Delta Chain Research Articles

B-248 Enhanced Detection of of Hemoglobin Variants in Clinical Research using Dried Blood Spot and HRAM MS

Abstract Background Hemoglobinopathies are one of the most prevalent genetic disorders, with more than 330,000 affected births occurring annually. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. However, there are challenges to differentiate isomers (i.e., between Hb C and Hb E), very similar masses, or new variants due to their lack of resolution. Mass spectrometry (MS) gained popularity in clinical research because of its robustness and versatility in analyzing a wide range of samples and analytes from small molecules to proteins. Here, we present the top-down analysis for enhanced detection of various hemoglobin variants using Orbitrap Exploris for clinical research. Methods Proteins were directly extracted from dried blood spots (DBS) and analyzed by Thermo Scientific™ Orbitrap Exploris™ 240 with BioPharma Option coupled to Vanquish HPLC system. A 3.2 mm disc was punched from the DBS cards to 96 well plate and 50 uL water was added to re-hydrate. For protein precipitation, 150 uL acetonitrile was added and incubated at -20 ⁰C for 15 minutes. After micro-centrifugation, 160 uL of supernatant was removed and 70 uL water was added to resuspend. For LC-MS analysis, 10 uL of supernatant was diluted 8 times with mobile phase A and transferred to another 96-well plate. LC separation was performed on MAbPac RP column 4 um, 1 x 100 mm, and data-dependent acquisition mode was operated. Results With direct protein extraction from DBS, the entire sample preparation time is less than 1 hour. The MAbPac column showed its capability of separating between different chains with minor overlap between normal beta and HbS beta chains. Data processing was performed by ProSightPD, resulting in ∼45% sequence coverage for both normal Hb and HbS beta chains. The mass difference between normal beta and Hb S beta chains is about 30 Da, which results in about 1.6 m/z difference at a charge state of 19. This mass difference can often be hindered by other proteins or interferences from the matrix if the mass analyzer doesn’t have sufficient resolving power. Here we successfully applied Orbitrap resolution at 120,000 to isolate these similar masses for accurate variants or subunits identification including normal alpha, normal beta, Hb S, and delta chains. The confirmation of the delta chain can be beneficial since its abundance is usually less than 0.5% in adults, which shows potential for detecting low abundant variants using this method. Also, the calibration curve was generated to demonstrate the method's feasibility which is not a typical quantitation method for hemoglobinopathies. For quantitation, three most abundant m/z values from 2 charge states were added to TraceFinder for peak area extraction and normal Hb beta chain was used as an alternative internal standard for data processing. The 3-day inter/intraday analysis generated great reproducibility with CV < 10% and LOQ was determined to be 2.5 mg/mL with R2 values >0.994. Conclusions Successful demonstration of a single-step protein extraction directly from DBS and top-down analysis of Hb variant for clinical research.

Characterization of a novel chicken γδ TCR-specific marker

Chickens are a species with a high number of γδ T cells in various tissues. Despite their abundance, γδ T cells are poorly characterized in chickens, partially due to a lack of specific reagents to characterize these cells. Up until now, the TCR1 clone has been the only γδ T cell-specific monoclonal antibody (mAb) in chickens and additional reagents for γδ T cell subsets are needed. In order to address this issue, new mAb were generated in our laboratory by immunizing mice with in vitro cultured γδ T cells. In an initial flow cytometric screen a new mAb, clone “8D2”, displayed an interesting staining pattern that mirrored γδ TCR up- and downregulation in the γδ T cell line D4 over time, prompting us to characterize this antibody further. We compared the expression of the unknown 8D2 epitope in combination with TCR1 staining across various primary cells. In splenocytes, peripheral blood lymphocytes and intestinal epithelial cells, 8D2 consistently labeled a subset of TCR1+ cells. To determine, whether specific γδ T cell receptors were recognized by 8D2, we sorted γδ T cells according to their 8D2 and TCR1 expression and analyzed their TCR V(D)J gene usage by TCR profiling. Strikingly, sorted 8D2+ cells preferentially expressed Vγ3 genes, whereas the TCR Vγ genes used by TCR1+ 8D2- cells were more variable. γδ TCR in 8D2+ cells were most frequently comprised of gamma chain VJ genes TRGV3-8 and TRGJ3, and delta chain VDJ genes TRDV1-2, TRDD2, TRDJ1. To confirm binding of 8D2 to specific γδ TCR, the preferentially utilized combination of TRG and TRD was expressed in HEK293 cells in combination with CD3, demonstrating surface binding of the 8D2 mAb to this Vγ3 γδ TCR-expressing cell line. Conversely, HEK293 cells expressing either Vγ1 or Vγ2 TCR did not react with 8D2. In conclusion, 8D2 is a novel tool for identifying specific Vγ3 bearing γδ T cells.

Serum lipidomic study of long-chain fatty acids in psoriasis patients prior to and after anti-IL-17A monoclonal antibody treatment by quantitative GC‒MS analysis with in situ extraction

BackgroundLong-chain fatty acids (LCFAs) are involved in regulating multiple physiological processes as signalling molecules. Gas chromatography–mass spectrometry (GC–MS) is widely used to quantify LCFAs. However, current quantitative methods for LCFAs using GC–MS have demonstrated complicated issues. Psoriasis is a chronic inflammatory skin disease, and its pathogenesis may be related to the overproduction of interleukin-17A (IL-17A). Clinical efficacy of anti-IL-17A monoclonal antibody (mAb) treatment in psoriasis patients has been demonstrated. Recent studies suggest that LCFAs play varying roles in the pathogenesis of psoriasis. However, more comprehensive research is needed to illuminate the mechanism of LCFAs in psoriasis.MethodsThe established in situ derivatization method for analysing LCFAs with a GC–MS platform was utilized to conduct serum lipidomics analysis of healthy volunteers and psoriasis patients receiving pretherapy and posttreatment with of anti-IL-17A mAb. Imiquimod (IMQ)-treated wild type (WT) and T-cell receptor delta chain knock-out (Tcrd−/−) mice were used to investigate the correlation between IL-17A and abnormal changes in LCFAs in psoriasis patients.ResultsA rapid and sensitive in situ extraction derivatization method for quantifying LCFAs using GC–MS was established. Serum lipidomic results showed that psoriasis patients had higher levels of saturated fatty acids (SFAs) and ω-6 polyunsaturated fatty acids (PUFAs) but lower levels of monounsaturated fatty acids (MUFAs) and ω-3 PUFAs than healthy individuals, indicating impaired serum LCFA metabolism. Anti-IL-17A mAb treatment affected most of these LCFA changes. Analysis of LCFAs in IMQ-treated mice showed that LCFAs increased in the serum of WT mice, while there were no significant changes in the Tcrd−/− mice. SFAs increased in IMQ-treated WT mice, while MUFAs showed the opposite trend, and PUFAs did not change significantly.ConclusionsThis study presented a dependable method for quantifying LCFAs that enhanced sensitivity and reduced analysis time. The lipidomic analysis results showed that anti-IL-17A mAb not only ameliorated skin lesions in psoriasis patients but also affected abnormal LCFAs metabolism. Furthermore, the study indicated a potential correlation between IL-17A and abnormal LCFA metabolism in psoriasis patients, which was supported by the alterations in serum LCFAs observed in IMQ-treated WT and Tcrd−/− mice.

A Novel Strategy to Produce CAR-Γδ T Cells Via Targeted Knockout By CRISPR/Cas9 and Targeted Knockin By AAV

Chimeric antigen receptor (CAR)-γδ T cells are promising in immunotherapy owing to the dual recognition system of γδ T cells, which is potential to exhibit stronger anti-tumor effect. Up to date, established CAR-γδ T cells exhibit poor anti-tumor efficiency and are commonly generated using lentiviral (LV) or gamma-retroviral (γRV) vectors, which may increase the risk of insertional mutagenesis. Nowadays, clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been widely used to cleave the genomic sequence precisely, as well as Adeno-associated Virus (AAV) being used to deliver DNA templates for target sequence knock-in (KI) mediated by homology-directed repair, which could contribute to achieve the accurate insertion of CAR at a specific site. Furthermore, the endogenous promoter of TCR has been used to express CAR molecules with stable expression and better antitumor function in the production of CAR-αβ T cells. In the present study, we established and optimized the strategy to generate CAR-γδ T cells, combining the site-specific knock-out (KO) targeting the TCR delta chain constant region (TRDC) site of γδ T cells using CRISPR/Cas9 and site-specific CAR knock-in using AAV. Firstly, we screened out a functional guide RNA (gRNA) targeting the γδ TCR TRDC locus by prediction tools and validation in cell line model experiments, and achieved an INDEL frequency of 31.8%, which showed a high activity of gRNA. Then, we optimized the option to increase KO efficiency including exploring appropriate Cas9 amount, ratio of gRNA to Cas9 and cell number titration. Besides, we designed AAV vector carrying CAR sequence targeting the gRNA aiming site selected above (Figure1A). After that, we used CRISPR/Cas9 and AAV to knock out TCR and knock in CAR sequence in γδ T cells. We tried different electroporation procedures (X001, T007, U014, T023) and cell culture conditions (cultured in medium containing zoledronic acid (ZOL)/ IL2 for 5 days or for 10 days) to select out the optimal procedure to acquire high KO/KI efficiency and high cell proliferation rate. When we used PBMC being cultured in medium containing zoledronic acid (ZOL)/ IL2 for 5 days (Day 5) to generate CAR-γδ T cells, cells electroporated using X001 procedure showed the best TCR KO efficiency of ~80%（p<0.05）and CAR expression of ~20% (p< 0.01), as well as keeping the highest cell proliferation rate than other procedures (p<0.0001) (Figure 1B and 1C). If we used PBMC being cultured in medium containing zoledronic acid (ZOL)/ IL2 for 10 days (Day 10) to generate CAR-γδ T cells, U014 program also exhibited a relative high CAR expression and the significant quicker cell growth than others in the later period (p<0.01) (Figure 1B and 1C). However, the later proliferation rate of CAR-γδ T cells generated at Day 5 was superior to that generated at Day10. Thus, we determined that a 5 days' cell culture and the use of X001 program were the optimal condition to generate CAR-γδ T cells. Besides, we found that KI efficiency is positively correlative with Multiplicity of infection (MOI). While MOI comes to 1.2×10 5, the KI efficiency couldn't be increased (Figure 1D). Finally, we evaluated the anti-tumor function of CAR-γδ T cells . Antigen stimulated CAR-γδ T cells significantly released TNFα and INFγ, but failed to produce IL2 (Figure 1E). Besides, CAR-γδ T cells could significantly lysis different tumor cells even at a low effector/target ratio (E/T) (Figure 1G). Interestingly, the proportion of memory phenotype (CD62L + ) in CAR-γδ T cells after being stimulated was comparable to that of CAR-γδ T cells without stimulation, indicating the potential of the great anti-tumor activity of CAR-γδ T cells after being stimulated in tumor environment (Figure 1F). Our new method successfully generated CAR-γδ T cells with a CAR expression of 20% and in a high proliferation rate, which was determined to exert effective anti-tumor effects.

The Proteomic Signature of Extracellular Vesicles from Sickle Cell Anemia Patients Provides Insights into Their Possible Role in the Pathophysiology of the Disease

Sickle cell anemia (SCA) is marked by hemolysis and vaso-occlusion, which contribute to various other complications observed in the disease. Extracellular vesicles (EVs), released by activated cells, serve as carriers of various markers, proteins, and RNA derived from their parent cells. These structures have the ability to transfer their cargo to the recipient cells, modulating their activity. Despite EVs involvement in physiological processes, there is growing evidence of their role in certain blood diseases, making them a subject of extensive research in recent years. In sickle cell disease (SCD), EVs have been previously associated with hemolysis markers, leg ulcers, and endothelial cell activation and several other authors have emphasized the significant role of EVs in its pathophysiology. However, despite these advances, the precise protein content of these EVs, which may hold the key to unraveling their involvement in disease complications, remains largely unexplored. Here, we aimed to characterize the proteomic profile of EVs from patients with SCA. We conducted a comparative analysis, comparing the differentially abundant proteins in the SCA group to those in the control group. Additionally, we focused on analyzing the distinct protein profiles that were uniquely present in the SCA group studied. Nine healthy volunteers (HC) and nineteen patients with SCA were included in the study, with three receiving no treatment, twelve undergoing hydroxyurea (HU) therapy, and four on HU+Crizanlizumab (CRIZ) combination treatment. EVs were isolated from plasma using a combination of differential centrifugation and size exclusion chromatography techniques, and their quantification was performed using nano-tracking analysis. The protein profile of EVs was assessed through mass spectrometry after sample digestion and desalination in a developed protocol for EVs research. The obtained data were analyzed using MaxQuant and Perseus software. To determine differentially abundant proteins between the HC and SCA groups, statistical tests such as ANOVA followed by Benjamini-Hochberg correction were applied, considering a significance level of p<0.05. Additionally, gene ontology analysis was conducted to identify the relevant biological processes (FDR <0,05) associated with the differentially abundant proteins. Firstly, we identified 41 unique proteins in the SCA group. The relevant biological processes associated with these proteins are primarily related to erythroid differentiation, inflammation, cellular response to oxidative stress, and the upregulation of signaling pathways mediated by TNF and other cytokines. These findings align with the characteristic pathophysiology of SCA. The proteins found in higher concentrations in EVs of SCA patients compared to HC group (p<0,05) were mainly related to endocytosis and exocytosis, inflammation, immune response and coagulation process. Moreover, we observed higher levels of hemoglobin chains in SCA EVs. The delta chain was increased in the SCA group with no treatment in relation to the HC group and the SCA group with HU. Beta and alpha chains were also found in increased levels in the SCA group without HU compared to the HC group, SCA with HU and SCA with HU+CRIZ. Additionally, both transferrin (TF) and the transferrin receptor (TFRC-CD71) were increased in all three subgroups of SCA patients compared to the HC group. The identification of various types of hemoglobin in these EVs holds significant importance. This observation aligns with the potential role of these structures in promoting the generation of reactive oxygen species (ROS), activating endothelial cells, and reducing nitric oxide levels in individuals with SCA. Such findings may provide valuable insights into the underlying mechanisms contributing to the pathophysiology of SCA and its associated complications. Moreover, our data shows higher levels of Tenascin-C in the EVs of SCA patients (all subgroups) (p<0,05), a protein related to tissue damage and inflammation that has not yet been described in the pathophysiology of the disease. In summary, we access, for the first time, the protein content of EVs from patients with SCA. The data described here are novel and could help to elucidate the putative functions of EVs in the context of SCA providing valuable insights for future investigations and potential therapeutic strategies.

Nonergodic one-magnon magnetization dynamics of the antiferromagnetic delta chain

Nonergodic one-magnon magnetization dynamics of the antiferromagnetic delta chain

Quantum spin nematic liquid in the low-dimensional anisotropic magnets -S=1/2 delta spin chain with the anisotropic ferromagnetic interaction in magnetic field-

The magnetization process of the S=1/2S=1/2 delta chain with the anisotropic ferromagnetic interaction is investigated using the numerical diagonalization of finite-size clusters. It is found that the spin nematic liquid phase appears in higher magnetization region, as well as the SDW liquid one in lower region.

Abstract 2187: Epigenetic monitoring of gamma delta T-cells

Abstract Human gamma delta T cells (γδ-T cells) are non-conventional T lymphocytes expressing a TCR heterodimer comprised of gamma (γ) and delta (δ) chains. They constitute an essential part of the innate immune system playing a pivotal role at the front line of the host's defense against pathogens. γδ-T cells have received particular attention as they exert multiple functions under non-infectious settings, most notably in carcinogenesis, where they can initiate a potent HLA-independent anti-tumor activity. Owing to these attributes, γδ-T cells appear to be a promising target for designing cell-based anti-cancer immunotherapies. For such strategies - which involve manipulation of γδ-T cells, e.g. by activation, tumor-targeting, or genetic modification - accurate and quantitative cell-counting tools are indispensable. The gold standard for immune cell quantification to date is still flow cytometry (FCM), although this technology is associated with demanding sample logistics as it requires suspensions of viable cells and presents challenges to standardization amongst different sites, cytometers and operators. In recent years, a huge body of evidence has been accumulated demonstrating epigenetic qPCR (Epiontis ID®) as a powerful and FCM-equivalent methodology for cell quantification and immune monitoring, which is not affected with such limitations. Here we present a DNA methylation-specific, real-time PCR-based assay for the precise quantification of γδ-T cells in blood, tissues and formalin fixed and paraffin embedded (FFPE) samples. The qPCR assay targets a differentially methylated region in the gene encoding the constant region of the T cell receptor gamma chain (TRGC2). This marker region was shown being entirely unmethylated in γδ-T cells, but appearing fully methylated in conventional CD4+ and CD8+ T cells, in CD56+ NK and CD19+ B cells, in CD14+ monocytes and CD15+ granulocytes. Validation of the assay according to ICH guidelines and ISO17025 standards demonstrated a robust performance and stability, linear measurements in the operational range (between 0% and 100% methylation), a definite target cell type specificity and a good correlation to paralleled flow cytometric analyses. The intra- and inter-assay precision is below 15% and 20%, respectively and the lower limit of quantification, above where standard precision of measurements can be achieved, lies at 74 unmethylated target molecules. Cell type-specificity was shown using blood from heathy donors, the applicability in cancerous samples still has to be confirmed.In summary the epigenetic, γδ-T cell-specific assay posted here may represent a useful tool for immune monitoring in clinical settings assisting the development of innovative γδ-T cell-based immunotherapies. Citation Format: Sven Olek, Udo Baron, Isabell Janack, Kati Bourquain, Stefan Kärst, Deborah Phippard, Laura Lozza, Ciro Novaes Lino Rosa. Epigenetic monitoring of gamma delta T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2187.

Gamma delta T-cell reconstitution after allogeneic HCT: A platform for cell therapy.

Allogeneic Hematopoietic stem cell transplantation (allo-HCT) is a curative platform for several hematological diseases. Despite its therapeutic benefits, the profound immunodeficiency associated with the transplant procedure remains a major challenge that renders patients vulnerable to several complications. Today, It is well established that a rapid and efficient immune reconstitution, particularly of the T cell compartment is pivotal to both a short-term and a long-term favorable outcome. T cells expressing a TCR heterodimer comprised of gamma (γ) and delta (δ) chains have received particular attention in allo-HCT setting, as a large body of evidence has indicated that γδ T cells can exert favorable potent anti-tumor effects without inducing severe graft versus host disease (GVHD). However, despite their potential role in allo-HCT, studies investigating their detailed reconstitution in patients after allo-HCT are scarce. In this review we aim to shed lights on the current literature and understanding of γδ T cell reconstitution kinetics as well as the different transplant-related factors that may influence γδ reconstitution in allo-HCT. Furthermore, we will present data from available reports supporting a role of γδ cells and their subsets in patient outcome. Finally, we discuss the current and future strategies to develop γδ cell-based therapies to exploit the full immunotherapeutic potential of γδ cells in HCT setting.

Optimizing storage of RDF archives using bidirectional delta chains

Linked Open Datasets on the Web that are published as RDF can evolve over time. There is a need to be able to store such evolving RDF datasets, and query across their versions. Different storage strategies are available for managing such versioned datasets, each being efficient for specific types of versioned queries. In recent work, a hybrid storage strategy has been introduced that combines these different strategies to lead to more efficient query execution for all versioned query types at the cost of increased ingestion time. While this trade-off is beneficial in the context of Web querying, it suffers from exponential ingestion times in terms of the number of versions, which becomes problematic for RDF datasets with many versions. As such, there is a need for an improved storage strategy that scales better in terms of ingestion time for many versions. We have designed, implemented, and evaluated a change to the hybrid storage strategy where we make use of a bidirectional delta chain instead of the default unidirectional delta chain. In this article, we introduce a concrete architecture for this change, together with accompanying ingestion and querying algorithms. Experimental results from our implementation show that the ingestion time is significantly reduced. As an additional benefit, this change also leads to lower total storage size and even improved query execution performance in some cases. This work shows that modifying the structure of delta chains within the hybrid storage strategy can be highly beneficial for RDF archives. In future work, other modifications to this delta chain structure deserve to be investigated, to further improve the scalability of ingestion and querying of datasets with many versions.

Highly multiplexed immune repertoire sequencing links multiple lymphocyte classes with severity of response to COVID-19

SummaryBackgroundDisease progression of subjects with coronavirus disease 2019 (COVID-19) varies dramatically. Understanding the various types of immune response to SARS-CoV-2 is critical for better clinical management of coronavirus outbreaks and to potentially improve future therapies. Disease dynamics can be characterized by deciphering the adaptive immune response.MethodsIn this cross-sectional study we analyzed 117 peripheral blood immune repertoires from healthy controls and subjects with mild to severe COVID-19 disease to elucidate the interplay between B and T cells. We used an immune repertoire Primer Extension Target Enrichment method (immunoPETE) to sequence simultaneously human leukocyte antigen (HLA) restricted T cell receptor beta chain (TRB) and unrestricted T cell receptor delta chain (TRD) and immunoglobulin heavy chain (IgH) immune receptor repertoires. The distribution was analyzed of TRB, TRD and IgH clones between healthy and COVID-19 infected subjects. Using McFadden's Adjusted R2 variables were examined for a predictive model. The aim of this study is to analyze the influence of the adaptive immune repertoire on the severity of the disease (value on the World Health Organization Clinical Progression Scale) in COVID-19.FindingsCombining clinical metadata with clonotypes of three immune receptor heavy chains (TRB, TRD, and IgH), we found significant associations between COVID-19 disease severity groups and immune receptor sequences of B and T cell compartments. Logistic regression showed an increase in shared IgH clonal types and decrease of TRD in subjects with severe COVID-19. The probability of finding shared clones of TRD clonal types was highest in healthy subjects (controls). Some specific TRB clones seems to be present in severe COVID-19 (Figure S7b). The most informative models (McFadden´s Adjusted R2=0.141) linked disease severity with immune repertoire measures across all three cell types, as well as receptor-specific cell counts, highlighting the importance of multiple lymphocyte classes in disease progression.InterpretationAdaptive immune receptor peripheral blood repertoire measures are associated with COVID-19 disease severity.FundingThe study was funded with grants from the Berlin Institute of Health (BIH).

γδ Tcells license immature B cells to produce a broad range of polyreactive antibodies.

Immature autoreactive B cells are present in all healthy individuals, but it is unclear which signals are required for their maturation into antibody-producing cells. Inducible depletion of γδ Tcells show that direct interaction between γδ Tcells and immature B cells in the spleen support an "innate" transition to mature B cells with a broad range of antigen specificities. IL-4 production of γδ Tcells and cell-to-cell contact via CD30L support B cell maturation and induce genes of the unfolded protein response and mTORC1 signaling. Eight days after invivo depletion of γδ Tcells, increased numbers of B cells are already stuck in the transitional phase and express increased levels of IgD and CD21. Absence of γδ Tcells leads also to reduced levels of serum anti-nuclear autoantibodies, making γδ Tcells an attractive target to treat autoimmunity.

Overcoming biases in causal inference of molecular interactions.

Computer inference of biological mechanisms is increasingly approachable due to dynamically rich data sources such as single-cell genomics. Inferred molecular interactions can prioritize hypotheses for wet-lab experiments to expedite biological discovery. However, complex data often come with unwanted biological or technical variations, exposing biases over marginal distribution and sample size in current methods to favor spurious causal relationships. Considering function direction and strength as evidence for causality, we present an adapted functional chi-squared test (AdpFunChisq) that rewards functional patterns over non-functional or independent patterns. On synthetic and three biology datasets, we demonstrate the advantages of AdpFunChisq over 10 methods on overcoming biases that give rise to wide fluctuations in the performance of alternative approaches. On single-cell multiomics data of multiple phenotype acute leukemia, we found that the T-cell surface glycoprotein CD3 delta chain may causally mediate specific genes in the viral carcinogenesis pathway. Using the causality-by-functionality principle, AdpFunChisq offers a viable option for robust causal inference in dynamical systems. The AdpFunChisq test is implemented in the R package 'FunChisq' (2.5.2 or above) at https://cran.r-project.org/package=FunChisq. All other source code along with pre-processed data is available at Code Ocean https://doi.org/10.24433/CO.2907738.v1. Supplementary materials are available at Bioinformatics online.

The NLPHL Tumor Microenvironment Is Markedly Enriched in the Tigit and PD-1 Signalling Axes Compared to Classical Hodgkin Lymphoma

The NLPHL Tumor Microenvironment Is Markedly Enriched in the Tigit and PD-1 Signalling Axes Compared to Classical Hodgkin Lymphoma

Single-Cell Transcriptional Profiling of Zebrafish Hematopoiesis Offers Insight into Early Lymphocyte Development and Reveals Novel Immune Cell Populations

The advent of single-cell RNA sequencing (scRNA-seq) has greatly expanded our appreciation for cell state diversity beyond classical developmental hierarchies and simple population subsets. In particular, rich transcriptional heterogeneity has been observed within immune cell populations leading to the identification of novel cell types. Except for a few notable exceptions, intensive work in this area has largely been confined to mammals. Much of the transcriptional profiling of blood cell development in zebrafish has failed to capture early stages of lymphocyte development as the majority of research has not included thymus datasets or captured sufficient marrow B cells to explore developmental trajectories. To gain insight into T and B cell development in the zebrafish and immune cell diversity more broadly, we performed scRNA-seq using 10x Genomics Next GEM technology on adult zebrafish kidney marrows (n = 5 biological replicates) in addition to whole juvenile thymi at 4 weeks post-fertilization (wpf) (n = 4 technical replicates) and whole adult thymi at 3-4 months post-fertilization (n = 4 biological replicates). After filtering out low quality cellular barcodes, 34,492 kidney marrow cells and 35,268 thymus cells remained for analysis. With respect to T cell development, we identified putative early thymic progenitors from their clustering with hematopoietic stem and progenitor cells and shared transcriptional signatures, including the enrichment of CD34, CSF1R, FLI1, and DNMT3B human orthologs. Multiple subsets of thymic and marrow T cells were characterized, including a subset of gamma delta T cells readily identified by their expression of T-cell receptor gamma and delta chain components and expression of a SOX13 ortholog in addition to a Th2-like population expressing IL4, IL13, and GATA3 orthologs. Among other immune cell populations, rich transcriptional diversity was present. Two distinct populations of B cells, largely mutually exclusive for ighd and ighz expression (dual detection <1%), were present across all datasets, including the 4 wpf thymi, a surprisingly early time point in zebrafish B cell ontogeny. A clustering of the adult datasets demonstrated ighz predominance in the thymus (63% of thymic B cells), whereas the majority of marrow B cells (85%) fell within the ighd cluster. Stages of B cell development were also clearly evident, with the earliest B cell progenitors expressing orthologs of human PAX5, DNTT, RAG1, and RAG2, in addition to sid1, an understudied gene proposed to be orthologous to VPREB1. The expression of dntt was notably absent from more mature subsets of rag1 and rag2+ B cell progenitors, analogous to its expression in mammals. Transcriptional signatures unique but reminiscent of mammalian dendritic cell subsets were identified: Plasmacytoid-like dendritic cells characterized by high expression of TLR7, TLR9, and IRF8 orthologs and conventional-like dendritic cells characterized by high expression of CKB, BATF3, and ZNF366 were present in both marrow and thymus datasets, suggestive of greater dendritic-like cell diversity in the zebrafish than previously appreciated. These findings illustrate the power of single-cell transcriptional profiling for illuminating immune cell development and heterogeneity in the zebrafish, demonstrating increasing parallels to the mammalian system. DisclosuresZon: Celularity: Consultancy; Branch Biosciences: Current holder of individual stocks in a privately-held company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Amagma Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; CAMP4 Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; Cellarity: Consultancy.

Screening for hemoglobinopathy with capillary electrophoresis in adult patients

Abstract Hemoglobinopathy screening is frequently needed in adult patients, including prenatal carrier screen, workup of unexplained anemia, and bone marrow donor and recipient screening. However, the preferred test method for screening of hemoglobinopathy is not well established due to limited guidance from professional societies. American College of Obstetricians and Gynecologists’ Committee on Genetics recommended hemoglobin electrophoresis as the screening method of hemoglobinopathy in pregnancy; nevertheless, electrophoresis employs various methodologies, including acid gel electrophoresis, alkaline gel electrophoresis, and capillary electrophoresis (CE) with alkaline buffer. For other adult patient populations who need hemoglobinopathy screening, no clear guidelines dictate the method of choice. A previous study has shown that CE captures major hemoglobinopathies with comparable performance to high-performance liquid chromatography (HPLC) in pediatric patients but no study has investigated using CE alone in adult patient screening. In this retrospective study, we evaluated the utility of CE as a screening method to rule out clinically significant hemoglobin variants. During eight months, 312 adult patients without previously identified hemoglobin variants had hemoglobinopathy screening performed using a comprehensive testing algorithm. This cascade algorithm screens for hemoglobinopathy using both CE (Capillarys, Sebia, Paris, France) and HPLC (laboratory-developed test) with reflex to more advanced variant identification such as mass spectrometry and genetic analyses. Categories of abnormal findings were reviewed to determine if hemoglobinopathy can be identified by using CE only. The patient population mainly consists of pregnant women and anemic patients with hematologic malignancies with an average age of 42. Out of the 312 screened patients, 47 had abnormal results. The most frequent condition was elevated hemoglobin F (N=25) ranging 2-5% seen in leukemia patients on chemotherapy attributed to bone marrow stress. Eight cases of beta plus thalassemia (featuring hemoglobin A2 &amp;gt;4%) and 3 cases of hemoglobin C trait were identified in patients with little to mild clinical manifestations (red blood cell indices suggesting anemia). Decreased hemoglobin A2 fraction was observed in 7 patients, and potential causes were alpha thalassemia or iron deficiency. Other less common hemoglobinopathies included heterozygote A2 prime (N=3, a benign delta chain variant that migrates separately from hemoglobin A2 on CE) and hemoglobin G-Philadelphia (N=1). All of the abnormal results are identifiable by CE alone, although HPLC and more advanced methods help confirm the diagnosis. Our study shows that CE as the first line of screening method would rule out major hemoglobinopathies in adults. There have been reports that rare but clinically significant hemoglobin variants like hemoglobin Malmo may not be detected by CE, and therefore, certain pre-test probability factors need to be considered when testing for hemoglobinopathies, such as race/ethnicity background, family history, red blood cell indices, and iron deficiency status.

Quantifying the Pathophysiology of Beta-Thalassemia Using a Novel in-Silico Model of Red Blood Cell Production and Lifespan

Background: From a qualitative perspective the pathophysiology of beta-thalassemia (BT) is generally well understood. Reduction in hemoglobin (Hb) beta-chain synthesis relative to the normal rate of alpha-chain synthesis causes a free alpha-chain excess (ACE) that is toxic to the RBC and its precursors. Circulating RBCs are hemolyzed in the spleen, shortening their lifespan from the normal value of 120 days. RBC precursors in the bone marrow undergo early apoptosis leading to reduced RBC production relative to other anemic states (so called “ineffective erythropoiesis”). Together with low MCH levels these mechanisms lead to Hb levels ranging from 10 - 12 g/dL in BT trait, 7 - 10 g/dL in non-transfusion dependent (NTD) BT, below 7 g/dL in transfusion-dependent (TD) BT at baseline and above 9 - 10.5 g/dL in TD patients receiving regular transfusions administered every 2 to 5 weeks. To quantify the mechanisms responsible for the different BT phenotypes and the response to transfusion in TD patients, we have developed an in-silico model of RBC production and lifespan.Methods: The theoretical amount of ACE per cell was estimated by assuming that the alpha-chain level corresponds to an MCH of 30 pg and the non-alpha chain level (beta, gamma and delta chains) corresponds to the MCH of the BT phenotype, which varies from ~12 to 20 pg in TD BT to ~16 to 23 pg in NTD BT and ~18 to 24 pg in BT trait. The in-silico model consists of a bone marrow module - representing the production and aging of reticulocytes, Hb synthesis and entry to the bloodstream (equivalent to the RBC production rate, PRBC); a circulation module - representing the total blood volume (TBV), conversion of reticulocytes to mature RBCs, attainment of the final MCH, normal aging of RBCs, and a hemolytic process in BT (with rate constant kH that depends on the ACE level); and a transfusion module - representing the volume of transfused blood (VTF) with hematocrit H, a normal RBC age-distribution, and a separate hemolytic process (with rate constant kH_Tf). Reticulocyte lifespans in the bone marrow and circulation were set to ~3 days and ~1 day, respectively, but can vary oppositely at high PRBC (stress erythropoiesis). A key assumption of the model is that PRBC is down-regulated by the Hb level according to a sigmoidal Hill function, which was calibrated using PRBC data from normal subjects and patients with sickle cell disease, and was assumed to have a maximum value 10-fold the normal PRBC rate. In BT the PRBC function is also modulated by a factor FBT, dependent on the ACE level. Additional data from BT patients were used to derive the kH and FBT values. The model was expressed in ordinary differential equations using the Berkeley Madonna platform (Version 8.3.18).Results: To simulate the spectrum of BT phenotypes, MCH levels were varied from 15 pg to the normal value of 30 pg and the ACE levels, kH and FBT values were computed from the model. The simulated steady-state dependences of Hb vs. MCH (Figure 1A) and Reticulocyte count vs. Hb level (Figure 1B) are in good agreement with published literature data. For virtual patients (VPs) with MCH levels of 23, 21 and 18 pg, typical of BT trait, NTD and TD phenotypes, respectively, the estimated ACE levels were 0.22, 0.28 and 0.38 fmole/cell; kH values were 0.01, 0.019 and 0.037 day-1 and values of FBT were 0.65, 0.6 and 0.55. The effective RBC lifespans for these VPs were 68, 47 and 23 days, respectively. Regular transfusions (VTF = 15 mL/kg; H = 0.6) were simulated over 24 weeks for a TD VP (MCH = 18 pg; baseline Hb = 6.61 g/dL) to maintain the Hb level above 9 g/dL. In Figure 2A, where kH_Tf = 0 (no hemolysis of transfused blood), the transfusion interval stabilizes at 3.71 weeks. In Figure 2B, where kH_Tf = 0.0165 day-1(an effective RBC lifespan of 50.7 days), the transfusion interval stabilizes at 2 weeks. Reticulocyte counts decrease markedly in both cases. To achieve a ~3 g/dL increment in the Hb level, TBV must equal 100 mL/kg (~40% greater than normal).Conclusions: Our in-silico model explains the phenotypic variation of Hb levels and reticulocyte counts in BT in terms of the effects of ACE on the hemolysis rate constant (kH) and the fractional reduction in RBC production (FBT). The 2-week transfusion interval required in some patients to maintain Hb above 9 g/dL is explained by a shortened lifespan of the transfused blood. More clinical data would be helpful to confirm and refine these observations to better understand the key drivers of disease pathology in BT. [Display omitted] DisclosuresMazer:Roche: Employment, Equity Ownership. Hermosilla:Roche: Employment. Koerner:Roche: Employment.

Proteomic analysis of drug-susceptible and multidrug-resistant nonreplicating Beijing strains of Mycobacterium tuberculosis cultured in vitro

The existence of latent tuberculosis infection (LTBI) is one of the main obstacles hindering eradication of tuberculosis (TB). To better understand molecular mechanisms and explore biomarkers for the pathogen during LTBI, we cultured strains of Mycobacterium tuberculosis (Mtb) under stress conditions, mimicking those in the host granuloma intracellular environment, to induce entry into the non-replicating persistence stage. The stresses included hypoxia, low pH (5.0), iron deprivation (100 μM of 2, 2’˗dipyridyl) and nutrient starvation (10% M7H9 medium). Three Mtb strains were studied: two clinical isolates (drug-susceptible Beijing (BJ) and multidrug-resistant Beijing (MDR-BJ) strains) and the reference laboratory strain, H37Rv. We investigated the proteomics profiles of these strains cultured in stressful conditions and then validated the findings by transcriptional analysis. NarJ (respiratory nitrate reductase delta chain) was significantly up-regulated at the protein level and the mRNA level in all three Mtb strains. The narJ gene is a member of the narGHJI operon encoding all nitrate reductase subunits, which play a role in nitrate metabolism during the adaptation of Mtb to stressful intracellular environments and the subsequent establishment of latent TB. The identification of up-regulated mRNAs and proteins of Mtb under stress conditions could assist development of biomarkers, drug targets and vaccine antigens.

Estimation for Entropy and Parameters of Generalized Bilal Distribution under Adaptive Type II Progressive Hybrid Censoring Scheme.

Entropy measures the uncertainty associated with a random variable. It has important applications in cybernetics, probability theory, astrophysics, life sciences and other fields. Recently, many authors focused on the estimation of entropy with different life distributions. However, the estimation of entropy for the generalized Bilal (GB) distribution has not yet been involved. In this paper, we consider the estimation of the entropy and the parameters with GB distribution based on adaptive Type-II progressive hybrid censored data. Maximum likelihood estimation of the entropy and the parameters are obtained using the Newton–Raphson iteration method. Bayesian estimations under different loss functions are provided with the help of Lindley’s approximation. The approximate confidence interval and the Bayesian credible interval of the parameters and entropy are obtained by using the delta and Markov chain Monte Carlo (MCMC) methods, respectively. Monte Carlo simulation studies are carried out to observe the performances of the different point and interval estimations. Finally, a real data set has been analyzed for illustrative purposes.

Adaptive Granularity Based Last-Level Cache Prefetching Method with eDRAM Prefetch Buffer for Graph Processing Applications

The emergence of big data processing and machine learning has triggered the exponential growth of the working set sizes of applications. In addition, several modern applications are memory intensive with irregular memory access patterns. Therefore, we propose the concept of adaptive granularities to develop a prefetching methodology for analyzing memory access patterns based on a wider granularity concept that entails both cache lines and page granularity. The proposed prefetching module resides in the last-level cache (LLC) to handle the large working set of memory-intensive workloads. Additionally, to support memory access streams with variable intervals, we introduced an embedded-DRAM based LLC prefetch buffer that consists of three granularity-based prefetch engines and an access history table. By adaptively changing the granularity window for analyzing memory streams, the proposed model can swiftly and appropriately determine the stride of memory addresses to generate hidden delta chains from irregular memory access patterns. The proposed model achieves 18% and 15% improvements in terms of energy consumption and execution time compared to global history buffer and best offset prefetchers, respectively. In addition, our model reduced the total execution time and energy consumption by approximately 6% and 2.3%, compared to those of the Markov prefetcher and variable-length delta prefetcher.