The exacerbation of GVHD by CD73 deficiency cannot be explained by differences in numbers of Tregs or allostimulation by DCs. (A) Lethally irradiated Cd73^+/+ or Cd73^−/− C57BL/6 mice were transplanted with 5 × 10⁶ BM cells and 15 × 10⁶ spleen cells from Cd73^+/+ or Cd73^−/− BALB/c mice, respectively. On day 6, the numbers of donor Treg (CD4⁺CD25⁺Foxp3⁺) in spleen were determined by flow cytometry (mean ± SD; n = 5). P > .05. In other experiments, lethally irradiated Cd73^+/+ BALB/c mice were transplanted with 5 × 10⁶ BM cells and 3 × 10⁵ CD4⁺/CD8⁺ T cells from Cd73^+/+ or Cd73^−/− C57BL/6 mice. Lymphocytes were isolated from the colon on day 8 and analyzed for the numbers of donor Treg (mean ± SD; n = 3). P > .05. The results are representative of 2 independent experiments. (B) In vitro expansion of CFSE-labeled WT BALB/c CD4⁺ T cells stimulated with BMDCs derived from a Cd73^+/+ or Cd73^−/− C57BL/6 mouse. Left panels: Representative histograms from one of 3 independent experiments show CFSE intensity of WT CD4⁺ cells after 5 days at different coculture ratios. Right panel: Comparison of the percentages of proliferating WT CD4⁺ T cells stimulated with either Cd73^+/+ or Cd73^−/− BMDCs for 5 days (mean ± SEM). P > .05. (C) Twenty-four hours after lethal irradiation, spleens were isolated from Cd73^+/+ and Cd73^−/− C57BL/6 mice, and the expression of CD86 on CD11c⁺CD11b⁺B220⁻ DCs was analyzed by flow cytometry. Histograms are representative of 2 independent experiments. Shaded and solid line histograms represent staining of DCs from nonirradiated mice with isotype control and anti-CD86 mAb, respectively. The dotted line histogram represents staining of DCs from irradiated mice with anti-CD86 mAb. The data are summarized as mean ± SD; mean fluorescence intensity (MFI) of CD86 staining (n = 3). P > .05.