OR11

Combination of TCR-deficient CAR-Tregs and non-mitogenic antiCD3 to promote transplant tolerance

T Blein(1) S Charbonnier(1) N Ayas(1) B Larmathée(1) H Waldmann(4) L Chatenoud(3) I André(1) J Zuber(1,2)

1:INSERM UMR 1163, Institut Imagine, Paris Descartes University-Sorbonne Paris Cité, Paris, France; 2:Assistance Publique–Hôpitaux de Paris, Hôpital Necker, Service des Maladies du Rein et du Métabolisme, Transplantation et Immunologie Clinique, Paris, France; 3:Institut Necker-Enfants Malades, CNRS UMR8253, Inserm UMR1151, Paris, France; 4:Sir William Dunn School of Pathology, Oxford, Royaume-Uni

CAR-regulatory T cells therapy holds promises for inducing long-term transplant tolerance while getting rid of immunosuppressive drugs. Our strategy aims to enhance the proportion of donor-specific CAR-Tregs, while debulking alloreactive T cells. We hypothesize that TCR-deficient CAR-Tregs would act synergistically with anti-CD3 therapy.

TCR-deficient HLA-A2-targeted CAR-Tregs were manufactured through lentiviral transduction and CRISPR-Cas9 gene editing. In a first model, NSG mice received HLA-A2+PBMC with CAR-Tregs, TCR+ or TCR-. In a second model, a mix of TCR+CAR-T and TCR-deficient CAR-Tregs, expressing different luciferases was adoptively transferred to mice, transplanted with HLA-A2+ skin grafts. Anti-CD3-induced depletion of TCR+ cells was assessed by in vivo bioluminescence and cytometry.

TCR-deficient CAR-Tregs, despite the absence of the TCR/CD3 complex, could still be activated in a HLA A2-specific manner. These cells retained key phenotypic and epigenetic Treg markers. Transcriptomic analysis revealed that gene editing predominantly affected TRAC-related genes, with minimal impact on other pathways. TCR+ cells, unlike their TCR- counterparts, were selectively eliminated from blood and lymphoid organs upon anti-CD3. Similarly, anti-CD3 induced a selective depletion of TCR+ CAR-T infiltrating HLA-A2+ skin allografts, while TCR-deficient CAR-Tregs persisted within the allografts. Ongoing transcriptomic profiling and imaging mass cytometry analysis of skin allografts are being conducted to further evaluate the effectiveness of this combination strategy and to map the location of graft-resident CAR-Tregs.

Our data demonstrate that anti-CD3 therapy can selectively advantage TCR-deficient CAR-Treg cells over resident TCR+ T cells in vivo. This approach significantly enhances the therapeutic potential of CAR-Tregs, while reducing the number of cells required.