High-dimensional single-cell phenotyping unveils persistent differences in immune cell profile between severe and moderate seasonal influenza

Johanna Eva Bodin^1,2,3*Gro Tunheim³Anja B Kristoffersen²Tove K Herstad³Eleonora Vianello⁴Mariëlle C Haks⁴Suzanne Van Veen⁴Torgun Wæhre⁵Anne-Marte Bakken Kran^6,7Sarah L Lartey⁸Fan Zhou⁸Rebecca Cox^8,9*Tom H.M. Ottenhoff⁴Anne Ma Dyrhol-Riise^10,5Unni C Nygaard³Fredrik Oftung³Siri Mjaaland³

• ¹Norwegian Institute of Public Health (NIPH), Oslo, Norway
• ²Department of Method Development and Analytics, Division of Infection Control and Environmental Health, Norwegian Institute of Public Health (NIPH), Oslo, Norway
• ³Section of Immunology, Department of Method Development and Analytics, Norwegian Institute of Public Health, Oslo, Norway., Oslo, Norway
• ⁴Department of Infectious Diseases, Leiden University Medical Center (LUMC), Leiden, Netherlands
• ⁵Department of Infectious Diseases, Oslo University Hospital, Oslo, Nordland, Norway
• ⁶Division of Infection Control, Department of Infectious Disease Registries, Norwegian Institute of Public health, Oslo, Norway., Oslo, Norway
• ⁷Department of Microbiology, Oslo University Hospital, Oslo, Oslo, Norway
• ⁸Influenza Centre, Department of Clinical Science, University of Bergen, Bergen, Norway, Bergen, Norway
• ⁹Department of Microbiology, Haukeland University Hospital, Bergen, Hordaland, Norway
• ¹⁰Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway

Influenza virus with pandemic potential and possible burden of post-viral sequelae is a global concern.To prepare for future pandemics and development of improved vaccines, it is vital to identify the immunological changes underlying influenza disease severity.We combined unsupervised high-dimensional single-cell mass cytometry with gene expression analyses, plasma CXCL13 measurements, and antigen-specific immune cell assays to characterize the immune profiles of hospitalized patients with severe and moderate seasonal influenza disease during active infection and at six months follow-up. Age-matched healthy donors were used as controls.Severe disease was associated with a distinct immune profile, including lower frequencies of ICOS + MAIT cells, CXCR5 + memory B and CD4 + CXCR5 + CD95 + ICOS + and CD8 + CXCR3 + CD95 + PD-1 + TIGIT + memory T cells, as well as lower CD4 gene expression. Higher frequencies of CD16 + CD161 + NK cells, CD169 + monocytes, CD123 +/-dendritic cells, CD38 high plasma cells, and high CXCL13 plasma levels were also associated with severe disease. Alterations in immune cell subpopulations persisted at convalescence for the severely ill patients only.Our results indicated a reduction in regulatory MAIT cells, memory T and B cells and an increase in inhibitory subpopulations of monocytes and NK cells in severe influenza that persisted at convalescence. These immune cell alterations were associated with higher age and presence of several underlying conditions that may contribute to frailty. This study illustrates the power and sensitivity of highdimensional single-cell analyses in identifying potential cellular biomarkers of disease severity after influenza infection.