The advent of large molecule therapeutics has revolutionized treatment options for previously unmet medical needs. This advent has also led to an increased impact of immunogenicity on drug efficacy and safety. In order to maximize the potential of large molecule therapeutics, immunogenicity-related liabilities must be identified as early in development as possible, using an integrated risk assessment that takes into account the various cell types and processes involved. Here, we describe the development of an ex vivo B-cell immunogenicity assay, to capture a key component of the immune response that has been missing from previously published ex vivo immunogenicity assays. Plasmablasts/plasma cells were preferentially expanded in this assay, a subset of which were drug-specific and presented drug-specific peptides on MHC Class II. This assay represents an important tool in the immunogenicity risk assessment toolkit, to allow liabilities to be identified and mitigated early in the drug development process.
Copyright © 2025 Looney, Ducret, Steiner, Dernick, Hartman, Siegel, Hickling, Odermatt and Marban-Doran.

The complete array of genes required for terminal erythroid differentiation remains unknown. To address this knowledge gap, we perform a genome-scale CRISPR knock-out screen in the human erythroid progenitor cell line HUDEP-2 and validate candidate regulators of erythroid differentiation in a custom secondary screen. Comparison of sgRNA abundance in the CRISPR library, proerythroblasts, and orthochromatic erythroblasts, resulted in the identification of genes that are essential for proerythroblast survival and genes that are required for terminal erythroid differentiation. Among the top genes identified are known regulators of erythropoiesis, underscoring the validity of this screen. Notably, using a Log2 fold change of <-1 and false discovery rate of <0.01, the screen identified 277 genes that are required for terminal erythroid differentiation, including multiple genes not previously nominated through GWAS. NHLRC2, which was previously implicated in hemolytic anemia, was a highly ranked gene. We suggest that anemia due to NHLRC2 mutation results at least in part from a defect in erythroid differentiation. Another highly ranked gene in the screen is VAC14, which we validated for its requirement in erythropoiesis in vitro and in vivo. Thus, data from this CRISPR screen may help classify the underlying mechanisms that contribute to erythroid disorders.
© 2025. The Author(s).

The mechanisms of chemical pleurodesis are still not fully explained. We aimed to evaluate the feasibility of using primary biopsy-derived human mesothelial cells to establish an in vitro culture and to assess the response of pleural mesothelial cells to different sclerosing agents. Talc, povidone-iodine, doxycycline, and TGF-β were used at different doses to stimulate pleural mesothelial cells. After 6 and 24 h, mRNA expression of interleukin (IL)-1β, IL-6, IL-8, TGF-β, MCP-1, IL-17A, and MMP9 was measured in cultured cells, and the protein level of IL-1β, IL-6, and IL-8 was measured in the culture supernatant. The most pronounced response was observed after talc exposure. It was expressed as an increase in IL-1β concentration in culture supernatant after 24 h of higher talc dose stimulation compared to 6 h of stimulation (17.14 pg/ml [11.96-33.32 pg/ml] vs. 1.84 pg/ml [1.81-1.90 pg/ml], p = 0.02). We showed that culture pleural mesothelial cells isolated from pleura biopsy specimens is feasible. Inflammatory responses of mesothelial cells to different sclerosants were highly variable with no consistent pattern of mesothelium reaction neither in terms of different sclerosing agents nor in the time of the most significant reaction. We demonstrated that pro-inflammatory mesothelial response includes an increase in IL-1β mRNA expression and protein production. This may suggest the role of IL-1β in the formation and maintenance of the inflammatory response during pleurodesis.
© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.

Cellular metabolism governs the susceptibility of CD4 T cells to HIV-1 infection. Multiple early post-fusion steps of HIV-1 replication are restricted in resting peripheral blood CD4 T cells; however, molecular mechanisms that underlie metabolic control of these steps remain undefined. Here, we show that mTOR activity following T cell stimulatory signals overcomes metabolic restrictions in these cells by enabling the expansion of dNTPs to fuel HIV-1 reverse transcription (RT), as well as increasing acetyl-CoA to stabilize microtubules that transport RT products. We find that catalytic mTOR inhibition diminishes the expansion of pools of both of these metabolites by limiting glucose and glutamine utilization in several pathways, thereby suppressing HIV-1 infection. We demonstrate how mTOR-coordinated biosyntheses enable the early steps of HIV-1 replication, add metabolic mechanisms by which mTOR inhibitors block HIV-1, and identify some metabolic modules downstream of mTOR as druggable targets for HIV-1 inhibition.
Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.

Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34+ lineage- hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.