Clinical-Grade Expanded Regulatory T Cells Are Enriched with Highly Suppressive Cells Producing IL-10, Granzyme B, and IL-35

In the setting of T cell-depleted, full-haplotype mismatched transplantation, adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) can prevent graft-versus-host disease (GVHD) and improve post-transplantation immunologic reconstitution and is associated with a powerful graft-versus-leukemia effect. To improve the purity and the quantity of the infused Tregs, good manufacturing practices (GMP)-compatible expansion protocols are needed. Here we expanded Tregs using an automated, clinical-grade protocol. Cells were extensively characterized in vitro, and their efficiency was tested in vivo in a mouse model. Tregs were selected by CliniMacs (CD4⁺CD25⁺, 94.5 ± 6.3%; FoxP3⁺, 63.7 ± 11.5%; CD127⁺, 20 ± 3%; suppressive activity, 60 ± 7%), and an aliquot of 100 × 10⁶ was expanded for 14 days using the CliniMACS Prodigy System, obtaining 684 ± 279 × 10⁶ cells (CD4⁺CD25⁺, 99.6 ± 0.2%; FoxP3⁺, 82 ± 8%; CD127⁺, 1.1 ± 0.8%; suppressive activity, 75 ± 12%). CD39 and CTLA4 expression levels increased from 22.4 ± 12% to 58.1 ± 13.3% (P < .05) and from 20.4 ± 6.7% to 85.4 ± 9.8% (P < .01), respectively. TIM3 levels increased from .4 ± .05% to 29 ± 16% (P < .05). Memory Tregs were the prevalent population, whereas naive Tregs almost disappeared at the end of the culture. mRNA analysis displayed significant increases in CD39, IL-10, granzyme B, and IL-35 levels at the end of culture period (P < .05). Conversely, IFNγ expression decreased significantly by day +14. Expanded Tregs were sorted according to TIM3, CD39, and CD62L expression levels (purity >95%). When sorted populations were analyzed, TIM3⁺ cells showed significant increases in IL-10 and granzyme B (P < .01) .When expanded Tregs were infused in an NSG murine model, mice that received Tcons only died of GVHD, whereas mice that received both Tcons and Tregs survived without GVHD. GMP grade expanded cells that display phenotypic and functional Treg characteristics can be obtained using a fully automated system. Treg suppression is mediated by multiple overlapping mechanisms (eg, CTLA-4, CD39, IL-10, IL-35, TGF-β, granzyme B). TIM3⁺ cells emerge as a potentially highly suppressive population. © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.