Rapamycin and abundant TCR stimulation are required for the generation of stable human induced regulatory T cells

Abstract

Objectives Regulatory T cells (Tregs) are a vital sub‐population of CD4+ T cells with major roles in immune tolerance and homeostasis. Given such properties, the use of regulatory T cells for immunotherapies has been extensively investigated, with a focus on adoptive transfer of ex vivo expanded natural Tregs (nTregs). For immunotherapies, induced Tregs (iTregs), generated in vitro from naïve CD4+ T cells, provide an attractive alternative, given the ease of generating cell numbers required for clinical dosage. While the combination of TGF‐β, ATRA and rapamycin has been shown to generate highly suppressive iTregs, the challenge for therapeutic iTreg generation has been their instability. Here, we investigate the impact of rapamycin concentrations and α‐CD3/CD28 bead ratios on human iTreg stability. Methods We assess iTregs generated with various concentrations of rapamycin and differing ratios of α‐CD3/CD28 beads for their differentiation, stability, expression of Treg signature molecules and T helper effector cytokines, and Treg‐specific demethylation region (TSDR) status. Results iTregs generated in the presence of TGF‐β, ATRA, rapamycin and a higher ratio of α‐CD3/CD28 beads were highly suppressive and stable upon in vitro re‐stimulation. These iTregs exhibited a similar expression profile of Treg signature molecules and T helper effector cytokines to nTregs, in the absence of TSDR demethylation. Conclusion This work establishes a method to generate human iTregs which maintain stable phenotype and function upon in vitro re‐stimulation. Further validation in pre‐clinical models will be needed to ensure its suitability for applications in adoptive transfer.

Induction of stable human FOXP3+ Tregs by a parasite‐derived TGF‐β mimic

“…The majority of published protocols for TGF‐β‐induction of human iTregs from naïve CD4 + T cells use between 1 and 5 ng mL −1 of TGF‐β . 21 , 22 , 23 We tested TGF‐β at concentrations from 1 to 100 ng mL −1 and confirmed that maximal FOXP3 expression (as determined by both the percentage of positive cells and MFI) had occurred by 1 ng mL −1 (dose curve data in Figure 1 ). This equates to 0.04 n m, as activated TGF‐β exists as a 25 kDa dimer, thus 1 ng mL −1 TGF‐β was selected as our comparison condition for the study.…”

“… 32 Future studies should investigate the epigenetic and functional effects on Tregs induced with Hp‐ TGM without CD28 co‐stimulation. However, as recent studies of iTregs generated by TGF‐β and all‐ trans retinoic acid and rapamycin 22 ; or TGF‐β and small molecule inhibitors 23 ; or lentiviral transfer of FOXP3 33 ; have found that the iTregs had robust and stable suppressive function without acquiring a methylated TSDR, this feature may not be essential for suppressive function of iTregs. Nevertheless, our data showing increased H3K27ac marks in the FOXP3 locus in Hp ‐TGM‐induced Tregs suggest that this molecule may drive epigenetic changes that promote Treg stability 34 and warrant further investigation.…”

Nanoparticles Engineered as Artificial Antigen-Presenting Cells Induce Human CD4+ and CD8+ Tregs That Are Functional in Humanized Mice

“…This associates with significant costs and specific technical requirements ( 25 ). Additionally, repeated treatments for the patient are often required, since ex vivo -expanded Tregs can become instable over time ( 26 ). Finally, chronic inflammation in autoimmune patients promotes the reversal of the phenotype of the transferred Tregs into T effector cells ( 27 ), and Treg potency may decrease over time ( 28 ).…”

Eras of designer Tregs: Harnessing synthetic biology for immune suppression

“…For example, the beneficial effects of rapamycin (sirolimus) on Tregs has been extensively explored 170,171 . Rapamycin can also synergize with TGF‐β1 and/or retinoic acid to promote Treg induction from Tconvs 172,173 . Accordingly, in a mouse model of heterotopic heart transplant, A2‐CAR Tregs synergized with rapamycin to prolong allograft survival compared to either monotherapy 174 .…”

“…170,171 Rapamycin can also synergize with TGF-β1 and/or retinoic acid to promote Treg induction from Tconvs. 172,173 Accordingly, in a mouse model of heterotopic heart transplant, A2-CAR Tregs synergized with rapamycin to prolong allograft survival compared to either monotherapy. 174 Furthermore, 4-1BB-CAR Tregs, which are more likely to destabilize into FOXP3 − Helios − as a result of mTOR pathway overactivation, can be restabilized using rapamycin and vitamin C. 175 Another drug, cyclophosphamide, which is used to prevent acute GVHD after hematopoietic stem cell transplant and condition people for CAR T cell therapy, may also have beneficial effects on Tregs.…”