Decoding multi-compartmental immune pathways in antibody-mediated and dose-stratified murine TRALI via high-dimensional cytometry and targeted proteomics Free

Transfusion-related acute lung injury (TRALI) remains one of the most severe and under-treated complications of blood transfusion. The immunological pathways are complex and not fully resolved, and specific therapies are not available, necessitating the need to better understand the underlying pathophysiology. Although preclinical murine models, particularly those involving major histocompatibility complex (MHC)-I antibodies, have significantly advanced our understanding of antibody-mediated TRALI, most mechanistic insights have generally been focused on isolated immune components or pulmonary pathology. TRALI is a systemic reaction with inflammation and inter-compartmental immune crosstalk playing a critical role, however, the broader immunological landscape across multiple organs remains poorly defined. It is still unclear whether TRALI-antibody mediated immune responses escalate in a linear dose-dependent manner or whether discrete threshold effects drive tissue-specific pathology.

To address this gap, we established a dose-stratified murine model of antibody-mediated TRALI and combined it with high-dimensional spectral cytometry and targeted proteomics. This approach enables immune profiling across lung, blood, and lymphoid tissues, providing a foundation for understanding TRALI as a coordinated systemic response culminating in pulmonary endothelial injury. Male BALB/c mice (n=10/group) were primed with low-dose LPS (0.1 mg/kg) to mimic pre-inflammation, followed 24 hours later by intravenous injection anti–MHC-I (clone 34-1-2S) at 0.5, 1.0, 2.5, or 4.5 mg/kg. Controls received PBS or LPS alone. TRALI severity was assessed by clinical scoring (respiratory distress, cyanosis, pain), body temperature, survival, and lung wet/dry weight ratio as a measure of pulmonary edema. Mice reaching humane endpoints were euthanized; remaining animals were sacrificed 90 minutes post-challenge. Single-cell suspensions from blood, lung, and spleen were analyzed by spectral flow cytometry using three customized multicolor panels: (1) innate inflammatory subsets (CD11b, CD11c, CD3, Ly6G, CD172a, CD69, F4/80, CD8a); (2) complement deposition and regulation (C1q, C3b, C5aR1, C5b-9/MAC, CRRY, CD59) along with endothelial cell identification via CD31; and (3) Fcγ receptor expression (FcγRI–IV) and adaptive immune populations (TCRβ⁺, B220⁺, NK1.1⁺). In parallel, plasma and lung homogenates were analyzed using the Olink® Target 48 Mouse Cytokine panel to quantify a broad range of inflammatory mediators including IL-1β, IL-6, IL-8, TNF, IL-10, IFN-γ, chemokines (CXCL1, CCL2), and immune checkpoint ligands (PD-L1, CTLA-4).

Injection of anti-MHC-I, after LPS priming, induced a clear dose-dependent increase in physiological lung injury, reflecting TRALI. Survival declined from 90% at 0.5 mg/kg to 50% and 40% at 2.5 and 4.5 mg/kg, respectively, while control and LPS-only groups showed 100% survival. Lung wet/dry weight ratios rose from 4.6 ± 0.2 (controls) to 9.8 ± 0.4 (high-dose), and temperature loss correlated with edema severity and mortality (r = 0.78, p < 0.01). Preliminary spectral cytometry revealed dose- and tissue-specific immune shifts, with increased neutrophil and macrophage activation in the lung and a decrease in circulating monocytes. Initial data suggest complement deposition (C1q, C3b, C5b-9) on pulmonary endothelium increases above 1.0 mg/kg MHC-I. Fcγ receptor expression (FcγRI–III) showed dose-dependent shifts, with early indications of increased activating receptor levels in the lung, aligning with enhanced local immune activation. These patterns are under further investigation and will be integrated with ongoing Olink® proteomics to assess dose-dependent changes in soluble mediators linked to TRALI severity.

This study represents, to the best of our knowledge, the first antibody dose-stratified, multi-compartmental map of pathogenic immune responses specific for antibody-mediated TRALI. By integrating high-dimensional cytometry with targeted proteomics, we aim to further dissect and refine these emerging profiles and support the identification of mechanistic pathways in the antibody-mediated TRALI pathogenesis which may exploited for diagnostic or therapeutic purposes.