[Study on Down-regulation of Interleukin-1ß Secretion by Inhibiting ABCC1/MRP1 Transporter].

OBJECTIVE: To screen interleukin (IL)-1ß secretion-related membrane transporters by macrophage experiment in vitro and conventional knockout mice. METHODS: THP-1 cell line was differentiated to obtain human THP-1-derived macrophages, and the primary macrophages were obtained from human peripheral blood. FVB wild-type mice with the same sex and age were used as the controls of MRP1 knockout mice. The macrophages in abdominal cavity and bone marrow of mice were cultivated. The cells were treated with ABCC1/MRP1, ABCG2/BCRP, ABCB1/P-gp, OATP1B1, and MATE transporter inhibitors, then stimulated by lipopolysaccharide and adenosine triphosphate. The secretion level of IL-1ß was detected by ELISA, Western blot, and immunofluorescence. RESULTS: After inhibiting ABCC1/MRP1 transporter, the secretion of IL-1ß decreased significantly, while inhibition of the other 4 transporters had no effect. In animal experiment, the level of IL-1ß secreted by macrophages in bone marrow of MRP1 knockout mice was significantly lower than control group (P < 0.05). CONCLUSION: ABCC1/MRP1 transporter is a newly discovered IL-1ß secretion pathway, which is expected to become a new target for solving clinical problems such as cytokine release syndrome.

A novel safer CD19CAR with shRNA interference of IFN-γ can reduce multiple cytokine levels without significantly compromising its killing efficacy.

Cytokine release syndrome (CRS) is a great challenge for the application of anti-CD19 CAR-T cell therapy. The aim of this study was to investigate the effect of knocking down interferon gamma (IFN-γ) by shRNA as a potential strategy to reduce the cytokine storms. A newly designed short hairpin interference RNA of IFN-γ (shIFN-γ) in CD19CAR gene was constructed. Several cellular model systems of approach using Nalm-6 cell lines including Nalm-6CD19pos and Nalm-6CD19neg with or without monocytes and endothelial cells were used to analyze the different levels of cytokines after shIFN-γ-anti-CD19CAR-T cell targeted therapy. The activity of this novel CD19CAR-T was evaluated both in vitro and in NSG mouse model. The killing efficacy of shIFN-γ-anti-CD19CAR-T at the E:T ratio of 2:1 was similar to that of regular anti-CD19CAR-T at the E:T ratio of 1:1. The IFN-γ level in the shIFN-γ-anti-CD19CAR-T cell group was (2673.1 ± 307.4) pg/ml at the E:T ratio of 2:1 which was significantly lower than that ((8261.5 ± 345.5) pg/ml) in the regular anti-CD19CAR-T group at the E:T ratio of 1:1. Cytotoxicity experiments in vitro showed significantly reduced concentrations of IFN-γ, IL-6 and TNFα in the shIFN-γ-anti-CD19CAR-T cell group compared to regular anti-CD19CAR-T cell group. Both regular anti-CD19CAR and shIFN-γ-CD19CAR-T exerted bystander killing effect in vitro. We conclude that shIFN-γ-anti-CD19CAR-T cells can reduce the generation of cytokine storms without significantly compromising their therapeutic efficacy in the preclinical setting. In mouse model, 3 × 106 shIFN-γ-anti-CD19CAR-T cells/mouse generated the similar killing efficacy to that with 2 × 106 regular anti-CD19CAR-T cells/mouse.

A scoring system based on fusion genes to predict treatment outcomes of the non-acute promyelocytic leukemia pediatric acute myeloid leukemia.

Background: Fusion genes are considered to be one of the major drivers behind cancer initiation and progression. Meanwhile, non-acute promyelocytic leukemia (APL) pediatric patients with acute myeloid leukemia (AML) in children had limited treatment efficacy. Hence, we developed and validated a simple clinical scoring system for predicting outcomes in non-APL pediatric patients with AML. Method: A total of 184 non-APL pediatric patients with AML who were admitted to our hospital and an independent dataset (318 patients) from the TARGET database were included. Least absolute shrinkage and selection operation (LASSO) and Cox regression analysis were used to identify prognostic factors. Then, a nomogram score was developed to predict the 1, 3, and 5 years overall survival (OS) based on their clinical characteristics and fusion genes. The accuracy of the nomogram score was determined by calibration curves and receiver operating characteristic (ROC) curves. Additionally, an internal verification cohort was used to assess its applicability. Results: Based on Cox and LASSO regression analyses, a nomogram score was constructed using clinical characteristics and OS-related fusion genes (CBFß::MYH11, RUNX1::RUNX1T1, KMT2A::ELL, and KMT2A::MLLT10), yielded good calibration and concordance for predicting OS of non-APL pediatric patients with AML. Furthermore, patients with higher scores exhibited worse outcomes. The nomogram score also demonstrated good discrimination and calibration in the whole cohort and internal validation. Furthermore, artificial neural networks demonstrated that this nomogram score exhibits good predictive performance. Conclusion: Our model based on the fusion gene is a prognostic biomarker for non-APL pediatric patients with AML. The nomogram score can provide personalized prognosis prediction, thereby benefiting clinical decision-making.

[Effect of Human Bone Marrow Mesenchymal Stem Cells with Ectopic High OCT4 Expression on T Lymphocyte Function].

OBJECTIVE: To explore the effect of human bone marrow mesenchymal stem cells (MSCs) with ectopic high OCT4 expression on T-cell proliferation, activation and secretion in vitro. METHODS: Peripheral blood mononuclear cells were isolated from healthy children. Anti-CD3 and anti-CD28 monoclonal antibodies were used to activate T lymphocytes, which were stimulated by interleukin (IL)-2 for one week in vitro. Then MSCs with ectopic high OCT4 expression (MSC-OCT4) were co-cultured with activated T lymphocytes. After one week of co-culture, the supernatant was collected and the levels of Th1/Th2 cytokines [IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α and interferon (IFN)-γ] were determined by flow cytometry. The lymphocytes after one week of co-culture were collected and counted by Countstar software. After the proportions of activated/inactivated T cell subsets were determined by flow cytometry, the absolute lymphocyte counts were calculated and expressed as mean ± standard deviation. RESULTS: Compared with control T cell alone culture group, the proliferation of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells were significantly inhibited in MSC group and MSC-OCT4 group. Compared with MSC, MSC-OCT4 could inhibit CD3+CD8+ T cell proliferation better (P =0.049), and mainly inhibited early T cell activation. Compared with control T cell alone culture group, the levels of IL-2 and INF-γ were significantly down-regulated both in MSC group and MSC-OCT4 group.After co-culture with T cells for one week, the level of IL-6 significantly increased in MSC group and MSC-OCT4 group compared with that before co-culture. Compared with control MSC group, MSC-OCT4 group had higher viable cell numbers after 1 week of co-culture (P =0.019), and could resist the inhibition of proliferation by higher concentration of mitomycin C. CONCLUSION: Both MSC and MSC-OCT4 can inhibit the proliferation and activation of IL-2-stimulated T cells in vitro. After overexpression of OCT4, MSC has better proliferation ability in vitro and can inhibit the proliferation of CD3+CD8+ T cells more effectively, which may have a better and more lasting immunosuppressive ability to regulate the balance of Th1/Th2.

[Advances in Structural Designs of Chimeric Antigen Receptor T Cells--Review].

Although chimeric antigen receptor (CAR)-T therapy has produced remarkable clinical responses for patients with relapsed or refractory hematological malignancies, setbacks were experienced, including antigen escape and heterogeneity, its efficacy and safety issues. In recent years, researchers at home and abroad are addressing the current obstacles by digging deeply into structural optimization of CAR gene in order to solve the problems of CAR-T cell therapy. In this review, we mainly illustrate the ectodomain structure, transmemberane domain, and endodomain structure, and new designs which promote persistence of CAR-T cells in vivo, so as to provide new ideas for improving the safety and the efficacy of CAR-T cell therapy.

U2AF1 expression is a novel and independent prognostic indicator of childhood T-lineage acute lymphoblastic leukemia.

INTRODUCTION: U2AF1 gene is associated with various types of hematological malignancies in adults. However, the expression level of U2AF1 gene and its prognostic significance are unclear in pediatric ALL patients. The study aimed to study the mRNA level of U2AF1 in pediatric ALL patients and its clinical relevance with long-term survival. METHODS: We quantitatively determined U2AF1 gene expression at diagnosis in 132 children with ALL by real-time PCR. According to the patients' median U2AF1 value, the patients' samples were classified into low U2AF1 and high U2AF1 expression groups. Twenty-two bone marrow samples from 22 patients with ITP were recruited as control. The correlation between the expression level of U2AF1 and clinical treatment outcome was analyzed. RESULTS: Pediatric patients with ALL showed higher U2AF1 mRNA levels than controls (P = .034). The relapse rates of patients in low U2AF1 levels group were obviously higher than those of U2AF1 high expression group (28.8% vs 12.1%, P = .030). Patients of low U2AF1 expression presented worse 5-year EFS than those of high U2AF1 expression (60% vs 81%, P = .035). For T-ALL, patients with low U2AF1 mRNA level showed lower BM blast percentages (P = .031), worse EFS (37.8% vs 92.3%, P = .003), and CIR (62.2% vs 7.7%, P = .003) than those in high U2AF1 expression group. Multivariate analysis confirmed low U2AF1 mRNA level could be used as an independent risk indicator of poor EFS and CIR of children with T-ALL. CONCLUSION: Low U2AF1 mRNA level is related to inferior prognosis and can be served as a prognostic indicator for risk stratification in children with T-ALL.