Ex Vivo Generation of Regulatory T Cells: Characterization and Therapeutic Evaluation in a Model of Chronic Colitis

Abstract

Naturally occurring regulatory T cells (nTregs; CD4+CD25+Foxp3+) are capable of suppressing the chronic inflammation observed in a variety of different animal models of autoimmune and chronic inflammatory diseases such as inflammatory bowel diseases, diabetes, and arthritis. A major limitation in exploring how and where nTregs exert their suppression in vivo is the relative paucity of these regulatory cells. Although several laboratories have described different methods to expand flow-purified nTregs or convert conventional/naïve T cells (CD4+Foxp3−) to Foxp3-expressing “induced” Tregs (iTregs; CD4+Foxp3+) ex vivo, we have found that many of these approaches are encumbered with their own limitations. Therefore, we sought to develop a relatively simple ex vivo method to generate large numbers of Foxp3-expressing iTregs that can be used to evaluate their trafficking properties, suppressive activity, and therapeutic efficacy in a mouse model of chronic gut inflammation in vivo. We present a detailed protocol demonstrating that polyclonal activation of conventional CD4+ T cells in the presence of IL-2, TGFβ, and all trans retinoic acid induces >90% conversion of these T cells to Foxp3-expressing iTregs as well as promotes a three- to fourfold increase in proliferation following a 4-day incubation period in vitro. This protocol enhances modestly the surface expression of the gut-homing adhesion molecule CCR9 but not α4β7. Furthermore, we provide preliminary data demonstrating that these iTregs are significantly more potent at suppressing T-cell activation in vitro and are equally effective as freshly isolated nTregs at attenuating chronic colitis in vivo. Finally, we report that this protocol has the potential to generate 30–40 million iTregs from one healthy mouse spleen.

Regulatory T cells are expanded by Teriparatide treatment in humans and mediate intermittent PTH ‐induced bone anabolism in mice

“…This was disclosed by experiments in which unstimulated splenic CD4+CD25-cells purified from iPTH treated mice were found to express lower levels of the negative regulator of TGFβ signaling SMAD7 as compared to CD4+CD25-cells from vehicle treated mice (supplemental figure 3A). To ascertain the functional relevance of this finding, splenic CD4+CD25-cells from vehicle or iPTH treated mice were stimulated in vitro with CD3/CD28 Ab, IL-2 and TGFb at 2.5 ng/ml for 72 hours to induce their differentiation into Tregs (Karlsson, Robinson-Jackson et al, 2011). Measurements of phosphorylated SMAD2 and SMAD3 (pSMAD2 and pSMAD3) at the end of the culture period revealed higher concentrations of pSMAD2 and pSMAD3 (supplemental figure 3B) in cells from iPTH treated mice as compared to those from control mice, suggesting that conventional CD4+ T cells from iPTH treated mice have a higher sensitivity to TGFb.…”

“…In vitro generation of induced Tregs. The generation was carried out as described (Karlsson et al, 2011) . Briefly, purified splenic CD4+CD25-T cells from WT mice at the concentration of one million each ml completed RPMI-1640 medium were cultured in plates coated with anti-CD3 Ab (10 µg/ml) in the presence of recombinant human IL-2 (100 U/ml), TGFβ1 (20 ng/ml) and all trans retinoic acid (1 pmol/ml) for 4 days.…”

A Functional Polymorphism of Ptpn22 Is Associated with Type 1 Diabetes in the BioBreeding Rat

“…It has previously been demonstrated that the Gimap5 null mutation of the BBDP strain severely compromises the function and survival of CD4 + 25 + Foxp3 + Tregs once they exit the thymus (29). To circumvent this problem, we generated induced Tregs (iTregs) from ex vivo naive CD4 + 25 2 T cells congenic for rat Ptpn22 as previously described (14,30). To examine whether differences in TCR signaling due to Ptpn22 allelic variation can affect the generation of iTregs, we stimulated cells with anti-CD28 and increasing concentrations of anti-CD3 mAbs in the presence of IL-2 and TGF-b1 for 3 d. After activation, cells were assessed for Foxp3 expression.…”

Ex-vivo iTreg differentiation revisited: Convenient alternatives to existing strategies

“…Due to their critical roles in suppressing effector T-cell activation, they have been extensively studied in the recent years. Both rodent and human studies largely depend on obtaining sufficient numbers of Tregs, which is often challenging due to the low abundance of thymic-derived (tTregs) or peripherally-induced (pTregs) Tregs (Karlsson et al, 2011; Shevach and Thornton, 2014). Therefore, alternative strategies including in-vitro differentiation and expansion of Tregs have been proposed (Chen et al, 2003; Fantini et al, 2007).…”

“…Several strategies employing FACS sorting and/or magnetic enrichment of CD4 + T-cell populations have been published in the recent years. Currently, FACS sorting of non-Treg CD4 + T-cells is considered as the gold standard technique for obtaining pure starting populations while magnetic separation techniques have been shown to generate variable success rates which are almost always lower than sorting (Karlsson et al, 2011; Flaherty and Reynolds, 2015). However, FACS sorting requires access to advanced sorting facilities that are costly and often require scheduling in advance.…”