Tsuda, H. | bioRxiv Science

bioRxiv2026

NK Cells Effectively Mediating Antibody-Mediated Kidney Allograft Rejection Requires a Specific Activation Receptor and Graft Expression of the Ligand

Acute antibody-mediated rejection (aABMR) is an important cause of clinical kidney graft injury and failure. Transcripts associated with NK cell activation in graft biopsies are diagnostic of aABMR, but mechanisms underlying NK cell activation during ABMR remain poorly understood. In contrast to the long-term (> 60 days) survival of complete MHC-mismatched kidney allografts in wild type C57BL/6 mice, B6.CCR5-/- recipients develop high titers of donor-specific antibody (DSA) with allograft rejection between days 18 to 28 post-transplant. This has allowed investigation of mechanisms underlying NK cell activation within kidney allografts during aABMR. DSA titers first became detectable in B6.CCR5-/- (H-2b) recipients of A/J (H-2a) kidney allografts at day 8 and peaked on day 15 post-transplant and was accompanied by a parallel increase in mRNA levels of Rae-1e, a ligand for the NK cell activation receptor NKG2D. A/J kidneys in B6.CCR5-/-NKG2D-/- recipients and A/J.Rae-1e-/- kidneys in B6.CCR5-/- recipients survived >60 days, despite high serum DSA levels. Flow cytometric analysis of allograft infiltrating cells in B6.CCR5-/- recipients on day 15 post-transplant revealed inflammatory monocyte and NK cell infiltration and NK cell activation to proliferate and express CD107a, a marker of cytotoxic function. These features of aABMR were absent or markedly reduced by recipient NKG2D- or donor graft Rae-1e-deficiency. These findings suggest that interference with expression of allograft Rae-1e or recipient NK cell NKG2D abrogates aABMR despite persistently high DSA levels and that aABMR requires coordination between infiltrating NK cell and inflammatory monocyte activation within the kidney allograft.

immunology↗

bioRxiv2025

Developing a Novel In Vitro Toxicity Assay for Predicting Inhalation Toxicity in Rats

The development of alternative in vitro methods for assessing acute inhalation toxicity is a critical step toward reducing animal testing and aligns with the principles of the 3Rs (replacement, reduction, and refinement). In this study, we developed and optimized a neutral red uptake (NRU) assay using human lung adenocarcinoma cells (A549) as a predictive model (A549-NRU) for acute inhalation toxicity. To improve assay efficiency and robustness, we introduced two key modifications: the incubation time was reduced to 15 minutes to enable rapid and high-throughput screening, and for chemicals reactive with polystyrene 6-well glass plates were used to prevent chemical-induced degradation and ensure assay consistency. LC50 values were determined for 49 chemicals and compared with reported LC50 values from 4-hour rat inhalation studies. A significant positive correlation was observed between A549-NRU-derived LC50 values and in vivo LC50 values for water-soluble compounds and chemicals containing aldehyde, ketone, alcohol, ether, and epoxide functional groups, suggesting that in vivo LC50 values may be predictable using the A549-NRU assay. Additionally, A549-NRU LC50 values showed significant negative correlations with molecular weight and octanol-water partition coefficients, indicating that chemicals with higher values tended to be less cytotoxic in vitro. Importantly, the A549-NRU assay demonstrated stronger correlation with in vivo LC50 values than the conventional NRU assay using mouse 3T3 fibroblast cells. These findings support the use of the A549-NRU assay to estimate starting doses for in vivo studies, and potentially as an in vitro alternative for predicting acute inhalation toxicity. Impact statementThe optimized A549-NRU assay demonstrates predictive potential for inhalation toxicity while reducing reliance on animal testing. This model serves as a human-relevant alternative for estimating starting doses for in vivo inhalation toxicity studies.

pharmacology and toxicology↗