488 - Development and validation of an ELIspot assay to qualitatively assess adaptive immune function in murine models of sepsis

Publication Number: 488.4536

Mahil Rao, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Coralville, IA, United States; Elvia E.. Silva, Sandia National Laboratories, Livermore, CA, United States; Mohammad Heidarian, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, United States; Thomas S. Griffith, University of Minnesota Medical School, Minneapolis, MN, United States; Vladimir Badovinac, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, United States

Background: Sepsis remains a leading cause of morbidity and mortality worldwide. A significant number of early-phase survivors develop prolonged immune dysregulation. The development of effective immunomodulatory therapies has been hampered by the inability to assess immune function in a clinically-meaningful timeframe.

Objective: Develop a modified enzyme-linked immunospot (ELISpot) assay to rapidly assess adaptive immune function in three different murine sepsis models.

Design/Methods: Peripheral blood and splenocytes were harvested from young C57/BL6 mice (either healthy or following a septic event) housed in specific-pathogen free conditions. Cells were seeded into ELISpot plates in media supplemented with various immunoadjuvants and placed in a tissue culture chamber. At end of the stimulation period, cells were removed and “spots” developed using standard ELISpot techniques. Plates were imaged using an EntryS6 machine from CTL/ImmunoSpot and the number and size of spots in each well was quantitated. In parallel, IFN-γ production was measured in these cells using flow cytometry with intracellular staining.

Results: The number of cells seeded, concentration of anti-CD3/CD28 antibody, and duration of stimulation all demonstrate a sigmoidal relationship with the number of IFN-γ positive spots formed in young immunologically-naïve C57BL/6 mice. The greatest dynamic range is achieved with seeding of 400,000 cells and stimulation for 4 hours. Harvested cells did not show spontaneous production of IFN-γ under any conditions but anti-CD3/anti-CD28 antibodies production of IFN-γ spots. The induction of sepsis did not significantly alter the response to any of the stimulants tested. The patterns of frequency and magnitude of IFN-γ production determined by flow cytometry mirrored that observed using ELIspot. Sources of IFN-γ included naïve CD8 T cells, antigen-experienced CD8+ T cells, CD4 cells, and NK cells.

Conclusion(s): ELISpot can be used to rapidly assess the frequency of IFN-γ cells following ex vivo stimulation with immunoadjuvants. Data obtained using ELIspot mirrors that obtained using conventional flow cytometry. The frequency of IFN-γ producing cells is dependent on the number of cells seeded, the time of stimulation, and the dose of CD3-CD28 antibody used. Induction of sepsis does not significantly alter IFN-γ production at 5 days post event.