Dysregulated NK cell gene expression defines the enduring symptoms of long COVID-19

Urvi RayAntje Schulze SeltingRoshan Priyarangana PereraZhiqi YangVladislav LysenkovSiri GöpelMichael BitzerMadhuri S SalkerStephan OssowskiOlaf RiessNicolas CasadeiYogesh Singh^*

• University Hospital and Faculty of Medicine, University of Tübingen, Tübingen, Germany

Objective: Long-Term COVID-19 Syndrome (LTCS) or "long COVID" is a debilitating post-viral condition affecting approximately 2–8% of individuals after SARS-CoV-2 infection. It manifests typically ≥3 months post-infection with symptoms persisting for at least 2 months, including fatigue, pulmonary dysfunction, and cognitive impairment, in the absence of alternative diagnoses. The biological mechanisms underlying LTCS remain poorly defined, yet emerging evidence implicates immune dysregulation. Methods: We profiled plasma antibodies and cytokines from healthy controls (HC, N = 66), convalescents (CONV, N = 24), and LTCS patients (N = 94), followed by multiparametric 14-color flow cytometry of PBMCs from HC (N = 9), CONV (N = 6), and LTCS (N = 23) participants. To gain mechanistic insight, we performed single-cell transcriptomic profiling (scRNA-seq) on PBMCs from HC (N = 8), CONV (N = 6), and LTCS (N = 32) individuals. Results: LTCS patients exhibited elevated anti–SARS-CoV-2 IgG (Spike S1/RBD/N) titres compared to HC, but displayed significantly reduced systemic cytokine levels, including IFN-γ, TNF-α, IL-6, and IL-10. Flow cytometry revealed marked depletion of CD56⁺CD16⁺ NK cells and CD56⁺CD3⁺ NKT cells, accompanied by altered T-cell activation states. scRNA-seq confirmed NK type I cell loss and uncovered broad transcriptional reprogramming with up-regulation of PDCD4, CHD1, CXCR4, and SLC7A5 and down-regulation of TGFBR3, RIPOR2, and MBNL1. Gene-set enrichment analyses indicated activation of circadian and translational programs and suppression of olfactory receptor, neurotransmitter receptor, and GABA-gated ion-channel pathways. Functional assays validated reduced NK-cell inflammatory capacity in LTCS participants. Conclusion: LTCS is characterized by systemic cytokine attenuation and a quantitative and functional NK-cell deficit coupled to neuro-sensory pathway suppression. These findings identify NK cells as key sentinels of LTCS pathophysiology and highlight an NK-centric neuro-immune axis as a promising target for biomarker discovery and therapeutic intervention.