Clara Sánchez-Menéndez ¹ Olivia De La Calle-Jiménez ¹ Elena Mateos ¹ Lorena Vigón ¹ Daniel Fuertes ² MARIA-ARANZAZU MURCIANO-ANTON ³ Esther San Jose ⁴ Valentin Garcia-Gutierrez ⁵ Miguel Cervero ⁶ Montserrat Torres ¹ Mayte Coiras ^1*

• ¹ Carlos III Health Institute (ISCIII), Madrid, Spain
• ² Escuela Técnica Superior de Ingeniería de Telecomunicación, Universidad Politécnica de Madrid., Madrid, Madrid, Spain
• ³ Centro de Salud Pedro Laín Entralgo, Madrid, Asturias, Spain
• ⁴ European University of Madrid, Villaviciosa de Odón, Madrid, Spain
• ⁵ Ramón y Cajal University Hospital, Madrid, Madrid, Spain
• ⁶ Alfonso X el Sabio University, Villanueva de la Cañada, Madrid, Spain

Introduction: After mild COVID-19 that does not require hospitalization, some individuals develop persistent symptoms that may worsen over time, producing a multisystemic condition termed Post-COVID condition (PCC). Among other disorders, PCC is characterized by persistent changes in the immune system that may not be solved several months after COVID-19 diagnosis. Methods: People with PCC were recruited to determine the distribution and functionality of CD4+ T helper (Th) subsets in comparison with individuals with mild, severe, and critical presentations of acute COVID-19 to evaluate their contribution as risk or protective factors for PCC. Results: People with PCC showed low levels of Th1 cells, similar to individuals with severe and critical COVID-19, although these cells presented a higher capacity to express IFN in response to stimulation. Th2/Th1 correlation was negative in individuals with acute forms of COVID-19, but there was no significant Th2/Th1 correlation in people with PCC. Th2 cells from people with PCC presented high capacity to express IL-4 and IL-13, which are related to low ventilation and death associated with COVID-19. Levels of proinflammatory Th9 and Th17 subsets were significantly higher in people with PCC in comparison with acute COVID-19, being Th1/Th9 correlation negative in these individuals, which probably contributed to a more pro-inflammatory than antiviral scenario. Th17 cells from approximately 50% of individuals with PCC had no capacity to express IL-17A and IL-22, similar to individuals with critical COVID-19, which would prevent clearing extracellular pathogens. Th2/Th17 correlation was positive in people with PCC, which in the absence of negative Th1/Th2 correlation could also contribute to the proinflammatory state. Finally, Th22 cells from most individuals with PCC had no capacity to express IL-13 or IL-22, which could increase tendency to reinfections due to impaired epithelial regeneration. Discussion: People with PCC showed skewed polarization of CD4+ Th subsets with altered functionality that was more similar to individuals with severe and critical presentations of acute COVID-19 than to people who fully recovered from mild disease. New strategies aimed at reprogramming the immune response and redirecting CD4+ Th cell polarization may be necessary to reduce the proinflammatory environment characteristic of PCC.