Comprehensive analysis of human monocyte subsets using full-spectrum flow cytometry and hierarchical marker clustering.

Introduction: Exploring monocytes' roles within the tumor microenvironment is crucial for crafting targeted cancer treatments. Methods: This study unveils a novel methodology utilizing four 20-color flow cytometry panels for comprehensive peripheral immune system phenotyping, specifically targeting classical, intermediate, and non-classical monocyte subsets. Results: By applying advanced dimensionality reduction techniques like t-distributed stochastic neighbor embedding (tSNE) and FlowSom analysis, we performed an extensive profiling of monocytes, assessing 50 unique cell surface markers related to a wide range of immunological functions, including activation, differentiation, and immune checkpoint regulation. Discussion: This in-depth approach significantly refines the identification of monocyte subsets, directly supporting the development of personalized immunotherapies and enhancing diagnostic precision. Our pioneering panel for monocyte phenotyping marks a substantial leap in understanding monocyte biology, with profound implications for the accuracy of disease diagnostics and the success of checkpoint-inhibitor therapies. Key findings include revealing distinct marker expression patterns linked to tumor progression and providing new avenues for targeted therapeutic interventions.

Diagnostic and prognostic value of ferroptosis-related genes in patients with Myelodysplastic neoplasms.

This study delves into the emerging role of ferroptosis in Myelodysplastic Neoplasms (MDS) and aims to identify a prognostic ferroptosis-related gene signature for MDS. Utilizing RNA-seq data and clinical information from the Gene Expression Omnibus database, the researchers extracted ferroptosis-related genes from the FerrDb website and conducted differential expression analysis using the 'limma' package in R. Hub ferroptosis-related genes in MDS were screened using the "RandomForest" and "carat" R packages. Kaplan -Meier and Cox regression analyses were employed to assess the prognostic role of three identified hub genes (BNIP3, MDM2, and RRM2). Receiver operator characteristic curve analysis confirmed the diagnostic efficacy of these genes. The study delved further into immune infiltration correlations, ncRNA-transcription factor coregulatory network analysis, and the identification of potential therapeutic drugs targeting hub ferroptosis-related genes in MDS. The researchers constructed a 3-gene signature-based risk score using datasets GSE58831 and GSE19429, demonstrating high accuracy (AUC > 0.75) in both datasets for survival prediction in MDS. A nomogram analysis reinforced the prognostic value of the risk-scoring model. Immunological analysis revealed an association between the risk score and immune infiltration. Quantitative reverse transcription polymerase chain reaction (qPCR) data indicated significant expression differences in MDM2, RRM2, and BNIP3 between MDS and healthy bone marrow samples. Notably, MDM2 and RRM2 showed decreased expression, while BNIP3 exhibited increased expression in MDS samples. This comprehensive study concludes that BNIP3, MDM2, and RRM2 hold diagnostic and prognostic significance in MDS and provide valuable insights into immune cell landscapes and potential therapeutic avenues for this condition.

Mesenchymal stem cells reversibly de-differentiate myofibroblasts to fibroblast-like cells by inhibiting the TGF-ß-SMAD2/3 pathway.

BACKGROUND: Myofibroblasts (MFB), one of the major effectors of pathologic fibrosis, mainly derived from the activation of fibroblast to myofibroblast transition (FMT). Although MFBs were historically considered terminally differentiated cells, their potential for de-differentiation was recently recognized and implied with therapeutic value in treating fibrotic diseases, for instance, idiopathic pulmonary fibrosis (IPF) and post allogeneic hematopoietic stem cell transplantation bronchiolitis obliterans (BO). During the past decade, several methods were reported to block or reverse MFB differentiation, among which mesenchymal stem cells (MSC) have demonstrated potential but undetermined therapeutic values. However, the MSC-mediated regulation of FMT and underlying mechanisms remained largely undefined. METHOD: By identifying TGF-ß1 hypertension as the pivotal landmark during the pro-fibrotic FMT, TGF-ß1-induced MFB and MSC co-culture models were established and utilized to investigate regulations by MSC on FMT in vitro. Methods including RNA sequencing (RNA-seq), Western blot, qPCR and flow cytometry were used. RESULT: Our data revealed that TGF-ß1 readily induced invasive signatures identified in fibrotic tissues and initiated MFB differentiation in normal FB. MSC reversibly de-differentiated MFB into a group of FB-like cells by selectively inhibiting the TGF-ß-SMAD2/3 signaling. Importantly, these proliferation-boosted FB-like cells remained sensitive to TGF-ß1 and could be re-induced into MFB. CONCLUSION: Our findings highlighted the reversibility of MSC-mediated de-differentiation of MFB through TGF-ß-SMAD2/3 signaling, which may explain MSC's inconsistent clinical efficacies in treating BO and other fibrotic diseases. These de-differentiated FB-like cells are still sensitive to TGF-ß1 and may further deteriorate MFB phenotypes unless the pro-fibrotic microenvironment is corrected.

Targeting Bcl-6 prevents sclerodermatous chronic graft-versus-host disease by abrogating T follicular helper differentiation in mice.

BACKGROUND: Chronic graft-versus-host disease (cGVHD) is the most common cause of non-relapse mortality (NRM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). CD4+ follicular helper T (Tfh) cells, specialized providers of T cell help to B cells, play a vital role in GVHD pathogenesis. B-cell lymphoma-6 (Bcl-6) transcription factor has been shown to be required for Tfh-mediated germinal center reactions. In this study, we would like to evaluate the effect of Bcl-6 on Tfh function in sclerodermatous cGVHD and the efficacy of Bcl-6 inhibitors (Bcl-6i) for treating a minor histocompatibility complex (miHC) mismatch model of sclerodermatous cGVHD (scl-cGVHD). METHODS: A minor histocompatibility haploidentical model of scl-cGVHD was established and received intraperitoneal injection of 79-6, a small-molecule inhibitor of Bcl-6. The clinical manifestations and survival times of cGVHD mice were recorded. The histological assessment was performed by hematoxylin-eosin (HE) and Masson's trichrome staining on the skin and lung tissues. Tfh cells and germinal center B cells in the spleen and peripheral blood were detected by flow cytometry. The cellular markers were immunostained in different organs. ELISA was performed to detect cytokine secretion. RESULTS: Bcl-6 inhibition by 79-6 improved the clinical manifestation of scl-cGVHD mice and prolonged their survival. The histopathologic damage, particular the fibrotic changes of scl-cGVHD mice was significantly relieved after 79-6 treatment. Furthermore, 79-6 treatment not only suppressed the development and function of Tfh and Tph cells in the peripheral blood, but also reduced the survival of Tfh cells in the spleen. Moreover, 79-6 decreased the frequency of GC plasmocytes accompanied by a reduction in IL-21. CONCLUSIONS: Our study demonstrates that Bcl-6 inhibitor could prevent murine sclerodermatous chronic graft-versus-host disease by abrogating T follicular helper differentiation and suppressing the function of GC B cells, indicating that Bcl-6 inhibition may be a potential treatment for patients with cGVHD.

Incidence and effect of secondary cardiac amyloidosis on outcomes of patients with t(11;14) multiple myeloma.

Background: The t(11;14)(q13;32) is a common chromosome translocation in multiple myeloma (MM), but its prognostic value remains controversial. Immunoglobulin light chain amyloidosis is commonly secondary to multiple myeloma, which can rapidly cause heart failure and high mortality. We aimed to investigate the prevalence of secondary cardiac amyloidosis in MM patients with t(11;14) and to evaluate its impact on survival outcomes. Methods: We retrospectively identified 52 MM patients with t(11;14) in our center between October 2015 and April 2022. The associations between cardiac amyloidosis and clinical and biological parameters were statistically analyzed, and the impacts of concomitant of cardiac amyloidosis on survival and prognosis of MM patients with t(11;14) were also assessed. Results: Concomitant presence of cardiac amyloidosis was observed in 15 (28.8%) of all cases. Patients with cardiac amyloidosis had significantly higher NT-proBNP (p = 0.002) and higher hs-cTnT (p < 0.001), while the patients without cardiac amyloidosis had higher percentage of bone marrow plasma cells (p = 0.027), higher incidence of hemoglobin <80 g/L (p = 0.021) and bone destruction (p < 0.001). The median overall survival (OS) for all patients was 33.4 months after a median follow-up of 23.8 months. The amyloidosis group showed a significantly shorter OS than the non-amyloidosis group (15.3 vs. 41.8 months, p < 0.001). Besides, patients harboring NT-proBNP >1,800 pg/ml (p < 0.001) or hs-cTnT ≧40 pg/ml (p = 0.001) or light chain (LC) only isotype (p = 0.033) had a significantly shorter mean OS compared with patients with lower NT-proBNP or hs-cTnT or other M-protein isotype. Univariate analyses showed that NT-proBNP >1,800 pg/ml, hs-cTnT ≧40 pg/ml, LC only isotype, and concomitant presence of cardiac amyloidosis were independently associated with shorter OS, while NT-proBNP >1,800 pg/ml still retained the prognostic value for OS in multivariate analyses. Conclusion: The t(11;14) MM patients with coexisting cardiac amyloidosis may represent a distinct clinical entity that confers a poor outcome. These findings may have important clinical and biological implications.

Gene Mutations Related to Glucocorticoid Resistance in Pediatric Acute Lymphoblastic Leukemia.

Objective: To investigate the correlation between gene mutations and glucocorticoid resistance in pediatric acute lymphoblastic leukemia (ALL). Methods: A total of 71 children with ALL admitted to our center between September 2019 and September 2021 were enrolled. DNA obtained from bone marrow or peripheral blood samples at initial diagnosis was used for genetic testing via whole exome sequencing. Meanwhile, patient clinical information was collected. Subsequently, the correlations of gene mutations with clinical features and glucocorticoid resistance were analyzed. Results: Of the 71 children enrolled, 61 (85.9%) had B-cell ALL (B-ALL) and 10 (14.1%) had T-cell ALL (T-ALL). The five genes with the highest mutation frequency in B-ALL were TTN (24.4%), FLT3 (14.6%), TP53 (14.6%), MUC16 (9.8%), and EPPK1 (9.8%). In contrast, those with the highest frequency in T-ALL were NOTCH1 (54.5%), FBXW7 (27.3%), TTN (27.3%), MUC16 (27.3%), and PHF6 (18.2%). Upon statistical analysis, TTN and NOTCH1 mutations were found to be associated with prednisone resistance. Further, TTN and MUC16 mutations were associated with a lower age at diagnosis, and NOTCH1 mutations were associated with T-ALL in female patients. Leukocyte counts and LDH levels did not differ based on the presence of any common gene mutation, and no association between these gene mutations and overall survival was observed. Conclusions: Our study is the first to demonstrate the association between TTN mutation and glucocorticoid resistance in ALL. Our findings could guide strategies for overcoming drug resistance and aid in the development of drug targets.

The novel HLA-A*69:01:04 allele identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-A*69:01:04 differs from HLA-A*69:01:01:01 in exon 2 at position 336 by a single synonymous mutation.

Lower BCL11B expression is associated with adverse clinical outcome for patients with myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is an aggressive and genetically heterogeneous disease with poor prognosis. Cellular immune disorder is a common characteristic of this disease and is thought to be related to clinical outcome. Alterations in T cell clonal expansion and T cell dysfunction has been detected in MDS patients. Little is known about whether there are immune biomarkers to evaluate the T cell alterations with clinical outcome. Previous studies have demonstrated that B-cell leukemia/lymphoma 11B (BCL11B) plays an important role in regulating T cell development and proliferation. In this study, the prognostic value of BCL11B for MDS patients was explored by analyzing RNA-seq data from 270 patients in two datasets in the Gene Expression Omnibus (GEO) database and real-time quantitative PCR data (qRT-PCR) of 31 bone marrow (BM) samples of MDS and 6 BM samples of patients with MDS progress to secondary acute myeloid leukemia (sAML) from our clinical center. The results demonstrated that BCL11B is significantly down-regulated in MDS patients as compared with healthy individuals (HIs). Importantly, lower BCL11B expression was found in MDS patients who were of high/very high risk, older than 60 y, or male and patients with sAML. Furthermore, low BCL11B expression appeared to be associated with poor overall survival (OS) for MDS patients, though the data were not yet significant enough at this point. In addition, BCL11B low-expressing MDS patients had shorter restricted mean survival time (RMST) than those with high BCL11B expression. Interestingly, BCL11B positively correlated with naive and activated memory CD4 + T cells, CD8 + T cells, and the T cell receptor complex genes CD3E and CD3G, but it negatively correlated with regulatory T cells (Treg). Additionally, co-occurrence of low BCL11B expression and CD3E and CD3G was associated with poor OS and shorter RMST. In conclusion, lower BCL11B expression in BM samples of MDS patients was associated with adverse clinical outcome.

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

The original article [1] contains an error in authorship whereby author, Robert Weinkove's name is mistakenly inverted. The configuration noted in this Correction article should be considered instead along with author's updated affiliation.

Phenotype of mesenchymal stem cells from patients with myelodyplastic syndrome maybe partly modulated by decitabine.

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.

Emerging role of C5a/C5aR IL-17A axis in cGVHD.

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.

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

BACKGROUND: Anti-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. METHODS: 1928zT2 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. RESULTS: 1928zT2 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. CONCLUSIONS: Our 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 REGISTRATION: ClinicalTrials.gov identifier NCT02822326 . Date of registration: July 4, 2016.

Premature exhaustion of mesenchymal stromal cells from myelodysplastic syndrome patients.

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.

Analysis of the expression of PHTF1 and related genes in acute lymphoblastic leukemia.

BACKGROUND: Previous study showed that downregulated BCL11B expression in T cell acute lymphoblastic leukemia (T-ALL) cell line Molt-4 inhibited cell proliferation and induce apoptosis, which may be related to PHTF1 gene overexpression. The objective of this study was to investigate the expression of PHTF1 and related genes in ALL and further explore its function in T-ALL cell lines. METHODS: 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 (including T-ALL and B-ALL) and healthy individuals (HIs). Inhibition and overexpression of PHTF1 by lentiviral transduction were performed using the Molt-4 and Jurkat cell lines. Cell growth and apoptosis were measured by the Cell Counting Kit-8 assay and flow cytometry, respectively. Upon PHTF1 overexpression, the BCL11B, FEM1B and Apaf-1 gene expression levels were determined by real-time PCR. RESULTS: PHTF1 overexpression was found in both T-ALL (p = 0.004) and B-ALL (p < 0.001) groups compared with HIs group. A trend toward a negative correlation between the PHTF1 and BCL11B genes was detected for the T-ALL group, while positively correlated expression was found for the PHTF1 and BCL11B genes in HIs (P = 0.001). FEM1b and Apaf-1 overexpression was found in recently diagnosed ALL patients compared with HIs (p < 0.05). Positively correlated expression was found for the PHTF1, FEM1b and Apaf-1 genes in patients with ALL (p < 0.05) and HIs (p < 0.05). Direct up-regulation of PHTF1 expression inhibited the proliferation of Jurkat and Molt-4 cells and effectively induced apoptosis in Molt-4 cells. Direct inhibition of PHTF1 expression had no significant effect on the proliferation or apoptosis of Jurkat and Molt-4 cells. FEM1b and Apaf-1 overexpression, which did not obviously alter the BCL11B expression level, was detected in PHTF1-transduced T-ALL cell lines. CONCLUSIONS: PHTF1 overexpression is responsible for regulating cell proliferation and apoptosis in T-ALL cell lines. PHTF1 may be a tumor-suppressor like gene and a therapeutic target for triggering the PHTF1-FEM1b-Apaf-1 apoptosis pathway.