Figure 5. New CSB-MLR coculture conditions support scale up of Treg expansion in healthy PBMC.

HC PBMC were co-cultured with irradiated PBMC or SPL stimulators in the presence of belatacept-mediated CSB using standard culture conditions (1×10⁶ cells/mL and 1R:1S ratio) or new optimized conditions (1.5×10⁶ cells/mL and 2R:1S ratio)

(A) Percent inhibition of proliferation (measured by ³H-thymidine incorporation) on day 6 of primary CSB-MLR was not significantly different using standard or new CSB-MLR co-culture conditions with either PBMC (left) or SPL (right) as stimulators. (n=10–34)

(B) Under new CSB-MLR culture conditions, measurement of CD25^highFoxp3⁺ Treg frequency, expressed as percentage of CD3⁺CD4⁺ T cells, prior to MLR (baseline) and after primary CSB-MLR (CSB) demonstrated similar Treg expansion using PBMCs (n=5) or SPL (n=10) as stimulators.

(C-D) Characterization of CSB-MLR Treg manufactured using different culture conditions. Conventional CD4 T cells (Tconv) and natural Treg without CSB (baseline Treg) were used as controls. (C) Representative histograms and MFI values of the Treg marker Foxp3 in CSB-Treg (CD4⁺CD25^highFoxp3⁺) showed no differences in Foxp3 expression. (D) TSDR demethylation in CSB-Tregs isolated by multicolor fluorescence-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. All CSB-Tregs showed comparable high demethylation pattern. (Tconv n=1; baseline Treg n=5; CSB-Treg n=2–5)

Horizontal lines are medians, boxes are 25^th and 75^th percentiles, and whiskers are maximum and minimum values. The p-values were calculated using unpaired T test with Welch’s correction (A) and paired t-test (B) with significant p-values highlighted in bold. HC: healthy controls; MLR: mixed lymphocyte reaction; CSB: costimulatory blockade; SPL: splenocytes; Std: standard culture conditions; New: new optimized culture conditions; MFI: mean fluorescence intensity; Tconv: conventional CD4 T cells; TSDR: Treg-specific demethylated region; Treg: regulatory T cell