Responsiveness Of Cells Research Articles

Bacteria-derived nanovesicles enhance tumour vaccination by trained immunity.

Trained immunity enhances the responsiveness of immune cells to subsequent infections or vaccinations. Here we demonstrate that pre-vaccination with bacteria-derived outer-membrane vesicles, which contain large amounts of pathogen-associated molecular patterns, can be used to potentiate, and enhance, tumour vaccination by trained immunity. Intraperitoneal administration of these outer-membrane vesicles to mice activates inflammasome signalling pathways and induces interleukin-1β secretion. The elevated interleukin-1β increases the generation of antigen-presenting cell progenitors. This results in increased immune response when tumour antigens are delivered, and increases tumour-antigen-specific T-cell activation. This trained immunity increased protection from tumour challenge in two distinct cancer models.

Leonurine ameliorates the STAT3 pathway through the upregulation of SHP-1 to retard the growth of hepatocellular carcinoma cells

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that directs the transcription of genes involved in the promotion of cell survival and proliferation, inflammation, angiogenesis, invasion, and migration. Overactivation of STAT3 is often witnessed in human cancers, thereby making it a good target in oncology. Herein the efficacy of Leonurine (Leo), a bioactive alkaloid present in Herba leonuri, was investigated for its STAT3-inhibitory potential in hepatocellular carcinoma (HCC) cells. Leo downregulated the persistent as well as IL-6-driven activation of STAT3. Leo abrogated the nuclear localization and DNA interacting ability of STAT3. Leo was also found to impart STAT3 inhibition by mitigating the activation of upstream kinases such as JAK1, JAK2, and Src both in constitutive and IL-6 inducible systems. Leo curbed the STAT3-driven luciferase gene expression and the depletion of STAT3 resulted in the reduced responsiveness of HCC cells to Leo. Pervanadate exposure counteracted Leo-induced STAT3 inhibition suggesting the involvement of a protein tyrosine phosphatase. SHP-1 was significantly elevated upon Leo exposure whereas the depletion of SHP-1 was found to revert the effect of Leo on STAT3. Leo induced apoptosis and also significantly potentiated the cytotoxic effect of paclitaxel, doxorubicin, and sorafenib. Leo was found to be non-toxic up to the dose of 10 mg/kg in NCr nude mice. In conclusion, Leo was demonstrated to induce cytotoxicity in HCC cells by mitigating the persistent of activation of STAT3 pathway.

IL-15-dependent immune crosstalk between natural killer cells and dendritic cells in HIV-1 elite controllers

As the principal effector cell population of the innate immune system, natural killer (NK) cells may make critical contributions to natural, immune-mediated control of HIV-1 replication. Using genome-wide assessments of activating and inhibitory chromatin features, we demonstrate here that cytotoxic NK (cNK) cells from elite controllers (ECs) display elevated activating histone modifications at the interleukin 2 (IL-2)/IL-15 receptor β chain and the BCL2 gene loci. These histone changes translate into increased responsiveness of cNK cells to paracrine IL-15 secretion, which coincides with higher levels of IL-15 transcription by myeloid dendritic cells in ECs. The distinct immune crosstalk between these innate immune cell populations results in improved IL-15-dependent cNK cell survival and cytotoxicity, paired with a metabolic profile biased toward IL-15-mediated glycolytic activities. Together, these results suggest that cNK cells from ECs display a programmed IL-15 response signature and support the emerging role of innate immune pathways in natural, drug-free control of HIV-1.

Cellular Dynamics Following CAR T Cell Therapy Are Associated with Response, Resistance and Cytokine Release Syndrome in Relapsed/Refractory Multiple Myeloma

Introduction The introduction of B cell maturation antigen (BCMA)-targeting chimeric antigen receptor (CAR) T cells revolutionized the treatment of relapsed/refractory multiple myeloma (RRMM). While long-term follow-up of CD19 CAR T cells demonstrated a connection between CAR T cell expansion and persistence with outcome, very little is known about persistence and effects on bystander cells of BCMA-targeting CAR T cells. Methods We analyzed 27 RRMM patients (pts) treated with Idecabtagene vicleucel (Ide-cel) at our center. Pts were grouped based on their remission evaluated 100 days after infusion into responders (partial response or better, n=16) or non-responders (progressive disease, n=11). Peripheral blood (pb) was drawn at day of leukapheresis, at day of infusion/after lymphodepletion (LD, d0) and at several time points following Ide-cel infusion (d7, d14, d30 and d100). In order to reflect a comprehensive picture of the longitudinal dynamics of CAR and non-CAR T cells, we subjected pb mononuclear cells to advanced flow cytometry. Additionally, we assessed the impact of cellular dynamics on the occurrence of cytokine release syndrome (CRS), tocilizumab application and the incidence of cytopenia. Results The median number of infused CAR T cells was 430x10 6 (range: 248.8 - 489.1x10 6) with one exception (25.7x10 6). There was no correlation between the median number of infused cells and in vivo expansion of CAR T cells after infusion as well as response or the development of CRS or cytopenia. Significant differences between responders and non-responders were already detected on day of leukapheresis: While non-responders showed similar numbers of CD4+ and CD8+ T cells, responders had significantly higher CD8+ T cell counts as compared to CD4+ T cells (p<0.05, Figure 1). Additionally, significantly reduced percentages of effector memory CD8+ T cells were detected in responders as compared to non-responders. The latter group showed significantly increased numbers of CD3+CD8+ T cells at the day of infusion indicating a less effective LD in non-responders. Following CAR T cell infusion, peak expansion occurred two weeks after infusion and was significantly higher in responders as compared to non-responders (p<0.001, Figure 2). CD3+ CAR T cell compartment predominantly comprised of CD8+ T cells regardless of response (median: 85%). Analysis of the distribution of CAR and non-CAR T cells after infusion revealed that the majority of CD4+ T cells in responders and non-responders were untransfected. Similar results were detected for CD8+ T cells in non-responders. However, in responders CD8+ CAR T cells approximated CD8+ non-CAR T cells with no significant differences on d7 and d14. With regard to T cell differentiation, almost the entire CD4+ T cells exhibited either a central memory or effector memory phenotype after CAR T cell infusion. Shortly after CAR T cell infusion, effector memory CD8+ T cells outbalanced all other CD8+ T cell subtypes. Most pts had no detectable CAR T cells 100 days after infusion, regardless of remission. Overall, 22 pts (81%) developed CRS shortly after CAR T cell infusion and 7 of these (32%) required tocilizumab treatment. Whereas percentages of CD3+ CAR T cells were significantly elevated in both CRS positive groups (p<0.05) shortly after infusion, absolute numbers were significantly increased only in CRS positive pts without tocilizumab. Analysis of blood counts following CAR T cell infusion revealed that cytopenias persisted longer in CRS pts treated with tocilizumab, which was also reflected by significantly increased numbers of days below thresholds for all cell lines (anemia, thrombocytopenia and leukopenia, p<0.01). Even though the occurrence of CRS and especially the treatment with tocilizumab affected blood cell composition, it did not correlate to response. Conclusion We demonstrate that BCMA-targeting CAR T cells only transiently persist in pb after infusion. Still, initial CAR T cell expansion, predominantly caused by effector memory CD8+ CAR T cells, was linked to response and CRS in RRMM pts. A correlation between CRS and tocilizumab administration with prolonged cytopenias following CAR T cell infusion was also observed. Our data provide first evidence that responders and non-responders can early be distinguished by differential cellular composition in CAR and non-CAR T cell compartments with distinct features already present at day of apheresis.

Mitochondrial Fitness and Immune Function of Natural Killer Cells Are Impaired By the Multiple Myeloma (MM) Tumor Cells: The Role of MM Microenvironment Interactions

The immune system including Natural Killer (NK) activity was shown to be impaired in active MM. We and others have previously demonstrated the important role of the bone marrow (BM) microenvironment in MM pathophysiology, however, data on its effects on NK activity in MM is limited. We first assessed the phenotype of the BM-derived NK cells. BM-NK from MM pts (n=10) showed an exhausted phenotypic signature with significantly reduced expression of CD16 (p<0.01), and increased expression of exhaustion markers CD39 (p<0.001) and CD57 (p<0.001) compared to BM-NK cells from healthy controls (n=8). Furthermore, MM-BM NK cells demonstrated reduced mitochondrial membrane potential, suggesting impaired mitochondrial fitness. The dysfunction of the mitochondria led to deficiency of ATP synthesis; mitochondrial ATP levels were reduced in NK cells from MM BM samples in comparison to the normal BMs (p<0.001). Next, the functional response of BM NK cells to the stimulation with IL-2 (2000 IU/ml for 7 and 14 days) was evaluated in vitro. NK cells from MM BM demonstrated reduced proliferation capacity, increased CD39, reduced expression of activating receptors NKp44 and NKp30, and reduced mitochondrial membrane potential upon the activation with IL-2. The impaired mitochondrial fitness correlated with reduced levels of NKp44, indicating a potential link between the decreased mitochondrial activity and the reduced functionality of NK cells. Indeed, in vitro, the cytotoxic activity of IL-2-stimulated MM-BM NK cells against MM cell lines was significantly diminished compared to healthy IL-2-activated BM-NK cells. Glucose and fatty acids (FA) are key substrates of the mitochondrial bioenergetic pathways and essential for NK cell activation. Of note, glucose uptake was increased comparably upon IL-2 stimulation in NK cells from both MM patients and healthy donors. In contrast, MM-BM NK cells demonstrated reduced FA uptake in response to IL-2 stimulation compared to BM NK cells from healthy donors. These data indicate impaired mitochondrial and NK cell properties in the BM MM niche. Next, we established an in vitro model to interrogate the relationship between NK cell mitochondrial and immune dysfunction within the MM BM microenvironment. Specifically, we cultured MM cell lines (n=5) with the NK92 cell line or primary IL-2-activated or not activated blood-derived NK cells, and assessed NK cell mitochondrial fitness, metabolic activity, and immune functional properties. Co-culture with MM cells significantly impaired mitochondrial mass and mitochondrial membrane potential in NK cells, decreased mitochondrial ATP levels, while upregulating MM mitochondrial ROS in NK cells, indicating that MM cells induce mitochondrial damage and impair ATP synthesis in NK cells. Mitochondrial fitness and energy production are pivotal for NK cell persistence and cytotoxicity. In accordance, direct interaction with MM cells suppressed NK cell proliferation in vitro, inducing G0/G1 cell cycle arrest. Moreover, MM-induced mitochondrial impairment in NK cells correlated with reduced responsiveness of MM cells to NK-induced killing, while short-term exposure to MM cells significantly diminished the anti-myeloma cytotoxic activity of NK cells. Accordingly, CD39 expression was upregulated, while NKp44 levels significantly decreased in NK cells upon interaction with MM cells. To further characterize the molecular mechanisms associated with MM-mediated NK cell mitochondrial impairment, we assessed the gene expression profile of MM-exposed sorted NK92 cells versus unexposed cells. A distinct metabolic signature characterized by reduced expression of genes involved in the mitochondrial activity was detected in MM-exposed NK cells. Thus, genes involved in respiratory activity and mitochondrial dynamics were downregulated post-MM exposure. Furthermore, a profound increase in fructose-1,6-biphosphatase 1 (FBP1) expression was detected in MM-exposed NK cells. FBP1 is the rate-limiting enzyme for gluconeogenesis that simultaneously inhibits glycolysis, possibly interfering with NK cell metabolic activity and immune function. Altogether, our data suggest that MM BM microenvironment and direct interactions with malignant plasma cells impair NK mitochondrial fitness, diminishing energy production, increasing oxidative stress, and driving exhaustion with reduced anti-tumor capacity in NK cells.

TIGIT+ NK cells in combination with specific gut microbiota features predict response to checkpoint inhibitor therapy in melanoma patients

BackgroundComposition of the intestinal microbiota has been correlated to therapeutic efficacy of immune checkpoint inhibitors (ICI) in various cancer entities including melanoma. Prediction of the outcome of such therapy, however, is still unavailable. This prospective, non-interventional study was conducted in order to achieve an integrated assessment of the connection between a specific intestinal microbiota profile and antitumor immune response to immune checkpoint inhibitor therapy (anti-PD-1 and/or anti-CTLA-4) in melanoma patients.MethodsWe assessed blood and stool samples of 29 cutaneous melanoma patients who received immune checkpoint inhibitor therapy. For functional and phenotypical immune analysis, 12-color flow cytometry and FluoroSpot assays were conducted. Gut microbiome was analyzed with shotgun metagenomics sequencing. To combine clinical, microbiome and immune variables, we applied the Random Forest algorithm.ResultsA total of 29 patients was analyzed in this study, among whom 51.7% (n = 15) reached a durable clinical benefit. The Immune receptor TIGIT is significantly upregulated in T cells (p = 0.0139) and CD56high NK cells (p = 0.0037) of responders. Several bacterial taxa were associated with response (e.g. Ruminococcus torques) or failure (e.g. Barnesiella intestinihominis) to immune therapy. A combination of two microbiome features (Barnesiella intestinihominis and the Enterobacteriaceae family) and one immune feature (TIGIT+ CD56high NK cells) was able to predict response to ICI already at baseline (AUC = 0.85; 95% CI: 0.841–0.853).ConclusionsOur results reconfirm a link between intestinal microbiota and response to ICI therapy in melanoma patients and furthermore point to TIGIT as a promising target for future immunotherapies.

Novel Pi3kδ Inhibitor Roginolisib Synergizes with the Bcl-2 Inhibitor Venetoclax in Hematological Malignancies

Roginolisib (IOA-244) is a first in class allosteric modulator and non-ATP competitive, selective PI3Kδ inhibitor currently in a Phase 1 clinical study in lymphoma and solid tumors. Initial structural and biochemical studies identified unique chemical structure, excellent selectivity, and excellent PK properties for roginolisib as compared to earlier PI3Kδ inhibitors, while the clinical trial has demonstrated a favorable safety profile. Consistent with prior PI3Kδ inhibitors, roginolisib inhibits the in vitro growth of lymphoma cells. In contrast to other PI3Kδ inhibitors, roginolisib activity is correlated with the expression levels of PIK3CD, suggesting specific and on target cancer cell-intrinsic effects (PMID: 37066023). To identify rational combination strategies, initial pharmacological screening of 474 compounds in combination with roginolisib was carried out using a MTT assay in the lymphoma cell lines HH (cutaneous T cell lymphoma, CTCL) and SP-53 (mantle cell lymphoma, MCL). Initial screening identified that roginolisib synergized with the bcl-2 inhibitor venetoclax in the HH cell line with a combination index value (CI) of 0.003 by the Chou-Talalay method. To further validate this finding, the combination of roginolisib with venetoclax or with another bcl-2 inhibitor S55746 was tested in a broad range of lymphoma cell lines including GRANTA519, JVM2, SP49 (MCL); FARAGE, TMD8 (diffuse large B cell lymphoma); MEC1 (chronic lymphocytic leukemia); MJ (cutaneous T cell lymphoma); and YT (NK lymphoma). The combination showed synergism in all the cell lines (CI 0.81 - 0.05) except SP49 (CI- 1.3). Similarly, the combination of roginolisib with S55746 showed synergism in most of the cell lines (CI- 0.46 - 0.089), additive effect in SP49 (CI- 1) and antagonistic effect in GRANTA519 (CI- 1.3) and FARAGE (CI- 2.17). To assess the tumor-intrinsic effect of roginolisib in patient derived CLL cells, PBMCs isolated from CLL patients responding to prior BTK inhibitor (i) (n=4) or progressed on BTKi (n=6) were tested to check the effect of roginolisib on BCR signalling. Cells were treated with increasing doses of roginolisib from 0.625 μM to 5 μM in the presence of anti-IgM. Other BCRi such as idelalisib, duvelisib and acalabrutinib were used at 5 μM concentrations as positive controls. Roginolisib showed a dose dependent suppression of the activity of BCR downstream kinases such as AKT and ERK. The suppression of phosphorylation of AKT and ERK were comparable with other BCRi tested. Additionally, the apoptosis analysis by annexin/7AAD showed comparable activity of roginolisib with idelalisib, duvelisib or acalabrutinib at 5 μM concentration. To investigate the possible synergy of roginolisib with venetoclax in CLL patient samples who are BTKi responders (n=6) or BTKi progressors (n=8), apoptosis analysis was carried out in patient PBMCs. Viability of CLL cells following 48h of ex vivo treatment with increasing concentrations of roginolisib (0.625 to 5 μM) and venetoclax (1 to 8 nM) alone or in combination was tested. Both the BTKi responders and progressors showed strong synergy with the combination, with slightly better cell killing activity in BTKi progressors ( p=0.007) than responders ( p=0.019). To understand the mechanism by which roginolisib increases the sensitivity to venetoclax, changes in the expression levels of various Bcl-2 family proteins that have been implicated in venetoclax resistance such as Mcl-1, Bcl-xL, Bcl-2, A1 and BIM were measured by immunoblotting. Changes in expression were evaluated in PBMCs from both the BTKi responders (n=8) and progressors (n=7) after 24h treatment with 0.625 and 5 μM concentrations of roginolisib. A significant dose dependent downregulation of Mcl-1 was observed compared to DMSO treated cells in both BTKi responders ( p=0.0033) and progressors ( p= 0.0025). In addition, a significant increase in proapoptotic BIM EL was also observed at both the 0.625 μM ( p=0.0054) and 5 μM ( p=0.0194) concentrations compared to DMSO. In summary, the novel PI3Kδ selective inhibitor roginolisib shows tumor cell killing on various hematologic malignancies including CLL cells from patients with disease progression on BTK inhibitors. In addition, roginolisib synergized with venetoclax in lymphoma cell lines and CLL patient samples. Our data support extending this combination strategy to clinical trials in hematological malignancies.

CD56 brightCD16 -Perforin - regulatory Natural Killer Cells Associated with the Suppression of Chronic Graft-Versus-Host Disease Prevent CD4 + T Cell Proliferation through PD-1 and LAG-3 Dependent Pathways

Background: Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity and mortality after Hematopoietic Stem Cell Transplantation (HSCT). Previously, in large cohorts of HSCT patients we identified increased numbers of CD56 bright, granzyme B -, perforin - NK cells (NK reg) to be associated with a lack of cGvHD development. Transcriptome analysis of HSCT patient samples identified a unique transcriptome among the CD56 bright NK reg cells which associated with patients who failed to develop cGvHD, including overexpression of genes IL7R, GPR183, and Granzyme K, and low expression of genes such as perforin, granzyme B, and CD16. We then utilized the transcriptome information and further cell phenotyping to determine the optimal sorting approach for viable NK reg cells to allow for functional analysis by the sorting of CD56 brightCD3 -CD16 - cells. Functional analysis suggested that the NK reg population suppresses CD4 + T cells through a non-cytolytic, and direct cell-cell contact-dependent mechanism, though the exact receptor and ligand interaction of which CD4 + T cell populations are impacted is unknown. We hypothesized that the NK reg cell suppressive mechanism may be more specific than the more widely studied regulatory cell population, T reg cells, by suppressing CD4 + T cell subpopulations involved in inflammatory responses. Methodology: To investigate the suppressive capacity of NK reg cells, the cells were isolated from healthy donor peripheral blood and co-cultured with allogeneic CD4 + T cells, CD8 + T cells, T reg cells, T reg cells and dendritic cells, B cells, or NK cells for 96hrs. After 96hrs, the proliferation and viability of the responder cells were evaluated via proliferation dye dilution, and 7-AAD dye, respectively. To determine if the CD4 + T cell suppression is specific to a T helper (T h) cell subset we stained the co-cultured cells with a T h1/T h2/T h17 Phenotyping Kit. Further, PD-1 and LAG-3 neutralizing antibodies were added to the NK reg/CD4 + T cell co-culture to determine the receptor dependence. All samples were acquired with the BD FACSymphony Flow Cytometer and the data was analyzed via Kaluza software. Results: CD56 brightCD16 -perforin - NK regcellsstrongly suppress CD4 + T cell proliferation (approximately 96% suppression of CD4 + T cell proliferation at the 1:1 ratio of NK reg cells to CD4 + T cells). The contact-dependent mechanism of NK reg suppression of CD4 + T cell proliferation was significantly decreased when blocking either the PD-1 (15% decrease in suppression, p=0.03), or LAG-3 (29% decrease in suppression, p=0.04) receptors, at the 1:2 ratio of NK reg cells to CD4 + T cells. When both receptors are blocked the inhibition of suppressive effect is comparable to that of the LAG-3 blocking antibody being added individually (30% decrease in suppression, p=0.03). The suppressive mechanism of NK reg cells was observed to be selective in that they strongly suppress CD4 + T cell proliferation, but do not result in statistically significant suppression of CD8 + T cell, T reg cell, B cell, NK cell, or a specific T h cell subset proliferation (p>0.05). Further, though the average proliferation of the T reg cells co-cultured with NK reg cells and dendritic cells compared to just NK reg cells was greater (127% proliferation compared to 100% proliferation, respectively), we did not observe NK reg cells to induce a statistically significant proliferative effect towards the T reg cells in the presence of dendritic cells. Conclusion: As a result of our studies, we have confirmed the NK reg cell immune suppressive function towards CD4 + T cell proliferation, a main contributor to cGvHD development. Further, we demonstrated a PD-1/LAG-3-dependent direct contact mechanism of NK reg cell suppression, which is selective of total CD4 + T cells, with a lack of suppressive effect towards CD8 + T cells, T reg cells, B cells, and NK cells. The results of these studies contribute to our better understanding of how NK reg cells may induce a cell-specific suppressive function to promote immune tolerance, providing potential cell therapeutic applications for enhancing NK reg cell suppressive function through increasing PD-1/LAG-3 ligand or receptor expression on NK reg cells.

Elucidation of GPR55-Associated Signaling behind THC and LPI Reducing Effects on Ki67-Immunoreactive Nuclei in Patient-Derived Glioblastoma Cells.

GPR55 is involved in many physiological and pathological processes. In cancer, GPR55 has been described to show accelerating and decelerating effects in tumor progression resulting from distinct intracellular signaling pathways. GPR55 becomes activated by LPI and various plant-derived, endogenous, and synthetic cannabinoids. Cannabinoids such as THC exerted antitumor effects by inhibiting tumor cell proliferation or inducing apoptosis. Besides its effects through CB1 and CB2 receptors, THC modulates cellular responses among others via GPR55. Previously, we reported a reduction in Ki67-immunoreactive nuclei of human glioblastoma cells after GPR55 activation in general by THC and in particular by LPI. In the present study, we investigated intracellular mechanisms leading to an altered number of Ki67+ nuclei after stimulation of GPR55 by LPI and THC. Pharmacological analyses revealed a strongly involved PLC-IP3 signaling and cell-type-specific differences in Gα-, Gβγ-, RhoA-ROCK, and calcineurin signaling. Furthermore, immunochemical visualization of the calcineurin-dependent transcription factor NFAT revealed an unchanged subcellular localization after THC or LPI treatment. The data underline the cell-type-specific diversity of GPR55-associated signaling pathways in coupling to intracellular G proteins. Furthermore, this diversity might determine the outcome and the individual responsiveness of tumor cells to GPR55 stimulation by cannabin oids.

A third (booster) dose of the inactivated SARS-CoV-2 vaccine elicits immunogenicity and T follicular helper cell responses in people living with HIV.

This study sought to explore the immunogenicity of a booster dose of an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in people living with human immunodeficiency virus (HIV) and identify the factors affecting the magnitude of anti-SARS-CoV-2 antibody levels. A total of 34 people living with HIV (PLWH) and 34 healthy donors (HD) were administered a booster dose of the same SARS-CoV-2 vaccine. Anti-SARS-CoV-2 antibody and immunoglobulin G (IgG) levels were measured using the SARS-CoV-2 S protein neutralizing antibody Enzyme-Linked Immunosorbent Assay (ELISA) and 2019-nCov IgG Chemiluminescent Immunoassay Microparticles, respectively. Spearman correlation analysis was used to measure the correlation between laboratory markers and neutralizing antibody and IgG levels. Peripheral blood mononuclear cells (PBMCs) were extracted from each subject using density gradient centrifugation and the numbers of memory T and T follicular helper (Tfh) cells were determined using flow cytometry. PLWH had a marked reduction in CD4 and B cell levels that was accompanied by a lower CD4/CD8 T cell ratio. However, those who received a supplementary dose of inactivated SARS-CoV-2 vaccines exhibited antibody positivity rates that were analogous to levels previously observed. The booster vaccine led to a reduction in IgG and neutralizing antibody levels and the amplitude of this decline was substantially higher in the PLWH than HD group. Correlation analyses revealed a strong correlation between neutralizing antibody levels and the count and proportion of CD4 cells. Anti-SARS-CoV-2 IgG antibody levels followed a similar trend. The expression of memory T and Tfh cells was considerably lower in the PLWH than in the HD group. PLWH had an attenuated immune response to a third (booster) administration of an inactivated SARS-CoV-2 vaccine, as shown by lower neutralizing antibody and IgG levels. This could be attributed to the reduced responsiveness of CD4 cells, particularly memory T and cTfh subsets. CD4 and cTfh cells may serve as pivotal markers of enduring and protective antibody levels. Vaccination dose recalibration may be critical for HIV-positive individuals, particularly those with a lower proportion of CD4 and Tfh cells.

MODL-18. ESTABLISHMENT OF A NOVEL HUMANIZED PERIPHERAL BLOOD MONONUCLEAR CELL (HU-PBMC) MOUSE MODEL TO EVALUATE LYMPHOCYTE INVOLVEMENT IN GLIOBLASTOMA AND ITS TREATMENT

Abstract Glioblastoma (GBM) has traditionally been classified as an ‘immune cold’ tumor, characterized by strong immunosuppression, low mutational burden, and few presentable antigens. However, lack of standard-of-care improvement and the discovery of targetable immune-modulatory biomarkers like PD-1/PD-L1 and CTLA4 in the GBM microenvironment has prompted clinical trials to explore immune checkpoint inhibition (ICI) efficacy in patients, with moderate responses in specific patient subsets. To better characterize ICI in GBM, and to match prospective patients to suitable therapeutics, it is necessary to establish preclinical GBM mouse models that incorporate the lymphocytic milieu as xenograft and syngeneic models are unable to replicate the immune responses seen in humans. We have established a novel humanized PBMC mouse model that recapitulates human GBM pathology in the brain, supports and maintains systemic and intracranial human T cell engraftment in the presence of GBM tumors, and allows for a suitable timeline for preclinical testing of therapeutics prior to onset of GVHD. In our study, orthotopic GBM-bearing NSG mice showed successful human CD45+ engraftment in the blood (~20%) and brain (~5%) 2 weeks post intravenous implantation of hu-PBMCs. Restricted tumor growth was observed, indicating initial responsiveness of human immune cells to GBM. Engraftment remained unaffected upon treatment with temozolomide (GBM standard of care), and persisted over 6 weeks (brain ~40%, blood ~70%) before GVHD onset (week 7). ~90% of the hCD45+ population was CD3+, indicating robust T cell engraftment, which was further characterized into CD4+ and CD8+ subsets and by activation status (HLA-DR, CD45RA/RO, PD-1, FOXP3 etc) within the tumor-bearing brain and blood. Anti-PD-1 inhibitor efficacy was also tested in engrafted animals to observe ICI potential in GBM. This humanized mouse provides a powerful translational model to evaluate T cell responses in tumor pathology and immunotherapy in GBM and potentially other primary or secondary brain malignancies.

Single-Cell Transcriptomics Points to a Role of Stemness and Bone Marrow Niche Interactions in the Poor Response to Induction Therapy in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis with a 15-20% relapse rate. The induction therapy response is the strongest predictor of relapse and shows great variation between patients. However, whether intra-patient leukemic cell heterogeneity plays a role for the induction therapy response has not yet been investigated. We studied 13 patients with T-ALL (11 pediatric and 2 young adult, median follow-up 37 months). Of these, three were good responders (end of induction (EOI) minimal residual disease (MRD) undetectable/non-quantifiable by flowcytometry and PCR), while ten had a poor response (EOI MRD>0.01%, (range 0.05-97%). Of the ten poor responders, three experienced relapse and one died from resistant disease. Bone marrow samples from diagnosis (n=13) and paired samples from relapse (n=3) or MRD (n=4, MRD >5%) were subjected to combined single-cell RNA sequencing, single-cell surface marker profiling by antibody-derived tag (ADT)-sequencing, and single-cell TCR sequencing, using the 10X Genomics platform. A published healthy bone marrow dataset was used for cell annotation and clear distinction was observed between healthy cell subsets and leukemic blasts. TCR sequencing data revealed a hyperexpanded clone in six patients, with identical clonal sequences at diagnosis and relapse(n=2)/MRD(n=1). Surface marker profiling using a panel of 134 surface antigens showed good correspondence between leukemia-associated immunophenotypes found by ADT-sequencing and by flow cytometry for 16 common clinically used markers. To examine factors associated with a poor therapy response, we compared average gene expression of leukemic cells in poor and good responders at diagnosis using differential gene expression (DGE) and gene set enrichment analysis (GSEA). At diagnosis, poor responders showed upregulation of genes involved in extracellular matrix (ECM) degradation/organization, including the adhesion molecules ITGA5 and PECAM1, and MMP11, while showing downregulation of cell cycle genes. Accordingly, the fraction of cells predicted to be in G0/G1 phase at diagnosis of poor responders positively correlated with EOI MRD (r=0.52, p=0.07, Spearman's rank correlation). To investigate if this high fraction of quiescent cells in poor responders was associated with a more stem-like cell state, we examined the expression of a published hematopoietic stem cell (HSC) signature (Forsberg et al, 2010) that we previously selected for investigation in BCP-ALL (Modvig et al, Molecular Oncology, 2022). Indeed, patients with a poor response had a higher percentage of leukemic blasts with an HSC signature module score above zero (p=0.028, Kolmogorov-Smirnov, distribution in Figure 1), suggestive of stemness as a contributing factor to therapy resistance. To define molecular changes in leukemic blasts during therapy, we compared the leukemic blast gene expression profiles in matched diagnostic and MRD samples using DGE and GSEA. This revealed that the leukemic blasts within the first month of therapy downregulated cell cycle genes, while upregulating chemokine receptors, such as CXCR4. To identify specific genes associated with high MRD, we combined genes upregulated in poor responders (n=314, p<0.01, DGE) with genes associated with EOI MRD levels (n=98, r>0.8 and p<0.001, rank regression). This resulted in a 30-gene signature, comprising several genes previously associated with cancer stem cells, such as MICAL3, ELF4, and NLRP3, and genes encoding proteins involved in cancer drug resistance. These included sialyl transferases, acid ceramidase ( ASAH1) and zyxin ( ZYX). ZYX silencing was previously shown to downregulate the anti-apoptotic protein BCL-2 in leukemia. Accordingly, the percentage of cells with detectable BCL2 expression at diagnosis correlated with EOI MRD (r=0.58, p=0.04), supporting a role of BCL-2 inhibitor therapy in T-ALL. In conclusion, our data suggest that therapy-resistant cells are more stem-like and quiescent at diagnosis, display upregulation of genes involved in bone marrow niche ECM organization and adhesion, and genes related to drug resistance. Likewise, residual leukemic blast following therapy show less proliferation and CXCR4 upregulation. Thus, single-cell transcriptomics provides important molecular insights into therapy-resistant leukemic cells and could comprise a valuable prognostic tool in T-ALL.

Modakafusp Alfa Demonstrates Type I Interferon-Mediated Innate and Adaptive Immune Enhancement in a Phase 1/2 Study in Patients with Relapsed and/or Refractory Multiple Myeloma (RRMM)

Modakafusp alfa (moda), a novel immunocytokine, is an innate immunity enhancer comprising two attenuated interferon (IFN) α2b molecules fused to an anti-CD38 IgG4 monoclonal antibody backbone, driving preferential IFNα signalling in CD38-expressing innate and adaptive immune cells as well as myeloma cells. Here, we present pharmacodynamic (PD) data from the first-in-human Phase 1/2 study of moda as a monotherapy in patients with RRMM (iinnovate-1; NCT03215030). A total of 37 patients treated with moda at 1.5 or 3 mg/kg every 4 weeks were included in the analysis. Peripheral blood (PB) and bone marrow (BM) samples were collected during screening or pre-dose on cycle 1 day 1 (C1D1) and at multiple timepoints after treatment. BM and PB samples were evaluated by cytometry time of flight (CyTOF) and bulk RNA sequencing (RNAseq) to assess pharmacodynamic changes in immune cell populations, cell activation, and gene expression. Administration of moda led to enhanced innate immune cell activation and cytotoxic function as demonstrated by increased proportions of CD69+ and granzyme B+ in peripheral NK cells analyzed by CyTOF. This was accompanied by a decrease in the proportion of TIGIT+ NK cells, indicating reduced inhibitory signaling. Furthermore, enhanced proliferation (%Ki67+) of NK cells was observed in both BM and PB. Moda also impacted myeloid cell populations in BM and PB. Dendritic cells (DCs) and monocytes in PB showed increased surface expression of the co-stimulatory molecule CD86, suggesting a potential for enhanced antigen presentation and co-stimulation ability. Increased proliferation (%Ki67) of DCs and monocytes was detected in both PB and BM, accompanied by a decrease in numbers of peripheral DCs which may indicate recruitment or homing to secondary lymphoid organs. Additionally, RNAseq analysis revealed that treatment with moda led to upregulation of CD68 expression in both PB and BM, indicating a proinflammatory M1 macrophage type response. Finally, CyTOF analysis also indicated that moda significantly impacted adaptive immunity. We observed enhanced activation (%CD69, %PD1 and %CD40L) and cytotoxic function (%granzyme B) of peripheral CD8 T cells, and increased CD8 T cell proliferation (%Ki67 CD8) in both PB and BM samples. Enhanced activation of peripheral CD4 T cells (%CD69, %CD40L) was also detected. Consistent with activation of the adaptive immune system, there was a reduction in naïve CD8 T cells accompanied by an increase in CD8 T effector memory cells in PB. Analysis of exhaustion markers (TIGIT, PD-1) in cycle 1 (C1D8, C1D15, and C2D1 predose) showed that moda did not induce CD8 T cell exhaustion in either PB or BM, and there was no change in FoxP3+ Treg cells. Moda-induced innate and adaptive immune cell phenotypic changes were evaluated for correlation with clinical response at timepoints with sufficient number of evaluable samples. While none of the associations were statistically significant after correcting for multiplicity testing (using the false discovery rate method), we observed trends warranting further investigation. There was a greater increase of CD40L+ NK cells and PD1+ CD8 cells in responders vs. non-responders at C1D2. Additionally, at C1D15 responders showed a lower increase in proliferation (%Ki67+) of classical monocytes, NK cells, and myeloid DCs in PBas compared to non-responders. A similar trend was also observed in the BM classical monocytes. Even though the peak of Ki67 expression was observed at C1D8, diminished increases of proliferating cells at C1D15 in responders was a surprising observation that needs further consideration. The PD biomarker data from this first-in-human clinical trial demonstrated that treatment with moda enhances both innate and adaptive immune cell activation in PB and BM. Importantly, moda-mediated immune activation did not result in T cell exhaustion during cycle 1, as evaluated in both PB and BM. We have observed interesting trends of immune phenotypic changes that may correlate with response. Follow-up analyses of the correlation of biomarkers with clinical response are in progress and will be presented. Further clinical trials are underway (iinnovate-2, NCT05556616; iinnovate-3, NCT05590377) to evaluate moda's novel immune activating mechanism in combination with standard of care anti-myeloma therapies.

Inflammatory Change of FoxP3 + Regulatory T Cells in Human Acute Graft-Versus-Host Disease: Implication in Immunopathogenesis

FoxP3 + regulatory T (Treg) cells are a subset of CD4 + T cells that are requisite for control of immune responses in various settings, thereby preventing excessive inflammation and maintaining homeostasis of the immune system. Paradoxically, in several autoimmune disorders, it has been suggested that Treg cells undergo inflammatory conversion to produce effector cytokines promoting tissue inflammation. Although Treg cells are known to play an important role in the pathogenesis of acute graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT), such inflammatory changes have not been focused on so far especially in the setting of human acute GvHD. Herein, in a prospective cohort of patients with hematologic malignancies who were treated with allogeneic SCT, we investigated the quantitative and qualitative changes of Treg cells in subjects with acute GvHD, including production of inflammatory cytokines from Treg cells. We also studied a cellular mechanism for production of effector cytokines by Treg cells and clinical significance of inflammatory changes of Treg cells. We first determined the frequency of Treg cells in the peripheral blood of patients with acute GvHD and compared it with that of patients who did not develop acute GvHD. Percentage of CD25 hiCD127 loFoxP3 + Treg cells among CD4 + T cells was significantly lower in patients with acute GvHD. When we analyzed for the composition of Treg cell subsets in more detail, we observed that the proportion of CD45RA -FoxP3 hi (actively suppressive) Treg cells was decreased and the proportion of CD45RA -FoxP3 lo (cytokine-producing non-suppressive) Treg cells was increased in patients with acute GvHD. Next, we assessed the production of inflammatory cytokines from Treg cells following T cell receptor (TCR) stimulation of peripheral blood lymphocytes with anti-CD3/CD28 antibody, using intracellular cytokine staining. As a result, we found that a remarkable proportion of CD4 +CD25 hiFoxP3 + Treg cells of acute GvHD patients produced TNF-α upon TCR stimulation. Interestingly, a significant proportion of TNF-α-producing Treg cells also secreted IL-17A simultaneously. These findings are consistent with the increased CD45RA -FoxP3 lo fraction among the Treg cell subsets. To explore a potential mechanism underlying inflammatory change of Treg cells, we quantified a variety of soluble factors in systemic circulation before and after onset of acute GvHD. The concentration of IL-6 and IL-21 was significantly higher in the serum drawn after onset of acute GvHD compared to baseline, suggesting that these Th17-polarizing cytokines might be responsible for the inflammatory conversion of Treg cells during acute GvHD. We further checked the suppressive activity of TNF-α-producing CD4 +CD25 hiFoxP3 + Treg cells of acute GvHD patients. TNF-α + Treg cells of acute GvHD patients expressed lower level of FoxP3 than the TNF-α - counterpart. Similarly, the percentage of CD39 + cells was lower in TNF-α + Treg cells than in the TNF-α - counterpart. In fact, Treg cell suppressive activity during acute GvHD was significantly reduced compared with that of pre-SCT control when non-Treg CD4 + T cells or CD8 + T cells were used as responder cells. Finally, we examined the clinical significance of TNF-α-producing CD4 +CD25 hiFoxP3 + Treg cells in acute GvHD patients. The frequency of TNF-α + cells among circulating Treg cells significantly correlated with the severity of acute GvHD, with higher proportion of TNF-α + Treg cells in patients with the higher grade of acute GvHD. This result indicates that the pathologic conversion of Treg cells to produce TNF-α contributes to immune-mediated tissue injury during acute GvHD. Taken together, during human acute GvHD, Treg cells undergo functional alteration to produce proinflammatory cytokines including TNF-α and IL-17A and to exhibit the reduced suppressive activity. TNF-α production by Treg cells is closely associated with the clinical severity of acute GvHD.

Sting Activation Improves T-Cell Engaging Immunotherapy of Acute Myeloid Leukemia

Background: Despite emerging therapeutic options for Acute myeloid leukemia (AML) patients, for the majority of relapsed/refractory patients the outcome remains poor. Allogeneic stem cell transplantation has proven the power of T cells in eradicating residual leukemic cells, but alternative strategies based on T-cell recruiting bispecifics have so far failed to induce sustained responses. Resistance is mediated by the immunosuppressive tumor-microenvironment and secretion of immune dampening metabolites by AML cells. We hypothesized that combining a CD33-directed T cell engager (CD33 BiTE ® molecule or AMG 330) with a cGAS-STING agonist has the potential to augment anti-leukemic activity. Methods: In vitro co-culture assays of human T cells and AML cell lines for up to 72 h were performed to study the effect of AMG 330 in combination with the physiological STING-agonist 2‘-3‘ cGAMP (cGAMP). The cytotoxicity against the AML cell lines HL-60, MOLM-13 and OCI-AML-3 was assessed by flow cytometry. T-cell proliferation, T-cell degranulation (CD107a), as well as Granzyme B and TRAIL expression were characterized by flow cytometry. The secretion of effector cytokines (IFNy, TNF, IL-2, IFNa2a, CXCL10) was analyzed via CBA and ELISA assays. To better understand how AMG 330 and cGAMP work together, immunoblotting, transcriptomic analysis (bulk RNA-sequencing) and comprehensive CRISPRCas9 knockout-studies were carried out. Results: We observed a significant increase in AMG 330-mediated cytotoxicity of AML target cells upon co-application of cGAMP (Fig1A). The latter observation was enhanced upon prolongation of the culture and was also notable at low effector-to-target ratios. Monotherapy or addition of cGAMP by itself exerted only minor cytotoxicity against AML cells. The increase in AMG 330 mediated cytotoxicity through cGAMP was accompanied by an increase in T-cell proliferation, degranulation, Granzyme B secretion and TRAIL expression. Moreover, we observed a strong increase in secretion of proinflammatory cytokines (IFNy, IL-2 and IFNa2a). Bulk RNA-sequencing of AMG 330 and cGAMP stimulated co-cultures revealed type-I-IFN signature in the target (HL-60) and the effector cell (T cells) cell population. In addition, distinct phenotypes were found in each cell population: T cells demonstrated a marked increase in TNFa, IFNy and GZMB expression through the co-treatment, whereas HL-60 cells displayed an IFNy signaling signature (Fig1B). Importantly, the responsiveness of AML target cells to STING activation in terms of interferon stimulated gene (ISG)-induction was increased by the co-treatment, evidenced by further upregulation of STAT1, IRF7, WARS, CXCL11, IFI16, RSAD2 and CXCL10. An increase in type-I-IFN production and subsequent ISG-induction could also be confirmed on the protein level by measuring IFNα2a and CXCL-10. Knockout-studies revealed that the cGAMP enhanced AMG 330-mediated cytotoxicity was fully dependent on target cell intrinsic STING and IRF3 signaling, but also dependent on functional IFNy and TNFa signaling. Notably, the increased IFNy production by T cells upon cGAMP addition was dependent on functional target cell STING and IFNy signaling. These observations imply a crosstalk between effector and target cells and an intricate interdependence between target cell intrinsic IFNγ- and STING-signaling. Of note, the deletion of the IFNy-receptor in AML target cells also reduced AMG 330-dependent cytotoxicity under steady state conditions. Finally, we demonstrated that the combination of IFNy, TNFa and cGAMP sufficed to induce an apoptotic cell death of AML cells, similar to the cGAMP containing T-cell-HL-60 cocultures. Conclusion: We propose a novel mechanism by which AMG 330-activated T cells prime and sensitize AML target cells in a forward feedback loop towards STING activation, leading to increased type-I-IFN production and induction of ISGs. The beneficial effect of physiological cGAMP in enhancing AMG 330-mediated cytotoxicity was accompanied by the pronounced expression of effector cytokines and an overall cytotoxic T-cell phenotype. We established a key role for interferon gamma in AMG 330-mediated cytotoxicity of AML cells and in rendering AML cells responsive to STING agonism. We are currently conducting in vivoexperiments to further validate our findings.

C9orf142 transcriptionally activates MTBP to drive progression and resistance to CDK4/6 inhibitor in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) presents the most challenging subtype of all breast cancers because of its aggressive clinical phenotypes and absence of viable therapy targets. In order to identify effective molecular targets for treating patients with TNBC, we conducted an integration analysis of our recently published TNBC dataset of quantitative proteomics and RNA-Sequencing, and found the abnormal upregulation of chromosome 9 open reading frame 142 (C9orf142) in TNBC. However, the functional roles of C9orf142 in TNBC are unclear. In vitro and in vivo functional experiments were performed to assess potential roles of C9orf142 in TNBC. Immunoblotting, real-time quantitative polymerase chain reaction (RT-qPCR),and immunofluorescent staining were used to investigate the expression levels of C9orf142 and its downstream molecules. The molecular mechanisms underlying C9orf142-regulated mouse double minute 2 (MDM2)-binding protein (MTBP) were determined by chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. In TNBC tissues and metastatic lymph nodes, we observed that C9orf142 exhibited abnormal up-regulation, and its elevated expression was indicative of unfavorable prognosis for TNBC patients. Both in vitro and in vivo functional experiments demonstrated that C9orf142 accelerated TNBC growth and metastasis. Further mechanism exploration revealed that C9orf142 transcriptionally activated MTBP, thereby regulating its downstream MDM2/p53/p21 signaling axis and the transition of cell cycle from G1 to S phase. Functional rescue experiment demonstrated that knockdown of MTBP attenuated C9orf142-mediated tumour growth and metastasis. Furthermore, depletion of C9orf142 remarkably increased the responsiveness of TNBC cells to CDK4/6 inhibitor abemaciclib. Together, these findings unveil a previously unrecognized effect of C9orf142 in TNBC progression and responsiveness to CDK4/6 inhibitor, and emphasize C9orf142 as a promising intervention target for TNBC treatment.

Small RNA Sequencing Reveals a Distinct MicroRNA Signature between Glucocorticoid Responder and Glucocorticoid Non-Responder Primary Human Trabecular Meshwork Cells after Dexamethasone Treatment.

Glucocorticoids (GCs) are known to regulate several physiological processes and are the mainstay in the management of inflammatory eye diseases. The long-term use of GC causes raised intraocular pressure (IOP) or ocular hypertension (OHT) in about 30-50% of the susceptible individuals depending on the route of administration, and can lead to steroid-induced secondary glaucoma. The present study aims to understand the role of microRNAs (miRNAs) in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using small RNA sequencing. The human organ-cultured anterior segment (HOCAS) model was used to identify whether donor eyes were from GC-responders (GC-R; n = 4) or GC-non-responders (GC-NR; n = 4) following treatment with either 100 nM dexamethasone (DEX) or ethanol (ETH) for 7 days. The total RNA was extracted from cultured HTM cells with known GC responsiveness, and the differentially expressed miRNAs (DEMIRs) were compared among the following five groups: Group #1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR; #3: overlapping DEGs between Group #1 and #2; #4: Unique DEMIRs of GC-R; #5: Unique DEMIRs of GC-NR; and validated by RT-qPCR. There were 13 and 21 DEMIRs identified in Group #1 and Group #2, respectively. Seven miRNAs were common miRNAs dysregulated in both GC-R and GC-NR (Group #3). This analysis allowed the identification of DEMIRs that were unique to GC-R (6 miRNAs) and GC-NR (14 miRNAs) HTM cells, respectively. Ingenuity Pathway Analysis identified enriched pathways and biological processes associated with differential GC responsiveness in HTM cells. This is the first study to reveal a unique miRNA signature between GC-R and GC-NR HTM cells, which raises the possibility of developing new molecular targets for the management of steroid-OHT/glaucoma.

Fabrication of Polypropylene Nanoplastics Via Thermal Oxidation Reaction for Human Cells Responsiveness Studies.

With the current worldwide increasing use of plastics year by year, nanoplastics (NPs) have become a global threat to environmental and public health concerns. Among plastics, polypropylene (PP) is widely used in industrial and medical applications. Owing to the lack of validated detection methods and standard materials for PP NPs, understanding the impact of PP NPs on the environmental and biological systems is still limited. Here, isotactic polypropylene (iPP) was fabricated into oxidized polypropylene micro/nanoplastics (OPPs) via a thermal oxidation using hydrogen peroxide (H2O2) under various heating temperatures. The resulting OPPs were investigated in terms of the size distribution, surface chemistry, morphology, and thermal property as well as their concentration-dependent cytotoxicity to a human intestinal epithelial cell line (Caco-2), which could be a route to uptake NPs into the body through the food chain. The average diameters of the OPPs decrease with increasing reaction temperature. The OPPs obtained at 175 °C (OPP175) were spherical in shape and had a rough surface, with size distributions of approximately 0.14 ± 0.02 μm. A significant increase in the carbonyl content of the oxidized product was confirmed by Fourier transform infrared and X-ray photoelectron spectroscopy analyses. Caco-2 cells were exposed to OPP175 in a dose-dependent manner, and a significant loss of cell viability occurred at the concentration of 100 μg/mL. Thus, this study provides a fundamental approach for the fabrication of a model of NPs for the urgently demanded in vitro and in vivo studies to assess the potential impact of NPs on biological systems.

The role of KDM4A-mediated histone methylation on temozolomide resistance in glioma cells through the HUWE1/ROCK2 axis.

Temozolomide(TMZ) resistance presents a significant challenge in the treatment of gliomas. Although lysine demethylase 4A (KDM4A) has been implicated in various cancer-related processes, its role in TMZ resistance remains unclear. This study aims to elucidate the contribution of KDM4A to TMZ resistance in glioma cells and its potential implications for glioma prognosis. We assessed the expression of KDM4A in glioma cells (T98G and U251MG) using qRT-PCR and Western blot assays. To explore the role of KDM4A in TMZ resistance, we transfected siRNA targeting KDM4A into drug-resistant glioma cells. Cell viability was assessed using the CCK-8 assay and the TMZ IC50 value was determined. ChIP assays were conducted to investigate KDM4A, H3K9me3, and H3K36me3 enrichment on the promoters of ROCK2 and HUWE1. Co-immunoprecipitation confirmed the interaction between HUWE1 and ROCK2, and we examined the levels of ROCK2 ubiquitination following MG132 treatment. Notably, T98G cells exhibited greater resistance to TMZ than U251MG cells, and KDM4A displayed high expression in T98G cells. Inhibiting KDM4A resulted in decreased cell viability and a reduction in the TMZ IC50 value. Mechanistically, KDM4A promoted ROCK2 transcription by modulating H3K9me3 levels. Moreover, disruption of the interaction between HUWE1 and ROCK2 led to reduced ROCK2 ubiquitination. Inhibition of HUWE1 or overexpression of ROCK2 counteracted the sensitization effect of si-KDM4A on TMZ responsiveness in T98G cells. Our findings highlight KDM4A's role in enhancing TMZ resistance in glioma cells by modulating ROCK2 and HUWE1 transcription and expression through H3K9me3 and H3K36me3 removal.

Elsholtzia ciliata Essential Oil Exhibits a Smooth Muscle Relaxant Effect.

A recent in vivo study in pigs demonstrated the hypotensive properties of essential oil extracted from the blossoming plant Elsholtzia ciliata. This study was designed to examine the effect of E. ciliata essential oil (EO) on smooth muscle contraction. Tension measurements were performed on prostate strips and intact aortic rings isolated from rats. Results showed that EO caused a concentration-dependent reduction in phenylephrine-induced contraction of both the prostate and aorta, with a more pronounced inhibitory effect in the prostate. The IC50 of EO for the prostate was 0.24 ± 0.03 µL/mL (n = 10) and for the aorta was 0.72 ± 0.11 µL/mL (n = 4, p < 0.05 vs. prostate). The chromatographic analysis identified elsholtzia ketone (10.64%) and dehydroelsholtzia ketone (86.23%) as the predominant compounds in the tested EO. Since both compounds feature a furan ring within their molecular structure, other furan ring-containing compounds, 2-acetylfuran (2AF) and 5-methylfurfural (5MFF), were examined. For the first time, our study demonstrated the relaxant effects of 2AF and 5MFF on smooth muscles. Further, results showed that EO, 2AF, and 5MFF altered the responsiveness of prostate smooth muscle cells to phenylephrine. Under control conditions, the EC50 of phenylephrine was 0.18 ± 0.03 µM (n = 5), while in the presence of EO, 2AF, or 5MFF, the EC50 values were 0.81 ± 0.3 µM (n = 5), 0.89 ± 0.11 µM (n = 5), and 0.69 ± 0.23 µM (n = 4), respectively, p < 0.05 vs. control. Analysis of the affinity of EO for α1-adrenergic receptors in the prostate suggested that EO at a certain range of concentrations has a competitive antagonistic effect on α1-adrenergic receptors. In conclusion, EO elicits a relaxant effect on smooth muscles which may be related to the inhibition of α1-adrenoreceptors.