The anti-atherosclerotic effect of Paeonol against the lipid accumulation in macrophage-derived foam cells by inhibiting ferroptosis via the SIRT1/NRF2/GPX4 signaling pathway.

Atherosclerosis (AS) is the underlying cause of many severe vascular diseases and is primarily characterized by abnormal lipid metabolism. Paeonol (Pae), a bioactive compound derived from Paeonia Suffruticosa Andr., is recognized for its significant role in reducing lipid accumulation. Our research objective is to explore the link between lipid buildup in foam cells originating from macrophages and the process of ferroptosis, and explore the effect and mechanism of Pae on inhibiting AS by regulating ferroptosis. In our animal model, ApoE-deficient mice, which were provided with a high-fat regimen to provoke atherosclerosis, were administered Pae. The treatment was benchmarked against simvastatin and ferrostatin-1. The results showed that Pae significantly reduced aortic ferroptosis and lipid accumulation in the mice. In vitro experiments further demonstrated that Pae could decrease lipid accumulation in foam cells induced by oxidized low-density lipoprotein (LDL) and challenged with the ferroptosis inducer erastin. Crucially, the protective effect of Pae against lipid accumulation was dependent on the SIRT1/NRF2/GPX4 pathway, as SIRT1 knockdown abolished this effect. Our findings suggest that Pae may offer a novel therapeutic approach for AS by inhibiting lipid accumulation through the suppression of ferroptosis, mediated by the SIRT1/NRF2/GPX4 pathway. Such knowledge has the potential to inform the creation of novel therapeutic strategies aimed at regulating ferroptosis within the context of atherosclerosis.

Paeonol Attenuates Atherosclerosis by Inhibiting Vascular Smooth Muscle Cells Senescence via SIRT1/P53/TRF2 Signaling Pathway.

Atherosclerosis is a chronic inflammatory disease leading to various vascular diseases. Vascular smooth muscle cell (VSMC) senescence promotes atherosclerotic inflammation and the formation of plaque necrosis core, in part through telomere damage mediated by a high-fat diet. Our previous research found that paeonol, a potential anti-inflammatory agent extracted from Cortex Moutan, could significantly improve VSMCs dysfunction. However, the impact of paeonol on the senescence of VSMCs remains unexplored. This study presents the protective effects of paeonol on VSMCs senescence, and its potential activity in inhibiting the progression of atherosclerosis in vivo and in vitro. Sirtuin 1 (SIRT1) is a nuclear deacetylase involved in cell proliferation, senescence, telomere damage, and inflammation. Here, SIRT1 was identified as a potential target of paeonol having anti-senescence and anti-atherosclerosis activity. Mechanistic studies revealed that paeonol binds directly to SIRT1 and then activates the SIRT1/P53/TRF2 pathway to inhibit VSMCs senescence. Our results suggested that SIRT1-mediated VSMCs senescence is a promising druggable target for atherosclerosis, and that pharmacological modulation of the SIRT1/P53/TRF2 signaling pathway by paeonol is of potential benefit for patients with atherosclerosis.

Integration of metabolomics and transcriptomics reveals metformin suppresses thyroid cancer progression via inhibiting glycolysis and restraining DNA replication.

The anti-tumoral effects of metformin have been widely studied in several types of cancer, including thyroid cancer; however, the underlying molecular mechanisms remain poorly understood. As an oral hypoglycemic drug, metformin facilitates glucose catabolism and disrupts metabolic homeostasis. Metabolic reprogramming, particularly cellular glucose metabolism, is an important characteristic of malignant tumors. This study aimed to explore the therapeutic effects of metformin in thyroid cancer and the underlying metabolic mechanism. In the present study, it was shown that metformin reduced cell viability, invasion, migration, and EMT, and induced apoptosis and cell cycle G1 phase arrest in thyroid cancer. Transcriptome analysis demonstrated that the differentially expressed genes induced by metformin were involved in several signaling pathways including apoptosis singling pathways, TGF-ß signaling, and cell cycle regulation in human thyroid cancer cell lines. In addition, the helicase activity of the CDC45-MCM2-7-GINS complex and DNA replication related genes such as RPA2, RAD51, and PCNA were downregulated in metformin-treated thyroid cancer cells. Moreover, metabolomics analysis showed that metformin-induced significant alterations in metabolic pathways such as glutathione metabolism and polyamine synthesis. Integrative analysis of transcriptomes and metabolomics revealed that metformin suppressed glycolysis by downregulating the key glycolytic enzymes LDHA and PKM2 and upregulating IDH1 expression in thyroid cancer. Furthermore, the anti-tumor role of metformin in thyroid cancer in vivo was shown. Together these results show that metformin plays an anti-tumor role by inhibiting glycolysis and restraining DNA replication in thyroid cancer.

MicroRNA-142-3P suppresses the progression of papillary thyroid carcinoma by targeting FN1 and inactivating FAK/ERK/PI3K signaling.

OBJECTIVES: miR-142-3P is a tumor suppressor in various malignant cancers. However, the function of miR-142-3P in papillary thyroid carcinoma (PTC) remains to be elucidated. The aim of this study was to explore the function and mechanism of miR-142-3P in PTC. METHODS: Real Time Quantitative PCR (RT-qPCR) was used to assess the expression of miR-142-3P and Fibronectin 1 (FN1) in PTC. The correlation between FN1 and miR-142-3P expression was analyzed by Spearman's correlation analysis. Cell Counting Kit 8 (CCK8), 5-ethynyl-2'-deoxyuridine (EDU) assay, cell migration and invasion assay and wound healing measures evaluated the effect of miR-142-3P and FN1 on cell proliferation, migration and invasion. Dural Luciferase reported gene assay evaluated the interaction between miR-142-3P and 3' untranslated region (UTR) of FN1. The Epithelial-Mesenchymal-Transition (EMT) and apoptosis related marker genes were measured using western blot analysis (WB). RESULTS: miR-142-3P was significantly decreased in both PTC specimens and relevant cell lines. Functionally, miR-142-3P inhibited cell proliferation, migration, invasion and EMT, and induced the cell apoptosis in PTC. In addition, miR-142-3P bound directly with 3' UTR of FN1 and negatively regulated the expression of FN1 in PTC. FN1 expression is elevated in PTC, and its aberrant high correlated with declines in recurrence-free survival (RFS). Moreover, FN1 promoted cell proliferation, migration, invasion and EMT, induced cell apoptosis in PTC cells. Depletion of FN1 rescues the effect of miR-142-3P inhibitor on cell proliferation, invasion, apoptosis and EMT via inactivating Focal Adhesion Kinase (FAK)/Extracellular Signal-Regulated Kinase (ERK) / Phosphoinostide 3-kinase (P13K) signaling. CONCLUSION: miR-142-3P suppressed cell proliferation, migration, invasion and EMT through modulating FN1/FAK/ERK/PI3K signaling in PTC, suggesting it as a potential therapeutic target for PTC.

The prognostic and biology of tumour-infiltrating lymphocytes in the immunotherapy of cancer.

Tumour immunotherapy has achieved remarkable clinical success in many different types of cancer in the past two decades. The outcome of immune checkpoint inhibitors in cancer patients has been linked to the quality and magnitude of T cell, NK cell, and more recently, B cell within the tumour microenvironment, suggesting that the immune landscape of a tumour is highly connected to patient response and prognosis. It is critical to understanding tumour immune microenvironments for identifying immune modifiers of cancer progression and developing cancer immunotherapies. The infiltration of solid tumours by immune cells with anti-tumour activity is both a strong prognostic factor and a therapeutic goal. Recent approaches and applications of new technologies, especially single-cell mRNA analysis in dissecting tumour microenvironments have brought important insights into the biology of tumour-infiltrating immune cells, revealed a remarkable degree of cellular heterogeneity and distinct patterns of immune response. In this review, we will discuss recent advances in the understanding of tumour infiltrated lymphocytes, their prognostic benefit, and predictive value for immunotherapy.

Separation of a new triterpenoid saponin together with six known ones from Clematis tangutica (Maxim.) Korsh and evaluation of their cytotoxic activities.

A new triterpenoid saponin, 3-O-ß-D-allopyranosyl (1→3)-α-L-rhamnopyranosyl (1→2)-α-L-arabinopyranosyl hederagenin 28-O-α-L-rhamnopyranosyl (1→4)-ß-D-glucopyranosyl (1→6)-ß-D-glucopyranosyl ester (IV), together with six known ones Hederacholichiside F (I), Tanguticoside B (II), Tauroside St-H1 (III), Hederoside H1 (V), Kalopanaxsaponin G (VI), Hederasaponin B (VII) were separated from Clematis tangutica (Maxim.) Korsh. Their cytotoxic activities were evaluated. Saponins IV (new compound) and I showed selective inhibitory activities against HGC-27 with IC50 values of 20.17 and 66.18 µM. Saponin VII exhibited extensive inhibitory action against HGC-27, Hela and SK-OV-3 with IC50 values of 16.47-71.36 µM. Saponin III showed selective inhibitory activity against SK-OV-3 with the IC50 value of 48.70 µM. All isolated saponins were inactive (IC50 >150 µM) to GES-1.

A UHPLC-Q-TOF-MS-based serum and urine metabolomics approach reveals the mechanism of Gualou-Xiebai herb pair intervention against atherosclerosis process in ApoE-/- mice.

Atherosclerosis (AS) is a metabolic disorder commonly correlated with a high-fat diet (HFD). There are many endogenous metabolic changes associated with AS development. Gualou-Xiebai (GLXB) is a traditional Chinese medicine herb pair that has been used to treat AS. However, the mechanism of GLXB herb pair on the process of AS is still essentially unknown. In this study, aortic histopathological examination and biochemical analyses were used to validate the anti-atherosclerotic effects of GLXB herb pair on ApoE-/- mice during the disease course of AS. The mechanism of GLXB herb pair were performed by metabolomics approach based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). As a result, GLXB herb pair has protective effects on AS lesion development and improves blood lipid levels in ApoE-/- mice. A total of 34, 39, and 49 metabolites were found to be profoundly altered in the 9-week, 14-week, and 19-week model groups compared with the corresponding control groups. Among them, 16, 18, and 18 metabolites showed a trend toward normal levels after pharmacological intervention. Metabolic pathway analysis found that GLXB herb pair mainly affects glycerophospholipid metabolism, pentose and glucuronate interconversions in 9 weeks; linoleic acid metabolism, cysteine and methionine metabolism, and arachidonic acid metabolism in 14 weeks; arachidonic acid metabolism and pentose and glucuronate interconversions in 19 weeks. The results demonstrated that GLXB herb pair mainly played a therapeutic role by regulating glycerophospholipid metabolism and pentose and glucuronate interconversions in the whole process of AS.

Modulation of intracellular kinase signaling to improve TIL stemness and function for adoptive cell therapy.

INTRODUCTION: Adoptive cellular therapy with tumor-infiltrating lymphocytes (TIL) has demonstrated promising clinical benefits in several solid tumors, but the efficacy of this therapy might be compromised by the "prone-to-exhaustion" phenotype of TIL and poor persistence in vivo. This calls for a robust expansion process to produce a large number of cells for clinical usage while at the same time maintaining favorable anti-tumor function and memory phenotype. Previous studies showed that the PI3K-AKT signaling pathway plays a key role in the regulation of T cell activation, differentiation and memory formation. METHOD: We modulated the PI3K-AKT pathway in TIL isolated from cervical and ovarian cancer by application of AKT or PI3K inhibitors or CRISPR knockout of AKT1 and/or AKT2, and characterized their effects on TIL phenotype and effector function. Mechanistic study was further performed with RNA-seq analysis of AKT1/2 KO TIL in comparison to control TIL. RESULT: The inhibition of either PI3K or AKT led to an increase in the population of effector CD8+ T cells with upregulation of activation markers, elevated CD39- CD69- memory T cells, and significantly enhanced cytotoxicity when cocultured with tumor cell lines and patient-derived tumor samples. Moreover, dual knockout of AKT1 and AKT2 largely phenocopies the functional impact of AKT or PI3K inhibition on TIL. This result was further validated by RNA-seq analysis indicating that AKT1/2 ablation primarily regulates T cell differentiation and function-related programs. CONCLUSION: Modulation of PI3K-AKT signaling represents a promising strategy to enhance TIL stemness and cytotoxicity and improve the clinical outcome of current TIL-based therapy to treat solid tumors.

Tuning the Spin State of the Iron Center by Bridge-Bonded Fe-O-Ti Ligands for Enhanced Oxygen Reduction.

Exploring functional substrates and precisely regulating the electronic structures of atomic metal active species with moderate spin state are of great importance yet remain challenging. Hereon, we provide an axial Fe-O-Ti ligand regulated spin-state transition strategy to improve the oxygen reduction reaction (ORR) activity of Fe centers. Theoretical calculations indicate that Fe-O-Ti ligands in FeN3 O-O-Ti can induce a low-to-medium spin-state transition and optimize O2 adsorption by FeN3 O. As a proof-of-concept, the oriented catalyst was prepared from atomic-Fe-doped polymer-like quantum dots and ultrathin o-terminated MXene. The optimal catalyst exhibits an intrinsic activity that is almost 5 times higher than the control sample (without axial Fe-O-Ti ligands). It also delivers a superior performance in Zn-air batteries and H2 /O2 anion exchange membrane fuel cells in a wide-temperature range.

Separation of five flavone glycosides including two groups with similar polarities from Dracocephalum tanguticum by a combination of three high-speed counter-current chromatography modes.

The separation of compounds with similar polarities is challenging. In the present study, five flavone glycosides, including two groups with similar polarities, were obtained from Dracocephalum tanguticum by three high-speed counter-current chromatography modes, including flow rate conversion mode, recycling mode, and heart-cut mode. With flow rate conversion mode, compounds 3 and 4 with similar polarities and compound 5 were separated by high-speed counter-current chromatography with ethyl acetate/methanol/water (5.0% acetic acid) (8:2:10, v/v) system. The flow rate was controlled as: 1.8 mL/min for 0-160 min, 2.2 mL/min for 160-200 min, and 2.5 mL/min for 200-400 min. However, compounds 1 and 2 with similar polarities were not separated due to the similar distributive properties. Then, a recycling and heart-cut mode were introduced to improve the separation efficiency. The heart-cut mode was introduced in the second and third cycles, and compounds 1 and 2 were well separated in the fourth cycle. Consequently, five flavone glycosides, including two groups with similar polarities were obtained and identified as cosmosiin (1), pedaliin (2), quercetin-3-O-rutinoside (3), pedaliin-6''-acetate (4), and sorbifolin-6-O-ß-glucopyranoside (5). The current strategy provides a reference for separating compounds with similar polarities from a crude sample.

Perspectives of tumor-infiltrating lymphocyte treatment in solid tumors.

Tumor-infiltrating lymphocyte (TIL) therapy is a type of adoptive cellular therapy by harvesting infiltrated lymphocytes from tumors, culturing and amplifying them in vitro and then infusing back to treat patients. Its diverse TCR clonality, superior tumor-homing ability, and low off-target toxicity endow TIL therapy unique advantages in treating solid tumors compared with other adoptive cellular therapies. Nevertheless, the successful application of TIL therapy currently is still limited to several types of tumors. Herein in this review, we summarize the fundamental work in the field of TIL therapy and the current landscape and advances of TIL clinical trials worldwide. Moreover, the limitations of the current TIL regimen have been discussed and the opportunities and challenges in the development of next-generation TIL are highlighted. Finally, the future directions of TIL therapy towards a broader clinical application have been proposed.

Peripheral eosinophil counts predict efficacy of anti-CD19 CAR-T cell therapy against B-lineage non-Hodgkin lymphoma.

Rationale: The onset of cytokine release syndrome (CRS) and in vivo persistence of anti-CD19 chimeric antigen receptor T (CAR-T) cells after infusion correlate with clinical responsiveness. However, there are no known baseline biomarkers that can predict the prognosis of patients with B-lineage non-Hodgkin lymphoma (B-NHL). The aim of this study was to identify blood cell populations associated with beneficial outcomes in B-NHL patients administered CAR-T cell immunotherapies. Methods: We enumerated peripheral blood and CAR-T cells by retrospectively analyzing three CAR-T cell trials involving 65 B-NHL patients. We used a preclinical model to elucidate the eosinophil mechanism in CAR-T cell therapy. Results: During an observation period up to 30 mo, B-NHL patients with higher baseline eosinophil counts had higher objective response rates than those with low eosinophil counts. Higher baseline eosinophil counts were also significantly associated with durable progression-free survival (PFS). The predictive significance of baseline eosinophil counts was validated in two independent cohorts. A preclinical model showed that eosinophil depletion impairs the intratumoral infiltration of transferred CAR-T cells and reduces CAR-T cell antitumor efficacy. Conclusion: The results of this study suggest that peripheral eosinophils could serve as stratification biomarkers and a recruitment machinery to facilitate anti-CD19 CAR-T cell therapy in B-NHL patients.

The toxicity of cell therapy: Mechanism, manifestations, and challenges.

Adoptive cell therapy (ACT), including tumor-infiltrating lymphocytes (TILs), T cell receptor engineered T cell (TCR-T), and chimeric antigen receptor engineered T cell (CAR-T), has shown significant clinical benefits for cancer treatment. However, all of these ACT therapies are associated with toxicities from mild to life threatening in clinic. Common ACT-related toxicities include cytokine release syndrome (CRS) resulting from immune activation, neurological toxicity, on-target/off tumor or off-target toxicities, and toxicities associated with lymphodepletion preconditioning and high does IL-2 administration. This review summarizes clinical manifestations of adverse events associated with ACT treatment and discusses the underlying pathological mechanisms. Moreover, challenges and opportunities of managing ACT-related toxicities have been discussed to give an indication of how to improve the safety of ACT treatment without dampening the therapeutic effect.

[Inhibitory effect of paeonol on aortic endothelial inflammation in atherosclerotic rats by up-regulation of caveolin-1 expression and suppression of NF-κB pathway].

To explore whether paeonol can play an anti-atherosclerotic role by regulating the expression of aortic caveolin-1 and affecting NF-κB pathway, so as to inhibit the inflammatory response of vascular endothelium in atherosclerotic rats. The atherosclerotic model of rats was induced by high-fat diet and vitamin D_2. The primary culture of vascular endothelial cells(VECs) was carried out by tissue block pre-digestion and adherent method. The injury model of VECs was induced by lipopolysaccharide(LPS), and filipin, a small concave protein inhibitor, was added for control. HE staining was used to observe pathological changes of aorta. TNF-α, IL-6 and VCAM-1 were detected by ELISA. Western blot assay was used to detect the protein expression levels of caveolin-1 and p65 in aorta and VECs. The results showed that as compared with model group, paeonol significantly reduced aortic plaque area and lesion degree in rats, decreased the level of serum TNF-α, IL-6 and VCAM-1 in the rats and enhanced the relative expression level of caveolin-1, decreased p65 expression conversely(P<0.05 or P<0.01). In vitro, as compared to model group, paeonol obviously improved cell morphology, decreased the secretion of TNF-α, IL-6 and VCAM-1 in VECs, increased caveolin-1 expression, and decreased p65 protein expression(P<0.05 or P<0.01). Furthermore, filipin could reverse the effect of paeonol on expression of inflammatory factors and proteins(P<0.05 or P<0.01). According to the results, it was found that paeonol could play the role of anti-atherosclerosis by up-regulating the expression of caveolin-1 and inhibiting the activation of NF-κB pathway to reduce vascular inflammation in atherosclerotic rats.

Hypoxia-induced apoptosis of cardiomyocytes is restricted by ginkgolide B-downregulated microRNA-29.

Ginkgolide B exerts a cardioprotective function against ischemia-caused apoptosis in myocardial infarction. Here we sought out to address a functional mechanism associated with microRNA-29 (miR-29). Rat cardiomyocytes (H9c2 cells) were cultured in ginkgolide B-conditioned medium prior to hypoxic induction. To construct miR-29-overexpressed cells, miR-29 mimic was transfected into H9c2 cells. The cells were harvested for assaying survivability and apoptosis by CCK-8 and FITC-Annexin V staining methods. Western blot was applied to identify apoptotic hallmarks and signaling transducers. RT-PCR was carried out for investigating miR-29 expression. Cardiomyocytes were sensitive to hypoxic apoptosis, while ginkgolide B intensified the abilities of cardiomyocytes to resist hypoxia by increasing survivability and repressing apoptosis. Specifically, ginkgolide B repressed Bax and cleaved caspase 3 while enhanced Bcl-2. Ginkgolide B buffered the expression of miR-29 induced by hypoxia. However, ginkgolide B showed a slight role in survivability and apoptosis in the cells overexpressing miR-29. Meanwhile, ginkgolide B triggered the phosphorylation of PI3 K and AKT, as well as induced Sp1, while this beneficial role was abrogated in the cells treated by miR-29 mimic. Our results confirmed that ginkgolide B might have therapeutic significance by repressing hypoxic apoptosis. Ginkgolide B-elicited miR-29 inhibition might be the basis of this beneficial role.

LDAI-ISPS: LncRNA-Disease Associations Inference Based on Integrated Space Projection Scores.

Long non-coding RNAs (long ncRNAs, lncRNAs) of all kinds have been implicated in a range of cell developmental processes and diseases, while they are not translated into proteins. Inferring diseases associated lncRNAs by computational methods can be helpful to understand the pathogenesis of diseases, but those current computational methods still have not achieved remarkable predictive performance: such as the inaccurate construction of similarity networks and inadequate numbers of known lncRNA-disease associations. In this research, we proposed a lncRNA-disease associations inference based on integrated space projection scores (LDAI-ISPS) composed of the following key steps: changing the Boolean network of known lncRNA-disease associations into the weighted networks via combining all the global information (e.g., disease semantic similarities, lncRNA functional similarities, and known lncRNA-disease associations); obtaining the space projection scores via vector projections of the weighted networks to form the final prediction scores without biases. The leave-one-out cross validation (LOOCV) results showed that, compared with other methods, LDAI-ISPS had a higher accuracy with area-under-the-curve (AUC) value of 0.9154 for inferring diseases, with AUC value of 0.8865 for inferring new lncRNAs (whose associations related to diseases are unknown), with AUC value of 0.7518 for inferring isolated diseases (whose associations related to lncRNAs are unknown). A case study also confirmed the predictive performance of LDAI-ISPS as a helper for traditional biological experiments in inferring the potential LncRNA-disease associations and isolated diseases.

TAK1 mediates microenvironment-triggered autocrine signals and promotes triple-negative breast cancer lung metastasis.

Triple-negative breast cancer (TNBC) is a highly metastatic subtype of breast cancer that has limited therapeutic options. Thus, developing novel treatments for metastatic TNBC is an urgent need. Here, we show that nanoparticle-mediated delivery of transforming growth factor-ß1-activated kinase-1 (TAK1) inhibitor 5Z-7-Oxozeaenol can inhibit TNBC lung metastasis in most animals tested. P38 is a central signal downstream of TAK1 in TNBC cells in TAK1-mediated response to multiple cytokines. Following co-culturing with macrophages or fibroblasts, TNBC cells express interleukin-1 (IL1) or tumor necrosis factor-α (TNFα), respectively. Compared to TAK1 inhibition, suppressing IL1 signaling with recombinant IL1 receptor antagonist (IL1RA) is less efficient in reducing lung metastasis, possibly due to the additional TAK1 signals coming from distinct stromal cells. Together, these observations suggest that TAK1 may play a central role in promoting TNBC cell adaptation to the lung microenvironment by facilitating positive feedback signaling mediated by P38. Approaches targeting the key TAK1-P38 signal could offer a novel means for suppressing TNBC lung metastasis.

CAR-T Cells Surface-Engineered with Drug-Encapsulated Nanoparticles Can Ameliorate Intratumoral T-cell Hypofunction.

One limiting factor of CAR T-cell therapy for treatment of solid cancers is the suppressive tumor microenvironment (TME), which inactivates the function of tumor-infiltrating lymphocytes (TIL) through the production of immunosuppressive molecules, such as adenosine. Adenosine inhibits the function of CD4+ and CD8+ T cells by binding to and activating the A2a adenosine receptor (A2aR) expressed on their surface. This suppression pathway can be blocked using the A2aR-specific small molecule antagonist SCH-58261 (SCH), but its applications have been limited owing to difficulties delivering this drug to immune cells within the TME. To overcome this limitation, we used CAR-engineered T cells as active chaperones to deliver SCH-loaded cross-linked, multilamellar liposomal vesicles (cMLV) to tumor-infiltrating T cells deep within the immune suppressive TME. Through in vitro and in vivo studies, we have demonstrated that this system can be used to effectively deliver SCH to the TME. This treatment may prevent or rescue the emergence of hypofunctional CAR-T cells within the TME. Cancer Immunol Res; 6(7); 812-24. ©2018 AACR.

In Vivo Expansion and Antitumor Activity of Coinfused CD28- and 4-1BB-Engineered CAR-T Cells in Patients with B Cell Leukemia.

Several recent clinical trials have successfully incorporated a costimulatory domain derived from either CD28 or 4-1BB with the original CD3ζ T cell activating domain to form second-generation chimeric antigen receptors (CARs) that can increase the responsiveness and survival of CAR-engineered T (CAR-T) cells. However, a rigorous assessment of the individual benefits of these costimulatory components relative to the in vivo performance of infused T cells in patients is still lacking. Therefore, we have designed a study that allows us to investigate and compare the impact of different costimulatory signal domains on CAR-T cells in vivo. Patients with B cell leukemia were infused with a mixture of two types of CD19-specific CAR-T cells, individually bearing CD28 (28ζ) and 4-1BB (BBζ) costimulatory signaling domains. We found that such a clinical procedure was feasible and safe. Complete remission (CR) was observed in five of seven enrolled patients, with two patients exhibiting durable CR lasting more than 15 months. The in vivo expansion pattern of 28ζ and BBζ CAR-T cells varied significantly among individual patients. These results confirm a feasible method of comparing different CAR designs within individual patients, potentially offering objective insights that may facilitate the development of optimal CAR-T cell-based immunotherapies.