Longitudinal immune profiling uncovers Regulatory T cell signatures associated with the progression of COVID-19

Camila Kossack^1*Felipe Bravo^2,3Francisco Fuentes-Villalobos²Claudio Quevedo⁴Matias Medina¹Raul Riquelme⁵MARIA LUISA RIOSECO⁵MARIO ANDRES CALVO⁶Karina Pino-Lagos⁷Felipe Aguilera⁴Maria Ines Barria^1*Jose Luis Garrido^1,3*

• ¹Universidad San Sebastian - Campus Pichi Pelluco, Puerto Montt, Chile
• ²Universidad de Concepcion Departamento de Microbiologia, Concepción, Chile
• ³Ichor Biologics LLC, New York, United States
• ⁴Universidad de Concepcion Facultad de Ciencias Biologicas, Concepción, Chile
• ⁵Hospital Puerto Montt Dr Eduardo Schutz Schroeder, Puerto Montt, Chile
• ⁶Universidad Austral de Chile Facultad de Medicina, Valdivia, Chile
• ⁷Universidad de los Andes Facultad de Medicina, Santiago, Chile

The role of Regulatory T cells (Tregs) in severe COVID-19 remains unclear. Some authors reported that Tregs increased in peripheral circulation, while other investigators reported that these cells decreased in severe COVID-19 patients. The expression of FoxP3 in T regs remains inconsistent and controversial as well. These observations have been made using immune phenotyping via flow cytometry and sequencing of T cells; however, none of these data provide a clear indication of what the Tregs are doing in this chaotic scenario of a hyperactivated immune response. Here, we aim to progressively characterize the Treg compartment during acute COVID-19 in a longitudinal cohort of patients who present mild or severe disease. Interestingly, gene expression analysis revealed a significant downregulation of genes related to regulatory pathways that involve T regs' functional activities (FOXP3, CTLA4, IL2RA, TNFRSF4, NT5E, PDCD1), which was consistent among the severe patients analyzed. In contrast, we found an increase in the expression of these genes in mild patients. This finding was further confirmed by phenotyping analysis, where we observed significant differences in CD25+CD127-Treg cells between mild and severe patients, which were positively associated with CTLA-4 and PD-1 inhibitory markers. Surprisingly, these results did not correlate with the expression of FoxP3. Furthermore, a high frequency of CD25+CD127-Treg cells was associated with young, mild patients. In contrast, low frequency of CD25+CD127-cells and higher frequency of FoxP3 positive cells were associated with elderly patients. Finally, a Treg functional assay showed a poor capacity of suppression of Tregs obtained from severe compared to mild patients. Our findings offer critical insights into the role of Tregs in SARS-CoV-2, with implications that extend beyond this viral infection. Understanding how Tregs contribute to immune responses in COVID-19 could inform therapeutic strategies to modulate immune regulation in infectious diseases more broadly.