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.

Adoptive transfer of FOXP3 + Tregs to prevent or cure multiple autoimmune diseases or GVHD has proven to be highly efficacious in animal models and human [23] [24] [25] . But a major obstacle to overcome is to achieve a consistent and highly purified FOXP3 + Treg product after ex vivo expansion in a cost-effective manner. In this study, we use LAP as a unique cell surface marker that distinguishes bona fide Tregs from activated FOXP3 + and FOXP3 − nonTregs and show that it is feasible to sort expanded FOXP3 + Tregs from nonTregs based on expression of LAP. We obtain a post-sort FOXP3 purity of greater than 90% which would be ideal for cellular immunotherapy. Furthermore, we find that ex vivo expanded LAP + Tregs with aAPCs maintain TCR Vβ repertoire diversity, instead of clonal expansion due to selective cell death. Similar data has been detected in expanded cord blood (CB) Tregs: the process of ex vivo expansion did not skew the CB Tregs TCR Vβ repertoire [26] . Although it is possible to obtain a highly purified FOXP3 + population after re-stimulation of the expanded CD25 hi cells, it has also been shown that the effector T cells can transiently possess this feature, particularly after repeated in vitro stimulation [27] . Moreover, several studies have pointed out the potential plasticity of Tregs and caution that this instability could result in the generation of pathogenic memory T cells in vivo [28] . In our study, a quantitative assay to assess the anergic state of Tregs is used to detect the cytokine secretion. Only minimal percentages of IL2, IFNγ, IL17, IL4, IL13 and IL10 are detected in the purified LAP + Tregs as compared to CD25 + and LAP‾ population. This method offers the opportunity to reduce most of the cytokine secreting cells in our culture compared with rapamycin culture system, which still has >50% of cells secreting IL4 (both FOXP3 + and FOXP3 − cells) after the fourth re-stimulation [4] . In this study, only minimal percentages of LAP + Treg cells secrete IL-10. While the LAP + cells are mainly GARP + cells, the immunosuppressive role of LAP + might be through GARP/LAP complexes. Glycoprotein A repetitions predominant (GARP), also known as leucine rich repeat containing 32 (LRRC32), plays important role in immunosuppressive function of Tregs by inhibiting T effector cell activity [29] . Targeting GARP by siRNA or monoclonal antibodies specific for GARP inhibit immunosuppressive activity of Treg in vitro and in vivo [30, 31] . However, the role of LAP/GARP on Tregs remains unclear and controversial. Deletion of GARP on mouse Tregs is not sufficient to inhibit the growth of transplanted tumors. They found the suppressive function of Tregs was not affected by knockout of garp in Tregs [32] . Further studies are needed to reveal the mechanisms responsible for suppressive function of LAP + Tregs.