Fig. 1: Polyclonal versus alloantigen-specific expansion of donor Treg generates cell products with similar phenotypic properties but different TCR diversities.

a Schematic presentation of in vitro expansion strategies. b In vitro expansion rates of polyclonal (poly) and alloantigen-specific (allo) Treg cultures (n = 12 each). Two-tailed t-test. c Barplot of Tcrb clonotype diversities (iSI, inverse Simpson index) in independent isolations of C57BL/6 Treg from spleen (fresh, d0) or expanded Treg (allo, poly, d11–14) as measured by UMI-based sequencing of 5’RACE products of Tcrb mRNAs. Bars represent means ± SEM of n = 6 independent experiments, significant differences between groups are indicated above bars (one-way ANOVA with Tukey’s post hoc test). Individual data points are shown as dots. d FACS analysis of the expression of canonical Treg markers FoxP3 and Helios, as well as Ki-67 as a marker for proliferation. Representative expression profiles of polyclonal or alloantigen-specific in vitro expanded Treg (upper panels) and percent marker-positive cells in five independent Treg cultures (lower panels) are shown. Representative gating examples for each marker are shown in Supplementary Fig. 1e. Bars in the lower panels represent means ± SD of the indicated number of independent biological replicates. Individual data points are shown as dots. e Scatter plot indicating genes differentially expressed in allo versus polyTreg (as measured by RNA-seq, n = 6 independent experiments each). Treg-signature genes (based on²⁶ plus additional markers, listed in Supplementary Table 1) are labeled. f Heatmap comparing expression levels of Treg signature genes in independent isolations of C57BL/6 Treg from spleen (fresh, d0) or in vitro expanded Treg (allo, poly). b–f Source data are provided as a Source Data file.