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. 2014 Jan 24;9(1):e87265.
doi: 10.1371/journal.pone.0087265. eCollection 2014.

Egr3 induces a Th17 response by promoting the development of γδ T cells

Rose M Parkinson  1 , Samuel L Collins  2 , Maureen R Horton  2 , Jonathan D Powell  1
Affiliations

Egr3 induces a Th17 response by promoting the development of γδ T cells

Rose M Parkinson et al. PLoS One. .

Abstract

The transcription factor Early Growth Response 3 (Egr3) has been shown to play an important role in negatively regulating T cell activation and promoting T cell anergy in Th1 cells. However, its role in regulating other T helper subsets has yet to be described. We sought to determine the role of Egr3 in a Th17 response using transgenic mice that overexpress Egr3 in T cells (Egr3 TG). Splenocytes from Egr3 TG mice demonstrated more robust generation of Th17 cells even under non-Th17 skewing conditions. We found that while Egr3 TG T cells were not intrinsically more likely to become Th17 cells, the environment encountered by these cells was more conducive to Th17 development. Further analysis revealed a considerable increase in the number of γδ T cells in both the peripheral lymphoid organs and mucosal tissues of Egr3 TG mice, a cell type which normally accounts for only a small fraction of peripheral lymphocytes. Consistent with this marked increase in peripheral γδ T cells, thymocytes from Egr3 TG mice also appear biased toward γδ T cell development. Coculture of these Egr3-induced γδ T cells with wildtype CD4+ T cells increases Th17 differentiation, and Egr3 TG mice are more susceptible to bleomycin-induced lung inflammation. Overall our findings strengthen the role for Egr3 in promoting γδ T cell development and show that Egr3-induced γδ T cells are both functional and capable of altering the adaptive immune response in a Th17-biased manner. Our data also demonstrates that the role played by Egr3 in T cell activation and differentiation is more complex than previously thought.

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Conflict of interest statement

Competing Interests: Jonathan Powell is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. CD4+ T cells from Egr3 TG mice are biased toward Th17 differentiation.
A-B) Splenocytes from 10-12 week old Egr3 WT and Egr3 TG mice were stimulated for 2 days with soluble anti-CD3 either with (Th17) or without (Th0) the addition of skewing cytokines. A) IL-17 production by Egr3 WT and Egr3 TG CD4+ T cells within the splenic population after restimulation. B) IL-17A, IL-17F, IL-21, RORγt, IL-23, and IL-6 mRNA expression in unstimulated (day 0), overnight stimulated (day 1) or previously activated and restimulated (day 6) splenocytes from Egr3 WT and Egr3 TG mice. Cells stimulated for mRNA were not skewed and mRNA expression was determined by qRT-PCR done in triplicate and normalized to 18s expression. C) Naive CD4+ T cells from Egr3 WT and Egr3 TG mice were stimulated with anti-CD3 and anti-CD28 for 2 days with or without skewing cytokines as in A, then restimulated and assessed for IL-17 production. Data are representative of 3 independent experiments, each using 2 mice per genotype. *p≤0.05, ** p≤0.01, ***p≤0.005
Figure 2
Figure 2. Increased number of γδ T cells in Egr3 TG mice.
A-D) Splenocytes or mucosal lymphocytes harvested from untreated Egr3WT or Egr3TG mice were counted and stained for surface expression of CD3, CD4, CD8 and either γδTCR or a specific Vγ chain. A) CD3 expression shown as a fraction of total lymphocytes (left panels). Percentages of CD4+ T cells, CD8+ T cells, and CD4- CD8- T cells, shown as a fraction of CD3+ gated cells (center panels). γδTCR and CD8 expression shown as fraction of CD4- T cells (right panels). B) CD4- CD8- (DN) cells (left panel) or CD4- CD8- (DN) γδTCR+ and CD8+ γδTCR+ cells (center panel) shown as a percentage of total T cells (CD3+ gated splenocytes), with each dot representing a separate mouse. Averaged absolute numbers of T cell subsets per mouse spleen (right panel). Data in A and B are representative of 4 independent experiments, each using 2-3 mice per genotype. Absolute numbers in B are averaged from 4 experiments where splenocytes from 2-3 mice were pooled. C) γδ T cells found in the spleen, lung, small intestine, or colon of Egr3 WT or Egr3 TG mice, shown as a percentage of the CD3+ gated population. D) Percentage of cells expressing Vγ1, Vγ4, or Vγ7 chains, shown as a fraction of the CD3+ gated population. Data in C and D are representative of 3 independent experiments, each using lymphocytes pooled from 5-6 mice per genotype. ***p ≤ 0.005
Figure 3
Figure 3. Thymocyte selection in Egr3 TG mice favors γδ T cells.
A-B) Thymocytes harvested from littermate Egr3 WT and Egr3 TG mice were stained for surface marker expression or lysed for mRNA quantification. A) CD4 vs. CD8 expression was measured to determine double negative, double positive, and single positive subsets (left panels). DN1, DN2, DN3, and DN4 subsets shown as a percentage of the double negative fraction (right panels). B) γδT cells shown as a percentage of thymocytes (left panels) and C) γδT cells shown as an absolute number. D) Expression of Id3 mRNA in DN1, DN2, DN3, DN4 thymocytes assessed by qRT-PCR and normalized to 18s expression. Data in A-D are representative of 3 independent experiments, each using 2 mice per genotype. **p ≤ 0.01, ***p ≤ 0.005
Figure 4
Figure 4. Egr3 TG γδ T cells produce IL-17 and skew CD4+ T cells to a Th17 phenotype.
A) Splenocytes were stimulated and rested, and IL-17 production by CD8 γδ and DN γδ T cells was measured on day 6 after short restimulation (flow plots). The percentage of each subset producing IL-17 (top bar graph) and absolute number of IL-17 producing cells of each subset (bottom bar graph) are shown as an average of 4 independent experiments, each using 2 mice per genotype. B) Purified CD4+ T cells or γδ T cells were stimulated directly ex vivo with plate bound anti-CD3 and soluble anti-CD28 for 16 hours. IL-17A, IL-17F, IL-6, and IL-23R mRNA expression was measured by qRT-PCR done in triplicate and normalized to 18s expression. Data are representative of 3 independent experiments, each using 2 mice per genotype. C) IL-17 production measured as a percentage of either the Vγ1-expressing subset (left panels) or the Vγ4-expressing subset (right panels) of γδ T cells in splenocyte stimulation. Data are representative of 1 experiment using 5 mice per genotype. D) Egr3 WT CD4+ T cells were stimulated for 2 days with plate bound anti-CD3 and soluble anti-CD28 either alone (Th0), with the skewing cytokines TGFβ, IL-6, and IL-23 (Th17), or with equal numbers of Egr3 TG γδ T cells. IL-17 production by was assessed on day 6 after restimulation. CD4 and Thy1.1 expression shows the gating strategy used in this experiment (top row). IL-17 expression by the CD4+ Thy1.1- gated Egr3 WT T cells (bottom row). The overlay flow plot (top row, right) shows both the Egr3 WT CD4+ Thy1.1- T cells and Egr3 TG Thy1.1+ gates from the co-culture. Plots are representative of 3 independent experiments, which are averaged in the bar graph (bottom row, right). *p≤ 0.05, ** p≤ 0.01, ***p≤ 0.005
Figure 5
Figure 5. Egr3 TG mice are more susceptible in a model of pulmonary fibrosis.
Egr3 WT or Egr3 TG mice were challenged with intratracheal bleomycin at day 0 and monitored for A) weight loss and B) survival. Data in A and B are compiled from 4 independent experiments. C) Separate mice were challenged as in A and sacrificed at day 5 to determine lymphocyte infiltration in the bronchaeolar lavage (BAL) and lung as well as the absolute number of IL-17-secreting cell infiltrates in each compartment. Data in C are representative of 3 independent experiments. *p≤ 0.05, ***p≤ 0.005
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