Research ArticleIMMUNOLOGY

Inhibition of two-pore channels in antigen-presenting cells promotes the expansion of TNFR2-expressing CD4+Foxp3+ regulatory T cells

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Science Advances  30 Sep 2020:
Vol. 6, no. 40, eaba6584
DOI: 10.1126/sciadv.aba6584
  • Fig. 1 In vitro treatment with TPC inhibitors up-regulates tmTNF expression on APCs.

    (A to E) Raw264.7 cells, DC2.4 cells, mouse BMDCs, or human MoDCs were treated with 1 μM tetrandrine. After 48 hours, tmTNF expression was analyzed by FCM (A and B) or by Western blot (C) [total TNF in (E), Raw264.7 cell lysate]. Supernatant TNF levels were determined by enzyme-linked immunosorbent assay (ELISA) (D). MFI, mean fluorescence intensity. (F to H) BMDCs were treated with 100 μM diltiazem (DIL), nimodipine (NIM), verapamil (VER), or Ned 19 (NED) for 48 hours. tmTNF expression was analyzed by FCM (F and G). Supernatant TNF levels were determined by ELISA (H). (I to K) Mouse DC2.4 cells were transfected with TPCN1-specific (I and J) and TPCN2-specific (I and K) siRNA. After 48 hours, tmTNF was analyzed by FCM. Data were presented as means ± SEM (n = 3 to 6 independent experiments). By comparison with vehicle control group, *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., no significant differences.

  • Fig. 2 BMDCs pretreated with tetrandrine selectively stimulate the in vitro proliferation of Tregs.

    (A to C) BMDCs pretreated with tetrandrine (DC-TET) or control (DC-Ctrl) were cocultured with CFSE-labeled WT CD4 T cells, with or without IL-2 or anti-TNF antibody. After 72 hours, proliferation of Tregs or Teffs was assessed by CFSE dilution assay. (A) Typical FCM histogram (the number indicated proportion of replicating cells) and division index of Tregs (B) and Teffs (C) were shown. (D and E) WT or TNFR2-deficient CD4 cells were labeled with CFSE and cocultured with DC-Ctrl or DC-TET for 72 hours, and division index of Tregs (D) or Teffs (E) was determined by FCM. Data were displayed as means ± SEM; n = 3 independent experiments. Compared with the indicated group, *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., no significant differences.

  • Fig. 3 In vivo treatment with tetrandrine expands Tregs in a TNF/TNFR2-dependent manner.

    (A to E) WT mice were injected with tetrandrine (TET; intraperitoneally) for 3 days. Spleen and lymph nodes were harvested 24 hours after the last injection. tmTNF expression on CD11c+ cells (A), proportion of Tregs in splenic CD4 cells (B), number of Tregs (C), and expression of Ki-67 (D) and TNFR2 (E) by splenic Tregs were analyzed by FCM. (F to J) Three types of gene KO mice, including TNFR1 KO (F and H), TNFR2 KO (G and I), or TNF KO (J) mice, were injected with tetrandrine (intraperitoneally) for 3 days. tmTNF expression on splenic CD11c+ cells (F and G), proportion of Tregs in splenic CD4 cells, and number of Tregs in spleen (H to J) were analyzed by FCM. For typical FCM plots, the number indicated proportion of gated cells. Data (means ± SEM) were representative of three separate experiments with similar results (B and C, n = 6; G and J, n = 4; others, n = 3 mice). Compared with vehicle control, *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., no significant differences.

  • Fig. 4 Knockdown of TPCs induces Treg proliferation.

    (A to E) WT mice were intraperitoneally injected with TPC siRNAs or nontargeting control for 2 days, and 24 hours later, tmTNF expression on splenic CD11c+ cells (A), proportion and number of splenic Tregs (B and C), and expression of Ki-67 (D) or TNFR2 (E) by Tregs were analyzed by FCM. (F to K) Two types of gene KO mice, including TNFR1 (F to H) or TNFR2 KO mice (I to K), were intraperitoneally injected with TPC siRNAs for 2 days. tmTNF expression on splenic CD11c+ cells (F and I) and proportion and number of Tregs in the spleen (G, H, J, and K) were analyzed by FCM. (L and M) WT mice were intraperitoneally injected with lentivirus encoding Tpcn1 or Tpcn2 or control. Twenty-four hours later, mice were treated with tetrandrine for 3 days. The proportion of Tregs in splenic CD4 cells (L) and the number of splenic Tregs (M) were analyzed by FCM. For typical FCM plots, the number indicated proportion of gated cells. Data (means ± SEM, n = 3 mice) were representative of three separate experiments. Compared with the indicated group, *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., no significant differences.

  • Fig. 5 Tetrandrine inhibits colitis by promoting expansion of Tregs.

    (A) Schematic diagram of experimental procedure. In brief, naïve CD4 T cells (Teffs, 4 × 105 cells per mouse) from Ly5.1 B6 mice (CD45.1+) alone or together with CD4+Foxp3/GFP+ cells (Tregs, CD45.2+, 2 × 104 cells per mouse) were injected intraperitoneally into Rag1−/− mice. The mice were treated with tetrandrine (100 mg/kg per day, intraperitoneally) or vehicle control twice a week, starting from the second day after cell injection, for 5 weeks. The mouse colon and spleen were harvested on week 8 after cell transfer. (B to E) Proportion of Tregs (CD45.2+) in colonic CD4 cells before (B, left) or 8 weeks after transfer (B, middle and right), number of splenic Tregs (C), TNFR2 expression on colonic Tregs (D), and tmTNF expression on colonic CD11c+ cells (E) were analyzed by FCM. (F) Hematoxylin and eosin staining of colon. (G) Histological score. (H) Length of colon. (I) Body weight change (% of initial). Arrows indicated inflammatory cell infiltrates. Data (means ± SEM) shown (n = 4 to 5 mice) were representative of three separate experiments. Compared with the indicated group, *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., no significant differences.

  • Fig. 6 Inhibition of TPC down-regulates mTACE expression on DCs.

    (A to C) BMDCs or Raw264.7 cells (D) were treated with 1 μM tetrandrine or vehicle for 48 hours. (A and B) The surface expression of TACE was analyzed by FCM. (C) TACE mRNA was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). (D) Membrane or total levels of TACE were analyzed by Western blot. (E and F) WT mice were intraperitoneally injected with nilotinib and/or tetrandrine for 3 days. After 24 hours, the proportion of Tregs in splenic CD4 cells (E) and the number of Tregs in the spleen (F) were analyzed by FCM. (G and H) BMDCs were treated with 100 μM diltiazem (DIL), nimodipine (NIM), verapamil (VER), or Ned 19 (NED). After 48 hours, the surface expression of TACE was analyzed by FCM. (I to K) BMDCs were treated with TPCN1- or TPCN2-specific siRNA or nontargeting control. The surface expression of TACE was determined by FCM. Data were represented as means ± SEM (B to D, H, J, and K: n = 3 to 6 independent experiments; E and F: n = 3 mice, representative of three separate experiments with similar results). Compared with the indicated group, *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., no significant differences.

Supplementary Materials

  • Supplementary Materials

    Inhibition of two-pore channels in antigen-presenting cells promotes the expansion of TNFR2-expressing CD4+Foxp3+ regulatory T cells

    Tianzhen He, De Yang, Xiao-Qing Li, Mengmeng Jiang, Md Sahidul Islam, Shaokui Chen, Yibo Chen, Yang Yang, Chon-Kit Chou, Anna L. Trivett, Joost J. Oppenheim, Xin Chen

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