Our study demonstrated for the first time that the combination of rapamycin and DON had additive effects on immune cells in vitro and in vivo. We revealed that rapamycin and DON additively suppressed CD4+ T cell proliferation, and both of them inhibited Th17 cell differentiation. In myeloid cells, DON strongly inhibited DC and macrophage differentiation but facilitated the differentiation of G-MDSCs, while rapamycin enhanced the immunosuppressive ability of G-MDSCs and mildly suppressed DC and macrophage differentiation. Thus, rapamycin and DON additively induced the expansion and immunosuppressive effect of G-MDSCs. Finally, we demonstrated that the combined treatment with rapamycin and DON significantly suppressed arthritis in SKG mice compared to rapamycin or DON monotherapy.

Our study is also the first to demonstrate that inhibiting both mTOR and glutamine metabolism had an additive effect on CD4+ T cells. Activating naïve T cells with anti-CD3 and CD28 mAbs causes a rapid increase in glutamine uptake that is dependent on the amino acid transporters such as ASCT2 (SLC1A5) and LAT139,40. Glutamine uptake also positively regulates differentiation into Th17 and Th1 cells40,41,42. Inhibiting mTORC1, which plays a significant role in activating CD4+ T cells and differentiating Th1 and Th17 cells, with rapamycin decreased the differentiation of Th1 and Th17 cells43,44. Although rapamycin could enhance the generation of induced Treg cells45,46,47, inhibition of both mTORC1 and mTORC2 was required for the generation of Treg cells in the absence of exogenous TGF-β in a previous report34. Thus, in addition to mTOR, glutamine and other amino acids are also important in activating CD4+ T cells. These two pathways seem to be closely connected, since amino acid inhibition regulates mTOR activation. In our study, the combination of rapamycin and DON additively suppressed CD4+ T cell proliferation. However, although both rapamycin and DON suppressed Th17 cell differentiation, the combination of DON and rapamycin did not have an additive effect. In addition, rapamycin but not DON promoted Treg cell differentiation in vitro. These results suggested that rapamycin and DON have similar but not identical effects on CD4+ T cell activation and differentiation. Prolonged treatment of rapamycin in vitro (more than 24 hrs) and in vivo (up to 6 weeks) showed the suppression of mTORC2 as well as mTORC148,49. Although immunologic functions of mTORC2 are less understood rather than mTORC1, we assumed rapamycin suppressed both mTORC1 and mTORC2 in vivo judging from the treatment period (almost 6 weeks). In the Western blotting analysis of the proliferating CD4+ T cells stimulated for 24 hrs with anti-CD3 and CD28 mAbs, we found that 1 μM rapamycin suppressed strongly both mTORC1 and mTORC2, but 5 μM DON suppressed mTORC1 partially and mTORC2 slightly. It has been reported that in ASCT2 (a glutamine transporter) deficient CD4+ T cells, TCR and CD28-mediated mTORC1 activation was severely attenuated and recovered by an excessive amount of glutamine, while the activation of mTORC2 was not affected39. The difference of mTORC2 suppression between rapamycin and DON could explain the difference of Treg differentiation in vitro. In addition, among various concentrations of rapamycin (0, 0.1, 1, 10, 100, and 1000 nM), rapamycin more than 1 nM (which had been shown to inhibit both mTORC1 and mTORC2) plus DON significantly more suppressed CD4+ T cell proliferation compared to DON, but 0.1 nM (which had been shown to inhibit mTORC1 selectively) rapamycin plus DON showed slightly additive suppression compared to DON in vitro. From these results, we speculated that DON affected CD4+ T cells through the suppression of mTOR, dominantly mTORC1, but also through mTOR-independent mechanisms which possibly contributed to the additive effect on CD4+ T cell proliferation.

We showed that DON had a stronger effect than rapamycin on inhibiting the differentiation of DCs and macrophages and facilitating the differentiation of MDSCs, suggesting that glutamine metabolism is essential for both DC and macrophage differentiation. The mechanism by which glutamine metabolism influences the differentiation of DCs is not clear; however, mTOR is upregulated in the differentiation of DCs and M1 macrophages4,10,11,12.

Although the metabolism of MDSCs has not been clarified, several studies have reported on the influence of rapamycin on MDSCs. Rapamycin prolongs graft survival and ameliorates AKI by inducing MDSCs, primarily G-MDSCs24,25,26. mTOR is reported to govern M-MDSCs in mouse allograft and tumor models, and glycolysis via mTOR activation is essential for M-MDSCs to differentiate and acquire immunosuppressive ability25. The same study found that the proportion of G-MDSCs was not influenced by an mTOR inhibitor or a cell-specific mTOR knockout. We found that both DON and rapamycin inhibited the differentiation of M-MDSCs and facilitated the differentiation of G-MDSCs. Intriguingly, DON had stronger effects on myeloid cell differentiation than on G-MDSC differentiation. Thus, glutamine metabolism may be essential for the differentiation of DCs, macrophages, and M-MDSCs, but not G-MDSCs.

We showed that treating in vitro–generated G-MDSCs with rapamycin increased their immunosuppressive effect and enhanced their expression of TGF-β and PD-L1. Rapamycin has been shown to upregulate immunosuppressive ability in both MDSC subtypes24,26. The adoptive transfer of rapamycin-treated MDSCs upregulated serum TGF-β1 levels in an AKI model24. In an experimental autoimmune encephalomyelitis, elevated PD-L1 expression mediated the protective effects of MDSCs, whereas no Arg-1 or NO was detected in G-MDSCs22. Likewise, Arg-1, iNOS, and ROS were not upregulated in rapamycin-treated MDSCs in our study. Taken together with previous studies, our results indicate that rapamycin facilitates the differentiation of G-MDSCs and promotes their immunosuppressive ability, possibly by upregulating PD-L1 expression and TGF-β production. Given that DON doesn’t increase the immunosuppressive ability of G-MDSCs, rapamycin’s effect on G-MDSCs may be independent of glutamine metabolism.

We demonstrated that the inhibitors rapamycin and DON had additive and therapeutic effects in an arthritis mouse model. Several reports describe strategies that simultaneously inhibit two or more metabolic pathways. Simultaneously inhibiting mTOR and glutamine metabolism suppressed brain tumor proliferation50. A triple-therapy regimen consisting of DON, the glycolytic inhibitor 2-DG, and metformin was effective for GVHD by suppressing CD4+ and CD8+ effector T cells51. Metformin is a commonly used oral hypoglycemic drug that inhibits mTOR and mitochondrial respiratory complex I, activates AMPK, and promotes fatty acid oxidation. This triple therapy profoundly suppressed activation of the mTOR pathway, as assessed by phosphorylated S6K. We at first thought that rapamycin and DON suppressed the same metabolic pathway, and that DON might inhibit mTOR signaling by reducing the influx of amino acids. However, the suppression of mTOR by DON was weaker than rapamycin in the proliferating CD4+ T cells. Thus, we speculate that the additive therapeutic effects we observed in the SKG arthritis model might be explained by the inhibition of glutamine metabolism at the same time that mTOR signaling and mTOR-induced glycolysis were also inhibited, but further investigation is necessary to determine the metabolic profiles involved.

Our in vitro and in vivo findings agree in some but not all points. Rapamycin and DON additively inhibited CD4+ T cell proliferation; when administered separately, rapamycin and DON inhibited Th17 differentiation both in vitro and in vivo. This point of agreement between the in vitro and in vivo results suggested that the additive effect of rapamycin and DON acts primarily on lymphocytes in SKG mice.

On the other hand, rapamycin-treated mice had higher proportions of total MDSCs and G-MDSCs than did the other groups, including those treated with both DON and rapamycin in vivo, while treatment with both rapamycin and DON promoted G-MDSC differentiation in vitro. Since MDSCs are induced by inflammatory cytokines or mediators in arthritic mice16, this point of discord might reflect residual inflammation in the rapamycin-treated group. Another possible explanation for this discord is the dosage of DON. DON decreased Th17 /CD4+ T cell ratio both in vitro and in vivo, but did not decrease CD4+ T cell counts in vivo in our series of experiments, suggesting that the dose of DON we used in vivo was lower than that of in vitro experiments.

In this study, DON significantly suppressed arthritis even when administered alone. As a glutamine analog, DON acts as an inhibitor of various glutamine-utilizing enzymes involved in several important metabolic pathways, such as purine, pyrimidine, and amino acid synthesis as well as glutaminase in the first step of glutamine metabolism. We confirmed a glutaminase 1 inhibitor, compound 968 (C968), also suppressed CD4+ T cell proliferation similarly with DON, indicating the crucial role for glutamine metabolism. (Suppl. Fig. 1) We have previously reported that C968 suppressed the proliferation of fibroblast-like synoviocytes derived from RA patients (RA-FLS) and ameliorated clinical arthritis in SKG mice33. We have found that DON also suppressed the proliferation of RA-FLS in a dose-dependent manner (data not shown). In the immunohistochemistry of the hind paw, the extents of the proliferation of synoviocytes as well as myeloid cells and T lymphocytes were suppressed in Rapa, DON, and Rapa + DON groups. Thus, we speculate that both rapamycin and DON attenuates arthritis by directly suppressing synovial cell proliferation as well as affecting immune cells in SKG mice. When DON was tested in clinical trials for cancer in the 1980s, researchers reported that it had severe dose-limiting toxicity with nausea and vomiting and without any obvious efficacy as a monotherapy50. In recent years, however, recognition of the importance of glutamine metabolism and glycolysis in cancer has spurred new interest in therapies that inhibit several metabolic pathways simultaneously51,52,53. In the present study, adding rapamycin to DON did not increase the proportion of dead cells in vitro, and no toxicity was observed for the combination therapy in vivo, suggesting that these drugs might be clinically feasible.

In summary, combined treatment with rapamycin and DON additively ameliorated arthritis in SKG mice, mainly by suppressing CD4+ T cell proliferation and Th17 differentiation. The simultaneous inhibition of mTOR and glutamine metabolism may represent a novel therapeutic strategy for RA.