Chimeric antigen receptor T (CAR-T) cell therapy targeting T cell tumors still faces many challenges, one of which is its fratricide due to the target gene expressed on CAR-T cells. Despite this, these CAR-T cells can be expanded in vitro by extending the culture time and effectively eliminating malignant T cells. However, the mechanisms underlying CAR-T cell survival in cell subpopulations, the molecules involved, and their regulation are still unknown. We performed single-cell transcriptome profiling to investigate the fratricidal CAR-T products (CD26 CAR-Ts and CD44v6 CAR-Ts) targeting T cells, taking CD19 CAR-Ts targeting B cells from the same donor as a control. Compared with CD19 CAR-Ts, fratricidal CAR-T cells exhibit no unique cell subpopulation, but have more exhausted T cells, fewer cytotoxic T cells, and more T cell receptor (TCR) clonal amplification. Furthermore, we observed that fratricidal CAR-T cell survival was accompanied by target gene expression. Gene expression results suggest that fratricidal CAR-T cells may downregulate their human leukocyte antigen (HLA) molecules to evade T cell recognition. Single-cell regulatory network analysis and suppression experiments revealed that exhaustion mediated by critical regulatory factors may contribute to fratricidal CAR-T cell survival. Together, these data provide valuable and first-time insights into the survival of fratricidal CAR-T cells.
© 2024 The Authors.

Senescent cells can spread the senescent phenotype to other cells by secreting senescence-associated secretory phenotype factors. The resulting paracrine senescent cells make a significant contribution to the burden of senescent cell accumulation with age. Previous efforts made to characterize paracrine senescence are unreliable due to analyses being based on mixed populations of senescent and non-senescent cells. Here, we use dipeptidyl peptidase-4 (DPP4) as a surface maker to isolate senescent cells from mixed populations. Using this technique, we enrich the percentage of paracrine senescence from 40% to 85%. We then use this enriched culture to characterize DPP4+ primary and paracrine senescent cells. We observe ferroptosis dysregulation and ferrous iron accumulation as a common phenomenon in both primary and paracrine senescent cells. Finally, we identify ferroptosis induction and ferrous iron-activatable prodrug as a broad-spectrum senolytic approach to ablate multiple types of primary and paracrine senescent cells.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Circulating large "preplatelets" undergo fission via barbell platelet intermediates into two smaller, mature platelets. In this study, we determine whether preplatelets and/or barbells are equivalent to reticulated/immature platelets by using ImageStream flow cytometry and super-resolution microscopy. Immature platelets, preplatelets, and barbells were quantified in healthy and thrombocytopenic mice, healthy human volunteers, and patients with immune thrombocytopenia or undergoing chemotherapy. Preplatelets and barbells were 1.9% ± 0.18%/1.7% ± 0.48% (n = 6) and 3.3% ± 1.6%/0.5% ± 0.27% (n = 12) of total platelet counts in murine and human whole blood, respectively. Both preplatelets and barbells exhibited high expression of major histocompatibility complex class I with high thiazole orange and Mitotracker fluorescence. Tracking dye experiments confirmed that preplatelets transform into barbells and undergo fission ex vivo to increase platelet counts, with dependence on the cytoskeleton and normal mitochondrial respiration. Samples from antibody-induced thrombocytopenia in mice and patients with immune thrombocytopenia had increased levels of both preplatelets and barbells correlating with immature platelet levels. Furthermore, barbells were absent after chemotherapy in patients. In mice, in vivo biotinylation confirmed that barbells, but not all large platelets, were immature. This study demonstrates that a subpopulation of large platelets are immature preplatelets that can transform into barbells and undergo fission during maturation.
© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.

Human fetal progenitor tenocytes (hFPT) produced in defined cell bank systems have recently been characterized and qualified as potential therapeutic cell sources in tendon regenerative medicine. In view of further developing the manufacture processes of such cell-based active pharmaceutical ingredients (API), the effects of hypoxic in vitro culture expansion on key cellular characteristics or process parameters were evaluated. To this end, multiple aspects were comparatively assessed in normoxic incubation (i.e., 5% CO2 and 21% O2, standard conditions) or in hypoxic incubation (i.e., 5% CO2 and 2% O2, optimized conditions). Experimentally investigated parameters and endpoints included cellular proliferation, cellular morphology and size distribution, cell surface marker panels, cell susceptibility toward adipogenic and osteogenic induction, while relative protein expression levels were analyzed by quantitative mass spectrometry. The results outlined conserved critical cellular characteristics (i.e., cell surface marker panels, cellular phenotype under chemical induction) and modified key cellular parameters (i.e., cell size distribution, endpoint cell yields, matrix protein contents) potentially procuring tangible benefits for next-generation cell manufacturing workflows. Specific proteomic analyses further shed some light on the cellular effects of hypoxia, potentially orienting further hFPT processing for cell-based, cell-free API manufacture. Overall, this study indicated that hypoxic incubation impacts specific hFPT key properties while preserving critical quality attributes (i.e., as compared to normoxic incubation), enabling efficient manufacture of tenocyte-based APIs for homologous standardized transplant products.

Coronavirus induced disease 2019 (COVID-19) can be complicated by severe organ damage leading to dysfunction of the lungs and other organs. The processes that trigger organ damage in COVID-19 are incompletely understood.
Samples were donated from hospitalized patients. Sera, plasma, and autopsy-derived tissue sections were examined employing flow cytometry, enzyme-linked immunosorbent assays, and immunohistochemistry.
Here, we show that severe COVID-19 is characterized by a highly pronounced formation of neutrophil extracellular traps (NETs) inside the micro-vessels. Intravascular aggregation of NETs leads to rapid occlusion of the affected vessels, disturbed microcirculation, and organ damage. In severe COVID-19, neutrophil granulocytes are strongly activated and adopt a so-called low-density phenotype, prone to spontaneously form NETs. In accordance, markers indicating NET turnover are consistently increased in COVID-19 and linked to disease severity. Histopathology of the lungs and other organs from COVID-19 patients showed congestions of numerous micro-vessels by aggregated NETs associated with endothelial damage.
These data suggest that organ dysfunction in severe COVID-19 is associated with excessive NET formation and vascular damage.
Deutsche Forschungsgemeinschaft (DFG), EU, Volkswagen-Stiftung.
Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.