FA Treg cells show lower suppression potential. (A) FA deficiency does not alter Treg cell number in native mice. Splenocytes from mice with the indicated genotypes were subjected to flow cytometry analysis for CD4, CD25, Foxp3 staining. CD4⁺ cells were gated for analysis of CD25⁺Foxp3⁺ cell portion. Representative flow graph (left) and quantification (right) are shown. Results are means plus or minus SD of 2 independent experiments (n = 6 per group). (B) FA Treg cells fail to suppress Teff cell proliferation in vitro. Carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled WT CD4⁺CD25⁻ Teff cells were cocultured with CD4⁺CD25⁺ cells freshly isolated from either WT or Fanca^−/−, Fancd2^−/− mice at a ratio of 2:1 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum for 5 days followed by flow cytometry analysis for CFSE retention. Data were analyzed by FlowJo software. Representative flow graph (left) and quantification (right) are shown. Results are means plus or minus SD of 3 independent experiments (n = 9 per group). (C) FA Treg cells are less suppressive in preventing GVHD in vivo. Lethally irradiated Balb/c recipients were transplanted with 5 × 10⁶ TCD BMCs from WT C57BL/6 animals alone, or with sorted WT Teff cells (CD4⁺CD25⁻, 5 × 10⁵) plus or minus equal numbers of sorted CD4⁺CD25⁺ Treg cells from either WT C57BL/6, Fanca^−/−. Survival of recipients was monitored by Kaplan-Meier curve method. Each group includes 7 to 10 mice. (D) No difference in cell number of donor-derived Treg cells. Splenocytes isolated from recipients described in (C) were subjected to Flow cytometric analysis for H-2^b, CD4, Foxp3 staining. Donor H-2^b+ cells were gated for CD4⁺Foxp3⁺ cell portion. Representative flow graph (left) and quantification (right) are shown.