Figure 6. New CSB-MLR co-culture conditions with SPL as stimulators support Treg expansion in post-LTx PBMCs.

PBMCs from post-LTx patients were co-cultured using new MLR conditions (1.5×10⁶ cells/mL and 2R:1S ratio) with irradiated SPL as stimulators in the presence of belatacept-mediated CSB.

(A) CSB-MLR demonstrated expected decreases in proliferation, measured by ³H-thymidine incorporation on day 6, showed an overall median percentage inhibition of 76% (range, 88 – 66%).

(B) Frequencies of CD25^highFoxp3⁺ Tregs expressed as percentage of CD3⁺CD4⁺ T cells at day 0 (baseline) and day 3 CSB-MLR (CSB) showed an overall 1.9-fold median Treg expansion.

(C) Individual flow cytometry plots demonstrate the increase in CD25 expression and the frequency of CD25^highFoxp3⁺ Tregs within CD3⁺CD4⁺ gate prior to MLR (baseline) and at day 3 after primary CSB-MLR culture (CSB).

(D-E) Characterization of CSB-MLR Treg from post-LTx PBMC samples. Conventional CD4⁺ T cells (Tconv) and natural Tregs without CSB (baseline Treg) from post-LTx patients were used as controls. (D) Representative histograms and MFI values of the Treg marker Foxp3 in post-LTx CSB-Tregs (CD4⁺CD25^highFoxp3⁺) showed high Foxp3 expression. (E) TSDR demethylation in CSB-Treg isolated by multicolor fluoresce-activated cell sorting (CD4⁺CD25^highCD127⁻). The demethylation rates were translated into a color code from yellow (0%) through green (50%) up to blue (100%). Each rectangle represents the demethylation of one CpG motif. Post-LTx CSB-Treg showed retention of high TSDR demethylation. (Tconv n=2; baseline Treg n=3; CSB-Treg n=3).

HC: healthy controls; PBMC: peripheral blood mononuclear cells; CSB: costimulatory blockade; MLR: mixed lymphocyte reaction; post-LTx: post liver transplant; SPL: splenocytes; cpm: counts per minute; Treg: regulatory T cell. MFI: mean fluorescence intensity; Tconv: conventional CD4⁺ T cells; TSDR: Treg-specific demethylated region; Treg: regulatory T cell