Naringin ameliorates obesity via stimulating adipose thermogenesis and browning, and modulating gut microbiota in diet-induced obese mice.

Naringin, a natural flavanone primarily found in citrus fruits, has garnered increased attention due to its recognized antioxidative, anti-inflammatory, and cardioprotective attributes. However, the functions of naringin in regulating energy expenditure are poorly understood. In the present study, we observed that twelve weeks of naringin supplementation substantially reshaped the metabolic profile of high-fat diet (HFD)-fed mice, by inhibiting body weight gain, reducing liver weight, and altering body compositions. Notably, naringin exhibited a remarkable capacity to augment whole-body energy expenditure of the tested mice by enhancing the thermogenic activity of brown adipose tissue (BAT) and stimulating browning of inguinal white adipose tissue (iWAT). Furthermore, our results showed naringin supplementation modified gut microbiota composition, specifically increasing the abundance of Bifidobacterium and Lachnospiraceae_bacterium_28-4, while reducing the abundance of Lachnospiraceae_bacterium_DW59 and Dubosiella_newyorkensis. Subsequently, we also found naringin supplementation altered fecal metabolite profile, by significantly promoting the production of taurine, tyrosol, and thymol, which act as potent activators of thermoregulation. Interestingly, the metabolic effects of naringin were abolished upon gut microbiota depletion through antibiotic intervention, concurrently leading the disappearance of naringin-induced thermogenesis and protective actions on diet-induced obesity. This discovery revealed a novel food-driven cross-sectional communication between gut bacteria and adipose tissues. Collectively, our data indicate that naringin supplementation stimulates BAT thermogenesis, alters fat distribution, promotes the browning process, and consequently inhibits body weight gain; importantly these metabolic effects require the participation of gut bacteria.

Chitosan-Delivered Chlorantraniliprole for Pest Control: Preparation Optimization, Deposition Behavior, and Application Potential.

Chitosan has emerged as a promising biopolymer carrier for the sustained release of pesticides owing to its good biocompatibility, biodegradability, and bioactivity. In this work, a controlled-release formulation of insecticide chlorantraniliprole was fabricated through coprecipitation-based synchronous encapsulation with chitosan, where the optimum preparation conditions, storage stability, deposition behavior, and application potential were investigated. Preparation of optimization data from response surface methodology showed high correlation coefficient (R2) of 0.9875 and adjusted coefficient (Radj2) of 0.9715. The resulting formulation displayed good loading content of 28.39%, high encapsulation efficiency of 75.71%, and good storage stability. Compared with the commercial suspension concentrate, the formulation exhibited better wettability and retention behaviors on plant leaves. Excitingly, effective control against one species of mealybug genus Paraputo Laing (outside the killing spectrum) on the Hippeastrum reticulatum plant was successfully achieved by spraying the controlled-release formulation at different time intervals. This work indicates the good potential of the developed formulation in expanding the application scope of chlorantraniliprole, which shows a new strategy for sustainable pest management.

Oxamate enhances the efficacy of CAR-T therapy against glioblastoma via suppressing ectonucleotidases and CCR8 lactylation.

BACKGROUND: Chimeric antigen receptor (CAR)-T immunotherapy fails to treat solid tumors due in part to immunosuppressive microenvironment. Excess lactate produced by tumor glycolysis increases CAR-T immunosuppression. The mechanism of lactate inducing the formation of immunosuppressive microenvironment remains to be further explored. METHODS: Immunocyte subpopulations and molecular characteristics were analyzed in the orthotopic xenografts of nude mice using flow cytometry assay and immunohistochemical staining after oxamate, a lactate dehydrogenase A (LDHA) inhibitor, and control T or CAR-T cells injection alone or in combination. RT-qPCR, western blot, flow cytometry, immunofluorescence, luciferase reporter assay, chromatin immunoprecipitation and ELISA were performed to measure the effect of lactate on the regulation of CD39, CD73 and CCR8 in cultured glioma stem cells, CD4 + T cells or macrophages. RESULTS: Oxamate promoted immune activation of tumor-infiltrating CAR-T cells through altering the phenotypes of immune molecules and increasing regulatory T (Treg) cells infiltration in a glioblastoma mouse model. Lactate accumulation within cells upregulated CD39, CD73 and CCR8 expressions in both lactate-treated cells and glioma stem cells-co-cultured CD4 + T cells and macrophages, and intracellular lactate directly elevated the activities of these gene promotors through histone H3K18 lactylation. CONCLUSIONS: Utilizing lactate generation inhibitor not only reprogramed glucose metabolism of cancer stem cells, but also alleviated immunosuppression of tumor microenvironment and reduced tumor-infiltrating CAR-Treg cells, which may be a potential strategy to enhance CAR-T function in glioblastoma therapy.

A spike-targeting bispecific T cell engager strategy provides dual layer protection against SARS-CoV-2 infection in vivo.

Neutralizing antibodies exert a potent inhibitory effect on viral entry; however, they are less effective in therapeutic models than in prophylactic models, presumably because of their limited efficacy in eliminating virus-producing cells via Fc-mediated cytotoxicity. Herein, we present a SARS-CoV-2 spike-targeting bispecific T-cell engager (S-BiTE) strategy for controlling SARS-CoV-2 infection. This approach blocks the entry of free virus into permissive cells by competing with membrane receptors and eliminates virus-infected cells via powerful T cell-mediated cytotoxicity. S-BiTE is effective against both the original and Delta variant of SARS-CoV2 with similar efficacy, suggesting its potential application against immune-escaping variants. In addition, in humanized mouse model with live SARS-COV-2 infection, S-BiTE treated mice showed significantly less viral load than neutralization only treated group. The S-BiTE strategy may have broad applications in combating other coronavirus infections.

Acetyl-11-Keto-ß-Boswellic Acid Accelerates the Repair of Spinal Cord Injury in Rats by Resisting Neuronal Pyroptosis with Nrf2.

The primary aim of this study is to delve into the potential of Acetyl-11-keto-ß-boswellic acid (AKBA) in ameliorating neuronal damage induced by acute spinal cord injury, as well as to unravel the intricate underlying mechanisms. A cohort of 40 Sprague-Dawley rats was meticulously categorized into four groups. Following a seven-day oral administration of AKBA, damaged spinal cord samples were meticulously procured for Nissl staining and electron microscopy to assess neuronal demise. Employing ELISA, immunofluorescence, Western blot (WB), and quantitative polymerase chain reaction (qPCR), the modulatory effects of AKBA within the context of spinal cord injury were comprehensively evaluated. Furthermore, employing an ex vivo extraction of spinal cord neurons, an ATP + LPS-induced pyroptotic injury model was established. The model was subsequently subjected to Nrf2 inhibition, followed by a battery of assessments involving ELISA, DCFH-DA staining, flow cytometry, immunofluorescence, and WB to decipher the effects of AKBA on the spinal cord neuron pyroptosis model. By engaging the Nrf2-ROS-NLRP3 pathway, AKBA exerted a repressive influence on the expression of the pyroptotic initiator protein Caspase-1, thereby mitigating the release of GSDMD and alleviating pyroptosis. Additionally, AKBA demonstrated the ability to attenuate the release of IL-18 and IL-1ß, curbing neuronal loss and expediting the restorative processes within the context of spinal cord injury. Our study elucidates that AKBA can reduce spinal cord neuronal apoptosis, providing a basis for the development of AKBA as a clinical treatment for spinal cord injury.

Lactobacillus plantarum MH-301 as an effective adjuvant to isotretinoin in the treatment of acne vulgaris: a randomized and open-label trail.

Introduction: Acne vulgaris is a common chronic inflammatory skin disease originating in the sebaceous gland units of the skin follicles. Isotretinoin is presently the primary choice for the treatment of acne vulgaris. However, it could induce several adverse reactions like diarrhea, cheilitis, headache, elevated triglyceride levels and risk of inflammatory bowel disease and depression. Hence, it is imperative to seek an alternative therapy. Methods: One hundred five patients were randomly divided into 3 groups, and received a baseline treatment of oral doxycycline for the initial 4 weeks. Group I received isotretinoin oral for 12 weeks; Group P received oral Lactobacillus plantarum MH-301 treatment for 12 weeks; Group IP received combined treatment with oral probiotics and oral isotretinoin for 12 weeks. The number of skin lesions was recorded at 0, 4, 8, and 12 weeks during the treatment to compare the efficacy of each intervention, and skin and fecal samples were collected from patients at 12 weeks for high-throughput sequencing to explore the microbiota differences between various groups. Results: Our results revealed that the combination of L. plantarum MH-301 with isotretinoin significantly reduced the number of skin lesions in patients compared to using L. plantarum MH-301 and isotretinoin alone (p < 0.001). Additionally, skin microbiome High-throughput analysis indicated the restorative effects of L. plantarum MH-301 on skin microbial diversity while also observing a reduction in the main microbiota of skin lesions, Propionibacterium and Corynebacterium. Meanwhile, gut microbiome High-throughput analysis showed that it could regulate disorders of the intestinal microbiota and increased the abundance of probiotics such as Lactobacillus, Bifidobacterium, Coprococcus and Bacteroides genera. Conclusion: In conclusion, L. plantarum MH-301 could be used in combination with isotretinoin for optimal results in the treatment of acne vulgaris. The research conducted provides theoretical and data support for the adjuvant effect of L. plantarum in the treatment of acne vulgaris. Clinical Trial Registration: [ClinicalTrials.gov], identifier (ChiCTR2200063499).

Association of hematologic response and assay sensitivity on the prognostic impact of measurable residual disease in acute myeloid leukemia: a systematic review and meta-analysis.

Measurable residual disease (MRD) is associated with relapse and survival in acute myeloid leukemia (AML). We aimed to quantify the impact of MRD on outcomes across clinical contexts, including its association with hematologic response and MRD assay sensitivity. We performed systematic literature review and meta-analysis of 48 studies that reported the association between MRD and overall survival (OS) or disease-free survival (DFS) in AML and provided information on the MRD threshold used and the hematologic response of the study population. Among studies limited to patients in complete remission (CR), the estimated 5-year OS for the MRD-negative and MRD-positive groups was 67% (95% Bayesian credible interval [CrI], 53-77%) and 31% (95% CrI, 18-44%), respectively. Achievement of an MRD-negative response was associated with superior DFS and OS, regardless of MRD threshold or analytic sensitivity. Among patients in CR, the benefit of MRD negativity was highest in studies using an MRD cutoff less than 0.1%. The beneficial impact of MRD negativity was observed across MRD assays and timing of MRD assessment. In patients with AML in morphological remission, achievement of MRD negativity is associated with superior DFS and OS, irrespective of hematologic response or the MRD threshold used.

Tannin Reduces the Incidence of Polyspermic Penetration in Porcine Oocytes.

Tannin (TA) improves porcine oocyte cytoplasmic maturation and subsequent embryonic development after in vitro fertilization (IVF). However, the mechanism through which TA blocks polyspermy after IVF remains unclear. Hence, the biological function of organelles (cortical granule [CG], Golgi apparatus, endoplasmic reticulum [ER], and mitochondria) and the incidence of polyspermic penetration were examined. We found no significant difference in oocyte nuclear maturation among the 1 µg/mL, 10 µg/mL TA, and control groups. Moreover, 100 µg/mL TA significantly reduced 1st polar body formation rate compared to the other groups. Additionally, 1 and 10 µg/mL TA significantly increased the protein levels of GDF9, BMP15, and CDK1 compared to the control and 100 µg/mL TA groups. Interestingly, 1 and 10 µg/mL TA improved the normal distribution of CGs, Golgi, ER, and mitochondria by upregulating organelle-related gene expression and downregulating ER stress (CHOP) gene expression. Simultaneously, 1 and 10 µg/mL TA significantly increased the proportion of normal fertilized oocytes (2 pronuclei; 2 PN) and blastocyst formation rate compared to the control, as well as that of 100 µg/mL TA after IVF by upregulating polyspermy-related genes. In conclusion, TA during IVM enhances 2PN and blastocyst formation rates by regulating organelles' functions and activities.

Bayesian sparse modeling to identify high-risk subgroups in meta-analysis of safety data.

Meta-analysis allows researchers to combine evidence from multiple studies, making it a powerful tool for synthesizing information on the safety profiles of new medical interventions. There is a critical need to identify subgroups at high risk of experiencing treatment-related toxicities. However, this remains quite challenging from a statistical perspective as there are a variety of clinical risk factors that may be relevant for different types of adverse events, and adverse events of interest may be rare or incompletely reported. We frame this challenge as a variable selection problem and propose a Bayesian hierarchical model which incorporates a horseshoe prior on the interaction terms to identify high-risk groups. Our proposed model is motivated by a meta-analysis of adverse events in cancer immunotherapy, and our results uncover key factors driving the risk of specific types of treatment-related adverse events.

Ramelteon Reduces Oxidative Stress by Maintenance of Lipid Homeostasis in Porcine Oocytes.

This study aimed to determine the underlying mechanism of ramelteon on the competence of oocyte and subsequent embryo development in pigs during in vitro maturation (IVM). Our results showed that the cumulus expansion index was significantly lower in the control group compared to the ramelteon groups (p < 0.05). Moreover, supplementation of 10−11 and 10−9 M ramelteon significantly increased the cumulus expansion and development-related genes expression, and reduced apoptosis in cumulus cells (p < 0.05). In oocytes, the nuclear maturation rate was significantly improved in 10−11, 10−9, and 10−7 M ramelteon groups compared to the control (p < 0.05). Additionally, the level of intracellular GSH was significantly increased and ROS was significantly decreased in ramelteon-supplemented groups, and the gene expression of oocyte development and apoptosis were significantly up- and down-regulated by 10−11 and 10−9 M ramelteon (p < 0.05), respectively. The immunofluorescence results showed that the protein levels of GDF9, BMP15, SOD1, CDK1, and PGC1α were significantly increased by 10−11 M ramelteon compared to the control (p < 0.05). Although there was no significant difference in cleavage rate, the blastocyst formation rate, total cell numbers, and hatching/-ed rate were significantly improved in 10−11 M ramelteon group compared to the control (p < 0.05). Furthermore, embryo development, hatching, and mitochondrial biogenesis-related genes were dramatically up-regulated by 10−11 M ramelteon (p < 0.05). In addition, the activities of lipogenesis and lipolysis in oocytes were dramatically increased by 10−11 M ramelteon compared to the control (p < 0.05). In conclusion, supplementation of 10−11 M ramelteon during IVM improved the oocyte maturation and subsequent embryo development by reducing oxidative stress and maintenance of lipid homeostasis.

Evaluation of Hybrid VMAT Advantages and Robustness Considering Setup Errors Using Surface Guided Dose Accumulation for Internal Lymph Mammary Nodes Irradiation of Postmastectomy Radiotherapy.

Objectives: Setup error is a key factor affecting postmastectomy radiotherapy (PMRT) and irradiation of the internal mammary lymph nodes is the most investigated aspect for PMRT patients. In this study, we evaluated the robustness, radiobiological, and dosimetric benefits of the hybrid volumetric modulated arc therapy (H-VMAT) planning technique based on the setup error in dose accumulation using a surface-guided system for radiation therapy. Methods: We retrospectively selected 32 patients treated by a radiation oncologist and evaluated the clinical target volume (CTV), including internal lymph node irradiation (IMNIs), and considered the planning target volume (PTV) margin to be 5 mm. Three different planning techniques were evaluated: tangential-VMAT (T-VMAT), intensity-modulated radiation therapy (IMRT), and H-VMAT. The interfraction and intrafraction setup errors were analyzed in each field and the accumulated dose was evaluated as the patients underwent daily surface-guided monitoring. These parameters were included while evaluating CTV coverage, the dose required for the left anterior descending artery (LAD) and the left ventricle (LV), the normal tissue complication probability (NTCP) for the heart and lungs, and the second cancer complication probability (SCCP) for contralateral breast (CB). Results: When the setup error was accounted for dose accumulation, T-VMAT (95.51%) and H-VMAT (95.48%) had a higher CTV coverage than IMRT (91.25%). In the NTCP for the heart, H-VMAT (0.04%) was higher than T-VMAT (0.01%) and lower than IMRT (0.2%). However, the SCCP (1.05%) of CB using H-VMAT was lower than that using T-VMAT (2%) as well as delivery efficiency. And T-VMAT (3.72) and IMRT (10.5).had higher plan complexity than H-VMAT (3.71). Conclusions: In this study, based on the dose accumulation of setup error for patients with left-sided PMRT with IMNI, we found that the H-VMAT technique was superior for achieving an optimum balance between target coverage, OAR dose, complication probability, plan robustness, and complexity.

T cell receptor ß-chain-targeting chimeric antigen receptor T cells against T cell malignancies.

The success of chimeric antigen receptor (CAR) T cells in treating B cell malignancies comes at the price of eradicating normal B cells. Even though T cell malignancies are aggressive and treatment options are limited, similar strategies for T cell malignancies are constrained by the severe immune suppression arising from bystander T cell aplasia. Here, we show the selective killing of malignant T cells without affecting normal T cell-mediated immune responses in vitro and in a mouse model of disseminated leukemia. Further, we develop a CAR construct that carries the single chain variable fragment of a subtype-specific antibody against the variable TCR ß-chain region. We demonstrate that these anti-Vß8 CAR-T cells are able to recognize and kill all Vß8+ malignant T cells that arise from clonal expansion while sparing malignant or healthy Vß8- T cells, allowing sufficient T cell-mediated cellular immunity. In summary, we present a proof of concept for a selective CAR-T cell therapy to eradicate T cell malignancies while maintaining functional adaptive immunity, which opens the possibility for clinical development.

On Bayesian modeling of censored data in JAGS.

BACKGROUND: Just Another Gibbs Sampling (JAGS) is a convenient tool to draw posterior samples using Markov Chain Monte Carlo for Bayesian modeling. However, the built-in function dinterval() for censored data misspecifies the default computation of deviance function, which limits likelihood-based Bayesian model comparison. RESULTS: To establish an automatic approach to specifying the correct deviance function in JAGS, we propose a simple and generic alternative modeling strategy for the analysis of censored outcomes. The two illustrative examples demonstrate that the alternative strategy not only properly draws posterior samples in JAGS, but also automatically delivers the correct deviance for model assessment. In the survival data application, our proposed method provides the correct value of mean deviance based on the exact likelihood function. In the drug safety data application, the deviance information criterion and penalized expected deviance for seven Bayesian models of censored data are simultaneously computed by our proposed approach and compared to examine the model performance. CONCLUSIONS: We propose an effective strategy to model censored data in the Bayesian modeling framework in JAGS with the correct deviance specification, which can simplify the calculation of popular Kullback-Leibler based measures for model selection. The proposed approach applies to a broad spectrum of censored data types, such as survival data, and facilitates different censored Bayesian model structures.

Efficacy of front-line immunochemotherapy for follicular lymphoma: a network meta-analysis of randomized controlled trials.

Front-line treatment for follicular lymphoma has evolved with the introduction of maintenance therapy, bendamustine (Benda), obinutuzumab (G), and lenalidomide (Len). We conducted a random-effects Bayesian network meta-analysis (NMA) of phase 3 randomized controlled trials (RCTs) to identify the regimens with superior efficacy. Progression-free survival (PFS) was compared between 11 modern regimens with different immunochemotherapy and maintenance strategies. G-Benda-G resulted in with the best PFS, with an HR of 0.41 compared to R-Benda, a surface under the cumulative ranking curve (SUCRA) of 0.97, a probability of being the best treatment (PbBT) of 72%, and a posterior ranking distribution (PoRa) of 1 (95% BCI 1-3). This was followed by R-Benda-R4 (HR = 0.49, PbBT = 25%, PoRa = 2) and R-Benda-R (HR = 0.60, PbBT = 3%, PoRa = 3). R-CHOP-R (HR = 0.96) and R-Len-R (HR = 0.97) had similar efficacy to R-Benda. Bendamustine was a better chemotherapy backbone than CHOP either with maintenance (R-Benda-R vs R-CHOP-R, HR = 0.62; G-Benda-G vs G-CHOP-G, HR = 0.55) or without maintenance therapy (R-Benda vs R-CHOP, HR = 0.68). Rituximab maintenance improved PFS following R-CHOP (R-CHOP-R vs R-CHOP, HR = 0.65) or R-Benda (R-Benda-R vs R-Benda, HR = 0.60; R-Benda-R4 vs R-Benda, HR = 0.49). In the absence of multi-arm RCTs that include all common regimens, this NMA provides an important and useful guide to inform treatment decisions.

Type I Interferon Promotes Antitumor T Cell Response in CRPC by Regulating MDSC.

BACKGROUND: Metastatic castration-resistant prostate cancer (CRPC) is the leading cause of death among prostate cancer patients. Here, our aim was to ascertain the immune regulatory mechanisms involved in CRPC development and identify potential immunotherapies against CRPC. METHODS: A CRPC model was established using Myc-CaP cells in immune-competent FVB mice following castration. The immune cell profile of the tumor microenvironment (TME) was analyzed during CRPC development. Different immunotherapies were screened in the CRPC tumor model, and their efficacies and underlying mechanisms were investigated in vitro and in vivo. RESULTS: During CRPC development, the proportion of granulocytic myeloid-derived suppressor cells (G-MDSCs) in the TME increased. Among the immunotherapies tested, IFNα was more effective than anti-PD-L1, anti-CTLA-4, anti-4-1BB, IL-2, and IL-9 in reducing Myc-CaP CRPC tumor growth. IFNα reduced the number of G-MDSCs both in vitro during differentiation and in vivo in CRPC mice. Furthermore, IFNα reduced the suppressive function of G-MDSCs on T cell proliferation and activation. CONCLUSION: G-MDSCs are crucial to effective immunotherapy against CRPC. Treatment with IFNα presents a promising therapeutic strategy against CRPC. Besides the direct inhibition of tumor growth and the promotion of T cell priming, IFNα reduces the number and the suppressive function of G-MDSCs and restores T cell activation.

Engineering mesenchymal stromal cells with neutralizing and anti-inflammatory capability against SARS-CoV-2 infection.

The emergence of the novel human severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to the pandemic of coronavirus disease 2019 (COVID-19), which has markedly affected global health and the economy. Both uncontrolled viral replication and a proinflammatory cytokine storm can cause severe tissue damage in patients with COVID-19. SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2) as its entry receptor. In this study, we generated ACE2 extracellular domain-Fc and single-chain variable fragment-interleukin 6 (IL-6) single-chain variable fragment against IL-6 receptor (scFv-IL6R)-Fc fusion proteins to differentially neutralize viruses and ameliorate the cytokine storm. The human ACE2 (hACE2)1-740-Fc fusion protein showed a potent inhibitory effect on pseudo-typed SARS-CoV-2 entry and a good safety profile in mice. In addition, scFv-IL6R-Fc strongly blocked IL-6 signal activation. We also established a mesenchymal stromal cell (MSC)-based hACE21-740-Fc and scFv-IL6R-Fc delivery system, which could serve as a potential therapy strategy for urgent clinical needs of patients with COVID-19.

USP22 deficiency in melanoma mediates resistance to T cells through IFNγ-JAK1-STAT1 signal axis.

Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated 9 (Cas9)-mediated loss-of-function screens are powerful tools for identifying genes responsible for diverse phenotypes. Here, we perturbed genes in melanoma cells to screen for genes involved in tumor escape from T cell-mediated killing. Multiple interferon gamma (IFNγ) signaling-related genes were enriched in melanoma cells resistant to T cell killing. In addition, deletion of the deubiquitinating protease ubiquitin specific peptidase 22 (USP22) in mouse melanoma (B16-OVA) cells decreased the efficacy of T cell-mediated killing, both in vitro and in vivo, while overexpression enhanced tumor-cell sensitivity to T (OT-I) cell-mediated killing. USP22 deficiency in both mouse and human melanoma cells showed impaired sensitivity to interferon pathway and USP22 was positively correlated with key molecules of interferon pathway in clinical melanoma samples. Mechanistically, USP22 may directly interact with signal transducer and activator of transcription 1 (STAT1), deubiquitinate it, and improve its stability in both human and mouse melanoma cells. Our findings identified a previously unknown function of USP22 and linked the loss of genes in tumor cells that are essential for escaping the effector function of CD8+ T cells during immunotherapy.

Bcl-2 Enhances Chimeric Antigen Receptor T Cell Persistence by Reducing Activation-Induced Apoptosis.

PURPOSE: To evaluate the potential added value of integrating anti-apoptotic molecules for improving the anti-tumor activity of CAR-T cells. METHODS: Four small molecules inhibiting apoptosis were tested for their ability to prevent activated induced CAR-T cell death. Five CD20-targeting, CD137 (4-1BB) and CD3ζ integrated CAR-T cells (20BBZ) with constitutively expressed anti-apoptotic genes were established, and we screened out the strongest proliferation enhancer: Bcl-2. The memory subtype and the exhaustion markers of CAR-T cells were analyzed. The anti-tumor activities of Bcl-2 integrating CAR-T cells (20BBZ-Bcl-2) were evaluated in vitro and in a mouse xenograft lymphoma model. CONCLUSION: The 20BBZ-Bcl-2 CAR-T cells showed improved proliferation ability compared to 20BBZ CAR-T cells in vitro. In addition, activation-induced apoptosis was reduced in the 20BBZ-Bcl-2 CAR-T cells. Consistent with the enhanced proliferation in vitro, 20BBZ-Bcl-2 CAR-T cells exhibited improved anti-tumor activity in a mouse xenograft lymphoma model.

4-1BB Signaling Boosts the Anti-Tumor Activity of CD28-Incorporated 2nd Generation Chimeric Antigen Receptor-Modified T Cells.

While chimeric antigen receptor-modified T (CAR-T) cells have shown great success for the treatment of B cell leukemia, their efficacy appears to be compromised in B cell derived lymphoma and solid tumors. Optimization of the CAR design to improve persistence and cytotoxicity is a focus of the current CAR-T study. Herein, we established a novel CAR structure by adding a full length 4-1BB co-stimulatory receptor to a 28Z-based second generation CAR that targets CD20. Our data indicated that this new 2028Z-4-1BB CAR-T cell showed improved proliferation and cytotoxic ability. To further understand the mechanism of action, we found that constitutive 4-1BB sensing significantly reduced the apoptosis of CAR-T cells, enhanced proliferation, and increased NF-κB pathway activation. Consistent with the enhanced proliferation and cytotoxicity in vitro, this new structure of CAR-T cells exhibited robust persistence and anti-tumor activity in a mouse xenograft lymphoma model. This work provides evidence for a new strategy to optimize the function of CAR-T against lymphoma.