Figure 3.

Hematopoietic reconstitution replaces >95% of resident CD45⁺ cells. We assessed the efficiency of hematopoietic chimerism in our experimental protocol by making MHC class I mismatched chimeras. Bone marrow from B6 mice (MHC haplotype b) or HeOuJ mice (MHC haplotype k) were transferred into irradiated HeJ mice (MHC haplotype k). Two months after reconstitution, spleens were isolated from chimeric mice. Single-cell suspensions from spleen were labeled with antibodies to CD45 as well as antibodies specific to the MHC Class I molecules (H-2K) of the k (H-2K^k) or b (H-2K^b) haplotype. Staining was analyzed on a BD FACScalibur. Plots show MHC staining observed on cells that are CD45⁺ (gated). In the case of mice receiving HeOuJ bone marrow (a), ∼96% of the CD45⁺ cells were of the HeOuJ origin. In the case of mice receiving the B6 bone marrow (b), ∼91% of the cells stained for the B6 Class I molecule. Although ∼2% of the cells fell within the bottom right quadrant, suggesting a staining by the k haplotype, there was no distinct population corresponding to the MHC-expressing cells (compare bottom right quadrants in a). This allows us to conclude that our protocols were successful in generating at least 90-95% donor-derived hematopoietic chimerism with no significant contribution from the recipient toward this compartment. Although this analysis was done using MHC-mismatched chimeras involving a protocol for depletion of resident NK cells before reconstitution, those WT and HeJ mice that were used to construct the experimental chimeras did not involve a mismatch at the MHC locus.