A novel Human Immune System mouse model to investigate long-term efficacy and stability of human CAR-Tregs.
BLEIN T. 1, JOSHI A. 1, CHARBONNIER S. 1, AYAS N. 1, ANDRÉ I. 1, ZUBER J. 1,2
1 INSERM UMR 1163, Institut Imagine, Paris Descartes University-Sorbonne Paris Cité, Paris, France, Paris 15, France; 2 Assistance Publique–Hôpitaux de Paris, Hôpital Necker, Service des Maladies du Rein et du Métabolisme, Transplantation et Immunologie Clinique, Paris, France, Paris 15, France
Solid organ transplantation remains the best therapeutic option for life-threatening organ failure, yet is associated with severe complications inherent to life-long immunosuppression. In this respect, CAR-Treg therapy holds much promise to promote transplant tolerance. Several groups, including ours, have demonstrated the efficacy of CAR-Tregs at preventing acute Graft-vs-Host disease (GVHD) in humanized mice. However, these models, based on the transfer of human mature T cells, preclude any assessment in the long-term. In contrast, Human Immune System (HIS) mice, with fully reconstituted human myeloid and lymphoid compartments, are powerful tools to investigate chronic immune responses. However, HIS mice lack Tregs. We thus aimed to create a new HIS model to assess long-term effect and stability of human CAR-Tregs.
To this end, HLA-A2-targeted-CAR-Tregs were generated from HLA-A2-negative CD4+CD25brightCD127lowCD45RA+ naïve Tregs, isolated from the same cord blood (CB) as the CD34-positive hematopoietic stem cells (HSCs). To get rid of interfering xenoreactive TCR, TRAC gene was disrupted with CRISPR-Cas9.
We first demonstrated that CB-derived CAR-Tregs maintained high expression of FOXP3 and HELIOS, and could be expanded through CAR stimulation after TRAC deletion. We next generated HIS mice through intrahepatic inoculation of human HSCs in NSG-SGM3 neonates. HIS mice generated from CB-derived HSCs demonstrated greater human chimerism than HIS mice obtained with mobilized adult HSCs. At least 30% of multilineage human chimerism was observed at week 16 post HSC injection, including B, T and myeloid cells. These mice were able to reject skin allograft from allogeneic HLA-A2-expressing RAG2-/- BL/6 donors, but not syngenic grafts. Adoptive transfer of HLA-A2 CAR-Tregs allowed us to track them in vivo through bioluminence and to monitor their efficacy at preventing acute and chronic graft rejection.
We developed a new HIS model to assess CAR-Treg fate and long-term outcomes on the path toward clinical development.