Background:

Infusion of ex vivo expanded regulatory T cells (Treg) is a promising therapy to prevent allograft rejection. Trafficking of Treg to allografts and secondary (2^ry) lymphoid tissues is essential for their optimum therapeutic efficacy. We assessed the persistence and migration of autologous ex vivo expanded donor-reactive Treg (drTreg) infused after transplantation (Tx) in nonhuman primates.

Methods:

Heart allograft recipient (n=3) and donor monkeys were MHC-mismatched. Immunosuppression was based on lymphodepletion using anti-thymocyte globulin (ATG) and FK506/rapamycin maintenance. CD4⁺CD25⁺CD127^–Foxp3⁺ drTreg were expanded ex vivo using activated donor B cells, then labelled with either CFSE or VPD450 before infusion. drTreg were infused weekly, either early (n=2), total 2 infusions at 20-74 x10⁶/kg, or late (n=1) total 4 infusions at 116-182 x10⁶/kg, post-Tx. Sequential monitoring of drTreg in peripheral blood was performed. Recipients were electively euthanized one week after the last drTreg infusion. Frequencies of drTreg among live CD45⁺ leukocytes in the native organs, 2^ry lymphoid tissues and grafts were assessed by flow cytometry.

Results:

In all recipients, peripheral blood drTreg maintained high CD25 and low CD127 expression. drTreg infused early post-Tx (n=2) were detected in kidney (0.05% & 2.36%) and liver (0.24% & 2%), but not in lung. drTreg were also detected in axillary (0.05% & 0.36%), mesenteric (0.04% & 0.21%), inguinal (0.02% & 0.32%) lymph nodes (LNs), spleen (0.22% & 0.76%), bone marrow (0.05% & 0.43%), but not in thymus. drTreg infused late post-Tx (n=1) were detected in kidney (0.49%), liver (2.35%), and lung (0.66%). drTreg were also detected in axillary (3.6%), mesenteric (1.04%), inguinal (2.9%) LNs, spleen (2.74%), bone marrow (0.87%), and thymus (0.09%). Notably, higher frequencies of drTreg were detected in heart grafts (0.81%, 0.34% & 0.25%) in comparison to native hearts (0.05%, 0.19% & 0.05%). In all tissues, readily detectable drTreg maintained high CD25 and low CD127 expression. Foxp3 expression and lineage specific transcription factors are currently being evaluated.

Conclusions:

These findings suggest that drTreg preferentially migrate to organ allografts in nonhuman primates, and that delayed and frequent infusion of drTreg may be necessary to maintain sufficient numbers after Tx.

CITATION INFORMATION: Ezzelarab M., Hong Z., Zahorchak A., Lien L., Nakao T., Dai H., van der Windt D., Perez-Gutierrez A., Bhama J., Thomson A. Persistence and Homing of Autologous Donor-Reactive Ex-Vivo Expanded Regulatory T Cells to Heart Allografts in Lymphodepleted Recipient Monkeys Am J Transplant. 2017;17 (suppl 3).

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