Figure 4.

moDC XBP-1s direct human iTreg differentiation via TGFβ. T cells were cultured with B-I09 or DMSO pre-treated moDCs (moDC:T cell ratio of 1:30), and additional B-I09 (20μM) or DMSO (0.1%) was added once on day 0. (A) Percentage of Tregs (CD4⁺, CD127⁻, CD25⁺, Foxp3⁺) in the 5-day co-cultures, with representative contour plots shown (B). Means from 6 independent experiments are shown. (C) The suppressive capacity of harvested moDC-allostimulated Tregs was tested at different ratios of Treg to T cell responders stimulated by fresh allogeneic moDCs (moDC:responder T cell ratio 1:30) in alloMLRs. No additional B-I09 or DMSO was added. Graph shows mean percent T effector (Teff) proliferation measured by Ki-67. Means from 3 independent experiments are shown. To generate inducible Tregs, Treg-depleted CD4⁺ Tconv were stimulated by B-I09 or DMSO-pretreated allogeneic moDCs at a moDC:T cell ratio of 1:30. B-I09 or DMSO was added once on day 0. Magnetic bead enriched natural Tregs (CD4⁺, CD25⁺) were similarly treated in allogeneic Treg:moDC co-cultures. Treg populations were evaluated by flow cytometry on day +5. Means from 4 iTreg and 4 nTreg independent experiments are shown, paired t-test. Percentage and absolute number of iTreg (D,E) and nTreg (F,G) are shown. (H) Representative contour plots are shown for iTreg and nTreg. (I) Representative contour plots show that adding recombinant human TGFβ rescues iTreg generation in Treg-depleted alloMLRs treated with B-I09 vs. DMSO. 1 representative experiment of 2 independent studies is shown. (J) T cell proliferation (MTS colormetric assay) at day +5 among Treg-depleted alloMLRs treated with B-I09 or DMSO, with recombinant human TGFβ added as indicated. Replicate means from 4 independent experiments are shown, Dunnett's test.