Adverse Long-Term Outcomes and an Immune Suppressed Endotype in Sepsis Patients with Reduced Interferon-γ ELISpot: A Multicenter, Prospective Observational Study

Barrios, Evan A.; Mazer, Monty B.; McGonagill, Patrick; Bergmann, Christian B.; Goodman, Michael D.; Gould, Robert W.; Rao, Mahil; Polcz, Valerie; Davis, Ruth; Del Toro, Drew; Dirain, Marvin; Dram, Alexandra; Hale, Lucas; Heidarian, Mohammad; Kucaba, Tamara A.; Lanz, Jennifer P.; McCray, Ashley; Meszaros, Sandra; Miles, Sydney; Nelson, Candace; Rocha, Ivanna; Silva, Elvia E; Ungaro, Ricardo; Walton, Andrew; Xu, Julie; Zeumer-Spataro, Leilani; Drewry, Anne M.; Liang, Muxuan; Bible, Letitia E.; Loftus, Tyler; Turnbull, Isaiah; Efron, Philip A.; Remy, Kenneth E.; Brakenridge, Scott; Badovinac, Vladimir P.; Griffith, Thomas S.; Moldawer, Lyle L.; Hotchkiss, Richard S.; Caldwell, Charles C.

doi:10.1101/2023.09.13.23295360

Cited by 4 publications

(1 citation statement)

References 59 publications

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“…Another major advantage of ELISpot is that it can independently assess the functional status of the two major arms of immunity (10,11). For example, the functional state of adaptive immunity can be evaluated by anti-CD3/CD28 mAb stimulated IFN-γ production while innate immunity can be assessed by lipopolysaccharide (LPS) stimulated monocyte production of TNF-α.…”

Section: Introductionmentioning

confidence: 99%

Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot

Unsinger,

Osborne,

Walton

et al. 2023

Preprint

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BackgroundThe inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The ELISpot assay is afunctionalbioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis on whether the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity.MethodsMice were made septic using sublethal cecal ligation and puncture (CLP). Blood and spleens were harvested serially andex vivoIFN-γ and TNF-α production were compared by ELISpot and ELISA. The capability of ELISpot to detect changes in innate and adaptive immunity due toin vivoimmune therapy with dexamethasone, IL-7, and arginine was also evaluated.ResultsELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example dexamethasone, arginine, and IL-7 in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and ELISA results tended to parallel one another although some differences were noted.ConclusionELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow thein vivoeffects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.

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Section: Introductionmentioning

confidence: 99%

Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot

Unsinger,

Osborne,

Walton

et al. 2023

Preprint

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

Ungaro,

Xu,

Kucaba

et al. 2024

Preprint

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Background: Sepsis 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. Methods: Heparinized 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 IFNg and TNFa production as measured by ELISpot. Results: We 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. Conclusions: The 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.

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Determining potential immunomodulatory drug efficacy in sepsis using ELISpot

Walton,

Mazer,

Remy

et al. 2024

Preprint

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PurposeThis study evaluated the ability of ELISpot to identify immuno-modulatory drug therapies for their potential efficacy in patients with sepsis.MethodsELISpot was performed using diluted whole blood from 61 septic patients and 48 healthy matched controls. Innate and adaptive immunity were evaluated byex vivostimulated production of TNF-α and IFN-γ respectively. Potential drug efficacy was determined by the drugs’ effects to increase or decrease the number of cytokine-producing cells and amount of cytokine produced per cell as determined by spot size and intensity. The corticosteroid dexamethasone was evaluated for its ability to down modulate TNF-α and IFN-γ production. The TLR7/8 agonist resiquimod (R848) and T-cell stimulants IL-7 and anti-PD-1 mAb were tested for their ability to enhance immune responses in sepsis.ResultsSpontaneous production of TNF-α and IFN-γ varied among healthy subjects and septic patients. LPS or resiquimod stimulation increased total TNF-α production in septic patients by 1,648% and 1,929% respectively. Conversely, dexamethasone diminished the responses to LPS or resiquimod by 71% and 61% respectively. IL-7, but not anti-PD-1 mAb markedly increased IFN-γ production in both healthy subjects (127%) and septic patients (79%). Dexamethasone also reduced anti-CD3/CD28 mAb stimulated IFN-γ production by 54%; while IL-7 ameliorated dexamethasone-induced suppression. IL-7 significantly enhanced lymphocyte function in over 90% of septic patients.ConclusionELISpot can reveal host immune response patterns and the effects of drugs to selectively down– or up-regulate patient immunity. Furthermore, the ability of ELISpot to detect the effect of specific immuno-modulatory drugs to independently regulate the innate and adaptive host response could enable precision-based immune drug therapies in sepsis.

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Adverse Long-Term Outcomes and an Immune Suppressed Endotype in Sepsis Patients with Reduced Interferon-γ ELISpot: A Multicenter, Prospective Observational Study

Cited by 4 publications

References 59 publications

Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot

Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot

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

Determining potential immunomodulatory drug efficacy in sepsis using ELISpot

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