Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks.

Due to their immunomodulatory properties and in vitro differentiation ability, human mesenchymal stromal cells (hMSCs) have been investigated in more than 1000 clinical trials over the last decade. Multiple studies that have explored the development of gene-modified hMSC-based products are now reaching early stages of clinical trial programmes. From an engineering perspective, the challenge lies in developing manufacturing methods capable of producing sufficient doses of ex vivo gene-modified hMSCs for clinical applications. This work demonstrates, for the first time, a scalable manufacturing process using a microcarrier-bioreactor system for the expansion of gene-modified hMSCs. Upon isolation, umbilical cord tissue mesenchymal stromal cells (UCT-hMSCs) were transduced using a lentiviral vector (LV) with green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) transgenes. The cells were then seeded in 100 mL spinner flasks using Spherecol microcarriers and expanded for seven days. After six days in culture, both non-transduced and transduced cell populations attained comparable maximum cell concentrations (≈1.8 × 105 cell/mL). Analysis of the culture supernatant identified that glucose was fully depleted after day five across the cell populations. Lactate concentrations observed throughout the culture reached a maximum of 7.5 mM on day seven. Immunophenotype analysis revealed that the transduction followed by an expansion step was not responsible for the downregulation of the cell surface receptors used to identify hMSCs. The levels of CD73, CD90, and CD105 expressing cells were above 90% for the non-transduced and transduced cells. In addition, the expression of negative markers (CD11b, CD19, CD34, CD45, and HLA-DR) was also shown to be below 5%, which is aligned with the criteria established for hMSCs by the International Society for Cell and Gene Therapy (ISCT). This work provides a foundation for the scalable manufacturing of gene-modified hMSCs which will overcome a significant translational and commercial bottleneck. KEY POINTS: • hMSCs were successfully transduced by lentiviral vectors carrying two different transgenes: GFP and VEGF • Transduced hMSCs were successfully expanded on microcarriers using spinner flasks during a period of 7 days • The genetic modification step did not cause any detrimental impact on the hMSC immunophenotype characteristics.

Recent advances in the therapeutic efficacy of hepatocyte growth factor gene-modified mesenchymal stem cells in multiple disease settings.

Mesenchymal stem cell (MSC) therapy is considered a new treatment for a wide range of diseases and injuries, but challenges remain, such as poor survival, homing and engraftment rates, thus limiting the therapeutic efficacy of the transplanted MSCs. Many strategies have been developed to enhance the therapeutic efficacy of MSCs, such as preconditioning, co-transplantation with graft materials and gene modification. Hepatocyte growth factor (HGF) is secreted by MSCs, which plays an important role in MSC therapy. It has been reported that the modification of the HGF gene is beneficial to the therapeutic efficacy of MSCs, including diseases of the heart, lung, liver, urinary system, bone and skin, lower limb ischaemia and immune-related diseases. This review focused on studies involving HGF/MSCs both in vitro and in vivo. The characteristics of HGF/MSCs were summarized, and the mechanisms of their improved therapeutic efficacy were analysed. Furthermore, some insights are provided for HGF/MSCs' clinical application based on our understanding of the HGF gene and MSC therapy.

Composite CD79A/CD40 co-stimulatory endodomain enhances CD19CAR-T cell proliferation and survival.

Adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have led to impressive clinical outcomes in B cell malignancies. Beyond induction of remission, the persistence of CAR-T cells is required to prevent relapse and provide long-term disease control. To improve CAR-T cell function and persistence, we developed a composite co-stimulatory domain of a B cell signaling moiety, CD79A/CD40, to induce a nuclear translocating signal, NF-κB, to synergize with other T cell signals and improve CAR-T cell function. CD79A/CD40 incorporating CD19CAR-T cells (CD19.79a.40z) exhibited higher NF-κB and p38 activity upon CD19 antigen exposure compared with the CD28 or 4-1BB incorporating CD19CAR-T cells (CD19.28z and CD19.BBz). Notably, we found that CD19.79a.40z CAR-T cells continued to suppress CD19+ target cells throughout the co-culture assay, whereas a tendency for tumor growth was observed with CD19.28z CAR-T cells. Moreover, CD19.79a.40z CAR-T cells exhibited robust T cell proliferation after culturing with CD19+ target cells, regardless of exogenous interleukin-2. In terms of in vivo efficiency, CD19.79a.40z demonstrated superior anti-tumor activity and in vivo CAR-T cell proliferation compared with CD19.28z and CD19.BBz CD19CAR-T cells in Raji-inoculated mice. Our data demonstrate that the CD79A/CD40 co-stimulatory domain endows CAR-T cells with enhanced proliferative capacity and improved anti-tumor efficacy in a murine model.

B cell receptor ligation induces display of V-region peptides on MHC class II molecules to T cells.

The B cell receptors (BCRs) for antigen express variable (V) regions that are enormously diverse, thus serving as markers on individual B cells. V region-derived idiotypic (Id) peptides can be displayed as pId:MHCII complexes on B cells for recognition by CD4+ T cells. It is not known if naive B cells spontaneously display pId:MHCII in vivo or if BCR ligation is required for expression, thereby enabling collaboration between Id+ B cells and Id-specific T cells. Here, using a mouse model, we show that naive B cells do not express readily detectable levels of pId:MHCII. However, BCR ligation by Ag dramatically increases physical display of pId:MHCII, leading to activation of Id-specific CD4+ T cells, extrafollicular T-B cell collaboration and some germinal center formation, and production of Id+ IgG. Besides having implications for immune regulation, the results may explain how persistent activation of self-reactive B cells induces the development of autoimmune diseases and B cell lymphomas.

Insights on the Functional Role of Beta-Glucans in Fungal Immunity Using Receptor-Deficient Mouse Models.

Understanding the host anti-fungal immunity induced by beta-glucan has been one of the most challenging conundrums in the field of biomedical research. During the last couple of decades, insights on the role of beta-glucan in fungal disease progression, susceptibility, and resistance have been greatly augmented through the utility of various beta-glucan cognate receptor-deficient mouse models. Analysis of dectin-1 knockout mice has clarified the downstream signaling pathways and adaptive effector responses triggered by beta-glucan in anti-fungal immunity. On the other hand, assessment of CR3-deficient mice has elucidated the compelling action of beta-glucans in neutrophil-mediated fungal clearance, and the investigation of EphA2-deficient mice has highlighted its novel involvement in host sensing and defense to oral mucosal fungal infection. Based on these accounts, this review focuses on the recent discoveries made by these gene-targeted mice in beta-glucan research with particular emphasis on the multifaceted aspects of fungal immunity.

New Era of Immunotherapy in Pediatric Brain Tumors: Chimeric Antigen Receptor T-Cell Therapy.

Immunotherapy, including chimeric antigen receptor (CAR) T-cell therapy, immune checkpoint inhibitors, cancer vaccines, and dendritic cell therapy, has been incorporated as a fifth modality of modern cancer care, along with surgery, radiation, chemotherapy, and target therapy. Among them, CAR T-cell therapy emerges as one of the most promising treatments. In 2017, the first two CAR T-cell drugs, tisagenlecleucel and axicabtagene ciloleucel for B-cell acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL), respectively, were approved by the Food and Drug Administration (FDA). In addition to the successful applications to hematological malignancies, CAR T-cell therapy has been investigated to potentially treat solid tumors, including pediatric brain tumor, which serves as the leading cause of cancer-associated death for children and adolescents. However, the employment of CAR T-cell therapy in pediatric brain tumors still faces multiple challenges, such as CAR T-cell transportation and expansion through the blood-brain barrier, and identification of the specific target antigen on the tumor surface and immunosuppressive tumor microenvironment. Nevertheless, encouraging outcomes in both clinical and preclinical trials are coming to light. In this article, we outline the current propitious progress and discuss the obstacles needed to be overcome in order to unveil a new era of treatment in pediatric brain tumors.

Gene Modified CAR-T Cellular Therapy for Hematologic Malignancies.

With advances in the understanding of characteristics of molecules, specific antigens on the surface of hematological malignant cells were identified and multiple therapies targeting these antigens as neoplasm treatments were developed. Among them, chimeric antigen receptor (CAR) T-cell therapy, which got United States Food and Drug Administration (FDA) approval for relapsed/refractory (r/r) diffuse large B-cell lymphoma (DLBCL) as well as for recurrent acute lymphoblastic leukemia (ALL) within the past five years, and for r/r mantle cell lymphoma (MCL) this year, represents one of the most rapidly evolving immunotherapies. Nevertheless, its applicability to other hematological malignancies, as well as its efficacy and persistence are fraught with clinical challenges. Currently, more than one thousand clinical trials in CAR T-cell therapy are ongoing and its development is changing rapidly. This review introduces the current status of CAR T-cell therapy in terms of the basic molecular aspects of CAR T-cell therapy, its application in hematological malignancies, adverse reactions during clinical use, remaining challenges, and future utilization.

Altered Peptide Ligands Impact the Diversity of Polyfunctional Phenotypes in T Cell Receptor Gene-Modified T Cells.

The use of T cell receptor (TCR) gene-modified T cells in adoptive cell transfer has had promising clinical success, but often, simple preclinical evaluation does not necessarily accurately predict treatment efficacy or safety. Preclinical studies generally evaluate one or a limited number of type 1 cytokines to assess antigen recognition. However, recent studies have implicated other "typed" T cells in effective anti-tumor/viral immunity, and limited functional evaluations may underestimate cross-reactivity. In this study, we use an altered peptide ligand (APL) model and multi-dimensional flow cytometry to evaluate polyfunctionality of TCR gene-modified T cells. Evaluating six cytokines and the lytic marker CD107a on a per cell basis revealed remarkably diverse polyfunctional phenotypes within a single T cell culture and among peripheral blood lymphocyte (PBL) donors. This polyfunctional assessment identified unexpected phenotypes, including cells producing both type 1 and type 2 cytokines, and highlighted interferon γneg (IFNγneg) antigen-reactive populations overlooked in our previous studies. Additionally, APLs skewed functional phenotypes to be less polyfunctional, which was not necessarily related to changes in TCR-peptide-major histocompatibility complex (pMHC) affinity. A better understanding of gene-modified T cell functional diversity may help identify optimal therapeutic phenotypes, predict clinical responses, anticipate off-target recognition, and improve the design and delivery of TCR gene-modified T cells.

Timing of erythropoietin modified mesenchymal stromal cell transplantation for the treatment of experimental bronchopulmonary dysplasia.

The aim of this study is to optimize the timing of erythropoietin gene modified mesenchymal stem cells (EPO-MSCs) transplantation for bronchopulmonary dysplasia (BPD). Three weeks post-operation, the results indicated that the damage of airway structure and apoptosis were significantly decreased, the proliferation was increased in three EPO-MSCs transplantation groups as compared with BPD mice. Moreover, the inflammation cytokines were improvement in early EPO-MSCs injection mice than in BPD mice, but there was no significant difference between late injection and BPD groups. Furthermore, the protein expression ratio of p-p38/p38MAPK was down-regulation in early mice but not in late transplantation mice. Our findings suggest that EPO-MSCs maybe attenuate BPD injury in early than in late administration by inhibiting inflammation response through down-regulation of the p38MAPK signalling pathway.

EPO modified MSCs can inhibit asthmatic airway remodeling in an animal model.

There was no effective measures can be obtained at present to reverse or prevent airway remodeling. We investigated the therapeutic effect of Erythropoietin (EPO) gene modified mesenchymal stem cells (MSCs) on asthmatic airway remodeling and the possible underlied molecular mechanisms. EPO gene was transfected into MSCs via lentivirus vector. The transfected cells (EPO-MSCs) were identified by flow cytometry and the EPO secreting function was detected by PCR and Western blot. MSCs or EPO-MSCs were administrated to albumin (OVA)-induced chronic asthmatic mouse model via tail veins. The asthmatic phenotype was analyzed. Number of cells in bronchoalveolar lavage fluid (BALF) was counted using a hemocytometer. Histological findings of airways were evaluated by microscopic examination. The concentrations of interleukin 4(IL-4), interleukin 5(IL-5), and interleukin 13(IL-13) in lung homogenate were determined by ELISA. The activation state of transforming growth factor-ß 1 (TGF-ß1), Transforming growth factor beta-activated kinase 1 (TAK1), and p38 Mitogen Activated Protein Kinase (p38MAPK) signaling was detected by Real-Time PCR and Western blotting. EPO-MSCs were successfully constructed. EPO-MSCs showed a more potently suppressive effect on local asthmatic airway inflammation and the level of IL-4, IL-5, and IL-13 in lung tissue than MSCs. Moreover, the numbers of goblet cells, the thicknesses of smooth muscle layer, collagen density, percentage of proliferating cell nuclear antigen positive (PCNA+ ) mesenchymal cells, and von Willebrand factor positive(vWF+ ) vessels were also significantly inhibited by EPO-MSCs. Furthermore, EPO-MSCs could downregulate the expression of TGF-ß1, TAK1, and p38MAPK in lung tissue both in mRNA level and in protein level. EPO gene modified MSCs may more efficiently attenuate asthmatic airway remodeling, which maybe related with the downregulation of TGF-ß1-TAK1-p38MAPK pathway activity.

Increased Energy Expenditure, Lipolysis and Hyperinsulinemia Confer Resistance to Central Obesity and Type 2 Diabetes in Mice Lacking Alpha2α-Adrenoceptors.

The alpha2A-adrenoceptors (α2A-ARs) are Gi-coupled receptors, which prejunctionally inhibit the release of norepinephrine (NE) and epinephrine (Epi), and postjunctionally inhibit insulin secretion and lipolysis. We have earlier shown that α2A-/- mice display sympathetic hyperactivity, hyperinsulinemia and improved glucose tolerance. Here we employed α2A-/- mice and placed the mice on a high-fat diet (HFD) to test the hypothesis that lack of α2A-ARs protects from diet-induced obesity and type 2 diabetes (T2D). In addition, a high-caloric diet was combined with running wheel exercise to test the interaction of diet and exercise. HFD was obesogenic in both genotypes, but α2A-/- mice accumulated less visceral fat than the wild-type controls, were protected from T2D, and their insulin secretion was unaltered by the diet. Lack of α2A-ARs is associated with an increased sympatho-adrenal tone, which resulted in increased energy expenditure and fat oxidation rate potentiated by HFD. Fittingly, α2A-/- mice displayed enhanced lipolytic responses to Epi, and increased faecal lipids suggesting altered fat mobilization and absorption. Subcutaneous white fat appeared to be thermogenically more active (measured as Ucp1 mRNA expression) in α2A-/- mice, and brown fat showed an increased response to NE. Exercise was effective in reducing total body adiposity and increasing lean mass in both genotypes, but there was a significant diet-genotype interaction, as even modestly increased physical activity combined with lack of α2A-AR signalling promoted weight loss more efficiently than exercise with normal α2A-AR function. These results suggest that blockade of α2A-ARs may be exploited to reduce visceral fat and to improve insulin secretion.

[Strategical consideration of the interaction between tumor microenvironment and tumor immunity].

In cancer immunotherapy, the importance of tumor microenvironment (TME) has recently been highlighted. TME, where trafficked T lymphocytes with tumoricidal activity, particularly endowed by dendritic cells in the sentinel lymph nodes, suppress cancer cells, consists of multiple cellular components, such as fibroblasts, macrophages, myeloid-derived suppressor cells, monocytes, neutrophils, regulatory T cells, and NK cells, and non-cellular components, such as extracellular matrix and blood and lymphatic vessels. These stroma-forming components have been demonstrated to be mutually interrelated and to provide pro-cancer propensity. The recent clinical success of immune checkpoint inhibitors in cancer treatment has again highlighted the importance of controlling the immunosuppressive milieu of TME to achieve an improved clinical outcome. Simultaneously, understanding the unsuccessful clinical efficacy mediated by adoptively transferred cancer antigen receptor gene-modified T cells against solid tumors has similarly drawn a conclusion. This review presents some recent and notable progresses in cancer immunotherapy, thus aiming at an opportunity to understand the interrelation between tumor immunity in cancer-bearing host and TME.

Mesenchymal stem cell therapy: A promising cell-based therapy for treatment of myocardial infarction.

For decades, mesenchymal stem (MSCs) cells have been used for cardiovascular diseases as regenerative therapy. This review is an attempt to summarize the types of MSCs involved in myocardial infarction (MI) therapy, as well as its possible mechanisms effects, especially the paracrine one in MI focusing on the studies (human and animal) conducted within the last 10 years. Recently, reports showed that MSC therapy could have infarct-limiting effects after MI in both experimental and clinical trials. In this context, various types of MSCs can help cardiac regeneration by either revitalizing the cardiac stem cells or revascularizing the arteries and veins of the heart. Furthermore, MSCs could produce paracrine growth factors that increase the survival of nearby cardiomyocytes, as well as increase angiogenesis through recruitment of stem cell from bone marrow or inducing vessel growth from existing capillaries. Recent research suggests that the paracrine effects of MSCs could be mediated by extracellular vesicles including exosomes. Exosomal microRNAs (miRNAs) released by MSCs are promising therapeutic hotspot target for MI. This could be attributed to the role of miRNA in cardiac biology, including cardiac regeneration, stem cell differentiation, apoptosis, neovascularization, cardiac contractility and cardiac remodeling. Furthermore, gene-modified MSCs could be a recent promising therapy for MI to enhance the paracrine effects of MSCs, including better homing and effective cell targeted tissue regeneration. Although MSC therapy has achieved considerable attention and progress, there are critical challenges that remains to be overcome to achieve the most effective successful cell-based therapy in MI.

Critical biological parameters modulate affinity as a determinant of function in T-cell receptor gene-modified T-cells.

T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

Generation of CMAHKO/GTKO/shTNFRI-Fc/HO-1 quadruple gene modified pigs.

As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG1-Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc/hHO-1 that will be suitable for xenotransplantation by overcoming hyperacute, acute and anti-inflammatory rejection.

Strategies to genetically engineer T cells for cancer immunotherapy.

Immunotherapy is one of the most promising and innovative approaches to treat cancer, viral infections, and other immune-modulated diseases. Adoptive immunotherapy using gene-modified T cells is an exciting and rapidly evolving field. Exploiting knowledge of basic T cell biology and immune cell receptor function has fostered innovative approaches to modify immune cell function. Highly translatable clinical technologies have been developed to redirect T cell specificity by introducing designed receptors. The ability to engineer T cells to manifest desired phenotypes and functions is now a thrilling reality. In this review, we focus on outlining different varieties of genetically engineered T cells, their respective advantages and disadvantages as tools for immunotherapy, and their promise and drawbacks in the clinic.

Gene Therapy for the Treatment of Primary Immune Deficiencies.

The use of gene therapy in the treatment of primary immune deficiencies (PID) has advanced significantly in the last decade. Clinical trials for X-linked severe combined immunodeficiency, adenosine deaminase deficiency (ADA), chronic granulomatous disease, and Wiskott-Aldrich syndrome have demonstrated that gene transfer into hematopoietic stem cells and autologous transplant can result in clinical improvement and is curative for many patients. Unfortunately, early clinical trials were complicated by vector-related insertional mutagenic events for several diseases with the exception of ADA-deficiency SCID. These results prompted the current wave of clinical trials for primary immunodeficiency using alternative retro- or lenti-viral vector constructs that are self-inactivating, and they have shown clinical efficacy without leukemic events thus far. The field of gene therapy continues to progress, with improvements in viral vector profiles, stem cell culturing techniques, and site-specific genome editing platforms. The future of gene therapy is promising, and we are quickly moving towards a time when it will be a standard cellular therapy for many forms of PID.

Translation of Data from Animal Models of Cancer to Immunotherapy of Breast Cancer and Chronic Lymphocytic Leukemia.

The field of clinical oncology has been revolutionized over the past decade with the introduction of many new immunotherapies the existence of which have depended to a large extent on experimentation with both in vitro analysis and the use of various animal models, including gene-modified mice. The discussion below will review my own laboratory's studies, along with those of others in the field, on cancer immunotherapy. Our own studies have predominantly dwelt on two models of malignancy, namely a solid tumor model (breast cancer) and lymphoma. The data from our own laboratory, and that of other scientists, highlights the novel information so obtained, and the evidence that application of such information has already had an impact on immunotherapy of human oncologic diseases.

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.