The Pivotal Role of Immune Functional Assays in Deciphering Immune Function Alterations

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1 Abstract Recently, immune function assessment has gained prominence in clinical settings. Immune functional assays (IFAs), involving in vitro stimulation, offer a relevant approach to complement traditional immunomonitoring methods which, while widely used, do not fully capture functional immune capabilities. Despite growing interest in IFAs, their added value remains unclear. To address this gap, our study aimed to determine if insights from IFAs could be replicated with unstimulated immunoprofiling. Using the same analytical pipeline, we compared transcriptomic profiles (Nanostring®) between stimulated (TruCulture®) and unstimulated (PaxGene™) samples from i) patients with an overstimulated immune system 3-4 days post-sepsis onset, and ii) patients undergoing immune reconstitution 6-months post-allogeneic hematopoietic stem cell transplantation (allo-HSCT). In sepsis, post-stimulation transcriptomic profiles revealed immune clusters linked to disease severity and outcomes, surpassing traditional markers like mHLA-DR, while baseline analyses failed to generate clinically relevant stratification. Similarly, allo-HSCT patients’ post-stimulation data revealed immune heterogeneity and treatment-related alterations not detected using baseline transcriptomic or cellular profiles alone. Our findings emphasize the value of IFAs in uncovering functional immune alterations that unstimulated assessments may miss, which could offer deeper insights into immune dysfunction. This study supports IFAs as complementary tools to current clinical practices, enhancing patient management with a functional view of immune system dynamics.

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