Suxia Geng

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.

Analysis of the expression pattern of the BCL11B gene and its relatives in patients with T-cell acute lymphoblastic leukemia

BackgroundIn a human T-cell acute lymphoblastic leukemia (T-ALL) cell line (Molt-4), siRNA-mediated suppression of BCL11B expression was shown to inhibit proliferation and induce apoptosis, functions which may be related to genes involved in apoptosis (such as TNFSF10 and BCL2L1) and TGF-β pathways (such as SPP1 and CREBBP).MethodsThe expression levels of the above mentioned genes and their correlation with the BCL11B gene were analyzed in patients with T-ALL using the TaqMan and SYBR Green I real-time polymerase chain reaction technique.ResultsExpression levels of BCL11B, BCL2L1, and CREBBP mRNA in T-ALL patients were significantly higher than those from healthy controls (P < 0.05). In T-ALL patients, the BCL11B expression level was negatively correlated with the BCL2L1 expression level (rs = -0.700; P< 0.05), and positively correlated with the SPP1 expression level (rs = 0.683; P< 0.05). In healthy controls, the BCL11B expression level did not correlate with the TNFSF10, BCL2L1, SPP1, or CREBBP expression levels.ConclusionsOver-expression of BCL11B might play a role in anti-apoptosis in T-ALL cells through up-regulation of its downstream genes BCL2L1 and CREBBP.

Expression and distribution of PPP2R5C gene in leukemia

BackgroundRecently, we clarified at the molecular level novel chromosomal translocation t(14;14)(q11;q32) in a case of Sézary syndrome, which caused a rearrangement from TRAJ7 to the PPP2R5C gene. PPP2R5C is one of the regulatory B subunits of protein phosphatase 2A (PP2A). It plays a crucial role in cell proliferation, differentiation, and transformation. To characterize the expression and distribution of five different transcript variants of the PPP2R5C gene in leukemia, we analyzed the expression level of PPP2R5C in peripheral blood mononuclear cells from 77 patients with de novo leukemia, 26 patients with leukemia in complete remission (CR), and 20 healthy individuals by real-time PCR and identified the different variants of PPP2R5C by RT-PCR.FindingsSignificantly higher expression of PPP2R5C was found in AML, CML, T-ALL, and B-CLL groups in comparison with healthy controls. High expression of PPP2R5C was detected in the B-ALL group; however, no significant difference was found compared with the healthy group. The expression level of PPP2R5C in the CML-CR group decreased significantly compared with that in the de novo CML group and was not significantly different from the level in the healthy group. By using different primer pairs that covered different exons, five transcript variants of PPP2R5C could be identified. All variants could be detected in healthy samples as well as in all the leukemia samples, and similar frequencies and distributions of PPP2R5C were indicated.ConclusionsOverexpression of PPP2R5C in T-cell malignancy as well as in myeloid leukemia cells might relate to its proliferation and differentiation. Investigation of the effect of target inhibition of this gene might be beneficial to further characterization of molecular mechanisms and targeted therapy in leukemia.

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.

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.

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.

A novel generation 1928zT2 CAR T cells induce remission in extramedullary relapse of acute lymphoblastic leukemia

BackgroundAnti-CD19 chimeric antigen receptor (CAR) T cells have shown promise in the treatment of B cell acute lymphocytic leukemia (B-ALL). However, its efficacy in B-ALL patients with extramedullary involvement is limited due to poor responses and neurotoxicity. Here, we utilized a third generation of CAR T cell vector, which contains the Toll/interleukin-1 receptor (ITR) domain of Toll-like receptor 2 (TLR2), to generate 1928zT2 T cells targeting CD19, and evaluated the efficacy of 1928zT2 T cells in relapse or refractory B-ALL patients with extramedullary involvement.Methods1928zT2 T cells were generated by 19-28z-TLR2 lentiviral vector transfection into primary human T lymphocytes. The anti-leukemia effect of 1928zT2 T cells were determined by killing assays and in xenografts. Three patients diagnosed as relapse or refractory ALL with extramedullary involvement were infused with 1928zT2 T cells, and the clinical responses were evaluated by BM smear, B-ultrasonography, PET/CT, histology, flow cytometry, qPCR, ELISA, and luminex assay.Results1928zT2 T cells exhibited enhanced effector function against CD19+ leukemic cells in vitro and in a xenograft model of human extramedullary leukemia. Notably, the 1928zT2 T cells eradicated extramedullary leukemia and induced complete remission in the three relapse and refractory ALL patients without serious adverse effects. 1928zT2 T cells expanded robustly in the circulation of these three patients and were detected in the cerebrospinal fluid of patient 3. These three patients experienced cytokine release syndrome (CRS) with grade 2 or 3, which remitted spontaneously or after tocilizumab treatment. None of the three patients suffered neurotoxicity or needed further intensive care.ConclusionsOur results demonstrate that 1928zT2 T cells with TLR2 incorporation augment anti-leukemic effects, particularly for eradicating extramedullary leukemia cells, and suggest that the infusion of 1928zT2 T cells is an encouraging treatment for relapsed/refractory ALL patients with extramedullary involvement.Trial registrationClinicalTrials.gov identifier NCT02822326. Date of registration: July 4, 2016.

Attenuation of cGVHD by C5a/C5aR blockade is associated with increased frequency of Treg

C5aR signaling plays an important role in the regulation of T cell activation and alloimmune responses in chronic graft-versus-host disease (cGVHD). However, direct evidence of this modulation and the efficacy of C5aR blockade in the treatment of cGVHD have not been demonstrated. We observed higher expression of C5aR on both monocytes and T cells of patients with cGVHD compared with healthy controls and non-GVHD patients after allogeneic hematopoietic stem cell transplantation. Our data also demonstrated a significant negative correlation between C5aR expression and regulatory T cells (Treg) frequency in cGVHD patients, indicating a potential role of C5aR in the generation and regulation of Treg. In addition, an in vitro experiment revealed C5aR deficiency promoted the development of Treg whereas C5a activation abolished the differentiation of Treg. Importantly, we found C5aR blockade by PMX53 attenuated the pathology of cGVHD and improved the survival of cGVHD mice. PMX53 had a direct regulatory effect on Treg commitment and increased TGF-β1 expression. Thus, C5aR signaling may induce and intensify cGVHD by down-regulating Treg induction. The modulation of C5aR activation by PMX53 may provide a potential therapy for cGVHD.

Effects of the combination of decitabine and homoharringtonine in SKM-1 and Kg-1a cells

The methylation inhibitor decitabine (DAC) has great therapeutic value for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, DAC monotherapy is associated with relatively low rates of overall response and complete remission. Previous studies have shown promising results for combination treatment regimens including DAC. Homoharringtonine (HHT), an alkaloid from Chinese natural plants and Cephalotaxus, has demonstrated potential for leukemia treatment. Our studies have suggested that the combination of DAC and HHT has synergistic effects for inhibiting the viability of SKM-1 and Kg-1a cells. This combination leads to enhanced inhibition of colony formation and apoptosis induction compared with DAC alone in SKM-1 but not Kg-1a cells. Only high-dose DAC and HHT significantly up-regulate caspase-3 and caspase-9 and inhibit BCL-XL in the SKM-1 cell line. The combined effects of DAC plus HHT on apoptosis may not only depend on regulation of the apoptosis-related genes we examined but others as well. HHT had no demethylation effects, and HHT in combination with DAC had no enhanced effects on hypomethylation and DNMT1, DNMT3A and DNMT3B mRNA expression in SKM-1 cells. Overall, these results suggest that DAC used in combination with HHT may have clinical potential for MDS treatment.

Role of Toll-like receptor 4 signaling in cutaneous chronic graft-versus-host disease.

Cutaneous damage is one of the characterized manifestations in chronic graft‐versus‐host disease (cGVHD). When local effective immunity in the skin is altered to a dysimmune reaction, cutaneous injuries occur. Toll‐like receptor 4 signaling is regarded as a central mediator of inflammation and organ injury. In this study, we found that TLR4 mRNA in peripheral blood from patients with cutaneous cGVHD was markedly increased compared with that from non‐GVHD patients and healthy controls. In addition, NF‐κB expression, TLR4 downstream signaling, and TLR4‐mediated cytokines, including IL‐6 and ICAM‐1, were upregulated. Moreover, ICAM‐1 was widely distributed in skin biopsies from patients with cutaneous cGVHD. We also found that LPS induced TLR4‐mediated NF‐κB activation and IL‐6 and ICAM‐1 secretion in human fibroblasts in vitro. Thus, TLR4, NF‐κB, IL‐6, and ICAM‐1 contribute to the inflammatory response that occurs in cutaneous cGVHD, indicating the TLR4 pathway may be a novel target for cutaneous cGVHD therapy.