Multiparametric Investigation Of Spike-Protein Specific T-cell Cytokine Expression Profile In Children With Symptomatic COVID-19 Or Multisystem Inflammatory Syndrome

ABSTRACT

Background. Data regarding cell-mediated immunological differences in children across COVID-19 clinical spectrum are limited. We prospectively investigated Spike-protein specific cellular immunity in children with symptomatic COVID-19 or MIS-C, by single cell cytokine expression profiling. Methods. PBMCs fromchildrenwithMIS-C, symptomaticCOVID-19 (onemonth after hospitalization) and healthy controls were isolated prospectively. Cell suspensions were divided into two quantitatively equal samples (a negative control-unstimulated and a positive-stimulated).Cells stimulationwas performed usingSARS-CoV-2 Spike antigenic peptidesmix (Peptivator SARS-CoV-2 Prot-S). Cells of each sample were stained with fluorochrome monoclonal antibodies against 8 surface markers (CD3, CD4, CD8, CD14, CD19, CD137, CD197, CD45RA) and 6 intracellular markers (IL-4, IL-2, IL-17, IFN-gamma, TNF-alpha, Granzyme B). Viability was assessed by 7AAD. Stained cell preparations were analysed using 13 colour Flow Cytometry (DX Flex, Beckman Coulter). Flow cytometric analysis was performed using Kaluza 2.1 Software. Results. Sixteen children (4 MIS-C, 8 post-COVID-19 and 4 healthy controls) were included in the study. The mean age (+/-SD) of the participants was 11.22 years (+/-3.48). Children with MIS-C had significantly higher mean (+/-SD) values of CD8+IFN-gamma/million CD3+ [226.68 (134.92) vs 45.43 (57.28);P 0.033] and median (IQR) of CD8+IFN-gamma/ml [156.38 (184.13) vs 26.34 (82.36);P 0.033] compared to symptomatic COVID-19 children. Compared to healthy controls, MIS-C patients had significantly higher median (IQR) values of CD4+IL-2/million CD3+ [923.12 (3777.95) vs 97.59 (71.71);P 0.042] and CD4+IL-2/ml median (IQR) [626.33 (2744.98) vs 169.3 (173.91);P 0.042]. No other significant differences were observed regarding other immunological markers in 3 study groups. Conclusion. IFN-gamma production by CD8+ T cells is highly expressed in children with MIS-C compared to hospitalized children one month after acute COVID-19 and could consist possible immunological biomarker. Further studies are needed in order to elucidate the pathophysiological basis of these findings.