Targeting dendritic cell activation: the therapeutic impact of paeoniflorin in cortosteroid-dependent dermatitis management.

This study investigates the mechanism through which paeoniflorin inhibits TSLP expression to regulate dendritic cell activation in corticosteroid-dependent dermatitis treatment. Utilizing databases like TCMSP, we identified paeoniflorin's components, targets, and constructed networks. Molecular docking and gene enrichment analysis helped pinpoint key targets and pathways affected by paeoniflorin. In vitro and in vivo models were used to study CD80, CD86, cytokines, T-cell activation, skin lesions, histopathological changes, TSLP, CD80, and CD86 expression. Our study revealed paeoniflorin's active constituent targeting IL-6 in corticosteroid-dependent dermatitis. In vitro experiments demonstrated reduced TSLP expression, CD80, CD86, and cytokine secretion post-paeoniflorin treatment. In vivo, paeoniflorin significantly decreased skin lesion severity, cytokine levels, TSLP, CD80, and CD86 expression. The study highlights paeoniflorin's efficacy in inhibiting TSLP expression and suppressing dendritic cell activation in corticosteroid-dependent dermatitis, suggesting its potential as a therapeutic intervention. Additionally, it offers insights into the complex molecular mechanisms underlying paeoniflorin's anti-inflammatory properties in treating corticosteroid-dependent dermatitis.

The effect of gD-derived peptides on T cell immune response mediated by BTLA-HVEM protein complex in melanoma patients.

Introduction: The effector function of T cells is regulated via immune checkpoints, activating or inhibiting the immune response. The BTLA-HVEM complex, the inhibitory immune checkpoint, may act as one of the tumor immune escape mechanisms. Therefore, interfering with the binding of these proteins can prove beneficial in cancer treatment. Our study focused on peptides interacting with HVEM at the same place as BTLA, thus disrupting the BTLA-HVEM interaction. These peptides' structure and amino acid sequences are based on the gD protein, the ligand of HVEM. Here, we investigated their immunomodulatory potential in melanoma patients. Methods: Flow cytometry analyses of activation, proliferation, and apoptosis of T cells from patients were performed. Additionally, we evaluated changes within the T cell memory compartment. Results: The most promising compound - Pep(2), increased the percentages of activated T cells and promoted their proliferation. Additionally, this peptide affected the proliferation rate and apoptosis of melanoma cell line in co-culture with T cells. Discussion: We conclude that the examined peptide may act as a booster for the immune system. Moreover, the adjuvant and activating properties of the gD-derived peptide could be used in a combinatory therapy with currently used ICI-based treatment. Our studies also demonstrate that even slight differences in the amino acid sequence of peptides and any changes in the position of the disulfide bond can strongly affect the immunomodulatory properties of compounds.

Design of Crosslinking Antibodies For T-Cell Activation: Experimental and Computational Analysis of PD-1/CD137 Bispecific Agents.

Bispecific and multispecific agents have become increasingly utilized in cancer treatment and immunotherapy, yet their complex design parameters present a challenge in developing successful therapeutics. Bispecifics that crosslink receptors on two opposing cells can provide specific activation of a receptor only when these cells are in close spatial proximity, such as an immune cell and cancer cell in a tumor. These agents, including T cell activating bispecifics, can avoid off-tumor toxicity through activation only in the tumor microenvironment by utilizing a tumor target to cluster T-cell receptors for a selective costimulatory signal. Here, we investigate a panel of PD-1/CD137 targeted Humabody VH domains to determine the key factors for T cell activation, such as affinity, valency, expression level, domain orientation, and epitope location. Target expression is a dominant factor determining both specificity and potency of T cell activation. Given an intrinsic expression level, the affinity can be tuned to modulate the level of activation and IC50 and achieve specificity between low and high expression levels. Changing the epitope location and linker length showed minor improvements to activation at low expression levels, but increasing the valency for the target decreased activation at all expression levels. By combining non-overlapping epitopes for the target, we achieved higher receptor activation at low expression levels. A kinetic model was able to capture these trends, offering support for the mechanistic interpretation. This work provides a framework to quantify factors for T cell activation by cell-crosslinking bispecific agents and guiding principles for the design of new agents.

BCG priming followed by a novel interleukin combination activates Natural Killer cells to selectively proliferate and become anti-tumour long-lived effectors.

The short-lived nature and heterogeneity of Natural Killer (NK) cells limit the development of NK cell-based therapies, despite their proven safety and efficacy against cancer. Here, we describe the biological basis, detailed phenotype and function of long-lived anti-tumour human NK cells (CD56highCD16+), obtained without cell sorting or feeder cells, after priming of peripheral blood cells with Bacillus Calmette-Guérin (BCG). Further, we demonstrate that survival doses of a cytokine combination, excluding IL18, administered just weekly to BCG-primed NK cells avoids innate lymphocyte exhaustion and leads to specific long-term proliferation of innate cells that exert potent cytotoxic function against a broad range of solid tumours, mainly through NKG2D. Strikingly, a NKG2C+CD57-FcεRIγ+ NK cell population expands after BCG and cytokine stimulation, independently of HCMV serology. This strategy was exploited to rescue anti-tumour NK cells even from the suppressor environment of cancer patients' bone marrow, demonstrating that BCG confers durable anti-tumour features to NK cells.

Targeting BTLA with the peptide inhibitor HVEM(14-39) - A new way to restore the activity of T cells in melanoma.

The complex of B- and T-lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) plays a critical role in immune regulation and has emerged as a promising therapeutic target for cancer treatment. In this study, we investigated the potential of the peptide inhibitor HVEM(14-39) to restore peripheral T cell activity in patients with advanced melanoma. In these patients, CD8+ T cells downregulated BTLA expression and increased HVEM expression upon activation. The addition of HVEM(14-39) reduced the percentage of BTLA+ CD8+ T cells and increased the subpopulation of HVEM+ CD8+ T cells. Additionally, HVEM(14-39) enhanced T cell activation, proliferation, and the shift toward effector memory T cell subpopulations. Finally, this peptide affected the proliferation rate and late apoptosis of melanoma cell line in co-culture with T cells. These findings suggest that HVEM(14-39) can overcome T cell exhaustion and improve antitumor responses. Peptide-based immunotherapy targeting the BTLA-HVEM complex offers a promising alternative to monoclonal antibody-based therapies, with the potential for fewer side effects and higher treatment efficacy.

Senotherapeutic Peptide 14 Suppresses Th1 and M1 Human T Cell and Monocyte Subsets In Vitro.

Inflammation contributes to the onset and exacerbation of numerous age-related diseases, often manifesting as a chronic condition during aging. Given that cellular senescence fosters local and systemic inflammation, senotherapeutic interventions could potentially aid in managing or even reducing inflammation. Here, we investigated the immunomodulatory effects of the senotherapeutic Peptide 14 (Pep 14) in human peripheral blood mononuclear cells (PBMCs), monocytes, and macrophages. We found that, despite failing to significantly influence T cell activation and proliferation, the peptide promoted a Th2/Treg gene expression and cytokine signature in PBMCs, characterized by increased expression of the transcription factors GATA3 and FOXP3, as well as the cytokines IL-4 and IL-10. These observations were partially confirmed through ELISA, in which we observed increased IL-10 release by resting and PHA-stimulated PBMCs. In monocytes from the U-937 cell line, Pep 14 induced apoptosis in lipopolysaccharide (LPS)-stimulated cells and upregulated IL-10 expression. Furthermore, Pep 14 prevented LPS-induced activation and promoted an M2-like polarization in U-937-derived macrophages, evidenced by decreased expression of M1 markers and increased expression of M2 markers. We also showed that the conditioned media from Pep 14-treated macrophages enhanced fibroblast migration, indicative of a functional M2 phenotype. Taken together, our findings suggest that Pep 14 modulates immune cell function towards an anti-inflammatory and regenerative phenotype, highlighting its potential as a therapeutic intervention to alleviate immunosenescence-associated dysregulation.

Priming with LSD1 inhibitors promotes the persistence and antitumor effect of adoptively transferred T cells.

The antitumor efficacy of adoptively transferred T cells is limited by their poor persistence, in part due to exhaustion, but the underlying mechanisms and potential interventions remain underexplored. Here, we show that targeting histone demethylase LSD1 by chemical inhibitors reshapes the epigenome of in vitro activated and expanded CD8+ T cells, and potentiates their antitumor efficacy. Upon T cell receptor activation and IL-2 signaling, a timely and transient inhibition of LSD1 suffices to improve the memory phenotype of mouse CD8+ T cells, associated with a better ability to produce multiple cytokines, resist exhaustion, and persist in both antigen-dependent and -independent manners after adoptive transfer. Consequently, OT1 cells primed with LSD1 inhibitors demonstrate an enhanced antitumor effect in OVA-expressing solid tumor models implanted in female mice, both as a standalone treatment and in combination with PD-1 blockade. Moreover, priming with LSD1 inhibitors promotes polyfunctionality of human CD8+ T cells, and increases the persistence and antitumor efficacy of human CD19-CAR T cells in both leukemia and solid tumor models. Thus, pharmacological inhibition of LSD1 could be exploited to improve adoptive T cell therapy.

Regulation of T Lymphocyte Functions through Calcium Signaling Modulation by Nootkatone.

Recent advancements in understanding the intricate molecular mechanisms underlying immunological responses have underscored the critical involvement of ion channels in regulating calcium influx, particularly in inflammation. Nootkatone, a natural sesquiterpenoid found in Alpinia oxyphylla and various citrus species, has gained attention for its diverse pharmacological properties, including anti-inflammatory effects. This study aimed to elucidate the potential of nootkatone in modulating ion channels associated with calcium signaling, particularly CRAC, KV1.3, and KCa3.1 channels, which play pivotal roles in immune cell activation and proliferation. Using electrophysiological techniques, we demonstrated the inhibitory effects of nootkatone on CRAC, KV1.3, and KCa3.1 channels in HEK293T cells overexpressing respective channel proteins. Nootkatone exhibited dose-dependent inhibition of channel currents, with IC50 values determined for each channel. Nootkatone treatment did not significantly affect cell viability, indicating its potential safety for therapeutic applications. Furthermore, we observed that nootkatone treatment attenuated calcium influx through activated CRAC channels and showed anti-proliferative effects, suggesting its role in regulating inflammatory T cell activation. These findings highlight the potential of nootkatone as a natural compound for modulating calcium signaling pathways by targeting related key ion channels and it holds promise as a novel therapeutic agent for inflammatory disorders.

Antigenic Peptide-Thioredoxin Fusion Chimeras for In Vitro Stimulus of CD4+ TCR+ Jurkat T Cells.

Study of CD4+ T cell response and T cell receptor (TCR) specificity is crucial for understanding etiology of immune-mediated diseases and developing targeted therapies. However, solubility, accessibility, and stability of synthetic antigenic peptides used in T cell assays may be a critical point in such studies. Here we present a T cell activation reporter system using recombinant proteins containing antigenic epitopes fused with bacterial thioredoxin (trx-peptides) and obtained by bacterial expression. We report that co-incubation of CD4+ HA1.7 TCR+ reporter Jurkat 76 TRP cells with CD80+ HLA-DRB1*01:01+ HeLa cells or CD4+ Ob.1A12 TCR+ Jurkat 76 TRP with CD80+ HLA-DRB1*15:01+ HeLa cells resulted in activation of reporter Jurkat 76 TPR after addition of recombinant trx-peptide fusion proteins, containing TCR-specific epitopes. Trx-peptides were comparable with corresponding synthetic peptides in their capacity to activate Jurkat 76 TPR. These data demonstrate that thioredoxin as a carrier protein (trx) for antigenic peptides exhibits minimal interference with recognition of MHC-specific peptides by TCRs and consequent T cell activation. Our findings highlight potential feasibility of trx-peptides as a reagent for assessing the immunogenicity of antigenic fragments.

Exploring the Structural Attributes of Yoda1 for the Development of New-Generation Piezo1 Agonist Yaddle1 as a Vaccine Adjuvant Targeting Optimal T Cell Activation.

Piezo1, a mechano-activated ion channel, has wide-ranging physiological and therapeutic implications, with the ongoing development of specific agonists unveiling cellular responses to mechanical stimuli. In our study, we systematically analyzed the chemical subunits in Piezo1 protein agonist Yoda1 to comprehend the structure-activity relationship and push forward next-generation agonist development. Preliminary screening assays for Piezo1 agonism were performed using the Piezo1-mCherry-transfected HEK293A cell line, keeping Yoda1 as a positive control. We introduce a novel Piezo1 agonist Yaddle1 (34, 0.40 µM), featuring a trifluoromethyl group, with further exploration through in vitro studies and density functional theory calculations, emphasizing its tetrel interactions, to act as an ambidextrous wedge between the domains of Piezo1. In contrast to the poor solubility of the established agonist Yoda1, our results showed that the kinetic solubility of Yaddle1 (26.72 ± 1.8 µM at pH 7.4) is 10-fold better than that of Yoda1 (1.22 ± 0.11 µM at pH 7.4). Yaddle1 (34) induces Ca2+ influx in human CD4+ T cell, suggesting its potential as a vaccine adjuvant for enhanced T cell activation.

Optimization of polydimethylsiloxane (PDMS) surface chemical modification and formulation for improved T cell activation and expansion.

Adoptive T cell therapy has undergone remarkable advancements in recent decades; nevertheless, the rapid and effective ex vivo expansion of tumor-reactive T cells remains a formidable challenge, limiting their clinical application. Artificial antigen-presenting substrates represent a promising avenue for enhancing the efficiency of adoptive immunotherapy and fostering T cell expansion. These substrates offer significant potential by providing flexibility and modularity in the design of tailored stimulatory environments. Polydimethylsiloxane (PDMS) silicone elastomer stands as a widely utilized biomaterial for exploring the varying sensitivity of T cell activation to substrate properties. This paper explores the optimization of PDMS surface modification and formulation to create customized stimulatory surfaces with the goal of enhancing T cell expansion. By employing soft PDMS elastomer functionalized through silanization and activating agent, coupled with site-directed protein immobilization techniques, a novel T cell stimulatory platform is introduced, facilitating T cell activation and proliferation. Notably, our findings underscore that softer modified elastomers (Young' modulus E∼300 kPa) exhibit superior efficacy in stimulating and activating mouse CD4+ T cells compared to their stiffer counterparts (E∼3 MPa). Furthermore, softened modified PDMS substrates demonstrate enhanced capabilities in T cell expansion and Th1 differentiation, offering promising insights for the advancement of T cell-based immunotherapy.

Formyl peptide enhances cancer immunotherapy by activating antitumoral neutrophils, and T cells.

Neutrophils are heterogeneous and plastic, with the ability to polarize from antitumour to protumour phenotype and modulate tumour microenvironment components. While some advances have been made, the neutrophil-targeting therapy remains underexplored. Activation of formyl peptide receptors (FPRs) by formylated peptides is needed for local control of infection through the recruitment of activated neutrophils while the potential contribution of antitumour activity remains underexplored. Here, we demonstrate that neutrophils can be harnessed to suppress tumour growth through the action of the formyl peptide (FP) on the formyl peptide receptor (FPR). Mechanistically, FP efficiently recruits neutrophils to produce reactive oxygen species production (ROS), resulting in the direct killing of tumours. Antitumour functions disappeared when neutrophils were depleted by anti-Ly6G antibodies. Interestingly, extensive T-cell activation was observed in mouse tumours treated with FP, showing the potential to alter the immune suppressed tumour microenvironment (TME) and further sensitize mice to anti-PD1 therapy. Transcriptomic and flow cytometry analyses revealed the mechanisms of FP-sensitized anti-PD1 therapy, mainly including stimulated neutrophils and an altered immune-suppressed tumour microenvironment. Collectively, these data establish FP as an effective combination partner for sensitizing anti-PD1 therapy by stimulating tumour-infiltrated neutrophils.

Comparative analysis of whole plant, flower and root extracts of Chamomilla recutita L. and characteristic pure compounds reveals differential anti-inflammatory effects on human T cells.

Introduction: Chronic inflammation is a hallmark of chronic wounds and inflammatory skin diseases. Due to a hyperactive and prolonged inflammation triggered by proinflammatory immune cells, transitioning to the repair and healing phase is halted. T cells may exacerbate the proinflammatory milieu by secreting proinflammatory cytokines. Chamomilla recutita L. (chamomile) has been suggested for use in several inflammatory diseases, implying a capability to modulate T cells. Here, we have characterized and compared the effects of differently prepared chamomile extracts and characteristic pure compounds on the T cell redox milieu as well as on the migration, activation, proliferation, and cytokine production of primary human T cells. Methods: Phytochemical analysis of the extracts was carried out by LC-MS/MS. Primary human T cells from peripheral blood (PBTs) were pretreated with aqueous or hydroethanolic chamomile extracts or pure compounds. Subsequently, the effects on intracellular ROS levels, SDF-1α induced T cell migration, T cell activation, proliferation, and cytokine production after TCR/CD3 and CD28 costimulation were determined. Gene expression profiling was performed using nCounter analysis, followed by ingenuity pathway analysis, and validation at protein levels. Results: The tested chamomile extracts and pure compounds differentially affected intracellular ROS levels, migration, and activation of T cells. Three out of five differently prepared extracts and two out of three pure compounds diminished T cell proliferation. In line with these findings, LC-MS/MS analysis revealed high heterogeneity of phytochemicals among the different extracts. nCounter based gene expression profiling identified several genes related to T cell functions associated with activation and differentiation to be downregulated. Most prominently, apigenin significantly reduced granzyme B induction and cytotoxic T cell activity. Conclusion: Our results demonstrate an anti-inflammatory effect of chamomile- derived products on primary human T cells. These findings provide molecular explanations for the observed anti-inflammatory action of chamomile and imply a broader use of chamomile extracts in T cell driven chronic inflammatory diseases such as chronic wounds and inflammatory skin diseases. Importantly, the mode of extract preparation needs to be considered as the resulting different phytochemicals can result in differential effects on T cells.

The STING agonist IMSA101 enhances chimeric antigen receptor T cell function by inducing IL-18 secretion.

As a strategy to improve the therapeutic success of chimeric antigen receptor T cells (CART) directed against solid tumors, we here test the combinatorial use of CART and IMSA101, a newly developed stimulator of interferon genes (STING) agonist. In two syngeneic tumor models, improved overall survival is observed when mice are treated with intratumorally administered IMSA101 in addition to intravenous CART infusion. Transcriptomic analyses of CART isolated from tumors show elevated T cell activation, as well as upregulated cytokine pathway signatures, in particular IL-18, in the combination treatment group. Also, higher levels of IL-18 in serum and tumor are detected with IMSA101 treatment. Consistent with this, the use of IL-18 receptor negative CART impair anti-tumor responses in mice receiving combination treatment. In summary, we find that IMSA101 enhances CART function which is facilitated through STING agonist-induced IL-18 secretion.

Glutamine supplementation improves the activity and immunosuppressive action of induced regulatory T cells in vitro and in vivo.

BACKGROUND: Glutamine is crucial for the activation and efficacy of T cells, and may play a role in regulating the immune environment. This study aimed to investigate the potential role of glutamine in the activation and proliferation of induced regulatory T cells (iTregs). METHODS: CD4+CD45RA+T cells were sorted from peripheral blood mononuclear cells and cultured to analyze iTreg differentiation. Glutamine was then added to the culture system to evaluate the effects of glutamine on iTregs by determining oxidative phosphorylation (OXPHOS), apoptosis, and cytokine secretion. Additionally, a humanized murine graft-versus-host disease (GVHD) model was constructed to confirm the efficacy of glutamine-treated iTregs in vivo. RESULTS: After being cultured in vitro, glutamine significantly enhanced the levels of Foxp3, CTLA-4, CD39, CD69, IL-10, TGF-ß, and Ki67 (CTLA-4, IL-10, TGF-ß are immunosuppressive markers of iTregs) compared with that of the control iTregs (P < 0.05). Furthermore, the growth curve showed that the proliferative ability of glutamine-treated iTregs was better than that of the control iTregs (P < 0.01). Compared with the control iTregs, glutamine supplementation significantly increased oxygen consumption rates and ATP production (P < 0.05), significantly downregulated Annexin V and Caspase 3, and upregulated BCL2 (P < 0.05). However, GPNA significantly reversed the effects of glutamine (P < 0.05). Finally, a xeno-GVHD mouse model was successfully established to confirm that glutamine-treated iTregs increased the mice survival rate, delayed weight loss, and alleviated colon injury. CONCLUSION: Glutamine supplementation can improve the activity and immunosuppressive action of iTregs, and the possible mechanisms by which this occurs are related to cell proliferation, apoptosis, and OXPHOS.

Ex vivo expansion and activation of Vγ9Vδ2 T cells by CELMoDs in combination with zoledronic acid.

As multiple myeloma (MM) progresses, immune effector cells decrease in number and function and become exhausted. This remains an insurmountable clinical issue that must be addressed by development of novel modalities to revitalize anti-MM immunity. Human Vγ9Vδ2 T (Vδ2+ γδ T) cells serve as the first line of defense against pathogens as well as tumors and can be expanded ex vivo from peripheral blood mononuclear cells (PBMCs) upon treatment with amino-bisphosphonates in combination with IL-2. Here, we demonstrated that next-generation immunomodulators called cereblon E3 ligase modulators (CELMoDs), as well as lenalidomide and pomalidomide, expanded Th1-like Vδ2+ γδ T cells from PBMCs in the presence of zoledronic acid (ZA). However, the expansion of Th1-like Vδ2+ γδ T cells by these immunomodulatory drugs was abolished under IL-2 blockade, although IL-2 production was induced in PBMCs. BTN3A1 triggers phosphoantigen presentation to γδ T-cell receptors and is required for γδ T-cell expansion and activation. ZA but not these immunomodulatory drugs upregulated BTN3A1 in monocytes. These results suggest that immunomodulatory drugs and ZA have cooperative roles in expansion of Th1-like Vδ2+ γδ T cells, and provide the important knowledge for clinical application of human Vδ2+ γδ T cells as effector cells.

Toosendanin inhibits T-cell proliferation through the P38 MAPK signalling pathway.

In recent years, immunosuppressants have shown significant success in the treatment of autoimmune diseases. Therefore, there is an urgent need to develop additional immunosuppressants that offer more options for patients. Toosendanin has been shown to have immunosuppressive activity in vitro as well as effects on autoimmune hepatitis (AIH) in vivo. Toosendanin did not induce apoptosis in activated T-cells and affect the survival rate of naive T-cells. Toosendanin did not affect the expression of CD25 or secretion of IL-2 by activated T-cells, and not affect the expression of IL-4 and INF-γ. Toosendanin did not affect the phosphorylation of STAT5, ERK, AKT, P70S6K. However, toosendanin inhibited proliferation of anti-CD3/anti-CD28 mAbs-activated T-cells with IC50 of (10 ± 2.02) nM. Toosendanin arrested the cell cycle in the G0/G1 phase, significantly inhibited IL-6 and IL-17A secretion, promoted IL-10 expression, and inhibited the P38 MAPK pathway. Finally, toosendanin significantly alleviated ConA-induced AIH in mice. In Summary, toosendanin exhibited immunosuppressive activity in vivo and in vitro. Toosendanin inhibits the proliferation of activated T-cells through the P38 MAPK signalling pathway, significantly suppresses the expression of inflammatory factors, enhances the expression of anti-inflammatory factors, and effectively alleviates ConA-induced AIH in mice, suggesting that toosendanin may be a lead compound for the development of novel immunomodulatory agents with improved efficacy and reduced toxicity.

Activates B lymphocytes and enhanced immune response: A promising adjuvant based on PLGA nanoparticle to improve the sensitivity of ZEN monoclonal antibody.

In preparing monoclonal antibodies by hybridoma cell technology, the quality of B lymphocytes used for cell fusion directly affects the sensitivity of monoclonal antibodies. To obtain B-lymphocytes producing high-quality specific antibodies for cell fusion during the immunization phase of the antigen, we prepared a TH2-Cell stimulatory delivery system as a novel adjuvant. Astragalus polysaccharide has a good ability to enhance antigenic immune response, and it was encapsulated in biocompatible materials PLGA as an immunostimulatory factor to form the delivery system (APS-PLGA). The preparation conditions of APSP were optimized using RSM to attain the highest utilization of APS. Immunization against ZEN-BSA antigen using APSP as an adjuvant to obtain B lymphocytes producing ZEN-specific antibodies for cell fusion. As results present, APSP could induce a stronger TH2 immune response through differentiating CD4 T cells and promoting IL-4 and IL-6 cytokines. Moreover, it could slow down the release efficiency of ZEN-BSA and enhance the targeting of ZEN-BSA to lymph nodes in vivo experiments. Ultimately, the sensitivity of mouse serum ZEN-specific antibodies was enhanced upon completion of immunization, indicating a significant upregulation of high-quality B lymphocyte expression. In the preparation of monoclonal antibodies, the proportion of positive wells for the first screening was 60%, and the inhibition rates of the antibodies were all similar (>50%). Then we obtained the ZEN monoclonal antibody with IC50 of 0.049 ng/mL, which was more sensitive than most antibodies prepared under conventional adjuvants. Finally, a TRFIAS strip assay was preliminarily established with a LOD value of 0.246 ng/mL.

Zinc Ionophore Pyrithione Mimics CD28 Costimulatory Signal in CD3 Activated T Cells.

Zinc is an essential trace element that plays a crucial role in T cell immunity. During T cell activation, zinc is not only structurally important, but zinc signals can also act as a second messenger. This research investigates zinc signals in T cell activation and their function in T helper cell 1 differentiation. For this purpose, peripheral blood mononuclear cells were activated via the T cell receptor-CD3 complex, and via CD28 as a costimulatory signal. Fast and long-term changes in intracellular zinc and calcium were monitored by flow cytometry. Further, interferon (IFN)-γ was analyzed to investigate the differentiation into T helper 1 cells. We show that fast zinc fluxes are induced via CD3. Also, the intracellular zinc concentration dramatically increases 72 h after anti-CD3 and anti-CD28 stimulation, which goes along with the high release of IFN-γ. Interestingly, we found that zinc signals can function as a costimulatory signal for T helper cell 1 differentiation when T cells are activated only via CD3. These results demonstrate the importance of zinc signaling alongside calcium signaling in T cell differentiation.

Nonclinical Investigation of Cytokine Mitigation Strategies for T-cell-Engaging Bispecifics in the Cynomolgus Macaque.

SUMMARY: T-cell-directed cancer therapies such as T-cell-engaging bispecifics (TCBs) are commonly associated with cytokine release syndrome and associated clinical signs that can limit their tolerability and therapeutic benefit. Strategies for reducing cytokine release are therefore needed. Here, we report on studies performed in cynomolgus monkeys to test different approaches for mitigating cytokine release with TCBs. A "priming dose" as well as subcutaneous dosing reduced cytokine release compared with intravenous dosing but did not affect the intended T-cell response to the bispecific. As another strategy, cytokines or cytokine responses were blocked with an anti-IL-6 antibody, dexamethasone, or a JAK1/TYK2-selective inhibitor, and the effects on toxicity as well as T-cell responses to a TCB were evaluated. The JAK1/TYK2 inhibitor and dexamethasone prevented CRS-associated clinical signs on the day of TCB administration, but the anti-IL-6 had little effect. All interventions allowed for functional T-cell responses and expected damage to target-bearing tissues, but the JAK1/TYK2 inhibitor prevented the upregulation of activation markers on T cells, suggesting the potential for suppression of T-cell responses. Our results suggest that short-term prophylactic dexamethasone treatment may be an effective option for blocking cytokine responses without affecting desired T-cell responses to TCBs.