tTregs accumulate in all lymphoid organs of PTCy-treated chimeras, whereas generation of pTregs is most prominent in lymph nodes. Chimeras were constructed as in Figure 2, except that the GVH inocula contained 1.8 × 10⁶ sorted CD4⁺ Foxp3^GFP/RFP– cells (Tcons), supplemented with 2 × 10⁵ CD4⁺Foxp3^RFP/GFP+ (Tregs). Designated groups received PBS or PTCy (200 mg/kg IP) on day +3 after alloBMT. Animals were euthanized on day +28 and blood and organs processed and analyzed by multicolor flow cytometry. (A) Percentages of tTregs and pTregs in peripheral blood, spleen, CLNs, and mesenteric lymph nodes (MLNs). (B) Representative flow data plots gated on CD45.2⁺ CD3⁺CD4⁺ lymphocytes from CLNs and MLNs of a control and a PTCy-treated mouse depict proportions of Foxp3⁺ tTregs and pTregs. (C) Total counts of tTregs and pTregs in the CLNs, MLNs, and spleens of control and PTCy-treated chimeras. Data shown here represent 2 independent experiments with at least 10 animals per group.