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. 2010 Dec 2;116(23):4829-37.
doi: 10.1182/blood-2010-03-272153. Epub 2010 Aug 16.

Low-strength T-cell activation promotes Th17 responses

Harriet A Purvis  1 , Jeroen N Stoop, Jelena Mann, Steven Woods, Anne E Kozijn, Sophie Hambleton, John H Robinson, John D Isaacs, Amy E Anderson, Catharien M U Hilkens
Affiliations

Low-strength T-cell activation promotes Th17 responses

Harriet A Purvis et al. Blood. .

Abstract

We show that the strength of T-cell stimulation determines the capability of human CD4(+) T cells to become interleukin-17 (IL-17) producers. CD4(+) T cells received either high- (THi) or low (TLo)-strength stimulation via anti-CD3/CD28 beads or dendritic cells pulsed with superantigen in the presence of pro-Th17 cytokines IL-1β, transforming growth factor β, and IL-23. We found that TLo, but not THi, stimulation profoundly promoted Th17 responses by enhancing both the relative proportion and total number of Th17 cells. Titration of anti-CD3 revealed that low TCR signaling promoted Th17 cells, but only in the presence of anti-CD28. Impaired IL-17 production in THi cells could not be explained by high levels of Foxp3 or transforming growth factor β-latency-associated peptide expressed by THi cells. Nuclear factor of activated T cells was translocated to the nucleus in both THi and TLo cells, but only bound to the proximal region of the IL-17 promoter in TLo cells. The addition of a Ca(2+) ionophore under TLo conditions reversed the pro-Th17 effect, suggesting that high Ca(2+) signaling impairs Th17 development. Although our data do not distinguish between priming of naive T cells versus expansion/differentiation of memory T cells, our results clearly establish an important role for the strength of T-cell activation in regulating Th17 responses.

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Figures

Figure 1
Figure 1
Low-strength T-cell stimulation favors Th17 responses. (A-D) Human CD4+ T cells were cultured under THi or TLo conditions for 6 days. (A-B) THi and TLo cells were restimulated with PMA/ionomycin for 5 hours, and expression of IL-17 and IFN-γ were determined by intracellular flow cytometry. Plots of 1 example of 24 independent experiments are shown (A). (B) Percentages of IL-17 and IFN-γ single and double producers; data of 24 independent experiments are shown. (C) THi and TLo cells were restimulated with PMA/ionomycin for 24 hours, and cytokines were measured by ELISA (n = 3). (D) Expression of IL-17 and ROR-γt in TLo cells was determined by intracellular flow cytometry. One representative of 3 independent experiments is shown. (E) Mouse CD4+ T cells were cultured under THi or TLo conditions, and expression of IL-17 and IFN-γ were determined by intracellular flow cytometry (n = 6). (F) Human CD4+ T-cells were cultured under TLo conditions in the presence (“TLo”) or absence (“w/o cytokines”) of IL-1β, IL-23, and TGF-β for 6 days. Expression of IL-17 and IFN-γ were determined by intracellular flow cytometry. Results are representative of 3 independent experiments. Horizontal bars represent the median value; error bars represent SEM. P values were calculated using the Wilcoxon test: *P < .05; ***P < .0001.
Figure 2
Figure 2
Low-strength T-cell stimulation by DCs favors Th17 responses. (A) Human CD4+ T cells were cultured for 6 days with allogeneic peptidoglycan-activated DCs at either a 1 DC:1 CD4+ T cell ratio (DC Hi) or 1 DC:50 CD4+ T cell ratio (DC Lo). Expression of IL-17 and IFN-γ was determined by intracellular flow cytometry. Plots are representative of 3 independent experiments. (B) Human CD4+ T cells were cultured with autologous DCs at a 1:10 DC:CD4+ T cell ratio with decreasing concentrations of SEB (1000-0 pg/mL) for 6 days. Expression of IL-17 and IFN-γ was determined by intracellular flow cytometry. Results are representative of 3 independent experiments.
Figure 3
Figure 3
TLo stimulation induces higher numbers of proliferating Th17 cells than THi stimulation. (A-B) Human CD4+ T cells were labeled with CFSE and cultured under THi or TLo conditions for 6 days. Proliferating IL-17+ (A) and IFN-γ+ (B) cells were assessed by intracellular flow cytometry. Plots are representative of 3 independent experiments. (C) The number of alive cells on day 6 was determined by cell counting using trypan blue, and the absolute number of IL-17+ cells was then calculated using the proportion of IL-17+ cells after intracellular flow cytometry (n = 3). Error bars represent SEM. P values were calculated using the Student t test: *P < .05. (D) Human CD4+ T cells were cultured under THi or TLo conditions for 3, 6, or 10 days. To generate day 10 cells, beads were removed at day 6 and cells were washed, replated, and cultured for 4 more days. Expression of IL-17 was determined by intracellular flow cytometry. Day 3 and 10 graphs: n = 3; day 6 graphs: n = 4. Error bars represent SEM. P values were calculated using the Mann-Whitney U test: *P < .05
Figure 4
Figure 4
Low signal strength through the TCR/CD3 complex favors Th17 cells only in the presence of anti-CD28. Human CD4+ T cells were cultured for 6 days at a 1:10 bead:CD4+ T cell ratio. Beads were loaded with decreasing concentrations (40-2.5 μg/mL) of anti-CD3 ± 10 μg/mL anti-CD28. Cells were restimulated with PMA/ionomycin for 5 hours, and expression of IL-17 and IFN-γ was determined by intracellular flow cytometry. Results are representative of 3 independent experiments.
Figure 5
Figure 5
TGF-β is required for high IL-17 production by TLo cells, and high latent TGF-β/LAP expression by THi cells does not inhibit Th17 responses. (A) Human CD4+ T cells were cultured under THi or TLo conditions for 6 days, and LAP expression was determined by flow cytometry. Plots are representative of 3 independent experiments. (B) Human CD4+ T cells were cultured under THi or TLo conditions with or without TGF-β in decreasing concentrations of the ALK5 inhibitor, SB505124 (5-0.05μM). Expression of IL-17 and IFN-γ were determined by flow intracellular cytometry on day 6 (n = 3). Error bars represent SEM. P values were calculated using one-way analysis of variance with Tukey correction: *P < .05.
Figure 6
Figure 6
Inverse relationship between Foxp3 and Th17 in TLo, but not THi, conditions. (A) Human CD4+ T cells were cultured under THi or TLo conditions for 6 days. Expression of IL-17 and Foxp3 was determined by intracellular flow cytometry. Plots are representative of 4 independent experiments. (B) Human CD4+ T cells were cultured under THi or TLo conditions for 3, 6, or 10 days. To generate day 10 cells, beads were removed at day 6 and cells were washed, replated, and cultured for 4 more days. Expression of IL-17 and Foxp3 were determined by intracellular flow cytometry (n = 3). IL-17 data shown in Figure 3D were plotted in this graph. (C) IPEX patient PBMCs were cultured under THi or TLo conditions. Expression of IL-17 and IFN-γ were determined by intracellular flow cytometry.
Figure 7
Figure 7
NFATc1 binds to the IL-17 promoter in TLo, but not THi, cells. Human CD4+ T cells were cultured under THi or TLo conditions for 6 days. Day 6 cells were either restimulated for 2 hours with PMA/ionomycin (+) or left untreated (-). (A) Cytoplasmic and nuclear lysates were prepared, and expression of NFATc1 was determined by Western blotting. Blots are representative of 3 independent experiments. (B) NFATc1 binding to the proximal IL-17 promoter was assessed by ChIP (n = 2). Error bars respresent SEM. (C) CD4+ T cells were cultured for 6 days under either THi or TLo conditions or TLo conditions +500nM ionomycin (TLo +) added daily for the first 4 days of culture. Expression of IL-17 was determined by intracellular flow cytometry (n = 3). Error bars represent SEM.
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