Scaffold protein Disc large homolog 1 is required for T-cell receptor-induced activation of regulatory T-cell function

Abstract

Foxp3(+)CD4(+)CD25(high) regulatory T cell (Treg) suppression of inflammation depends on T-cell receptor-mediated Nuclear Factor of Activated T cells c1 (NFATc1) activation with reduced Akt activity. We investigated the role of the scaffold protein Disc large homolog 1 (Dlgh1) in linking the T-cell receptor to this unique signaling outcome. The Treg immunological synapse (IS) recruited fourfold more Dlgh1 than conventional CD4(+) T-cell IS. Tregs isolated from patients with active rheumatoid arthritis, or treated with tumor necrosis factor-α, displayed reduced function and diminished Dlgh1 recruitment to the IS. Furthermore, Dlgh1 silencing abrogated Treg function, impaired NFATc1 activation, reduced phosphatase and tensin homolog levels, and increased Akt activation. Dlgh1 operates independently of the negative feedback pathway mediated by the related adapter protein Carma1 and thus presents an array of unique targets to selectively manipulate Treg function.

Fig. 1.

Dlgh1 is strongly recruited to IS in Tregs. Freshly FACS-sorted (A) and MACS bead (B) purified human blood CD4⁺CD25^hi (Treg) and CD4⁺CD25⁻ T cells or expanded umbilical cord blood (UCB)-derived Treg and CD4⁺CD25⁻ T cells (C) were introduced into bilayers containing both anti-CD3 (5 μg/mL) and ICAM-1 at 250 molecules per mm² (A and C) or anti-CD3 or ICAM-1 molecules alone (B), fixed at 8 min and permeabilized, stained with anti-Dlgh1 antibodies, and imaged by TIRFM. Shown are representative images. Dlgh1 staining was quantified by calculation of average fluorescence intensity in cells. Data are representative of three different experiments. P values were calculated by Mann–Whitney test.

Fig. 2.

Dlgh1 recruitment to IS correlates with Treg suppressive function. Freshly purified Tregs from healthy donors or RA patients were untreated (A) or TNF-α–treated (50 ng/mL for 24 h) (B), introduced to bilayers with anti-CD3 and ICAM-1, fixed, and imaged by TIRFM. Shown are representative images. Dlgh1 staining was quantified by calculation of average fluorescence intensity in cells. Data are representative of seven (A) or three (B) different experiments. P values were calculated by Mann–Whitney test.

Fig. 3.

Dlgh1 is required for Treg function. (A) Freshly purified human Tregs 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). After 48 h Dlgh1 expression was measured by Western blot analysis. (B) siRNA-transfected Tregs were mixed with CD4⁺CD25⁻ T cells at a 1:3 ratio and activated with anti-CD3/CD28 dynal beads. CD4⁺CD25⁻ T-cell proliferation was determined after 96 h by CFSE dilution. Representative experiment of three is shown. (C) The supernatants were analyzed for IFN-γ, IL-17, and IL-4 after 48 h. (D and E) Foxp3 expression was determined by flow cytometry 48 h after Treg transfection (D), and average of three different experiments is shown (E). (F and G) Some Tregs 48 h after transfection were introduced to bilayers with anti-CD3 and ICAM-1, fixed, stained for PKC-θ, and imaged by confocal microscopy (F) or treated with PKC-θ inhibitor, C-20 (1 μM, 30 min), washed, and then mixed with untreated CD4⁺CD25⁻ T cells at ratio 1:3 (G). Average of three (E–G) or four (A and C) different experiments are shown. P values were calculated by t test. *P < 0.05.

Fig. 4.

Dlgh1 controls Treg function via p38/NFAT and PTEN/Akt signaling pathways. (A, B, D, and E) siRNA-transfected Tregs (48 h after transfection) were activated (A, B, and E) or not (D) by immobilized anti-CD3 antibodies (5 μg/mL) and lysed. (A, D, and E) PTEN levels as well as p38 and AKT phosphorylation were determined by Western blot analysis. Numbers represent the intensity of specific bands divided by intensity of loading controls and multiplied by 100. Representative results of three independent experiments are shown. (B) NFATc1 activation (Left) and p50-specific binding to NF-κB consensus sequence (Right) were tested by ELISA. Average of three independent experiments is shown. (C) Dlgh1 coimmunoprecipitates with PTEN, but not with control IgG, in freshly purified human Tregs. Representative results of two independent experiments are shown. (F) Dlgh1 is required for activation of p38 and NFAT, which synergizes with Foxp3 and provides a positive feed-forward signal for Treg function. In addition, Dlgh1 interaction with PTEN inhibits AKT pathway that negatively regulates Treg function. P values were calculated by t test. *P < 0.05.