Retinoic Acid and Rapamycin Differentially Affect and Synergistically Promote the Ex Vivo Expansion of Natural Human T Regulatory Cells

Tatiana N Golovina,Tatiana Mikheeva,Jeffrey A Bluestone,James L Riley,Todd M Brusko,Bruce R Blazar,Derya Unutmaz

doi:10.1371/journal.pone.0015868

Abstract

Natural T regulatory cells (Tregs) are challenging to expand ex vivo, and this has severely hindered in vivo evaluation of their therapeutic potential. All trans retinoic acid (ATRA) plays an important role in mediating immune homeostasis in vivo, and we investigated whether ATRA could be used to promote the ex vivo expansion of Tregs purified from adult human peripheral blood. We found that ATRA helped maintain FOXP3 expression during the expansion process, but this effect was transient and serum-dependent. Furthermore, natural Tregs treated with rapamycin, but not with ATRA, suppressed cytokine production in co-cultured effector T cells. This suppressive activity correlated with the ability of expanded Tregs to induce FOXP3 expression in non-Treg cell populations. Examination of CD45RA+ and CD45RA− Treg subsets revealed that ATRA failed to maintain suppressive activity in either population, but interestingly, Tregs expanded in the presence of both rapamycin and ATRA displayed more suppressive activity and had a more favorable epigenetic status of the FOXP3 gene than Tregs expanded in the presence of rapamycin only. We conclude that while the use of ATRA as a single agent to expand Tregs for human therapy is not warranted, its use in combination with rapamycin may have benefit.

Highlights

FOXP3-expressing, natural T regulatory cells (Tregs) cells play a crucial role in maintaining immune homeostasis by attenuating aberrant immune responses
Decreased Treg yields were not observed when human Tregs were expanded in the presence of All trans retinoic acid (ATRA), supporting the notion that ATRA could overcome the major drawback of using rapamycin to promote ex vivo Treg expansion
It should be noted that Tregs expanded in the presence of both RAPA and ATRA grew to cells cultured in the presence of RAPA alone, indicating that the anti-proliferative effects of RAPA were not overcome by ATRA

Summary

Introduction

FOXP3-expressing, natural Tregs cells play a crucial role in maintaining immune homeostasis by attenuating aberrant immune responses. Given the seriousness of these issues regarding the stability and function of ex vivo expanded Tregs, many investigators have sought culture conditions that promote the stable expansion of fully functional natural Tregs These investigations have focused on identifying costimulatory pathways and soluble reagents that best enable expansion of functional human Tregs. Rapamycin has been shown to promote memory cell formation [3] which may translate into long term side effects of persistent memory Tregs in some adoptive Treg cell therapy applications These drawbacks have provided the rationale to search for agents that will equal or better rapamycin in terms of generating pure expanded Treg populations, while eliminating the rapamycin-imposed decrease in overall Treg expansion

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Results

Conclusion