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Hypomethylating agents (HMAs) including azacitidine (AZA) represent the only FDA-approved first-line treatments for patients with chronic myelomonocytic leukemia (CMML). However, the mechanism by which HMAs produce therapeutic responses (e.g., hematological improvement) remains unclear. Bone marrow mesenchymal stromal cells (MSCs) play a crucial role in regulating the self-renewal, survival and differentiation of hematopoietic stem and progenitor cells (HSPCs). Recently, the identified sensitivity of patient-derived MSCs to HMAs underlines a critical yet unexplored role of MSCs in regulating post-HMA efficacies. By utilizing high-throughput approaches including targeted exon sequencing, DNA methylation profiling, and RNA sequencing, our present study aims to delineate the modifications and consequences of AZA on CMML-MSCs. Demonstrated by integrated multi-omics analysis, our results reveal that cytogenetically independent CMML-MSCs exhibit strikingly high amenability to AZA. Through selectively de-methylating/methylating CpGs of 1395 sensitive genes, AZA re-activates HSPC-supportive signatures and enhances the protective effect of CMML-MSCs on healthy HSPCs. Along with the reconstitution of the dysregulated methylome/transcriptome, AZA partially restored the impaired functions of CMML-MSCs to support healthy hematopoiesis in long-term co-culture conditions. Our findings suggested a niche-dependent mechanism of post-HMA recovery of normal hematopoiesis. Identifying AZA-sensitive genes and functions may be of particular value in developing niche-targeting strategies in treating myeloid malignancies.

Objective. AML is a heterogeneous disease both in genomic and proteomic backgrounds, and variable outcomes may appear in the same cytogenetic risk group. Therefore, it is still necessary to identify new antigens that contribute to diagnostic information and to refine the current risk stratification. Methods. The expression of C-type lectin-like molecule-1 (CLL-1) in AML blasts was examined in 52 patients with newly diagnosed or relapsed/refractory AML and was compared with two other classic markers CD33 and CD34 in AML, in order to assess the value of CLL-1 as an independent biomarker or in combination with other markers for diagnosis in AML. Subsequently, the value of CLL-1 as a biomarker for prognosis was assessed in this malignant tumor. Results. The results showed that CLL-1 was expressed on the cell surface of the majority of AML blasts (78.8%) and also expressed on leukemic stem cells in varying degree but absent on normal hematopoietic stem cells. Notably, CLL-1 was able to complement the classic markers CD33 or CD34. After dividing the cases into CLL-1high and CLL-1low groups according to cutoff 59.0%, we discovered that event-free survival and overall survival (OS) of the CLL-1low group were significantly lower than that of the CLL-1high group, and low CLL-1 expression seems to be independently associated with shorter OS. Conclusions. These preliminary observations identified CLL-1 as a biomarker for diagnosis and prognosis of AML.

The present meta-analysis was performed to evaluate the efficacy, toxicities of both hypomethylating agents (decitabine and azaciticine) in the treatment of CMML patients.All available cohort studies of patients with CMML treated with decitabine and azacitidine were identified. The primary endpoints of this meta-analysis were response to hypomethylating agents. Pooled estimates of treatment response and drug-related adverse events were calculated using fixed or random effect models.Fourteen studies with 600 CMML patients (decitabine: n=196; azacitidine: n=404) were identified and included for meta-analysis. HMAs yielded a pooled ORR estimate of 43% (95% CI: 36%-50%) in patients with CMML. Patients received either azacitidine or decitabine exhibited comparable incidence of ORR (43% vs. 45%, P=0.810), while significantly higher incidence of mCR was observed in patients treated with decitabine (23% vs. 10%, P=0.000). Decitabine treatment was also associated with higher incidence of transfusion independence (42% vs. 20%, P=0.044). Both HMAs led to objective hematologic or non-hematologic AEs (27%-43%), while dosage modification/delay were more frequent in patients treated with azacitidine (81% vs. 67%, P=0.021).This current study may provide preliminary data in evaluating the efficacy and safety of HMAs in patients with CMML. Decitabine and azacitidine are comparable effective and safe in treating CMML. However, it is necessary to point out that any comparison of decitabine and azacitidine with respect to clinical outcomes can only be done in the context of a randomized controlled trial.

Chimeric antigen receptor (CAR)-modified adoptive T-cell therapy is a promising immunotherapy for hematologic malignancies. However, the efficacy in extramedullary leukemia is still limited and new strategies with improved anti-tumor activity are highly needed. Since C3aR activation builds a bridge between innate immune and adaptive immune and it could trigger Th17 response, we hypothesized that C3aR incorporation as costimulatory domain would augment anti-tumor activity of CAR-T. To address this possibility, we introduced a genetically engineered construct, comprising the domain of C3aR into CAR-T cells. The generated BB-ζ-C3aR CAR-T exhibited potent cytolytic ability to eradicate various tumor cells expressing CD19 or BCMA in vitro. When administrated intravenously into the xenografts leukemia NCG mice who received CD19 + or BCMA + expressing tumor cells, BB-ζ-C3aR CAR-T cells reduced the tumor burden and improved the survival of mice. Of note, these BB-ζ-C3aR CAR-T cells could effectively eradicate subcutaneous CD19 + tumor cells, highlighting the potential therapy for extramedullary leukemia. Mechanistically, the BB-ζ-C3aR CAR-T cells preferred to exhibit tumor-killing Th17 phenotype and suppressed the tumor-tolerated Treg function. In addition, the induction of memory T cells phenotype in BB-ζ-C3aR CAR-T group indicated their long duration effects. Taken together, our findings suggest that the application of C3aR costimulation to boost CAR-T-cell activity is efficacious against aggressive tumor cells via Th17 expansion and memory T cell induction.

IntroductionDysbiosis of gut bacteria has been discovered in a large number of autoimmune diseases. However, the influence of the gut bacteria in the mice model of chronic sclerodermatous graft-versus-host disease (Scl-GVHD), a disease that resembles an autoimmune disease characterized by chronic inflammation of multiple organs, such as skin, remains elusive. Here, we explore the role of gut bacteria in an Scl-cGVHD mice model.MethodsWe established a mouse model of Scl-cGVHD, collected fecal flora, analyzed the composition, and diversity of intestinal flora using 16S rDNA amplicon sequencing, and detected the proportion of Treg and Th1 cells in splenocytes of Scl-cGVHD mice. To verify the immunoregulatory effect of Scl-cGVHD intestinal flora, we prepared bacterial extracts, co-cultured with splenocytes in vitro, and used flow cytometry to detect T cell differentiation and cytokine secretion.ResultsBy examining T-cell differentiation in splenocytes of cGVHD mice, we found that Treg cells were significantly reduced (15.27 ± 0.23 vs. 12.23 ± 0.47, p = 0.0045) and Th1 cells were increased (1.54 ± 0.18 vs. 6.68 ± 0.80, p = 0.0034) in cGVHD mice. Significant differences were observed in the composition and diversity of the gut bacteria in mice with Scl-cGVHD versus without GVHD. Analysis of mice fecal bacteria samples (n = 10, 5 Scl-cGVHD and 5 Non-GVHD) showed significant separation [R = 0.732, p = 0.015, non-parametric analysis (ANOSIM)] in Scl-cGVHD and non-GVHD mice. The abundance of the family and genus Ruminococcaceae bacteria decreased and the family Lachnospiraceae and limited to the species Lachnospiraceae_bacterium_DW17 increased in Scl-cGVHD mice. In vitro results of the cellular level study suggest that the bacteria extracts of gut microbiota from Scl-cGVHD mice modulated the splenic T cells toward differentiation into CD4+IFN-γ+ Th1 cells (14.37 ± 0.32 vs. 10.40 ± 2.19, p = 0.036), and the percentage of CD4+CD25+Foxp3+ Tregs decreased (6.36 ± 0.39 vs. 8.66 ± 0.07, p = 0.001) compared with the non-GVHD mice. In addition, the secretion of proinflammatory interferon- γ (IFN-γ) cytokine in the supplement of cellular culture was increased (4,898.58 ± 235.82 vs. 4,347.87 ± 220.02 pg/ml, p = 0.042) in the mice model of the Scl-cGVHD group, but anti-inflammatory interleukin (IL)-10 decreased (7,636.57 ± 608.05 vs. 9,563.56 ± 603.34 pg/ml, p = 0.018).ConclusionOur data showed the different composition and diversity of gut bacteria in the Scl-cGVHD mice. The dysbiosis of gut bacteria may regulate the differentiation ratio of Treg and Th1 cells, which was associated with Scl-cGVHD.

Sophisticated cross-talk between bone marrow mesenchymal stromal cells (BM MSCs) and haematopoietic/leukaemic stem cells in patients with myelodysplastic syndromes (MDS) and myeloid leukaemia have been emphasized in previous reports. However, mesenchymal elements in patients with chronic myelomonocytic leukaemia (CMML) were poorly investigated. By utilizing a parallel RNA-sequencing method, we investigated the transcriptional profile and functional defects of primary BM MSCs from patients with CMML for the first time. Within a 24-patient cohort, transcriptional and functional analysis reveals a prominent enrichment of WNT/β-catenin signalling and multiple biology processes. Deregulated expression of WNT/β-catnin factors CTNNB1, CMYC, LEF1, and FRZB is associated with impaired proliferation, senescence phenotype, and abnormal secretion in CMML MSCs. The impaired ability to support healthy CD34+ haematopoietic stem and progenitor cells (HSPCs) correlates with activation of WNT/β-catenin signalling in CMML MSCs. Furthermore, we observed an association between WNT/β-catenin factors and treatment response to hypomethylating agents (HMAs) in a cohort of patients with MDS/myeloproliferative neoplasms (MPNs). Taken together, our study provides evidence for transcriptional and functional abnormalities in CMML MSCs, and suggests potential prognostic value of evaluating WNT/β-catenin signalling in patients with CMML.

The concurrence of Myelodysplastic syndromes (MDS) and large granular lymphocyte leukemia (LGLL) has been reported in a small group of patients and might suggest an etiologic relationship rather than a simple coincidence. In this present study, clinicopathological features were detailed in ten cases of MDS concurrent with LGLL (MDS-LGLL). These cases included seven patients with T-LGLL, two with mixed-phenotype LGLL, and one with CLPD-NK. Subsequently, gene mutation screening for commonly myeloid-related or lymphoid-related genes was performed in MDS-LGLL patients by using next generation sequencing (NGS). The genes with the highest frequency of mutations were ASXL1 (3/10, 30%) and STAG2 (3/10, 30%) among a panel of 114 genes. LGLL-associated mutations of STAT3 (2/10, 20%) and STAT5b (1/10, 10%) were also detected. Moreover, whole-exome sequencing (WES) and gene ontology (GO) analysis for one patient in his different phases revealed increased enrichment of histone H3 lysine 4 (H3K4) mono-methylation (GO:0097692) pathway and decreased enrichment of translocation of ZAP-70 to immunological synapse (R-HAS-202430) pathway upon progression from MDS to MDS-LGLL.

The treatment outcomes of intermediate or high-risk myelodysplastic syndrome (MDS) remain unsatisfactory. This study was designed to evaluate the safety and efficacy of human leukocyte antigen (HLA)-mismatched hematopoietic stem cell micro-transplantation (MST) in patients with MDS. A total of 22 patients with MDS, ranging between the ages of 39 and 74, were enrolled in this study. Eleven patients were given decitabine (DAC), a DNA methyltransferase inhibitor, combined with HLA-mismatched MST (MST-DAC group), and the remaining patients were given decitabine only (DAC group). The median overall survival (OS) of the MST-DAC group was higher than that of the DAC group (24 vs. 14.3 months; HR 0.32; 95% CI: 0.11–0.96; p = 0.04), although it is a study with small samples. The overall response rate (ORR), marrow complete remission (mCR), plus hematological improvement (HI) rates of the MST-DAC group were higher than that of the DAC group (81.8 vs. 54.5%, p = 0.36; 63.6 vs. 27.3%, p = 0.09, respectively); however, there were no statistical differences between the two groups, which may be attributed to the limited number of cases evaluated in this study. No graft-vs.-host disease was observed in the MST-DAC group. Patients in the MST-DAC group demonstrated a slightly lower incidence of hematological and non-hematological adverse events (AEs). DAC combined with HLA-mismatched MST may provide a novel, effective, and safe treatment for use in intermediate or high-risk MDS pathologies.

Background Cytogenetic and molecular genetic abnormalities are now considered to be important factors affecting the prognosis of acute myeloid leukemia (AML), while variable outcomes may appear in the same risk group. Immunophenotyping has been the core in AML diagnosis, which is simple and universal application. The aim of this study was to explore and identify new immunophenotypic markers that related to the diagnosis and prognosis of AML.Methods The expression of C type lectin like molecule 1 (CLL‐1) in AML blasts were examined in 52 patients with newly diagnosed or relapsed/refractory AML, and was compared with other classic markers in AML, in order to assess the value of CLL-1 as an independent biomarker or in combination with other markers for diagnosis in AML. Subsequently, we assessed the value of CLL-1 as a biomarker for prognosis in this malignant tumor.Results The results showed that CLL 1 was expressed on the cell surface of the majority of AML blasts (78.8%), and also expressed on leukemic stem cells in varying degree but absent on normal hematopoietic stem cells. Notably, CLL 1 was able to complement the classic markers CD33 or CD34. After dividing the cases into CLL‐1high and CLL‐1low groups according to cutoff 59.0%, we discovered that event free survival and overall survival (OS) of the CLL‐1low group were significantly lower than the CLL‐1high group, and low CLL 1 expression seems to be independently associated with shorter OS.Conclusions These data identified CLL 1 as a potential biomarker for diagnosis and prognosis of AML.

Objective To illustrate the potential effects and mechanism of extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) on fibrosis in sclerodermatous chronic graft-versus-host-disease (cGVHD) models after allogeneic hematopoietic stem cell transplantation. Methods We first observed the therapeutic effects of MSC-EVs on a minor histocompatibility haploidentical model of sclerodermatous cGVHD and the function of MSC-EVs on skin fibrosis and macrophage activation and the related pro-fibrosis protein. Additionally, we observed the effects of MSC-EVs on B cells, the T follicular helper cell (TFH) and germinal center B cell (GC B cells) interaction and the ratio of B cell activation factor (BAFF) to B cells in vivo. Results MSC-EVs treatment could alleviate the cGVHD scores and fibrosis of skin in sclerodermatous cGVHD mice, and this was associated with a reduction macrophage percentage in the skin and spleen, and a reduction in macrophage infiltration and TGF-β and smad2 production in the skin. Additionally, MSC-EVs influence B cells immune response by blocking the TFH/GC B cells interaction and reducing the ratio of BAFF to B cells in vivo. Conclusion MSC-EVs prevent the fibrosis of sclerodermatous cGVHD mouse model by suppressing the activation of macrophages and B cells immune response.

Mesenchymal stem cells (MSCs) derived from myelodysplastic syndromes (MDSs) have been demonstrated to accelerate the progression of MDS. However, whether the phenotype of MSCs derived from MDS (MDS-MSCs) may be reversed and serve as a potential target for the treatment of MDS remains unclear. The present study investigated the functional alternations of MDS-MSCs following in vitro decitabine-treatment. Primary MSCs were cultured from the bone marrow aspirates of 28 patients with MDS. The impact on the growth of MDS-MSCs treated with decitabine was analyzed using the MTT assay. Changes in the gene expression levels of runt related transcription factor 2 (RUNX2), Sp7 transcription factor (SP7), cyclin dependent kinase inhibitor 1A (CDKN1A) and CD274 in MDS-MSCs following treatment with decitabine were analyzed by reverse transcription-quantitative polymerase chain reaction. The effects of decitabine on apoptosis and the cell cycle were examined using flow cytometry. The effect of decitabine on the immune regulation of MDS-MSCs was tested by the co-culture of MSCs with activated T cells in vitro. The results revealed that proliferation, apoptosis and the mRNA expression levels of RUNX2 and SP7 in MDS-MSCs did not significantly change following treatment with decitabine compared with control MDS-MSCs. However, treatment with decitabine resulted in a smaller population of cells in the G1 phase and an increase in the number of cells in the G2/M phase compared with control MDS-MSCs. This change was associated with decreased expression of CDKN1A in cells treated with decitabine compared with control cells. Notably, the ability of MDS-MSCs treated with decitabine to induce the differentiation of T cells into regulatory T cells was significantly reduced compared with control MDS-MSCs. This was associated with a decreased expression of CD274 in MDS-MSCs treated with decitabine compared with control MDS-MSCs. In conclusion, the phenotype of MSCs derived from patients with MDS was partially reversed by treatment with decitabine, presenting a potential therapeutic intervention.

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.

Currently, arsenic trioxide therapy is widely used for the treatment of acute promyelocytic leukemia (APL), relapsed and refractory adult T-cell leukemia/lymphoma and myelodysplastic syndrome. Regarding the broad antitumor activity of arsenic, certain studies have been undertaken to test its efficacy in treating acute T-cell lymphoblastic leukemia (T-ALL) cell lines and patients; however, to the best of our knowledge, no reports document that arsenic is able to induce the remission of T-ALL patients. The present study reports the case of young male patient diagnosed with T-ALL, with no significant response to common chemotherapy regimens, who finally achieved complete remission without minimal residual disease (as detected by flow cytometry) due to arsenic treatment. This result is encouraging, and the present study has shown that malignant TCRαβ+ cell clones can be detected at the molecular level using reverse transcription-polymerase chain reaction (PCR) combined with the GeneScan technique. The result is mainly based on the T-cell receptor (TCR) Vβ1 clone (a 190-base pair PCR product that with the same complementarity determining region 3 length can be detected for all samples collected during various statuses) and on undetectable TCR Vγ subfamily members, at the time of disease diagnosis. It is important to analyze the dynamically changing TCR pool in leukemia patients during therapy. Although the molecular mechanism through which arsenic contributes to malignant clone elimination remains unclear in the case presented, the use of arsenic is expected to be effective for clinically treating refractory and relapsed T-ALL patients.

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.

The aim of the present study was to conduct a retrospective analysis of efficacy and safety profiles of azacitidine (AZA) versus. decitabine (DAC) in Chinese patients with intermediate or higher-risk MDS, which was based on two clinical trials in a single center. A total of 40 included MDS patients diagnosed with refractory anemia with excess blast (RAEB) were from two independent clinic trials. Patients in each trial received either AZA (n = 19) or DAC (n = 21) respectively, and the effectiveness as well as the safety profile of the two drugs were compared. Patients treated with AZA showed a comparative efficacy to DAC group with regard to the overall response rate (73.7% versus. 76.2%, P = 0.86), overall survival (median: 19.3 versus. 20.8 months, P = 0.56), progression-free survival (median: 12.3 versus. 9.3 months, P = 0.43) and leukemia-free survival (median: 22.8 versus. 26.6 months, P = 0.62). Patients treated with DAC showed slightly higher incidence of severe hematological adverse events during the whole treatment. Comparing hematological AEs in each observation interval, a trend of higher percentage of neutropenia, leukopenia and anemia as well as treatment delays were seen during the first 6 cycles in the DAC group.

Chronic graft-versus-host disease (cGVHD) manifests with features characteristic of autoimmune disease with organs attacked by pathogenic Th17 cells. However, the mechanism of Th17 cells generation in the setting of cGVHD is still unclear. Here we defined C5a/C5aR-IL-17Aaxis as a novel signaling that required in the pathologies of cGVHD. We firstly found a positive link between complement activation and the Th17 cells in patients with cGVHD. C5a, a critical component of complements, promoted the generation of Th17 cells in vitro and inhibition of the receptor for C5a (C5aR) reduced the Th17-bias response. Of note, C5aR blockade by PMX53 could suppress the generation of IL-17A-expressing Th17 cells and retard the onset and progression of cGVHD in vivo. Overall, our results provide new mechanistic insights that activation of C5a-C5aR signaling was required for IL-17A-induced immune responses in cGVHD and define novel molecular targets for developing effective therapeutics for cGVHD.

Despite recent advances in the treatment of myelodysplastic syndrome (MDS), single-agent clinical effects remain unsatisfactory, and decitabine monotherapy is also associated with a relatively low rate of complete remission. To study the combined effects and mechanism of decitabine (DAC) and arsenic trioxide (ATO) on the human myelodysplastic cell line SKM-1,we used the MTS assay and CalcuSyn software to determine the cytotoxicity and potential synergistic effects, respectively. Furthermore, we determined apoptosis and measured the mRNA expression level of two genes that are considered main regulators of the apoptosis process. The results showed that DAC and/or ATO can inhibit the proliferation of SKM-1 cells and demonstrated significant synergy between the two agents (CI

Immune dysregulation has been implicated in myelodysplastic syndrome (MDS) pathogenesis. Refractory anemia with ringed sideroblasts (RARS) is a low-risk subtype of MDS. Our previous study has reported an abnormal γδ T cell receptor (TCR) repertoire in patients with intermediate or high-risk MDS. To characterize the status of T cell immunity in RARS, we investigated the distribution and clonality of the TCR Vδ repertoire and the expression of Foxp3 in patients with RARS. The number of expressed Vδ subfamily members in the RARS group (4.8±2.25) was significantly lower than that in the control group (7.6±0.52, p=0.0012). A significantly lower expression frequency for the Vδ4 (p=0.007), Vδ5 (p=0.0049) and Vδ7 subfamilies (p=0.0225) could be detected in the RARS group. The most frequent clonally expanded T cell subfamily member in the RARS group was Vδ7 (100%, 3/3). Foxp3 mRNA expression was significantly lower and higher than that in the controls in 60% and 40% RARS patients, respectively. In conclusion, marked restriction of the TCR Vδ subfamily expression pattern and great heterogeneity in Foxp3 expression were characteristics found in RARS. These results provide data regarding the immunodeficiency and immune reactive characteristics of patients with RARS, which may provide a basis for immunotherapy options.

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.

Table S1. Primers used for real-time PCR. (DOCX 17Â kb)

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.

Extracellular vesicles (EVs) secreted by mesenchymal stem cells (MSC-EVs) are taken more seriously as immunomodulatory and anti-inflammatory agents. We studied the therapeutic effects of MSC-EVs on allergic contact dermatitis (ACD), a typical T cell-mediated disorder. A contact hypersensitivity (CHS) mouse model for ACD was established and treated by intravenous MSC-EVs injection. We found that human umbilical cord MSC-EVs could significantly prevent the pathology of CHS, including reduced ear swelling and leukocyte infiltration. Injection of MSC-EVs significantly inhibited CD8+IFN-γ+ cytotoxic T (Tc1) cells and CD4+IFN-γ+ type 1 helper T (Th1) cells, and reduced the level of pro-inflammatory Tumor Necrosis Factor-alpha (TNF-α) and interferon gamma (IFN-γ), and induced CD4+CD25+Foxp3+ regulatory T cells (Tregs) and the level of anti-inflammatory IL-10. In vitro, MSC-EVs also suppressed Tc1 and Th1 cells and induced Tregs and the related cytokines, further indicating the immune regulatory role of MSC-EVs. Interestingly, PKH26-labeled MSC-EVs were found to be directly internalized by CD3+ T cells, resulting in reduced signal transducer and activator of transcription 1 (STAT1) protein levels in vitro. In summary, MSC-EVs can prevent the onset of CHS by inhibiting Tc1 and Th1 immune responses and inducing the Tregs phenotype in vivo and in vitro. The mechanism by which MSC-EVs influence CD3+ T cells might partially involve targeting STAT1 in vitro. Therefore, MSC-EVs are ideal candidates for cell-free immunomodulatory therapy for T cell-mediated diseases such as ACD.

Myelodysplastic syndrome (MDS) predominantly occurs in aging people. Over the past decades, the cellular and molecular pathologies of MDS cells have been intensively investigated. However, how the bone marrow stromal niches are altered during MDS development remains elusive. In this study, we attempted to isolate and characterize mesenchymal stromal cells (MSCs) from 30 MDS patients. We observed that only 9/30 bone marrow aspirations from MDS patients successfully formed a monolayer in vitro, while 17/17 bone marrow aspirations from normal donors (median age 45 years, range: 22-73 years) succeeded in this process. Compared to normal MSCs, the MDS MSCs showed premature exhaustion, including reduced osteogenic differentiation ability, slower passage rate, and extremely limited passage times. These functional defects were associated with downregulation of Osterix and Runx2 genes and increased cell cycle arrest and apoptosis. However, the premature exhaustion of MDS MSCs did not correlate with patients’ ages, indicating that natural aging is not the cause of dysfunction in MDS MSCs. Our result provides a strong rational to target prematurely exhausting MSCs in future MDS treatment.

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.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic stem cell diseases. Approximately 30% of patients with MDS will develop acute myeloid leukemia (AML). Immune dysregulation may contribute to MDS initiation and progression. The altered expression and clonal expansion of the Vβ repertoire were observed in patients with MDS. To further examine the characteristic features of γδ(+)T cells in MDS, we investigated the distribution pattern and clonal expansion capacity of the T-cell receptor (TCR) Vγ and Vδ repertoire in patients with refractory anemia with excess of blasts (RAEB) and compared the difference between groups of patients with RAEB and RAEB-AML. Thirty-one patients with newly diagnosed MDS-RAEB were enrolled, and 9 of the 31 patients with RAEB developed AML (RAEB-AML). The TCR Vγ subfamily expression frequencies were similar in the RAEB and RAEB-AML patient groups. The number of the TCR Vδ subfamilies expressed in the RAEB group was higher than that in the RAEB-AML group. In most cases, a significantly higher Vδ4 subfamily expression frequency (63.64%, 14/22) could be detected in the RAEB group, whereas only 11.11% (1/9) was found in the RAEB-AML group (p=0.0079). At least one clonally expanded TCR Vδ subfamily member was detected in all cases in both groups. Vδ3 was the most frequent clonally expanded T cell subfamily member found in the RAEB and RAEB-AML group, while the most frequent clonally expanded T cell subfamily member in the RAEB-AML group was Vδ8 (87.5%, 7/8), which was significantly higher than that in the RAEB group (42.86%, 9/21; p=0.0307). In conclusion, the TCR Vδ subfamily expression pattern exhibited a marked restriction in patients with RAEB-AML. The lower Vδ4 frequency and higher clonally expanded Vδ8 T cell alterations were the characteristic features found in RAEB-AML. These results provide new data regarding the immunodeficiency and immune reactive characteristics of patients with RAEB and RAEB-AML.

T-cell immunodeficiency is a common feature in cancer patients, which may relate to initiation and development of tumor. In expanding our previous observations in this area, we studied the repertoire of T-cell receptor beta variable region (TRBV) and T-cell proliferative history in CD4+ and CD8+ T cells from chronic myeloid leukemia (CML) patients. The expression and clonality analysis were performed by reverse transcription-polymerase chain reaction (RT-PCR) and GeneScan technique in peripheral blood mononuclear cells (PBMCs), CD4+ and CD8+ subsets of T cells. Nineteen CML cases in chronic phase were selected for this study and 17 healthy individuals served as controls. Marked restriction of TRBV repertoire was observed in both CD4+ and CD8+ T cells from CML. In most CML samples, clonally expanded T cells were identified in CD4+ and CD8+ T cells, predominantly in TRBV19 and TRBV21 (5/19) subfamilies. In conclusion, the restricted expression of TRBV subfamilies indicates the T-cell immunodeficiency in CML patients; however, clonally expanded T cells suggest a specific immune response to leukemia associated antigens.

To investigate the effects of Artesunate treatment on chronic graft-versus-host disease (cGVHD). Recipient BALB/cJ mice received 8 × 106 bone marrow cells with 8 × 106 spleen cells from B10.D2 mice. Artesunate solubilized in acetone was injected intraperitoneally every day at the dose of 1 mg/kg at Day 28 after transplatation. The clinical scores, survival times and histopathological damage were analyzed. The frequency of Th17 and Tregs in PB and spleens from the mice was evaluated by flow cytometry. In addition, we cultured the CD4+ T cells from the spleens of mice in vitro, and stimulated these cells with artesunate, the frequency of Th17 and Tregs in these splenocytes was evaluated by flow cytometry. Artesunate administration diminishes clinical and histopathological damage and improved the survival of cGVHD mice. Artesunate contributed to Tregs development (4.45±0.04 vs 8.40±0.23; 6.62±0.24 vs 10.48±0.48; p Disclosures No relevant conflicts of interest to declare.

Refractory chronic GVHD (cGVHD) is an important complication after allogeneic hematopoietic SCT and is prognostic of poor outcome. MSCs are involved in tissue repair and modulating immune responses in vitro and in vivo. From April 2005 to October 2008, 19 patients with refractory cGVHD were treated with MSCs derived from the BM of volunteers. The median dose of MSCs was 0.6 × 10(6) cells per kg body weight. Fourteen of 19 patients (73.7%) responded well to MSCs, achieving a CR (n=4) or a PR (n=10). The immunosuppressive agent could be tapered to less than 50% of the starting dose in 5 of 14 surviving patients, and five patients could discontinue immunosuppressive agents. The median duration between MSC administration and immunosuppressive therapy discontinuation was 324 days (range, 200-550 days). No patients experienced adverse events during or immediately after MSC infusion. The 2-year survival rate was 77.7% in this study. Clinical improvement was accompanied by the increasing ratio of CD5+CD19+/CD5-CD19+ B cells and CD8+CD28-/CD8+CD28+ T cells. In conclusion, transfusion of MSCs expanded in vitro, irrespective of the donor, might be a safe and effective salvage therapy for patients with steroid-resistant, cGVHD.

To revalidate the conversion factor（CF）for the conversion of BCR-ABL （P210）transcript levels to the international scale（BCR- ABLIS）in chronic myeloid leukemia（CML） which validated before.Peking University People's Hospital（PKUPH）prepared the exchange samples for revalidation of CFs of 15 laboratories which validated nine or eighteen months ago. The fresh BCR-ABL（P210）（+）bone morrow or peripheral blood nucleated cells were diluted with BCR-ABL （P210）（-）cells to achieve different BCR- ABL levels, totally 16 sets and 24 samples per set were prepared. TRIzol reagent was added in each tube. Each laboratory tested BCR-ABL transcript levels of one set of samples. Agreement between BCR-ABLIS of each laboratory and PKUPH was assessed by the Bland- Altman method. For laboratories which did not meet the criteria of revalidation, linear regression equation was derived after the samples with maximum BCR-ABL deviation were removed until R²0.98, then new CF was calculated.10 laboratories met the revalidation criteria with both bias within ±1.4 fold and 95% limits of agreement within ±6 folds, and their CFs still could be used for accurately conversion of BCR-ABLIS. New CFs were recalculated as of 1.8-6.3 folds of their previous CFs in 5 laboratories not met the criteria.Revalidation of CF by sample exchange among laboratories was necessary for accurate and continuous application of BCR-ABLIS, which not only tested the validity of CF acquired before but also calculated new available CFs for those with invalid CFs.

Background: In the 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, which may be related to PHTF1 gene expression, and the FEM1B and Apaf-1 genes may be downstream of PHTF1. In this study, we analyzed the expression level of PHTF1 and related genes in patients with ALL to clarify the role of the PHTF1-FEM1b-Apaf-1 pathway in hematologic malignancies. Methods: Fifteen newly diagnosed and untreated patients with AML, fourteen newly diagnosed and untreated patients with CML in chronic phase, twenty-two newly diagnosed and untreated patients with ALL, and six newly diagnosed and untreated patients with CLL were recruited. Peripheral blood mononuclear cells (PBMCs) from ten healthy individuals (HIs) served as controls. PBMCs were separated using the Ficoll-Hypaque gradient centrifugation method. All procedures were conducted in accordance with the guidelines of the Medical Ethics committees of the Health Bureau of Guangdong Province, China.Real-time PCR was used to determine the gene expression level of PHTF1 in hematologic malignancies. The PHTF1, BCL11B, FEM1B and Apaf-1 gene expression levels and correlations were analyzed in patients with primary ALL and healthy individuals (HIs). Results: PHTF1 overexpression was found in recently diagnosed AML (p Conclusions: PHTF1 acts as a tumor suppressor gene, and its overexpression might be related to cell proliferation, inhibition and apoptosis. PHTF1 and BCL11B gene disorders may contribute to T-ALL pathogenesis. PHTF1 might be a therapeutic target for triggering the PHTF1-FEM1b-Apaf-1 apoptosis pathway in primary acute lymphoblastic leukemia. Acknowledgment The project was sponsored by grants from National Natural Science Foundation of China (No. 81100384, No. 81270648, No.91129720, and No. 30771980) Disclosures No relevant conflicts of interest to declare.