Frequency of regulatory T cells after early-gestation IUHCT. Allogeneic transplantation was performed at 14 DPC by injection of WBM cells into the vitelline vein (A), which results in long-term (B), multilineage (C) donor cell chimerism. All fetuses from a litter were injected. To account for the effect of maternal milk-borne antibodies on engraftment, all litters were fostered immediately after birth with naive Balb/c dams. Of the 69 fetuses injected, 48 (70%) survived to birth and 46 (67%) survived to weaning at 4 weeks of age. Among these survivors, 42 (91%) macroengrafted. (D) To evaluate the possible contribution of regulatory T cells to tolerance and engraftment, B6^FoxP3GFP WBM cells were injected into Balb/c^FoxP3GFP fetuses at 14 DPC, and the resulting chimeric mice were analyzed at multiple times after birth. Donor- and host-derived traditional Tregs (E) and type 1 regulatory T cells (Tr1 cells) (F) were first measured as a percentage of CD4⁺ cells in the spleen, LNs, BM, and PB at 4 weeks of age and compared with uninjected, age-matched B6^FoxP3GFP and Balb/c^FoxP3GFP controls. The frequency of traditional Tregs (G) and Tr1 cells (H) was then assessed in the spleen at additional times. Early-gestation IUHCT was next performed using the B6^IL-10GFP→Balb/c strain combination. (I) The percentage of donor-derived CD4⁺ cells expressing IL-10 was measured in the spleen, BM, LN, and PB at 4 weeks of age. Mean and standard deviation are included in addition to individual data points. Data were analyzed using ANOVA with multiple comparisons with statistically significant differences indicated by * (P < .05).