Macrophage-based cancer cellular therapy has gained substantial interest. However, the capability of engineered macrophages to target cancer heterogeneity and modulate adaptive immunity remains unclear. Here, exploiting the myeloid antibody-dependent cellular phagocytosis biology and phagocytosis checkpoint blockade, we report the enhanced synthetic phagocytosis receptor (eSPR) that integrate FcRγ-driven phagocytic chimeric antigen receptors (CAR) with built-in secreted CD47 blockers. The eSPR engineering empowers macrophages to combat tumor antigen heterogeneity. Transduced by adenoviral vectors, eSPR macrophages are intrinsically pro-inflammatory imprinted and resist tumoral polarization. Transcriptomically and phenotypically, eSPR macrophages elicit a more favorable tumor immune landscape. Mechanistically, eSPR macrophages in situ stimulate CD8 T cells via phagocytosis-dependent antigen cross-presentation. We also validate the functionality of the eSPR system in human primary macrophages.

Since the blood and lymphatic endothelium regulates homeostasis and inflammation during health and disease, establishment of vascularized Organ-on-Chip platforms with blood and lymphatic endothelial cells (BEC/LEC) is a pre-requisite to further advance the field of tissue engineering. Here, we aimed to determine whether characteristics of BECs and LECs cultured under flow in a multi-organ-chip (MOC) are influenced by shear stress or inflammation. Dermis-derived primary BECs and LECs were used to endothelialize a MOC followed by culture for up to 14 days at lymphatic and blood flow rates. Under blood flow, both cell types changed morphology, aligned in flow direction, and showed close cell-cell contacts as in in vivo blood vasculature. Under lymphatic flow, neither BEC nor LEC aligned, and both showed a cobblestone-appearance with limited intercellular contacts similar to lymphatics. Cells retained their cell type-specific phenotype and cytokine secretion profiles. CCL21 expression in LECs was rescued by flow, but diminished again with TNFα exposure, together with the LEC-specific markers PROX1 and TFF3. Homeostatic cytokine secretion was higher in BECs, but the response to TNFα was more pronounced in LECs. Results indicate that BEC and LEC phenotype and cytokine secretion is mostly an intrinsic property with only morphology and CCL21 being influenced by flow.

Squalene-based emulsion (SE) adjuvants like MF59 and AS03 are used in protein subunit vaccines against influenza virus (e.g., Fluad, Pandemrix, Arepanrix) and SARS-CoV-2 (e.g., Covifenz, SKYCovione). We demonstrate the critical role of uric acid (UA), a damage-associated molecular pattern (DAMP), in triggering immunogenicity by SE adjuvants. In mice, SE adjuvants elevated DAMP levels in draining lymph nodes. Strikingly, inhibition of UA synthesis reduced vaccine-induced innate immunity, subsequently impairing optimal antibody and T cell responses. In vivo treatment with UA crystals elicited partial adjuvant effects. In vitro stimulation with UA crystals augmented the activation of dendritic cells (DCs) and B cells and altered multiple pathways in these cells, including inflammation and antigen presentation in DCs and cell proliferation in B cells. In an influenza vaccine model, UA contributed to protection against influenza viral infection. These results demonstrate the importance of DAMPs, specifically the versatile role of UA in the immunogenicity of SE adjuvants, by regulating DCs and B cells.

The lymphatic vasculature plays essential roles in fluid balance, immunity, and lipid transport. Chronic, low-grade inflammation in peripheral tissues develops when lymphatic structure or function is impaired, as observed during aging. While aging has been associated with a broad range of heart pathophysiology, its effect on cardiac lymphatic vasculature has not been characterized. Here, we analyzed cardiac lymphatics in aged 20-month-old mice versus young 2-month-old mice. Aged hearts showed reduced lymphatic vascular density, more dilated vessels, and increased inflammation and fibrosis in peri-lymphatic zones. As exercise has shown benefits in several different models of age-related heart disease, we further investigated the effects of aerobic training on cardiac lymphatics. Eight weeks of voluntary wheel running attenuated age-associated adverse remodeling of the cardiac lymphatics, including reversing their dilation, increasing lymph vessel density and branching, and reducing perilymphatic inflammation and fibrosis. Intravital lymphangiography demonstrated improved cardiac lymphatic flow after exercise training. Our findings illustrate that aging leads to cardiac lymphatic dysfunction, and that exercise can improve lymphatic health in aged animals.

B cells exclusively expressing IgD on the cell surface (IgD+IgM- B cells) have been identified in mammals, where they seem to play a still not well-defined role in peripheral tolerance. These cells have also been reported in catfish (Ictalurus punctatus) peripheral blood and in several mucosal tissues of rainbow trout (Oncorhynchus mykiss), including gut, gills and skin. As in mammals, the precise function of these cells remains obscure, yet, in rainbow trout mucosal surfaces, these cells have been shown to be differentiated to plasma-like cells. Interestingly, in the gills, these IgD+IgM- B cells expressed high levels of the CC chemokine receptor 7 (CCR7), receptor that in mammals controls the migration of B and T cells to secondary lymphoid organs. In this work, we have established that this is also true for the trout skin, where CCR7 defines a specific subset of IgD+IgM- B cells that are further differentiated to a plasma-like profile than those not expressing CCR7. These findings increase the current understanding of this enigmatic B cell population and point to CCR7 as a key differentiation marker for these cells.

Decades of basic and translational research have led to a momentum shift in dissecting the relationship between immune cells and cancer. This culminated in the emergence of breakthrough immunotherapies that paved the way for oncologists to manage certain hard-to-treat cancers. The application of high-throughput techniques of genomics, transcriptomics, and proteomics was conclusive in making and expediting the manufacturing process of cancer vaccines. Using the latest research technologies has also enabled scientists to interpret complex and multiomics data of the tumour mutanome, thus identifying new tumour-specific antigens to design new generations of cancer vaccines with high specificity and long-term efficacy. Furthermore, combinatorial regimens of cancer vaccines with immune checkpoint inhibitors have offered new therapeutic approaches and demonstrated impressive efficacy in cancer patients over the last few years. In the present review, we summarize the current state of cancer vaccines, including their potential therapeutic effects and the limitations that hinder their effectiveness. We highlight the current efforts to mitigate these limitations and highlight ongoing clinical trials. Finally, a special focus will be given to the latest milestones expected to transform the landscape of cancer therapy and nurture hope among cancer patients.

Both type I interferon (IFN-I) and CD4+ T-cell help are required to generate effective CD8+ T-cell responses to cancer. We here outline based on existing literature how IFN-I signaling and CD4+ T-cell help are connected. Both impact on the functional state of dendritic cells (DCs), particularly conventional (c)DC1. The cDC1s are critical for crosspresentation of cell-associated antigens and for delivery of CD4+ T-cell help for cytotoxic T-lymphocyte (CTL) effector and memory differentiation. In infection, production of IFN-I is prompted by pathogen-associated molecular patterns (PAMPs), while in cancer it relies on danger-associated molecular patterns (DAMPs). IFN-I production by tumor cells and pDCs in the tumor micro-environment (TME) is often limited. IFN-I signals increase the ability of migratory cDC1s and cDC2s to transport tumor antigens to tumor-draining lymph nodes (tdLNs). IFN-I also enables cDC1s to form and sustain the platform for help delivery by stimulating the production of chemokines that attract CD4+ and CD8+ T cells. IFN-I promotes delivery of help in concert with CD40 signals by additive and synergistic impact on cross-presentation and provision of critical costimulatory and cytokine signals for CTL effector and memory differentiation. The scenario of CD4+ T-cell help therefore depends on IFN-I signaling. This scenario can play out in tdLNs as well as in the TME, thereby contributing to the cancer immunity cycle. The collective observations may explain why both IFN-I and CD4+ T-cell help signatures in the TME correlate with good prognosis and response to PD-1 targeting immunotherapy in human cancer. They also may explain why a variety of tumor types in which IFN-I signaling is attenuated, remain devoid of functional CTLs.

T cell activation is the final key event (KE4) in the adverse outcome pathway (AOP) of skin sensitization. However, validated new approach methodologies (NAMs) for evaluating this step are missing. Accordingly, chemicals that activate an unusually high frequency of T cells, as does the most prevalent metal allergen nickel, are not yet identified in a regulatory context. T cell reactivity to chemical sensitizers might be especially relevant in real-life scenarios, where skin injury, co-exposure to irritants in chemical mixtures, or infections may trigger the heterologous innate immune stimulation necessary to induce adaptive T cell responses. Additionally, cross-reactivity, which underlies cross-allergies, can only be assessed by T cell tests. To date, several experimental T cell tests are available that use primary naïve and memory CD4+ and CD8+ T cells from human blood. These include priming and lymphocyte proliferation tests and, most recently, activation-induced marker (AIM) assays. All approaches are challenged by chemical-mediated toxicity, inefficient or unknown generation of T cell epitopes, and a low throughput. Here, we summarize solutions and strategies to confirm in vitro T cell signals. Broader application and standardization are necessary to possibly define chemical applicability domains and to strengthen the role of T cell tests in regulatory risk assessment.

The successful development of germinal centers (GC) relies heavily on innate mechanisms to amplify the initial inflammatory cascade. In addition to their role in antigen presentation, innate cells are essential for the redirection of circulating lymphocytes toward the draining lymph node (dLN) to maximize antigen surveillance. Sphingosine-1-Phosphate (S1P) and its receptors (S1PR1-5) affect various aspects of immunity; however, the role of S1PR4 in regulating an immune response is not well understood. Here we use a footpad model of localized TH1 inflammation to carefully monitor changes in leukocyte populations within the blood, the immunized tissue, and the dLN. Within hours of immunization, neutrophils failed to adequately mobilize and infiltrate into the footpad tissue of S1PR4-/- mice, thereby diminishing the local vascular changes thought to be necessary for redirecting circulating cells toward the inflamed region. Neutrophil depletion with anti-Ly6G antibodies significantly reduced early tissue edema as well as the redirection and initial accumulation of naïve lymphocytes in dLN of WT mice, while the effects were less prominent or absent in S1PR4-/- dLN. Adoptive transfer experiments further demonstrated that the lymphocyte homing deficiencies in vivo were not intrinsic to the donor S1PR4-/- lymphocytes, but were instead attributed to differences within the S1PR4-deficient host. Reduced cell recruitment in S1PR4-/- mice would seed the dLN with fewer antigen-respondent lymphocytes and indeed, dLN hypertrophy at the peak of the immune response was severely diminished, with attenuated GC and activation pathways in these mice. Histological examination of the S1PR4-/- dLN also revealed an underdeveloped vascular network with reduced expression of the leukocyte tethering ligand, PNAd, within high endothelial venule regions, suggesting inadequate growth of the dLN meant to support a robust GC response. Thus, our study reveals that S1PR4 may link early immune modulation by neutrophils to the initial recruitment of circulating lymphocytes and downstream expansion and maturation of the dLN, thereby contributing to optimal GC development during an adaptive response.

During tuberculosis (TB), migration of dendritic cells (DCs) from the site of infection to the draining lymph nodes is known to be impaired, hindering the rapid development of protective T-cell-mediated immunity. However, the mechanisms involved in the delayed migration of DCs during TB are still poorly defined. Here, we found that infection of DCs with Mycobacterium tuberculosis (Mtb) triggers HIF1A-mediated aerobic glycolysis in a TLR2-dependent manner, and that this metabolic profile is essential for DC migration. In particular, the lactate dehydrogenase inhibitor oxamate and the HIF1A inhibitor PX-478 abrogated Mtb-induced DC migration in vitro to the lymphoid tissue-specific chemokine CCL21, and in vivo to lymph nodes in mice. Strikingly, we found that although monocytes from TB patients are inherently biased toward glycolysis metabolism, they differentiate into poorly glycolytic and poorly migratory DCs compared with healthy subjects. Taken together, these data suggest that because of their preexisting glycolytic state, circulating monocytes from TB patients are refractory to differentiation into migratory DCs, which may explain the delayed migration of these cells during the disease and opens avenues for host-directed therapies for TB.

Past research has identified that cancer cells sustain several cancer hallmarks by impairing function of the endolysosomal system (ES). Thus, maintaining the functional integrity of endolysosomes is crucial, which heavily relies on two key protein families: soluble hydrolases and endolysosomal membrane proteins. Particularly members of the TPC (two-pore channel) and TRPML (transient receptor potential mucolipins) families have emerged as essential regulators of ES function as a potential target in cancer therapy. Targeting TPCs and TRPMLs has demonstrated significant impact on multiple cancer hallmarks, including proliferation, growth, migration, and angiogenesis both in vitro and in vivo. Notably, endosomes and lysosomes also actively participate in various immune regulatory mechanisms, such as phagocytosis, antigen presentation, and the release of proinflammatory mediators. Yet, knowledge about the role of TPCs and TRPMLs in immunity is scarce. This prompts a discussion regarding the potential role of endolysosomal ion channels in aiding cancers to evade immune surveillance and destruction. Specifically, understanding the interplay between endolysosomal ion channels and cancer immunity becomes crucial. Our review aims to comprehensively explore the current knowledge surrounding the roles of TPCs and TRPMLs in immunity, whilst emphasizing the critical need to elucidate their specific contributions to cancer immunity by pointing out current research gaps that should be addressed.

Tumor immune microenvironment (TIME) and its indications for lung cancer patient prognosis and therapeutic response have become new hotspots in cancer research in recent years. Tumor cells, immune cells, various regulatory factors, and their interactions in the TIME have been suggested to commonly influence lung cancer development and therapeutic outcome. The heterogeneity of TIME is composed of dynamic immune-related components, including various cancer cells, immune cells, cytokine/chemokine environments, cytotoxic activity, or immunosuppressive factors. The specific composition of cell subtypes may facilitate or hamper the response to immunotherapy and influence patient prognosis. Various markers have been found to stratify the patient prognosis or predict the therapeutic outcome. In this article, we systematically reviewed the recent advancement of TIME studies in lung adenocarcinoma (LUAD) using single-cell RNA sequencing (scRNA-seq) techniques, with specific focuses on the roles of TIME in LUAD development, TIME heterogeneity, indications of TIME in patient prognosis and therapeutic response during immunotherapy and drug resistance. The main findings in TIME heterogeneity and relevant markers or models for prognosis stratification and response prediction have been summarized. We hope that this review provides an overview of TIME status in LUAD and an inspiration for future development of strategies and biomarkers in LUAD treatment.

Despite major advances in therapeutic platforms, most patients with multiple myeloma (MM) eventually relapse and succumb to the disease. Among the novel therapeutic options developed over the past decade, genetically engineered T cells have a great deal of potential. Cellular immunotherapies, including chimeric antigen receptor (CAR) T cells, are rapidly becoming an effective therapeutic modality for MM. Marrow-infiltrating lymphocytes (MILs) derived from the bone marrow of patients with MM are a novel source of T cells for adoptive T-cell therapy, which robustly and specifically target myeloma cells. In this review, we examine the recent innovations in cellular immunotherapies, including the use of dendritic cells, and cellular tools based on MILs, natural killer (NK) cells, and CAR T cells, which hold promise for improving the efficacy and/or reducing the toxicity of treatment in patients with MM.

CD40, a novel immunomodulatory cancer therapy target, is expressed by B cells, macrophages, and dendritic cells (DCs) and mediates cytotoxic T cell priming through the CD40 ligand. Some tumors show promising responses to monotherapy or combination therapy with agonistic anti-CD40 antibodies. The development of improved anti-CD40 antibodies makes CD40 activation an innovative strategy in cancer immunotherapy. In this review, we trace the history of CD40 research and summarize preclinical and clinical findings. We emphasize the ongoing development of improved anti-CD40 antibodies and explore strategies for effective combination therapies. Guided by predictive biomarkers, future research should identify patient populations benefiting the most from CD40 activation.

The transient receptor potential (TRP) ion channels act as cellular sensors and mediate a plethora of physiological processes, including somatosensation, proliferation, apoptosis, and metabolism. Under specific conditions, certain TRP channels are involved in inflammation and immune responses. Thus, focusing on the role of TRPs in immune system cells may contribute to resolving inflammation. In this review, we discuss the distribution of five subfamilies of mammalian TRP ion channels in immune system cells and how these ion channels function in inflammatory mechanisms. This review provides an overview of the current understanding of TRP ion channels in mediating inflammation and may offer potential avenues for therapeutic intervention.

Acute kidney injury (AKI) is characterized by a rapid decline in renal function and is associated with a high morbidity and mortality rate. At present, the underlying mechanisms of AKI remain incompletely understood. Immune disorder is a prominent feature of AKI, and dendritic cells (DCs) play a pivotal role in orchestrating both innate and adaptive immune responses, including the induction of protective proinflammatory and tolerogenic immune reactions. Emerging evidence suggests that DCs play a critical role in the initiation and development of AKI. This paper aimed to conduct a comprehensive review and analysis of the role of DCs in the progression of AKI and elucidate the underlying molecular mechanism. The ultimate objective was to offer valuable insights and guidance for the treatment of AKI.

The Helios protein (encoded by the IKZF2 gene) is a member of the Ikaros transcription family and it has recently been proposed as a promising biomarker for systemic lupus erythematosus (SLE) disease progression in both mouse models and patients. Helios is beginning to be studied extensively for its influence on the T regulatory (Treg) compartment, both CD4+ Tregs and KIR+/Ly49+ CD8+ Tregs, with alterations to the number and function of these cells correlated to the autoimmune phenomenon. This review analyzes the most recent research on Helios expression in relation to the main immune cell populations and its role in SLE immune homeostasis, specifically focusing on the interaction between T cells and tolerogenic dendritic cells (tolDCs). This information could be potentially useful in the design of new therapies, with a particular focus on transfer therapies using immunosuppressive cells. Finally, we will discuss the possibility of using nanotechnology for magnetic targeting to overcome some of the obstacles related to these therapeutic approaches.

Despite major improvements in immunotherapeutic strategies, the immunosuppressive tumor microenvironment remains a major obstacle for the induction of efficient antitumor responses. In this study, we show that local delivery of a bispecific Clec9A-PD-L1 targeted type I interferon (AcTaferon, AFN) overcomes this hurdle by reshaping the tumor immune landscape.Treatment with the bispecific AFN resulted in the presence of pro-immunogenic tumor-associated macrophages and neutrophils, increased motility and maturation profile of cDC1 and presence of inflammatory cDC2. Moreover, we report empowered diversity in the CD8+ T cell repertoire and induction of a shift from naive, dysfunctional CD8+ T cells towards effector, plastic cytotoxic T lymphocytes together with increased presence of NK and NKT cells as well as decreased regulatory T cell levels. These dynamic changes were associated with potent antitumor activity. Tumor clearance and immunological memory, therapeutic immunity on large established tumors and blunted tumor growth at distant sites were obtained upon co-administration of a non-curative dose of chemotherapy.Overall, this study illuminates further application of type I interferon as a safe and efficient way to reshape the suppressive tumor microenvironment and induce potent antitumor immunity; features which are of major importance in overcoming the development of metastases and tumor cell resistance to immune attack. The strategy described here has potential for application across to a broad range of cancer types.

In this review we discuss an underexposed mechanism in the adaptive immune system where B cell and T cell immunity collaborate. The main function of B cell immunity is the generation of antibodies which are well known for their high affinity and antigen-specificity. Antibodies can bind antigens in soluble form making so-called immune complexes (ICs) or can opsonize antigen-exposing cells or particles for degradation. This leads to well-known effector mechanisms complement activation, antibody-dependent cytotoxicity and phagocytosis. What is less realized is that antibodies can play an important role in the targeting of antigen to dendritic cells (DCs) and thereby can drive T cell immunity. Here we summarize the studies that described this highly efficient process of antibody-mediated antigen uptake in DCs in vitro and in vivo. Only very low doses of antigen can be captured by circulating antibodies and subsequently trapped by DCs in vivo. We studied the handling of these ICs by DCs in subcellular detail. Upon immune complex engulfment DCs can sustain MHC class I and II antigen presentation for many days. Cell biological analysis showed that this function is causally related to intracellular antigen-storage compartments which are functional endolysosomal organelles present in DCs. We speculate that this function is immunologically very important as DCs require time to migrate from the site of infection to the draining lymph nodes to activate T cells. The implications of these findings and the consequences for the immune system, immunotherapy with tumor-specific antibodies and novel vaccination strategies are discussed.

Corneal transplant rejection primarily occurs because of the T helper 1 (Th1) effector cell-mediated immune response of the host towards allogeneic tissue. The evidence suggests that type 1 migratory conventional CD103+ dendritic cells (CD103+DC1) acquire an immunosuppressive phenotype in the tumor environment; however, the involvement of CD103+DC1 in allograft survival continues to be an elusive question of great clinical significance in tissue transplantation. In this study, we assess the role of CD103+DC1 in suppressing Th1 alloreactivity against transplanted corneal allografts. The immunosuppressive function of CD103+DC1 has been extensively studied in non-transplantation settings. We found that host CD103+DC1 infiltrates the corneal graft and migrates to the draining lymph nodes to suppress alloreactive CD4+ Th1 cells via the programmed death-ligand 1 axis. The systemic depletion of CD103+ DC1 in allograft recipients leads to amplified Th1 activation, impaired Treg function, and increased rate of allograft rejection. Although allograft recipient Rag1 null mice reconstituted with naïve CD4+CD25- T cells efficiently generated peripheral Treg cells (pTreg), the CD103+DC1-depleted mice failed to generate pTreg. Furthermore, adoptive transfer of pTreg failed to rescue allografts in CD103+DC1-depleted recipients from rejection. These data demonstrate the critical role of CD103+DC1 in regulating host alloimmune responses.

Immune responses rely on the rapid and coordinated migration of leukocytes. Whereas it is well established that single-cell migration is often guided by gradients of chemokines and other chemoattractants, it remains poorly understood how these gradients are generated, maintained, and modulated. By combining experimental data with theory on leukocyte chemotaxis guided by the G protein-coupled receptor (GPCR) CCR7, we demonstrate that in addition to its role as the sensory receptor that steers migration, CCR7 also acts as a generator and a modulator of chemotactic gradients. Upon exposure to the CCR7 ligand CCL19, dendritic cells (DCs) effectively internalize the receptor and ligand as part of the canonical GPCR desensitization response. We show that CCR7 internalization also acts as an effective sink for the chemoattractant, dynamically shaping the spatiotemporal distribution of the chemokine. This mechanism drives complex collective migration patterns, enabling DCs to create or sharpen chemotactic gradients. We further show that these self-generated gradients can sustain the long-range guidance of DCs, adapt collective migration patterns to the size and geometry of the environment, and provide a guidance cue for other comigrating cells. Such a dual role of CCR7 as a GPCR that both senses and consumes its ligand can thus provide a novel mode of cellular self-organization.

The CCR7 receptor allows dendritic cells to sense the chemokine CCL19. It also depletes CCL19 from the environment by endocytosis, leading to local gradients that can steer cells accurately and robustly through tissues, even over long distances (see related Research Article by Alanko et al.).

In the emerging era of cancer immunotherapy, immune checkpoint blockades (ICBs) and adoptive cell transfer therapies (ACTs) have gained significant attention. However, their therapeutic efficacies are limited due to the presence of cold type tumors, immunosuppressive tumor microenvironment, and immune-related side effects. On the other hand, dendritic cell (DC)-based vaccines have been suggested as a new cancer immunotherapy regimen that can address the limitations encountered by ICBs and ACTs. Despite the success of the first generation of DC-based vaccines, represented by the first FDA-approved DC-based therapeutic cancer vaccine Provenge, several challenges remain unsolved. Therefore, new DC vaccine strategies have been actively investigated. This review addresses the limitations of the currently most adopted classical DC vaccine and evaluates new generations of DC vaccines in detail, including biomaterial-based, immunogenic cell death-inducing, mRNA-pulsed, DC small extracellular vesicle (sEV)-based, and tumor sEV-based DC vaccines. These innovative DC vaccines are envisioned to provide a significant breakthrough in cancer immunotherapy landscape and are expected to be supported by further preclinical and clinical studies.

Despite widespread study of dendritic cell (DC)-based cancer immunotherapies, the in vivo postinjection fate of DC remains largely unknown. Due in part to a lack of quantifiable imaging modalities, this is troubling as the amount of DC migration to secondary lymphoid organs correlates with therapeutic efficacy. Magnetic particle imaging (MPI) has emerged as a suitable modality to quantify in vivo migration of superparamagnetic iron oxide (SPIO)-labeled DC. Herein, we describe a popliteal lymph node (pLN)-focused MPI scan to quantify DC in vivo migration accurately and consistently.

Adenovirus (Ad)-transduced SPIO+ (Ad SPIO+) and SPIO+ C57BL/6 bone marrow-derived DC were generated and assessed for viability and phenotype, then fluorescently labeled and injected into mouse hind footpads (n = 6). Two days later, in vivo DC migration was quantified using whole animal, pLN-focused, and ex vivo pLN MPI scans.

No significant differences in viability, phenotype and in vivo pLN migration were noted for Ad SPIO+ and SPIO+ DC. Day 2 pLN-focused MPI quantified DC migration in all instances while whole animal MPI only quantified pLN migration in 75% of cases. Ex vivo MPI and fluorescence microscopy confirmed that pLN MPI signal was due to originally injected Ad SPIO+ and SPIO+ DC.

We overcame a reported limitation of MPI by using a pLN-focused MPI scan to quantify pLN-migrated Ad SPIO+ and SPIO+ DC in 100% of cases and detected as few as 1000 DC (4.4 ng Fe) in vivo. MPI is a suitable preclinical imaging modality to assess DC-based cancer immunotherapeutic efficacy.

Tracking the in vivo fate of DC using noninvasive quantifiable magnetic particle imaging can potentially serve as a surrogate marker of therapeutic effectiveness.

• Adenoviral-transduced and iron oxide-labeled dendritic cells are in vivo migration competent. • Magnetic particle imaging is a suitable modality to quantify in vivo dendritic cell migration. • Magnetic particle imaging focused field of view overcomes dynamic range limitation.

Tolerogenic dendritic cells (tolDCs) are a promising strategy to treat autoimmune diseases since they have the potential to re-educate and modulate pathological immune responses in an antigen-specific manner and, therefore, have minimal adverse effects on the immune system compared to conventional immunosuppressive treatments. TolDC therapy has demonstrated safety and efficacy in different experimental models of autoimmune disease, such as multiple sclerosis (MS), type 1 diabetes (T1D), and rheumatoid arthritis (RA). Moreover, data from phase I clinical trials have shown that therapy with tolDCs is safe and well tolerated by MS, T1D, and RA patients. Nevertheless, various parameters need to be optimized to increase tolDC efficacy. In this regard, one important parameter to be determined is the most appropriate route of administration. Several delivery routes, such as intravenous, subcutaneous, intraperitoneal, intradermal, intranodal, and intraarticular routes, have been used in experimental models as well as in phase I clinical trials. This review summarizes data obtained from preclinical and clinical studies of tolDC therapy in the treatment of MS, T1D, and RA and their animal models, as well as data from the context of cancer immunotherapy using mature peptide-loaded DC, and data from in vivo cell tracking experiments, to define the most appropriate route of tolDC administration in relation to the most feasible, safest, and effective therapeutic use.

Immunotherapy has achieved revolutionary success in clinics, but it remains challenging for treating hepatocellular carcinoma (HCC) characterized by high vascularization. Here, it is reported that metal-organic framework-801 (MOF-801) can be employed as a stimulator of interferon genes (STING) through Toll-like receptor 4 (TLR4) not just as a drug delivery carrier. Notably, cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) and 5, 6-dimethylxanthenone-4-acetic acid (DMXAA) STING agonist with vascular disrupting function coordinates with MOF-801 to self-assemble into a nanoparticle (MOF-CpG-DMXAA) that effectively delivers CpG ODNs and DMXAA to cells for synergistically improving the tumor microenvironment by reprogramming tumor-associated macrophages (TAMs), promoting dendritic cells (DCs) maturation, as well as destroying tumor blood vessels. In HCC-bearing mouse models, it is demonstrated that MOF-CpG-DMXAA triggers systemic immune activation and stimulates robust tumoricidal immunity, resulting in a superior immunotherapeutic efficiency in orthotopic and recurrent HCC.

DNA-based materials have attracted interest due to the tunable structure and encoded biological functionality of nucleic acids. A simple and general approach to synthesize DNA-based materials with fine control over morphology and bioactivity is important to expand their applications. Here, we report the synthesis of DNA-based particles via the supramolecular assembly of tannic acid (TA) and DNA. Uniform particles with different morphologies are obtained using a variety of DNA building blocks. The particles enable the co-delivery of cytosine-guanine adjuvant sequences and the antigen ovalbumin in model cells. Intramuscular injection of the particles in mice induces antigen-specific antibody production and T cell responses with no apparent toxicity. Protein expression in cells is shown using capsules assembled from TA and plasmid DNA. This work highlights the potential of TA as a universal material for directing the supramolecular assembly of DNA into gene and vaccine delivery platforms.

This review summarizes recent findings related to the role of the sympathetic nervous system (SNS) in pathogenesis of multiple sclerosis (MS) and its commonly used experimental model - experimental autoimmune encephalomyelitis (EAE). They indicate that noradrenaline, the key end-point mediator of the SNS, acting through β-adrenoceptor, has a contributory role in the early stages of MS/EAE development. This stage is characterized by the SNS hyperactivity (increased release of noradrenaline) reflecting the net effect of different factors, such as the disease-associated inflammation, stress, vitamin D hypovitaminosis, Epstein-Barr virus infection and dysbiosis. Thus, the administration of propranolol, a non-selective β-adrenoceptor blocker, readily crossing the blood-brain barrier, to experimental rats before the autoimmune challenge and in the early (preclinical/prodromal) phase of the disease mitigates EAE severity. This phenomenon has been ascribed to the alleviation of neuroinflammation (due to attenuation of primarily microglial activation/proinflammatory functions) and the diminution of the magnitude of the primary CD4+ T-cell autoimmune response (the effect associated with impaired autoantigen uptake by antigen presenting cells and their migration into draining lymph nodes). The former is partly related to breaking of the catecholamine-dependent self-amplifying microglial feed-forward loop and the positive feedback loop between microglia and the SNS, leading to down-regulation of the SNS hyperactivity and its enhancing influence on microglial activation/proinflammatory functions and the magnitude of autoimmune response. The effects of propranolol are shown to be more prominent in male EAE animals, the phenomenon important as males (like men) are likely to develop clinically more severe disease. Thus, these findings could serve as a firm scientific background for formulation of a new sex-specific immune-intervention strategy for the early phases of MS (characterized by the SNS hyperactivity) exploiting anti-(neuro)inflammatory and immunomodulatory properties of propranolol and other relatively cheap and safe adrenergic drugs with similar therapeutic profile.

Methylthioadenosine phosphorylase (MTAP) deficiency occurs in various malignancies and is associated with poor survival in cancer patients. However, the mechanisms underlying tumour progression due to MTAP loss are yet to be elucidated. Utilizing integrated analyses of the transcriptome, proteome and secretome, we demonstrated that MTAP deficiency alters tumour-intrinsic, immune-related pathways and reprograms cytokine profiles towards a tumour-favourable environment. Additionally, MTAP-knockout cells exhibited a marked increase in the immune checkpoint protein PD-L1. Upon co-culturing primary T cells with cancer cells, MTAP loss-mediated PD-L1 upregulation inhibited T cell-mediated killing activity and induced several T cell exhaustion markers. In two xenograft tumour models, we showed a modest increase in average volume of tumours derived from MTAP-deficient cells than that of MTAP-proficient tumours. Surprisingly, a remarkable increase in tumour size was observed in humanized mice bearing MTAP-deficient tumours, as compared to their MTAP-expressing counterparts. Following immunophenotypic characterization of tumour-infiltrating leukocytes by mass cytometry analysis, MTAP-deficient tumours were found to display decreased immune infiltrates with lower proportions of both T lymphocytes and natural killer cells and higher proportions of immunosuppressive cells as compared to MTAP-expressing tumour xenografts. Taken together, our results suggest that MTAP deficiency restructures the tumour immune microenvironment, promoting tumour progression and immune evasion.

The adaptive immune response is under circadian control, yet, why adaptive immune reactions continue to exhibit circadian changes over long periods of time is unknown. Using a combination of experimental and mathematical modeling approaches, we show here that dendritic cells migrate from the skin to the draining lymph node in a time-of-day-dependent manner, which provides an enhanced likelihood for functional interactions with T cells. Rhythmic expression of TNF in the draining lymph node enhances BMAL1-controlled ICAM-1 expression in high endothelial venules, resulting in lymphocyte infiltration and lymph node expansion. Lymph node cellularity continues to be different for weeks after the initial time-of-day-dependent challenge, which governs the immune response to vaccinations directed against Hepatitis A virus as well as SARS-CoV-2. In this work, we present a mechanistic understanding of the time-of-day dependent development and maintenance of an adaptive immune response, providing a strategy for using time-of-day to optimize vaccination regimes.

E3 ligases regulate key processes, but many of their roles remain unknown. Using Perturb-seq, we interrogated the function of 1,130 E3 ligases, partners and substrates in the inflammatory response in primary dendritic cells (DCs). Dozens impacted the balance of DC1, DC2, migratory DC and macrophage states and a gradient of DC maturation. Family members grouped into co-functional modules that were enriched for physical interactions and impacted specific programs through substrate transcription factors. E3s and their adaptors co-regulated the same processes, but partnered with different substrate recognition adaptors to impact distinct aspects of the DC life cycle. Genetic interactions were more prevalent within than between modules, and a deep learning model, comβVAE, predicts the outcome of new combinations by leveraging modularity. The E3 regulatory network was associated with heritable variation and aberrant gene expression in immune cells in human inflammatory diseases. Our study provides a general approach to dissect gene function.

Resection of the tumor-draining lymph -node (TDLN) represents a standard method to identify metastasis for several malignancies. Interestingly, recent preclinical studies indicate that TDLN resection diminishes the efficacy of immune checkpoint inhibitor-based cancer immunotherapies. Thus, accurate preclinical identification of TDLNs is pivotal to uncovering the underlying immunological mechanisms. Therefore, we validated preclinically, and clinically available non-invasive in vivo imaging approaches for precise TDLN identification.

For visualization of the lymphatic drainage into the TDLNs by non-invasive in vivo optical imaging, we injected the optical imaging contrast agents Patent Blue V (582.7 g mol^-1) and IRDye® 800CW polyethylene glycol (PEG; 25,000-60,000 g mol^-1), subcutaneously (s.c.) in close proximity to MC38 adenocarcinomas at the right flank of experimental mice. For determination of the lymphatic drainage and the glucose metabolism in TDLNs by non-invasive in vivo PET/magnetic resonance imaging (PET/MRI), we injected the positron emission tomography (PET) tracer (2-deoxy-2[¹⁸F]fluoro-D-glucose (¹⁸F-FDG) [181.1 g mol^-1]) in a similar manner. For ex vivo cross-correlation, we isolated TDLNs and contralateral nontumor-draining lymph nodes (NTDLNs) and performed optical imaging, biodistribution, and autoradiography analysis.

The clinically well-established Patent Blue V was superior for intraoperative macroscopic identification of the TDLNs compared with IRDye® 800CW PEG but was not sensitive enough for non-invasive in vivo detection by optical imaging. Ex vivo Patent Blue V biodistribution analysis clearly identified the right accessory axillary and the proper axillary lymph node (LN) as TDLNs, whereas ex vivo IRDye® 800CW PEG completely failed. In contrast, functional non-invasive in vivo ¹⁸F-FDG PET/MRI identified a significantly elevated uptake exclusively within the ipsilateral accessory axillary TDLN of experimental mice and was able to differentiate between the accessory axillary and the proper LN. Ex vivo biodistribution and autoradiography confirmed our in vivo ¹⁸F-FDG PET/MRI results.

When taken together, our results demonstrate the feasibility of ¹⁸F-FDG-PET/MRI as a valid method for non-invasive in vivo, intraoperative, and ex vivo identification of the lymphatic drainage and glucose metabolism within the TDLNs. In addition, using Patent Blue V provides additive value for the macroscopic localization of the lymphatic drainage both visually and by ex vivo optical imaging analysis. Thus, both methods are valuable, easy to implement, and cost-effective for preclinical identification of the TDLN.

Dendritic cells (DCs) are prime targets for vaccination and immunotherapy. However, limited control over antigen presentation at a desired maturation status in these plastic materials remains a fundamental challenge in efficiently orchestrating a controlled immune response. DC-derived extracellular vesicles (EVs) can overcome some of these issues, but have significant production challenges. Herein, we employ a unique chemically-induced method for production of DC-derived extracellular blebs (DC-EBs) that overcome the barriers of DC and DC-derived EV vaccines. DC-EBs are molecular snapshots of DCs in time, cell-like particles with fixed stimulatory profiles for controlled immune signalling. DC-EBs were produced an order of magnitude more quickly and efficiently than conventional EVs and displayed stable structural integrity and antigen presentation compared to live DCs. Multi-omic analysis confirmed DC-EBs are majorly pure plasma membrane vesicles that are homogeneous at the single-vesicle level, critical for safe and effective vaccination. Immature vs. mature molecular profiles on DC-EBs exhibited molecularly modulated immune responses compared to live DCs, improving remission and survival of tumor-challenged mice via generation of antigen-specific T cells. For the first time, DC-EBs make their case for use in vaccines and for their potential in modulating other immune responses, potentially in combination with other immunotherapeutics.

Dendritic cells (DCs) are crucial for the initiation and regulation of adaptive immune responses. When encountering immune stimulus such as bacterial and viral infection, parasite invasion and dead cell debris, DCs capture antigens, mature, acquire immunostimulatory activity and transmit the immune information to naïve T cells. Then activated cytotoxic CD8⁺ T cells directly kill the infected cells, while CD4⁺ T helper cells release cytokines to aid the activity of other immune cells, and help B cells produce antibodies. Thus, detailed insights into the DC maturation process are necessary for us to understand the working principle of immune system, and develop new medical treatments for infection, cancer and autoimmune disease. This review summarizes the DC maturation process, including environment sensing and antigen sampling by resting DCs, antigen processing and presentation on the cell surface, DC migration, DC-T cell interaction and T cell activation. Application of advanced imaging modalities allows visualization of subcellular and molecular processes in a super-high resolution. The spatiotemporal tracking of DCs position and migration reveals dynamics of DC behavior during infection, shedding novel lights on DC biology.

The purpose of this study was to evaluate magnetic particle imaging (MPI) as a method for the in vivo tracking of dendritic cells (DC). DC are used in cancer immunotherapy and must migrate from the site of implantation to lymph nodes to be effective. The magnitude of the ensuing T cell response is proportional to the number of lymph node-migrated DC. With current protocols, less than 10% of DC are expected to reach target nodes. Therefore, imaging techniques for studying DC migration must be sensitive and quantitative. Here, we describe the first study using MPI to detect and track DC injected into the footpads of C57BL/6 mice migrating to the popliteal lymph nodes (pLNs).

DC were labelled with Synomag-D™ and injected into each hind footpad of C57BL/6 mice (n = 6). In vivo MPI was conducted immediately and repeated 48 h later. The MPI signal was measured from images and related to the signal from a known number of cells to calculate iron content. DC numbers were estimated by dividing iron content in the image by the iron per cell measured from a separate cell sample. The presence of SPIO-labeled DC in nodes was validated by ex vivo MPI, histology, and fluorescence microscopy.

Day 2 imaging showed a decrease in MPI signal in the footpads and an increase in signal at the pLNs, indicating DC migration. MPI signal was detected in the left pLN in four of the six mice and two of the six mice showed MPI signal in the right pLN. Ex vivo imaging detected signal in 11/12 nodes. We report a sensitivity of approximately 4000 cells (0.015 µg Fe) in vivo and 2000 cells (0.007 µg Fe) ex vivo.

Here, we describe the first study to use MPI to detect and track DC in a migration model with immunotherapeutic applications. We also bring attention to the issue of resolving unequal signals within close proximity, a challenge for any pre-clinical study using a highly concentrated tracer bolus that shadows nearby lower signals.

Despite advancements in cancer immunotherapy, heterogeneity in tumor response impose barriers to successful treatments and accurate prognosis. Effective therapy and early outcome detection are critical as toxicity profiles following immunotherapies can severely affect patients' quality of life. Existing imaging techniques, including positron emission tomography, computed tomography, magnetic resonance imaging, or multiplexed imaging, are often used in clinics yet suffer from limitations in the early assessment of immune response. Conventional strategies to validate immune response mainly rely on the Response Evaluation Criteria in Solid Tumors (RECIST) and the modified iRECIST for immuno-oncology drug trials. However, accurate monitoring of immunotherapy efficacy is challenging since the response does not always follow conventional RECIST criteria due to delayed and variable kinetics in immunotherapy responses. Engineered nanomaterials for immunotherapy applications have significantly contributed to overcoming these challenges by improving drug delivery and dynamic imaging techniques. This review summarizes challenges in recent immune-modulation approaches and traditional imaging tools, followed by emerging developments in three-in-one nanoimmunotheranostic systems co-opting nanotechnology, immunotherapy, and imaging. In addition, a comprehensive overview of imaging modalities in recent cancer immunotherapy research and a brief outlook on how nanotheranostic platforms can potentially advance to clinical translations for the field of immuno-oncology is presented.

Menthol, a cyclic terpene alcohol, plays a critical role in overcoming the blood-brain barrier and stratum corneum barrier. Herein, we innovatively propose a menthol nanoliposome (Men-nanoLips) that can dramatically increase lymph node accumulation of the dendritic cell (DC)-based anti-tumor vaccines. Specifically, Men-nanoLips efficiently enhanced lymphatic endothelial cell (EC) barrier permeability by reducing the expression of tight junction proteins. And interestingly, Men-nanoLips not only up-regulated the expression of CCR7 in DCs but also increased the secretion of CCL21 in lymphatic ECs. Moreover, Men-nanoLips promoted DC vaccine maturation as evidenced by increasing the expression of costimulatory molecules and up-regulating the pseudopodia-like protein. With those complementary mechanisms provided by Men-nanoLips, the number of the B16 whole-tumor cell lysate-loaded DCs that target the draining LN enhanced remarkably and significantly boosted the treatment efficacy of DC anti-tumor vaccines. Therefore, we concluded that Men-nanoLips could be instructive for increasing LN homing of DC vaccines.