The pivotal role of immune functional assays in deciphering immune function alterations.

Growing evidence suggests that conventional immunomonitoring alone may not be sufficient to fully capture the complexity of immune dysfunctions. Immune functional assays (IFAs) have therefore emerged as valuable complementary tools, offering functional insights that extend beyond traditional phenotypic or quantitative approaches. Nevertheless, although in vitro stimulation represents a central component of IFAs, its specific contribution has never been rigorously evaluated, raising the critical question of whether this step is truly essential for detecting clinically relevant immune dysfunctions. To address this question, the present study compared gene expression levels (Nanostring) obtained from samples stimulated (TruCulture) or unstimulated (PaxGene) using the same analytical pipeline, in two distinct clinical settings: immune reconstitution following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and sepsis progression. In allo-HSCT patients, post-stimulation data revealed immune heterogeneity and alterations related to ongoing immunosuppressive treatment or infectious event, not detected using unstimulated transcriptomic or cellular profiles alone. Similarly, post-stimulation transcriptomic profiles in patients with sepsis revealed immune clusters linked to disease severity and outcomes, surpassing traditional markers like mHLA-DR, while analyses from the unstimulated datasets failed to generate clinically relevant stratification. These findings emphasize the value of IFAs in uncovering immune function alterations that unstimulated assessments may miss, which could offer deeper insights into immune dysfunction. This study supports the use of IFAs as complementary tools to current clinical practices to enhance patient management by offering a functional view of immune system dynamics.