Clinical and basic implications of dynamic T cell receptor clonotyping in hematopoietic cell transplantation

TCR repertoire diversification constitutes a foundation for successful immune reconstitution after allogeneic hematopoietic cell transplantation (allo-HCT). Deep TCR V beta sequencing of 135 serial specimens from a cohort of 35 allo-HCT recipients/donors was performed to dissect posttransplant TCR architecture and dynamics. Paired analysis of clonotypic repertoires showed a minimal overlap with donor expansions. Rarefied and hyperexpanded clonotypic patterns were hallmarks of T cell reconstitution and influenced clinical outcomes. Donor and pretransplant TCR diversity as well as divergence of class I human leukocyte antigen genotypes were major predictors of recipient TCR repertoire recovery. Complementary determining region 3-based specificity spectrum analysis indicated a predominant expansion of pathogen- and tumor-associated clonotypes in the late post-allo-HCT phase, while autoreactive clones were more expanded in the case of graft-versus-host disease occurrence. These findings shed light on post-allo-HCT adaptive immune reconstitution processes and possibly help in tracking alloreactive responses

The similarity of class II HLA genotypes defines patterns of autoreactivity in idiopathic bone marrow failure disorders

Abstract Idiopathic aplastic anemia (IAA) is a rare autoimmune bone marrow failure (BMF) disorder initiated by a human leukocyte antigen (HLA)-restricted T-cell response to unknown antigens. As in other autoimmune disorders, the predilection for certain HLA profiles seems to represent an etiologic factor; however, the structure-function patterns involved in the self-presentation in this disease remain unclear. Herein, we analyzed the molecular landscape of HLA complexes of a cohort of 300 IAA patients and almost 3000 healthy and disease controls by deeply dissecting their genotypic configurations, functional divergence, self-antigen binding capabilities, and T-cell receptor (TCR) repertoire specificities. Specifically, analysis of the evolutionary divergence of HLA genotypes (HED) showed that IAA patients carried class II HLA molecules whose antigen-binding sites were characterized by a high level of structural homology, only partially explained by specific risk allele profiles. This pattern implies reduced HLA binding capabilities, confirmed by binding analysis of hematopoietic stem cell (HSC)-derived self-peptides. IAA phenotype was associated with the enrichment in a few amino acids at specific positions within the peptide-binding groove of DRB1 molecules, affecting the interface HLA-antigen-TCR β and potentially constituting the basis of T-cell dysfunction and autoreactivity. When analyzing associations with clinical outcomes, low HED was associated with risk of malignant progression and worse survival, underlying reduced tumor surveillance in clearing potential neoantigens derived from mechanisms of clonal hematopoiesis. Our data shed light on the immunogenetic risk associated with IAA etiology and clonal evolution and on general pathophysiological mechanisms potentially involved in other autoimmune disorders.Peer reviewe

TET2 mutations as a part of DNA dioxygenase deficiency in myelodysplastic syndromes

Decrease in DNA dioxygenase activity generated by TET2 gene family is crucial in myelodysplastic syndromes (MDS). The general downregulation of 5-hydroxymethylcytosine (5-hmC) argues for a role of DNA demethylation in MDS beyond TET2 mutations, which albeit frequent, do not convey any prognostic significance. We investigated TETs expression to identify factors which can modulate the impact of mutations and thus 5-hmC levels on clinical phenotypes and prognosis of MDS patients. DNA/RNA-sequencing and 5-hmC data were collected from 1665 patients with MDS and 91 controls. Irrespective of mutations, a significant fraction of MDS patients exhibited lower TET2 expression, whereas 5-hmC levels were not uniformly decreased. In searching for factors explaining compensatory mechanisms, we discovered that TET3 was upregulated in MDS and inversely correlated with TET2 expression in wild type cases. Although TET2 was reduced across all age groups, TET3 levels were increased in a likely feedback mechanism induced by TET2 dysfunction. This inverse relationship of TET2 and TET3 expression also corresponded to the expression of L-2-hydroxyglutarate dehydrogenase, involved in agonist/antagonist substrate metabolism. Importantly, elevated TET3 levels influ-enced the clinical phenotype of TET2 deficiency whereby the lack of compensation by TET3 (low TET3 expression) was associated with poor outcomes of TET2 mutant carriers

A Machine Learning Model of Response to Hypomethylating Agents in Myelodysplastic Syndromes

Hypomethylating agents (HMA) prolong survival and improve cytopenias in individuals with higher-risk myelodysplastic syndrome (MDS). Only 30-40% of patients, however, respond to HMAs, and responses may not occur for more than 6 months after HMA initiation. We developed a model to more rapidly assess HMA response by analyzing early changes in patients’ blood counts. Three institutions’ data were used to develop a model that assessed patients’ response to therapy 90 days after the initiation using serial blood counts. The model was developed with a training cohort of 424 patients from2 institutions and validated on an independent cohort of 90 patients. The final model achieved an area under the receiver operating characteristic curve (AUROC) of 0.79 in the train/test group and 0.84 in the validation group. The model provides cohort-wide and individual- level explanations for model predictions, and model certainty can be interrogated to gauge the reliability of a given prediction

A Zebrafish Cell Culture Assay for the Identification of MicroRNA Targets

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate gene expression at the posttranscriptional level. Studies have shown that zebrafish miRNAs play a key role in embryo development, tissue fate establishment, and differentiation by interacting with specific targets, usually in the 3′UTR of the mRNA. Identification of the target sequence is fundamental to elucidating miRNA function. Since bioinformatics can predict hundreds of potential targets for each miRNA, experimental validation of the actual target site is required. Although recent studies have employed the HEK293 cell line to investigate mammalian miRNA targets, our results have shown that the cell line is not suitable for studies of zebrafish miR-430b miRNA. In this article we describe a convenient in vitro assay system that involves the use of zebrafish cell cultures and a luciferase reporter construct to evaluate miR-430b target sites. The cell culture-based assay could be used to validate target sequences of other zebrafish miRNAs

Demethylation of Non-CpG Sites in DNA Is Initiated by TET2 5-Methylcytosine Dioxygenase

In the mammalian genome, cytosine methylation predominantly occurs at CpG sites. In addition, a number of recent studies have uncovered extensive C5 cytosine methylation (5mC) at non-CpG (5mCpH, where H = A/C/T) sites. Little is known about the enzyme responsible for active demethylation of 5mCpH sites. Using a very sensitive and quantitative LC–MS/MS method, we demonstrate that the human TET2, an iron (II)- and 2OG-dependent dioxygenase, which is a frequently mutated gene in several myeloid malignancies, as well as in a number of other types of cancers, can oxidize 5mCpH sites in double-stranded DNA in vitro. Similar to oxidation of 5mCpG, oxidation of 5mC at CpH sites produces 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) bases in DNA. After 5mCpG, which is the most preferred substrate, TET2 prefers 5mCpC as a substrate, followed by 5mCpA and then 5mCpT. Since the TDG/BER pathway and deformylation or decarboxylation of 5fC or 5caC, respectively, can convert 5fCpH and 5caCpH to an unmodified cytosine base in DNA, our results suggest a novel demethylation pathway of 5mCpH sites initiated by TET2 dioxygenase

The Re-Discovery of Gender Inequality: EU–China trade

Christa Wichterich argues that in China, gender differences have been re-discovered in the course of liberalization, privatization and the marketization of the whole economy. Women's contributions to the economy are a comparative advantage of China competing in the world market. At the same time, gender has become a significant marker in the creation of new social classes in post-communist China, and the long standing claim of socialist policies for equal rights got subordinated to the imperative of fast economic growth. She suggests these processes were initiated by the Chinese government's ‘open door’-policies to set up a market economy; after China's World Trade Organization accession, they are increasingly driven by a complex interaction between domestic policies, and foreign trade and investment policies. Development (2007) 50, 83–89. doi:10.1057/palgrave.development.1100412

Combined Effect of Weak Magnetic Fields and Anions on Arsenite Sequestration by Zerovalent Iron: Kinetics and Mechanisms

In this study, the effects of major anions (e.g., ClO₄^–, NO₃^–, Cl^–, and SO₄^2–) in water on the reactivity of zerovalent iron (ZVI) toward As­(III) sequestration were evaluated with and without a weak magnetic field (WMF). Without WMF, ClO₄^– and NO₃^– had negligible influence on As­(III) removal by ZVI, but Cl^– and SO₄^2– could improve As­(III) sequestration by ZVI. Moreover, the WMF-enhancing effect on As­(III) removal by ZVI was minor in ultrapure water. A synergetic effect of WMF and individual anion on improving As­(III) removal by ZVI was observed for each of the investigated anion, which became more pronounced as the concentration of anion increased. Based on the extent of enhancing effects, these anions were ranked in the order of SO₄^2– > Cl^– > NO₃^– ≈ ClO₄^– (from most- to least-enhanced). Furthermore, the inhibitory effect of HSiO₃^–, HCO₃^–, and H₂PO₄^– on ZVI corrosion could be alleviated taking advantage of the combined effect of WMF and SO₄^2–. The coupled influence of anions and WMF was associated with the simultaneous movement of anions with paramagnetic Fe²⁺ to keep local electroneutrality in solution. Our findings suggest that the presence of anions is quite essential to maintaining or stimulating the WMF effect