Single-cell and multi-omic characterization of ex vivo expanded ASTRLs from stable kidney transplant recipients reveals a regulatory T cell phenotype

Therapeutic application of ex vivo expanded regulatory T cells is a promising approach to prolong allograft survival. In this work we performed a detailed characterization of a preclinical heterogenous antigen specific T enriched regulatory cell line (ASTRL) expanded ex vivo from PBMC of stable kidney transplant recipients. We used three different approaches: scRNA-seq, flow cytometry and mass cytometry, to compare pre-expansion PBMC to post-expansion ASTRL. Results show the CD4⁺ T cell compartment in ASTRL clonally expanded in response to donor antigen stimulation and showed decreased TCR diversity. ASTRL CD4⁺ T cells demonstrated a Treg associated transcriptome with upregulated CD39 and TIGIT together with other classical Treg genes like IL2RA, IKZF4, TNFRSF9, CXCR6, DUSP10 and HLA-DRA. Comparison of differentially expressed genes (DEGs) in ASTRL with classical Treg gene signatures showed strong overlap of genes associated with both peripheral and uterine Tregs together with a Th2-like Treg transcriptomic profile. In conclusion the CD4⁺ T cell compartment of ASTRL acquire a regulatory T cell transcriptomic profile in response to donor antigen specific stimulation. This suggests a promising approach towards the development of a regulatory cell therapy in organ transplantation.