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

Background Sepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients has generally relied on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision. Methods An ex vivo whole blood enzyme-linked immunosorbent (ELISpot) assay for cellular production of interferon-γ (IFN-γ) was evaluated in 107 septic and 68 non-septic patients from five academic health centers using blood samples collected on days 1, 4 and 7 following ICU admission. Results Compared with 46 healthy subjects, unstimulated and stimulated whole blood IFNγ expression were either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole blood IFNγ expression was significantly reduced on ICU days 1, 4 and 7 (all p<0.05), due to both significant reductions in total number of IFNγ−producing cells and amount of IFNγ produced per cell (all p<0.05). Importantly, IFNγ total expression on day 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6 and procalcitonin. Septic patients with low IFNγ expression were older and had lower ALC and higher sPD-L1 and IL-10 concentrations, consistent with an immune suppressed endotype. Conclusions A whole blood IFNγ ELISpot assay can both identify septic patients at increased risk of late mortality, and identify immune-suppressed, sepsis patients. Trial Registry Because the study is a prospective observational study, and not a clinical trial, registration with clinical trials.gov is not required. ### Competing Interest Statement Evan A, Barrios, M.D. reports institutional support for salary from a National Institute of General Medical Sciences training grant in burns, trauma and sepsis (T32 GM-008721) (PAE). Monty B. Mazer, M.D. is a member of Immune Functional Diagnostics, LLC and receives no direct financial compensation. Immune Functional Diagnostics, LLC is developing predictive metrics in critical illness and this technology is evaluated in this research. Christian B. Bergmann, M.D. is supported by grants from the Deutsche Forschungsgemeinschaft (German Research Foundation) (BE 7016/1-1). Michael D. Goodman, M.D. reports institutional support for salary from the National Institute of General Medical Sciences (R01 GM-124156) and the Department of Defense (G102983-6263608307-1). Valerie Polcz, M.D. reports institutional support for salary from a National Institute of General Medical Sciences training grant in burns, trauma and sepsis (T32 GM-008721) (PAE). Isaiah Turnbull, M.D is a member of Immune Functional Diagnostics, LLC and receives no direct financial compensation. Immune Functional Diagnostics, LLC is developing predictive metrics in critical illness and this technology is evaluated in this research. He is also supported by R35 GM-133756 from the National Institute of General Medical Sciences. Tyler Loftus, M.D. is supported by R01 GM-149657 from the National Institute of General Medical Sciences. Philip A. Efron, M.D. is supported by grants R35 GM-140806, T32 GM-008721 and RM GM-139690 awarded by the National Institute of General Medical Sciences. Kenneth E. Remy, M.D., M.S. is a member of Immune Functional Diagnostics, LLC and receives no direct financial compensation. Immune Functional Diagnostics, LLC is developing predictive metrics in critical illness and this technology is evaluated in this research. Scott Brakenridge, M.D., M.S.C.S. is funded by R35 GM-134880 from the National Institute of General Medical Sciences. Dr. Brakenridge and the University of Florida may receive royalty income based on a technology developed by Dr. Brakenridge and others and licensed by Washington University in St. Louis to IFDx LLC. That technology is evaluated in this research. Vladimir P. Badovinac Ph.D.is funded by R35 GM-134880 from the National Institute of General Medical Sciences. Thomas S. Griffith, Ph.D. is funded by R35 GM-140881 from the National Institute of General Medical Sciences and the recipient of a Research Career Scientist award (IK6BX006192) from the Department of Veteran Affairs. Lyle L. Moldawer, Ph.D. is supported by NIH grants RM1 GM-139690, R01GM-132364 and RF1 NS128626. Dr. Moldawer and the University of Florida may receive royalty income based on a technology developed by Dr. Moldawer and others and licensed by Washington University in St. Louis to IFDx LLC. That technology is evaluated in this research. Richard S. Hotchkiss, M.D. Dr. Hotchkiss and Washington University in St. Louis may receive royalty income based on a technology developed by Dr. Hotchkiss and others and licensed by Washington University in St. Louis to IFDx LLC. That technology is evaluated in this research. He is also supported by R35 GM-126928, awarded by the National Institute of General Medical Sciences. Charles C. Caldwell, Ph.D. Dr. Caldwell and the University of Cincinnati may receive royalty income based on a technology developed by Dr. Caldwell and others and licensed by Washington University in St. Louis to IFDx LLC. That technology is evaluated in this research. No other relationships or activities that could appear to have influenced the submitted work. ### Clinical Protocols ### Funding Statement This work was directly supported by R01 GM-132364, awarded by the National Institute of General Medical Sciences, including a supplement (R01 GM-132364-03S1) in 2022-2024. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Institutional review board (IRB-01) at the University of Florida gave ethical approval for this work (IRB# 202000924, approval 6/5/2020). Under NIH guidelines, the University of Florida IRB served as the sponsoring IRB for this study, and IRBs at the University of Cincinnati and Washington University of St. Louis ceded oversight of the study to the University of Florida. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available by request from the following site: * ALC : absolute lymphocyte count AMA : discharged against medical advice AUROC : area under the receiver-operator curve CINS : critically ill, nonseptic cohort of patients CTSI : UF Clinical and Translational Science Institute ELISpot : enzyme-linked immunospot assay HLA-DR : human leukocyte antigen DR isotype IFNγ : interferon-γ IL-6 : interleukin 6 IL-10 : interleukin 10 IPR : in-patient rehabilitation LTAC : long-term acute care facility mAb : monoclonal antibody PBMC : peripheral blood mononuclear cell SEPSIS : critically ill, sepsis cohort of patients SFU : ELISpot spot forming units SNF : specialized nursing facility SOFA : sequential organ failure assessment score sPD-L1 : soluble programmed death ligand-1 SS : ELISpot spot size TE : ELISpot total expression