Channel plan: control of adaptive immune responses by pannexins.

The development of mammalian adaptive (i.e., B and T cell-mediated) immune responses is tightly controlled at transcriptional, epigenetic, and metabolic levels. Signals derived from the extracellular milieu are crucial regulators of adaptive immunity. Beyond the traditionally studied cytokines and chemokines, many other extracellular metabolites can bind to specialized receptors and regulate T and B cell immune responses. These molecules often accumulate extracellularly through active export by plasma membrane transporters. For example, mammalian immune and non-immune cells express pannexin (PANX)1-3 channels on the plasma membrane, which release many distinct small molecules, notably intracellular ATP. Here, we review novel findings defining PANXs as crucial regulators of T and B cell immune responses in disease contexts such as cancer or viral infections.

Ginsenoside Rg3 targets glycosylation of PD-L1 to enhance anti-tumor immunity in non-small cell lung cancer.

Background: Reactivate the T cell immunity by PD-1/PD-L1 checkpoint blockade is widely used in non-small cell lung cancer (NSCLC) patients, while the post-translational modification of Programmed death ligand-1 (PD-L1) is commonly existed in various cancer cells, thus increases the complexity and difficulty in therapy development. Ginsenoside Rg3 is an active component of traditional Chinese herb Ginseng with multiple pharmacological effects including immune regulation. However, the effect on the glycosylation of PD-L1 is unknown. Methods: NSCLC cell lines were tested for glycosylation of PD-L1, and the potential mechanisms were investigated. Tumor cell-T cell coculture experiment was conducted and the activation of T cells and cytotoxicity were measured by flow cytometry. In vivo xenograft mouse tumor model was used to investigate the effects of Rg3 on PD-L1-mediated immunosuppression and tumor growth. Results: Here, we identified PD-L1 is widely N-linked glycosylated in NSCLC cell lines, while Rg3 could inhibit the glycosylation of PD-L1 by downregulating the EGFR signaling and further activate GSK3b-mediated degradation, thus resulted in reduced PD-L1 expression. Moreover, the inhibition of PD-L1 glycosylation promoted the activation and cytotoxicity of T cells under coculture condition. In addition, Rg3 could decrease the tumor volume and enhance anti-tumor T cell immunity as evidence by the upregulated expression of Granzyme B and perforin in CD8+T cells, along with elevated serum IL-2, IFN-g and TNF-a level in Rg3-treated mice. Conclusions: These results suggest that Rg3 inhibits PD-L1 glycosylation and thus enhance anti-tumor immunity, which provide new therapeutic insight into drug discovery.

Localized ablative immunotherapy enhances antitumor immunity by modulating the transcriptome of tumor-infiltrating Gamma delta T cells.

Gamma delta T cells (γδT cells) play crucial roles in the immune response against tumors, yet their functional dynamics under different cancer therapies remain poorly understood. Laser Ablative Immunotherapy (LAIT) is a novel cancer treatment modality combining local photothermal therapy (PTT) and intratumoral injection of an immunostimulant, N-dihydrogalactochitosan (glycated chitosan, GC). LAIT has been shown to induce systemic antitumor immune responses in pre-clinical studies and clinical trials, eradicating both treated local tumors and untreated distant metastases. In this study, we used LAIT to treat breast tumors in a mouse model and investigated the effects of LAIT on tumor-infiltrating γδT cells using single-cell RNA sequencing (scRNAseq). We characterized the γδT cells from tumors in control, PTT, GC, and LAIT (PTT + GC) groups, by identifying six distinct subtypes: activated, cytotoxic, cycling cytotoxic, IFN-enriched, antigen-presenting, and IL17-producing γδT cells. Differential gene expression analysis revealed that LAIT significantly upregulated genes associated with T cell activation, leukocyte adhesion, and interferon signaling in treated tumor tissues while downregulating genes involved in protein folding and stress responses. LAIT also uniquely increased the proportion of IL17-producing γδT cells, which correlated with prolonged survival in breast cancer patients, as analyzed using TCGA data. Furthermore, the transcriptomic profiles of γδT cells in LAIT-treated tumors closely resembled those in immune checkpoint inhibitor (ICI)-treated patients, suggesting potential synergistic effects. Our findings indicate that LAIT modulates the γδT cell transcriptome, enhancing their antitumor capabilities and providing a basis for combining LAIT with ICI therapy to improve cancer treatment outcomes.

Enhancing Tumor-Specific immunity with SLdacA: A attenuated Salmonella-mediated c-di-AMP delivery system targeting the STING pathway.

The STING agonist stimulates an anti-tumor immune response by activating T cells, but its limited tumor-targeting specificity poses risks of cytokine storms or autoimmune reactions. Conversely, attenuated Salmonella typhimurium △ppGpp (S.t△ppGpp) exhibits superior tumor-targeting specificity and potent anti-tumor immunogenicity. However, the anti-tumor effects of Salmonella carrying STING agonists remain underexplored. In this study, we engineered a strain called SLdacA, utilizing S.t△ppGpp as a carrier, to produce c-di-AMP. This engineered strain effectively enhances dendritic cell maturation and M1-type macrophage polarization by inducing type I interferon production, thereby recruiting and activating effector T cells against tumor progression. This process is regulated by the STING/type I interferon pathway. Our findings indicate that utilizing S.t△ppGpp as a delivery vehicle for STING agonists holds promise as a strategy for synergistic bacterial-mediated immunotherapy.

Dissociation of LAG-3 inhibitory cluster from TCR microcluster by immune checkpoint blockade.

Lymphocyte activation gene (Lag)-3 is an inhibitory co-receptor and target of immune checkpoint inhibitor (ICI) therapy for cancer. The dynamic behavior of Lag-3 was analyzed at the immune synapse upon T-cell activation to elucidate the Lag-3 inhibitory mechanism. Lag-3 formed clusters and co-localized with T-cell receptor microcluster (TCR-MC) upon T-cell activation similar to PD-1. Lag-3 blocking antibodies (Abs) inhibited the co-localization between Lag-3 and TCR-MC without inhibiting Lag-3 cluster formation. Lag-3 also inhibited MHC-II-independent stimulation and Lag-3 Ab, which did not block MHC-II binding could still block Lag-3's inhibitory function, suggesting that the Lag-3 Ab blocks the Lag-3 inhibitory signal by dissociating the co-assembly of TCR-MC and Lag-3 clusters. Consistent with the combination benefit of PD-1 and Lag-3 Abs to augment T-cell responses, bispecific Lag-3/PD-1 antagonists effectively inhibited both cluster formation and co-localization of PD-1 and Lag-3 with TCR-MC. Therefore, Lag-3 inhibits T-cell activation at TCR-MC, and the target of Lag-3 ICI is to dissociate the co-localization of Lag-3 with TCR-MC.

VISTA Emerges as a Promising Target against Immune Evasion Mechanisms in Medulloblastoma.

BACKGROUND: Relapsed medulloblastoma (MB) poses a significant therapeutic challenge due to its highly immunosuppressive tumor microenvironment. Immune checkpoint inhibitors (ICIs) have struggled to mitigate this challenge, largely due to low T-cell infiltration and minimal PD-L1 expression. Identifying the mechanisms driving low T-cell infiltration is crucial for developing more effective immunotherapies. METHODS: We utilize a syngeneic mouse model to investigate the tumor immune microenvironment of MB and compare our findings to transcriptomic and proteomic data from human MB. RESULTS: Flow cytometry reveals a notable presence of CD45hi/CD11bhi macrophage-like and CD45int/CD11bint microglia-like tumor-associated macrophages (TAMs), alongside regulatory T-cells (Tregs), expressing high levels of the inhibitory checkpoint molecule VISTA. Compared to sham control mice, the CD45hi/CD11bhi compartment significantly expands in tumor-bearing mice and exhibits a myeloid-specific signature composed of VISTA, CD80, PD-L1, CTLA-4, MHCII, CD40, and CD68. These findings are corroborated by proteomic and transcriptomic analyses of human MB samples. Immunohistochemistry highlights an abundance of VISTA-expressing myeloid cells clustering at the tumor-cerebellar border, while T-cells are scarce and express FOXP3. Additionally, tumor cells exhibit immunosuppressive properties, inhibiting CD4 T-cell proliferation in vitro. Identification of VISTA's binding partner, VSIG8, on tumor cells, and its correlation with increased VISTA expression in human transcriptomic analyses suggests a potential therapeutic target. CONCLUSIONS: This study underscores the multifaceted mechanisms of immune evasion in MB and highlights the therapeutic potential of targeting the VISTA-VSIG axis to enhance anti-tumor responses.

T-cell specific in vivo gene delivery with DART-AAVs targeted to CD8.

One of the biggest challenges for in vivo gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs (DART-AAVs) displaying DARPins specific for human and murine CD8. Insertion of DARPins into the GH2/GH3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in high selectivity for CD8-positive T cells with unimpaired gene delivery activity. Remarkably, the capsid core structure was unaltered with protruding DARPins detectable. In complex primary cell mixtures, including donor blood or systemic injections into mice, the CD8-targeted AAVs were by far superior to unmodified AAV2 and AAV6 in terms of selectivity, target cell viability, and gene transfer rates. In vivo, up to 80% of activated CD8+ T cells were hit upon a single vector injection into conditioned humanized or immunocompetent mice. While gene transfer rates decreased significantly under non-activated conditions, genomic modification selectively in CD8+ T cells was still detectable upon Cre delivery into indicator mice. In both mouse models, selectivity for CD8+ T cells was close to absolute with exceptional detargeting from liver. The CD8-AAVs described here expand strategies for immunological research and in vivo gene therapy options.

S-adenosyl-L-methionine supplementation alleviates aortic dissection by decreasing inflammatory infiltration.

Methionine, an indispensable amino acid crucial for dietary balance, intricately governs metabolic pathways. Disruption in its equilibrium has the potential to heighten homocysteine levels in both plasma and tissues, posing a conceivable risk of inducing inflammation and detriment to the integrity of vascular endothelial cells. The intricate interplay between methionine metabolism, with a specific focus on S-adenosyl-L-methionine (SAM), and the onset of thoracic aortic dissection (TAD) remains enigmatic despite acknowledging the pivotal role of inflammation in this vascular condition. In an established murine model induced by ß-aminopropionitrile monofumarate (BAPN), we delved into the repercussions of supplementing with S-adenosyl-L-methionine (SAM) on the progression of TAD. Our observations uncovered a noteworthy improvement in aortic dissection and rupture rates, accompanied by a marked reduction in mortality upon SAM supplementation. Notably, SAM supplementation exhibited a considerable protective effect against BAPN-induced degradation of elastin and the extracellular matrix. Furthermore, SAM supplementation demonstrated a robust inhibitory influence on the infiltration of immune cells, particularly neutrophils and macrophages. It also manifested a notable reduction in the inflammatory polarization of macrophages, evident through diminished accumulation of MHC-IIhigh macrophages and reduced expression of inflammatory cytokines such as IL1ß and TNFα in macrophages. Simultaneously, SAM supplementation exerted a suppressive effect on the activation of CD4 + and CD8 + T cells within the aorta. This was evidenced by an elevated proportion of CD44- CD62L + naïve T cells and a concurrent decrease in CD44 + CD62L- effector T cells. In summary, our findings strongly suggest that the supplementation of SAM exhibits remarkable efficacy in alleviating BAPN-induced aortic inflammation, consequently impeding the progression of thoracic aortic dissection.

Validation of the C-X-C chemokine receptor 3 (CXCR3) as a target for PET imaging of T cell activation.

PURPOSE: CXCR3 is expressed on activated T cells and plays a crucial role in T-cell recruitment to the tumor microenvironment (TME) during cell-based and immune checkpoint inhibitor (ICI) immunotherapy. This study utilized a 64Cu-labeled NOTA-α-CXCR3 antibody to assess CXCR3 expression in the TME and validate it as a potential T cell activation biomarker in vivo. PROCEDURES: CXCR3+ cells infiltrating MC38 tumors (B57BL/6 mice, untreated and treated with αPD-1/αCTLA-4 ICI) were quantified using fluorescence microscopy and flow cytometry. A commercial anti-mouse CXCR3 antibody (α-CXCR3) was site-specifically conjugated with 2,2,2-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA) and radiolabeled with 64Cu. Saturation binding of [64Cu]Cu-NOTA-α-CXCR3 was investigated using CHO cells stably transfected with murine CXCR3. Biodistribution and PET imaging studies both at baseline and after 1 to 3 cycles of ICI, respectively, were carried out using different molar activities (10 GBq/µmol to 300 GBq/µmol) of [64Cu]Cu-NOTA-α-CXCR3. RESULTS: Flow cytometry analysis at baseline confirmed the presence of CXCR3 + T-cells in MC38 tumors, which was significantly increased at day five after ICI (treated 33.8 ± 17.4 vs. control 8.8 ± 6.2 CD3+CXCR3+ cells/mg). These results were qualitatively and quantitatively confirmed by immunofluorescence of tumor cryoslices. In vivo PET imaging of MC38 tumor bearing mice before, during and after ICI using [64Cu]Cu-NOTA-α-CXCR3 (Kd = 3.3 nM) revealed a strong dependence of CXCR3-specificity of tracer accumulation in secondary lymphoid organs on molar activity. At 300 GBq/µmol (1.5 µg of antibody/mouse), a specific signal was observed in lymph nodes (6.33 ± 1.25 control vs. 3.95 ± 1.23%IA/g blocking) and the spleen (6.04 ± 1.02 control vs. 3.84 ± 0.79%IA/g blocking) at 48 h p.i. Spleen-to-liver ratios indicated a time dependent systemic immune response showing a steady increase from 1.08 ± 0.19 (untreated control) to 1.54 ± 0.14 (three ICI cycles). CONCLUSIONS: This study demonstrates the feasibility of in vivo imaging of CXCR3 upregulation under immunotherapy using antibodies. However, high molar activities and low antibody doses are essential for sensitive detection in lymph nodes and spleen. Detecting therapy-induced changes in CXCR3+ T cell numbers in tumors was challenging due to secondary antibody-related effects. Nonetheless, CXCR3 remains a promising target for imaging T cell activation, with anticipated improvements in sensitivity using alternative tracers with high affinities and favorable pharmacokinetics.

Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8+ T lymphocytes.

Introduction: CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Methods: Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. Results: We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Discussion: Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.

Case report: Successful treatment of intestinal leiomyositis in a dog using adjunctive intravenous immunoglobulin.

A 10-year-old spayed female Dachshund presented with abdominal pain and generalized severe ileus. An exploratory laparotomy was performed, confirming a severe ileus of undetermined origin. Multiple intestinal biopsy results confirmed acute intestinal leiomyositis. Immunohistochemistry (IHC) stains confirmed a T-cell predominant inflammatory infiltrate. Intravenous immunoglobulin (hIVIG) was administered prior to immunosuppressive therapy. Within 10 days of hIVIG treatment, functional peristaltic activity returned, and symptoms resolved. Long-term management, including the use of mycophenolate, resulted in sustained functional peristaltic recovery. Further studies are needed to explore the potential benefits of hIVIG treatment in the stabilization phase of this commonly fatal, treatment-refractory disease.

A general pHLA-CD80 scaffold fusion protein to promote efficient antigen-specific T cell-based immunotherapy.

Inadequate antigen-specific T cells activation hampers immunotherapy due to complex antigen presentation. In addition, therapeutic in vivo T cell expansion is constrained by slow expansion rates and limited functionality. Herein, we introduce a model fusion protein termed antigen-presenting cell-mimic fusion protein (APC-mimic), designed to greatly mimicking the natural antigen presentation pattern of antigen-presenting cells and directly expand T cells both in vitro and in vivo. The APC-mimic comprises the cognate peptide-human leukocyte antigen (pHLA) complex and the co-stimulatory marker CD80, which are natural ligands on APCs. Following a single stimulation, APC-mimic leads to an approximately 400-fold increase in the polyclonal expansion of antigen-specific T cells compared with the untreated group in vitro without the requirement for specialized antigen-presenting cells. Through the combination of single-cell TCR sequencing (scTCR-seq) and single-cell RNA sequencing (scRNA-seq), we identify an approximately 600-fold monoclonal expansion clonotype among these polyclonal clonotypes. It also exhibits suitability for in vivo applications confirmed in the OT-1 mouse model. Furthermore, T cells expanded by APC-mimic effectively inhibits tumor growth in adoptive cell transfer (ACT) murine models. These findings pave the way for the versatile APC-mimic platform for personalized therapeutics, enabling direct expansion of polyfunctional antigen-specific T cell subsets in vitro and in vivo.

Engineering a tumor-selective prodrug T-cell engager bispecific antibody for safer immunotherapy.

T-cell engaging (TCE) bispecific antibodies are potent drugs that trigger the immune system to eliminate cancer cells, but administration can be accompanied by toxic side effects that limit dosing. TCEs function by binding to cell surface receptors on T cells, frequently CD3, with one arm of the bispecific antibody while the other arm binds to cell surface antigens on cancer cells. On-target, off-tumor toxicity can arise when the target antigen is also present on healthy cells. The toxicity of TCEs may be ameliorated through the use of pro-drug forms of the TCE, which are not fully functional until recruited to the tumor microenvironment. This can be accomplished by masking the anti-CD3 arm of the TCE with an autoinhibitory motif that is released by tumor-enriched proteases. Here, we solve the crystal structure of the antigen-binding fragment of a novel anti-CD3 antibody, E10, in complex with its epitope from CD3 and use this information to engineer a masked form of the antibody that can activate by the tumor-enriched protease matrix metalloproteinase 2 (MMP-2). We demonstrate with binding experiments and in vitro T-cell activation and killing assays that our designed prodrug TCE is capable of tumor-selective T-cell activity that is dependent upon MMP-2. Furthermore, we demonstrate that a similar masking strategy can be used to create a pro-drug form of the frequently used anti-CD3 antibody SP34. This study showcases an approach to developing immune-modulating therapeutics that prioritizes safety and has the potential to advance cancer immunotherapy treatment strategies.

Reverse vaccinology approach for identification of epitopes from E1 protein as peptide vaccine against HCV: A proof of concept.

The development of effective vaccines against Hepatitis C Virus (HCV) remains a global health priority and challenge. In this study, we employed an integrative approach combining computational epitope prediction with experimental validation to identify immunogenic peptides targeting the E1 glycoprotein of HCV. In the present report, computational data from various epitope prediction algorithms such as IEDB and SYFPEITHI, followed by molecular dynamics (MD) simulations and immuno-informatics analysis is presented. Through computational screening, we identified potential epitope candidates, with QVRNSSGLY (P3) and QLFTFSPRH (P7) emerging as promising candidates. MD simulations revealed stable interactions between these epitopes and MHC molecule, further validated by free energy estimations using MMPBSA method. Immuno-informatics analysis supported these findings, showing high binding potential and immunogenicity scores for the selected peptides. Subsequent synthesis and characterization of epitope peptides confirmed their structural integrity and purity required for conducting immune activation assays. Experimental immunological assays carried out in this study involved epitope peptide induced activation of CD8 + and CD4 + T cells from healthy human subjects and HCV- recovered patients. Data from experimental validation revealed significant cytokine release upon exposure to epitope peptides, particularly TNF-a, IL-6, and GM-CSF, indicative of robust immune responses. Notably, peptides P3 and P7 exhibited the most pronounced cytokine induction profiles, underscoring their potential as vaccine candidates. Further investigations addressing the mechanism of action of these epitope peptides under preclinical and clinical settings may help in developing effective vaccine against HCV.

Spiky Gold Nanoparticles, a Nanoscale Approach to Enhanced Ex Vivo T-Cell Activation.

While existing synthetic technologies for ex vivo T-cell activation face challenges like suboptimal expansion rates and low effectiveness, artificial antigen-presenting cells (aAPCs) hold great promise for enhanced T-cell based therapies. In particular, gold nanoparticles (AuNPs), known for their biocompatibility, ease of synthesis, and versatile surface chemistry, are strong candidates for use as nanoscale aAPCs. In this study, we developed spiky AuNPs with branched geometries to present activating ligands to primary human T-cells. The special structure of spiky AuNPs enhances biomolecule loading capacity and significantly improves T-cell activation through multivalent binding of costimulatory ligands and receptors. Our spiky AuNPs outperform existing systems including Dynabeads and soluble activators by promoting greater polyclonal expansion of T-cells, boosting sustained cytokine production, and generating highly functional T-cells with reduced exhaustion. In addition, spiky AuNPs effectively activate and expand CD19 CAR-T cells while demonstrating increased in vitro cytotoxicity against target cells using fewer effector cells than Dynabeads. This study underscores the potential of spiky AuNPs as a powerful tool, bringing new opportunities to adoptive cell therapy applications.

Immune cell landscapes are associated with high-grade serous ovarian cancer survival.

High-grade serous ovarian cancer (HGSOC) is an aggressive disease known to develop resistance to chemotherapy. We investigated the prognostic significance of tumor cell states and potential mechanisms underlying chemotherapy resistance in HGSOC. Transcriptome deconvolution was performed to address cellular heterogeneity. Kaplan-Meier survival curves were plotted to illustrate the outcomes of patients with varying cellular abundances. The association between gene expression and chemotherapy response was tested. After adjusting for surgery status and grading, several cell states exhibited a significant correlation with patient survival. Cell states can organize into carcinoma ecotypes (CE). CE9 and CE10 were proinflammatory, characterized by higher immunoreactivity, and were associated with favorable survival outcomes. Ratios of cell states and ecotypes had better prognostic abilities than a single cell state or ecotype. A total of 1265 differentially expressed genes were identified between samples with high and low levels of C9 or CE10. These genes were partitioned into three co-expressed modules, which were associated with tumor cells and immune cells. Pogz was identified to be linked with immune cell genes and the chemotherapy response of paclitaxel. Collectively, the survival of HGSOC patients is correlated with specific cell states and ecotypes.

Human T-cell lymphotropic virus type 1 (HTLV-1) grip on T-cells: investigating the viral tapestry of activation.

INTRODUCTION: Human T-cell Lymphotropic virus type 1 (HTLV-1) belongs to retroviridae which is connected to two major diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and Adult T-cell leukemia/lymphoma (ATLL). This study aims to investigate the mRNA expressions of key proteins correlated to T-cell activation in asymptomatic carriers (ACs) HTLV-1 infected patients, shedding light on early molecular events and T-cell activation following HTLV-1 infection. MATERIAL AND METHODS: The study involved 40 participants, including 20 ACs and 20 healthy subjects. Blood samples were collected, ELISA assessment for screening and confirmation with PCR for Trans-activating transcriptional regulatory protein (Tax) and HTLV-1 basic leucine zipper factor (HBZ) of the HTLV-1 were done. mRNA expressions of C-terminal Src kinase (CSK), Glycogen Synthase Kinase-3 Beta (GSK3ß), Mitogen-Activated Protein Kinase 14 (MAP3K14 or NIK), Phospholipase C Gamma-1 (PLCG1), Protein Tyrosine Phosphatase non-Receptor Type 6 (PTPN6) and Mitogen-Activated Protein Kinase Kinase Kinase-7 (SLP-76) and Mitogen-Activated Protein Kinase14 (MAP3K7 or TAK1) were assayed using RT-qPCR. Statistical analyses were performed using PRISM and SPSS software. RESULTS: While there were no significant upregulation in CSK and PTPN6 in ACs compared to healthy individuals, expression levels of GSK3ß, MAP3K14, PLCG1, SLP-76, and TAK1 were significantly higher in ACs compared to healthy subjects which directly contributes to T-cell activation in the HTLV-1 ACs. CONCLUSION: HTLV-1 infection induces differential mRNA expressions in key proteins associated with T-cell activation. mRNAs related to T-cell activation showed significant upregulation compared to PTPN6 and CSK which contributed to T-cell regulation. Understanding these early molecular events in ACs may provide potential markers for disease progression and identify therapeutic targets for controlling viral replication and mitigating associated diseases. The study contributes novel insights to the limited literature on T-cell activation and HTLV-1 pathogenesis.

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.

Reinforcement learning-guided control strategies for CAR T-cell activation and expansion.

Reinforcement learning (RL), a subset of machine learning (ML), could optimize and control biomanufacturing processes, such as improved production of therapeutic cells. Here, the process of CAR T-cell activation by antigen-presenting beads and their subsequent expansion is formulated in silico. The simulation is used as an environment to train RL-agents to dynamically control the number of beads in culture to maximize the population of robust effector cells at the end of the culture. We make periodic decisions of incremental bead addition or complete removal. The simulation is designed to operate in OpenAI Gym, enabling testing of different environments, cell types, RL-agent algorithms, and state inputs to the RL-agent. RL-agent training is demonstrated with three different algorithms (PPO, A2C, and DQN), each sampling three different state input types (tabular, image, mixed); PPO-tabular performs best for this simulation environment. Using this approach, training of the RL-agent on different cell types is demonstrated, resulting in unique control strategies for each type. Sensitivity to input-noise (sensor performance), number of control step interventions, and advantages of pre-trained RL-agents are also evaluated. Therefore, we present an RL framework to maximize the population of robust effector cells in CAR T-cell therapy production.

Counterproductive effects of anti-CD38 and checkpoint inhibitor for the treatment of NK/T cell lymphoma.

Introduction: Natural killer/T cell lymphoma (NKTL) is an aggressive malignancy associated with poor prognosis. This is largely due to limited treatment options, especially for relapsed patients. Immunotherapies like immune checkpoint inhibitors (ICI) and anti-CD38 therapies have shown promising but variable clinical efficacies. Combining these therapies has been suggested to enhance efficacy. Methods: We conducted a case study on a relapsed NKTL patient treated sequentially with anti-CD38 followed by ICI (anti-PD1) using cytometry analyses. Results and Discussion: Our analysis showed an expected depletion of peripheral CD38+ B cells following anti-CD38 treatment. Further analysis indicated that circulating anti-CD38 retained their function for up to 13 weeks post-administration. Anti-PD1 treatment triggered re-activation and upregulation of CD38 on the T cells. Consequently, these anti-PD1-activated T cells were depleted by residual circulating anti-CD38, rendering the ICI treatment ineffective. Finally, a meta-analysis confirmed this counterproductive effect, showing a reduced efficacy in patients undergoing combination therapy. In conclusion, our findings demonstrate that sequential anti-CD38 followed by anti-PD1 therapy leads to a counterproductive outcome in NKTL patients. This suggests that the treatment sequence is antithetic and warrants re-evaluation for optimizing cancer immunotherapy strategies.