*Purpose: Conferring alloantigen-specificity to ex vivo expanded CD4+CD25+Foxp3+ Tregs increases their capacity to counteract effector alloimmune responses following adoptive transfer into transplant recipients. This has been achieved in humans by: 1) expanding Tregs in the presence of donor B cells (donor-reactive DR-Tregs); 2) culturing Tregs in the presence of donor material and co-stimulation blockade (CSB-Tregs); and 3) transducing Tregs with a HLA-A2-specific chimeric antigen receptor specific (CAR-Tregs). All 3 modalities of manufactured Treg products are currently undergoing clinical development. We lack, however, a clear understanding of the relative potency of each of these strategies. Our goal here was to directly compare the 3 manufacture approaches both in vitro and in vivo.

*Methods: Allogenic material (PBMCs or B cells) was isolated from HLA-A2+ positive and HLA-II mismatched donors. CSB Tregs were generated by culturing HLA-A2 negative PBMCs with irradiated allogenic donor PBMCs in the presence of CD80 (2D10.4) and CD86 (IT2.2) antibodies for 14 days. For DR-Tregs and CAR Tregs, we FACS isolated CD4+CD25+CD127- Tregs and co-cultured them with irradiated CD40L-activated donor B cells on the day of isolation at a ratio of 1 to 4 and transduced them with an anti-HLA-A2 second-generation CAR construct, respectively. In addition, we generated polyclonal (PC) non-antigen specific Tregs by expanding FACS isolated Tregs with CD3/CD28 beads. Antigen-specificity and suppressive function of the cell products were evaluated in vitro by performing activation, proliferation, and T cell suppression assays 14 days post isolation. Therapeutic efficacy was assessed in vivo in a xenogeneic GvHD model in humanized NSG mice.

*Results: As compared to PC Tregs, all 3 strategies increased the proportion of alloantigen-specific Tregs within the manufactured cell product, with the highest level being observed in the CAR-Treg products (6-fold higher than in DR-Tregs). The differences in the precursor frequency of alloreactive Tregs were consistent with the results of the suppression assays, in which on a cell-per-cell basis CAR-Tregs exhibited the strongest effects, followed by DR-Tregs and CSB-Tregs. In vivo, infusion of CAR-Tregs together with HLA-A2+ PBMCs into irradiated NSG mice resulted in most prolongation of recipient survival.

*Conclusions: The CAR technology is both more efficient and more powerful than non-engineered strategies in conferring alloantigen specificity to ex vivo expanded Treg products. This information will be essential to adequately interpret data derived from ongoing clinical trials.

« Back to 2022 American Transplant Congress