Abstract
A crucial issue for Treg-based immunotherapy is to maintain a bona fide Treg phenotype as well as suppressive function during and after ex vivo expansion. Several strategies have been applied to harness Treg lineage stability. For instance, CD28 superagonist stimulation in vitro, in the absence of CD3 ligation, is more efficient in promoting Treg proliferation, and prevention of pro-inflammatory cytokine expression, such as IL-17, as compared to CD3/CD28-stimulated Treg. Addition of the mTOR inhibitor rapamycin to Treg cultures enhances FOXP3 expression and Treg stability, but does impair proliferative capacity. A tumor necrosis factor receptor 2 (TNFR2) agonist antibody was recently shown to favor homogenous expansion of Treg in vitro. Combined stimulation with rapamycin and TNFR2 agonist antibody enhanced hypo-methylation of the FOXP3 gene, and thus promoting Treg stability. To further explore the underlying mechanisms of rapamycin and TNFR2 agonist-mediated Treg stability, we here stimulated FACS-sorted human Treg with a CD28 superagonist, in the presence of rapamycin and a TNFR2 agonist. Phenotypic analysis of expanded Treg revealed an autocrine loop of TNFα–TNFR2 underlying the maintenance of Treg stability in vitro. Addition of rapamycin to CD28 superagonist-stimulated Treg led to a high expression of TNFR2, the main TNFR expressed on Treg, and additional stimulation with a TNFR2 agonist enhanced the production of soluble as well as membrane-bound TNFα. Moreover, our data showed that the expression of histone methyltransferase EZH2, a crucial epigenetic modulator for potent Treg suppressor function, was enhanced upon stimulation with CD28 superagonist. Interestingly, rapamycin seemed to downregulate CD28 superagonist-induced EZH2 expression, which could be rescued by the additional addition of TNFR2 agonist antibody. This process appeared TNFα-dependent manner, since depletion of TNFα using Etanercept inhibited EZH2 expression. To summarize, we propose that an autocrine TNFα–TNFR2 loop plays an important role in endorsing Treg stability.
Highlights
CD4+FOXP3+ regulatory T cells (Treg) inhibit autoreactive effector T cells (Teff) and are important for immune homeostasis
We found that the harnessing effect of rapamycin and tumor necrosis factor receptor 2 (TNFR2) agonist on Treg stability was achieved through an autocrine loop of TNFα via TNFR2, whereby rapamycin enhanced TNFR2 expression and TNFR2 agonist increased the production of TNFα
Stimulation of Treg using CD28 superagonist mAb (CD28-SA) significantly enhanced the expression of TNFR2 (92.5 ± 2.9 vs. 70.5 ± 3.1% for medium control, p < 0.05), while the addition of Rap to CD28-SA stimulated Treg resulted in the highest expression level of TNFR2, both in frequency (95.5 ± 1.7%; p < 0.01) and in median fluorescence intensity [median fluorescent intensity (MFI), 18.9 ± 3.9 vs. 2.1 ± 0.2 for medium control; p < 0.001] (Figure 1A)
Summary
CD4+FOXP3+ regulatory T cells (Treg) inhibit autoreactive effector T cells (Teff) and are important for immune homeostasis. Treg lineage stability is defined by a stable expression of the transcription factor FOXP3, a highly demethylated Treg-specific demethylation region (TSDR), potent suppressive capacity and lack of pro-inflammatory cytokine production [2, 3]. Standard expansion protocols using anti-CD3/anti-CD28 mAb-coated microbeads plus exogenous rhIL-2 lead to high cell yields [4, 5] and reveal Treg plasticity, whereby Treg loose FOXP3 and start producing IL-17A and IFNγ [6,7,8]. It is of interest to note that the combined addition of a tumor necrosis factor receptor 2 (TNFR2) agonistic monoclonal antibody and rapamycin rescues rapamycin-mediated inhibition of Treg proliferation and leads to a highly homogenous Treg phenotype as well as a stable suppressive function upon expansion [19, 20]
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