Figure 1.
Suppressor ability of iT-reg derived from CD4+CD45RO+ memory (CD4mem) and from CD4+CD45RA+ naïve (CD4naive) cells.
The ability of iT-reg obtained from CD4mem and CDnaive-derived Th17 cells was evaluated after 4-day co-culture by 3H-thymidine incorporation in 5 healthy subjects. Mean (+SEM) percentage suppression of CD4 effectors by CD4mem or CD4naive iT-reg before and after exposure to IL-6, IL-1β and rTGF-β. CD4mem but not CD4naive iT-reg maintain their suppressor ability after exposure to Th17 driving cytokines. *P≤0.05.
Figure 2.
Phenotypic properties of supTh17.
Phenotype of CD4mem at baseline and of Th17, obtained from CD4mem after 3-day exposure to IL-6+IL-1β+rTGF-β; iT-reg, obtained following exposure of Th17 to high concentration IL-2 and T-cell expander; and supTh17, obtained upon iT-reg exposure to IL-6+IL-1β+rTGF-β. Cell phenotype was determined in 12 healthy subjects. (A) Representative flow cytometry plots of CD4 (X axis) and IL-17, CD25 and FOXP3 (Y axis) fluorescence. (B) Representative histogram depicting RORC fluorescence in CD4mem at baseline, Th17 and supTh17; representative flow cytometry plots of CD4 (X axis) and CCR6, IL-23R and IL-22 (Y axis) fluorescence. Compared to prototypic Th17, supTh17 display higher frequencies of IL-17+, FOXP3+ and IL-22+ lymphocytes, express similar levels of RORC and contain comparable numbers of CCR6+ cells.
Figure 3.
The ability of Th17, iT-reg and supTh17 cells to control CD4 target cell proliferation was evaluated after 4-day co-culture by 3H-thymidine incorporation in 10 healthy subjects. (A) Mean (+SEM) percentage inhibition of CD4 effector cell proliferation by Th17, iT-reg and supTh17 cells. (B) The ability of Th17, iT-reg and supTh17 cells to control CD4 target cell IL-17 and IFNγ production was evaluated after 4-day co-culture by intracellular cytokine staining in 10 healthy subjects. Mean (+SEM) percentage inhibition of CD4 effector cell IL-17 and IFNγ production by Th17, iT-reg and supTh17 cells. Compared to prototypic Th17, supTh17 exerted more effective control over CD4 cell proliferation and pro-inflammatory cytokine production. *P≤0.05; **P≤0.01.
Figure 4.
Expression of CD39 and CD73 ectonucleotidases and associated ectoenzymatic activity.
(A) Mean (+SEM) frequency of (A) CD39+ cells, (B) CD39 mean fluorescence intensity (MFI) and of (C) CD39+CD73+ cells within CD4mem at baseline and within Th17, iT-reg and supTh17. Results from 12 healthy subjects are shown. *P≤0.05; **P≤0.01; ***P≤0.001. (D) CD39 ADPase enzymatic activity was assessed by TLC following incubation of Th17, iT-reg and supTh17 with [14C] radiolabeled ADP substrates. A representative of 3 independent experiments is shown. In accordance with high levels of CD39 and CD73, supTh17 generate AMP, adenosine and its derivative inosine.
Figure 5.
Adenosinergic effects on cell immune phenotype and function.
(A) Mean (+SEM) frequency of CD39+ cells and of CD73+, FOXP3+ and IL-17+ lymphocytes within them in CD4mem at baseline, Th17, iT-reg and supTh17. Results from n = 12 healthy subjects. (B) Mean (+SEM) inhibition of CD4 T-cell proliferation by Th17, iT-reg and supTh17 in the absence or presence of adenosine. Adenosine boosts expression of CD39 and CD73 and enhances the suppressor properties of iT-reg, while not having any effect on supTh17. *P≤0.05.
Figure 6.
Purinergic molecular signatures of supTh17 cells.
(A) Relative mRNA expression of A1, A2A, A2B, A3 receptors by Th17, iT-reg and supTh17 was determined by quantitative real-time PCR in 10 healthy subjects. Results are expressed as mean+SEM. (B) Expression of ADA was determined by immunoblot analysis. One representative of 3 independent experiments is shown. Mean (+SEM) ADA densities noted in Th17, iT-reg and supTh17 cells are also shown. (C) Mean (+SEM) CD26 MFI in Th17, iT-reg and supTh17 cells obtained from 5 healthy subjects was evaluated by flow cytometry. A representative histogram of CD26 fluorescence in CD4mem at baseline, Th17, iT-reg and supTh17 is shown. (D) Mean (+SEM) relative mRNA expression of PDE4A and PDE4B was determined by quantitative real-time PCR in 10 healthy subjects. supTh17 uniquely express low levels of A2A adenosine receptor, exhibit ADA activity associated with CD26 but do not substantially up-regulate levels of PDE. *P≤0.05; ***P≤0.001.
Figure 7.
Demonstration of supTh17 cells in healthy subjects and associated decreases in Crohn’s disease.
The frequency of CD4+IL-17+ and of supTh17 was determined in PBMCs and LPMCs by flow cytometry. supTh17 were identified by initially gating CD4+CD45RO+ cells within PBMCs or LPMCs and then by determining the proportion of CD39+IL-17+ and FOXP3+ within them. Mean (+SEM) frequency of (A) CD4+IL-17+ and of (B) supTh17 cells in the circulation and in the lamina propria. Mean (+SEM) frequency of supTh17 positive for (C) Stat-3 and for (D) TNF-α and IL-2 in the circulation and in the lamina propria. Healthy subjects: n = 17; Crohn’s: n = 25; *P≤0.05; **P≤0.01; ***P≤0.001.
Figure 8.
SupTh17, iT-reg and purinergic control of T-cell immune responses.
Both supTh17 and iT-reg cells have the capacity to suppress effector T-cells (Teff) by generating adenosine. In a manner distinct from iT-reg which are anergic, however, supTh17 express low levels of A2A receptor and exhibit nucleoside scavenging ecto-enzymatic activity. These properties confer on supTh17 an important intrinsic resistance to suppressive effects of adenosine, which may develop in parallel with prolonged cellular activation in accordance with memory T-cell status. These differences suggest that supTh17 might undergo conversion and be recruited as suppressor-type cells in the later evolution of immune responses where these cells may persist at sites of resolving injury.