We previously reported that bone marrow-derived CD8⁺/TCR^– graft facilitating cells (FC) significantly enhance engraftment of HSC and prevent GVHD in mouse recipients. We have presently characterized a similar human CD8⁺/TCR^– FC that comprised 1.48%±0.32% of total G-CSF-mobilized peripheral blood mononuclear cells. Approximately 55% of FC express CD56^dim/- and the remaining express CD56^bright. CD56^dim/- FC contain CD3⁺ cells and CD19⁺ cells that highly express CXCR4. CD56^bright FC contain CD11c⁺ and CD11b⁺ cells. To evaluate engraftment potential of human CD56^dim/- or CD56^bright FC subpopulations of human HSC, we transplanted 100,000 HSC alone or plus 300,000 CD56^dim/- or CD56^bright FC into NOD/SCID/IL2rΓ^null (NSG) mice conditioned with 325 cGy of TBI. Only 34% of recipients of HSC alone engrafted compared to 65% of HSC plus CD56^bright FC and 78% of HSC plus CD56^dim/- FC. Donor chimerism in PB at one month was 1.1%±0.8%, 1.6%±0.4%, and 4.1%±1.3% respectively. Donor chimerism of HSC alone recipients at 6 months was 3.8%±3.5% in PB, 2.9%±1.3% in BM and 12.3%±9.8% in spleen. In contrast, HSC plus CD56^bright FC or CD56^dim/- FC exhibited durable donor chimerism and showed higher level of donor chimerism in PB (8.8%±6.4% or 9.1%±6.0%), in BM (14.5%±7.2% or 11.6%±4.8%), and in spleen (8.2%±3.3% or 26.3%±11.3%). To evaluate if FC enhance homing of HSC to BM, HSC plus CD56^bright or CD56^dim/- FC were transplanted into NSG mice supralethally conditioned with 1100 cGy of TBI. BM cells were harvested from recipients at 16 hours after transplantation and placed in colony forming cell (CFC) assay. Recipients of HSC plus CD56^dim/- FC generated significantly more colonies compared with HSC plus CD56^bright FC or HSC alone (P<0.05), indicating that CD56^dim/- FC enhance homing of donor HSC and progenitors to the BM. To test if CD56^dim/- FC promote HSC differentiation, HSC were incubated with CD56^dim/- FC for 18hrs and cultured for 14 days in CFC assay. HSC plus CD56^dim/- FC generated significantly more colonies compared with HSC alone (P=0.038), suggesting that human CD56^dim/- FC have a direct effect on clonogenicity of HSC. Our data indicate that: 1) human CD8⁺/TCR^– FC are heterogeneous; 2) CD56^dim/- FC enhance homing of HSC to BM and promote HSC clonogenicity; and 3) both CD56^dim/- and CD56^bright FC maintain a durable and high level of donor chimerism in PB, BM, spleen.

Bozulic, L.: Employee, Regenerex. Ildstad, S.: Other, Regenerex, a Biotech Start-Up Company, Equity/Employee. Colvin-Adams, M.: Other, Regenerex, a Biotech Start-Up Company, Equity/Employee.

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