Graft CD8⁺TCR^– facilitating cells (FC) enhance engraftment of hematopoietic stem cells (HSC) in allogeneic mouse recipients without causing graft-versus-host disease. FC induce antigen-specific CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Treg) in vivo. The major subpopulation in murine FC resembles plasmacytoid precursor dendritic cells. Treatment of mice with Flt3 ligand (FL) results in a significant increase in FC in peripheral blood (PB), and FL-expanded-PB FC enhance HSC engraftment. The number of CD8⁺TCR^– FC and p-pre DC FC are significantly decreased in FL-KO mice. In the present study, we evaluated whether FL-KO FC facilitate engraftment of HSC in allogeneic mouse recipients (C57BL/6 to BALB/c). HSC were sorted from the bone marrow (BM) of donor C57BL/6 (B6) mice, and CD8⁺TCR^– FC were sorted from donor FL-KO (B6 background) BM. 10,000 HSC and 30,000 FL-KO FC were transplanted into BALB/c recipients conditioned with 750 cGy of total body irradiation (TBI). Control mice received 10,000 HSC with or without 30,000 B6 FC. Twenty-nine percent (2 of 7) of BALB/c recipients of B6 HSC alone engrafted, with 33% donor chimerism (range 0.8% to 65%). Twenty percent (1 of 5) BALB/c recipients of B6 HSC + FL-KO B6 FC engrafted. In contrast, 53% (8 of 15) of recipients transplanted with B6 HSC + B6 FC engrafted, with an average of 39% donor chimerism (range 1% to 80%). We also tested the number and function of CD4⁺CD25⁺Foxp3⁺ Treg in FL-KO mice. The percentage of Treg in PB and BM of FL-KO mice were significantly decreased compared with B6 mice (p<0.05). The level of chemokine receptor CXCR3 expressed on Treg from FL-KO PB and spleen were significantly decreased compared with Treg from B6 mice (p < 0.05). To test the function of FL-KO Treg in facilitation of engraftment of HSC, Treg were sorted from the spleen of FL-KO mice. 10,000 B6 HSC + 100,000 FL-KO Treg were transplanted into BALB/c recipients conditioned with 750 cGy of TBI. Only 38% of mice transplanted with HSC + FL-KO Treg engrafted (n=8), with 3% donor chimerism. In contrast, 100% of recipients of HSC + B6 Treg engrafted (n = 10), with 62% donor chimerism. Taken together, our data suggest that: 1) the number of FC and p-preDC was significantly decreased in FL-KO mice; 2) the number of Treg and CXCR3⁺ Treg in PB and spleen was significantly decreased in FL-KO mice; 3) the function of FC from FL-KO BM or Treg from FL-KO spleen was impaired in facilitation of engraftment of allogeneic HSC.

CITATION INFORMATION: Huang Y, Xu H, Miller T, Wen Y, Ildstad S. The Function of CD8⁺TCR^– Facilitating Cells and Regulatory T Cells Is Impaired in Flt3-Ligand Knockout Mice. Am J Transplant. 2016;16 (suppl 3).

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