Rapamycin Combined with TGF-β Converts Human Invariant NKT Cells into Suppressive Foxp3+ Regulatory Cells

Abstract

Invariant NKT (iNKT) cells constitute a versatile T cell subset with important regulatory functions, which are thought to result essentially from their capacity to promptly produce cytokines that influence the Th1/Th2 balance. In this study, we report that these cells can also express Foxp3, an important transcriptional regulator associated with suppressive activity, once they have been exposed to TGF-β. Foxp3 was expressed by iNKT cells from both peripheral and cord blood. CD4+ iNKT cells acquired Foxp3 expression preferentially, although a lower proportion of their CD4− counterpart also became positive. All Foxp3+ iNKT cells displayed CD25 but not necessarily CTLA4 or GITR, regardless of the upregulation of these markers in the presence of TGF-β. Exposure to TGF-β decreased IL-4 and IFN-γ production while increasing IL-10, independently from Foxp3 expression. IL-17 was not detected. TGF-β induced high levels of Foxp3, but no suppressor activity, which emerged only in the presence of rapamycin. Peripheral and cord blood Foxp3+ iNKT cells suppressed the proliferation of conventional autologous and heterologous CD4+ T cells equally, in a cell contact-dependent and Ag-independent manner. Our findings demonstrate that human iNKT cells become suppressive in the presence of TGF-β plus rapamycin, thus adding a new facet to their complex functional properties.

mTOR Inhibition Per Se Induces Nuclear Localization of FOXP3 and Conversion of Invariant NKT (iNKT) Cells into Immunosuppressive Regulatory iNKT Cells

“…In this study, we show that both IL-10 and rapamycin promote FOXP3 expression in iNKT cell lines, whereas in contrast to previous reports using ex vivo cells (4)(5)(6), stimulation of these lines in the presence of TGF-b did not result in increased levels of FOXP3 in iNKT cells. Complementing recent data (6), we show that a suppressive phenotype was only induced when iNKT cell lines were cultured in the presence of rapamycin, irrespective of the additional presence of IL-10 or TGF-b.…”

“…We showed that FOXP3 could be induced in iNKT cells upon culture with IL-10 and rapamycin but not upon culture with TGF-b as previously described (6). There are several reasons that may account for this observed discrepancy.…”

“…First, Moreira-Teixeira et al (6) used iNKT cells derived from cord blood and freshly isolated PBMC, whereas we used iNKT cell lines. Freshly isolated PBMC, and cord blood cells in particular, have been suggested to be more plastic and perhaps easier to convert into a Treg phenotype with TGF-b compared with iNKT cell lines (6,22). In this study, we focused on the use of iNKT cell lines, as these are more reflective of the iNKT cell populations eventually used for future in vivo adoptive transfer studies.…”

“…Upon activation, iNKT cells rapidly produce a wide variety of pro-and anti-inflammatory cytokines, and it is the balance between these immunostimulatory and immunoregulatory cytokines that appears to determine the outcome of ensuing immune responses, for example, in the setting of autoimmunity, infection, transplantation, and cancer (1)(2)(3). Of interest, recent studies demonstrated the induction of FOXP3 expression in iNKT cells upon exposure to TGF-b (4,5) and the induction of an immunosuppressive phenotype in the additional presence of the mammalian target of rapamycin (mTOR) inhibitor rapamycin in iNKT cells derived from cord blood or PBMC (6). Although expression of FOXP3 is classically associated with CD4…”

“…+ regulatory T cells (Tregs), which represent a functionally distinct lineage of immunoregulatory T cells that is critically dependent on this transcription factor (7,8), human conventional T cells can transiently express FOXP3 upon activation (9,10), as can iNKT cells upon exposure to TGF-b, as mentioned above (4)(5)(6).…”