Human in vitro-induced regulatory T cells display Dlgh1dependent and PKC-θ restrained suppressive activity

Abstract

In vitro induced human regulatory T cells (iTregs) have demonstrated in vivo therapeutic utility, but pathways regulating their function have not been elucidated. Here, we report that human iTregs generated in vitro from naïve cord blood cells preferentially recruit Disc large homolog 1 (Dlgh1) and exclude protein kinase C (PKC)-θ from immunological synapses formed on supported lipid bilayers with laterally mobile ICAM-1 and anti-CD3 mAb. Also, iTregs display elevated Dlgh1 overall and Dlgh1-dependent p38 phosphorylation, higher levels of phosphatase and tensin homolog (PTEN), and diminished Akt phosphorylation. Pharmacological interruption of PKC-θ increases and Dlgh1 silencing decreases the ability of iTregs to suppress interferon-γ production by CD4⁺CD25^- effector T cells (Teff). Comparison with expanded cord blood-derived CD4⁺CD25^hi tTreg and expanded Teffs from the same donors indicate that iTreg are intermediate between expanded CD4⁺CD25^hi tTregs and Teffs, whereas modulation of suppressive activities by PKC-θ and Dlgh1 signaling pathways are shared.

Figure 1

Dlgh1 and PKC-θ are recruited differently to IS in tTregs, iTregs and Teffs. MACS bead purified human CD4⁺CD25^hi (tTreg), CD4⁺CD25⁻ T cells activated by TGF-β and Rapamycin (iTregs) and CD4⁺CD25⁻ T cells (Teffs) were introduced into bilayers containing both anti-CD3 (5 µg/ml) and ICAM-1 at 250 molecules/mm fixed at 20 min and permeabilized, stained with anti-Dlgh1 (a) and anti-PKC-θ (b) antibodies and imaged by TIRFM. The panels show representative images. Dlgh1 and PKC-θ staining was quantified by calculation of average fluorescence intensity in cells. Data are representative of two different experiments. P values were calculated using Mann-Whitney test.

Figure 2

Side view of Dlgh1 and PKC-θ intracellular distribution in tTreg, iTreg and Teffs. The cells were activated using lipid bilayers as mentioned above, for 10 min at 37 °C, fixed and processed for Dlgh1 and PKC-θ immunofluorescence. Cells were imaged using line scanning confocal microscope, and images were 3D projected to reveal intracellular distribution of aforementioned proteins. Anti-CD3 (pseudocolored red) marks synaptic and proximal areas, while the proteins have been pseudocolored green. Note the enriched distribution of Dlgh1 at the proximal and of PKC-θ towards distal side in tTreg (left panels), and a reduction of this distribution in iTreg (middle panels). The panels show representative images. Dlgh1 and PKC-θ staining was quantified by calculation of average fluorescence intensity in cells. Data are representative of two different experiments. P values were calculated using Mann-Whitney test.

Figure 3

Intracellular levels of Dlgh1 and PKC-θ were determined by intracellular staining by FACS (a) or by Western Blot (b) in tTregs, iTregs and Teffs. Cells were activated by immobilized anti-CD3 antibodies (5 μg/ml) and lysed. Phosphorylation of p38 (c) and AKT (d) as well as PTEN protein levels (e) was determined by Western Blot. Data are representative of 3 different experiments, the bar charts (B-E) are means ± SEM. *P < 0.05 was calculated by t test. Full-length gels are included in Supplementary Figure 2.

Figure 4

The suppressive function of both tTregs and iTregs is concurrently regulated via Dlgh1/PKC-θ signaling pathways. Treg subsets, tTregs and iTregs were transfected with small interfering RNA (siRNA) targeting Dlgh1 or with control siRNA by AMAXA and plated in presence of IL-2 (300 IU/ml) (a) or pre-treated with PKC-θ inhibitor (AEB071) at 10 μM for 30 min and washed three times (b). SiRNA-transfected (a) or AEB071-treated (b) Tregs were mixed with CD4⁺ CD25⁻ T cells at 1:3 ratio and plated on immobilized anti-CD3 antibodies (5 μg/ml). The supernatants were analyzed for IFN-γ secretion after 48 hours by ELISA. Data are representative of two different experiments.