A novel costimulatory molecule gene-modified leukemia cell-derived exosome enhances the anti-leukemia efficacy of DC vaccine in mouse models.

OBJECTIVES: Leukemia cell-derived exosomes (LEXs), carrying leukemia cell-specific antigens, can serve as a source of antigen for dendritic cell (DC) vaccine loading. However, LEX-targeted DC-based vaccines have demonstrated limited antitumor immune effects in clinical trials, attributed to the low immunogenicity of LEXs and the scant levels of costimulatory molecules on DCs. The costimulatory molecules CD80 and CD86, which are crucial to DC function, play a significant role in enhancing immune efficacy. In this study, we explored the anti-leukemia immune response of costimulatory molecule gene-modified LEX-targeted DCs (LEX-8086) in vitro and in animal models. METHODS: DCs were incubated with LEX-8086 to produce LEX-8086-targeted DCs (DCsLEX-8086). ELISA, cytotoxicity assays and flow cytometry utilized to assess the antitumor efficacy of DCsLEX8086 in vitro. Flow cytometry was used to evaluate the immunomodulatory function of DCsLEX8086 in animal models. RESULTS: Our findings indicated that LEX-8086 enhanced the maturation and antigen-presenting ability of DCs. Immunization with DCsLEX8086 significantly activated CD8+ T cells and boosted the CTL response in vitro. More importantly, DCsLEX-8086 effectively suppressed tumor growth and exerted anti-leukemia effects in both prophylactic and therapeutic animal models. Furthermore, DCsLEX-8086 promoted the proportion of CD4+ T cells, CD8+ T cells and M1 macrophages in the tumor environments both prophylactically and therapeutically. Treatment with DCsLEX-8086 showed no significant difference in the levels of M2 macrophages but decreased the proportion of Tregs within the tumor bed during therapeutic experiments. CONCLUSION: The results suggested that DCsLEX-8086 induces a more effective anti-leukemia immunity compared to DCsLEX-null in vivo and in vitro. DCsLEX-8086 might achieve antitumor effects by elevating the numbers of CD4+ T cells, CD8+ T cells, and M1 macrophages in tumors. Our findings indicate that DCsLEX-8086 could be leveraged to develop a new, highly effective vaccine for anti-leukemia immunity.

Endothelial-derived small extracellular vesicles support B-cell acute lymphoblastic leukemia development.

PURPOSE: The bone marrow niche plays an important role in leukemia development. However, the contributions of different niche components to leukemia development and their underlying mechanisms remain largely unclear. METHOD: Cre/LoxP-based conditional knockout technology was used to delete VPS33B or ANGPTL2 gene in niche cells. Murine B-ALL model was established by overexpressing the N-Myc oncogene in hematopoietic stem progenitor cells. The frequency of leukemia cells and immunophenotypic B220+ CD43+ LICs was detected by flow cytometry. SEVs was isolated by sequential centrifugation and mass spectrometry was performed to analyze the different components of SEVs. Immunoprecipitation and western blot were used to measure the interaction of VPS33B and ANGPTL2. RESULTS: Here, we showed that specific knockout of vascular protein sorting 33b (Vps33b) in endothelial cells (ECs), but not megakaryocytes or mesenchymal stem cells, resulted in a significant decrease in the secretion of small extracellular vesicles (SEVs) and a delay in the development of B-cell lymphoblastic leukemia (B-ALL). Vps33b knockdown endothelial cells contained much lower levels of SEVs that contained angiopoietin-like protein 2 (ANGPTL2) than the control cells. Importantly, conditional knockout of Angptl2 in ECs significantly delayed B-ALL progression. Moreover, C-terminal region of ANGPTL2 (aa247-471) could directly interact with Sec1-like domain 1 of VPS33B (aa1-aa146). We further demonstrated that the point mutations R399H and G402S in ANGPTL2 led to a dramatic decrease in the secretion of ANGPTL2-SEVs. We also showed that wild-type ANGPTL2-containing SEVs, but not mutant ANGPTL2-containing SEVs, significantly enhanced B-ALL development. CONCLUSION: In summary, our findings indicate that the secretion of ANGPTL2-containing SEVs in ECs sustains the leukemogenic activities of B-ALL cells, which is fine-tuned by the direct interaction of VPS33B and ANGPTL2. These findings reveal that niche-specific SEVs play an important role in B-ALL development.

Mesenchymal stem cell-derived exosomes can alleviate GVHD and preserve the GVL effect in allogeneic stem cell transplantation animal models.

Background: Mesenchymal stem cells (MSCs) can alleviate graft-versus-host disease (GVHD) in hematopoietic stem cell transplantation (HSCT). MSCs-derived exosomes (MEXs) can mirror the biological function of their parent cells. Whether MEXs can alleviate GVHD like their parent cells or not is unclear. In this study, we investigate the effects of MEXs on GVHD and graft-versus-leukemia (GVL) effect in vitro and in HSCT animal models. Method: MSCs were produced using bone marrow mononuclear cells (MNCs), and MEXs were separated from the supernatants of MSCs. Electron microscopy, western blot, and nanoparticle tracking analysis (NTA) were used to determine the characteristics of MEXs. The immunomodulatory function of MEXs and their effects on GVHD and GVL were examined in vitro and in vivo. Result: Like other cell-type derived exosomes, our data revealed that MEXs were also disc-shaped vesicles with a diameter of 100-200 nm under electron microscopy and were positive for the exosomal hallmark proteins. MEXs can notably inhibit the expression of costimulatory molecules and functional cytokine secretion of dendritic cells (DCs). Meanwhile, MEXs can exert suppressive effects on T lymphocyte proliferation and activation. Moreover, MEXs can also encourage the polarization of macrophages toward the M2 type. In animal HSCT models, MEXs can promote the differentiation of Treg cells in spleens, decrease the GVHD score, increase the survival rate of mice, and preserve the cytotoxic antileukemia effects of CD8+ T lymphocytes from recipient mice. Conclusion: These findings showed that MEXs exert their effects by inhibiting the immunomodulatory function of DCs, macrophages, and T lymphocytes. In the animal model, MEXs ameliorate the clinical symptoms of GVHD, while maintaining the antitumor effects of CD8+ T lymphocytes. Therefore, it can be inferred that MEXs can separate GVHD from GVL in HSCT. Our study suggests that MEXs have broad clinical application potential in the prevention and treatment of GVHD in HSCT in the near future.

P2X1 enhances leukemogenesis through PBX3-BCAT1 pathways.

How bone marrow niches regulate leukemogenic activities of leukemia-initiating cells (LICs) is unclear. The present study revealed that the metabolic niche component, ATP, efficiently induced ion influx in LICs through its ligand-gated ion channel, P2X1. P2X1 deletion impaired LIC self-renewal capacities and resulted in an approximately 8-fold decrease in functional LIC numbers in a murine acute myeloid leukemia (AML) model without affecting normal hematopoiesis. P2X1 phosphorylation at specific sites of S387 and T389 was essential for sustaining its promoting effects on leukemia development. ATP-P2X1-mediated signaling upregulated the PBX3 level to transactivate BCAT1 to maintain LIC fates. P2X1 knockdown inhibited the proliferation of both human AML cell lines and primary cells. The P2X1 antagonist sufficiently suppressed AML cell proliferation. These results provided a unique perspective on how metabolic niche factor ATP fine-tunes LIC activities, which may benefit the development of strategies for targeting LICs or other cancer stem cells.

A novel costimulatory molecule gene-modified leukemia cell-derived exosome-targeted CD4+ T cell vaccine efficiently enhances anti-leukemia immunity.

Previous studies demonstrated that CD4+ T cells can uptake tumor antigen-pulsed dendritic cell-derived exosomes (DEXO), which harbor tumor antigen peptide/pMHC I complex and costimulatory molecules and show potent effects on inducing antitumor immunity. However, in preliminary study, CD4+ T cells targeted by leukemia cell-derived exosomes (LEXs) did not show the expected effects in inducing effective anti-leukemia immunity, indicating that LEX is poorly immunogenetic largely due to an inadequate costimulatory capacity. Therefore, LEX-based anti-leukemia vaccines need to be optimized. In this study, we constructed a novel LEX-based vaccine by combining CD4+ T cells with costimulatory molecules gene-modified LEXs, which harbor upregulated CD80 and CD86, and the anti-leukemia immunity of CD80 and CD86 gene-modified LEX-targeted CD4+ T cells was investigated. We used lentiviral vectors encoding CD80 and CD86 to successfully transduced the L1210 leukemia cells, and the expression of CD80 and CD86 was remarkably upregulated in leukemia cells. The LEXs highly expressing CD80 and CD86 were obtained from the supernatants of gene-transduced leukemia cells. Our data have shown that LEX-CD8086 could promote CD4+ T cell proliferation and Th1 cytokine secretion more efficiently than control LEXs. Moreover, CD4+ TLEX-CD8086 expressed the acquired exosomal costimulatory molecules. With acquired costimulatory molecules, CD4+ TLEX-CD8086 can act as APCs and are capable of directly stimulating the leukemia cell antigen-specific CD8+ CTL response. This response was higher in potency compared to that noted by the other formulations. Furthermore, the animal study revealed that the CD4+ TLEX-CD8086 significantly inhibited tumor growth and prolonged survival of tumor-bearing mice than other formulations did in both protective and therapeutic models. In conclusion, this study revealed that CD4+ TLEX-CD8086 could effectively induce more potential anti-leukemia immunity than LEX-CD8086 alone, suggesting that the utilization of a costimulatory molecule gene-modified leukemia cell-derived exosome-targeted CD4+ T cell vaccine may have promising potential for leukemia immunotherapy.

Distinct roles but cooperative effect of TLR3/9 agonists and PD-1 blockade in converting the immunotolerant microenvironment of irreversible electroporation-ablated tumors.

Irreversible electroporation (IRE) is a new cancer ablation technology, but methods to improve IRE-induced therapeutic immunity are only beginning to be investigated. We developed a mouse model bearing large primary (300 mm3) and medium distant (100 mm3) EG7 lymphomas engineered to express ovalbumin (OVA) as a nominal tumor antigen. We established experimental protocols including IRE alone and IRE combined with Toll-like receptor (TLR)3/9 agonists (poly I:C/CpG) (IRE + pIC/CpG), PD-1 blockade (IRE + PD-1 blockade), or both (IRE + Combo) to investigate therapeutic effects on primary and distant EG7 tumors and conversion-promoting effects on the immunotolerant tumor microenvironment (TME). We demonstrated that IRE alone simulated very weak OVA-specific CD8+ T cell responses and did not inhibit primary tumor growth. IRE + pIC/CpG synergistically stimulated more efficient OVA-specific CD8+ T cell responses and primary tumor growth inhibition than IRE + PD-1 blockade. IRE + pIC/CpG played a major role in the modulation of immune cell profiles but a minor role in the downregulation of PD-L1 expression in the TME and vice versa for IRE + PD-1 blockade. IRE + Combo cooperatively induced potent OVA-specific CD8+ T cell immunity and rescued exhausted intratumoral CD8+ T cells, leading to eradication of not only primary tumors but also untreated concomitant distant tumors and lung metastases. IRE + Combo efficiently modulated immune cell profiles, as evidenced by reductions in immunotolerant type-2 (M2) macrophages, myeloid-derived suppressor-cells, plasmacytoid dendritic cells, and regulatory T cells and by increases in immunogenic M1 macrophages, CD169+ macrophages, type-1 conventional dendritic cells, and CD8+ T cells, leading to conversion of immunotolerance in not only primary TMEs but also untreated distant TMEs. IRE + Combo also showed effective therapeutic effects in two breast cancer models. Therefore, our results suggest that IRE + Combo is a promising strategy to improve IRE ablation therapy in cancer.

Construction of lncRNA-related ceRNA regulatory network in diabetic subdermal endothelial cells.

Long non-coding RNAs (lncRNAs) were considered to be involved in vascular complications in diabetes mellitus, but still only limited knowledge in this regard has been obtained. Herein, we further explored the roles of lncRNAs and mRNAs in diabetic vasculopathy (DV) through conducting bioinformatics analysis using data set downloaded from GEO database. The differentially expressed lncRNAs and mRNAs were identified by edge package. GO enrichment analysis and KEGG pathway analysis were performed based on clusterprofiler package. The relationship between lncRNA and miRNA was predicted using starBase database, and the potential mRNAs targeted by miRNAs were predicted by TargetScan, miRTarbase and miRDB database. The string database was used to analyze the protein-protein interaction (PPI). As a result, a total of 12 lncRNAs and 711 mRNAs were found to be differentially expressed in the diabetic subdermal endothelial cells compared with normal controls. A ceRNA network was established, which was composed of seven lncRNA nodes, 49 miRNA nodes, 58 mRNA nodes and 183 edges, and MSC-AS1 and LINC01550 may serve as key nodes. GO function enrichment analysis showed enrichments of epithelial cell proliferation, intercellular junction, and cell adhesion molecule binding. KEGG pathway analysis revealed 33 enriched pathways. PPI protein interaction analysis identified 57 potential ceRNA-related proteins. Overall, this study suggests that multiple lncRNAs, specifically MSC-AS1 and LINC01550, may play an important role in DV development and they are like to be developed as the therapeutic targets for DV. However, further experiments in vitro and in vivo should be conducted to validate our results.

Effect of the infusion ration between frozen plasma and plasma substitutes on the prognosis of adult patients with major burn in shock stage. / ä¼‘å ‹æœŸå†°å†»è¡€æµ†ä¸Žè¡€æµ†ä»£ç”¨å“è¾“æ³¨æ¯”ä¾‹å¯¹å¤§é¢ç§¯çƒ§ä¼¤æˆå¹´æ‚£è€ é¢„åŽçš„å½±å“.

OBJECTIVES: In recent years, it has been reported that the anti-shock effect of plasma substitutes in adult patients with major burn in shock stage is not good. However, due to the shortage of clinical frozen plasma supply, it is impossible to guarantee that frozen plasma is used as colloidal solution for anti-shock treatment. The purpose of this study is to explore the effect of the infusion ration between frozen plasma and plasma substitutes on the prognosis of adult patients with major burn in shock stage. METHODS: This study enrolled 586 adult patients with major burn by selecting the hospitalization burn patients, who had been hospitalized at the Jiangxi province burn center from September 2014 to April 2019. The patients with the infusion ratio of frozen plasma to plasma substitutes ≥2꞉1 at 48 hours after admission were included in the experimental group, otherwise they were included in the control group. The basic clinical data and clinical prognosis indicator in the 2 groups were compared. Logistic univariate regression analysis was used to screen the influential factors of 30-day mortality in adult patients with major burn, and logistic multivariate regression analysis was used to obtain independent risk and protective factors; Kaplan-Meier method was used to draw the survival curve of the 2 groups, and log-rank test was used to compare the 30-day survival rate of the 2 groups. RESULTS: There were significant differences in the infusion volume of frozen plasma and plasma substitutes between the 2 groups at 48 hours after admission (both P<0.05). The duration of mechanical ventilation in the experimental group was shorter than that in the control group, the percentage of continuous renal replacement therapy (CRRT) in the experimental group was lower than that in the control group, and the in-hospital mortality and 30-day mortality in the experimental group were lower than those in the control group, the differences were statistically significant (all P<0.05). The percentage of burn area and total body surface area (TBSA) was an independent risk factor for 30-day mortality of adult patients with major burn (OR=1.228, 95% CI 1.010 to 1.439, P=0.039), while the infusion ration between of frozen plasma and plasma substitutes 48 hours after admission was an independent protective factor (OR=0.016, 95% CI 0.001 to 0.960, P=0.023). The 30-day survival rate of the experimental group was significantly higher than that in the control group (P<0.001). CONCLUSIONS: Infusion ration between frozen plasma to plasma substitutes at 48 hours after admission is an independent protective factor for 30-day mortality of adult patients with major burn. In the early stage of adult patients with major burn, frozen plasma should be used as the anti-shock therapy as far as possible (frozen plasma꞉plasma substitute ≥2꞉1) to improve the prognosis and reduce the of 30-day mortality.

Bone marrow niche ATP levels determine leukemia-initiating cell activity via P2X7 in leukemic models.

How particular bone marrow niche factors contribute to the leukemogenic activities of leukemia-initiating cells (LICs) remains largely unknown. Here, we showed that ATP levels were markedly increased in the bone marrow niches of mice with acute myeloid leukemia (AML), and LICs preferentially localized to the endosteal niche with relatively high ATP levels, as indicated by a sensitive ATP indicator. ATP could efficiently induce the influx of ions into LICs in an MLL-AF9-induced murine AML model via the ligand-gated ion channel P2X7. P2x7 deletion led to notably impaired homing and self-renewal capacities of LICs and contributed to an approximately 5-fold decrease in the number of functional LICs but had no effect on normal hematopoiesis. ATP/P2X7 signaling enhanced the calcium flux-mediated phosphorylation of CREB, which further transactivated phosphoglycerate dehydrogenase (Phgdh) expression to maintain serine metabolism and LIC fates. P2X7 knockdown resulted in a markedly extended survival of recipients transplanted with either human AML cell lines or primary leukemia cells. Blockade of ATP/P2X7 signaling could efficiently inhibit leukemogenesis. Here, we provide a perspective for understanding how ATP/P2X7 signaling sustains LIC activities, which may benefit the development of specific strategies for targeting LICs or other types of cancer stem cells.

Enhanced immunogenicity of leukemia-derived exosomes via transfection with lentiviral vectors encoding costimulatory molecules.

BACKGROUND: Tumor cell-derived exosomes (TEXs) have been widely used to induce antitumor immune responses in animal models and clinical trials. Similarly, leukemia cell-derived exosomes (LEXs) can induce antileukemia immune responses in animal models. However, the antileukemia immunity induced by LEXs is less effective, which may be due to an inadequate costimulatory capacity. METHODS: In this study, we transduced L1210 leukemia cells with a lentiviral vector encoding two B7 costimulatory molecules (CD80, CD86) and obtained LEXs that highly expressed CD80 and CD86. The antileukemia immune response derived from these LEXs was examined in vitro and in vivo in animal models. RESULTS: We found that B7 gene-modified LEXs, including LEX-CD80, LEX-CD86, and LEX-8086, could significantly boost the expression of CD80 and CD86 in dendritic cells (DCs) and promote the secretion of functional cytokines such as TNF-α and IL-12. Moreover, these B7 gene-modified LEXs, particularly LEX-CD8086, could effectively induce CD4+ T cell proliferation, Th1 cytokine secretion, and an antigen-specific anti-leukemia cytotoxic T lymphocyte (CTL) response. Additional animal studies indicated that immunization with B7 gene-modified LEXs, in particular LEX-CD8086, could significantly retard tumor growth compared to the control LEXnull group. CONCLUSIONS: This study sheds light on the feasibility of obtaining LEXs that overexpress costimulatory molecules via genetically modified leukemia cells, thereby enhancing their anti-leukemia immunity and providing a potential therapeutic strategy that contributes to leukemia immunotherapy.

MCL-1 or BCL-xL-dependent resistance to the BCL-2 antagonist (ABT-199) can be overcome by specific inhibitor as single agents and in combination with ABT-199 in acute myeloid leukemia cells.

Aberrant over-expression of BCL-2 family proteins (BCL-2, BCL-xL, MCL-1) are associated with hematological malignancies. Antagonists of BCL-2 family proteins include BCL-2-selective inhibitor ABT-199, MCL-1-selective inhibitor A-1210477, BCL-xL-selective inhibitor A-1155463. In this study, we evaluated their potential inhibitory effectiveness. Our data showed that OCI-AML3 cells and U937 cells were resistant to BCL-2-selective inhibitor ABT-199 in vitro and in vivo, however, while OCI-AML3 cells were sensitive to MCL-1-selective inhibitor A-1210477 in vitro and in vivo, indicating that A-1210477 could counteract the resistance of AML cells to ABT-199 as a single agent in MCL-1-dependent AML cells. U-937 cell line and mouse model were resistant to A-1210477 or ABT-199, and expressed high level of BCL-xL, indicating that BCL-xL might play an important role in the resistance of A-1210477 or ABT-199. Besides, this study also showed that ABT-199 could synergize with A-1210477 in vitro or in vivo.

IL-10 gene-modified dendritic cells-induced type 1 T regulatory cells inhibit graft-versus-host disease while preserving graft-versus-leukemia effect.

To explore the role of type 1 regulatory T (Tr1) cells in allogeneic bone marrow transplantation (allo-BMT), we generated Tr1 cells from naïve CD4+ T cells in donor mice that were subjected to stimulation of recipient interleukin (IL)-10 gene-modified dendritic cells (DCs). In animal models, the severity of graft-versus-host disease (GVHD) in the group co-infused with DC-induced Tr1 cells was markedly milder than that in the groups without infusion of Tr1 cells. Moreover, the co-infusion Tr1 cells significantly prolonged the leukemia-free survival in tumor-bearing mice. Besides, Tr1-induced cells mediated the shift from T helper (Th)1 to Th2 cytokines in allo-BMT recipients. In Conclusions, our findings suggest that IL-10 gene-modified DC-induced Tr1 cells could inhibit GVHD while maintaining Graft-versus-leukemia (GVL) through mediating the shift of Th1/Th2 cytokines in an allo-BMT mice model.

Knockdown of the Hippo transducer YAP reduces proliferation and promotes apoptosis in the Jurkat leukemia cell.

Leukemia and lymphoma are common hematological malignancies in children and young adults, which pose a tremendous threat to the survival of these young patients worldwide, despite availability of various effective treatments. The Hippo pathway is a novel­signaling pathway that regulates organ size, cell proliferation, apoptosis and tumorigenesis. The chief component of this pathway is the transducer yes­associated protein (YAP) which is over­expressed in numerous categories of tumors. However, little is known about the effect of YAP in hematological malignancies. In the present study, YAP expression was screened in several leukemia and lymphoma cell lines, and high YAP expression was demonstrated in Jurkat cells. To further unravel its effect on the biological behavior of Jurkat cells, lentivirus transduced short hairpin RNA (shRNA) technique was used to silence YAP. As expected, the YAP­specific shRNA dramatically inhibited YAP expression at the mRNA and protein levels. Reduced leukemia cell proliferation and increased cell apoptosis were demonstrated in YAP knockdown Jurkat cells. It was also demonstrated that YAP knockdown resulted in deregulated expression of a cluster of downstream genes crucial to cell proliferation or apoptosis, including protein kinase B, B­cell lymphoma 2 (BCL2) and BCL2 like protein 1. Consequently, the results of the present study established that suppression of YAP expression serves an important role in Jurkat cell proliferation and apoptosis, which may serve as a potential therapeutic target.

Enhancement of Anti-Leukemia Immunity by Leukemia-Derived Exosomes Via Downregulation of TGF-ß1 Expression.

BACKGROUND/AIMS: Minimal residual leukemia cells (MRLs) are difficult to eradicate through traditional treatment and therefore remain to be a major threat to the long-term survival of leukemia patients. Tumor-derived exosomes (TEXs), which carry tumor associated antigens (TAA), may be a potential cell-free tumor vaccine for the specific eradication of MRLs. However, TEXs are intended to be less immunogenic due to exosomal TGF-ß1. To further optimize the efficacy of TEX-based vaccines, we investigated whether exosomes from TGF-ß1 silenced leukemia cells (LEXTGF-ß1si) had an increased potential to induce a specific antitumor effect compared with non-modified exosomes. METHODS: Exosomal TGF-ß1 was downregulated via lentiviral shRNA silencing of TGF-ß1 in leukemia cells. The characteristics of LEXTGF-ß1si were determined via electron microscopy, western blot analysis, and flow cytometry. The antitumor effect of LEXTGF-ß1si was evaluated by detecting the properties of LEXTGF-ß1si-pulsed dendritic cells (DCs), CD4+ T-cell proliferation, Th1 cytokine secretion, specific CTL activity, and NK cell function. Moreover, to verify the superiority of LEXTGF-ß1si immunization, LEXTGF-ß1si was subcutaneously injected into DBA/2 mice: either followed by tumor challenge or tumor bearing. RESULTS: The lentiviral shRNA silencing of TGF-ß1 in parental leukemia cells successfully downregulated the TGF-ß1 level in leukemia cell derived exosomes (LEX). LEXTGF-ß1si was uptaken by DCs and was more potent in promoting DC function by upregulating the surface expression of costimulatory factors and MHC class II molecules, while inducing the secretion of IL-12p70 and TNF-α. Furthermore, immunization with LEXTGF-ß1si facilitated CD4+ T-cell proliferation and Th1 cytokine secretion, and stimulated stronger specific cytotoxic lymphocyte (CTL) response and nature killer (NK) cell cytotoxicity more efficiently compared to non-modified LEX. In mice models, immunization with LEXTGF-ß1si resulted in a more potent capability to inhibit tumor growth and to prolong survival, suggesting that LEXTGF-ß1si was more effective in both protective and therapeutic antitumor tests than non-modified LEX. CONCLUSIONS: These data suggested that down-regulation of exosomal TGF-ß1 effectively induced potent anti-tumor immunity. Our strategy of optimizing exosome vaccine may have promising potential for leukemia immunotherapy.

Interleukin-10 Gene-Modified Dendritic Cell-Induced Type 1 Regulatory T Cells Induce Transplant-Tolerance and Impede Graft Versus Host Disease After Allogeneic Stem Cell Transplantation.

BACKGROUND/AIMS: Tr1 cells can induce peripheral tolerance to self- and foreign antigens, and have been developed as a therapeutic tool for the induction of tolerance to transplanted tissue. We explored the feasibility of generating Tr1 cells by using IL-10 gene-modified recipient DCs (DCLV-IL-10) to stimulate donor naive CD4+ T cells. We also investigated some biological properties of Tr1 cells. METHODS: DCLV-IL-10 were generated through DCs transduced with a lentivirus vector carrying the IL-10 gene, and Tr1 cells were produced by using DCLV-IL-10 to stimulate naive CD4+ T cells. The effects of Tr1 cells on T-cell proliferation and the occurrence of graft versus host disease (GVHD) following allogeneic stem-cell transplantation (allo-HSCT) were investigated. RESULTS: The DCLV-IL-10-induced Tr1 cells co-expressed LAG-3 and CD49b. Moreover, they also expressed CD4, CD25, and IL-10, but not Foxp3, and secreted significantly higher levels of IL-10 (1,729.36 ± 185.79 pg/mL; P < 0.001) and INF-γ (1,524.48 ± 168.65 pg/mL; P < 0.01) than the control T cells upon the stimulation by allogeneic DCs. Tr1 cells markedly suppressed T-lymphocyte proliferation and the mixed lymphocytic response (MLR) in vitro. The mice used in the allo-HSCT model had longer survival times and lower clinical and pathological GVHD scores than the control mice. CONCLUSION: IL-10 gene-modified DC-induced Tr1 cells may be used as a potent cellular therapy for the prevention of GVHD after allo-HSCT.

TGF-ß1-silenced leukemia cell-derived exosomes target dendritic cells to induce potent anti-leukemic immunity in a mouse model.

Tumor-derived exosomes (TEX) can induce a specific antitumor immune response and have been developed as a promising tumor vaccine. Despite promising preclinical data, TEX exhibit relatively low efficacy and limited clinical benefit in clinical trials. In the present study, we investigated whether exosomes from the TGF-ß1 silenced L1210 cells (LEXTGF-ß1si) can enhance the efficacy of DC-based vaccines. We silenced TGF-ß1 in L1210 cells with a lentiviral shRNA vector and prepared the LEXTGF-ß1si. It was shown that LEXTGF-ß1si can significantly decrease TGF-ß1 expression of dendritic cells (DC) and effectively promote their maturation and immune function. In addition, DC pulsed with LEXTGF-ß1si (DCLEX-TGF-ß1si) more effectively promoted CD4+ T cell proliferation in vitro and Th1 cytokine secretion and induced tumor-specific CTL response. This response was higher in potency compared to that noted by the other two formulations. Moreover, DCLEX-TGF-ß1si inhibited tumor growth more efficiently than other formulations did as the preventive or therapeutic tumor vaccine. Accordingly, these findings revealed that DCLEX-TGF-ß1si induced a more potent antigen-specific anti-leukemic immunity than DC pulsed with exosomes from non-manipulated L1210 cells. This indicated that the targeting of DC by LEXTGF-ß1si may be used as a promising approach for leukemia immunotherapy.

Sorting protein VPS33B regulates exosomal autocrine signaling to mediate hematopoiesis and leukemogenesis.

Certain secretory proteins are known to be critical for maintaining the stemness of stem cells through autocrine signaling. However, the processes underlying the biogenesis, maturation, and secretion of these proteins remain largely unknown. Here we demonstrate that many secretory proteins produced by hematopoietic stem cells (HSCs) undergo exosomal maturation and release that is controlled by vacuolar protein sorting protein 33b (VPS33B). Deletion of VPS33B in either mouse or human HSCs resulted in impaired exosome maturation and secretion as well as loss of stemness. Additionally, VPS33B deficiency led to a dramatic delay in leukemogenesis. Exosomes purified from either conditioned medium or human plasma could partially rescue the defects of HSCs and leukemia-initiating cells (LICs). VPS33B co-existed in exosomes with GDI2, VPS16B, FLOT1, and other known exosome markers. Mechanistically, VPS33B interacted with the GDI2/RAB11A/RAB27A pathway to regulate the trafficking of secretory proteins as exosomes. These findings reveal an essential role for VPS33B in exosome pathways in HSCs and LICs. Moreover, they shed light on the understanding of vesicle trafficking in other stem cells and on the development of improved strategies for cancer treatment.

CD274 promotes cell cycle entry of leukemia-initiating cells through JNK/Cyclin D2 signaling.

BACKGROUND: CD274 (programmed death ligand 1, also known as B7H1) is expressed in both solid tumors and hematologic malignancies and is of critical importance for the escape of tumor cells from immune surveillance by inhibiting T cell function via its receptor, programmed death 1 (PD-1). Increasing evidence indicates that functional monoclonal antibodies of CD274 may potently enhance the antitumor effect in many cancers. However, the role of CD274 in leukemia-initiating cells (LICs) remains largely unknown. METHODS: We established an MLL-AF9-induced acute myeloid leukemia (AML) model with wild-type (WT) and CD274-null mice to elucidate the role of CD274 in the cell fates of LICs, including self-renewal, differentiation, cell cycle, and apoptosis. RNA sequencing was performed to reveal the potential downstream targets, the results of which were further validated both in vitro and in vivo. RESULTS: In silico analysis indicated that CD274 level was inversely correlated with the overall survival of AML patients. In Mac-1+/c-Kit+ mouse LICs, CD274 was expressed at a much higher level than in the normal hematopoietic stem cells (HSCs). The survival of the mice with CD274-null leukemia cells was dramatically extended during the serial transplantation compared with that of their WT counterparts. CD274 deletion led to a significant decrease in LIC frequency and arrest in the G1 phase of the cell cycle. Interestingly, CD274 is not required for the maintenance of HSC pool as shown in our previous study. Mechanistically, we demonstrated that the levels of both phospho-JNK and Cyclin D2 were strikingly downregulated in CD274-null LICs. The overexpression of Cyclin D2 fully rescued the loss of function of CD274. Moreover, CD274 was directly associated with JNK and enhanced the downstream signaling to increase the Cyclin D2 level, promoting leukemia development. CONCLUSIONS: The surface immune molecule CD274 plays a critical role in the proliferation of LICs. The CD274/JNK/Cyclin D2 pathway promotes the cell cycle entry of LICs, which may serve as a novel therapeutic target for the treatment of leukemia.

Mucosal fluid evaporation is not the method of heat dissipation from fourth-degree laryngopharyngeal burns.

This study was designed to explore whether mucosal fluid evaporation represents a method of heat dissipation from thermal air inhalation injury and to assess laryngopharyngeal tissue damage according to heat quantity changes of dry air and vapour. Fifteen adult male beagles were divided into five groups to inhale heated air or vapour for 10 min as follows: control group (ordinary air), group I (91-110 °C heated air), group II (148-175 °C heated air), group III (209-227 °C heated air), and group IV (96 °C saturated vapour). The heat quantity changes of the dry air and vapour were calculated via thermodynamic formulas. The macroscopic and histological features of the laryngopharynxes were examined and assessed by various tissue damage grading systems. Group IV exhibited the most serious laryngopharyngeal damage, including cilia exfoliation, submucosal thrombosis, glandular atrophy, and chondrocyte degeneration, which is indicative of fourth-degree injury. The quality, heat quantity, and proportional reduction of heat quantity of vapour in group IV were all higher than those in the other groups. Furthermore, we found that mucosal fluid evaporation is not the method of heat dissipation from thermal air inhalation injury used by the airways. Laryngopharyngeal tissue damage depends chiefly on the heat quantity of vapour in the air.