Single and combined effect of retinoic acid and rapamycin modulate the generation, activity and homing potential of induced human regulatory T cells

Abstract

Adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Treg cells) has been successfully utilized to treat graft versus host disease and represents a promising strategy for the treatment of autoimmune diseases and transplant rejection. The aim of this study was to evaluate the effects of all-trans retinoic acid (atRA) and rapamycin (RAPA) on the number, phenotype, homing markers expression, DNA methylation, and function of induced human Treg cells in short-term cultures. Naive T cells were polyclonally stimulated and cultured for five days in the presence of different combinations of IL-2, TGF-β1, atRA and RAPA. The resulting cells were characterized by the expression of FOXP3, activation, surface and homing markers. Methylation of the Conserved Non-coding Sequence 2 was also evaluated. Functional comparison of the different culture conditions was performed by suppression assays in vitro. Culturing naive human T cells with IL-2/TGFβ1 resulted in the generation of 54.2% of Treg cells (CD4+CD25+FOXP3+) whereas the addition of 100 nM atRA increased the yield of Treg cells to 66% (p = 0.0088). The addition of RAPA did not increase the number of Treg cells in any of these settings. Treg cells generated in the presence of atRA had an increased expression of the β7 integrin to nearly 100% of the generated Treg cells, while RAPA treated cells showed enhanced expression of CXCR4. The differential expression of homing molecules highlights the possibility of inducing Treg cells with differential organ-specific homing properties. Neither atRA nor RAPA had an effect on the highly methylated CNS2 sites, supporting reports that their contribution to the lineage stability of Treg cells is not mediated by methylation changes in this locus. Treg cells generated in the presence of RAPA show the most potent suppression effect on the proliferation of effector cells.

Large-Scale Generation of Human Allospecific Induced Tregs With Functional Stability for Use in Immunotherapy in Transplantation

“…Several reports indicate that the presentation of antigens to T cells by immature DCs results in T cell anergy or Treg induction (22). In addition, there is evidence of previous studies describing iTreg generation (23,24); however, this is the first report in which a large amount of iTregs are obtained with specificity for the antigen of interest. To achieve this, a two-step strategy was used: first, the generation of allo iTregs in co-cultures between naïve T cells and MoDC, and second, isolation of the generated iTregs and their polyclonal expansion for 6 weeks (Figure 2A).…”

“…To date, there are a few published protocols on in vitro generation of human Tregs, most of them polyclonal and in short-term cultures (1-2 weeks) and some of them with no data on the methylation status of the FOXP3 gene (47)(48)(49). Other reports on polyclonal conversion showed that FOXP3 remains methylated or that Treg restimulation causes the loss of its expression (23,24,50). Among the studies describing long-term generation of allospecific iTregs, Tu et al reported the use of CD40L-stimulated B cells (NIH 3T3 cells transfected with the CD40 ligand; tCD40L NIH/3T3) as antigen-presenting cells.…”

Time-resolved transcriptome and proteome landscape of human regulatory T cell (Treg) differentiation reveals novel regulators of FOXP3

“…Here, the higher sensitivity of conventional T cells compared to Tregs to inhibition by Rapa [ 65 ] may allow for a preferential expansion of Treg-like cells despite lower total fractions of activated cells in the presence of Rapa and may explain the relatively high expression of iTreg candidate genes despite the lower CD25+ cell fraction. Very recently, Candia et al [ 66 ] re-sorted CD4 + CD25 high CD127 − cells from iTreg cultures prior to suppression assays and confirmed that human iTregs generated in the presence of Rapa had superior suppressive activity in vitro compared to the corresponding iTregs generated without Rapa. However, the in vivo relevance and functionality of iTregs is still controversial, and even iTregs generated with similar protocols are not always determined to be suppressive in vitro and/or in vivo.…”

“…For instance, Tregs residing in the gut, which may mostly comprise pTregs and may resemble ATRA-induced iTregs, are different from Tregs in other tissues [ 16 , 41 ]. In this regard, it is important to mention that ATRA and Rapa were shown to confer differential homing capacities to murine and human iTregs [ 66 , 71 ]. It is therefore conceivable that iTregs with a certain phenotype may be suppressive in one, but not in another disease model.…”

Dissecting the Multiplicity of Immune Effects of Immunosuppressive Drugs to Better Predict the Risk of de novo Malignancies in Solid Organ Transplant Patients

“…RAPA was also shown to preferentially promote the expansion of CD4 + CD25 high FOXP3 + Tregs as compared to the CD4 + CD25 neg FOXP3 + Treg subset (75). Intriguingly, RAPA enhances the expression of CXCR4, the ligand of stromal-derived-factor-1 (SDF-1), which is constitutively expressed in the bone marrow, suggesting that this drug may promote the development of Tregs with distinct homing properties (99).…”