Rapamycin Together With TGF-β1, IL-2 and IL-15 Induces The Generation Of Functional Regulatory γδT Cells From Human Peripheral Blood Mononuclear Cells

Gamma delta T cell, expressing gamma delta T cell receptor (gamma delta TCR), is considered as a group of unconventional T cells linking innate and adaptive immunity. Although gamma delta T cells only represent a minor population in human immune system, their functions are very broad. Recent researches have suggested that depending on the microenvironment, gamma delta T cells can assume features reminiscent of Th1, Th2, Th17 and regulatory T cells (Treg) as well as professional antigen present cells. Regulatory gamma delta T cell (gamma delta Treg) is a recently reported subset of gamma delta T cells characterized by both expressions of gamma delta TCR and forkhead/winged-helix family transcriptional repressor p3 (Foxp3), with potential immunosuppressive functions. Researches that focused on gamma delta Treg have showed the regulatory roles it has in preventing autoimmune response and relieving autoimmune diseases such as systemic lupus erythematosus (SLE), and the suppressive activity of breast tumor-derived gamma delta Tregs on innate and adaptive immunity, as well as the protective effects enhanced by gamma delta Tregs against xenogenic graft-versus-host disease in humanized mice found in our previous studies. All these observations suggest the important roles that gamma delta Treg plays in human immune system and its potential clinical applications. However, the further studies of gamma delta Treg are limited mainly due to its low quantities in vivo and the lack of methods to induce gamma delta Treg largely in vitro. It would be much significant if we found efficient induction and expansion methods for functional gamma delta Tregs.

As the mammalian target of rapamycin (mTOR) is an important integrative kinase that acts as a crucial negative regulator of Treg differentiation and expansion, which can inhibit the expression of Foxp3 induced by TGF-β1, and Rapamycin (Rapa) is an immunosuppressive agent which acts through the blockade of mTOR, here we studied whether Rapa, together with TGF-β1/IL-2/IL-15, could induce and expand gamma delta Tregs derived from human peripheral blood mononuclear cells (hPBMCs) efficiently in vitro, under the stimulation of zoledronic acid (ZOL). Our results demonstrated that Rapa, synergized with TGF-β1/IL-2/IL-15, could not only facilitate the generation of gamma delta Tregs largely in vitro, with the percentage of induced gamma delta Tregs increasing from 2.4±1.2% cultured without TGF-β1/IL-15 nor Rapa, to 55.2±8.2% with Rapa and TGF-β1/IL-2/IL-15 together, compared with 26.2±6.7% with TGF-β1/IL-2/IL-15 but no Rapa (as Figure 1A indicated), but also enhanced the Foxp3 and CD25 expression levels in Rapa-induced gamma delta Tregs (as Figure 1C). Other regulatory-associated molecules expressed in Rapa-induced gamma delta Tregs included CTLA-4, ICOS and HLA-DR, and approximately no CD127, which were somehow similar with conventional CD4+CD25+ Tregs and were related with strong immunosuppressive functions. Through the co-culturing with naïve T cells derived from hPBMCs in vitro, we found that thus-induced gamma delta Tregs displayed concentration-dependent suppressive activity against the proliferation of naïve T cells, and this suppressive function appeared much greater in Rapa-induced gamma delta Tregs, compared with those induced without Rapa (as Figure 1B indicated).

Figure 1

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Figure 1

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In conclusion, our results demonstrated that Rapa, combined with TGF-β1/IL-2/IL-15, could induce and expand gamma delta Tregs largely in vitro and thus-induced gamma delta Tregs expressed high levels of Foxp3 and CD25, and displayed significant immunosuppressive activities in vitro, which provided bases to some extent for the further studies on gamma delta Treg and its potential clinical applications.