Modeling Rett Syndrome Using TALEN-Edited MECP2 Mutant Cynomolgus Monkeys.

Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT.

Impact of tocilizumab on anti-CD19 chimeric antigen receptor T-cell therapy in B-cell acute lymphoblastic leukemia.

BACKGROUND: Tocilizumab is commonly used for the management of chimeric antigen receptor (CAR) T-cell therapy-associated cytokine release syndrome (CRS). However, it remains unknown whether tocilizumab or its dosage affects the efficacy and safety of CAR T-cell therapy. The objective of this multicenter retrospective study was to explore the impact of tocilizumab on CAR T-cell therapy. METHODS: In total, 93 patients with B-cell acute lymphoblastic leukemia (B-ALL) receiving humanized anti-CD19 CAR T cells were recruited from May 2016 to November 2022. Forty-five patients received tocilizumab (tocilizumab group), whereas 48 patients did not (nontocilizumab group). Thirteen patients received >1 dose of tocilizumab. The primary end point was the effect of tocilizumab on the efficacy and safety of CAR T cells. Additionally, proliferation, killing, and cytokine assays of CAR T cells were performed in vitro in the presence of tocilizumab. RESULTS: The median age of the patients was 33 years, with 47 males and 46 females. Patients in the tocilizumab group showed similar complete response (CR) rate, overall survival (OS), and event-free survival (EFS) compared with the nontocilizumab group. Compared with patients who received ≤1 dose of tocilizumab, receiving >1 dose of tocilizumab did not affect their CR rate, OS, or EFS. In the tocilizumab group, all patients experienced CRS and 26.7% experienced immune effector cell-associated neurotoxicity syndrome (ICANS). In the nontocilizumab group, 64.6% of patients experienced CRS and 8.3% experienced ICANS. Up to 75% of ICANS and 87.5% of grade ≥3 ICANS occurred in the tocilizumab group. In vitro, tocilizumab did not impair the proliferation and killing effects of CAR T cells. CONCLUSIONS: Tocilizumab does not affect the efficacy of CAR T cells but may increase the likelihood of ICANS.

Efficacy and safety of CD19-specific CAR T cell-based therapy in B-cell acute lymphoblastic leukemia patients with CNSL.

Few studies have described chimeric antigen receptor (CAR) T-cell therapy for patients with B-cell acute lymphoblastic leukemia (B-ALL) with central nervous system leukemia (CNSL) because of concerns regarding poor response and treatment-related neurotoxicity. Our study included 48 patients with relapsed/refractory B-ALL with CNSL to evaluate the efficacy and safety of CD19-specific CAR T cell-based therapy. The infusion resulted in an overall response rate of 87.5% (95% confidence interval [CI], 75.3-94.1) in bone marrow (BM) disease and remission rate of 85.4% (95% CI, 72.8-92.8) in CNSL. With a median follow-up of 11.5 months (range, 1.3-33.3), the median event-free survival was 8.7 months (95% CI, 3.7-18.8), and the median overall survival was 16.0 months (95% CI, 13.5-20.1). The cumulative incidences of relapse in BM and CNS diseases were 31.1% and 11.3%, respectively, at 12 months (P = .040). The treatment was generally well tolerated, with 9 patients (18.8%) experiencing grade ≥3 cytokine release syndrome. Grade 3 to 4 neurotoxic events, which developed in 11 patients (22.9%), were associated with a higher preinfusion disease burden in CNS and were effectively controlled under intensive management. Our results suggest that CD19-specific CAR T cell-based therapy can induce similar high response rates in both BM and CNS diseases. The duration of remission in CNSL was longer than that in BM disease. CD19 CAR T-cell therapy may provide a potential treatment option for previously excluded patients with CNSL, with manageable neurotoxicity. The clinical trials were registered at www.clinicaltrials.gov as #NCT02782351 and www.chictr.org.cn as #ChiCTR-OPN-16008526.

Anti-CD79b/CD3 bispecific antibody combined with CAR19-T cells for B-cell lymphoma treatment.

CD19 CAR-T (chimeric antigen receptor-T) cell immunotherapy achieves a remission rate of approximately 70% in recurrent and refractory lymphoma treatment. However, the loss or reduction of CD19 antigen on the surface of lymphoma cells results in the escape of tumor cells from the immune killing of CD19 CAR-T cells (CAR19-T). Therefore, novel therapeutic strategies are urgently required. In this study, an anti-CD79b/CD3 bispecific antibody (BV28-OKT3) was constructed and combined with CAR19-T cells for B-cell lymphoma treatment. When the CD19 antigen was lost or reduced, BV28-OKT3 redirected CAR19-T cells to CD79b+ CD19- lymphoma cells; therefore, BV28-OKT3 overcomes the escape of CD79b+ CD19- lymphoma cells by the killing action of CAR19-T cells in vitro and in vivo. Furthermore, BV28-OKT3 triggered the antitumor function of CAR- T cells in the infusion product and boosted the antitumor immune response of bystander T cells, markedly improving the cytotoxicity of CAR19-T cells to lymphoma cells in vitro and in vivo. In addition, BV28-OKT3 elicited the cytotoxicity of donor-derived T cells toward lymphoma cells in vitro, which depended on the presence of tumor cells. Therefore, our findings provide a new clinical treatment strategy for recurrent and refractory B-cell lymphoma by combining CD79b/CD3 BsAb with CAR19-T cells.

Strategies to optimize chimeric antigen receptor T-cell therapy in hematologic malignancies: Chinese experience.

Chimeric antigen receptor (CAR) T-cell therapy has emerged as a promising form of adoptive T-cell immunotherapy for selected hematologic malignancies including leukemia, lymphoma and multiple myeloma. China has become the country with the largest number of registered CAR T-cell trials. Despite the remarkable clinical outcomes achieved with CAR Tcell therapy, challenges such as disease relapse, the process of manufacturing the CAR T cells and safety have limited the therapeutic efficacy of CAR T cells in hematologic malignancies. In this period of innovation, several clinical trials have reported the design of CAR directed at new targets in hematologic malignancies. In this review, we comprehensively summarize the contemporary landscape and clinical development of CAR T-cell therapy in China. In addition, we present strategies for further improving the clinical utility of CAR T-cell therapy, such as increasing the efficacy and response duration, in hematologic malignancies.

Sirt1 Protects Subventricular Zone-Derived Neural Stem Cells from DNA Double-Strand Breaks and Contributes to Olfactory Function Maintenance in Aging Mice.

DNA damage is assumed to accumulate in stem cells over time and their ability to withstand this damage and maintain tissue homeostasis is the key determinant of aging. Nonetheless, relatively few studies have investigated whether DNA damage does indeed accumulate in stem cells and whether this contributes to stem cell aging and functional decline. Here, we found that, compared with young mice, DNA double-strand breaks (DSBs) are reduced in the subventricular zone (SVZ)-derived neural stem cells (NSCs) of aged mice, which was achieved partly through the adaptive upregulation of Sirt1 expression and non-homologous end joining (NHEJ)-mediated DNA repair. Sirt1 deficiency abolished this effect, leading to stem cell exhaustion, olfactory memory decline, and accelerated aging. The reduced DSBs and the upregulation of Sirt1 expression in SVZ-derived NSCs with age may represent a compensatory mechanism that evolved to protect stem cells from excessive DNA damage, as well as mitigate memory loss and other stresses during aging.

Prognostic impact of peripheral eosinophil counts in patients with diffuse large B-cell lymphoma receiving chimeric antigen receptor T-cell therapy.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T-cell therapy is a breakthrough treatment for patients with relapsed or refractory diffuse large B-cell lymphoma. However, many patients do not achieve remission or relapse after remission. Previous studies have demonstrated that eosinophils have synergistic anti-tumor effects with CD8+T cells and pre-CAR T-eosinophil counts are associated with the efficacy of CAR T cells. METHODS: We retrospectively analyzed the eosinophil counts of patients with diffuse large B-cell lymphoma and found it changed remarkably pre- and post-CAR T-cell therapy. RESULTS: Patients who achieved complete remission after CAR T-cell infusion had greater post-CAR T-eosinophil counts than those who did not. Kaplan-Meier curves showed that patients with greater eosinophil counts during the second month after CAR T-cell infusion had superior progression-free survival and overall survival compared with those with lower eosinophil counts. CONCLUSIONS: For patients who responded to CAR T-cell therapy, eosinophil counts also can be used to predict 6-month duration of response. In conclusion, the post-CAR T-eosinophil count is associated with the prognosis of patients treated with CAR T-cell therapy and can be used to clinically identify patients who can achieve longer remission after CAR T-cell infusion.

FLT3 inhibition upregulates HDAC8 via FOXO to inactivate p53 and promote maintenance of FLT3-ITD+ acute myeloid leukemia.

Internal tandem duplication (ITD) mutations within the FMS-like receptor tyrosine kinase-3 (FLT3) can be found in up to 25% to 30% of acute myeloid leukemia (AML) patients and confer a poor prognosis. Although FLT3 tyrosine kinase inhibitors (TKIs) have shown clinical responses, they cannot eliminate primitive FLT3-ITD+ AML cells, which are potential sources of relapse. Therefore, elucidating the mechanisms underlying FLT3-ITD+ AML maintenance and drug resistance is essential to develop novel effective treatment strategies. Here, we demonstrate that FLT3 inhibition induces histone deacetylase 8 (HDAC8) upregulation through FOXO1- and FOXO3-mediated transactivation in FLT3-ITD+ AML cells. Upregulated HDAC8 deacetylates and inactivates p53, leading to leukemia maintenance and drug resistance upon TKI treatment. Genetic or pharmacological inhibition of HDAC8 reactivates p53, abrogates leukemia maintenance, and significantly enhances TKI-mediated elimination of FLT3-ITD+ AML cells. Importantly, in FLT3-ITD+ AML patient-derived xenograft models, the combination of FLT3 TKI (AC220) and an HDAC8 inhibitor (22d) significantly inhibits leukemia progression and effectively reduces primitive FLT3-ITD+ AML cells. Moreover, we extend these findings to an AML subtype harboring another tyrosine kinase-activating mutation. In conclusion, our study demonstrates that HDAC8 upregulation is an important mechanism to resist TKIs and promote leukemia maintenance and suggests that combining HDAC8 inhibition with TKI treatment could be a promising strategy to treat FLT3-ITD+ AML and other tyrosine kinase mutation-harboring leukemias.

Harnessing A3G for efficient and selective C-to-T conversion at C-rich sequences.

BACKGROUND: Site-specific C>T DNA base editing has been achieved by recruiting cytidine deaminases to the target C using catalytically impaired Cas proteins; the target C is typically located within 5-nt editing window specified by the guide RNAs. The prototypical cytidine base editor BE3, comprising rat APOBEC1 (rA1) fused to nCas9, can indiscriminately deaminate multiple C's within the editing window and also create substantial off-target edits on the transcriptome. A powerful countermeasure for the DNA off-target editing is to replace rA1 with APOBEC proteins which selectively edit C's in the context of specific motifs, as illustrated in eA3A-BE3 which targets TC. However, analogous editors selective for other motifs have not been described. In particular, it has been challenging to target a particular C in C-rich sequences. Here, we sought to confront this challenge and also to overcome the RNA off-target effects seen in BE3. RESULTS: By replacing rA1 with an optimized human A3G (oA3G), we developed oA3G-BE3, which selectively targets CC and CCC and is also free of global off-target effects on the transcriptome. Furthermore, we created oA3G-BE4max, an upgraded version of oA3G-BE3 with robust on-target editing. Finally, we showed that oA3G-BE4max has negligible Cas9-independent off-target effects at the genome. CONCLUSIONS: oA3G-BE4max can edit C(C)C with high efficiency and selectivity, which complements eA3A-editors to broaden the collective editing scope of motif selective editors, thus filling a void in the base editing tool box.

Dnmt3a is downregulated by Stat5a and mediates G0/G1 arrest by suppressing the miR-17-5p/Cdkn1a axis in Jak2V617F cells.

BACKGROUND: Despite of the frequently reported Dnmt3a abormality in classical myeloproliferative neoplasms (cMPNs) patients, few research explores how the Dnmt3a is regulated by Jak2V617F mutation. In this study, we have investigated how the Dnmt3a is regulated by Jak2V617F mutation and its effects on downstream signaling pathways in cMPNs. METHODS: Specimens of Jak2V617F positive cMPN patients and normal controls were collected. Murine BaF3 cell line was used to construct cell models. Dual-Glo luciferase assays and chromatin immunoprecipitation (ChIP)-qPCR were performed to detect the impact of Stat5a on transcription activity of Dnmt3a. Soft agar colony formation assay and cell counting assay were performed to detect cell proliferation. BrdU staining and flow cytometry were used to investigate cell cycle distribution. Western blotting and quantitative reverse-transcription PCR (qPCR) were performed to detect the expression levels of genes. RESULTS: Firstly, the results of western blotting and qPCR revealed that compared with the control samples, Dnmt3a is downregulated in Jak2V617F positive samples. Then we explored the mechanism behind it and found that Dnmt3a is a downstream target of Stat5a, the transcription and translation of Dnmt3a is suppressed by the binding of aberrantly activated Stat5a with Dnmt3a promoter in Jak2V617F positive samples. We further revealed the region approximately 800 bp upstream of the first exon of the Dnmt3a promoter, which includes a gamma-activated sequence (GAS) motif of Stat5a, is the specific site that Stat5a binds to. Soft agar colony formation assay, cell counting assay, and BrdU staining and flow cytometry assay found that Dnmt3a in Jak2V617F-BaF3 cells significantly affected the cell proliferation capacity and cell cycle distribution by suppressing Cdkn1a via miR-17-5p/Cdkn1a axis and mediated G0/G1 arrest. CONCLUSIONS: Transcription and translation of Dnmt3a is downregulated by the binding of Stat5a with Dnmt3a promoter in Jak2V617F cells. The GAS motif at promoter of Dnmt3a is the exact site where the Stat5a binds to. Dnmt3a conducted G0/G1 arrest through regulating miR-17-5p/Cdkn1a axis. The axis of Stat5a/Dnmt3a/miR-17-5p/Cdkn1a potentially provides a treatment target for cMPNs.

DNA Damage Response in Quiescent Hematopoietic Stem Cells and Leukemia Stem Cells.

In humans, hematopoietic stem cells (HSCs) adopt unique responsive pathways counteracting with the DNA-damaging assaults to weigh the balance between the maintenance of normal stem cell poor for whole-life blood regeneration and the transformation to leukemia stem cells (LSCs) for leukemia initiation. LSCs also take actions of combating with the attack launched by externally therapeutic drugs that can kill most leukemic cells, to avoid extermination and promote disease relapse. Therefore, the collection of knowledge about all these underlined mechanisms would present a preponderance for later studies. In this chapter, the universal DNA damage response (DDR) mechanisms were firstly introduced, and then DDR of HSCs were presented focusing on the DNA double-strand breaks in the quiescent state of HSCs, which poses a big advantage in promoting its transformation into preleukemic HSCs. Lastly, the DDR of LSCs were summarized based on the major outcomes triggered by different pathways in specific leukemia, upon which some aspects for future investigations were envisioned under our currently limited scope of knowledge.

KU70 Inhibition Impairs Both Non-Homologous End Joining and Homologous Recombination DNA Damage Repair Through SHP-1 Induced Dephosphorylation of SIRT1 in T-Cell Acute Lymphoblastic Leukemia (T-ALL) [corrected].

BACKGROUND/AIMS: T-Cell Acute Lymphoblastic Leukemia (T-ALL) [corrected] is an aggressive disease which is highly resistant to chemotherapy. Studies show that enhanced ability of DNA damage repair (DDR) in cancer cells plays a key role in chemotherapy resistance. Here, we suggest that defect in DDR related genes might be a promising target to destroy the genome stability of tumor cells. METHODS: Since KU70 is highly expressed in Jurkat cells, one of the most representative cell lines of ATL, we knocked down KU70 by shRNA and analyzed the impact of KU70 deficiency in Jurkat cells as well as in NOD-SCID animal models by western blot, immunofluorescence, flow cytometry and measuring DNA repair efficiency. RESULTS: It is observed that silencing of KU70 resulted in accumulated DNA damage and impaired DDR in Jurkat cells, resulting in more apoptosis, decreased cell proliferation and cell cycle arrest. DNA damage leads to DNA double-strand breaks (DSBs), which are processed by either non-homologous end joining(NHEJ) or homologous recombination(HR). In our study, both NHEJ and HR are impaired because of KU70 defect, accompanied with increased protein level of SHP-1, a dephosphorylation enzyme. In turn, SHP-1 led to dephosphorylation of SIRT1, which further impaired HR repair efficiency. Moreover, KU70 deficiency prolonged survival of Jurkat-xenografted mice. CONCLUSION: These findings suggest that targeting KU70 is a promising target for ATL and might overcome the existing difficulties in chemotherapy.

SIRT1 Involved in the Regulation of Alternative Splicing Affects the DNA Damage Response in Neural Stem Cells.

BACKGROUND/AIMS: Alternative splicing and DNA damage exhibit cross-regulation, with not only DNA damage inducing changes in alternative splicing, but alternative splicing itself possibly modulating the DNA damage response (DDR). Sirt1, a prominent anti-aging player, plays pivotal roles in the DDR. However, few studies have examined alternative splicing with DNA damage in neural stem cells (NSCs) and, in essence, nothing is known about whether SIRT1 regulates alternative splicing. Hence, we investigated the potential involvement of Sirt1-mediated alternative splicing in the NSC DDR. METHODS: Genome-wide alternative splicing profiling was performed upon DNA damage induction and SIRT1 deletion. RESULTS: DNA damage caused genome-wide changes in alternative splicing in adult NSCs and Sirt1 deficiency dramatically altered DDR-related alternative splicing. In particular, extensive alternative splicing changes in DDR-related processes such as cell cycle control and DNA damage repair were observed; these processes were dramatically influenced by Sirt1 deficiency. Phenotypically, Sirt1 deficiency altered the proliferation and DNA repair of adult NSCs, possibly by regulating alternative splicing. CONCLUSION: SIRT1 helps to regulate alternative splicing, which itself affects the DDR of NSCs. Our findings provide novel insight into the mechanisms underlying the DDR in stem cells.

Synergistic action of 5Z-7-oxozeaenol and bortezomib in inducing apoptosis of Burkitt lymphoma cell line Daudi.

Treatment failure in cancer chemotherapy is largely due to the toxic effects of chemotherapeutic agents on normal cells/tissues. The proteasome inhibitor bortezomib has been successfully applied to treat multiple myeloma (MM), but there are some common adverse reactions in the clinic including peripheral neuropathy (PN). The TAK1 selective inhibitor 5Z-7-oxozeaenol has been widely studied in cancer therapy. Here, we investigated the potential synergy of bortezomib and 5Z-7-oxozeaenol in Burkitt's lymphoma (BL) cell lines. Cell viability assay showed that co-treatment of bortezomib at 8 nM, representing a one-eighth concentration for growth arrest, and 5Z-7-oxozeaenol at 2 µM, a dose that exhibited insignificant cytotoxic effects, synergistically induced apoptosis in the cell line Daudi. In parallel with the increasing dose of the bortezomib, and 5Z-7-oxozeaenol at 0.5 µM, lower colony formation efficiencies were seen in the cell line Daudi. Western blotting analysis verified that TAK1 inhibition by 5Z-7-oxozeaenol completely blocked JNK, p38, Erk, IKK, and IκB phosphorylation, which was almost instantly activated by TAK1 both directly or indirectly. Both agents synergistically prevented nuclear translocation of NF-κB, a characteristic of NF-κB inactivation. Moreover, a synergistic effect of bortezomib and 5Z-7-oxozeaenol on Western blotting analysis and flow cytometry was disclosed. Collectively, our results indicated that the proteasome inhibitor bortezomib and the TAK1 inhibitor 5Z-7-oxozeaenol displayed synergy on inhibiting BL cell apoptosis by inhibiting NF-κB activity.

Concise reviews: Characteristics and potential applications of human dental tissue-derived mesenchymal stem cells.

Recently, numerous types of human dental tissue-derived mesenchymal stem cells (MSCs) have been isolated and characterized, including dental pulp stem cells, stem cells from exfoliated deciduous teeth, periodontal ligament stem cells, dental follicle progenitor cells, alveolar bone-derived MSCs, stem cells from apical papilla, tooth germ progenitor cells, and gingival MSCs. All these MSC-like cells exhibit self-renewal, multilineage differentiation potential, and immunomodulatory properties. Several studies have demonstrated the potential advantages of dental stem cell-based approaches for regenerative treatments and immunotherapies. This review outlines the properties of various dental MSC-like populations and the progress toward their use in regenerative therapy. Several dental stem cell banks worldwide are also introduced, with a view toward future clinical application.

ß-Catenin overexpression is associated with gefitinib resistance in non-small cell lung cancer cells.

BACKGROUND: Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) presents great challenges in the treatment of non-small cell lung cancer (NSCLC) patients, while the mechanisms are still not well understood. The ß-catenin signaling pathway has been found to be associated with chemoresistance and can activate the EGFR and its downstream pathways. This study aimed to investigate the role of ß-catenin in acquired resistance to EGFR-TKIs in NSCLC cell lines. METHODS: The expression and transcriptional activity of ß-catenin were measured in both the NSCLC cell line PC9 and its sub-line PC9/AB(2) which has acquired resistance to gefitinib. Knockdown and overexpression of ß-catenin in the PC9/AB(2) and PC9 cells were performed. The cell survival rate and the activation of the EGFR and its downstream pathways were detected in the two cell lines after transfection. RESULTS: Nuclear translocation of ß-catenin was increased in the PC9/AB(2) cells and the baseline expression of members of the ß-catenin signaling pathway was also higher in the PC9/AB(2) cells. Knocking down the expression of ß-catenin increased the sensitivity of the PC9/AB(2) cells to gefitinib by blocking the activation of the EGFR downstream pathways, while ß-catenin overexpression improved PC9 cells resistance to gefitinib by enhancing the activation of the EGFR and its downstream signaling. CONCLUSION: ß-catenin plays an important role in acquired resistance to EGFR-TKIs in NSCLC cell lines and may be a potential therapeutic target for NSCLC patients who have failed to respond to targeted therapy.

Immune-related chemotactic factors were found in acute coronary syndromes by bioinformatics.

DNA microarray data for thrombus-related leukocyte from patients with acute coronary syndrome (ACS) was analyzed to acquire key genes associated with ACS. Microarray data set GSE19339, including four ACS patients' samples and four normal samples, were downloaded from Gene Expression Omnibus database. Raw data was pre-processed and differentially expressed genes (DEGs) were identified by Affy packages of R. The interaction network was established with STRING. DrugBank was retrieved to obtain relevant small molecules. A total of 487 differentially expressed genes were identified as DEGs between normal and disease samples. Among which, ten up-regulated genes belonging to chemokine family (CCL2, CCR1, CXCL3, CXCL2, CCL8, CXCL11, CCL7, IL10, CCL22 and CCL20) were related to inflammatory response. In addition, two inhibitors of CCL2 (L-Mimosine) were retrieved from the DrugBank database. Considering the roles of inflammatory response in the progression of ACS and the functions of the ten up-regulated genes, we speculated that these genes might be related to ACS. Moreover, the inhibitors could provide guidelines for future drug design acting on these genes.

Serine and Arginine-Rich Splicing Factor 3 Promotes the Activation of Quiescent Mouse Neural Stem Cells.

The quiescence and activation of adult stem cells are regulated by many kinds of molecular mechanisms, and RNA alternative splicing participates in regulating many cellular processes. However, the relationship between stem cell quiescence and activation regulation and gene alternative splicing has yet to be studied. In this study, we aimed to elucidate the regulation of stem cell quiescence and activation by RNA alternative splicing. The upregulated genes in activated mouse neural stem cells (NSCs), muscle stem cells, and hematopoietic stem cells were collected for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The genes from three tissue stem cells underwent Venn analysis. The mouse NSCs were used for quiescence and reactivation induction. The immunostaining of cell-specific markers was performed to identify cell properties. The reverse transcription-polymerase chain reaction and western blotting were used to detect the gene expression and protein expression, respectively. We found that the upregulated genes in activated stem cells from three tissues were all enriched in RNA splicing-related biological processes; the upregulated RNA splicing-related genes in activated stem cells displayed tissue differences; mouse NSCs were successfully induced into quiescence and reactivation in vitro without losing differentiation potential; serine and arginine-rich splicing factor 3 (Srsf3) was highly expressed in the activated mouse NSCs, and the overexpression of SRSF3 protein promoted the activation of quiescent mouse NSCs and increased the neural cell production. Our data indicate that the alternative splicing change may underline the transition of quiescence and activation of stem cells. The manipulation of the splicing factor may benefit tissue repair by promoting the activation of quiescent stem cells.

Tumor-derived exosomes induce initial activation by exosomal CD19 antigen but impair the function of CD19-specific CAR T-cells via TGF-ß signaling.

Tumor-derived exosomes (TEXs) enriched in immune suppressive molecules predominantly drive T-cell dysfunction and impair antitumor immunity. Chimeric antigen receptor (CAR) T-cell therapy has emerged as a promising treatment for refractory and relapsed hematological malignancies, but whether lymphoma TEXs have the same impact on CAR T-cell remains unclear. Here, we demonstrated that B-cell lymphoma-derived exosomes induce the initial activation of CD19-CAR T-cells upon stimulation with exosomal CD19. However, lymphoma TEXs might subsequently induce CAR T-cell apoptosis and impair the tumor cytotoxicity of the cells because of the upregulated expression of the inhibitory receptors PD-1, TIM3, and LAG3 upon prolonged exposure. Similar results were observed in the CAR T-cells exposed to plasma exosomes from patients with lymphoma. More importantly, single-cell RNA sequencing revealed that CAR T-cells typically showed differentiated phenotypes and regulatory T-cell (Treg) phenotype conversion. By blocking transforming growth factor ß (TGF-ß)-Smad3 signaling with TGF-ß inhibitor LY2109761, the negative effects of TEXs on Treg conversion, terminal differentiation, and immune checkpoint expression were rescued. Collectively, although TEXs lead to the initial activation of CAR T-cells, the effect of TEXs suppressed CAR T-cells, which can be rescued by LY2109761. A treatment regimen combining CAR T-cell therapy and TGF-ß inhibitors might be a novel therapeutic strategy for refractory and relapsed B-cell lymphoma.

CAR-Aptamers Enable Traceless Enrichment and Monitoring of CAR-Positive Cells and Overcome Tumor Immune Escape.

Chimeric antigen receptor (CAR)-positive cell therapy, specifically with anti-CD19 CAR-T (CAR19-T) cells, achieves a high complete response during tumor treatment for hematological malignancies. Large-scale production and application of CAR-T therapy can be achieved by developing efficient and low-cost enrichment methods for CAR-T cells, expansion monitoring in vivo, and overcoming tumor escape. Here, novel CAR-specific binding aptamers (CAR-ap) to traceless sort CAR-positive cells and obtain a high positive rate of CAR19-T cells is identified. Additionally, CAR-ap-enriched CAR19-T cells exhibit similar antitumor capacity as CAR-ab (anti-CAR antibody)-enriched CAR-T cells. Moreover, CAR-ap accurately monitors the expansion of CAR19-T cells in vivo and predicts the prognosis of CAR-T treatment. Essentially, a novel class of stable CAR-ap-based bispecific circular aptamers (CAR-bc-ap) is constructed by linking CAR-ap with a tumor surface antigen (TSA): protein tyrosine kinase 7 (PTK7) binding aptamer Sgc8. These CAR-bc-aps significantly enhance antitumor cytotoxicity with a loss of target antigens by retargeting CAR-T cells to the tumor in vitro and in vivo. Overall, novel CAR-aptamers are screened for traceless enrichment, monitoring of CAR-positive cells, and overcoming tumor cell immune escape. This provides a low-cost and high-throughput approach for CAR-positive cell-based immunotherapy.