AMD3100 leads to rapid and significant mobilization of Tregs into the peripheral blood. (A) Fold change in Tregs in the peripheral blood compared with baseline in animals treated with G-CSF, AMD3100, or G-CSF + AMD3100. Data were collected at baseline (before mobilization) and 2, 4, and 6 hours after treatment with AMD3100. Red squares represent G-CSF alone (n = 6); blue triangles, AMD3100 alone (n = 6); and green circles, G-CSF + AMD3100 (n = 6). Error bars represent SEM. The statistical analysis was performed as described in Figure 3. ns indicates not significant (P > .1). ^P < .1. *P < .05. **P < .01. ***P < .001. (B) Column-purified CD4⁺ T cells (left panel), flow-sorted putative non-Tregs (CD4⁺, CD25^low, CD127^high, middle panel), and flow-sorted putative Tregs (CD4⁺, CD25^high, CD127^low, right panel) were stained for CD4, CD25, and intracellular FoxP3, documenting that the putative Treg population (right panel) stained highly positive for FoxP3. (C) Expanded Tregs were stained for CD4, CD25, and either FoxP3 (right) or its corresponding isotype control (left). (D) CFSE MLR assay gated on either CD4⁺ T cells (top row) or CD8⁺ T cells (bottom row) in which the percentage of CFSE-labeled responder cells that have undergone at least one round of proliferation was quantified using FlowJo Version 9.3.1 flow cytometric analysis software. Left column: Responder PBMCs plus irradiated autologous PBMC stimulator cells. Middle column: Responder PBMCs plus irradiated allogeneic PBMC stimulator cells. Right column: Responder PBMCs plus irradiated allogeneic PBMC stimulator cells, plus CD3⁺, CD4⁺, CD25^high, CD127^low, FoxP3⁺ Tregs.