Figure 3. CRIg promotes iTreg generation in vitro.

(A) (left) Representative FACS plots depicting the generation of iTreg cells in the presence of CRIg-Ig, or control Ig, under various concentrations of TGF-β. (right) Statistics of multiple experiments. (B) Tconv cells were cultured in the condition of anti-CD3/CD28 and IL-2, anti-TGF-β neutralizing antibody (clone 1D11) and either control Ig, or CRIg-Ig. (C) Total splenocytes from 8-week-old NOD/BDC2.5/Thy1.1 mice were labeled with CTV and cultured with BDC2.5 mimotope (100 ng/ml) for 3 days. The generation of Treg cells were analyzed by intracellular Foxp3 staining. (D) iTreg generation in vitro as in (C) with the inclusion of CRIg⁺ or CRIg^- macrophages, sorted from peritoneal cavity. (E) Purified CD4⁺Foxp3 (GFP)^- T cells from NOD/BDC2.5/Foxp3^GFP/Thy1.1 mice were transferred into 4-week-old NOD mice, followed by i.p. injection of CRIg-Ig, or control Ig every other day for 2 weeks. (F) Purified CD4⁺Foxp3 (GFP)^- T cells from NOD/BDC2.5/Foxp3^GFP/Th1.1 mice were transferred into 4-week-old NOD or NOD/CRIg KO mice. The generation of Foxp3(GFP)⁺ cells in pancreatic islets, panLNs and inguinal LNs(ILNs) was analyzed 2 weeks after the transfer. (G) Flow cytometric analyses of Helios^- Treg cells from pancreas, colon and lung of NOD/CRIgKO and wildtype controls. (H, I) Purified CD4⁺ Foxp3(GFP)^- T cells were cultured with either control Ig or CRIg-Ig for 18 hr and analyzed for the phosphorylation of AKT (H) and ribosomal protein S6 (I). Dotted line, control Ig; solid line, CRIg-Ig. Data are representative of five (A), three (B–I) experiments. Student’s t-test was used. n.s., non-significant. *p<0.05; ***p<0.001; ****p<0.0001. Ctl, control; KO, CRIg knockout.

Figure 3—figure supplement 1. CRIg-Ig does not enhance TGF-β induced phosphorylation of Smad2/3.

CD4⁺ CD25^- cells were sorted and cultured with 0.2% of fetal bovine serum under iTreg differentiation condition. Phosphorylation of Smad2/3 was detected 2 hr after TGF-β stimulation by flow cytometry. Data are representative of three experiments. n.s., non-significant.

Figure 3—figure supplement 2. Experimental design and FACS profiles of in vivo pTreg generation promoted by CRIg-Ig.

(A) Schematic diagram depicting the experimental design. (B) Representative FACS profiles of gating strategy showing the engraftments of transferred T cells and the proportions of GFP⁺ cells. The top-right panel was negative control for GFP gating.