Fig 1.
IL-17 cytokines affect naïve and bulk CD4+ T-cells but show no effect on memory T-cells.
A. Schematic summary of the ex vivo treatment of bead-sorted bulk or memory CD4+ CD25- T-cells activated in media alone (control/baseline), IL-17A, IL-17F, or IL-17AF heterodimer. After activation, RNA was isolated and underwent mRNA sequencing. B. The number of significant genes upregulated and downregulated with IL-17A (yellow), IL-17F (orange) and IL-17AF (blue) within the indicated populations. Significance was defined as q-value < 0.05 and log2-fold change > 1 or < -1. C. Negatively selected CD4+25- T-cells were further sorted using either CD45RO or CD45RA magnetic beads resulting in bulk, naïve and memory T-cell populations, which were αCD3/αCD28-activated for 7 days in media alone (control) or in the presence of IL-17AF and then used as responder cells in flow cytometric suppression assays with autologous CD8+ T-cells as suppressors. ** p<0.005, *** p<0.001, n.s. = not significant by Paired Student T-test.
Fig 2.
IL-17RA/RC receptor expression does not account for the lack of memory cell response to IL-17.
A. IL17RA and IL17RC expression on ex vivo microbead-sorted CD4+ Naïve (CD45RA+ and CD45RO-) and CD4+ Memory (CD45RO+ and CD45RA-) T-cells (Mean+/-SEM; n = 9). B-C. IL17RA and IL17RC expression after 7-day differentiation cultures of Naïve CD4+ T cells into Th0 and Th17 conditions (Mean+/-SEM; n = 16). D. Ex vivo-purified memory (CD4+CD25-CD45RO+) and naïve (CD4+CD25-CD45RO-) T-cells from healthy donors were stained with CFSE, followed by a 7-day culture with autologous irradiated APCs, fixed αCD3 antibody, and +/- increasing numbers of CD8+ T-cells (at the indicated ratios). Column bars depict %proliferation (+/-SEM) of indicated CD4+ T-cells at the indicated CD4:CD8 ratios, showing similar proliferation at 1:0 and similar suppression at 1:0.5 and 1:1. **p<0.005, *** p<0.001, ****p<0.0001 by paired student t test; n = 6. E-F. Ex vivo-purified memory CD4+CD25-CD45RO+ T-cells from healthy donors were polarized under indicated differentiation conditions for 7 days and then placed in a 7-day CD8+ T-cell mediated suppression assay. (E) Column bars depict %suppression (+/-SEM) of indicated Th-subsets by CD8+ T-cells at the 1:0.5 CD4:CD8 ratio, showing greater sensitivity of “Th1 milieu”-exposed cells and greater resistance by “Th17 milieu”-exposed cells. (F) Paired %suppression data from the Th0 and Th17 suppression cultures. *p<0.05, ** p<0.005.
Fig 3.
Memory T-cell non-responsiveness to IL-17 is not affected by local auto/paracrine action of naïve T-cell cytokines.
Transwell cultures were set up in 24-well plates with transwell membrane inserts separating autologous presorted Memory and Naïve CD4+ T cells together (A) or individually (B and C), with indicated cytokine conditions (media alone, IL-17A, IL-17F, IL-17AF and IL-17A+IL-17F, panels D-H, respectively) stimulated with anti-CD3/anti-CD28-coated beads in culture for 7 days. On day 7, the cells were harvested, washed, stained with CFSE and subjected to CD8-mediated T-cell suppression assays with fixed anti-CD3 and anti-CD28 stimulation. Mean suppression +/- SEM for the 5 conditions is shown; n = 5 each; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 4.
Overlapping and unique expression patterns in IL-17-treated conditions.
Overlapping and unique upregulated genes in bulk CD4+CD25- T-cells in IL-17A, IL-17F, and IL-17AF conditions compared to media-only cells. B. Heatmap of upregulated genes in at least two of three conditions; clustering based on Euclidean distance with immune-related genes labeled. C. Overlapping and unique downregulated genes in bulk CD4+CD25- T-cells in IL-17A, IL-17F, and IL-17AF conditions compared to media-only cells. D. Heatmap of downregulated genes in at least two of three conditions; clustering based on Euclidean distance with immune-related genes labeled. E. Dispersion of differentially-regulated genes by condition, grey genes are common between at least two conditions and colored genes are unique to the indicated condition. F. Top 10 unique upregulated genes in each condition ordered by log2-fold change.
Fig 5.
Pathway induction following IL-17 exposure.
A. Overlapping and unique upregulated canonical pathway enrichment in bulk CD4+CD25- T-cells in IL-17A, IL-17F, and IL-17AF conditions compared to media-only cells. B. Bar chart of significantly (p < 0.05) altered in at least two of three conditions. C. Z-score of significantly enriched canonical pathways with non-unique pathways colored in grey. Top increased unique pathways labeled.
Fig 6.
Detailed analysis of significant IL-17AF mediated changes in cytokines and chemokines.
Volcano plot of differential genes identified comparing IL-17AF to media-only cells. Highlighted cells have q-value < 0.05 and log2-fold change > 1 or < -1. Selected genes labeled were in the top 20 genes by q-value. B. Filtered cytokine and chemokine genes with q-value < 0.05.
Fig 7.
ELISA measurements of selected cytokines and chemokines validate RNAseq analysis with significant differences between CD4+CD25- bulk T-cells at 48 hours but not between CD4+CD25-45RO+ memory T-cells at 48 hours or 7 days.
ELISAs were performed on supernatants frozen at -80 from cellular cultures of 48 hour bulk CD4+25- T-cell, 48-hour and 7-day memory CD4+25-45RO+ T-cells that were sent for RNA sequencing. N = 3. Data are +/- SEM. Statistical analysis is by paired Student t test *p<0.05, ** p<0.005.