Imbalanced immune cell network and suboptimal cell activation: signatures associated with disease severity in vaccine-naïve COVID-19 patients

Background COVID-19 vaccination has significantly reduced mortality and morbidity. Recent studies in unvaccinated people indicate a more complex immune response beyond just the cytokine storm. Understanding changes in the immune cell network is crucial for identifying vaccine-independent immune imbalances, especially in vaccine-naïve patients needing invasive mechanical ventilation (IMV). This knowledge could help improve vaccine development and find biomarkers linked to severe COVID-19. Methods Peripheral blood immune cells from vaccine-naïve COVID-19 patients from the first pandemic wave were classified into those who required IMV and those who did not (No-IMV). High-dimensional immune phenotyping was performed using multiparametric flow cytometry combined with FlowSOM clustering and UMAP for dimensionality reduction. Additionally, T-cell activation efficiency after polyclonal stimulation was evaluated in vitro . Results IMV patients, but not No-IMV, exhibited a marked disruption of immune cell networks, characterized by a loss of immune checkpoint (IC)-expressing T-cell subsets, particularly PD-1- and LAG-3-expressing T cells. Conversely, there was an increase in the frequency of T cells co-expressing molecules linked to inflammatory pathways (TNF/TNFR) and cell death (CD95L). These changes were also associated with reduced CD8 + T-cell activation capacity and the rise of non-conventional cytotoxic CD4 + T-cell subsets. In the B-cell compartment, IMV patients displayed depletion of CCR7+ subsets and decreased PD-1 expression. Additionally, higher frequencies of NK and NKT cells expressing TNF pathway-related molecules were observed. While classical monocyte subsets expressing ICs such as PD-L1, PD-L2, and TIM-3 remained stable, non-classical monocyte subsets showed altered IC expression. In contrast, No-IMV patients maintained a relatively balanced immune architecture. Conclusions Vaccine-naïve COVID-19 patients requiring IMV display an immune landscape distinct from that of No-IMV patients. IMV exhibits a profound imbalance in innate and adaptive immune cell networks, characterized by inflammatory skewing, loss of regulatory subsets, and impaired cytotoxic T-cell functionality, features not observed in No-IMV. These findings reveal coordinated immune alterations beyond cytokine hyperinflammation and identify cellular immune signatures associated with severe COVID-19.