Chimeric Regulatory T cells Enhance HSC Engraftment in An Antigen-Specific Fashion.

Abstract 2424

Poster Board II-401

We previously reported that CD8⁺TCR^- facilitating cells (FC) induce the generation of chimeric regulatory T cells (T_reg) in vivo. Transplantation of a mixture of CD8⁺/TCR^- FC and hematopoietic stem cells (HSC) into ablated recipients results in chimerism and tolerance. T_reg harvested from the spleen of chimeras (chimeric T_reg) potently increase long-term donor chimerism in secondary NOD recipient mice. Here, we evaluated whether chimeric T_reg enhance engraftment of hematopoietic stem cells (HSC) in an antigen-specific manner. To prepare mixed chimeras (B6 → NOD), NOD recipients were conditioned with 950 cGy TBI and transplanted with 10,000 B6 HSC and 1,000 NOD HSC plus 45,000 CD8⁺TCR^- B6 FC. At 5 weeks, CD8^-CD4⁺CD25^bright chimeric T_reg were sorted from spleens of the mixed chimeras (B6 → NOD). 100,000 chimeric T_reg were then mixed with 10,000 B6 HSC (donor-specific) + 10,000 B10.BR HSC (third-party) and transplanted into conditioned NOD recipients in competitive repopulation assays. NOD mice given HSC plus nonchimeric naïve B6 T_reg or HSC alone served as controls. Two of the four animals that received HSC alone engrafted and exhibited an average of 6.7% donor B6 chimerism at 30 days, 11.2% at 60 days, and 10.6% at 90 days. Three of five animals given HSC plus naïve B6 T_reg engrafted with 21.3% donor B6 chimerism at 30 days, 28.8% at 60 days, and 28.9% at 90 days. In contrast, eight of nine recipients of HSC + chimeric T_reg engrafted. These animals exhibited a significantly higher level of donor B6 chimerism, ranging from 56.3% at 30 days, 75.4% at 60 days to 85% at 90 days (P = 0.034). None of the recipients engrafted with the MHC-disparate third-party B10.BR HSC. We then assessed the suppressive function of chimeric T_regin vitro by using MLR suppressor cell assays. CD8^-/CD4⁺/CD25^bright T_reg were sorted from chimeric spleens 5 wks to 12 wks after HSC + FC transplantation. As shown in the Figure 1, T_reg from naïve B6 mice resulted in 1.9 fold; 1.3 fold and 1.1 fold inhibition of proliferation at 1:1, 1:0.25, 1:0.125 responder/T_reg ratios (n = 3). In contrast, chimeric T_reg potently suppressed T cell proliferation by 10.5 fold; 3.2 fold; and 1.7 fold at responder/T_reg ratios of 1:1, 1:0.25, 1:0.125 (n = 4). Chimeric T_reg significantly suppressed T cell proliferation at responder/T_reg ratios of 1:1 and 1:0.25 compared with naïve B6 T_reg (P < 0.05). NOD responder splenocytes remained hypoproliferative in response to B6 stimulator and chimeric T_reg compared with stimulator plus B6 T_reg, suggesting that chimeric T_reg are significantly more potent than naïve B6 T_reg in suppressing effector T cell proliferation in vitro. These data show that chimeric T_reg enhance donor B6 HSC engraftment but not third-party B10.BR HSC, demonstrating that chimeric T_reg function in vivo in an antigen-specific fashion. These data also show that the mechanism of FC function in vivo is associated with the establishment of an antigen-specific regulatory feedback loop.

Figure 1

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Figure 1

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