Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome characterized by the dysfunction of cytotoxic T cells (CTLs) and natural killer (NK) cells. The status of these immune cells during persistent inflammation remains unclear. This study aimed to investigate the immune cell subsets, the exhaustion and activation status, and the cytotoxic functions of the CTLs and NK cells in adult HLH. Furthermore, the features of immune cells in patients with Epstein-Barr virus-associated HLH (EBV-HLH) before and after treatment with programmed death-1 (PD-1) inhibitors were also analyzed.
Flow cytometry was utilized to evaluate the immune cell features in 75 patients with adult HLH.
This study observed skewed immune cell subsets, along with substantial exhaustion and activation in patients with adult HLH. Significant decreases were noted in NK, natural killer T (NKT), regulatory T cells (Tregs), CD4+ central memory T (TCM), CD8+ terminal effector memory T (TEMRA), and CD56dimCD16+ NK cells, while increases were found in the CD56brightCD16- and CD56dimCD16- NK subsets. The exhaustion markers (PD-1, TIGIT, and PD-L1) were elevated in CD8+ T cells and NK cells, and the activation markers (CD69, HLA-DR, and CD163) were higher in monocytes. An impaired IFN-γ production and a reduced degranulation function were also detected in CD8+ T cells and NK cells. In EBV-HLH patients treated with PD-1 inhibitors, we observed an increase in cytotoxic CD8+ T cells and CD56dimCD16+ NK cells and a decrease in CD56brightCD16- NK cells.
These findings suggest impairments in cytotoxic immune cells along with a widespread immune exhaustion and an aberrant activation in adult HLH. The results also indicate potential differentiation defects in CD8+ T cells and NK cells. Treatment with a PD-1 inhibitor may contribute to partial functional recovery and support viral clearance in EBV-HLH, which warrant further investigation.
Copyright © 2025 Qin, Zhou, Liu, Dai, Tang, Zhong, Chen, Zhu and Liu.

Propofol is a pharmaceutical agent commonly used as an intravenous anesthetic in surgical treatments and a sedative in intensive care. However, it is largely unknown how exposure to propofol affects the proliferation, invasion, and apoptosis of neoplastic cells in esophageal cancer. In this study, we sought to elucidate the impact of propofol exposure on the growth properties of human esophageal cancer cell lines in vitro. We treated two human esophageal cancer cell lines, KYSE30 and KYSE960, with up to 10 µg/mL of propofol for 12-36 h. The treated cells were then analyzed by cell proliferation assay, Matrigel invasion assay, quantification of caspase-3/7 and -9 activities, and cell staining with Annexin V and 7-aminoactinomycin D to detect early apoptosis and cell death, respectively, via flow cytometry. We found that 3-5 µg/mL propofol reduced the growth and Matrigel invasion of both cell lines in a dose-dependent manner. Executioner caspase-3/7, but not caspase-9 involved in intrinsic apoptosis pathway, was activated by cell exposure to 3-5 µg/mL propofol. In addition, 3-5 µg/mL propofol augmented early apoptosis in both cell lines and increased cell death in the KYSE30 cell line. In summary, exposure to propofol, at concentrations up to 5 µg/mL, led to the reduction of cell growth and Matrigel invasion, as well as the augmentation of apoptosis in esophageal cancer cell lines. These data will help define a methodology to safely utilize propofol, a common general anesthetic and sedative, with esophageal cancer patients.

The regenerative and immunomodulatory properties of multipotent mesenchymal stromal cells (MSCs) make them an intriguing asset for therapeutic applications. An off-the-shelf approach, using pre-expanded cryopreserved allogenic MSCs, bypasses many practical difficulties of cellular therapy. Reconstitution of a MSC product away from cytotoxic cryoprotectants towards a preferred administration solution might be favorable for several indications. Variations in MSC handling accompanied by a non-standardized use of reconstitution solutions complicate a general clinical standardization of MSC cellular therapies. In this study, we aimed to identify a simple and clinically compatible approach for thawing, reconstitution, and post-thaw storage of cryopreserved MSCs.
Human adipose tissue-derived MSCs were expanded in human platelet lysate (hPL) supplemented culture medium and cryopreserved using a dimethyl sulfoxide (DMSO)-based cryoprotectant. Isotonic solutions (saline, Ringer's acetate and phosphate buffered saline (PBS)) with or without 2% human serum albumin (HSA) were used as thawing, reconstitution, and storage solutions. MSCs were reconstituted to 5 × 106 MSCs/mL for evaluating MSC stability. Total MSC numbers and viability were determined using 7-aminoactinomycin D (7-AAD) and flow cytometry.
For thawing cryopreserved MSCs the presence of protein was proven to be essential. Up to 50% of MSCs were lost when protein-free thawing solutions were used. Reconstitution and post-thaw storage of MSCs in culture medium and widely used PBS demonstrated poor MSC stability (>40% cell loss) and viability (<80%) after 1 h of storage at room temperature. Reconstitution in simple isotonic saline appeared to be a good alternative for post-thaw storage, ensuring >90% viability with no observed cell loss for at least 4 h. Reconstitution of MSCs to low concentrations was identified as critical. Diluting MSCs to <105/mL in protein-free vehicles resulted in instant cell loss (>40% cell loss) and lower viability (<80%). Addition of clinical grade HSA could prevent cell loss during thawing and dilution.
This study identified a clinically compatible method for MSC thawing and reconstitution that ensures high MSC yield, viability, and stability. The strength of the method lies within the simplicity of implementation which offers an accessible way to streamline MSC therapies across different laboratories and clinical trials, improving standardization in this field.
© 2023 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.

Macrophage migration inhibitory factor (MIF) has been shown to promote disease progression in many malignancies, including multiple myeloma (MM). We previously reported that MIF regulates MM bone marrow homing and knockdown of MIF favors the extramedullary myeloma formation in mice. Here, based on MIF immunostaining of myeloma cells in paired intramedullary and extramedullary biopsies from 17 patients, we found lower MIF intensity in extramedullary MM (EMM) versus intramedullary MM (IMM). Flow cytometry and histology analysis in xenograft models showed a portion of inoculated human MM cells lost their MIF expression (MIFLow) in vivo. Of note, IMM had dominantly MIFHigh cells, while EMM showed a significantly increased ratio of MIFLow cells. Furthermore, we harvested the extramedullary human MM cells from a mouse and generated single-cell transcriptomic data. The developmental trajectories of MM cells from the MIFHigh to MIFLow state were indicated. The MIFHigh cells featured higher proliferation. The MIFLow ones were more quiescent and harbored abundant ribosomal protein genes. Our findings identified in vivo differential regulation of MIF expression in MM and suggested a potential pathogenic role of MIF in the extramedullary spread of disease.
Copyright © 2021 Xu, Yu, Zhao, Wang, Huang, Cui, Ding, Yang, Gao, Pan, Chang, Wu, Xiang, Gong, Shuai, Hou, Xie, Niu, Liu, Zhang, Liu, Zhang, Qu, Lin, Zhu, Zhao and Zheng.

ABSTRACT Embryonic stem cells (ESC) have the unique ability to differentiate into all three germ cell layers. ESC transition through different states of pluripotency in response to growth factor signals and environmental cues before becoming terminally differentiated. Here, we demonstrated, by a multi-omic strategy, that the deubiquitinase USP9X regulates the developmental potential of ESC, and their transition from a naive to a more developmentally advance, or primed, state of pluripotency. We show that USP9X facilitates developmental gene expression and induces modifications of the mitochondrial bioenergetics, including decreased routing of pyruvate towards its oxidation and reduced respiration. In addition, USP9X binds to the pluripotency factor ESRRB, regulates its abundance and the transcriptional levels of a subset of its target genes. Finally, under permissive culture conditions, depletion of Usp9X accelerates cell differentiation in all cell lineages. We thus identified a new regulator of naive pluripotency and show that USP9X couples ESRRB pluripotency transcriptional network and cellular metabolism, both of which are important for ESC fate and pluripotency.