffering from musculoskeletal diseases must cope with a diminished quality of life and an increased burden on medical expenses. The interaction of immune cells and mesenchymal stromal cells during bone regeneration is one of the key requirements for the restoration of skeletal integrity. While stromal cells of the osteo-chondral lineage support bone regeneration, an excessive accumulation of cells of the adipogenic lineage is thought to promote low-grade inflammation and impair bone regeneration. Increasing evidence indicates that pro-inflammatory signaling from adipocytes is responsible for various chronic musculoskeletal diseases. This review aims to summarize the features of bone marrow adipocytes by phenotype, function, secretory features, metabolic properties and their impact on bone formation. In detail, the master regulator of adipogenesis and prominent diabetes drug target, peroxisome proliferator-activated receptor γ (PPARG), will be debated as a potential therapeutic approach to enhance bone regeneration. We will explore the possibilities of using clinically established PPARG agonists, the thiazolidinediones (TZDs), as a treatment strategy to guide the induction of a pro-regenerative, metabolically active bone marrow adipose tissue. The impact of this PPARG induced bone marrow adipose tissue type on providing the necessary metabolites to sustain osteogenic-as well as beneficial immune cells during bone fracture healing will be highlighted.

The role of macrophages in non-small cell lung cancer and advancements in 3D co-cultures.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Traditional therapeutic approaches such as chemotherapy or radiotherapy have provided only a marginal improvement in the treatment of lung carcinomas. Inhibitors targeting specific genetic aberrations present in non-small cell lung cancer (NSCLC), the most common subtype (85%), have improved the prognostic outlook, but due to the complexity of the LC mutational spectrum, only a fraction of patients benefit from these targeted molecular therapies. More recently, the realization that the immune infiltrate surrounding solid tumors can foster tumor-promoting inflammation has led to the development and implementation of anticancer immunotherapies in the clinic. In NSCLC, one of the most abundant leukocyte infiltrates is macrophages. These highly plastic phagocytes, which are part of the cellular repertoire of the innate immunity, can have a pivotal role in early NSCLC establishment, malignant progression, and tumor invasion. Emerging macrophage-targeting therapies have been focused on the re-differentiation of the macrophages toward an antitumorigenic phenotype, depletion of tumor-promoting macrophage subtypes, or combination therapies combining traditional cytotoxic treatments with immunotherapeutic agents. The most extensively used models employed for the exploration of NSCLC biology and therapy have been 2D cell lines and murine models. However, studying cancer immunology requires appropriately complex models. 3D platforms, including organoid models, are quickly advancing powerful tools to study immune cell-epithelial cell interactions within the tumor microenvironment. Co-cultures of immune cells along with NSCLC organoids allow for an in vitro observation of the tumor microenvironment dynamics closely resembling in vivo settings. Ultimately, the implementation of 3D organoid technology into tumor microenvironment-modeling platforms might facilitate the exploration of macrophage-targeted therapies in NSCLC immunotherapeutic research, thus establishing a new frontier in NSCLC treatment.

Wound healing and microbiome, an unexpected relationship.

Skin wounds are common and represent a major public health and economical problem, with risks of complications and a significant negative impact on the quality of life of patients. Cutaneous wound healing is a tightly regulated process resulting in the restoration of tissue integrity. Wound healing involves the interaction of several skin, immune and vascular cells, growth factors and cytokines. However, external actors can play an important role in wound healing, such as the skin microbiome, which is the microbial commensal collection of bacteria, fungi and viruses inhabiting the skin. Indeed, recent advances have featured the interactions, within the wound environment, between different microbial species and between microbial species and the host immune system. This article reviews the relationship between the skin microbiome and the wound healing process. Although cutaneous wounds are a potential entry site for infection, the wound microbiome can have either a detrimental or a beneficial role on wound healing. Thus, targeting the skin microbiome could represent an essential part of wound healing management.

Study of Effector CD8+ T Cell Interactions with Cortical Neurons in Response to Inflammation in Mouse Brain Slices and Neuronal Cultures

Cytotoxic CD8+ T cells contribute to neuronal damage in inflammatory and degenerative CNS disorders, such as multiple sclerosis (MS). The mechanism of cortical damage associated with CD8+ T cells is not well understood. We developed in vitro cell culture and ex vivo brain slice co-culture models of brain inflammation to study CD8+ T cell-neuron interactions. To induce inflammation, we applied T cell conditioned media, which contains a variety of cytokines, during CD8+ T cell polyclonal activation. Release of IFNγ and TNFα from co-cultures was verified by ELISA, confirming an inflammatory response. We also visualized the physical interactions between CD8+ T cells and cortical neurons using live-cell confocal imaging. The imaging revealed that T cells reduced their migration velocity and changed their migratory patterns under inflammatory conditions. CD8+ T cells increased their dwell time at neuronal soma and dendrites in response to added cytokines. These changes were seen in both the in vitro and ex vivo models. The results confirm that these in vitro and ex vivo models provide promising platforms for the study of the molecular details of neuron-immune cell interactions under inflammatory conditions, which allow high-resolution live microscopy and are readily amenable to experimental manipulation.

Chimeric Newcastle Disease Virus Vectors Expressing Human IFN-γ Mediate Target Immune Responses and Enable Multifaceted Treatments.

The therapeutic potential of Newcastle disease virus (NDV) has been reported as both an oncolytic agent and a vaccine vector against many antigens. However, in the individuals already immunized with NDVs, second and subsequent administration does not provide substantial benefits. In this study, two types of recombinant chimeric NDVs using APMV-2 F and HN genes were generated. In rNDV-2HN, the wild-type NDV HN gene was replaced with the APMV-2 HN gene, and in rNDV-2F/2HN, both wild-type F and HN genes were replaced with APMV-2 F and HN genes, respectively. We enhanced the immune responses of these chimeric viruses by inserting the human IFN-γ gene. To examine the escape from NDV antiserum, each virus was treated with diluted NDV antiserum, and HEp-2 cells were infected with these virus particles. The two constructed chimeric viruses indicated notably lower virus-neutralizing titer compared to wild-type NDV and escaped the action of NDV antiserum. These two chimeric viruses infected both respiratory and colon cancer cell lines, indicating their potential as a cancer treatment tool. Chimeric viruses with enhanced immune responses can be considered a novel therapeutic strategy in cancer treatment that can be administered multiple times and used to enhance immune cells interaction.

A Sampling of Highlights from the Literature

The timing of immune interactions modulates antitumor responses (from LetsgomusicStyle via Wikimedia Commons)Immune cell interactions are critical to mounting effective antitumor responses. Wang et al. show that antitumor responses are coordinated by dendritic cell (DC)–CD8+ T-cell interactions and that expression of key circadian rhythm genes governs immune function, which is also affected by the time of day (day vs. night). Cervantes-Silva et al. reveal that DC antigen presentation is also dependent on expression of circadian rhythm genes, which regulate DC mitochondrial morphology and metabolism, and thus, affect T-cell priming. These studies highlight that induction of antitumor responses is a coordinated, rhythmic process, and a deeper understanding of immune rhythmicity can improve therapeutic strategies.Wang C, …, Scheiermann C. Nature 2022 December 5. DOI:10.1038/s41586-022-05605-0.Cervantes-Silva MP, …, Curtis AM. Nat Commun 2022 December 5;13:7217.IL2 is less active in the acidic tumor microenvironment (from BQUB17-ONadal via Wikimedia Commons)High-dose IL2 is approved for treating melanoma and renal cell carcinoma but is used infrequently because of limited efficacy and systemic on-target toxicity. Gaggero et al. find that the acidic pH of the tumor microenvironment (TME), created as a result of tumor production of lactic acid, reduces IL2 signaling in tumor-infiltrating CD8+ T cells, impairing the efficacy of high-dose IL2 therapy in preclinical models. A mutated IL2 variant (Switch-2) that binds IL2Rα with higher affinity at acidic pH than neutral pH shows enhanced antitumor activity with reduced on-target toxicity in mice. The data provide new insight into physicochemical properties of the TME that can impact immunotherapy efficacy and suggest ways to overcome this.Gaggero S, …, Mitra S. Sci Immuol 2022 December 2;7:eade5686.Eosinophils contrib-ute to the efficacy of ICB (from Blausen Medical via Wikimedia Commons)Understanding the mechanisms behind why immune checkpoint blockade (ICB) is not effective in many patients is key to improving treatment. Blomberg et al. find that eosinophils are enriched in ICB-responding patients with breast cancer. This observation was confirmed in a mouse model, and CD4+ T cells, IL5, and IL33 were identified as the driving factors for eosinophil enrichment. Increased eosinophil infiltration boosts eosinophil-dependent activation of CD8+ T cells, which in turn enhances efficacy of ICB. The data emphasize how immune cells other than T cells are affected by ICB and how they can be critical determinants of treatment efficacy.Blomberg OS, …, Kok M. Cancer Cell 2022 December 15;41:106–123.E10.Antitumor T-cell responses require two steps (from DENI070688 via Wikimedia Commons)Understanding T-cell differentiation can reveal insights into the mechanisms behind effective antitumor responses. Prokhnevska et al. demonstrate that T cells are activated via two key steps: first, initial priming and differentiation into a TCF-1+ stemlike phenotype in tumor-draining lymph nodes (TDLN); and second, acquisition of an effector program after infiltrating the tumor microenvironment (TME). Effector programming of CD8+ T cells is dependent on co-stimulation from antigen-presenting cells in the TME, as opposed to in the TDLNs. This process differs from canonical priming/activation of CD8+ T cells in other contexts and adds to our understanding of how T cells mount responses to cancer.Prokhnevska N, …, Kissick H. Immunity 2022 December 28;56:107–124.Eosinophils contribute to the efficacy of ICB (from Boghog2 via Wikimedia Commons)IL22 is known to facilitate tumorigenesis. Two new studies identify mechanisms by which IL22 promotes tumor metastasis and suggest the cytokine as a therapeutic target for preventing metastasis. Giannou et al. find that mice lacking IL22 and mice treated with an IL22-specific blocking antibody are protected from liver metastasis in a colorectal cancer model. Mechanistically, IL2 from liver-resident iNKT17 cells acts on endothelial cells, promoting cancer cell extravasation into the liver. In mouse models of lung and breast cancer, Briukhovetska et al. show that T helper–cell production of IL22 induces overexpression of CD155 on tumor cells. Binding of CD155 to CD226 on natural killer (NK) cells causes CD266 internalization and impaired NK-cell function, promoting metastasis.Giannou AD, …, Huber S. Immunity 2023 January 10;56:125–142.E12.Briukhovetska D, …, Kobold S. Immunity 2023 January 10;56:143–161.E11.Engineered live cancer cells can promote effective antitumor immunity (from Rita Elena Serda via NIH Flickr)The use of inactivated tumor cells as a therapeutic vaccine has been shown to provide limited clinical benefit. Chen et al. show that engineering live cancer cells to secrete IFNβ and GM-CSF, to be resistant to IFNβ, and to have a double kill switch (HSV1 thymidine kinase and rapamycin-activated caspase 9) yields therapeutic cancer cell–based vaccines that induce long-term antitumor immunity and improve survival in several preclinical models. The translatability of this approach is highlighted by the observation that similar engineering of an established human glioblastoma cell line yields a vaccine that reduces tumor growth and improves survival in a humanized model.Chen K-S, …, Shah K. Sci Trans Med 2023 January 4;14:eabo4778.

Association of Circulating Neutrophils with Relative Volume of Lipid-Rich Necrotic Core of Coronary Plaques in Stable Patients: A Substudy of SMARTool European Project

Coronary atherosclerosis is a chronic non-resolving inflammatory process wherein the interaction of innate immune cells and platelets plays a major role. Circulating neutrophils, in particular, adhere to the activated endothelium and migrate into the vascular wall, promoting monocyte recruitment and influencing plaque phenotype and stability at all stages of its evolution. We aimed to evaluate, by flow cytometry, if blood neutrophil number and phenotype-including their phenotypic relationships with platelets, monocytes and lymphocytes-have an association with lipid-rich necrotic core volume (LRNCV), a generic index of coronary plaque vulnerability, in a group of stable patients with chronic coronary syndrome (CCS). In 55 patients, (68.53 ± 1.07 years of age, mean ± SEM; 71% male), the total LRNCV in each subject was assessed by a quantitative analysis of all coronary plaques detected by computed tomography coronary angiography (CTCA) and was normalized to the total plaque volume. The expression of CD14, CD16, CD18, CD11b, HLA-DR, CD163, CCR2, CCR5, CX3CR1, CXCR4 and CD41a cell surface markers was quantified by flow cytometry. Adhesion molecules, cytokines and chemokines, as well as MMP9 plasma levels, were measured by ELISA. On a per-patient basis, LRNCV values were positively associated, by a multiple regression analysis, with the neutrophil count (n°/µL) (p = 0.02), neutrophil/lymphocyte ratio (p = 0.007), neutrophil/platelet ratio (p = 0.01), neutrophil RFI CD11b expression (p = 0.02) and neutrophil-platelet adhesion index (p = 0.01). Significantly positive multiple regression associations of LRNCV values with phenotypic ratios between neutrophil RFI CD11b expression and several lymphocyte and monocyte surface markers were also observed. In the bivariate correlation analysis, a significantly positive association was found between RFI values of neutrophil-CD41a+ complexes and neutrophil RFI CD11b expression (p < 0.0001). These preliminary findings suggest that a sustained increase in circulating neutrophils, together with the up-regulation of the integrin/activation membrane neutrophil marker CD11b may contribute, through the progressive intra-plaque accumulation of necrotic/apoptotic cells exceeding the efferocytosis/anti-inflammatory capacity of infiltrating macrophages and lymphocytes, to the relative enlargement of the lipid-rich necrotic core volume of coronary plaques in stable CAD patients, thus increasing their individual risk of acute complication.