Jianyu Weng

Mesenchymal Stromal Cells Treatment Attenuates Dry Eye in Patients With Chronic Graft-versus-host Disease

Cell therapy is a promising approach for the treatment of refractory ocular disease. This study investigated the efficacy of mesenchymal stromal cells (MSCs) for the treatment of dry eye associated with chronic graft-versus-host disease (cGVHD) and assessed the immunomodulatory effects of MSCs on regulatory CD8(+)CD28(-) T lymphocytes. A total of 22 patients with refractory dry eye secondary to cGVHD were enrolled. The symptoms of 12 out of 22 patients abated after MSCs transplantation by intravenous injection, improving in the dry eye scores, ocular surface disease index scores and the Schirmer test results. The clinical improvements were accompanied by increasing level of CD8(+)CD28(-) T cells, but not CD4(+)CD25(+) T cells, in the 12 patients who were treated effectively. They had significantly higher levels of Th1 cytokines (interleukin (IL)-2 and interferon-γ) and lower levels of Th2 cytokines (IL-10 and IL-4). In addition, CD8(+) T cells were prone to differentiation into CD8(+)CD28(-) T cells after co-culture with MSCs in vitro. In conclusion, transfusion of MSCs improved the clinical symptoms in patients (54.55%) with refractory dry eye secondary to cGVHD. MSCs appear to exert their effects by triggering the generation of CD8(+)CD28(-) T cells, which may regulate the balance between Th1 and Th2.

Gene expression profiles in BCL11B-siRNA treated malignant T cells

BackgroundDownregulation of the B-cell chronic lymphocytic leukemia (CLL)/lymphoma11B (BCL11B) gene by small interfering RNA (siRNA) leads to growth inhibition and apoptosis of the human T-cell acute lymphoblastic leukemia (T-ALL) cell line Molt-4. To further characterize the molecular mechanism, a global gene expression profile of BCL11B-siRNA -treated Molt-4 cells was established. The expression profiles of several genes were further validated in the BCL11B-siRNA -treated Molt-4 cells and primary T-ALL cells.Results142 genes were found to be upregulated and 109 genes downregulated in the BCL11B-siRNA -treated Molt-4 cells by microarray analysis. Among apoptosis-related genes, three pro-apoptotic genes, TNFSF 10, BIK, BNIP 3, were upregulated and one anti-apoptotic gene, BCL2L 1 was downregulated. Moreover, the expression of SPP 1 and CREBBP genes involved in the transforming growth factor (TGF-β) pathway was down 16-fold. Expression levels of TNFSF 10, BCL2L 1, SPP 1, and CREBBP were also examined by real-time PCR. A similar expression pattern of TNFSF 10, BCL2L 1, and SPP 1 was identified. However, CREBBP was not downregulated in the BLC11B-siRNA -treated Molt-4 cells.ConclusionBCL11B-siRNA treatment altered expression profiles of TNFSF 10, BCL2L 1, and SPP 1 in both Molt-4 T cell line and primary T-ALL cells.

Genome-wide analyses identify KLF4 as an important negative regulator in T-cell acute lymphoblastic leukemia through directly inhibiting T-cell associated genes

BackgroundKruppel-like factor 4 (KLF4) induces tumorigenesis or suppresses tumor growth in a tissue-dependent manner. However, the roles of KLF4 in hematological malignancies and the mechanisms of action are not fully understood.MethodsInducible KLF4-overexpression Jurkat cell line combined with mouse models bearing cell-derived xenografts and primary T-cell acute lymphoblastic leukemia (T-ALL) cells from four patients were used to assess the functional role of KLF4 in T-ALL cells in vitro and in vivo. A genome-wide RNA-seq analysis was conducted to identify genes regulated by KLF4 in T-ALL cells. Chromatin immunoprecipitation (ChIP) PCR was used to determine direct binding sites of KLF4 in T-ALL cells.ResultsHere we reveal that KLF4 induced apoptosis through the BCL2/BCLXL pathway in human T-ALL cell lines and primary T-ALL specimens. In consistence, mice engrafted with KLF4-overexpressing T-ALL cells exhibited prolonged survival. Interestingly, the KLF4-induced apoptosis in T-ALL cells was compromised in xenografts but the invasion capacity of KLF4-expressing T-ALL cells to hosts was dramatically dampened. We found that KLF4 overexpression inhibited T cell-associated genes including NOTCH1, BCL11B, GATA3, and TCF7. Further mechanistic studies revealed that KLF4 directly bound to the promoters of NOTCH1, BCL2, and CXCR4 and suppressed their expression. Additionally, KLF4 induced SUMOylation and degradation of BCL11B.ConclusionsThese results suggest that KLF4 as a major transcription factor that suppresses the expression of T-cell associated genes, thus inhibiting T-ALL progression.

Gene Expression Profiling-Based Identification of CD28 and PI3K as New Biomarkers for Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Currently, no reliable biomarkers are available to predict the onset or progression of cGVHD. Therefore, in this study, we collected peripheral blood mononuclear cells from four patients with cGVHD and four ones with non-GVHD after hematopoietic stem cell transplantation and employed Affymetrix GeneChip Human U133 Plus 2.0 microarrays to screen the genes differentially expressed in cGVHD versus non-GVHD groups, with the aim to identify potential clinical biomarkers to predict cGVHD risk or progression. Microarray analysis demonstrated that the expression of 3180 genes changed significantly in cGVHD versus non-GVHD, with 879 genes upregulated and 2301 genes downregulated. Among them we chose CD28 and PI3K as candidates for further verification. Flow cytometry and quantitative real-time polymerase chain reaction analysis confirmed the significant upregulation of CD28 and PI3K in samples from patients with cGVHD compared with patients with non-GVHD, respectively. In conclusion, our study suggested that the upregulation of CD28 and PI3K contributed to the onset and progression of cGVHD and provided evidence that CD28 and PI3K may serve as promising biomarkers for cGVHD.

Anti-GPC3-CAR T Cells Suppress the Growth of Tumor Cells in Patient-Derived Xenografts of Hepatocellular Carcinoma

Background The lack of a general clinic-relevant model for human cancer is a major impediment to the acceleration of novel therapeutic approaches for clinical use. We propose to establish and characterize primary human hepatocellular carcinoma (HCC) xenografts that can be used to evaluate the cytotoxicity of adoptive chimeric antigen receptor (CAR) T cells and accelerate the clinical translation of CAR T cells used in HCC. Methods Primary HCCs were used to establish the xenografts. The morphology, immunological markers, and gene expression characteristics of xenografts were detected and compared to those of the corresponding primary tumors. CAR T cells were adoptively transplanted into patient-derived xenograft (PDX) models of HCC. The cytotoxicity of CAR T cells in vivo was evaluated. Results PDX1, PDX2, and PDX3 were established using primary tumors from three individual HCC patients. All three PDXs maintained original tumor characteristics in their morphology, immunological markers, and gene expression. Tumors in PDX1 grew relatively slower than that in PDX2 and PDX3. Glypican 3 (GPC3)-CAR T cells efficiently suppressed tumor growth in PDX3 and impressively eradicated tumor cells from PDX1 and PDX2, in which GPC3 proteins were highly expressed. Conclusion GPC3-CAR T cells were capable of effectively eliminating tumors in PDX model of HCC. Therefore, GPC3-CAR T cell therapy is a promising candidate for HCC treatment.

Incorporation of a hinge domain improves the expansion of chimeric antigen receptor T cells

BackgroundMultiple iterations of chimeric antigen receptors (CARs) have been developed, mainly focusing on intracellular signaling modules. However, the effect of non-signaling extracellular modules on the expansion and therapeutic efficacy of CARs remains largely undefined.MethodsWe generated two versions of CAR vectors, with or without a hinge domain, targeting CD19, mesothelin, PSCA, MUC1, and HER2, respectively. Then, we systematically compared the effect of the hinge domains on the growth kinetics, cytokine production, and cytotoxicity of CAR T cells in vitro and in vivo.ResultsDuring in vitro culture period, the percentages and absolute numbers of T cells expressing the CARs containing a hinge domain continuously increased, mainly through the promotion of CD4+ CAR T cell expansion, regardless of the single-chain variable fragment (scFv). In vitro migration assay showed that the hinges enhanced CAR T cells migratory capacity. The T cells expressing anti-CD19 CARs with or without a hinge had similar antitumor capacities in vivo, whereas the T cells expressing anti-mesothelin CARs containing a hinge domain showed enhanced antitumor activities.ConclusionsHence, our results demonstrate that a hinge contributes to CAR T cell expansion and is capable of increasing the antitumor efficacy of some specific CAR T cells. Our results suggest potential novel strategies in CAR vector design.

The hENT1 and DCK genes underlie the decitabine response in patients with myelodysplastic syndrome

Decitabine is approved for the treatment of MDS, but resistance to this agent is common. To determine the mechanisms underlying decitabine resistance, we measured the mRNA expression of metabolism (hENT1, DCK, CDA) and apoptosis (BCL2L10) genes and found that the hENT1 mRNA level was significantly higher in response compared with non-response patients (P=0.004). Furthermore, the DCK level was significantly reduced for relapse (P=0.012) compared with those with continued marrow CR (P=0.222). These findings indicate that the decitabine metabolic pathway affects its therapeutic effects, lower hENT1 expression may induce primary resistance and down-regulated DCK expression may be related to secondary resistance.

Heterogeneity of CD34 and CD38 expression in acute B lymphoblastic leukemia cells is reversible and not hierarchically organized

The existence and identification of leukemia-initiating cells in adult acute B lymphoblastic leukemia (B-ALL) remain controversial. We examined whether adult B-ALL is hierarchically organized into phenotypically distinct subpopulations of leukemogenic and non-leukemogenic cells or whether most B-ALL cells retain leukemogenic capacity, irrespective of their immunophenotype profiles. Our results suggest that adult B-ALL follows the stochastic stem cell model and that the expression of CD34 and CD38 in B-ALL is reversibly and not hierarchically organized.

Toll-like receptor 2 costimulation potentiates the antitumor efficacy of CAR T Cells

Chimeric antigen receptor (CAR) T-cell immunotherapies have shown unprecedented success in treating leukemia but limited clinical efficacy in solid tumors. Here, we generated 1928zT2 and m28zT2, targeting CD19 and mesothelin, respectively, by introducing the Toll/interleukin-1 receptor domain of Toll-like receptor 2 (TLR2) to 1928z and m28z. T cells expressing 1928zT2 or m28zT2 showed improved expansion, persistency and effector function against CD19+ leukemia or mesothelin+ solid tumors respectively in vitro and in vivo. In a patient with relapsed B-cell acute lymphoblastic leukemia, a single dose of 5 × 104/kg 1928zT2 T cells resulted in robust expansion and leukemia eradication and led to complete remission. Hence, our results demonstrate that TLR2 signaling can contribute to the efficacy of CAR T cells. Further clinical trials are warranted to establish the safety and efficacy of this approach.

Persistent donor derived Vδ4 T cell clones may improve survival for recurrent T cell acute lymphoblastic leukemia after HSCT and DLI

The outcome for T-cell acute lymphoblastic leukemia (T-ALL) in relapse after hematopoietic stem cell transplantation (HSCT) is quite poor, while, both donor lymphocytes infusion (DLI) and adoptively infusion of γδ T cells in leukemia patients after HSCT have demonstrated good results in prolonging survival time of patients. Here, we reported a T-ALL case who experienced three relapses and received HSCT and DLI with an overall survival (OS) time lasting for more than seven years. Based on our previous identification of a leukemic and reactive clone in this patient, continual γδ T cell repertoire monitoring affirmed that the same Vδ5 leukemic clone existed in most samples from the patient, particularly including a sample taken at the time of the third T-ALL relapse, while it could not be detected in the donor sample. In addition, an identical Vδ4 monoclonal T cell that proliferated in the recipient for several years was confirmed to come from the donor graft, and its expression level significantly increased in third leukemia recurrence. These results indicate that clonally expanded Vδ4 T cells may represent a reconstituted γδ T cell repertoire after HSCT, which also hints to a relatively better outcome for this case. Based on this case study, we recommend DLI should be as a treatment strategy for patients who achieve CR or relapse from HSCT. Moreover, dynamically monitoring the TCR repertoire in patients who receive HSCT will benefit in supervising of malignant clone evolution and residue, identifying T cell clones mediate anti-infection, GvHD or GvL.