Tracking immune dysregulation in COVID-19: lymphocyte dynamics from hospitalization to recovery

A hallmark of COVID-19 patients is the reduction of the lymphocyte population accompanied by activation, senescence, and exhaustion markers. We investigated patients admitted to hospital wards who either recovered after a short hospitalization or progressed to critical illness. Patients (n=48) were recruited between May and September 2020; 19 healthy volunteers were enrolled as controls. Blood samples were collected on days 0, 3, and 7 of hospitalization and around 30 days after discharge (convalescence sample, CS30). Lymphocyte counts and extended immunophenotyping were performed by flow cytometry and analyzed using conventional and stochastic methods. At D0 and D7, total lymphocytes, natural killer cells, T cells, TCD4 cells, and TCD8 cells were lower in patients than in volunteers but were restored at CS30. The stochastic analysis identified 11 distinct clusters of lymphocytes, nine of them with significant differences between patients and healthy controls. Clusters of TCD8+ memory cells showing activation, senescence, and exhaustion were increased in patients during hospitalization and in the convalescence samples. In contrast, clusters 5 (TCD4+ Central Memory exhausted activated) and 7 (TCD4+ Central Memory exhausted) were decreased in patients during the disease compared to healthy controls. Overall, the conventional flow cytometry analyses corroborated the findings from the stochastic analysis, showing that effector memory (EM) and TEMRA subsets exhibited sustained markers of exhaustion and senescence, particularly in TCD8+ cells. Our findings reinforce lymphopenia, T cell activation, senescence, and exhaustion as essential immunological features of COVID-19; while cell counts fully recovered, lymphocytes remained dysfunctional in convalescent samples.