Deep Depletion Research Articles

Cutting-edge approaches to B-cell depletion in autoimmune diseases.

B-cell depletion therapy (BCDT) has been employed to treat autoimmune disease for ~20 years. Immunoglobulin G1 (IgG1) monoclonal antibodies targeting CD20 and utilizing effector function (eg, antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis) to eliminate B cells have historically been the predominant therapeutic approaches. More recently, diverse BCDT approaches targeting a variety of B-cell surface antigens have been developed for use in hematologic malignancies, including effector-function-enhanced monoclonal antibodies, chimeric antigen receptor T-cell (CAR-T) treatment, and bispecific T-cell engagers (TCEs). The latter category of antibodies employs CD3 engagement to augment the killing of target cells. Given the improvement in B-cell depletion observed with CAR-T and TCEs compared with conventional monospecific antibodies for treatment of hematologic malignancies and the recent case reports demonstrating therapeutic benefit of CAR-T in autoimmune disease, there is potential for these mechanisms to be effective for B-cell-mediated autoimmune disease. In this review, we discuss the various BCDTs that are being developed in autoimmune diseases, describing the molecule designs, depletion mechanisms, and potential advantages and disadvantages of each approach as they pertain to safety, efficacy, and patient experience. Additionally, recent advances and strategies with TCEs are presented to help broaden understanding of the potential for bispecific antibodies to safely and effectively engage T cells for deep B-cell depletion in autoimmune diseases.

Successful generation of fully human, second generation, anti-CD19 CAR T cells for clinical use in patients with diverse autoimmune disorders

BackgroundB-cell targeting chimeric antigen receptor (CAR) T-cell therapies, which lead to profound B-cell depletion, have been well-established in hematology-oncology. This deep B-cell depletion mechanism has prompted the exploration of their use in B-cell driven autoimmune diseases. We herein report on the manufacturing of KYV-101, a fully human anti-CD19 CAR T-cell therapy, derived from patients who were treated across a spectrum of autoimmune diseases. MethodsKYV-101 was manufactured from peripheral blood-derived mononuclear cells of 20 patients across 7 autoimmune disease types (neurological autoimmune diseases, n=13; rheumatological diseases, n=7). Patients ranged from 18–75 years of age. Duration of disease ranged from <1–23 years since diagnosis. Patients were heavily pretreated, and most were refractory to prior immunosuppressive treatments. Apheresis was collected across 9 sites, cryopreserved, and shipped to the manufacturing facility. Healthy donor apheresis samples were collected for manufacturing comparison. Manufacturing was performed using the CliniMACS Prodigy system. Cells were enriched for CD4+/CD8+ T cells, transduced with a 3rd generation lentiviral vector encoding the CAR, expanded in vitro, and harvested. Percent cell viability, T-cell purity, cellular expansion, and transduction efficiency were assessed. Activity was assessed using cytokine release assays for KYV-101 CAR T cells co-cultured with different CD19+/– target cell lines. ResultsKYV-101 was successfully manufactured for 100% of patients. Transduced cell populations were highly viable, with expansion ranging from 11–66 fold at Day 8, and were comparable across disease types. Healthy donor-derived controls displayed similar expansion ranges. High CAR expression and transduction rates were observed, ranging between 37–84% with low variation in transgene copy number (2 to 4 per cell). Cell viability of the final KYV-101 drug product ranged from 87–97%. KYV-101 displayed robust CD19-dependent and effector dose-related release of the pro-inflammatory cytokine IFN-γ. ConclusionsKYV-101 manufacturing yielded a CAR T-cell product with high viability and consistent composition and functionality, regardless of disease indication, pre-treatment, and heterogeneity of the incoming material. Cryopreservation of the apheresis and final drug product enabled widespread distribution. These results support the robustness of the manufacturing process for the fully human KYV-101 anti-CD19 CAR T-cell therapy drug product for patients across diverse autoimmune disease types.

Revealing Gliding-Induced Structural Distortion in High-Nickel Layered Oxide Cathodes for Lithium-Ion Batteries.

After charging to a high state-of-charge (SoC), layered oxide cathodes exhibit high capacities but suffer from gliding-induced structural distortions caused by deep Li depletion within alkali metal (AM) layers, especially for high-nickel candidates. In this study, we identify the essential structure of the detrimental H3 phase formed at high SoC to be an intergrowth structure characterized by random sequences of the O3 and O1 slabs, where the O3 slabs represent Li-rich layers and the O1 slabs denote Li-depleted (or empty) layers that glide from the O3 slabs. Moreover, we adopt two doping strategies targeting different doping sites to eliminate the formation of Li-vacant O1 slabs. First, we introduce direct transition metal (TM) pillars between TMO2 slabs achieved through dopants (e.g., Nb) positioned within AM layers, significantly improving the cycling stability. Second, we introduce indirect Li pillars achieved through dopants located at TM layers to adjust the Li-O bond strength. While this strategy can regulate the uniformity of Li at the slab level, it results in an uneven Li distribution at the particle scale, ultimately failing to enhance the electrochemical performance. Our established research strategy facilitates the realization of diverse pillars between TMO2 slabs through doping, thereby offering guidance for stabilizing high-capacity layered oxide cathodes at high SoC.

CD19-CAR T-cell therapy induces deep tissue depletion of B cells

ObjectivesCD19-targeting chimeric antigen receptor (CAR) T-cell therapy can induce long-term drug-free remission in patients with autoimmune diseases (AIDs). The efficacy of CD19-CAR T-cell therapy is presumably based on deep tissue...

Advancements and challenges in CAR T cell therapy in autoimmune diseases.

Chimeric antigen receptor (CAR) T cells are highly effective at targeting and eliminating cells of the B cell lineage. CAR T cell therapy has become a standard-of-care treatment for patients with relapsed or refractory B cell malignancies. In addition, the administration of genetically modified T cells with the capacity to deplete B cells and/or plasma cells has tremendous therapeutic potential in autoimmune diseases. In the past few years, CD19-based and B cell maturation antigen (BCMA)-based CAR T cell therapies have been applied to various B cell-mediated autoimmune diseases including systemic lupus erythematosus, idiopathic inflammatory myopathy, systemic sclerosis, neuromyelitis optica spectrum disorder, myasthenia gravis and multiple sclerosis. The scientific rationale behind this approach is that deep depletion of B cells, including autoreactive B cell clones, could restore normal immune function, referred to as an immune reset. In this Review, we discuss important aspects of CAR T cell therapy in autoimmune disease, including considerations relating to patient selection, safety, efficacy and medical management. These considerations are based on the early experiences of CAR T cell therapy in autoimmune diseases, and as the field of CAR T cell therapy in autoimmune diseases continues to rapidly evolve, these issues will remain subject to ongoing refinement and adaptation.

Estimating deep soil water depletion and availability under planted forest on the Loess Plateau, China

Deep soil water (DSW) plays a pivotal role in tree growth, susceptibility to drought-induced mortality, and belowground carbon and nutrient cycling. Assessing DSW depletion is essential for evaluating the resilience and sustainability of planted forests. But, due to the poor accessibility of deep soil layers, little is known about large scale DSW depletion. In this study, we leverage the concept that “plants are reliable indicators of deep soil water” to estimate DSW depletion in planted forests within the arid and semi-arid regions of the Chinese Loess Plateau (CLP). Our approach involves establishing a model that correlates forest age with DSW depletion. We then employ this model to estimate DSW depletion across the region, utilizing readily available data on the distribution of forest age and utilize the boundary models to consider the variability of DSW depletion estimated with forest age. Our results indicate that the model effectively estimates DSW depletion in planted forests, demonstrating a strong fit with an R2 of 0.71 and a low root mean square error (RMSE) of 332 mm. Notably, a substantial portion of the planted forest areas on the CLP has experienced DSW depletion from 800 mm to 1600 mm, and totaling 2.41 × 1010 m3 DSW depletion from 1995 to 2020 based on the general model. However, the available DSW in the existing planted forests on the CLP is estimated at only 1.73 × 1010 m3 by 2038. This suggests that there is potential risks and unsustainability for further afforestation efforts and carbon sequestration on the CLP under the current continuous afforestation measures. Our study holds significant implications for sustainable regional ecological management and quantifying water resources for carbon trading through afforestation.

Abstract 2730: Beyond antibodies and CAR-T: Topologically-engineered, super-dimeric antibody-like molecules with dual Fc domains for trispecific, bivalent targeting of CD19, CD20, and Fcgamma receptors

Abstract Background: Depletion of B cells has resulted in major therapeutic benefits in autoimmune diseases and hematological malignancies. Several FDA-approved B cell-depleting antibody-based therapies are available, including antibody-drug conjugates, T cell engagers, CAR-T cells, and antibody variants with enhanced effector functions through Fc engineering. However, a significant unmet need remains in both oncology and autoimmune disease. All currently approved B cell-depleting therapies target a single B cell antigen, increasing potential for incomplete depletion and emergence of escape mutants that give rise to relapse and treatment failure. Methods: Here we describe trispecific antibody-like molecules generated using GEM-DIMER technology. In addition to bivalent target binding of both CD19 and CD20, the GEM-DIMER candidates are designed to have two Fc domains to enable powerful effector functions via cooperative binding of Fcgamma receptors. The approach is based on incorporation of a super-dimerization domain into the hinge region of an immunoglobulin heavy chain, enabling the combination of all of the components of two IgG antibodies into a single molecule. Using this approach, we generated GEM-DIMER candidates from rituximab and FMC63, the parental anti-CD19 antibody for anti-CD19 scFv used in approved CAR-T cell therapies. Results: CD19/CD20-targeting GEM-DIMER molecules demonstrated binding to both CD19 and CD20 with affinities comparable to the individual parent antibodies. Importantly, CD19/CD20-targeting GEM-DIMER molecules demonstrated robust depletion of human B cells in overnight cultures of whole blood. Moreover, antibody-dependent cellular cytotoxicity (ADCC) was demonstrated in co-cultures of the human B cell lymphoma cell line Raji and human peripheral blood mononuclear cells (PBMC). As expected, due to the presence of dual Fc domains, CD19/CD20-targeting GEM-DIMER molecules exhibited enhanced binding to Fcgamma receptors, further explaining the robust effector functions observed. Conclusions: CD19/CD20-targeting GEM-DIMER molecules are promising candidates to provide efficient depletion of both CD19+ and CD20+ cells, providing potential for broad and deep depletion of B cells with reduced risk of emergence of antigen escape variants. These data support the advancement of CD19/CD20-targeting GEM-DIMER molecules in multiple indications where depletion of CD19+ and/or CD20+ B cells is needed. Preparations for clinical investigation are ongoing. Citation Format: Daniel J. Capon, Nelson L. Chan, Larisa Troitskaya, Marina Fomin, Ursula Edman, Brendon Frank, Benjamin Z. Capon, Brian Law, Steven J. Chapin, Gavin M. Lewis, Malcolm L. Gefter, Juha Punnonen. Beyond antibodies and CAR-T: Topologically-engineered, super-dimeric antibody-like molecules with dual Fc domains for trispecific, bivalent targeting of CD19, CD20, and Fcgamma receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2730.

The Fate of Soil Organic Carbon from Compost: A Pot Test Study Using Labile Carbon and 13c Natural Abundance

Recycled organic waste (OW) can be a valuable nutrient source for plant cultivation; however, knowledge is poor regarding its effect on soil carbon conservation, especially in the frame of organic-mineral fertilisation succession. In this study, four composts, green waste (GWC), anaerobically digested bio-waste (DC), sludge (SSC), and bio-waste (BWC), were compared (10 and 20 Mg volatile solids ha−1) in a ryegrass pot test over two growing cycles (112 + 112 days), along with an unamended control (Ctrl) and a chemical reference (Chem), with and without mineral nitrogen (N) fertilisation. At the end of the two growth cycles, the pot soil was analysed for total- (TOC) and labile-carbon (CL) as well as for 13C isotope natural abundance (δ13C and Δ13C vs. Chem). At day 112, the pot test showed that Ctrl and Chem gained poor TOC (8.48 g kg−1), lower than the compost at both 10 and 20 Mg volatile solids ha−1 (10.01 vs. 11.59 g kg−1). At day 224, a deep soil TOC depletion occurred in the pot soil treated with GWC, DC and BWC at both levels (-10 and -20). However, all the compost treatments showed more depleted soil d13C vs. the references, especially Chem, thus revealing relevant compost-derived carbon conservation. Regarding the compost treatments, the carbon management index (CMI) increased over time, indicating high soil functionality, also showing a good relationship with δ13C, suggesting a probable increase in relative lignin which could have been linked to carbon conservation and increased functionality.

CD19 CAR T-Cell Therapy in Autoimmune Disease — A Case Series with Follow-up

BackgroundTreatment for autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis often involves long-term immune suppression. Resetting aberrant autoimmunity in these diseases through deep depletion of B cells is a potential strategy for achieving sustained drug-free remission.MethodsWe evaluated 15 patients with severe SLE (8 patients), idiopathic inflammatory myositis (3 patients), or systemic sclerosis (4 patients) who received a single infusion of CD19 chimeric antigen receptor (CAR) T cells after preconditioning with fludarabine and cyclophosphamide. Efficacy up to 2 years after CAR T-cell infusion was assessed by means of Definition of Remission in SLE (DORIS) remission criteria, American College of Rheumatology–European League against Rheumatism (ACR–EULAR) major clinical response, and the score on the European Scleroderma Trials and Research Group (EUSTAR) activity index (with higher scores indicating greater disease activity), among others. Safety variables, including cytokine release syndrome and infections, were recorded.ResultsThe median follow-up was 15 months (range, 4 to 29). The mean (±SD) duration of B-cell aplasia was 112±47 days. All the patients with SLE had DORIS remission, all the patients with idiopathic inflammatory myositis had an ACR–EULAR major clinical response, and all the patients with systemic sclerosis had a decrease in the score on the EUSTAR activity index. Immunosuppressive therapy was completely stopped in all the patients. Grade 1 cytokine release syndrome occurred in 10 patients. One patient each had grade 2 cytokine release syndrome, grade 1 immune effector cell–associated neurotoxicity syndrome, and pneumonia that resulted in hospitalization.ConclusionsIn this case series, CD19 CAR T-cell transfer appeared to be feasible, safe, and efficacious in three different autoimmune diseases, providing rationale for further controlled clinical trials. (Funded by Deutsche Forschungsgemeinschaft and others.)

Direct characterization of deep soil water depletion reveals hydraulic adjustment of apple trees to edaphic changes

Deep soil water is important for trees to survive droughts in arid and semiarid regions, but often irreversibly decrease as tree age due to limitations in soil infiltration rates and precipitation. How adaptable is the hydraulic system of trees undergoing the depletion of deep soil moisture reserves? To answer this question, we examined 12 traits characterizing the hydraulic structure of apple tree branches along a plantation age gradient (7 - 26 years) under uniform soil and climatic conditions. We found that almost all deep soil available water was consumed (reduced by up to 98%) as apple trees aged, as a result, the fraction of roots located in water deficit layers increased by up to 70%. Reduced water availability in deep soil significantly increased xylem embolism resistance (P50) by 8%, without altering xylem-specific hydraulic conductivity, vessel diameter, or vessel density. A distinct adaptive response to permanently reduced deep soil water availability seems to occur primarily by increasing tree hydraulic safety with no reductions in hydraulic efficiency, through which mechanism trees may acclimatize to deep soil water reduction.

The role of а buffer layer at the contact with silicon in structures with an insulating gap made of a material replacing SiO2

The shape of high-frequency capacitance-voltage characteristics of structures with an insulating layer replacing silicon oxide is considered. It is shown that the buffer layer interaction on the interface with silicon substrates is related to the shielding of external electric fields by charges of the electron traps. It is found that the presence of two plateaus in the high-frequency capacitance-voltage characteristics does not necessarily indicate deep depletion and strong enrichment of a semiconductor. The presence of both capacitive plateaus in the case of strong screening can be explained by the pinning of the Fermi level of Si on electron traps of the buffer layer with a high concentration and a U-shaped spectrum. An algorithm is formulated for calculating the band bending in a semiconductor from the minimal values of the high-frequency capacitance of a sample in the region of depletion effects on the substrate. The theoretical construction was used for determining the characteristics of a structure with an insulating HfO2 layer and it was shown that in this case the buffer layer almost completely shields external electric fields, which prevents significant band bending in the silicon substrate.

Alterations in Immune Cell Composition during First-Line Therapy with Mosunetuzumab for Follicular or Marginal Zone Lymphoma

Background: Bispecific CD20xCD3 antibodies (BiAbs) that engage T-cells to eliminate CD20+ B-cells have emerged as a potent immunotherapy for the treatment of follicular (FL) and marginal zone (MZL) lymphomas. BiAbs have primarily been studied in patients with extensive prior immunosuppressive and lymphodepleting therapies, so their effects on the immune system without such exposures are not well understood. Unlike cellular therapies, BiAbs are administered continuously over time, spanning from 6 months to an indefinite period, and recent studies have raised concerns about T-cell exhaustion due to continuous BiAb exposure in B-lymphoblastic leukemia (Philipp et al, Blood 2022). Mosunetuzumab is a BiAb approved for treatment of relapsed/refractory FL. The ongoing phase 2 BrUOG-401 trial (NCT04792502) is investigating mosunetuzumab as first-line therapy for FL or MZL. Here, we present preliminary results focusing on treatment-induced changes in circulating immune cells. Methods: Patients with previously untreated, high-burden or symptomatic FL or MZL received subcutaneous mosunetuzumab for 8 planned cycles (C) with step-up dosing in C1 (day 1: 5mg, day 8 &amp; 15: 45 mg; then 45mg on day 1 of C4-C8). Patients without a complete response (CR) at interim assessment after C4 also received lenalidomide augmentation (10mg daily) during C5-8. Immune cell subsets were examined using multicolor flow cytometry (BD LSRII Analyzer). Peripheral blood samples were collected longitudinally before C1, on day 1 of C2 and C5, and 4-6 weeks after the end of therapy (EOT). Median cell abundance and marker expression were compared using generalized linear mixed models with patient and sample-level random effects. Analysis was performed in R (v.4.3.1) using the flowCore, flowStats and diffcyt packages; uncorrected P&amp;lt;0.05 was considered statistically significant. Results: As of July 2023, 13 patients (median age 62, 77% women, 77% FL, 23% MZL) were accrued in the trial. At data cutoff, 7 have completed therapy, 11 were evaluable for response, and 8 attained CR ( Fig. A). So far, only 1 patient started lenalidomide; samples collected while on lenalidomide were excluded from analysis. No instances of cytokine release syndrome (CRS) or neurotoxicity were observed. CR's occurred early (by C5). Mosunetuzumab resulted in a rapid, deep, and sustained depletion of circulating CD19+ B-cells. A significant decrease in circulating CD8+ T-cells was noted at cycle 2 (from median 20% to 12%, P&amp;lt;0.001; Fig. B), followed by recovery (19% at C5). Among the CD8+ subsets, the naïve CD8+ T-cells (CD45RA+CCR7+CD27+) decreased relatively at C5 &amp; EOT, while effector memory T-cells (CD45RA-CD45RO+CD27-CD62L-) increased. Notably, we observed a significant early, temporary, increase in the expression of LAG3 at C2 on CD8+ T-cells ( P=0.033), and a more pronounced increase in PD1 expression ( P=0.001), which persisted through the EOT ( Fig. B). No significant changes were observed in total abundance of CD4+ T-cells. However, CD4+CD25+CD127- Tregs notably increased at C2 ( P=0.008). This increase specifically driven by CD45RA+ “naïve” Tregs ( P&amp;lt;0.001 at C2), which remained elevated relative to CD45RO+ Tregs at C5 and EOT. All Tregs showed a significant increase in LAG3 expression at C2 ( P&amp;lt;0.001), which then subsided. In contrast to CD8+ T-cells, there was no significant increase in PD1 expression on CD4+ T-cells ( P=0.20 at C2). Throughout the therapy, there was a progressive increase in NK cells ( P=0.001 at EOT), with no significant shift in the relative proportions of CD16high or CD161high NK cells. Conclusions: In this initial investigation of immune cell changes in patients who received mosunetuzumab without prior chemotherapy exposure, we observed B-cell depletion and a temporary decrease in CD8+ cytotoxic T-cells (implying their migration out of the bloodstream), aligning with mosunetuzumab's mechanism of action. Additional data from this trial will aid in understanding whether the persistent increase in PD1+CD8+ T-cells and expansion of Tregs are linked to a functionally exhausted phenotype and whether lenalidomide could potentially reverse it. Although our observation is limited by sample size, it can still provide insights into optimizing the administration schedules of BiAb therapy in future trials (e.g., intermittent vs. continuous) or determining the most suitable timing for checkpoint inhibitor combinations.

The Cellular Impact, Immediate Targets and Mechanism of Action of NSD2 in t(4;14) Multiple Myeloma

INTRODUCTION: NSD2 is a potential therapeutic target in the 15% of cases of multiple myeloma that carry a t(4;14) translocation. As a consequence of the translocation, strong Ig enhancers are placed in proximity to NSD2 and FGFR3. FGFR3 is deleted at a subsequent time point in 25% of cases, while all cases retain NSD2 expression consistent with it likely being the key oncogenic driver. NSD2 is an H3K36 dimethyl transferase, a histone modification mark associated with active transcription, but the molecular targets and the mechanism by which NSD2 regulates its targets is unknown. The cellular function of NSD2 is also largely unknown, but previous shRNA knockdown studies have suggested it is involved in cell proliferation. The major limitation to understanding the role of NSD2 is the lack of an appropriate experimental system with which to investigate its molecular and cellular function. To address this deficit and to identify the primary direct targets of NSD2 as a means to elucidate its mechanism of action we have generated NSD2 dTAG and NSD2 KO cells together with NSD2 reconstitution systems. METHODS: We have utilized CRISPR gene editing to knock-in a dTAG in NSD2 in the t(4;14) KMS-11 cell line. This system enables the inducible and rapid degradation of NSD2 following the addition of a small-molecule degrader which is otherwise inert. We have also generated clonal NSD2 KO KMS-11 cells that allow us to observe and study the stable phenotypes of NSD2 KO. Because H3K36me2 deposited by NSD2 leads to induction of DNMT3A-mediated DNA methylation and suppression of PRC2-mediated H3K27me3, we have generated DNMT3A KO on top of NSD2dTAG/KO and have used two different PRC2 inhibitors to dissect the dependency of NSD2 function on such secondary modifications. To systematically dissect the dependency on NSD2 isoforms and its methyltransferase activity, we have reconstituted NSD2 in both NSD2 dTAG and NSD2 KO KMS-11 cells, including WT NSD2I WT, NSD2II WT, NSD2 Y1179A (LOF), NSD2 E1099K (GOF). To maximally match the timing of NSD2 perturbation, NSD2 reconstitution was generated in NSD2 dTAG and NSD2 KO cells in a Tet-ON and Tet-OFF expression system, respectively. Further we have generated KMS-11 cells overexpressing Dox-inducible H3.3K36M to investigate the consequences of abrogation of H3K36me2 by this dominant negative mutant of H3.3. We have done SLAM-seq to assess the transcriptional targets of NSD2 and will extend this with ATAC-seq and CUT&amp;Tag to identify the molecular targets of NSD2. RESULTS: We show that NSD2 is rapidly degraded within 1 h when a degrader is added and subsequently a significant reduction of H3K36me2 is seen by 24 hours although it takes more than 3 days to see its deep depletion. We observed a reciprocal increase of H3K27me3 as previously noted. Depletion of H3K36me2 and increase in H3K27me3 was also seen in the NSD2 KO KMS-11 cells. Paradoxically we didn't see an induction of apoptosis in response to the degradation of NSD2 or its KO. In sharp contrast to the previously published shRNA results only a mild inhibition of cell proliferation was observed in NSD2 KO cells and over an extended period of 3 weeks in the dTAG system. We didn't observe an effect of H3.3 K36M overexpression on cell proliferation although it abolished H3K36me2 very efficiently. These experiments demonstrate that NSD2 is not essential for viability of KMS-11 cells but is required for optimal cell growth, which is likely independent of its H3K36me2 methyltransferase activity. SLAMseq results show that NSD2 degradation in the dTAG system causes transcriptional changes of a specific set of newly synthesized mRNAs. ATAC-seq and CUT&amp;Tag experiments are being run and will be superimposed on the SLAM-seq data to identify immediate targets of NSD2. Subsequently, use of PRC2 inhibitors and DNMT3A KO cells in the dTAG cells and the NSD2 reconstitution experiments will uncover the mechanisms of how NSD2 controls the transcription of its targets. CONCLUSION: We show that NSD2 is not essential for viability of KMS-11 cells but is required for optimal cell growth and that NSD2 controls the transcription of a specific set of genes and does not exert a global impact on the transcriptome. We will further define the immediate targets of NSD2 by leveraging our SLAM-seq, ATAC-seq, and CUT&amp;Tag data in NSD2 dTAG/KO and NSD2-reconstituted cells and by blocking the crosstalk with other epigenetic marks (i.e. H3K27me3 and DNAme).

CTCF Is a Haploinsufficient Tumor Suppressor in B-Cell Lymphoma

Alteration of epigenetic regulators is a hallmark of diffuse large B cell lymphoma (DLBCL), the most common type of non-Hodgkin B cell malignancy. It has been recently shown that haploinsufficiency of the cohesin complex ATPase subunit Smc3 cooperates with the oncogene IμBcl6 to induce lymphoma in mice, by a mechanism that involves decreased intra-topological associating domain (TAD) chromosomal interactions and downregulation of tumor suppressor genes. The transcription factor CTCF is an obligate partner of the cohesin complex in defining TADs and regulatory gene loops, acting as the boundaries of TADs and restricting the interaction of promoters and enhancers. Alterations in CTCF have been shown in several cancer types, including lymphomas. In this study, we aim to define the role of CTCF during B-cell lymphoma and lymphomagenesis. We generated germinal center (GC) B cell-specific conditional knockout mice for CTCF by crossing the CTCF floxed mice to the Ighg1-Cre (Cγ1-Cre) allele. In these experiments, we compared haploinsufficient CTCF conditional knockout mice-in which only one allele of CTCF was knocked out-versus wild-type mice expressing the Cγ1-Cre. Eight days after immunization with the T-cell-dependent antigen sheep red blood cells, we excised spleens and surveyed B cell populations by flow cytometry and immunohistochemistry. Remarkably, CTCF haploinsufficient mice contained a similar percentage of follicular GC B cells compared to wild-type mice (Live B220 +CD95 +GL7 + or live B220 +CD95 +CD38 DIM splenocytes). However, CTCF haploinsufficient GCs displayed a bias in the GC subpopulations, namely an increased percentage of light zone centrocytes (LZ: GC + markers CXCR4 loCD86 hi) and decreased dark zone centroblasts (DZ: GC + markers CXCR4 hiCD86 lo). Because biases in GCs were an early indication of malignant transformation in previous studies, we wondered whether CTCF haploinsufficiency would cooperate with lymphoma oncogenes to induce B-cell lymphoma. To address the lymphomagenic potential of CTCF haploinsufficiency, we crossed CTCF conditional knockout mice to the vavP-Bcl2 mice that develop GC-derived follicular-like lymphoma with long latency. We harvested bone marrow blood cell precursors from Cγ1-Cre (“WT”), Cγ1-Cre;CTCF +/- (“CTCF”), vavP-BCL2 +;Cγ1-Cre (“BCL2”), and vavP-BCL2 +;Cγ1-Cre;CTCF +/- (“BCL2/CTCF”) and transplanted them into lethally irradiated C57BL6/J (30 per group). Mice that survived more than two weeks after irradiation and bone marrow transplant were considered stably engrafted and monitored for any signs of disease. Lethargic mice were sacrificed and macroscopically inspected for the presence of tumors. Survival analysis by Kaplan-Meier showed that BCL2/CTCF mice had the shortest survival from all four groups, having a median survival of 230 days, while survival for the BCL2 alone group did not fall below 50% up to day 370. Immunohistochemistry against B220 + was performed in tissue sampling from dying mice to confirm the cause of death. Curiously, CTCF haploinsufficiency accelerated BCL2-induced lymphomagenesis but did not affect the survival of IμBCL6-induced lymphoma. To explore the mechanistic basis of CTCF haploinsufficiency-induced lymphoma in mice, we FACS sorted DZ and LZ GC B-cells from 8-day immunized mice and explored global transcriptomics by RNA-sequencing. While the transition from DZ to LZ involves the regulation of around ~1,500 genes, we failed to detect changes in gene expression between wild-type and CTCF haploinsufficiency cells. ATAC-seq assay on the same cells showed little to no change in chromatin accessibility between cells from CTCF haploinsufficient and wild-type mice. TOBIAS footprinting analysis, which identifies biases in Tn5 transposase activity due to protein binding, showed that CTCF haploinsufficient cells maintained a deep depletion in CTCF binding. Hi-C chromosomal conformation analysis identified an overlapping set of gene regulatory loops that lose interactions and showed decreased signal by gene set enrichment analysis. In this study, we characterized the role of CTCF haploinsufficiency phenotypically and are currently exploring the molecular mechanisms that contribute to the acceleration of the lymphomagenesis induced by the BCL2 oncogene.

Local Electron Density Depletions in the Martian Upper Ionosphere Obtained From MARSIS: Comparison With ASPERA‐3, and MAVEN, and the Connection With Crustal Magnetic Fields

AbstractSteep electron density depletions in the Martian ionosphere are important, especially because they could be regions of significant plasma escape. Local electron density profiles of the Martian ionosphere obtained from the radar sounder onboard the Mars Express spacecraft often show large fluctuations. In some cases, these fluctuations are observed as deep depletions in the electron density. Investigation over the past 12 years of Mars Advanced Radar for Subsurface and Ionospheric Sounding data have revealed over 200 cases of density depletions where the lowest density is less than one fifth of the density in the surrounding regions. These density dips occur both on the dayside and the nightside, with an abundance between solar zenith angles of 50–70° and 100–120°. They occur at all altitudes in the ionosphere. Many of the depletions occur in the southern hemisphere where the crustal magnetic field is stronger and there are more regions where the crustal magnetic field is mostly vertical. In more than half of the cases, the depletion in the local electron density corresponds to a decrease in the electron flux measured with the Analyzer of Space Plasmas and Energetic Atoms electron spectrometer. Several different mechanisms could be responsible for the formation of the depletions, including regions of localized electric fields or magnetic fields creating areas of increased field pressure, escape of the plasma along magnetic field lines depleting density locally, and the density decrease around the photoelectron boundary followed by an increase in the plasma density due to plasma clouds.

Enhancement of Transient Current in MIS(p) Tunneling Diode with Reduced Reversed Bias Current by Oxide Removal at the Gate Edge

Transient behavior plays a critical role in electronic devices since it represents the potential to serve as memory applications. Recently, some transient current behavior of metal-insulator-semiconductor tunnel diode (MISTD) has been demonstrated with unconventional structures [1], [2]. However, due to the usage of multiple photomasks and the process complexity at the gate edge, these devices may have scale-down issues in advanced CMOS-compatible processes. In this work, a structure of edge-removed (ER) MISTD has been proposed. Compared to previous work [2], which removes surrounding oxide and Si substrate by trench forming, the ER MISTD improves the device performance by not damaging the Si substrate. The ER MISTD can remain the same transient current window (CW) while being operated at smaller write voltage and device area. With the advantages of a smaller device area, energy-efficient operations, and the simplicity of the fabrication process, the ER MISTD is promising to serve as dynamic memory applications.The schematics of the conventional co-planar and ER MISTD are shown in Fig. 1. With the removal of the surrounding gate oxide, ER MISTD demonstrates not only lower reverse bias current but also hysteresis displacement current in Fig. 2(a) compared to planar MISTD. The leakage current of ER MISTD is 3 orders of magnitude smaller than that of planar MISTD at VG = 2V. Fig. 2(b) shows the high-frequency capacitance-voltage measurement, which demonstrates enlarged deep depletion region of the ER MISTD. We further investigated the phenomenon by Sentaurus TCAD as shown in Fig. 3. The existence of oxide charges was also adopted in simulation. The deeper depletion phenomenon of ER MISTD can be attributed to the lack of minority carriers and oxide charges without the existence of surrounding gate oxide. Another evidence of the lack of minority carriers is shown in Fig. 4. The saturation current of ER MISTD remains lower while that of planar MISTD increases and eventually induces oxide breakdown around VG = 5V. It is also found that ER MISTD isn't in oxide breakdown when VG is over 40 V as shown in the inset of Fig. 4.Next, the enhanced two-state transient current behavior has been demonstrated in Fig. 5. The specific measurement steps are shown in the inset of Fig. 5(a) and 5(b). ER MISTD has better CW in both operations compared to planar MISTD. We further investigated the mechanism of the two operations.When VG is switched from 1V to 0V, the minority carriers (𝑄𝑖𝑛𝑣) would become excess carriers since no enough time for them to be recombined. Because ER MISTD has an insufficient supply of minority carriers due to the absence of gate edge fringing field, the electron tunnel current generated by excess carriers is smaller compared to planar MISTD. With a smaller electron tunnel current to compensate for the transient discharging current, the total transient read current of ER MISTD is larger than that of planar MISTD.When VG is switched from -0.5V to 0V, the depletion region is suddenly expanded and therefore generates extra displacement current. Since the change of depletion width of ER MISTD is larger, the positive displacement current is larger as well. Because the transient current is the summation of the positive discharging current and displacement current, the transient current of ER MISTD is larger.The endurance measurement of ER MISTD has been examined as shown in Fig. 6. Fig. 6(a) shows the endurance can be tested up to 5000 cycles between 1V and 0V without much degradation. Fig. 6(b) shows the endurance between 1V and -0.5V with 102 cycles.We further investigated the CW of two operations corresponding to oxide thickness. As shown in Fig. 7, when oxide becomes thicker, the transient behavior becomes larger due to the decrease of the tunneling probability. However, the CW would eventually decay when the oxide is too thick. This is because tunnel current will disappear at thicker oxide, the transient behavior is therefore reduced. The peak value of the CW is around EOT = 3.1 nm.In conclusion, ER MISTD shows enhanced transient current and reduced leakage current due to the enlarged deep depletion. We also proposed two-state memory operations and endurance measurement of ER MISTD. The correspondence of CW and oxide thickness has been investigated as well. Because of the above characteristics, ER MISTD shows its potential to serve as a dynamic memory application.Reference:[1] Huang S -W and Hwu J -G 2021 IEEE Trans. Electron Devices 68 6580-6585[2] Lin J -Y and Hwu J -G 2021 IEEE Trans. on Electron Devices 68 4189-4194 Figure 1

Influence of Outer Oxide Charges on the Electrostatics of Metal-Insulator-Semiconductor Tunnel Diode

Metal-Insulator-Semiconductor Tunnel Diode (MISTD) has an ultra-thin oxide with thickness less than 3.5 nm. When a MISTD is biased higher than a critical voltage V C, the device will be driven into the deep depletion region [1]. In this biasing condition, the electrostatics of the device will deviate from the behavior of traditional metal-oxide-semiconductor capacitors with thick oxide [1]. Thus, finding out the critical voltage V C is important to predict the electrostatics of a MISTD. In this work, we found that there is a systematic deviation of V C between the theoretical and the experimental values. A model considering the oxide-charge-induced lateral coupling (OCILC) is suggested as a possible mechanism leading to the deviation. With adding effective positive oxide charges of Q eff/q = 2.4-2.6×1011 cm-2, the calculated V C can fit well with the experimental observations.Figs. 1(a) and (b) are the band diagrams of MISTD biased at V TD < V C and V TD > V C, respectively, where V TD is the positive voltage applied on the metal. When V TD < V C, the generated electrons flow -J gen,bulk are able to follow the increase in the inversion charges and leakage current -J tunnel. In this situation, most of the applied V TD drops on the oxide, and the surface band bending of the silicon ψ s keeps around its initial value ψ 0, as shown in Fig. 1(a). In contrast, at V TD > V C, the generated electrons in original depletion region cannot follow the increase of the tunneling current. To keep the balancing of -J tunnel = -J gen,bulk, a large portion of V TD drops on the silicon and leads to an intense increase in ψ s, as shown in Fig. 1(b). The MISTD will operate under a significant non-equilibrium region under this bias condition. The balancing of -J tunnel = -J gen,bulk is finely discussed in [1].Ten MISTDs with distinct oxide thicknesses ranging from 23.7 to 34.4 Å were fabricated to extract V C. The device structure is shown in Fig. 2(a). The shape of the device is a circle with a diameter of 170 µm. From the measured high-frequency capacitance-voltage (HFCV) relations shown in Fig. 2(a), one can observe a flat region before a critical voltage and a quick decrease after the critical voltage. One can further extract surface band bending ψ s by HFCV [2]. The extracted q∆ψ/kT = q(ψ s-ψ 0)/kT is plotted in Fig. 2(b), where ∆ψ is the difference between ψ s and ψ 0. As the definition in [1], we extract V C at q∆ψ/kT = 3. The extract V C v.s. t ox is shown in Fig. 3 as solid symbols. At the same time, theoretical V C from the modeling proposed in [1] is also attached as a solid line. From Fig. 3, we observed a significant difference in V C between the modeling and the experimental.It is suggested that the OCILC causes the deviation of V C. The mechanism of OCILC is plotted in Fig. 4(a). The positive oxide charges outside the electrode will attract electrons at the silicon surface and form a lateral coupling channel. The potential applied on the electrode will laterally transport through the channel and collect the generated electrons from the lateral region. Finally, the balancing of electrons will become -J tunnel = -J gen,bulk-J gen,lateral, as shown in Fig. 4(b). Because of the extra supplement of electrons (-J gen,lateral), the MISTD can hold the quasi-equilibrium status to a higher voltage, and the V C increase. An improved model considering the effect of OCILC is proposed. The calculated result is plotted in Fig. 5. One can observe V C increase with Q eff. When positive Q eff/q is around 2.4-2.6×1011 cm-2, the calculated V C is close to the V C extract from the experimental.In conclusion, we suggested that the oxide charges outside the electrode play an important role in the electrostatics of MISTD. The mechanism of OCILC is proposed to explain the impact. A model considering OCILC is developed to calculate V C and the results fit well with the experimental results.This work was supported by the Ministry of Science and Technology of Taiwan, ROC, under Contract No. MOST 111-2221-E-192-MY3 and the National Science and Technology Council of Taiwan, ROC, under Contract No. NSTC 111-2622-8-002-001.[1] K. W. Lin, K. C. Chen and J. G. Hwu, An Analytical Model for the Electrostatics of Reverse-Biased Al/SiO₂/Si(p) MOS Capacitors With Tunneling Oxide, IEEE Trans. Electron Devices, 69, 1972, (2022)[2] E. H. Nicollian and J. R. Brews, MOS (Metal Oxide Semiconductor) Physics and Technology (Wiley, Hoboken) (1982) Figure 1

CLN-978, a novel half-life extended CD19/CD3/HSA-specific T cell-engaging antibody construct with potent activity against B-cell malignancies with low CD19 expression

BackgroundDespite significant progress in the development of T cell-engaging therapies for various B-cell malignancies, a high medical need remains for the refractory disease setting, often characterized by suboptimal target levels.MethodsTo...

Machine learning approaches to equilibrium burnup analysis for Molten Salt Reactor

During the operation of a liquid-fueled molten salt reactor (MSR), the deep depletion to an equilibrium state can be attained with online refueling and reprocessing techniques. However, the equilibrium burnup simulation of an MSR using current depletion codes requires considerable computational cost and time, and even fails when the equilibrium conditions are unsatisfied. To efficiently evaluate the specific characteristics of the equilibrium burnup in MSRs, a fast-filtering model for equilibrium state and a fast prediction model for equilibrium neutronic properties are developed based on the machine learning (ML) technique. A database of 4860 cases including the neutronic information of equilibrium and non-equilibrium of a fuel assembly with variable geometric parameters in an MSR is first generated, and then the neutronic properties of 232Th-233U, 232Th-EU (enriched uranium) and 232Th-Pu fuel cycles are evaluated. Based on the MSR database, fifteen different ML classification algorithms are implemented to establish the fast-filtering model for equilibrium burnup state. Moreover, a model for the fast prediction of equilibrium neutronic parameters of neutron flux (Φ) and breeding ratio (BR) is developed with fifteen ML regression algorithms. Considering the various performance metrics for measuring the predicted performances of ML models in the classification and regression, the LightGBM (LGBM) model is the most favorable for filtering the burnup state and predicting the neutronic parameters in MSRs. With the test data, the LGBM model can obtain the accuracy of about 0.98 for classification and the relative error of about 1.10% for regression.

Investigating Coronal Holes and CMEs as Sources of Brightness Depletion Detected in PSP/WISPR Images

The Parker Solar Probe (PSP) mission provides a unique opportunity to observe the solar corona from distances below 20 R ☉. In this work, we utilize white light images from the Wide-field Imager for Solar PRobe aboard the PSP from solar encounters 10 through 13 to examine the causes of brightness depletions of the corona during the rapid transit of PSP through the perihelia of its orbit. We analyze the effect of (1) coronal holes (CHs) and (2) energetic coronal mass ejection (CME) events on the observed brightness of the images. We speculate on the causes of the brightness depletions, ascribing them to the evacuation of (1) free electrons (reduced K-corona) and (2) interplanetary dust (reduced F-corona). In particular, we show that (1) the presence of CHs in all of the orbits is directly correlated with the depletion of the global white light emission recorded, and (2) a huge CME event in encounter 13 caused a very deep depletion in its wake that removed the electron content as well as some of the interplanetary dust.