Human LAP(+)GARP(+)FOXP3(+) regulatory T cells attenuate xenogeneic graft versus host disease

Wang, Huihui; Song, Hyo; Pham, Anha V.; Cooper, Laurence J.; Schulze, Janika J.; Olek, Sven; Tran, Dat Q.

We have demonstrated previously that iTregs lack the cardinal features of tTregs based on anergy and suppressive function [11] . While >90% FOXP3 + cells can be isolated by FACS-sorting, frequently the percentage of FOXP3 + T cells decreases to 75% after two weeks and 50% after three weeks of expansion [12] [13] [14] . Due to these limitations that have hindered the advancement of Treg analysis and therapy, we have developed an innovative technique to repurify Tregs from expanded cultures contaminated with activated nonTregs. By targeting the selective expression of latency-associated peptide (LAP), this novel method allows for expansion and consistent re-purification of sufficient quantity of highly purified, bona fide Tregs (>90% FOXP3), even from an initial limited blood volume of 5-10 ml [15] . Without this technique, it has been difficult to analyze and assess definitively the function of Tregs due to variable contaminants of nonTregs. Moreover, this method has the potential to optimize Treg therapy by permitting the manufacturing of a Treg product that is consistently >90% bona fide Tregs regardless of methods of expansion or donor's variability. Using the LAP marker, we can selectively isolate LAP + Tregs from any time point during expansion that are typically contaminated by >25% FOXP3 + and FOXP3 -nonTregs. This purity and selectivity will allow for separation of different subsets and definitive determination of Tregs function in GVHD. Another important factor is the cost effectiveness for production of a Treg product. In this study, we use CD25 + cells isolated from buffy coats with anti-CD25 microbeads. These CD25 + cells are expanded with OKT3-loaded KT64/86 artificial APC (aAPC) expressing CD86 and CD64 and re-purified based on LAP expression. We perform an analysis of the TCR repertoire, function, and phenotype within CD25 + cells and the subsets of LAP + Tregs and LAP − nonTregs isolated from expansion cultures. We demonstrate that LAP + Tregs after 4-week ex vivo expansion still maintain diverse TCR Vβ repertoire, are highly demethylated in the TSDR region, and possess suppressive function. In contrast, the LAP − nonTregs possess no suppressive function, while the CD25 + parent population had variable functionality depending on the level of expanded, contaminated nonTregs.