Resistance to regulatory T cell-mediated suppression in rheumatoid arthritis can be bypassed by ectopic foxp3 expression in pathogenic synovial T cells

Abstract

Increasing evidence suggests that regulatory T cell (Treg) function is impaired in chronic inflammatory diseases such as rheumatoid arthritis (RA). Here we demonstrate that Tregs are unable to modulate the spontaneous production of TNF-α from RA synovial cells cultured from the diseased synovium site. Cytokine (IL-2, IL-6, TNF-α) activated T cells (Tck), cells we previously demonstrated to mimic the effector function of pathogenic RA synovial T cells, contained Tregs that survived and divided in this cytokine environment; however, the up-regulation of key molecules associated with Treg function (CTLA-4 and LFA-1) was impaired. Furthermore, Tregs were unable to suppress the function of Tcks, including contact-dependent induction of TNF-α from macrophages, supporting the concept that impaired Treg function/responsiveness contributes to chronicity of RA. However, ectopic foxp3 expression in both Tcks and pathogenic RA synovial T cells attenuated their cytokine production and function, including contact-dependent activation of macrophages. This diminished response to cytokine activation after ectopic foxp3 expression involved inhibited NF-κB activity and differed mechanistically from that displayed endogenously in conventional Tregs. These results suggest that diseases such as RA may perpetuate owing to the inability of Tregs to control cytokine-activated T-cell function. Understanding the mechanism whereby foxp3 attenuates the pathogenic function of synovial T cells may provide insight into the mechanisms of chronicity in inflammatory disease and potentially reveal new therapeutic candidates.

Fig. 1.

RA MNC cytokine production is not modulated by Tregs. CD4⁺CD25⁺ blood Tregs from healthy donors were cocultured with RA synovial MNCs with soluble anti-CD3, and supernatants were harvested at 24 h for TNF-α by ELISA. A representative titratable experiment (A) and pooled data (B) (n = 19) are shown; and where RA MNCs spontaneously produced <500 pg/mL of TNF-α a representative titratable experiment (C) and pooled data (D) (n = 9) are illustrated. Similarly IL-6 (E) and IL-1β (F) levels were determined in RA MNC cultures with or without CD4⁺CD25⁺ Tregs. IFN-γ was not detectable in supernatants/cell lysates. **P < 0.01, ***P < 0.001. n.s., not significant.

Fig. 2.

Tregs do not suppress Teffs in cytokine-activated cultures. (A) Proportion of cells expressing foxp3 (mean ± SEM; n = 28) was determined before and after (8 d) stimulation with the Tck cytokine mixture by flow cytometry. (B) Carboxyfluorescein succinimidyl ester (CFSE) content of foxp3⁺ and foxp3⁻ populations of CD4⁺CD45RO⁺ cells at day 8 (n = 3). CD4⁺CD45RO⁺CD25⁻ (Teff) cells were cultured in the presence (gray bars) or absence (black bars) of CD4⁺CD45RO⁺CD25⁺ (Treg) cells at a 1:1 ratio and stimulated with either (C) anti-CD3 (1 μg/mL)/anti-CD28 (2.5 μg/mL) or (D) the Tck cytokine mixture. Proliferation was assessed by ³H-thymidine incorporation at 102–120 h. IFN-γ production was determined after 120 h (anti-CD3/anti-CD28) or 8 d (Tck cytokine mixture) (n > 3). Cells were stimulated for (E) 3 d with anti-CD3/anti-CD28 or (F) 8 d with Tck cytokine mixture, then cocultured for 8 h with autologous macrophages transduced with a TNF-α–luciferase reporter. Representative experiments of TNF-α levels (ELISA) and expression of TNF-α reporter are shown. **P < 0.01. n.s., not significant.

Fig. 3.

Cytokine stimulation does not induce fully activated Treg phenotype. CD4⁺CD45RO⁺ cells were isolated from PBLs by negative selection (5) and activated with either anti-CD3 for 48 h or Tck cytokine mixture for 8 d and expression of intracellular CTLA-4 (A), CD69 (B), CD18 (LFA-1 β-chain) (C), and CD11a (LFA-1 α-chain) (D) on foxp3^hi gated cells determined by flow cytometry (n = 4).

Fig. 4.

Ectopic foxp3 expression attenuates CD4+ effector function. CD4⁺ lymphocytes isolated from either blood of healthy donors (A–D) or RA synovial MNCs (E–G) were transduced with either pCCL or pCCL-foxp3 lentivirus. NGFR⁺ cells were restimulated with either anti-CD3 (5 μg/mL) for 24 h or the Tck cytokine mixture for 5 d. RA synovial CD4⁺ lymphocytes were restimulated with the Tck cytokine mixture. Ratios indicate T cell:macrophage proportions. Representative experiments of >n = 3 are shown and illustrate cytokine production from anti-CD3 activated (A) and cytokine-stimulated (B) T lymphocytes. TNF-α reporter gene and TNF-α protein levels are shown for cocultures with anti-CD3 (C) and cytokine-stimulated (D) T lymphocytes. (E) Phenotype of synovial CD4⁺ lymphocytes was determined after transduction with pCCL (black) or pCCL-foxp3 (blue) lentivirus. (F) IFN-γ production from RA CD4⁺ lymphocytes and (G) TNF-α reporter gene activity and protein levels from RA CD4⁺ lymphocyte:macrophage cocultures after transduction with pCCL-foxp3. *P < 0.05, **P < 0.01, ***P < 0.001, ^†cytokine concentration below limits of ELISA.

Fig. 5.

Ectopic foxp3 expression attenuates NF-κB transcription. Kinetics of p65 and STAT-3 phosphorylation were determined in Teffs and nTregs (A) and pCCL and pCCL-foxp3 (B) transduced cytokine-stimulated Teffs by flow cytometry (mean ± SEM of > n = 3). (C) CD4⁺ cells transduced with NF-κB-luciferase reporter lentivirus and pCCL/pCCLfo×p3 were stimulated with the Tck cytokine mixture for 2 h and luciferase activity determined (mean ± SEM, n = 4; *P < 0.05).