Search Results (75)

Autoimmune diseases arise from the failure of self-tolerance. The recognition of self-antigen peptide–MHC (pMHC) complexes by the T-cell receptor (TCR) is the fundamental event triggering autoimmune pathogenesis. While traditional immunosuppressants provide broad systemic effects, they often compromise global immunity. Emerging molecular strategies aim to selectively disrupt the trimolecular complex—comprising the TCR, the antigenic peptide, and the MHC molecule—to induce antigen-specific tolerance. This review highlights the pMHC–TCR interaction as the primary molecular checkpoint for antigen-specific intervention. We discuss the structural basis of these interactions and their potential to redefine the therapeutic landscape for autoimmune diseases (ADs). We examine the molecular drivers of tolerance breakdown—including genetic susceptibility, molecular mimicry, post-translational modifications (PTMs), and ectopic MHC II expression—that shape the autoreactive T-cell landscape. This review examines current advancements in biological and pharmacological interventions, such as pMHC-decorated nanoparticles and soluble pMHC, to reprogram pathogenic T-cell response. We also explored CAR-T therapy strategies for autoimmune diseases, such as CAR-Treg, designed to precisely modulate pMHC-TCR signaling. Collectively, these precision interventions in immunological synapse assembly during autoimmune response are considered the basis for safer, antigen-specific immunotherapy capable of restoring self-tolerance without global immunosuppression. Full article

Advances in multimodal therapy for high-risk neuroblastomas (NBs) have plateaued, prompting therapeutic initiatives to harness the immune system. NBs, however, are immunologically “cold” and a significant challenge to immunotherapy. Here, in a Jurkat lymphocyte cytotoxicity model, we describe an antigen-independent, cell-mediated mechanism for eliminating NB cells, first detected in PMA-activated low pcDNA-SH-SY5Y and high TrkAIII-SH-SY5Y TrkAIII-expressing cells, which are resistant to Jurkat elimination under normal conditions. Characterization of this mechanism through live cell imaging, adhesion assays, RT-PCR, Western blotting and indirect IF, employing a variety of inhibitors, indicates that it initiates with PMA-induced NB cell CCL2 expression. This results in CCL2 promotion of Jurkat CCR2b expression, CCL2/CCR2b-mediated Jurkat LFA-1 activation and the formation of cytotoxic lipid-raft LFA1/ICAM-1 immune synapses, through which Jurkat m-TRAIL combines with PMA-enhanced NB cell DR5/TRAIL-R2 expression to induce NB cell apoptosis. This mechanism is enhanced by the NB-associated oncoprotein TrkAIII through Shp/Src-regulated c-FLIP sequester and is PD-L1/PD-1-independent and resistant to osteoprotegerin. It eliminates both non-MYCN-amplified (SH-SY5Y and SK-N-SH) and MYCN-amplified (SMS-KCNR) NB cells that exhibit PMA-inducible CCL2 expression but not MYCN-amplified NB cells (IMR-32 and NB-1) that exhibit CCL2 repression, and is offset by reciprocal NB cell-induced Fas-mediated Jurkat cell apoptosis. These findings form a solid foundation for further pre-clinical development aimed at identifying clinically relevant physiological immune cell equivalents and alternative PKC activators, with the ultimate goal of translating this mechanism into an effective immune-therapeutic approach for the treatment of high-risk non-immunogenic NBs, especially NBs that exhibit CCL2 and TrkAIII expression. Full article

Insights into the state of individual cells within a living organism are essential for identifying diseases and abnormalities. The internal state of a cell is reflected in its morphological features and changes in the localization of intracellular molecules. Using this information, it is possible to infer the state of the cells with high precision. In recent years, technological advancements and improvements in instrument specifications have made large-scale analyses, such as single-cell analysis, more widely accessible. Among these technologies, imaging flow cytometry (IFC) is a high-throughput imaging platform that can simultaneously acquire information from flow cytometry (FCM) and cellular images. While conventional FCM can only obtain fluorescence intensity information corresponding to each detector, IFC can acquire multidimensional information, including cellular morphology and the spatial arrangement of proteins, nucleic acids, and organelles for each imaging channel. This enables the discrimination of cell types and states based on the localization of proteins and organelles, which is difficult to assess accurately using conventional FCM. Because IFC can acquire a large number of single-cell morphological images in a short time, it is well suited for automated classification using machine learning. Furthermore, commercial instruments that combine integrated imaging and cell sorting capabilities have recently become available, enabling the sorting of cells based on their image information. In this review, we specifically highlight practical applications of IFC in four representative areas: cell cycle analysis, protein localization analysis, immunological synapse formation, and the detection of leukemic cells. In addition, particular emphasis is placed on applications that directly contribute to elucidating molecular mechanisms, thereby distinguishing this review from previous general overviews of IFC. IFC enables the estimation of cell cycle phases from large numbers of acquired cellular images using machine learning, thereby allowing more precise cell cycle analysis. Moreover, IFC has been applied to investigate intracellular survival and differentiation signals triggered by external stimuli, to monitor DNA damage responses such as γH2AX foci formation, and more recently, to detect immune synapse formation among interacting cells within large populations and to analyze these interactions at the molecular level. In hematological malignancies, IFC combined with fluorescence in situ hybridization (FISH) enables high-throughput detection of chromosomal abnormalities, such as BCR-ABL1 translocations. These advances demonstrate that IFC provides not only morphological and functional insights but also clinically relevant genomic information at the single-cell level. By summarizing these unique applications, this review aims to complement existing publications and provide researchers with practical insights into how IFC can be implemented in both basic and translational research. Full article

The review compares the principles of organization of the brain and immune system, two important organs developed over 500 million years in multicellular organisms, including humans. It summarizes the latest results from research in neurosciences and immunology concerning intercellular communication. While in the brain, intercellular communication is primarily based on exchange of electrical signals, this is not the case in the immune system. The question, therefore, arises as to whether nature developed two entirely different systems of organization. It will be demonstrated that a few basic principles of brain and immune responses are organized in a different way. A majority of intercellular communications, however, such as the formation of synapses, are shown to have many similarities. Both systems are intimately interconnected to protect the body from the1 dangers of the outside and the inside world. During homeostasis, all systems are in regulatory balance. A new hypothesis states that the central systems surrounded by bone, namely the central nervous system (CNS) and the central immune system (CIS), are based on three types of stem cells and function in an open but autonomous way. T cell immune responses to antigens from blood and cerebrospinal fluid protect the system and maintain neuroimmune homeostasis. The newly discovered tunneling nanotubes and extracellular vesicles are postulated to play an important role in crosstalk with already known homeostasis regulators and help in cellular repair and the recycling of biologic material. Three examples are selected to illustrate dysfunctions of homeostasis, namely migraine, multiple sclerosis, and brain cancer. The focus on these different conditions provides deep insights into such neurological and/or immunological malfunctions. Technological advances in neurosciences and immunology can enable neuroimmunomodulation and the development of new treatment possibilities. Full article

Introduction: The interface between T cells and the tumor microenvironment, termed the ‘immunological synapse’, consists of multiple checkpoint protein pairs co-expressed on both sides of the synapse. mir-16, a microRNA from a widely known tumor-suppressor family of miRNAs, was previously shown by us to be downregulated in melanoma. As other miRNAs from this family have been shown to directly target checkpoint proteins, here we investigated whether miR-16 influences the expression patterns of checkpoint proteins in melanoma. Methods: Single-cell gene expression data from the melanoma microenvironment were retrieved from a public database. Melanoma cell lines were established from metastatic lesions and transiently transfected with an hsa-miR-16-5p-mimic RNA or a mir-16-expressing plasmid. The mRNA expression profiles were analyzed using an Affymetrix microarray. Direct targets of miR-16 were identified by luciferase reporter assays. Protein levels were assessed by Western blotting. Results: Bioinformatic analysis revealed that the expression levels of eight checkpoint mRNAs, known to be present on the melanoma side of the immunological synapse, were highly correlated. Four of these mRNAs contained putative binding sites for the miR-15/16 family. miR-16 expression was significantly reduced in melanoma cells, compared to normal melanocytes. Luciferase reporter assays demonstrated that miR-16 directly targets the 3′ untranslated regions (3′UTRs) of CD40, CD80. The mRNAs downregulated following miR-16 overexpression were highly enriched for genes involved in autophagy, vesicle-mediated transport, and the regulation of protein membrane localization. Among these, VTI1B and SMPD1 were confirmed to be direct targets of miR-16. Transient overexpression of miR-16 resulted in a significant reduction in SMPD1 and VTI1B levels in melanoma cell lines. Conclusions: Our findings suggest that miR-16 potentially modulates melanoma tumorigenesis, metastasis and immunogenicity by altering the composition of checkpoint proteins at the immunological synapse and by regulating cellular pathways associated with intracellular trafficking and transmembrane protein presentation. Full article

(1) Background: Single-cell multi-omics is a powerful method for the dissection and detection of complicated immunologic functions and synapses. However, most currently available technologies merge datasets of different omics from separate portions of the same sample to generate combined multi-omics. This process is a source of bias, mainly in the field of immunology on cells originating from pluripotent hematopoietic stem cells with high flexibility during maturation. (2) Methods: Although new multi-omics approaches have been developed to use the advantages of cellular and molecular barcoding and next-generation sequencing to solve this issue, one of the main current challenges is intracellular proteomics, which should be combined with other omics data with high importance for immune system studies. We designed this study to evaluate previously recommended minimal permeabilization and fixation methods on the quality and quantity of transcriptomics and proteomics data generated by the BD Rhapsody™ Single-Cell Analysis System. (3) Results: Our findings showed that high-throughput sequencing with advanced quality and read-out is required for the combination of multi-omics outcomes from a permeabilized single cell. Therefore, the HiseqX platform was selected for further analysis. The effect of immune stimulation was observed clearly as the separated clusters of helper and cytotoxic T cells using unsupervised clustering. Importantly, fixation and permeabilization did not affect the general expression profile of unstimulated cells. However, fixation and permeabilization were proved to negatively impact the detection of the whole transcriptome for single-cell assay. Nevertheless, about 60% of the transcriptomic signature of the stimulation was detected. If the measurement of combined surface and intracellular markers is required to be achieved, the modified fixation and permeabilization method is recommended because of a lower transcriptomic loss and more precise proteomic fingerprint detected. (4) Conclusions: The findings of this study support the potential possibility for integrating intracellular proteomics, which needs to be optimized and tested with newly designed oligonucleotide-tagged antibodies targeting intracellular proteins. Full article

T-cell-engaging bispecific antibodies (BsAbs) are monoclonal antibodies that redirect the cytotoxic activity of T-cells to target malignant neoplasms. B-cell non-Hodgkin lymphoma (B-NHL) is a heterogenous group of aggressive and indolent malignancies with significant therapeutic challenges due to high relapse rates and limited options for relapsed/refractory disease. BsAbs function by simultaneously binding to CD3 on endogenous T-cells and a tumor-associated antigen, creating an immunologic synapse which results in the death of the target cell. The widespread T-cell activation that occurs with BsAb administration can result in cytokine release syndrome and neurological adverse events. Mosunetuzumab, epcoritamab, and glofitamab are CD20-targeting BsAbs that have demonstrated promising single-agent activity in both indolent and aggressive B-NHL. BsAbs are now being evaluated in combination with other anti-lymphoma agents and in earlier lines of treatment, and the results of ongoing clinical trials involving these agents have the potential to reshape the treatment landscape for B-NHL. In this review, we describe the structural features, clinical data, and toxicity profile associated with the BsAbs currently used to treat B-NHL and then discuss ongoing studies and future directions for this exciting new class of therapeutic agents. Full article

The complement system is a pivotal component of innate immunity, extensively studied in vertebrates but also present in invertebrates. This study explores the existence of a terminal complement pathway in the tunicate Botryllus schlosseri, aiming to understand the evolutionary integration of innate and adaptive immunity. Through transcriptome analysis, we identified a novel transcript, BsITCCP, encoding a protein with both MACPF and LDLa domains—a structure resembling that of vertebrate C9 but with a simpler organization. Phylogenetic reconstruction positions BsITCCP between invertebrate perforins and vertebrate terminal complement proteins, suggesting an evolutionary link. Localization studies confirmed that bsitccp is transcribed in cytotoxic morula cells (MCs), which are also responsible for producing other complement components like BsC3, BsMBL, BsMASP, and BsBf. Functional assays demonstrated that bsitccp transcription is upregulated in response to nonself challenges and is dependent on BsC3 activity; inhibition of BsC3 led to a significant reduction in BsITCCP expression. Electron microscopy revealed that MCs form contact with perforated yeast cells, indicating a possible mechanism of cell lysis similar to the immunological synapse observed in vertebrates. These findings suggest that a C3-governed lytic complement pathway exists in B. schlosseri, challenging the assumption that a C5 ortholog is necessary for such a pathway. This work enhances our understanding of the evolution of the complement system and suggests that invertebrates possess a terminal complement complex capable of mediating cell lysis, regulated by C3. Future studies will focus on confirming the pore-forming ability of BsITCCP and its role in the immunological synapse. Full article

Background/Objectives: Tumor microenvironmental hypoxia is an established hallmark of solid tumors. It significantly contributes to tumor aggressiveness and therapy resistance and has been reported to affect the balance of activating/inhibitory surface receptors’ expression and activity on NK cells. In the current study, we investigated the impact of hypoxia on the surface expression of Siglec-7 and Siglec-9 (Sig-7/9) and their ligands in NK cells and tumor target cells. The functional consequence of Siglec blockage using nanoparticles specifically designed to target and block Sig-7/9 receptors on NK cell cytotoxicity was elucidated. Methods: CD56⁺ CD3⁻ NK cells were isolated from PBMCs along with an NK-92 clone and used as effector cells, while MCF-7 and K562 served as target cells. All cells were incubated under normoxic or hypoxic conditions for 24 h. To assess Siglec-7 and Siglec-9 receptor expression, U937, NK-92, and primary NK cells were stained with PE-labeled antibodies against CD328 Siglec-7/9. Interactions between Siglec-7/9 and their sialylated ligands, along with their functional impact on NK cell activity, were evaluated using polymeric nanoparticles coated with a sialic acid mimetic. Immunological synapse formation and live-cell imaging were performed with a ZEISS LSM 800 with Airyscan at 10× magnification for 24 h. Results: Our data indicate that hypoxia had no effect on the expression of Siglec-7/9 receptors by NK cells. In contrast, hypoxic stress resulted in an increase in Siglec-7 sialoglycan ligand expression by a sub-population of NK target cells. Using polymeric nanoparticles coated with a sialic acid mimetic that binds both Siglec-7 and -9 (Sig-7/9 NP), we demonstrated that incubation of these nanoparticles with NK cells resulted in increased immunological synapse formation, granzyme B accumulation, and killing of NK target cells. These studies indicate that hypoxic stress may have an impact on NK cell-based therapies and highlight the need to consider the hypoxic microenvironment for tumor-specific glycosylation. Conclusions: Our findings point to the role of Siglec–sialylated glycan interactions in hypoxic stress-induced NK cell dysfunction and recommend the potential integration of the manipulation of this axis through the targeting of Siglecs in future cancer immunotherapy strategies. Full article

Background: Although belonging to different branches of the immune system, cytotoxic CD8⁺ αβ T cells and γδ T cells utilize common cytolytic effectors including FasL, granzymes, perforin and granulysin. The effector proteins are stored in different subsets of lysosome-related effector vesicles (LREVs) and released to the immunological synapse upon target cell encounter. Notably, in activated cells, LREVs and potentially other vesicles are continuously produced and released as extracellular vesicles (EVs). Presumably, EVs serve as mediators of intercellular communication in the local microenvironment or at distant sites. Methods: EVs of activated and expanded cytotoxic CD8⁺ αβ T cells or γδ T cells were enriched from culture supernatants by differential and ultracentrifugation and characterized by nanoparticle tracking analyses and Western blotting. For a comparative proteomic profiling, EV preparations from both cell types were isobaric labeled with tandem mass tags (TMT10plex) and subjected to mass spectrometry analysis. Results: 686 proteins were quantified in EV preparations of cytotoxic CD8⁺ αβ T cells and γδ T cells. Both populations shared a major set of similarly abundant proteins, while much fewer proteins presented higher abundance levels in either CD8⁺ αβ T cells or γδ T cells. To our knowledge, we provide the first comparative analysis of EVs from cytotoxic CD8⁺ αβ T cells and γδ T cells. Full article

Fifty-two years have passed since President Nixon launched the “War on Cancer”. Despite unparalleled efforts and funds allocated worldwide, the outlined goals were not achieved because cancer treatment approaches such as chemotherapy, radiation therapy, hormonal and targeted therapies have not fully met the expectations. Based on the recent literature, a new direction in cancer therapy can be proposed which targets connections between cancer cells and their microenvironment by chemical means. Cancer–stromal synapses such as immunological synapses between cancer and immune cells provide an attractive target for this approach. Such synapses form ligand–receptor clusters on the interface of the interacting cells. They share a common property of involving intercellular clusters of spatially proximate and cooperatively acting proteins. Synapses provide the space for the focused intercellular signaling molecules exchange. Thus, the disassembly of cancer–stromal synapses may potentially cause the collapse of various tumors. Additionally, the clustered arrangement of synapse components offers opportunities to enhance treatment safety and precision by using targeted crosslinking chemical agents which may inactivate cancer synapses even in reduced concentrations. Furthermore, attaching a cleavable cell-permeable toxic agent(s) to a crosslinker may further enhance the anti-cancer effect of such therapeutics. The highlighted approach promises to be universal, relatively simple and cost-efficient. We also hope that, unlike chemotherapeutic and immune drugs that interact with a single target, by using supramolecular large clusters that include many different components as a target, the emergence of a resistance characteristic of chemo- and immunotherapy is extremely unlikely. Full article

Oncolytic viruses and combinatorial immunotherapy for cancer (this Special Issue) are both part of cancer treatment at IOZK. This review focusses on an individual multimodal cancer immunotherapy concept developed by IOZK, Cologne, Germany. The scientific rationale for employing three main components is explained: (i) oncolytic Newcastle disease virus, (ii) modulated electrohyperthermia and (iii) individual tumor antigen and oncolytic virus modified dendritic cell vaccine (IO-VAC^R). The strategy involves repeated cancer-immunity cycles evoked in cancer patients by systemic oncolytic virus exposure plus hyperthermia pretreatment to induce immunogenic cell death followed by intradermal IO-VAC^R vaccination. As an example of the experience at IOZK, we present the latest results from combining the immunotherapy with standard treatment of patients suffering from glioblastoma multiforme. The promising clinical results in terms of overall survival benefit of additional individualized multimodal immunotherapy are presented. The cancer-immunity cycle, as introduced 10 years ago, describes key important steps occurring locally at the sites of both tumor and draining lymph nodes. This view is extended here towards systemic events occuring in blood where immunogenic cell death-induced tumor antigens are transported into the bone marrow. For 20 years it has been known that bone marrow is an antigen-responsive organ in which dendritic cells present tumor antigens to T cells leading to immunological synapse formation, tumor antigen-specific T cell activation and memory T cell formation. Bone marrow is known to be the most prominent source of de novo cellular generation in the body and to play an important role for the storage and maintenance of immunological memory. Its systemic activation is recommended to augment cancer-immunity cycles. Full article

Bone marrow is known as the site of hematopoiesis. What is not being described in textbooks of immunology is the fact that bone marrow is not only a generative, but also an antigen-responsive, immune organ. It is also a major storage site for antigen-specific memory B and T cells. That bone marrow is a priming site for T cell responses to blood borne antigens was discovered exactly 20 years ago. This review celebrates this important discovery. The review provides a number of examples of medical relevance of bone marrow as a central immune system, including cancer, microbial infections, autoimmune reactions, and bone marrow transplantation. Bone marrow mesenchymal stem cell-derived stromal cells provide distinct bone marrow niches for stem cells and immune cells. By transmitting anti-inflammatory dampening effects, facilitating wound healing and tissue regeneration mesenchymal stem cells contribute to homeostasis of bone and other tissues. Based on the evidence presented, the review proposes that bone marrow is a multifunctional and protective immune system. In an analogy to the central nervous system, it is suggested that bone marrow be designated as the central immune system. Full article

Endocytic recycling regulates the cell surface receptor composition of the plasma membrane. The surface expression levels of the T cell receptor (TCR), in concert with signal transducing co-receptors, regulate T cell responses, such as proliferation, differentiation, and cytokine production. Altered TCR expression contributes to pro-inflammatory skewing, which is a hallmark of autoimmune diseases, such as systemic lupus erythematosus (SLE), defined by a reduced function of regulatory T cells (Tregs) and the expansion of CD4⁺ helper T (Th) cells. The ensuing secretion of inflammatory cytokines, such as interferon-γ and interleukin (IL)-4, IL-17, IL-21, and IL-23, trigger autoantibody production and tissue infiltration by cells of the adaptive and innate immune system that induce organ damage. Endocytic recycling influences immunological synapse formation by CD4⁺ T lymphocytes, signal transduction from crosslinked surface receptors through recruitment of adaptor molecules, intracellular traffic of organelles, and the generation of metabolites to support growth, cytokine production, and epigenetic control of DNA replication and gene expression in the cell nucleus. This review will delineate checkpoints of endosome traffic that can be targeted for therapeutic interventions in autoimmune and other disease conditions. Full article

Inflammatory bowel diseases (IBD) are complex chronic inflammatory disorders of the gastrointestinal (GI) tract. Recent evidence suggests that the gut-brain axis may be pivotal in gastrointestinal and neurological diseases, especially IBD. Here, we present the first proof of concept for a microfluidic technology to model bilateral neuro-immunological communication. We designed a device composed of three compartments with an asymmetric channel that allows the isolation of soma and neurites thanks to microchannels and creates an in vitro synaptic compartment. Human-induced pluripotent stem cell-derived cortical glutamatergic neurons were maintained in soma compartments for up to 21 days. We performed a localized addition of dendritic cells (MoDCs) to either the soma or synaptic compartment. The microfluidic device was coupled with microelectrode arrays (MEAs) to assess the impact on the electrophysiological activity of neurons while adding dendritic cells. Our data highlight that an electrophysiologic signal is transmitted between two compartments of glutamatergic neurons linked by synapses in a bottom-up way when soma is exposed to primed dendritic cells. In conclusion, our study authenticates communication between dendritic cells and neurons in inflammatory conditions such as IBD. This platform opens the way to complexification with gut components to reach a device for pharmacological compound screening by blocking the gut-brain axis at a mucosal level and may help patients. Full article