DNA Reaction Circuits to Establish Designated Biological Functions in Multicellular Community.

In multicellular organisms, individual cells are coordinated through complex communication networks to accomplish various physiological tasks. Aiming to establish new biological functions in the multicellular community, we used DNA as the building block to develop a cascade of nongenetic reaction circuits to establish a dynamic cell-cell communication network. Utilizing membrane-anchored amphiphilic DNA tetrahedra (TDN) as the nanoscaffold, reaction circuits were incorporated into three unrelated cells in order to uniquely regulate their sense-and-response behaviors. As a proof-of-concept, this step enabled these cells to simulate significant biological events involved in T cell-mediated anticancer immunity. Such events included cancer-associated antigen recognition and the presentation of antigen-presenting cells (APCs), APC-facilitated T cell activation and dissociation, and T cell-mediated cancer targeting and killing. By combining the excellent programmability and molecular recognition ability of DNA, our cell-surface reaction circuits hold promise for mimicking and manipulating many biological processes.

Lipidation of pneumococcal proteins enables activation of human antigen-presenting cells and initiation of an adaptive immune response.

Streptococcus pneumoniae remains a significant global threat, with existing vaccines having important limitations such as restricted serotype coverage and high manufacturing costs. Pneumococcal lipoproteins are emerging as promising vaccine candidates due to their surface exposure and conservation across various serotypes. While prior studies have explored their potential in mice, data in a human context and insights into the impact of the lipid moiety remain limited. In the present study, we examined the immunogenicity of two pneumococcal lipoproteins, DacB and MetQ, both in lipidated and non-lipidated versions, by stimulation of primary human immune cells. Immune responses were assessed by the expression of common surface markers for activation and maturation as well as cytokines released into the supernatant. Our findings indicate that in the case of MetQ lipidation was crucial for activation of human antigen-presenting cells such as dendritic cells and macrophages, while non-lipidated DacB demonstrated an intrinsic potential to induce an innate immune response. Nevertheless, immune responses to both proteins were enhanced by lipidation. Interestingly, following stimulation of dendritic cells with DacB, LipDacB and LipMetQ, cytokine levels of IL-6 and IL-23 were significantly increased, which are implicated in triggering potentially important Th17 cell responses. Furthermore, LipDacB and LipMetQ were able to induce proliferation of CD4+ T cells indicating their potential to induce an adaptive immune response. These findings contribute valuable insights into the immunogenic properties of pneumococcal lipoproteins, emphasizing their potential role in vaccine development against pneumococcal infections.

Carrier-free mRNA vaccine induces robust immunity against SARS-CoV-2 in mice and non-human primates without systemic reactogenicity.

Carrier-free naked mRNA vaccines may reduce the reactogenicity associated with delivery carriers; however, their effectiveness against infectious diseases has been suboptimal. To boost efficacy, we targeted the skin layer rich in antigen-presenting cells (APCs) and utilized a jet injector. The jet injection efficiently introduced naked mRNA into skin cells, including APCs in mice. Further analyses indicated that APCs, after taking up antigen mRNA in the skin, migrated to the lymph nodes (LNs) for antigen presentation. Additionally, the jet injection provoked localized lymphocyte infiltration in the skin, serving as a physical adjuvant for vaccination. Without a delivery carrier, our approach confined mRNA distribution to the injection site, preventing systemic mRNA leakage and associated systemic proinflammatory reactions. In mouse vaccination, the naked mRNA jet injection elicited robust antigen-specific antibody production over 6 months, along with germinal center formation in LNs and the induction of both CD4- and CD8-positive T cells. By targeting the SARS-CoV-2 spike protein, this approach provided protection against viral challenge. Furthermore, our approach generated neutralizing antibodies against SARS-CoV-2 in non-human primates at levels comparable to those observed in mice. In conclusion, our approach offers a safe and effective option for mRNA vaccines targeting infectious diseases.

DNA nanodevice as a multi-module co-delivery platform for combination cancer immunotherapy.

A major challenge in combining cancer immunotherapy is the efficient delivery of multiple types of immunological stimulators to elicit a robust anti-tumor immune response and reprogram the immunosuppressive tumor microenvironment (TME). Here, we developed a DNA nanodevice that was generated by precisely assembling three types of immunological stimulators. The doxorubicin (Dox) component induced immunogenic cell death (ICD) in tumor cells and enhanced phagocytosis of antigen-presenting cells (APCs). Exogenous double-stranded DNA (dsDNA) could act as a molecular adjuvant to activate the stimulator of interferon genes (STING) signaling in APCs by engulfing dying tumor cells. Interleukin (IL)-12 and small hairpin programmed cell death-ligand 1 (shPD-L1) transcription templates were designed to regulate TME. Additionally, for targeted drug delivery, multiple cyclo[Arg-Gly-Asp-(d-Phe)-Cys] (cRGD) peptide units on DNA origami were employed. The incorporation of disulfide bonds allowed the release of multiple modules in response to intracellular glutathione (GSH) in tumors. The nanodevice promoted the infiltration of CD8+ and CD4+ cells into the tumor and generated a highly inflamed TME, thereby enhancing the effectiveness of cancer immunotherapy. Our research results indicate that the nanodevice we constructed can effectively inhibit tumor growth and prevent lung metastasis without obvious systemic toxicity, providing a promising strategy for cancer combination treatment.

Programming peptide-oligonucleotide nano-assembly for engineering of neoantigen vaccine with potent immunogenicity.

Background: Neoantigen nanovaccine has been recognized as a promising treatment modality for personalized cancer immunotherapy. However, most current nanovaccines are carrier-dependent and the manufacturing process is complicated, resulting in potential safety concerns and suboptimal codelivery of neoantigens and adjuvants to antigen-presenting cells (APCs). Methods: Here we report a facile and general methodology for nanoassembly of peptide and oligonucleotide by programming neoantigen peptide with a short cationic module at N-terminus to prepare nanovaccine. The programmed peptide can co-assemble with CpG oligonucleotide (TLR9 agonist) into monodispersed nanostructures without the introduction of artificial carrier. Results: We demonstrate that the engineered nanovaccine promoted the codelivery of neoantigen peptides and adjuvants to lymph node-residing APCs and instigated potent neoantigen-specific T-cell responses, eliciting neoantigen-specific antitumor immune responses with negligible systemic toxicity. Furthermore, the antitumor T-cell immunity is profoundly potentiated when combined with anti-PD-1 therapy, leading to significant inhibition or even complete regression of established melanoma and MC-38 colon tumors. Conclusions: Collectively, this work demonstrates the feasibility and effectiveness of personalized cancer nanovaccine preparation with high immunogenicity and good biosafety by programming neoantigen peptide for nanoassembly with oligonucleotides without the aid of artificial carrier.

Novel JAK Inhibitors to Reduce Graft-Versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation in a Preclinical Mouse Model.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a highly effective, well-established treatment for patients with various hematologic malignancies and non-malignant diseases. The therapeutic benefits of allo-HCT are mediated by alloreactive T cells in donor grafts. However, there is a significant risk of graft-versus-host disease (GvHD), in which the donor T cells recognize recipient cells as foreign and attack healthy organs in addition to malignancies. We previously demonstrated that targeting JAK1/JAK2, mediators of interferon-gamma receptor (IFNGR) and IL-6 receptor signaling, in donor T cells using baricitinib and ruxolitinib results in a significant reduction in GvHD after allo-HCT. Furthermore, we showed that balanced inhibition of JAK1/JAK2 while sparing JAK3 is important for the optimal prevention of GvHD. Thus, we have generated novel JAK1/JAK2 inhibitors, termed WU derivatives, by modifying baricitinib. Our results show that WU derivatives have the potential to mitigate GvHD by upregulating regulatory T cells and immune reconstitution while reducing the frequencies of antigen-presenting cells (APCs) and CD80 expression on these APCs in our preclinical mouse model of allo-HCT. In addition, WU derivatives effectively downregulated CXCR3 and T-bet in primary murine T cells. In summary, we have generated novel JAK inhibitors that could serve as alternatives to baricitinib or ruxolitinib.

Surgical tumor-derived nanoplatform targets tumor-associated macrophage for personalized postsurgical cancer immunotherapy.

Directly activating CD8+ T cells within the tumor through antigen-presenting cells (APCs) hold promise for tumor elimination. However, M2-like tumor-associated macrophages (TAMs), the most abundant APCs in tumors, hinder CD8+ T cell activation due to inefficient antigen cross-presentation. Here, we demonstrated a personalized nanotherapeutic platform using surgical tumor-derived galactose ligand-modified cancer cell membrane (CM)-coated cysteine protease inhibitor (E64)-loaded mesoporous silica nanoparticles for postsurgical cancer immunotherapy. The platform targeted M2-like TAMs and released E64 within lysosomes, which reshaped antigen cross-presentation and directly activated CD8+ T cells, thus suppressing B16-OVA melanoma growth. Furthermore, this platform, in combination with anti-PD-L1 antibodies, enhanced the therapeutic efficacy and substantially inhibited 4T1 tumor growth. CMs obtained from surgically resected tumors were used to construct a personalized nanotherapeutic platform, which, in synergy with immune checkpoint blockade (ICB), effectively inhibited postsurgical tumor recurrence in 4T1 tumor. Our work offered a robust, safe strategy for cancer immunotherapy and prevention of postsurgical tumor recurrence.

CD4+CD25+ regulatory T cells ex vivo generated from autologous naïve CD4+ T cells suppress EAE progression.

CD4+CD25+ regulatory T cells (Tregs) play an important role in maintaining immune homeostasis in multiple sclerosis (MS). Hence, we aimed to explore the therapeutic efficacy and safety of adoptive cell therapy (ACT) utilizing induced antigen-specific Tregs in an animal model of MS, that is, in an experimental autoimmune encephalomyelitis (EAE) model. B cells from EAE model that were activated with soluble CD40L were used as antigen-presenting cells (APCs) to induce the differentiation of antigen-specific Tregs from naïve CD4 precursors, and then, a stepwise isolation of CD4+CD25highCD127low Tregs was performed using a flow sorter. All EAE mice were divided into Treg-treated group (2 × 104 cells in 0.2 mL per mouse, n = 14) and sham-treated group (0.2 mL normal saline (NS), n = 20), which were observed daily for clinical assessment, and for abnormal appearance for 6 weeks. Afterward, histological analysis, immunofluorescence and real-time PCR were performed. Compared to sham-treated mice, Treg-treated mice exhibited a significant decrease in disease severity scores and reduced inflammatory infiltration and demyelination in the spinal cord. Additionally, Tregs-treated mice demonstrated higher CCN3 protein and mRNA levels than sham-treated mice. The results of this preclinical study further support the therapeutic potential of this ACT approach in the treatment of MS.

Local administration of myeloid-derived suppressor cells prevents progression of immune-mediated dry eye disease.

Myeloid derived suppressor cells (MDSCs) are a heterogenous population of immature hematopoietic precursors with known immunoregulatory functions. The immunosuppressive role of MDSCs has been highlighted in several inflammatory ophthalmic disorders; however, their therapeutic application in suppressing the immune-mediated changes in dry eye disease (DED) has not been studied. We observed significant reduction in antigen presenting cell (APC) frequencies and their maturation in the presence of MDSCs. Moreover, co-culturing MDSCs with T helper 17 cells (Th17) resulted in reduced Th17 frequencies and their IL-17 expression. On the contrary, MDSCs maintained regulatory T cell frequencies and enhanced their function in-vitro. Furthermore, we delineated the role of interleukin-10 (IL-10) secreted by MDSCs in their immunoregulatory functions. We confirmed these results by flow cytometry analysis and observed that treatment with MDSCs in DED mice effectively suppressed the maturation of APCs, pathogenic Th17 response, and maintained Treg function and significantly ameliorated the disease. The results in this study highlight the potential therapeutic application of MDSCs in treating refractory DED.

Particle-Based Artificial Antigen-Presenting Cell Systems for T Cell Activation in Adoptive T Cell Therapy.

T cell-based adoptive cell therapy (ACT) has emerged as a promising treatment for various diseases, particularly cancers. Unlike other immunotherapy modalities, ACT involves directly transferring engineered T cells into patients to eradicate diseased cells; hence, it necessitates methods for effectively activating and expanding T cells in vitro. Artificial antigen-presenting cells (aAPCs) have been widely developed based on biomaterials, particularly micro- and nanoparticles, and functionalized with T cell stimulatory antibodies to closely mimic the natural T cell-APC interactions. Due to their vast clinical utility, aAPCs have been employed as an off-the-shelf technology for T cell activation in FDA-approved ACTs, and the development of aAPCs is constantly advancing with the emergence of aAPCs with more sophisticated designs and additional functionalities. Here, we review the recent advancements in particle-based aAPCs for T cell activation in ACTs. Following a brief introduction, we first describe the manufacturing processes of ACT products. Next, the design and synthetic strategies for micro- and nanoparticle-based aAPCs are discussed separately to emphasize their features, advantages, and limitations. Then, the impact of design parameters of aAPCs, such as size, shape, ligand density/mobility, and stiffness, on their functionality and biomedical performance is explored to provide deeper insights into the design concepts and principles for more efficient and safer aAPCs. The review concludes by discussing current challenges and proposing future perspectives for the development of more advanced aAPCs.

A vaccine targeting antigen-presenting cells through CD40 induces protective immunity against Nipah disease.

Nipah virus (NiV) has been recently ranked by the World Health Organization as being among the top eight emerging pathogens likely to cause major epidemics, whereas no therapeutics or vaccines have yet been approved. We report a method to deliver immunogenic epitopes from NiV through the targeting of the CD40 receptor of antigen-presenting cells by fusing a selected humanized anti-CD40 monoclonal antibody to the Nipah glycoprotein with conserved NiV fusion and nucleocapsid peptides. In the African green monkey model, CD40.NiV induces specific immunoglobulin A (IgA) and IgG as well as cross-neutralizing responses against circulating NiV strains and Hendra virus and T cell responses. Challenge experiments using a NiV-B strain demonstrate the high protective efficacy of the vaccine, with all vaccinated animals surviving and showing no significant clinical signs or virus replication, suggesting that the CD40.NiV vaccine conferred sterilizing immunity. Overall, results obtained with the CD40.NiV vaccine are highly promising in terms of the breadth and efficacy against NiV.

Mannosylated polyethylenimine-cholesterol-based nanoparticles for targeted delivery of minicircle DNA vaccine against COVID-19 to antigen-presenting cells.

DNA vaccines can be a potential solution to protect global health, triggering both humoral and cellular immune responses. DNA vaccines are valuable in preventing intracellular pathogen infections, and therefore can be explored against coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2). This work explored different systems based on polyethylenimine (PEI), functionalized for the first time with both cholesterol (CHOL) and mannose (MAN) to deliver parental plasmid (PP) and minicircle DNA (mcDNA) vectors encoding the receptor-binding domain (RBD) of SARS-CoV-2 to antigen-presenting cells (APCs). For comparative purposes, three different systems were evaluated: PEI, PEI-CHOL and PEI-CHOL-MAN. The systems were prepared at various nitrogen-to-phosphate group (N/P) ratios and characterized in terms of encapsulation efficiency, surface charge, size, polydispersity index (PDI), morphology, and stability over time. Moreover, in vitro transfection studies of dendritic cells (JAWS II) and human fibroblast cells were performed. Viability studies assured the biocompatibility of all nanocarriers. Confocal microscopy studies confirmed intracellular localization of systems, resulting in enhanced cellular uptake using PEI-CHOL and PEI-CHOL-MAN systems when compared with the PEI system. Regarding the RBD expression, PEI-CHOL-MAN was the system that led to the highest levels of transcripts and protein expression in JAWS II cells. Furthermore, the nanosystems significantly stimulated pro-inflammatory cytokines production and dendritic cell maturation in vitro. Overall, mannosylated systems can be considered a valuable tool in the delivery of plasmid DNA or mcDNA vaccines to APCs.

Role of Extracellular Vesicles in Immunity and Host Defense.

Extracellular vesicles (EVs) are membrane-bound structures released by cells and have become significant players in immune system functioning, primarily by facilitating cell-to-cell communication. Immune cells like neutrophils and dendritic cells release EVs containing bioactive molecules that modulate chemotaxis, activate immune cells, and induce inflammation. EVs also contribute to antigen presentation, lymphocyte activation, and immune tolerance. Moreover, EVs play pivotal roles in antimicrobial host defense. They deliver microbial antigens to antigen-presenting cells (APCs), triggering immune responses, or act as decoys to neutralize virulence factors and toxins. This review discusses host and microbial EVs' multifaceted roles in innate and adaptive immunity, highlighting their involvement in immune cell development, antigen presentation, and antimicrobial responses.

Clathrin controls bidirectional communication between T cells and antigen presenting cells.

In circulation, T cells are spherical with selectin enriched dynamic microvilli protruding from the surface. Following extravasation, these microvilli serve another role, continuously surveying their environment for antigen in the form of peptide-MHC (pMHC) expressed on the surface of antigen presenting cells (APCs). Upon recognition of their cognate pMHC, the microvilli are initially stabilized and then flatten into F-actin dependent microclusters as the T cell spreads over the APC. Within 1-5 min, clathrin is recruited by the ESCRT-0 component Hrs to mediate release of T cell receptor (TCR) loaded vesicles directly from the plasma membrane by clathrin and ESCRT-mediated ectocytosis (CEME). After 5-10 min, Hrs is displaced by the endocytic clathrin adaptor epsin-1 to induce clathrin-mediated trans-endocytosis (CMTE) of TCR-pMHC conjugates. Here we discuss some of the functional properties of the clathrin machinery which enables it to control these topologically opposite modes of membrane transfer at the immunological synapse, and how this might be regulated during T cell activation.

Peptide nanovaccine in melanoma immunotherapy.

Melanoma is an especially fatal neoplasm resistant to traditional treatment. The advancement of novel therapeutical approaches has gained attention in recent years by shedding light on the molecular mechanisms of melanoma tumorigenesis and their powerful interplay with the immune system. The presence of many mutations in melanoma cells results in the production of a varied array of antigens. These antigens can be recognized by the immune system, thereby enabling it to distinguish between tumors and healthy cells. In the context of peptide cancer vaccines, generally, they are designed based on tumor antigens that stimulate immunity through antigen-presenting cells (APCs). As naked peptides often have low potential in eliciting a desirable immune reaction, immunization with such compounds usually necessitates adjuvants and nanocarriers. Actually, nanoparticles (NPs) can provide a robust immune response to peptide-based melanoma vaccines. They improve the directing of peptide vaccines to APCs and induce the secretion of cytokines to get maximum immune response. This review provides an overview of the current knowledge of the utilization of nanotechnology in peptide vaccines emphasizing melanoma, as well as highlights the significance of physicochemical properties in determining the fate of these nanovaccines in vivo, including their drainage to lymph nodes, cellular uptake, and influence on immune responses.

Tsyn-Seq: a T-cell Synapse-Based Antigen Identification Platform.

Tools for genome-wide rapid identification of peptide-major histocompatibility complex targets of T-cell receptors (TCR) are not yet universally available. We present a new antigen screening method, the T-synapse (Tsyn) reporter system, which includes antigen-presenting cells (APC) with a Fas-inducible NF-κB reporter and T cells with a nuclear factor of activated T cells (NFAT) reporter. To functionally screen for target antigens from a cDNA library, productively interacting T cell-APC aggregates were detected by dual-reporter activity and enriched by flow sorting followed by antigen identification quantified by deep sequencing (Tsyn-seq). When applied to a previously characterized TCR specific for the E7 antigen derived from human papillomavirus type 16 (HPV16), Tsyn-seq successfully enriched the correct cognate antigen from a cDNA library derived from an HPV16-positive cervical cancer cell line. Tsyn-seq provides a method for rapidly identifying antigens recognized by TCRs of interest from a tumor cDNA library. See related Spotlight by Makani and Joglekar, p. 515.

Artificial Antigen-Presenting Cell Fabrication for Murine T Cell Expansion.

Antigen-presenting cells (APCs), such as dendritic cells and macrophages, have a unique ability to survey the body and present information to T cells via peptide-loaded major histocompatibility complexes (signal 1). This presentation, along with a co-stimulatory signal (signal 2), leads to activation and subsequent expansion of T cells. This process can be harnessed and utilized for therapeutic applications, but the use of patient-derived APCs can be complex and inefficient. Alternatively, artificial APCs (aAPCs) provide a simplified method to achieve T cell activation by presenting the two necessary stimulatory signals. This protocol describes the utilization of magnetic nanoparticles and stimulatory proteins to create aAPCs that can be employed for activating and expanding antigen-specific T cells for both basic and translational immunology and immunotherapy studies. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Protein and particle modification for aAPC fabrication Basic Protocol 2: aAPC validation by immunolabeling of conjugated protein Support Protocol 1: Quantification of aAPC stock concentration Basic Protocol 3: Determination of aAPC usage for murine CD8+ T cell activation Support Protocol 2: Isolation of murine CD8+ T cells.

Multimodal probing of T-cell recognition with hexapod heterostructures.

Studies using antigen-presenting systems at the single-cell and ensemble levels can provide complementary insights into T-cell signaling and activation. Although crucial for advancing basic immunology and immunotherapy, there is a notable absence of synthetic material toolkits that examine T cells at both levels, and especially those capable of single-molecule-level manipulation. Here we devise a biomimetic antigen-presenting system (bAPS) for single-cell stimulation and ensemble modulation of T-cell recognition. Our bAPS uses hexapod heterostructures composed of a submicrometer cubic hematite core (α-Fe2O3) and nanostructured silica branches with diverse surface modifications. At single-molecule resolution, we show T-cell activation by a single agonist peptide-loaded major histocompatibility complex; distinct T-cell receptor (TCR) responses to structurally similar peptides that differ by only one amino acid; and the superior antigen recognition sensitivity of TCRs compared with that of chimeric antigen receptors (CARs). We also demonstrate how the magnetic field-induced rotation of hexapods amplifies the immune responses in suspended T and CAR-T cells. In addition, we establish our bAPS as a precise and scalable method for identifying stimulatory antigen-specific TCRs at the single-cell level. Thus, our multimodal bAPS represents a unique biointerface tool for investigating T-cell recognition, signaling and function.

Novel Lipid Nanoparticles Stable and Efficient for mRNA Transfection to Antigen-Presenting Cells.

mRNA vaccines have emerged as a pivotal tool in combating COVID-19, offering an advanced approach to immunization. A key challenge with these vaccines is their need for extremely-low-temperature storage, which affects their stability and shelf life. Our research addresses this issue by enhancing the stability of mRNA vaccines through a novel cationic lipid, O,O'-dimyristyl-N-lysyl aspartate (DMKD). DMKD effectively binds with mRNA, improving vaccine stability. We also integrated phosphatidylserine (PS) into the formulation to boost immune response by promoting the uptake of these nanoparticles by immune cells. Our findings reveal that DMKD-PS nanoparticles maintain structural integrity under long-term refrigeration and effectively protect mRNA. When tested, these nanoparticles containing green fluorescent protein (GFP) mRNA outperformed other commercial lipid nanoparticles in protein expression, both in immune cells (RAW 264.7 mouse macrophage) and non-immune cells (CT26 mouse colorectal carcinoma cells). Importantly, in vivo studies show that DMKD-PS nanoparticles are safely eliminated from the body within 48 h. The results suggest that DMKD-PS nanoparticles present a promising alternative for mRNA vaccine delivery, enhancing both the stability and effectiveness of these vaccines.

Illuminating T cell-dendritic cell interactions in vivo by FlAsHing antigens.

Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.