McGonagill, P. W. | bioRxiv Science

bioRxiv2024

Development and optimization of a diluted whole blood ELISpot assay to test immune function

BackgroundSepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis ( SPIES) is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. MethodsHeparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFN{gamma} and TNF production as measured by ELISpot. ResultsWe demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24 hours at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4 hours allowing for a substantial decrease in processing time. ConclusionsThe data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients.

immunology↗

bioRxiv2021

Autoimmunity increases susceptibility to and mortality from sepsis

Our prior publication detailing how sepsis influences subsequent development of EAE presented a conceptual advance in understanding the post-sepsis chronic immunoparalysis state (Jensen et al., 2020). However, the reverse scenario (autoimmunity prior to sepsis) defines a high-risk patient population whose susceptibility to sepsis remains poorly defined. Herein, we present a retrospective analysis of University of Iowa Hospital and Clinics patients demonstrating increased sepsis incidence among MS, relative to non-MS, patients. To interrogate how autoimmune disease influences host susceptibility to sepsis well-established murine models of MS and sepsis, EAE and CLP, respectively, were utilized. EAE, relative to non-EAE, mice were highly susceptible to sepsis-induced mortality with elevated cytokine storms. These results were further recapitulated in LPS and S. pneumoniae sepsis models. This work highlights both the relevance of identifying highly susceptible patient populations and expands the growing body of literature that host immune status at the time of septic insult is a potent mortality determinant.

immunology↗