Research ArticleIMMUNOLOGY

Immune synapse instructs epigenomic and transcriptomic functional reprogramming in dendritic cells

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Science Advances  03 Feb 2021:
Vol. 7, no. 6, eabb9965
DOI: 10.1126/sciadv.abb9965
  • Fig. 1 psDCs enhance the expression of genes related with inflammation, cytokine response, and antiviral immunity.

    (A) Heatmap showing normalized gene expression values for a selection of 60 genes detected as differentially expressed [Benjamini and Hochberg (B-H)–adjusted P value < 0.05; abs(log2FC) > 1]. RNA-seq–based transcriptomic profile comparison of psDCs versus nsDCs (n = 3). (B) Enriched canonical pathways detected with IPA on the complete set of 1108 differentially expressed genes, after filtering with B-H–adjusted P value < 0.05 and abs(z score) > 1. The length of the bars represents enrichment significance [−log10(B-H P value)], and the red dashed line indicates the significance threshold. Bar colors represent “z-score” values (predicted activation state of the pathway, IPA); positive values (dark red) indicate higher activity in psDCs. Connected black dots represent parameter “ratio,” this is, the fraction of genes associated with any given pathway included in the collection of differentially expressed genes. NF-κB, nuclear factor κB; IL-8, interleukin-8. (C) Enriched biological process GO terms detected with clusterProfiler on the complete set of 1108 differentially expressed genes (right side of the circular plot). Only five selected terms are shown (B-H–adjusted P value < 1 × 10−8) and are connected to small rectangles representing differentially expressed genes associated to those terms (left side of the circular plot), colored according to the logFC value. (D) IPA network connecting functional terms “virus clearance” and “antiviral response” to associated differentially expressed genes. Genes up- or down-regulated in psDCs relative to nsDCs are colored in red and blue, respectively. (E) qPCR analysis of the gene expression levels indicated in psDCs versus nsDCs. Pulsed with or without OVA323–339 cognate OT-II peptide or LCMV GP61–80 control peptide [n = 7; Kruskal-Wallis test with Dunn’s posttest or one-way analysis of variance (ANOVA) test with Tukey’s posttest; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

  • Fig. 2 Gene expression of psDCs upon CD8+ T cell IS.

    qPCR analysis of the expression levels of the indicated genes in DCs cocultured with CD8+ OT-I T cells under the presence or absence of OVA257–264 cognate OT-I peptide or VACV B8R20–27 control peptide (n = 7; Kruskal-Wallis test and Dunn’s posttest or one-way ANOVA with Tukey’s posttest; **P < 0.01, ***P < 0.001, and ****P < 0.0001).

  • Fig. 3 Epigenomic signature of psDCs is modified upon T cell contact.

    (A) MA plot representing differential accessibility analysis results for ATAC-seq data. Differentially accessible sites (15,062) [with FDR < 0.05 and abs(log2FC) > 1] are symbolized as pink dots over a blue cloud that represents the complete consensus set of 98,731 ATAC-seq sites. X-axis values (“log concentration”) represent logarithmically transformed, normalized counts, averaged for all samples, for each site. Y-axis values represent log2(fold change) values for ATAC-seq counts in psDCs relative to nsDCs. Positive log2FC values indicate higher accessibility in psDCs. (B) Scatterplot relating log2(fold change) values for ATAC-seq counts and log2(fold change) values for RNA-seq counts. Dots represent the association between 1634 differentially accessible genomic sites and 632 differentially expressed genes that were located at the shortest distance of a differentially accessible site. (C) Enriched biological processes GO terms, detected with GREAT, were associated to differentially accessible site proximal genes. Only the top 12 terms, sorted by significance, are presented. (D) Peak representation of the DNA accessibility in psDCs (green) versus nsDCs (blue) within regions proximal to locus for the indicated genes. The height of the peak represents the counts per million from the ATAC-seq data (n = 2). (E) ChIP-qPCR assay of the coimmunoprecipitation H3K4me3 with the promoter regions of the indicated genes. Results are expressed as fold enrichment relative to a glyceraldehyde-3-phosphate dehydrogenase promoter region (n = 5, paired t test; *P < 0.05).

  • Fig. 4 IS enhances psDC migratory capacity in response to Ccr7.

    (A) Transwell assay of psDCs and nsDCs in response to CCL19 (100 ng/ml) for 3 hours. Medium without cytokines was set as a control (basal). Cell numbers were normalized to the initial input of cells (n = 6; Wilcoxon matched-paired signed ranks test; *P < 0.05). (B) Image showing the tracks and speed (color coded) of psDCs (green) versus nsDCs (red) in an under-agarose assay in response to a CCL19 gradient (focus on the right side of the image). (C) Quantification of the speed and straightness of the chemotactic movement of DCs in the under-agarose assay (n ≥ 44, Student’s t test; ****P < 0.0001). (D) Dot plot describing the gating strategy for the analysis of the number of CD11c+ (gated in the CD3 population) and CD45.1+ cells in psDCs and nsDCs. (E) Quantification of the number of migrated psDCs and nsDCs in the popliteal lymph node is shown (n = 5, paired t test; **P < 0.01).

Supplementary Materials

  • Supplementary Materials

    Immune synapse instructs epigenomic and transcriptomic functional reprogramming in dendritic cells

    Ana Alcaraz-Serna, Eugenio Bustos-Morán, Irene Fernández-Delgado, Diego Calzada-Fraile, Daniel Torralba, Ester Marina-Zárate, Erika Lorenzo-Vivas, Enrique Vázquez, Juliana Barreto de Alburquerque, Nora Ruef, Manuel José Gómez, Fátima Sánchez-Cabo, Ana Dopazo, Jens V. Stein, Almudena Ramiro, Francisco Sánchez-Madrid

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