Effector Molecules Research Articles

Transforming Bacterial Pathogens into Wonder Tools in Cancer Immunotherapy.

Cancer immunotherapy has revolutionized cancer treatment due to its precise, target-specific approach compared to conventional therapies. However, treating solid tumors remains challenging as these tumors are inherently immunosuppressive, and their tumor microenvironment (TME) often limits therapeutic efficacy. Interestingly, certain bacterial species offer a promising alternative by exhibiting an innate ability to target and proliferate within tumor environments. Bacterial structural and functional components can activate innate and adaptive immune responses, creating tumor-suppressive conditions that reduce tumor mass. Additionally, bacteria can deliver effector molecules directly into tumor cells, inducing apoptotic and necrotic cell death. Despite their potential, the use of bacteria in cancer immunotherapy poses risks due to possible toxicities and unpredictable in vivo behavior. Advances in genetic engineering have addressed these concerns by enabling the development of attenuated bacterial strains with enhanced anticancer properties for safer medical applications. This review highlights the role of bacteria in TME modulation, recent strategies to bioengineer bacterial pathogens as therapeutic tools, and the synergistic effects of combining bacteria with other immunotherapies. It also discusses the challenges and prospects of translating this innovative approach into clinical practice, offering a comprehensive overview of bacteria-based cancer immunotherapy's potential to reshape the future of cancer treatment.

Mechanisms and optimization strategies for the occurrence of antibody-drug conjugate toxicity

Antibody-drug conjugates (ADCs) have emerged as a promising class of targeted cancer therapies, with 13 ADCs approved by the FDA and over 140 undergoing clinical research. ADCs are designed to enhance the selective delivery of cytotoxic drugs to tumor cells through high-affinity monoclonal antibodies (mAbs), improving the therapeutic index of the drug. Despite their potential, ADCs face significant challenges related to toxicity, including hematologic, ocular, skin, and neurological side effects, which limit their clinical application. This review provides an in-depth analysis of the mechanisms underlying ADC toxicity, which includes both targeted and off-target effects that can damage normal cells and tissues. Strategies for optimizing ADC safety, such as improving antibody structure, effector molecules, linkers, and combination therapies, are discussed. The review emphasizes the need for continued research to address these challenges and reduce off-target toxicity while enhancing the efficacy of ADCs, paving the way for the development of safer and more effective next-generation ADC drugs.

GZMK-expressing CD8+ T cells promote recurrent airway inflammatory diseases.

Inflammatory diseases are often chronic and recurrent, and current treatments do not typically remove underlying disease drivers1. T cells participate in a wide range of inflammatory diseases such as psoriasis2, Crohn's disease3, oesophagitis4 and multiple sclerosis5,6, and clonally expanded antigen-specific T cells may contribute to disease chronicity and recurrence, in part by forming persistent pathogenic memory. Chronic rhinosinusitis and asthma are inflammatory airway diseases that often present as comorbidities7. Chronic rhinosinusitis affects more than 10% of the general population8. Among these patients, 20-25% would develop nasal polyps, which often require repeated surgical resections owing to a high incidence of recurrence9. Whereas abundant T cells infiltrate the nasal polyps tissue10,11, T cell subsets that drive the disease pathology and promote recurrence are not fully understood. By comparing T cell repertoires in nasal polyp tissues obtained from consecutive surgeries, here we report that persistent CD8+ T cell clones carrying effector memory-like features colonize the mucosal tissue during disease recurrence, and these cells characteristically express the tryptase Granzyme K (GZMK). We find that GZMK cleaves many complement components, including C2, C3, C4 and C5, that collectively contribute to the activation of the complement cascade. GZMK-expressing CD8+ T cells participate in organized tertiary lymphoid structures, and tissue GZMK levels predict the disease severity and comorbidities better than well-established biomarkers such as eosinophilia and tissue interleukin-5. Using a mouse asthma model, we further show that GZMK-expressing CD8+ T cells exacerbate the disease in a manner dependent on the proteolytic activity of GZMK and complements. Genetic ablation or pharmacological inhibition of GZMK after the disease onset markedly alleviates tissue pathology and restores lung function. Our work identifies a pathogenic CD8+ memory T cell subset that promotes tissue inflammation and recurrent airway diseases by the effector molecule GZMK and suggests GZMK as a potential therapeutic target.

DAMPs, PAMPs, NLRs, RIGs, CLRs and TLRs - Understanding the Alphabet Soup in the Context of Bone Biology.

The purpose of this review is to summarize the current understanding of cell-autonomous innate immune pathways that contribute to bone homeostasis and disease. Germ-line encoded pattern recognition receptors (PRRs) are the first line of defense against danger and infections. In the bone microenvironment, PRRs and downstream signaling pathways, that mount immune defense, interface intimately with the core cellular processes in bone cells to alter bone formation and resorption. The role of PRR engagement on bone remodeling has been best described as a result of activated macrophages secreting effector molecules that reshape the characteristics of bone-resident cells. However, it is being increasingly recognized that local bone resident-cells like osteoclasts and osteoblasts possess an arsenal of PRRs. The engagement of these PRRs by stimuli in the bone niche can drive cell-autonomous (aka cell-intrinsic) responses that, in turn, impact bone-remodeling dramatically, irrespective of immune cell effectors. Indeed, this vital role for cell-autonomous innate immune responses is evident in how reduced PRR activity within osteoclast progenitors correlates with their reduced differentiation and abnormal bone remodeling. Further, cell-intrinsic PRR activity has now been shown to influence the behavior of osteoblasts, osteocytes and other local immune/non-immune cell populations. However, distinct PRR families have varying impact on bone homeostasis and inflammation, emphasizing the importance of investigating these different nodes of innate immune signaling in bone cells to better identify how they synergistically and/or antagonistically regulate bone remodeling in the course of an immune response. Innate immune sensing within bone resident cells is a critical determinant for bone remodeling in health and disease.

Kangfuxin solution alleviates esophageal stenosis after endoscopic submucosal dissection: A natural ingredient strategy.

Esophageal stricture ranks among the most significant complications following endoscopic submucosal dissection (ESD). Excessive fibrotic repair is a typical pathological feature leading to stenosis after ESD. To examine the effectiveness and underlying mechanism of Kangfuxin solution (KFX) in mitigating excessive fibrotic repair of the esophagus post-ESD. Pigs received KFX at 0.74 mL/kg/d for 21 days after esophageal full circumferential ESD. Endoscopic examinations occurred on days 7 and 21 post-ESD. In vitro, recombinant transforming growth factor (TGF)-β1 (5 ng/mL) induced a fibrotic microenvironment in primary esophageal fibroblasts (pEsF). After 24 hours of KFX treatment (at 1.5%, 1%, and 0.5%), expression of α-smooth muscle actin-2 (ACTA2), fibronectin (FN), and type collagen I was assessed. Profibrotic signaling was analyzed, including TGF-β1, Smad2/3, and phosphor-smad2/3 (p-Smad2/3). Compared to the Control group, the groups treated with KFX and prednisolone exhibited reduced esophageal stenosis, lower weight loss rates, and improved food tolerance 21 d after ESD. After treatment, Masson staining revealed thinner and less dense collagen fibers in the submucosal layer. Additionally, the expression of fibrotic effector molecules was notably inhibited. Mechanistically, KFX downregulated the transduction levels of fibrotic functional molecules such as TGF-β1, Smad2/3, and p-Smad2/3. In vitro, pEsF exposed to TGF-β1-induced fibrotic microenvironment displayed increased fibrotic activity, which was reversed by KFX treatment, leading to reduced activation of ACTA2, FN, and collagen I. The 1.5% KFX treatment group showed decreased expression of p-Smad 2/3 in TGF-β1-activated pEsF. KFX showed promise as a therapeutic option for post-full circumferential esophageal ESD strictures, potentially by suppressing fibroblast fibrotic activity through modulation of the TGF-β1/Smads signaling pathway.

Using environment-sensitive tetramethylated thiophene-BODIPY fluorophores in DNA probes for studying effector-induced conformational changes of protein-DNA complexes.

The LutR protein represses the transcription of genes encoding enzymes for the utilization of l-lactate in Bacillus subtilis through binding to a specific DNA region. In this study, we employed oligonucleotide probes modified by viscosity-sensitive tetramethylated thiophene-BODIPY fluorophores to investigate the impact of selected metabolites on the LutR-DNA complex. Our goal was to identify the effector molecule whose binding alters the protein-DNA affinity, thereby enabling gene transcription. The designed DNA probes exhibited distinctive responses to the binding and release of the protein, characterized by significant alterations in fluorescence lifetime. Through this method, we have identified l-lactate as the sole metabolite exerting a substantial modulating effect on the protein-DNA interaction and thus confirmed its role as an effector molecule. Moreover, we showed that our approach was able to follow conformation changes affecting affinity, which were not captured by other methods commonly used to study the protein-DNA interaction, such as electro-mobility shift assays and florescence anisotropy binding studies. This work underlines the potential of environment-sensitive fluorophore-linked nucleotide modifications, i.e. dCTBdp, for studying the dynamics and subtle changes of protein-DNA interactions.

The Emerging Role of Schwann Cells in the Tumor Immune Microenvironment and Its Potential Clinical Application.

As the primary glial cells in the peripheral nervous system (PNS), Schwann cells (SCs) have been proven to influence the behavior of cancer cells profoundly and are involved in cancer progression through extensive interactions with cancer cells and other stromal cells. Indeed, the tumor microenvironment (TME) is a critical factor that can significantly limit the efficacy of immunotherapeutic approaches. The TME promotes tumor progression in part by reshaping an immunosuppressive state. The immunosuppressive TME is the result of the crosstalk between the tumor cells and the different immune cell subsets, including macrophages, natural killer (NK) cells, dendritic cells (DCs), lymphocytes, myeloid-derived suppressor cells (MDSCs), etc. They are closely related to the anti-tumor immune status and the clinical prognosis of cancer patients. Increasing research demonstrates that SCs influence these immune cells and reshape the formation of the immunosuppressive TME via the secretion of various cytokines, chemokines, and other effector molecules, eventually facilitating immune evasion and tumor progression. In this review, we summarize the SC reprogramming in TME, the emerging role of SCs in tumor immune microenvironment, and the underlying mechanisms involved. We also discuss the possible therapeutic strategies to selectively target SCs, providing insights and perspectives for future research and clinical studies involving SC-targeted treatment.

Engineering Adhesion of the Probiotic Strain Escherichia coli Nissle to the Fungal Pathogen Candida albicans.

Engineering live biotherapeutic products against fungal pathogens such as Candida albicans has been suggested as a means to tackle the increasing threat of fungal infections and the development of resistance to classical antifungal treatments. One important challenge in the design of live therapeutics is to control their localization inside the human body. The specific binding capability to target organisms or tissues would greatly increase their effectiveness by increasing the local concentration of effector molecules at the site of infection. In this study, we utilized surface display of carbohydrate binding domains to enable the probiotic E. coli Nissle 1917 to adhere specifically to the pathogenic yeast Candida albicans. Binding was quantified using a newly developed method based on the automated analysis of microscopic images. In addition to a rationally selected chitin binding domain, a synthetic peptide of identical length but distinct sequence also conferred binding. Efficient binding was specific to fungal hyphae, the invasive form of C. albicans, while the yeast form, as well as abiotic cellulose and PET particles, was only weakly recognized.

CD69+CD103+CD8+ tissue-resident memory T cells possess stronger anti-tumor activity and predict better prognosis in colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most prevalent cancers worldwide. Despite advancements in therapeutic methodologies, it still causes a high rate of patient mortality. CD8+ tissue-resident memory T (TRM) cells are strategically positioned to mediate effective anti-tumor responses. However, the characteristic surface molecules and functions of CD8+ TRM cells exhibit significant heterogeneity.MethodsThe roles and anti-tumor biological functions of different CD8+ TRM subsets in CRC were determined by clinical CRC samples, bioinformatics analysis, and in vitro experiments including co-culture experiments and transwell migration assays. The signaling pathways that synergistically regulate the differentiation of CD8+ TRM cells were identified by in vitro CD8+ T cell activation and inhibition assays, and the functioning transcription factors were predicted using the UCSC and JASPAR databases.ResultsWe found that different CD8+ TRM subsets existed in CRC tumor tissues, which were identified as CD69−CD103−CD8+ TRM, CD69+CD103−CD8+ TRM (SP CD8+ TRM), and CD69+CD103+CD8+ TRM (DP CD8+ TRM) subsets. Compared with SP CD8+ TRM cells, increased infiltration of DP CD8+ TRM cells predicted better prognosis and played a protective role mainly in tumor invasion and lymph node metastasis of CRC. DP CD8+ TRM cells expressed higher levels of effector molecules and exerted stronger anti-tumor effects in a FAS/FASL pathway-dependent manner. Additionally, DP CD8+ TRM cells secreted higher levels of CXCL13 and recruited B cells into tumor tissues through the CXCL13/CXCR5 signaling axis to form tertiary lymphoid structures, participating in anti-tumor immune responses. Notch and TGF-β signaling pathways synergistically regulate the differentiation of DP CD8+ TRM cells.ConclusionsWe clarified the roles and mechanisms of different CD8+ TRM subsets in CRC and identified that DP CD8+ TRM cells exert stronger anti-tumor effects and predict better prognosis, which provides ideas for developing new clinically available therapeutic targets.

Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

Toxoplasma gondii, an obligate intracellular parasite, control its host cell cycle through mechanisms that are not fully understood. Key effector molecules, including MYR1 and HCE1, play roles in translocating parasite proteins and inducing host cellular cyclin E1 overexpression, respectively. We investigated the early role of MYR1- and HCE1-driven host cell cycle arrest and DNA damage (up to 3 h p.i.). Our findings showed that T. gondii-infected cells experienced S-phase arrest and displayed double-strand DNA breaks as soon as 15 min p.i. This condition persisted until 3 h p.i., at which point we also observed increased host cell binucleation and micronuclei formation, both hallmarks of genomic instability. Furthermore, host cells responded to DNA damage by activating the ATM branch of the homologous recombination repair pathway. MYR1 was shown to be crucial, as TgΔmyr1 tachyzoites failed to induce S-phase arrest and DNA damage foci. In contrast, the absence of HCE1 did not produce these effects, suggesting that cyclin E1 expression was not involved. Also, DNA damage was demonstrated to be ROS-independent, suggesting that ROS did not trigger DNA damage. Our results suggest that T. gondii compromises host cellular DNA integrity depending on MYR1 shortly after infection, maintaining it over time.

Activation of the tick Toll pathway to control infection of Ixodes ricinus by the apicomplexan parasite Babesia microti.

The vector competence of blood-feeding arthropods is influenced by the interaction between pathogens and the immune system of the vector. The Toll and IMD (immune deficiency) signaling pathways play a key role in the regulation of innate immunity in both the Drosophila model and blood-feeding insects. However, in ticks (chelicerates), immune determination for pathogen acquisition and transmission has not yet been fully explored. Here, we have mapped homologs of insect Toll and IMD pathways in the European tick Ixodes ricinus, an important vector of human and animal diseases. We show that most genes of the Toll pathway are well conserved, whereas the IMD pathway has been greatly reduced. We therefore investigated the functions of the individual components of the tick Toll pathway and found that, unlike in Drosophila, it was specifically activated by Gram-negative bacteria. The activation of pathway induced the expression of defensin (defIR), the first identified downstream effector gene of the tick Toll pathway. Borrelia, an atypical bacterium and causative agent of Lyme borreliosis, bypassed Toll-mediated recognition in I. ricinus and also resisted systemic effector molecules when the Toll pathway was activated by silencing its repressor cactus via RNA interference. Babesia, an apicomplexan parasite, also avoided Toll-mediated recognition. Strikingly, unlike Borrelia, the number of Babesia parasites reaching the salivary glands during tick infection was significantly reduced by knocking down cactus. The simultaneous silencing of cactus and dorsal resulted in greater infections and underscored the importance of tick immunity in regulating parasite infections in these important disease vectors.

Functional Analysis of KPvRxLR27, a Novel Plasmopara viticola Effector from a Korean Isolate, and Its Role in Hypersensitive Response.

Plasmopara viticola causes grape downy mildew, one of the most notorious diseases of cultivated grapes that damage vineyards worldwide. The pathogen secretes various effector molecules to infect and modulate the host biological processes. In this study, we aimed to evaluate the roles of KPvRxLR27, an arginine-any amino acid-leucine-arginine (RxLR) effector isolated from P. viticola JN-9 from Jeonju (South Korea) with respect to the reported Bcl-2-associated X and inverted formin1in inducing cell death in non-host Nicotiana benthamiana and resistant grape host cultivars via Agrobacterium-mediated transient transformation. We found that, KPvRxLR27 induced programmed cell death in N. benthamiana and rapid hypersensitive response in resistant grape cultivars. Agroinfiltration assay revealed that putative N-glycosylation at the N186 amino acid sequence and nuclear localization signal motifs at the C-terminus were critical for the effector's cell death-inducing activity of KPvRxLR27. Overexpression assay revealed that KPvRxLR27 was abundantly expressed in the plasma membrane and nuclear regions and activated the accumulation of reactive oxygen species in N. benthamiana. Moreover, KPvRxLR27 expression was significantly delayed in the resistant cultivar than in the susceptible cultivar. Our results suggest KPvRxLR27 as a potential avirulence gene recognized by the host receptors to activate the host immune response-associated genes, providing valuable insights to enhance the pathogen resistance of commercial cultivars.

Post-translational digital data encoding into the genomes of mammalian cell populations.

High resolution cellular signal encoding is critical for better understanding of complex biological phenomena. DNA-based biosignal encoders alter genomic or plasmid DNA in a signal dependent manner. Current approaches involve the signal of interest affecting a DNA edit by interacting with a signal specific promoter which then results in expression of the effector molecule (DNA altering enzyme). Here, we present the proof of concept of a biosignal encoding system where the enzyme terminal deoxynucleotidyl transferase (TdT) acts as the effector molecule upon directly interacting with the signal of interest. A template independent DNA polymerase (DNAp), TdT incorporates nucleotides at the 3' OH ends of DNA substrate in a signal dependent manner. By employing CRISPR-Cas9 to create double stranded breaks in genomic DNA, we make 3'OH ends available to act as substrate for TdT. We show that this system can successfully resolve and encode different concentrations of various biosignals into the genomic DNA of HEK-293T cells. Finally, we develop a simple encoding scheme associated with the tested biosignals and encode the message "HELLO WORLD" into the genomic DNA of HEK-293T cells at a population level with 91% accuracy. This work demonstrates a simple and engineerable system that can reliably store local biosignal information into the genomes of mammalian cell populations.

The Sdp-SH3b2 domain contained in Lactobacillus johnsonii N6.2-derived extracellular vesicles inhibit murine norovirus replication.

The internalization of Lactobacillus johnsonii N6.2 extracellular vesicles (EVs) by cells results in a significant induction of the 2',5'-oligoadenylate synthetase (OAS) pathway. It also induces expression of IFI44L, MX1, MX2 and DDX60. In this work, we evaluated whether the antiviral response induced by L. johnsonii N6.2-derived EVs, has an inhibitory effect on an RNA viral insult using murine norovirus (MNV-1) as the viral infection model. We found that RAW 264.7 Macrophages treated with EVs significantly decreased the levels of MNV-1 genome. These results were consistent with an increase in expression of Oas1b, Oas2, Oasl, Mx1, Mx2 and Ifi44l (6 hours post infection). Out of six proteins enriched in EVs, we found that SH3b2 domain of Sdp was the only protein effector molecule able to recapitulate the activation of the OAS pathway. In C57BL6 mice, the administration of live L. johnsonii N6.2, EVs, and Sdp-SH3b2/liposomes significantly decreased MNV-1 titers in the distal ileum, in contrast to the controls with PBS and liposomes alone that did not affect MNV-1. These results establish that the SH3b2 domain of Sdp, which is enriched in L. johnsonii derived EVs, is an effector molecule in EVs that can orchestrate the control of viral infections in vivo.

Stressor-Actuated Proximity Labeling for Reporting Cellular Interaction.

Cell-cell interactions determine the activation state and function of cells. When host cells are exposed to stressors such as microorganisms, immune defense machinery is activated to release H2O2, providing direct evidence of the relevant cellular physiological processes. Inspired by the fact that peroxidase can catalyze proximity labeling in the presence of exogenous H2O2, a stressor-actuated proximity labeling (SAPL) strategy is developed to report the process information on cell-cell interactions by recording stress levels. The stressors are covalently modified with horseradish peroxidase (HRP) and the H2O2 released by the host cells in response to the stressors triggers HRP-based proximity labeling. Using a fungal mimic or live fungi as stressors, the stress levels of different host cells are compared by in situ imaging of the labeling signals. The ability to accumulate stress signals allows SAPL to more sensitively differentiate between interactions involving different macrophage phenotypes. SAPL is also a powerful tool for real-time, in situ monitoring of the effects of surface modifications on cellular interactions. Thus, the SAPL strategy represents a new perspective in the monitoring of cell-cell interactions using endogenous effector molecules.

GrB-Fc-KS49, an anti-EMP2 granzyme B fusion protein therapeutic alters immune cell infiltration and suppresses breast cancer growth

BackgroundGranzyme B (GrB) is a key effector molecule, delivered by cytotoxic T lymphocytes and natural killer cells during immune surveillance to induce cell death. Fusion proteins and immunoconjugates represent an...

Integrating T-cell inflammation features for prognosis in hepatocellular carcinoma: a novel predictive model.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death globally and accounts for 75% to 90% of primary liver cancer cases. The high mortality rate of HCC, coupled with the absence of reliable prognostic biomarkers, makes its treatment and prognosis evaluation challenging. The features of the T cell-inflamed microenvironment include active interferon (IFN)-γ signaling and the presence of cytotoxic effector molecules, antigen presentation, and T-cell activating cytokines. Although these features are closely associated with anticancer immunity, their specific roles in HCC remain unclear. This study aimed to investigate the role and prognostic significance of T-cell inflammation (TCI) in HCC patients, providing new insights for clinical diagnosis and treatment strategies. We integrated single-sample gene set enrichment analysis (ssGSEA) and weighted gene coexpression network analysis (WGCNA) to identify the genes associated with TCI at both the single-cell and bulk-transcriptome levels. The HCC TCI-related score (HTCIRS) was developed and assessed with 10 different machine learning algorithms and their combinations, which was followed by validation of the key gene KLF2 in clinical samples and tissue microarrays (TMAs). We identified 65 genes associated with TCI, of which 36 were significantly correlated with overall survival (OS). The HTCIRS demonstrated excellent performance in prognostic prediction, revealing differences in biological functions and immune cell infiltration between different risk groups within the tumor microenvironment (TME). Furthermore, KLF2 was identified to be linked to the prognosis of patients with HCC. The TCI-related score proposed in this study serves as an important tool for prognostic prediction and personalized treatment of patients with HCC, with KLF2 emerging as a potential biomarker for predicting the prognosis of patients with HCC.

Convergence of plant sterols and host eukaryotic cell-derived defensive lipids at the infectious pathogen-host interface.

Plant sterols (PSs) exhibit intrinsic functions such as antibacterial effects. Their effects simultaneously on both host-mediated and bacteria-mediated pathogenesis are not yet fully understood. We hypothesized that when absorptive cells, defensive cells and detoxer cells are cultured together, their convergent response to an infectious pathogen depends on the molecular mimicry between the ingested sterols and their own defensive lipids. A human triple cell co-culture model incorporating colonocytes, macrophages, and hepatocytes was established. Cocultures were stimulated with Klebsiella pneumoniae 52145 (Kp52145) in the presence of pure plant sterol (β-sitosterol, PS) for 6h. Changes in the structural health markers of the stimulated cocultured cells and their immune response and biochemical markers of pathogenicity were determined. PS significantly inhibited the secretion of cytokines induced by Kp52145. Cell viability was higher in the Kp52145+PS group compared to the Kp52145 alone group. PS decreased Kp52145-induced marker of pathogenicity (SOD), accompanied by reduced levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), mannose binding lectin (MBL), and pentraxin 3 (PTX3) which are the mediators and enzymes associated with the inflammatory response to an infectious-inflamed milieu. PS recovered Kp52145-decreased peroxidase (POX), catalase (CAT), complement component 3 (C3), and high-density lipoprotein cholesterol (HDL-C) values. Convergence of ingested plant sterols and host eukaryotic cell-derived defensive lipids mitigates the disruptive effects of bacterial toxic effector molecules. Structural or immunological similarities (molecular mimicry) between ingested plant sterols and host defensive lipids play an important role in modulating bacterial signalling that occurs at the pathogen-host interface and in the mitigation of infection- and inflammation-driven pathological processes.

Hypoimmunogenic CD19 CAR-NK cells derived from embryonic stem cells suppress the progression of human B-cell malignancies in xenograft animals.

Chimeric antigen receptor (CAR) engineered natural killer (NK) cells exhibit advantages such as MHC-independent recognition and strong anti-tumor functions. However, allogeneic CAR-NK cells derived from human tissues are heterogeneous and susceptible to clearance by hosts. We generated a B2M knockout, HLA-E and CD19 CAR ectopic expressing embryonic stem cell (ESC) line, which differentiated normally and gave rise to homogeneous CD19 CAR-NK (CD19 CAR-UiNK) cells using an organoid aggregate induction method. The CD19 CAR-UiNK were co-cultured with T cells or NK cells derived from peripheral blood mononuclear cells (PBMC) with the mismatched HLA to evaluate the immunogenicity of CD19 CAR-UiNK cells. We further assessed the therapeutic effects of CD19 CAR-UiNK cells on CD19+ tumor cells through in vitro cytotoxicity assays and in vivo animal models. The CD19 CAR-UiNK cells exhibited typical expression patterns of activating and inhibitory receptors, and crucial effector molecules of NK cells, similar to those of unmodified NK cells. In co-culture assays, the CD19 CAR-UiNK cells evaded allogeneic T cell response and suppressed allogeneic NK cell response. Functionally, the CD19 CAR-UiNK cells robustly secreted IFN-γ and TNF-α, and upregulated CD107a upon stimulation with Nalm-6 tumor cells. The CD19 CAR-UiNK cells effectively eliminated CD19+ tumor cells in vitro, including B-cell cancer cell lines and primary tumor cells from human B-cell leukemia and lymphoma. Further, the CD19 CAR-UiNK cells exhibited strong anti-tumor activity in xenograft animals. We offer a strategy for deriving homogeneous and hypoimmunogenic CD19 CAR-iNK cells with robust anti-tumor effects from ESCs. Our study has significant implications for developing hypoimmunogenic CD19 CAR-NK cell therapy using human ESC as an unlimited cell source.

The HSP90 Inhibitor Pimitespib Targets Regulatory T Cells in the Tumor Microenvironment.

Regulatory T (Treg) cells play key roles in cancer immunity by suppressing a range of antitumor immune responses and contributing to resistance to programmed death (PD)-1 blockade therapy. Given their critical roles in self-tolerance, local control of immunosuppression by Treg cells, such as in the tumor microenvironment (TME), has been intensively studied. Inhibition of heat shock protein 90 (HSP90), a chaperone with vital roles in regulating proteostasis in cancer cells, impedes cancer progression by interrupting oncogenic signaling pathways and potentially modulating antitumor immunity, but we have very little mechanistic insight into these immune modulatory effects. Here, we show that the number of Treg cells are selectively reduced by the HSP90 inhibitor pimitespib in animal models and patients with gastric cancer in a clinical trial (EPOC1704). Pimitespib reduced the highly immunosuppressive human FOXP3high effector Treg cells by inhibiting their proliferation and decreasing their expression of effector molecules, which improved the priming and activation of antigen-specific CD8+ T cells. Mechanistic studies revealed that pimitespib selectively degraded STAT5, a key transducer of the IL-2 signaling pathway, which is essential for Treg cell development and maintenance, and consequently compromised FOXP3 expression, leading to selective impairment of immunosuppression in the TME by Treg cells. Thus, pimitespib treatment combined with PD-1 blockade exhibited a far stronger antitumor effect than either treatment alone in animal models. Through these data, we propose that HSP90 inhibition is a promising therapeutic option for Treg cell-targeted cancer immunotherapy.