Search

Abstract: Acute myeloid leukemia (AML) is a molecularly heterogeneous disease. Based on cytogenetics and FISH, AML patients are stratified into three major risk categories: favourable, intermediate and unfavourable. However, prognostic stratification and treatment decision for the intermediate risk category, that mostly comprises AML patients with normal cytogenetics (CN-AML), has been difficult due to the clinical heterogeneity and scarce knowledge of the molecular alterations underlying this large AML subgroup. During the past decade, the identification of several mutations associated with CN-AML has resulted into important advances in the AML field. In this review, we address the biological features of the main mutations associated with CN-AML and the impact of next generation sequencing studies in expanding our knowledge of the molecular landscape of CN-AML. In addition, we outline the prognostic value of mutations for risk stratification of CN-AML patients and discuss the potential of mutations discovery process for developing new molecular targeted therapies. [Copyright &y& Elsevier]

Introduction: Haploidentical transplantation, with extensive T cell depletion to prevent GvHD, is associated with a high incidence of infection-related deaths. The key challenge is to improve immune recovery with allogeneic donor T cells without triggering GvHD. As T regulatory cells (Tregs) controlled GvHD in pre-clinical studies, the present study evaluated the impact of an infusion of donor CD4/CD25 + Tregs, followed by an inoculum of donor mature T cells (Tcons) and positively immunoselected CD34 + cells in the setting of haploidentical stem cell transplantation. Patients and methods: Twenty-eight patients were enrolled in this study (22 AML; 5 ALL; 1 NHL). All received immunoselected Tregs (CliniMACS, Miltenyi Biotec) followed by positively immunoselected CD34 + cells together with Tcons 4 days later. No GvHD prophylaxis was administered. Results: 26/28 patients engrafted. No acute GvHD developed in 24/26 patients; 2 developed ≥ grade II acute GvHD. No patient has developed chronic GvHD. CD4 and CD8 counts rapidly increased after transplant. Episodes of CMV reactivation were significantly fewer than in controls. Conclusions: In the setting of haploidentical transplantation infusion of Tregs makes administration of a high dose of T cells feasible. This strategy provides a long-term protection from GvHD and robust immune reconstitution. [Copyright &y& Elsevier]

Abstract: T-cell homeostasis is regulated by several molecules; among these, interleukin (IL)-7 plays an essential role in the survival and homeostatic proliferation of peripheral naive T cells. In a previous study, we investigated whether human mesenchymal stromal cells (MSCs) could be engineered with the IL-7 gene to produce functional level of this cytokine. In the present study, we analyzed the impact of different quantities of IL-7 produced by MSCs on the survival and proliferation of a negative immunoselected naive (CD3+/CD45RA+) T-cell population. Co-cultivation of peripheral naive T cells with MSCs producing low (16 pg/mL) or high (1000 pg/mL) IL-7 levels or in the presence of exogenous IL-7 (0.01 ng/mL and 100 ng/mL) maintained the CD3+/CD45RA+ naive T-cell phenotype. Chemokine receptor CCR7+ expression was also maintained among this T-cell population. Naive T-cell molecular characteristics were maintained as assessed by the Vβ spectratyping complexity score, which showed the maintenance of a broad T-cell repertoire. No Th1 or Th2 differentiation was observed, as assessed by interferon-γ or IL-4 accumulation. In contrast, only MSCs producing high amounts of IL-7 caused increased activation (CD25 31.2% ± 12% vs 10% ± 3.5%; P < .05), proliferation (CD71 17.8±7% vs 9.3%±3, P < .05), apoptosis (assessed by annexin V: 18.6% ± 5% vs 14.9% ± 2.6%; P > .05), and the phase S cell cycle (15% vs 6.9%, P > .05). Exogenous IL-7 exhibited no significant effect. In conclusion, we demonstrated that IL-7 produced by MSCs has a dose-independent effect on naive T-cell survival while exerting a dose-dependent effect on activation/proliferation. Due to the continuous production of IL-7 by engineered cells, our system is more efficacious than exogenous IL-7. [Copyright &y& Elsevier]

• Good manufacturing practice (GMP) automated expanded T regulatory cells (Tregs) are in vitro and in vivo characterized. • Tregs produce high amounts of IL-10, granzyme B, and IL-35. • Expanded Tregs protect mice from graft-versus-host disease. • TIM3+ cells emerge as a potentially highly suppressive population. In the setting of T cell-depleted, full-haplotype mismatched transplantation, adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) can prevent graft-versus-host disease (GVHD) and improve post-transplantation immunologic reconstitution and is associated with a powerful graft-versus-leukemia effect. To improve the purity and the quantity of the infused Tregs, good manufacturing practices (GMP)-compatible expansion protocols are needed. Here we expanded Tregs using an automated, clinical-grade protocol. Cells were extensively characterized in vitro, and their efficiency was tested in vivo in a mouse model. Tregs were selected by CliniMacs (CD4+CD25+, 94.5 ± 6.3%; FoxP3+, 63.7 ± 11.5%; CD127+, 20 ± 3%; suppressive activity, 60 ± 7%), and an aliquot of 100 × 106 was expanded for 14 days using the CliniMACS Prodigy System, obtaining 684 ± 279 × 106 cells (CD4+CD25+, 99.6 ± 0.2%; FoxP3+, 82 ± 8%; CD127+, 1.1 ± 0.8%; suppressive activity, 75 ± 12%). CD39 and CTLA4 expression levels increased from 22.4 ± 12% to 58.1 ± 13.3% (P <.05) and from 20.4 ± 6.7% to 85.4 ± 9.8% (P <.01), respectively. TIM3 levels increased from.4 ±.05% to 29 ± 16% (P <.05). Memory Tregs were the prevalent population, whereas naive Tregs almost disappeared at the end of the culture. mRNA analysis displayed significant increases in CD39, IL-10, granzyme B, and IL-35 levels at the end of culture period (P <.05). Conversely, IFNγ expression decreased significantly by day +14. Expanded Tregs were sorted according to TIM3, CD39, and CD62L expression levels (purity >95%). When sorted populations were analyzed, TIM3+ cells showed significant increases in IL-10 and granzyme B (P <.01).When expanded Tregs were infused in an NSG murine model, mice that received Tcons only died of GVHD, whereas mice that received both Tcons and Tregs survived without GVHD. GMP grade expanded cells that display phenotypic and functional Treg characteristics can be obtained using a fully automated system. Treg suppression is mediated by multiple overlapping mechanisms (eg, CTLA-4, CD39, IL-10, IL-35, TGF-β, granzyme B). TIM3+ cells emerge as a potentially highly suppressive population. © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. [ABSTRACT FROM AUTHOR]

We developed a good manufacturing practices–compatible expansion protocol to improve number and purity of regulatory T cells (Tregs) available for clinical trials. Six clinical-grade separation procedures were performed, followed by expansion with high-dose interleukin (IL)-2, anti-CD3/anti-CD28 TCR stimulation, and rapamycin for 19 days achieving a median of 8.5-fold (range, 6.25 to 13.7) expansion. FOXP3 expression was stably maintained over the culture period, while the percentage of CD127 was significantly reduced. The in vitro suppression assay showed a strong Mixed Lymphocytes Reaction inhibition. In vitro amplification did not induce any karyotypic modification. To evaluate the graft-versus-host disease (GVHD)/graft-versus-leukemia (GVL) bifunctional axis, expanded Tregs and conventional T cells (Tcons) were tested in NOD/SCID/IL2Rgnull mice injected with primary acute myeloid leukemia (AML) cells, AML cell line, acute lymphoid leukemia Philadelphia cell line, or Burkitt-like lymphoma cell line. All mice that received leukemia cells together with expanded Tregs and Tcons were rescued from leukemia and survived without GVHD, showing that Treg expansion procedure did not compromise GVHD control and the strong Tcon-mediated GVL activity. This report might represent the basis for treating high-risk leukemia and/or relapsed/refractory leukemia patients with high-dose Treg/Tcons. [ABSTRACT FROM AUTHOR]

Abstract: We selected T regulatory cells (Tregs) from standard leukapheresis using double-negative selection (anti-CD8 and anti-CD19) followed by positive selection (anti-CD25) and 72 procedures were performed. A median of 263×106 cells (range 143–470×106) were recovered with a mean of CD4+/CD25+ cells of 94.5±2.4% (36.5±18.6% CD4+/CD25+hi). FoxP3+ cells were equal to 79.8%±22.2. CD127+ cells were 12.5%±8.2. The inhibition assay showed an inhibition rate of 67±22. Cells isolated by means of this approach can be used in allogeneic hematopoietic stem cell transplantation to reduce the incidence and severity of GvHD without bystander inhibition of general immunity. [Copyright &y& Elsevier]

NPM1 is a ubiquitously expressed nucleolar phosphoprotein, the gene for which maps to chromosome 5q35 in close proximity to a commonly deleted region associated with (del)5q, a type of myelodysplastic syndrome (MDS). This region is also a frequent target of deletions in de novo and therapy-related MDS/acute myeloid leukemia. Previous studies have shown that Npm1+/- mice develop an MDS-like disease that transforms to acute myeloid leukemia over time. To better understand the mechanism by which NPM1 haploinsufficiency causes an MDS phenotype, we generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/- and Npm1+/- mice and demonstrated compromised neutrophil-specific gene expression in the MNPM1+/- cells. We attribute these observations to increased levels of the shorter, dominant negative leukemogenic isoform (p30) of CCAAT enhancer-binding protein α (C/EBPα). We show that this increase is caused, in part, by elevated levels of the activated translation initiation factor eIF4E, overexpression of which also increases translation of C/EBPαp30 in HEK293 cells. In a positive feedback loop, eIF4E expression is further elevated both at the mRNA and protein levels by C/EBPαp30 but not by the full-length C/EBPαp42. Re-expression of C/EBPαp42 or NPM1but not C/EBPαp30 inMNPM1 +/- cells partially rescues the myeloid phenotype. Our observations suggest that the aberrant feed-forward pathway that keeps eIF4E and C/EBPαp30 elevated in NPM1+/- cells contributes to the MDS phenotype associated with NPM1 deficiency. [ABSTRACT FROM AUTHOR]

Abstract: Glucocorticoid-Induced TNFR family Related gene (GITR), a Tumor Necrosis Factor Receptor Superfamily (TNFRSF) member involved in immune/inflammatory processes, has been previously shown to regulate T cell activation. To study GITR role in antigen presenting cells, we evaluated the capability of bone marrow derived dendritic cells (BMDC) from GITR−/− mice to stimulate the activation of CD4+CD25− T lymphocytes. We found that GITR−/− BMDC are weaker stimulators of T cell proliferation than GITR+/+ BMDC, either in syngenic or allogenic BMDC/T cell co-cultures. Expression of GITR in GITR−/− BMDC restored their ability to activate T cells while GITR silencing in GITR+/+ BMDC inhibited the capability to stimulate T cells. GITR−/− BMDC showed a reduced production of the pro-inflammatory cytokine IL-6 and an increased production of the anti-inflammatory cytokine IL-10. Notably, co-culture of CD4+CD25− cells with GITR−/− BMDC originated FoxP3+ cells, secreting IL-10 and TGF-β. Finally, in vivo injection of GITR−/− OVA-loaded BMDC led to a lower cell number and a lower activated cell number in draining lymph nodes than in GITR+/+ OVA-loaded BMDC injected mice. Together, these results indicate that GITR plays a role in regulating BMDC activity. [ABSTRACT FROM AUTHOR]

Summary: Centrosome abnormalities lead to genomic instability and are a common feature of many cancer cells. Here we show that mutations in morgana/chp-1 result in centrosome amplification and lethality in both Drosophila and mouse, and that the fly centrosome phenotype is fully rescued by the human ortholog of morgana. In mouse cells, morgana forms a complex with Hsp90 and ROCK I and II, and directly binds ROCK II. Morgana downregulation promotes the interaction between ROCK II and nucleophosmin (NPM), leading to an increased ROCK II kinase activity, which results in centrosome amplification. morgana +/− primary cells and mice display an increased susceptibility to neoplastic transformation. In addition, tumor tissue array histochemical analysis revealed that morgana is underexpressed in a large fraction of breast and lung human cancers. Thus, morgana/chp-1 appears to prevent both centrosome amplification and tumorigenesis. [Copyright &y& Elsevier]

Objective: Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Because multipotent mesenchymal stromal cells (MSCs) exert an immune regulatory function and suppress T-cell proliferation, this in vitro study investigated their role in Treg recruitment and function. Materials and Methods: Human MSCs and different T cell populations (CD3+, CD3+/CD45RA+, CD3+/CD45RO+, CD4+/CD25+, CD4+/CD25+/CD45RO+, CD4+/CD25+/CD45RA+) from healthy donors were cocultured for up to 15 days. Harvested lymphocytes were analyzed by flow cytometry and FoxP3 and CD127 expressions were measured by real-time polymerase chain reaction. Their regulatory activity was assessed. Results: We demonstrate MSC recruit Tregs from a fraction of CD3+ and from immunoselected CD3+/CD45RA+ and CD3+/CD45RO+ fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4+/CD25bright/FoxP3 subset and CD127 expression was downregulated. When purified Treg populations (CD4/CD25+, CD4/CD25+/CD45RA+, and CD4/CD25+/CD45RO+) are used in MSC cocultures, they maintain FoxP3 expression and CD127 expression is downregulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. Conclusions: In conclusion, our study demonstrates that MSCs recruit, regulate, and maintain T-regulatory phenotype and function over time. [Copyright &y& Elsevier]