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To further understand the impact of deficiency of the autoimmune regulator ( Aire ) gene during the adhesion of medullary thymic epithelial cells (mTECs) to thymocytes, we sequenced single-cell libraries (scRNA-seq) obtained from Aire wild-type (WT) ( Aire wt/wt ) or Aire -deficient ( Aire wt/mut ) mTECs cocultured with WT single-positive (SP) CD4 + thymocytes. Although the libraries differed in their mRNA and long noncoding RNA (lncRNA) profiles, indicating that mTECs were heterogeneous in terms of their transcriptome, UMAP clustering revealed that both mTEC lines expressed their specific markers, i.e., Epcam, Itgb4 , Itga6 , and Casp3 in resting mTECs and Ccna2, Pbk , and Birc5 in proliferative mTECs. Both cocultured SP CD4 + thymocytes remained in a homogeneous cluster expressing the Il7r and Ccr7 markers. Comparisons of the two types of cocultures revealed the differential expression of mRNAs that encode transcription factors ( Zfpm2, Satb1 , and Lef1 ), cell adhesion genes ( Itgb1 ) in mTECs, and Themis in thymocytes, which is associated with the regulation of positive and negative selection. At the single-cell sequencing resolution, we observed that Aire acts on both Aire WT and Aire -deficient mTECs as an upstream controller of mRNAs, which encode transcription factors or adhesion proteins that, in turn, are posttranscriptionally controlled by lncRNAs, for example, Neat1, Malat1, Pvt1, and Dancr among others. Under Aire deficiency, mTECs dysregulate the expression of MHC-II, CD80, and CD326 (EPCAM) protein markers as well as metabolism and cell cycle-related mRNAs, which delay the cell cycle progression. Moreover, when adhered to mTECs, WT SP CD4 + or CD8 + thymocytes modulate the expression of cell activation proteins, including CD28 and CD152/CTLA4, and the expression of cellular metabolism mRNAs. These findings indicate a complex mechanism through which an imbalance in Aire expression can affect mTECs and thymocytes during adhesion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Monteiro, Duarte, Machado, Mascarenhas, Palma, García, Nakaya, Cunha, Donadi and Passos.)

Objectives: To isolate cancer stem cells (CSC) from a metastatic oral squamous cell carcinoma (OSCC) cell line and investigate their in vitro and in vivo phenotypic characteristics., Materials and Methods: Subpopulations with individual staining intensities for CD44 and CD326 were isolated from the OSCC cell line LN-1A by FACS: CD44 Low /CD326 - (CSC-M 1 ), CD44 Low /CD326 High (CSC-E), and CD44 High /CD326 - (CSC-M 2 ). Proliferation, clonogenic potential, adhesion, migration, epithelial-mesenchymal transition markers, and sensitivity to cisplatin and TVB-3166 were analyzed in vitro. Tumor formation and metastasis were assessed by subcutaneous and orthotopic inoculations into BALB/c mice., Results: E-cadherin levels were higher in CSC-E cells while vimentin and Slug more produced by CSC-M 2 cells. CSC-M 1 and CSC-M 2 subpopulations showed higher proliferation, produced more colonies, and have stronger adhesion to the extracellular matrix. All cell lines established tumors; however, CSC-E and CSC-M 2 formed larger masses and produced more metastases., Conclusion: The CSC subpopulations here described show increased cancer capabilities in vitro, tumorigenic and metastatic potential in vivo, and may be exploited in the search for novel therapeutic targets for OSCC., (© 2024 Wiley Periodicals LLC.)

Background and Objectives: Bone marrow mesenchymal stromal cells (BM-MSCs) are key elements of the hematopoietic niche and participate in the regulatory mechanisms of hematopoietic stem cells (HSCs). Hematological diseases can affect MSCs and their functions. However, the dysregulations caused by sickle cell disease (SCD) are not fully elucidated. This work explored changes in BM-MSCs and their relationship with age using sickle cell mice (Townes-SS)., Materials and Methods: BM-MSCs were isolated from Townes-SS, and control groups 30- and 60-day-old Townes-AA and C57BL/6 J., Results: The BM-MSCs showed no morphological differences in culture and demonstrated a murine MSC-like immunophenotypic profile (Sca-1+, CD29+, CD44+, CD90.2+, CD31-, CD45-, and CD117-). Subsequently, all BM-MSCs were able to differentiate into adipocytes and osteocytes in vitro. Finally, 30-day-old BM-MSCs of Townes-SS showed higher expression of genes related to the maintenance of HSCs (Cxcl12, Vegfa, and Angpt1) and lower expression of pro-inflammatory genes (Tnfa and Il-6). However, 60-day-old BM-MSCs of Townes-SS started to show expression of genes related to reduced HSC maintenance and increased expression of pro-inflammatory genes., Conclusion: These results indicates age as a modifying factor of gene expression of BM-MSCs in the context of SCD.

Philadelphia-negative myeloproliferative neoplasms (MPN) are clonal hematological diseases associated with driver mutations in JAK2, CALR, and MPL genes. Moreover, several evidence suggests that chronic inflammation and alterations in stromal and immune cells may contribute to MPN's pathophysiology. We evaluated the frequency and the immunophenotype of peripheral blood monocyte subpopulations in patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (MF). Peripheral blood monocytes from PV (n = 16), ET (n = 16), and MF (n = 15) patients and healthy donors (n = 10) were isolated and submitted to immunophenotyping to determine the frequency of monocyte subpopulations and surface markers expression density. Plasma samples were used to measure the levels of soluble CD163, a biomarker of monocyte activity. PV, ET, and MF patients presented increased frequency of intermediate and non-classical monocytes and reduced frequency of classical monocytes compared to controls. Positivity for JAK2 mutation was significantly associated with the percentage of intermediate monocytes. PV, ET, and MF patients presented high-activated monocytes, evidenced by higher HLA-DR expression and increased soluble CD163 levels. The three MPN categories presented increased frequency of CD56 +  aberrant monocytes, and PV and ET patients presented reduced frequency of CD80/86 +  monocytes. Therefore, alterations in monocyte subpopulations frequency and surface markers expression pattern may contribute to oncoinflammation and may be associated with the pathophysiology of MPN., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is the only currently available curative treatment for sickle cell disease (SCD). Here, we comprehensively evaluated the reconstitution of T- and B-cell compartments in 29 SCD patients treated with allo-HSCT and how it correlated with the development of acute graft-versus-host disease (aGvHD)., Methods: T-cell neogenesis was assessed by quantification of signal-joint and β-chain TCR excision circles. B-cell neogenesis was evaluated by quantification of signal-joint and coding-joint K-chain recombination excision circles. T- and B-cell peripheral subset numbers were assessed by flow cytometry., Results: Before allo-HSCT (baseline), T-cell neogenesis was normal in SCD patients compared with age-, gender- and ethnicity-matched healthy controls. Following allo-HSCT, T-cell neogenesis declined but was fully restored to healthy control levels at one year post-transplantation. Peripheral T-cell subset counts were fully restored only at 24 months post-transplantation. Occurrence of acute graft-versus-host disease (aGvHD) transiently affected T- and B-cell neogenesis and overall reconstitution of T- and B-cell peripheral subsets. B-cell neogenesis was significantly higher in SCD patients at baseline than in healthy controls, remaining high throughout the follow-up after allo-HSCT. Notably, after transplantation SCD patients showed increased frequencies of IL-10-producing B-regulatory cells and IgM + memory B-cell subsets compared with baseline levels and with healthy controls., Conclusion: Our findings revealed that the T- and B-cell compartments were normally reconstituted in SCD patients after allo-HSCT. In addition, the increase of IL-10-producing B-regulatory cells may contribute to improve immune regulation and homeostasis after transplantation., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only currently available curative treatment for sickle cell disease (SCD). However, the effects of HSCT on SCD pathophysiology are poorly elucidated. Here, we assessed red blood cell (RBC) adhesiveness, intensity of hemolysis, vascular tone markers and systemic inflammation, in SCD patients treated with allogeneic HSCT. Thirty-two SCD patients were evaluated before and on long-term follow-up after HSCT. Overall survival was 94% with no severe (grade III-IV) graft- vs -host disease and a 22% rejection rate (graft failure). Hematological parameters, reticulocyte counts, and levels of lactate dehydrogenase (LDH), endothelin-1 and VCAM-1 normalized in SCD patients post-HSCT. Expression of adhesion molecules on reticulocytes and RBC was lower in patients with sustained engraftment. Levels of IL-18, IL-15 and LDH were higher in patients that developed graft failure. Increased levels of plasma pro-inflammatory cytokines, mainly TNF-α, were found in SCD patients long-term after transplantation. SCD patients with sustained engraftment after allo-HSCT showed decreased reticulocyte counts and adhesiveness, diminished hemolysis, and lower levels of vascular tonus markers. Nevertheless, systemic inflammation persists for at least five years after transplantation, indicating that allo-HSCT does not equally affect all aspects of SCD pathophysiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 de Azevedo, Costa, Lima, Maciel, Palma, Darrigo-Júnior, Setanni Grecco, Stracieri, Elias, Pieroni, Guerino-Cunha, Pinto, De Santis, Covas, Hermine, Simões, Oliveira and Malmegrim.)

Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent advances in molecular technologies allowed to classify MB in 4 major molecular subgroups: WNT, SHH, Group 3 and Group 4. In cancer research, cancer cell lines are important for examining and manipulating molecular and cellular process. However, it is important to know the characteristics of each cancer cell line prior to use, because there are some differences among them, even if they originate from the same cancer type. This study aimed to evaluate the similarities and differences among four human medulloblastoma cell lines, UW402, UW473, DAOY and ONS-76. The medulloblastoma cell lines were analyzed for (1) cell morphology, (2) immunophenotyping by flow cytometry for some specifics surface proteins, (3) expression level of adhesion molecules by RT-qPCR, (4) proliferative potential, (5) cell migration, and (6) in vivo tumorigenic potential. It was observed a relationship between cell growth and CDH1 (E-chaderin) adhesion molecule expression and all MB cell lines showed higher levels of CDH2 (N-chaderin) when compared to other adhesion molecule. ONS-76 showed higher gene expression of CDH5 (VE-chaderin) and higher percentage of CD144/VE-chaderin positive cells when compared to other MB cell lines. All MB cell lines showed low percentage of CD34, CD45, CD31, CD133 positive cells and high percentage of CD44, CD105, CD106 and CD29 positive cells. The DAOY cell line showed the highest migration potential, the ONS-76 cell line showed the highest proliferative potential and only DAOY and ONS-76 cell lines showed tumorigenic potential in vivo. MB cell lines showed functional and molecular differences among them, which it should be considered by the researchers in choosing the most suitable cellular model according to the study proposal.

Human multipotent mesenchymal stromal cells (MSCs) display immunoregulatory functions that can modulate innate and adaptive cellular immune responses. The suppressive and immunomodulatory activities of MSCs occur through the action of soluble factors that are constitutively produced and released by these cells or, alternatively, after MSC induction by stimuli of inflammatory microenvironments. However, to date the contribution of MSCs in the inflammatory microenvironment resulting from viral infection is unknown. In our study, we evaluated the MSC immunosuppressive effect on human T lymphotropic virus type 1 (HTLV-1) infected T lymphocytes. To evaluate if MSC immunoregulation can influence the proliferation of HTLV-1 infected T lymphocytes, we compared the proliferation of lymphocytes obtained from HTLV-1 infected and healthy individuals cocultured in the presence of MSCs. It was observed that the lymphoproliferative inhibition by MSCs on infected lymphocytes was similar compared to the cells obtained from healthy individuals. In addition, this suppressive effect was related to a significant increase of indoleamine-2,3-dioxygenase and prostaglandin E2 gene expression (p ≤ .05). Furthermore, the HTLV-1 pol gene was less expressed after coculturing with MSCs, suggesting that the MSC immunoregulation can have effective suppression on HTLV-1 infected T cells. In conclusion, this study suggests that MSCs could be involved in the immunomodulation of the HTLV-1 infected T lymphocytes.

Human T cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia/lymphoma. The development of HAM/TSP, a chronic neuroinflammatory disease, is correlated to complex interaction between the host immune response and the infecting virus. Tax expression plays an important role in HAM/TSP pathogenesis by activating various cellular genes, including the cytokines interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Exosomes have emerged as an important factor of cell-to-cell communication contributing to diverse cellular processes, including immune modulation. Considering the potential role of exosomes in modulating the immune response and inflammation, the main objective of this study was to examine if HTLV-1-infected cells produce exosomes carrying viral proteins or inflammatory molecules, which can participate in the chronic inflammation that is observed in patients with HAM/TSP. Exosomes were isolated from HTLV-1-infected cell line, evaluated for the tax mRNA presence, and tested for the ability to activate peripheral mononuclear cells (PBMC) in inducing an inflammatory immune response. We observed that the proinflammatory cytokines, IFN-γ and TNF-α, were upregulated in T cells after treatment of the PBMC with Tax-carrying exosomes compared to the negative control. Interleukin-4, Granzyme B, and Perforin did not show alterations. Taken together, these results suggest that exosomes carrying tax -mRNA isolated from HTLV-1-infected cells might induce the production of proinflammatory cytokines and activate T helper (Th) 1 , and not Th 2 -immune response. If this finding is further confirmed, this study may have impact on investigations on the pathogenesis of HAM-TSP and the inflammatory response involved in this disease.

The bone marrow (BM) biology during HTLV-1 infection is obscure. In this study, we investigated BM mononuclear cells and mesenchymal stromal cells (MSC) from HTLV-1 asymptomatic and symptomatic individuals. An infiltration of CD4 + T-cell lymphocytes in the BM of HTLV-1-infected individuals was observed when compared to healthy controls. The provirus detection in the BM CD4 + T cells confirmed the presence of integrated HTLV DNA. In regard to MSC, we observed that the number of fibroblast progenitor cells was lower in HTLV-1 infected individuals than in healthy controls. Isolated HTLV-1 infected BM-MSC demonstrated surface expression markers and in vitro differentiation potential similar to uninfected individuals. The presence of HTLV-1 proviral DNA in the BM-MSC of HTLV-1-infected patients was demonstrated but no p19 antigen was detected in supernatant from cultured MSC. We suppose that HTLV-1 infects human MSC probably by cell-to-cell contact from the infected CD4 + T-lymphocytes infiltrated into the bone marrow., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Adipose-derived stromal/stem cells (ASCs) are promising candidates for cell-based therapies. However, the lack of markers able to unequivocally identify these cells, the differential expression of cell surface molecules among stromal progenitors from different tissues and cellular alterations caused by culture are phenomena that need to be comprehensively addressed in order to improve ASC purification and consequently refine our knowledge about their function and therapeutic efficiency. In this study, we investigated the potential of CD271, a marker used for purification of bone marrow-derived mesenchymal stem cells, on enriching ASCs from CD34 + stromal cells of human adipose tissue. Putative ASC populations were sorted based on CD271 expression (CD45 - CD31 - CD34 + CD271 + and CD45 - CD31 - CD34 + CD271 - cells) and compared regarding their clonogenic efficiency, proliferation, immunophenotypic profile, and multilineage potential. To shed light on their native identity, we also interrogated the expression of key perivascular cell markers in freshly isolated cells. CD271 - cells displayed twofold higher clonogenic efficiency than CD271 + cells. Upon culture, the progeny of both populations displayed similar immunophenotypic profile and in vitro adipogenic and chondrogenic potentials, while CD271 + cells produced more calcified extracellular matrix. Interestingly, uncultured freshly isolated CD271 + cells displayed higher expression of pericyte-associated markers than CD271 - cells and localized in the inner region of the perivascular wall. Our results demonstrate that cells with in vitro ASC traits can be obtained from both CD271 + and CD271 - stromal populations of human adipose tissue. In addition, gene expression profiling and in situ localization analyses indicate that the CD271 + population displays a pericytic phenotype., (© 2017 Wiley Periodicals, Inc.)

Purpose: The fusion gene BCR-ABL has an important role to the progression of chronic myeloid leukemia (CML) and several signaling pathways have been characterized as responsible for the terminal blastic phase (BP). However, the initial phase, the chronic phase (CP), is long lasting and there is much yet to be understood about the critical role of BCR-ABL in this phase. This study aims to evaluate transcriptional deregulation in CD34+ hematopoietic cells (CD34+ cells) from patients with untreated newly diagnosed CML compared with CD34+HC from healthy controls., Methods: Gene expression profiling in CML-CD34 cells and CD34 cells from healthy controls were used for this purpose with emphasis on five main pathways important for enhanced proliferation/survival, enhanced self-renewal and block of myeloid differentiation., Results: We found 835 genes with changed expression levels (fold change ≥ ±2) in CML-CD34 cells compared with CD34 cells. These include genes belonging to PI3K/AKT, WNT/b-catenin, SHH, NOTCH and MAPK signaling pathways. Four of these pathways converge to MYC activation. We also identified five transcripts upregulated in CD34-CML patients named OSBPL9, MEK2, p90RSK, TCF4 and FZD7 that can be potential biomarkers in CD34-CML-CP., Conclusion: We show several mRNAs up- or downregulated in CD34-CML during the chronic phase.

Breast cancer is the most common cancer in women worldwide and metastatic dissemination is the principal factor related to death by this disease. Breast cancer stem cells (bCSC) are thought to be responsible for metastasis and chemoresistance. In this study, based on whole transcriptome analysis from putative bCSC and reverse engineering of transcription control networks, we identified two networks associated with this phenotype. One controlled by SNAI2, TWIST1, BNC2, PRRX1 and TBX5 drives a mesenchymal or CSC-like phenotype. The second network is controlled by the SCML4, ZNF831, SP140 and IKZF3 transcription factors which correspond to immune response modulators. Immune response network expression is correlated with pathological response to chemotherapy, and in the Basal subtype is related to better recurrence-free survival. In patient-derived xenografts, the expression of these networks in patient tumours is predictive of engraftment success. Our findings point out a potential molecular mechanism underlying the balance between immune surveillance and EMT activation in breast cancer. This molecular mechanism may be useful to the development of new target therapies.

High dose immunosuppression followed by autologous hematopoietic stem cell transplantation (AHSCT) induces prolonged clinical remission in multiple sclerosis (MS) patients. However, how patient immune profiles are associated with clinical outcomes has not yet been completely elucidated. In this study, 37 MS patients were assessed for neurological outcomes, thymic function and long-term immune reconstitution after AHSCT. Patients were followed for a mean (SD) of 68.5 (13.9) months post-transplantation and were retrospectively clustered into progression- and non-progression groups, based on Expanded Disease Status Scale (EDSS) outcomes at last visit. After AHSCT, both patient groups presented increased regulatory T-cell subset counts, early expansion of central- and effector-memory CD8(+)T-cells and late thymic reactivation. However, the non-progression group presented early expansion of PD-1(+)CD8(+)T-cells and of PD-1-expressing CD19(+) B-cells. Here, we suggest that along with increased numbers of regulatory T-cell subsets, PD-1 inhibitory signaling is one possible immunoregulatory mechanism by which AHSCT restores immune tolerance in MS patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)