マウス骨髄細胞からGM-CSFを添加し未熟樹状細胞を大量に培養した後、psolarenと紫外線を併用し(いわゆるPUVA療法の応用)制御性樹状細胞を大量に作製することに成功した。その細胞は、混合リンパ球反応(MLR)においてMHC非拘束性に抑制性の機能を有していた。メカニズムは、抗炎症性サイトカイン(IL-10やTGF-β)によるものではなく、細胞間の接触によって引き起こされていた。トリプトファン代謝による細胞増殖の抑制をみるため、indoleamine 2,3-dioxygenase (IDO)の発現を定量PCR法にて測定したところ、骨髄由来の樹状細胞に比べて5倍以上発現していた。IDOを発現し、MHC非拘束性に抑制機能を有するPUVA処理した樹状細胞による細胞療法は、致死的なGVHDに対して有用な治療法と成りうる可能性がある。Bone marrow-derived cultured DCs acquired tolerogenicity by PUVA (psoralen+UVA) treatment in mice. In MLR assays, strong tolerogenicity was observed when adding PUVA-DCs generated from the same strain of stimulator cells, or responder cells, or even from third party strain into MLR mixture. That is, the PUVA-treated DCs have tolerogenic function in a MHC-independent manner, in part, due to the reduced expression levels of CD80 and CD86. To clarify the mechanisms of how PUVA-treated DCs induce tolerogenicity in further detail, we performed MLR with the addition of neutralizing antibodies against IL-10 or TGF-b1 or both. Neutralization of immunosuppressive cytokines had no effects on MLR. We then showed that cell-to-cell contact between PUVA-DCs and alloreactive T-cells was needed to mediate the regulatory effect by transwell experiment. Next we compared the expression of the indoleamine 2,3-dioxygenase (IDO), which induces T-cell anergy by tryptophan depletion and by the production of metabolic byproducts collectively known as kynurenines, by real-time PCR between PUVA-DCs and BM-DCs. An increase IDO gene transcription level was observed in PUVA-DCs about 5 times more than in BM-DCs (p<0.01). In conclusion, PUVA-DCs acquired regulatory function in a MHC-independent manner by upregulation of IDO. Infusion of PUVA-treated DCs could have a great potential to treat lethal acute GVHD in clinical settings
