Figure 7.
Azithromycin (AZM) inhibits T-cell receptor (TCR) signaling in T cells, promotes immunomodulatory pathways, and impedes mitochondrial mRNA synthesis. (A) Thawed peripheral mononuclear blood cells (PBMCs) from 8 healthy donors (HDs) were treated with AZM or control (CTRL) for 24 hours before CD3/CD28 activation. Activation was stopped at 0, 3, 5, 10, and 30 minutes. To prevent batch effect on sample labeling, samples from each donor were barcoded with a mix of anti-CD45 antibodies and then pooled before mass cytometry staining procedure. Single cells were then clustered by means of FlowSOM algorithm according to phenotype antiges expression. Area under the receiver operating characteristic curve (AUC) of signaling mean signal intensity was computed. Cluster phenotypes are presented in supplemental Figure 18. (B) Dot plot depicting statistically significant difference in AUC between CTRL and AZM conditions. Individual differences at the 5 time points are presented in supplemental Figure 20. P values were computed by means of matched-paired Wilcoxon Rank Sum test. pSTAT6 is not depicted, because it was not statistically different. (C) CD3+-sorted cells from 10 HDs were treated with AZM or CTRL for 24 hours before CD3/CD28 activation. Analysis was performed 2 days after cells activation. The pie chart illustrates the number of living cells retrieved at the end of the single-cell RNA sequencing with the use of cell surface antigen embedding pipeline. Next, using cell surface antigen expression, 26 phenotypic cell clusters were identified as illustrated with the uniform manifold approximation and projection (UMAP). (D) UMAP highlighting retrieved cells from activated conditions. (E) Heatmap representing manually ordered and annotated cell clusters with the corresponding scaled surface antigen expression. Abundances of clusters in cells treated with AZM or CTRL are depicted on boxplots. For visualization purposes, square root transformation was applied on the x-axis. P values were computed by means of matched-paired Wilcoxon rank sum test. (F) Dot plot showing differentially expressed genes in each T-cell clusters from volcano plots is shown in supplemental Figure 21. Only statistically significant changes are depicted. P values were computed by means of Wilcoxon rank sum test and adjusted with false discovery rate.

Azithromycin (AZM) inhibits T-cell receptor (TCR) signaling in T cells, promotes immunomodulatory pathways, and impedes mitochondrial mRNA synthesis. (A) Thawed peripheral mononuclear blood cells (PBMCs) from 8 healthy donors (HDs) were treated with AZM or control (CTRL) for 24 hours before CD3/CD28 activation. Activation was stopped at 0, 3, 5, 10, and 30 minutes. To prevent batch effect on sample labeling, samples from each donor were barcoded with a mix of anti-CD45 antibodies and then pooled before mass cytometry staining procedure. Single cells were then clustered by means of FlowSOM algorithm according to phenotype antiges expression. Area under the receiver operating characteristic curve (AUC) of signaling mean signal intensity was computed. Cluster phenotypes are presented in supplemental Figure 18. (B) Dot plot depicting statistically significant difference in AUC between CTRL and AZM conditions. Individual differences at the 5 time points are presented in supplemental Figure 20. P values were computed by means of matched-paired Wilcoxon Rank Sum test. pSTAT6 is not depicted, because it was not statistically different. (C) CD3+-sorted cells from 10 HDs were treated with AZM or CTRL for 24 hours before CD3/CD28 activation. Analysis was performed 2 days after cells activation. The pie chart illustrates the number of living cells retrieved at the end of the single-cell RNA sequencing with the use of cell surface antigen embedding pipeline. Next, using cell surface antigen expression, 26 phenotypic cell clusters were identified as illustrated with the uniform manifold approximation and projection (UMAP). (D) UMAP highlighting retrieved cells from activated conditions. (E) Heatmap representing manually ordered and annotated cell clusters with the corresponding scaled surface antigen expression. Abundances of clusters in cells treated with AZM or CTRL are depicted on boxplots. For visualization purposes, square root transformation was applied on the x-axis. P values were computed by means of matched-paired Wilcoxon rank sum test. (F) Dot plot showing differentially expressed genes in each T-cell clusters from volcano plots is shown in supplemental Figure 21. Only statistically significant changes are depicted. P values were computed by means of Wilcoxon rank sum test and adjusted with false discovery rate.

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