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T cell-dependent bispecific antibodies (TDBs) are next-generation antibody therapies that link cancer cells and T cells to achieve potent antitumor effects. Despite the successful development of TDBs for hematological malignancies, their efficacy against solid tumors remains limited. Overcoming this challenge requires a deeper understanding of their mechanisms of action. While the basic process of immunological synapse (IS) formation and T cell activation by TDB is known, the detailed effects of IS on the bystander effect and T cell migration, both crucial for therapeutic efficacy, remain unclear. This study investigated these mechanisms using an EGFR/CD3 TDB (hEx3) and EGFR knockout cancer cells (KO). The results revealed that IS formation by TDB induced a bystander effect, leading to damage in surrounding KO, with the extent depending on the proportion of EGFR-positive wild-type cancer cells (WT) and the duration of co-culture. Furthermore, IS formation significantly enhanced T cell cytokine and chemokine secretion, promoting T cell migration. These findings provide critical insights into TDB efficacy mechanisms and highlight the importance of evaluating IS formation in developing new antibody drugs. Establishing a reliable system for assessing IS formation will be essential for advancing TDBs and other antibody-based therapies, particularly against solid tumors.

T cell-dependent bispecific antibody (TDB)-induced T cell activation, which can eliminate tumor cells independent of MHC engagement, is expected to be a novel breakthrough immunotherapy against refractory cancer. However, the mechanism of action of TDBs has not been fully elucidated thus far. We focused on TDB-induced T cell–tumor cell contact as an important initial step in direct T cell-mediated tumor cell killing via transport of cytotoxic cell proteases (e.g., granzymes) with or without immunological synapse formation. Using an anti-EGFR/CD3 TDB, hEx3, we visualized and quantified T cell–tumor cell contact and demonstrated a correlation between the degree of cell contact and TDB efficacy. We also found that cytokines, including interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα) secreted by activated T cells, damaged tumor cells in a cell contact-independent manner. Moreover, therapeutic experiences clearly indicated that hEx3, unlike conventional anti-EGFR antibodies, was effective against colorectal cancer (CRC) cells with mutant KRAS, BRAF, or PIK3CA. In a pharmacokinetic analysis, T cells spread gradually in accordance with the hEx3 distribution within tumor tissue. Accordingly, we propose that TDBs should have four action steps: 1st, passive targeting via size-dependent tumor accumulation; 2nd, active targeting via specific binding to tumor cells; 3rd, T cell redirection toward tumor cells; and 4th, TDB-induced cell contact-dependent (direct) or -independent (indirect) tumor cell killing. Finally, our TDB hEx3 may be a promising reagent against refractory CRC with an oncogenic mutation associated with a poor prognosis.

Successful subunit vaccination with recombinant proteins requires adjuvants. The glycolipid trehalose-dibehenate (TDB), a synthetic analog of the mycobacterial cord factor, potently induces Th1 and Th17 immune responses and is a candidate adjuvant for human immunization. TDB binds to the C-type lectin receptor Mincle and triggers Syk-Card9-dependent APC activation. In addition, interleukin (IL)-1 receptor/MyD88-dependent signaling is required for TDB adjuvanticity. The role of different innate immune cell types in adjuvant-stimulated Th1/Th17 responses is not well characterized. We investigated cell recruitment to the site of injection (SOI) and to the draining lymph nodes (dLNs) after immunization with the TDB containing adjuvant CAF01 in a protein-based vaccine. Recruitment of monocytes and neutrophils to the SOI and the dramatic increase in lymph node cellularity was partially dependent on both Mincle and MyD88. Despite their large numbers at the SOI, neutrophils were dispensable for the induction of Th1/Th17 responses. In contrast, CCR2-dependent monocyte recruitment was essential for the induction of Th1/Th17 cells. Transport of adjuvant to the dLN did not require Mincle, MyD88, or CCR2. Together, adjuvanticity conferred by monocytes can be separated at the cellular level from potential tissue damage by neutrophils.

Successful induction of protective T cells by recombinant protein vaccines requires adjuvants. The liposomal adjuvant system CAF01 induces robust Th17 responses in mice. CAF01 contains the synthetic glycolipid trehalose-6,6-dibehenate (TDB), whose recognition by the C-type lectin receptor Mincle is required for Th17 induction. In previous work, we identified a pivotal role of TNF in upregulation of Mincle expression in macrophages and Th17 adjuvanticity of CAF01. The question has remained on which cell type(s) TNF acts to mediate the Th17 adjuvanticity of CAF01, and whether TNF-induced Mincle upregulation is causative. We used conditional TNFR1-deficient mice to dissect cell type-specific contributions of TNF signaling in myeloid cells, DC and T cells to Th17 induction by the recombinant tuberculosis fusion protein H1 adjuvanted with CAF01. LysM-Cre-mediated deletion of TNFR1 on myeloid cells completely abrogated vaccine-induced Th17 differentiation, replicating the phenotype in mice deficient in TNF or treated with the TNF blocker Etanercept. In contrast, TNFR1 deletion in DC by Clec9a-Cre did not affect Th17 induction, and by CD11c-Cre only partially reduced Th17 cells. T cell-specific deletion of TNFR1 by Lck-Cre had no impact on Th17 differentiation after vaccination. TNFR1 was expressed highly, and deleted efficiently via LysM-Cre, in monocytes and in neutrophils. We recently showed that neutrophils are not required for the adjuvant effect of CAF01, but monocytes are essential. Therefore, we analyzed activation of monocytes by TDB and observed robust upregulation of Mincle expression and of the Th17-inducing cytokines IL-1β and IL-6, that was inhibited by Etanercept. Finally, we asked whether Th17 induction by TNF is causally linked to Mincle upregulation. Constitutive, TNF-independent transgenic Mincle expression partially restored Th17 induction by CAF01 despite TNF blockade. Thus, upregulation of Mincle by TNF plays a causal role, likely by enabling production of Th17-polarizing cytokines by myeloid cells upon enhanced sensing of the adjuvant component TDB.

The ultra-rapid satellite clock product based on the satellite clock batch estimation is commonly used for high-precision and reliable precise point positioning (PPP) services. In order to clarify the effect of different ranging errors on the satellite clock batch estimation accuracy, the source of the satellite clock bias induced by the batch observation model is classified into the initial clock bias (ICB) and time-dependent bias (TDB). In addition to the effect of the ICB and TDB, the analytic relationship between the observation redundancy and the satellite clock batch estimation accuracy are derived and verified. The suitable number of stations is suggested to be 40 for the satellite clock batch estimation to achieve the counterbalance between the efficiency and saturable accuracy. For the PPP based on the batch-estimated satellite clock, the impacts of the ICB and TDB on PPP are clarified. The satellite clock batch estimation and PPP experiments are carried out to investigate the impacts of the ICB and TDB on the satellite clock batch estimation accuracy and the PPP performance. The ICB causes a significant bias for the batch-estimated satellite clock. The TDB is impacted by the assimilation ability of the batch-estimated satellite clock to the satellite orbit error. The convergence time and the positioning accuracy after the convergence of PPP are primarily affected by the ICB and TDB, respectively.

Microglial activation has long been recognized as a hallmark of neuroinflammation. Recently, the bacillus Calmette-Guerin (BCG) vaccine has been reported to exert neuroprotective effects against several neurodegenerative disorders. Trehalose-6,6′-dibehenate (TDB) is a synthetic analogue of trehalose-6,6′-dimycolate (TDM, also known as the mycobacterial cord factor) and is a new adjuvant of tuberculosis subunit vaccine currently in clinical trials. Both TDM and TDB can activate macrophages and dendritic cells through binding to C-type lectin receptor Mincle; however, its action mechanism in microglia and their relationship with neuroinflammation are still unknown. In this article, we found that TDB inhibited LPS-induced M1 microglial polarization in primary microglia and BV-2 cells. However, TDB itself had no effects on IKK, p38, and JNK activities or cytokine expression. In contrast, TDB activated ERK1/2 through PLC-γ1/PKC signaling and in turn decreased LPS-induced NF-κB nuclear translocation. Furthermore, TDB-induced AMPK activation via PLC-γ1/calcium/CaMKKβ-dependent pathway and thereby enhanced M2 gene expressions. Interestingly, knocking out Mincle did not alter the anti-inflammatory and M2 polarization effects of TDB in microglia. Conditional media from LPS-stimulated microglial cells can induce in vitro neurotoxicity, and this action was attenuated by TDB. Using a mouse neuroinflammation model, we found that TDB suppressed LPS-induced M1 microglial activation and sickness behavior, but promoted M2 microglial polarization in both WT and Mincle −/− mice. Taken together, our results suggest that TDB can act independently of Mincle to inhibit LPS-induced inflammatory response through PLC-γ1/PKC/ERK signaling and promote microglial polarization towards M2 phenotype via PLC-γ1/calcium/CaMKKβ/AMPK pathway. Thus, TDB may be a promising therapeutic agent for the treatment of neuroinflammatory diseases.

The peptide P10 is a vaccine candidate for Paracoccidioidomycosis, a systemic mycosis caused by fungal species of the genus Paracoccidioides spp. We have previously shown that peptide P10 vaccination, in the presence of several different adjuvants, induced a protective cellular immune response mediated by CD4+ Th1 lymphocytes that was associated with the increased production of IFN-γ in mice challenged with a virulent isolate of Paracoccidoides brasiliensis. Cationic liposomes formulated with dioctadecyldimethylammonium and trehalose dibehenate (DDA/TDB, termed also CAF01–cationic adjuvant formulation) have been developed for safe administration in humans and CAF01 liposomes are utilized as an adjuvant for modulating a robust Th1/Th17 cellular response. We evaluated the efficacy of the adsorption of peptide P10 to CAF01 cationic liposomes and used the generated liposomes to vaccinate C57Bl/6 mice infected with P. brasiliensis. Our results showed that P10 was efficiently adsorbed onto CAF01 liposomes. The vaccination of infected mice with cationic liposomes formulated with DDA/TDB 250/50 µg/mL and 20 µg of P10 induced an effective cellular immune response with increased levels of Th17 cytokines, which correlated with significant decreases in the fungal burdens in lungs and protective granulomatous tissue responses. Hence, cationic liposomes of DDA/TDB 250/50 µg/mL with 20 µg of P10 are a promising therapeutic for safely and effectively improving the treatment of paracoccidioidomycosis.

Purpose The combination of delivery systems like cationic liposomes and immunopotentiators such as Toll-like receptor (TLR) ligands is a promising approach for rational vaccine adjuvant design. The purpose of this study was to investigate how the incorporation of the poorly soluble TLR4 agonist monophosphoryl lipid A (MPL) into cationic liposomes based on dimethyldioctadecylammonium (DDA) and trehalose 6,6′-dibehenate (TDB) influenced the physicochemical and immunological properties of the liposomes. Methods The DDA/TDB/MPL liposomes were characterized with regard to particle size, poly dispersity, surface charge, stability and thermodynamic properties. The adjuvant formulations were tested in vivo in mice using ovalbumin (OVA) as model antigen. Results Integration of MPL into the bilayer structure of DDA/TDB liposomes was evident from a decreased phase transition temperature, an improved membrane packing, and a reduction in surface charge. The particle size and favorable liposome storage stability were not affected by MPL. In mice, DDA/TDB/MPL liposomes induced an antigen-specific CD8⁺ T-cell response and a humoral response. Conclusions Enhancing the solubility of MPL by inclusion into the bilayer of DDA/TDB liposomes changes the membrane characteristics of the adjuvant system and provides the liposomes with CD8⁺ T-cell inducing properties without compromising humoral responses.

Heparin-binding hemagglutinin (HBHA), a mycobacterial cell surface protein, mediates adhesion to nonphagocytic cells and the dissemination of ( ) from the site of primary infection. Superior expression systems are required to obtain abundant proteins for the purpose of diagnosing infection or for the immunization. Here, HBHA was expressed by ( ) GS115 strain , and the immunogenicity of HBHA was evaluated. The gene of was cloned into the pPIC9K plasmid, which was good for electroporation into GS115 strain. Unlabeled HBHA protein was purified using a Sepharose CL-6B column, and its expression was confirmed using anti-HBHA polyclonal antibody from mouse serum. We injected C57BL/6 mice with HBHA/ dimethyldioctadecylammonium/trehalose 6,6'-dibehenate (HBHA/DDA/TDB) to investigate the immunogenicity of this potential vaccine. The results demonstrated that HBHA/DDA/TDB has the ability to induce high levels of HBHA-specific IgG antibody and its subclasses, as well as interferon-gamma, compared with injection of phosphate-buffered saline, DDA/TDB alone and Bacillus Calmette-Guérin (BCG) controls ( <0.05). Moreover, the ratio of IgG2a/IgG1 of the HBHA/DDA/TDB group was higher than that of the BCG group ( <0.05). HBHA with no label has excellent immunogenicity, and is suitable for evaluating the effectiveness to prevent infection.

Fourier transform theory is of central importance in a vast range of applications in physical science, engineering and applied mathematics. Providing a concise introduction to the theory and practice of Fourier transforms, this book is invaluable to students of physics, electrical and electronic engineering, and computer science. After a brief description of the basic ideas and theorems, the power of the technique is illustrated through applications in optics, spectroscopy, electronics and telecommunications. The rarely discussed but important field of multi-dimensional Fourier theory is covered, including a description of Computer Axial Tomography (CAT scanning). The book concludes by discussing digital methods, with particular attention to the Fast Fourier Transform and its implementation. This new edition has been revised to include new and interesting material, such as convolution with a sinusoid, coherence, the Michelson stellar interferometer and the van Cittert–Zernike theorem, Babinet's principle and dipole arrays.