Immunol. and unadjusted p-values are reported. Altogether, 50 variables had been assessed: (A) IgG1, IgG2, IgG3, IgG4, IgA, and IgM, and (B) FcR2A, FcR2B, FcR3A, and FcR3B against spike (S), receptor binding domains (RBD), and nucleocapsid (N) antigens. mass media-2.pdf (459K) GUID:?C4D434CB-1D02-40C5-A5E0-089FC80D5C7C Dietary supplement 3: Supplementary Amount 3. Gating technique for T cell stream cytometry. (A) Data provided in Amount 1 were attained utilizing a 15-color multiparameter stream Rabbit polyclonal to Osteocalcin cytometry panel. Occasions had been isolated from a period gate initial, accompanied by singlets. Practical cells were discovered, and Compact disc14 and Compact disc19 markers had been utilized to recognize B cells and monocytes, respectively. Gating proceeded from lymphocytes to another singlet gate then. From the next singlet gate, Compact disc56 was utilized to identify normal killer cells. In parallel, Compact disc3+ T cells in the singlet gate had been additional characterized using Compact disc1d- -Galactosylceramide (-GalCer) and MR1C5-(2-oxopropyl phenylamino)-6-D-ribitylaminouracil (5-OP-RU) tetramers to recognize invariant organic killer T cells and mucosal-associated invariant T cells, respectively, aswell as activation markers (HLA-DR and Compact disc38), and T cells (Skillet- and V2). Furthermore, Compact disc3+ T cells were examined for co-receptor usage with Compact disc4 and Compact disc8 markers also. Finally, storage populations had been individually gated for CD4+ Chlormezanone (Trancopal) and CD8+ cells using CD45RA and CCR7. (B) Data presented in Figures 3C5 were obtained using a 14-color multiparameter intracellular cytokine staining (ICS) flow cytometry panel. A time gate was applied to the events, and then viable CD3+ T cells were identified. CD14 Chlormezanone (Trancopal) and CD19 markers were used to exclude monocytes and B cells, and then a singlet gate was applied. Lymphocytes were then gated and analyzed for HLA-DR (activation), CD38 (activation), and CD4 and CD8 co-receptor expression. For CD4+ and CD8+ populations, cells were characterized for expression of IFN- (Th1), IL-2 (Th1), TNF (Th1), IL4/5/13 (Th2), IL-17 (Th17), CD40L (activation and B cell help), CD107a (degranulation), CD45RA (memory), and CCR7 (memory) expression. media-3.pdf (608K) GUID:?69D0BC74-C8FA-40F8-A345-5C8E506DF6E5 Supplement 4: Supplementary Figure 4. Cell frequencies of donor-unrestricted T cells, B cells, monocytes, and natural killer cells. Flow cytometric analysis of peripheral blood mononuclear cells (PBMC) was performed using a 15-color surface staining and phenotyping panel. (A) Frequencies and activation statuses of invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells were compared between hospitalized (purple) and non-hospitalized (green) subjects. Frequencies are displayed as percent of total T cells, and activation is calculated as the percentage total iNKT or MAIT cells that co-expressed HLA-DR and CD38. (B) B cells (CD19+), monocytes (CD14+), and natural killer (NK) cell (CD3-CD56+) frequencies are shown as percent of live cells and are compared between groups. (C) The frequency of activated (HLADR+CD38+) T cells is plotted against days since symptom onset for both hospitalized and non-hospitalized subjects. T cell frequencies were compared between groups using Mann-Whitney U tests, followed by correction for multiple hypothesis testing using the Bonferroni method. Median, 25th, and 75th quartiles are indicated in the violin plots. The black line on the scatter plot represents a best fit linear regression line, and the grey-shaded area represents the 95% confidence interval of the predicted mean. If not shown, p-values were not significantly different. media-4.pdf (224K) GUID:?C4E9F3AD-CD21-4BD7-A6CB-2F33F455A050 Supplement 5: Supplementary Figure 5. Convalescent COVID-19 subjects demonstrate both IFN- dependent and impartial CD4+ T cell responses following stimulation with SARS-CoV-2 protein antigens. Background subtracted magnitudes of responding CD4 T cells is usually displayed for each of the functional subsets identified by COMPASS in Physique 3A after stimulation with peptide pools targeting (A) S1, (B) S2, (C) nucleocapsid, and (D) envelope. Boxplots indicating median and interquartile range are shown for hospitalized (purple) and non-hospitalized (green) subjects. Cell frequencies were compared between groups using the Mann-Whitney U assessments followed by correction for multiple hypothesis testing using the Bonferroni method. Only significant p-values are indicated. media-5.pdf (164K) GUID:?43C009CE-11C9-4F78-805F-F3FDF36FE44A Supplement 6: Supplementary Figure 6. Validation of PLS-DA Model. The classification accuracy distributions of the model presented in Physique 6 was compared to unfavorable control models based on randomly selected or permuted data, by measuring the classification accuracies of each model in a five-fold cross-validation framework. (A) The violin plot shows the distributions of these classification accuracies for all those three models across cross-validation replicates. Model performs significantly better compared to permuted labels. The model is Chlormezanone (Trancopal) not able to outperform the randomly selected features because a substantial portion of the measured features (54%).