Figure 1. Expansion of cynomolgus monkey FoxP3⁺ Treg.

(A) Treg expansion protocol. Treg (CD4⁺CD25^hiCD127⁻) were flow-sorted from fresh PBMC or cryopreserved PBMC or LN cells on d 0, and expanded using NHP-specific expansion beads, high dose rhu IL-2 and rhu TGF-β. Simultaneously, conventional T effector cells (Teff; CD4⁺CD25⁻) were sorted and expanded using beads and IL-2, but without TGF-β. Assays were carried out on the days indicated by arrows.

(B) Strong expansion of Treg from fresh PBMC. Treg were flow-sorted from fresh PBMC (n=3 experiments), or cryopreserved (stored) PBMC (n=3) or LN cells (n=4), then expanded following the protocol indicated in (A). Treg isolated from fresh PBMC (continuous line) expanded at a much faster rate than Treg sorted from either stored PBMC (dashed line) or stored LN cells (broken lines).

(C) Significant up-regulation of FoxP3 in expanded Treg. Fresh PBMC were stained for CD3, CD4, CD25, CD127, and FoxP3. FoxP3 MFI was analyzed on fresh CD3⁺CD4⁺ T cells, on fresh CD4⁺CD25^hiCD127⁻ Treg and fresh CD4⁺CD25⁻ Teff. In the same experiment, expanded Treg and Teff were also stained, gating on CD4⁺ cells, and analyzed for FoxP3 at the end of round 1 and 2 of expansion. Expanded T cells showed increased FoxP3 expression, that was greater in expanded Treg than in expanded Teff (n=3 experiments for all conditions). *p<0.05.

(D, E) Expanded Treg exhibit strong suppressive function on CD4⁺ and CD8⁺ T cell proliferation. When sufficient cells were available, Treg were tested for suppressive function in CFSE-MLR, as described in the Materials and Methods. Expanded Treg (upper panels in D and black bars in E) showed strong suppressive capacity when added to bead-stimulated CD2⁺ autologous T cells, whereas expanded Teff (lower panels in D and gray bars in E) did not. Treg were strongly suppressive at ratios of up to 1Treg:4 CD2⁺ T cells. *p<0.05; **p<0.01. Data are representative of 3 experiments (D) and analyzed across experiments (E).