Centre for Transplant & Renal Research, Westmead Institute for Medical Research, University of Sydney, Westmead, NSW, Australia.
Cell Transplantation and Gene Therapy Institute of Central South University at the 3rd Xiangya Hospital, Changsha, Hunan, China.
Xenotransplantation. 2017 Sep;24(5). doi: 10.1111/xen.12326. Epub 2017 Aug 2.
For xenotransplantation to truly succeed, we must develop immunomodulatory strategies to suppress the xenoimmune response but by minimizing immunosuppression over the long term. Regulatory macrophages (Mreg) have been shown to suppress polyclonal T-cell proliferation in vitro and prolong allograft survival in vivo. However, the question of whether they are capable of suppressing xenoimmune responses remains unknown. This study assessed the potential of human Mreg to be used as an effective immunomodulatory method in xenotransplantation.
CD14+ monocytes selected from human peripheral blood mononuclear cells (PBMC) were cultured with macrophage colony-stimulating factor (M-CSF) for 7 days with IFN-γ added at day 6 for Mreg induction. Mreg phenotyping was performed by flow cytometric analysis, and the in vitro suppressive function was assessed by mixed lymphocyte reaction (MLR) using irradiated pig PBMC as the xenogeneic stimulator cells, human PBMC as responder cells, and autologous Mreg as suppressor cells. To assess mRNA expression of Mreg functional molecules indoleamine-2,3-dioxygenase (IDO), IL-10, inducible nitric oxide synthase (iNOS) and TGF-β were measured by real-time PCR. Supernatants were collected from the MLR cultures for IDO activity assay by high-performance liquid chromatography (HPLC). The effects of the IDO inhibitor 1-D/L-methyl-tryptophan (1-MT), iNOS inhibitor N -monomethyl-l-arginine (L-NMMA), and anti-IFN-γ or anti-TGF-β monoclonal antibody (mAb) treatment on Mreg suppressive capacity were tested from the supernatants of the MLR assays.
We demonstrated that induced Mreg with a phenotype of CD14 CD16 CD80 CD83 CD86 HLA-DR were capable of suppressing proliferating human PBMC, CD4+, and CD8+ T cells, even at a higher responder:Mreg ratio of 32:1 in a pig-human xenogeneic MLR. The strong suppressive potency of Mreg was further correlated with their upregulated IDO expression and activity. The IDO upregulation of Mreg was associated with an increased production of IFN-γ, an IDO stimulator, by xenoreactive responder cells in the xenogeneic MLR. While no effect on Mreg suppressive potency was detected by addition of the iNOS inhibitor L-NMMA or anti-TGF-β mAb into the MLR assays, inhibition of IDO activity by neutralizing IFN-γ or by IDO inhibitor 1-MT substantially impaired the capacity of Mreg to suppress the xenogeneic response, indicating the importance of upregulated IDO activity in Mreg-mediated suppression of the xenogeneic response in vitro.
This study demonstrates that human Mreg are capable of suppressing the xenoimmune response in vitro via IDO-involved mechanism(s), suggesting their potential role as an effective immunomodulatory tool in xenotransplantation.
为了使异种移植真正成功,我们必须开发免疫调节策略来抑制异种免疫反应,但同时要长期将免疫抑制作用降到最低。调节性巨噬细胞(Mreg)已被证明可以在体外抑制多克隆 T 细胞增殖,并延长同种异体移植物的存活时间。然而,它们是否能够抑制异种免疫反应仍不得而知。本研究评估了人源性 Mreg 作为异种移植中有效免疫调节方法的潜力。
从人外周血单核细胞(PBMC)中选择 CD14+单核细胞,用巨噬细胞集落刺激因子(M-CSF)培养 7 天,并在第 6 天添加 IFN-γ诱导 Mreg。通过流式细胞术分析进行 Mreg 表型鉴定,并通过混合淋巴细胞反应(MLR)评估体外抑制功能,使用辐照猪 PBMC 作为异种刺激细胞、人 PBMC 作为反应细胞、自体 Mreg 作为抑制细胞。通过实时 PCR 测量吲哚胺 2,3-双加氧酶(IDO)、白细胞介素 10(IL-10)、诱导型一氧化氮合酶(iNOS)和转化生长因子-β(TGF-β)等 Mreg 功能分子的 mRNA 表达。从 MLR 培养物中收集上清液,通过高效液相色谱法(HPLC)测定 IDO 活性。通过 MLR 测定上清液中的 IDO 抑制剂 1-D/L-甲基色氨酸(1-MT)、iNOS 抑制剂 N-单甲基-L-精氨酸(L-NMMA)和抗 IFN-γ或抗 TGF-β单克隆抗体(mAb)处理,测试 Mreg 抑制能力。
我们证明了具有 CD14+CD16+CD80+CD83+CD86+HLA-DR 表型的诱导型 Mreg 能够抑制增殖的人 PBMC、CD4+和 CD8+T 细胞,即使在猪-人异种 MLR 中反应细胞:Mreg 的比例高达 32:1 时,抑制作用仍很强。Mreg 的强抑制能力与它们上调的 IDO 表达和活性相关。Mreg 中的 IDO 上调与异种反应性反应细胞在异种 MLR 中产生更多的 IFN-γ(IDO 刺激剂)有关。在 MLR 测定中添加 iNOS 抑制剂 L-NMMA 或抗 TGF-β mAb 对 Mreg 抑制效力没有影响,但通过中和 IFN-γ或 IDO 抑制剂 1-MT 抑制 IDO 活性,显著损害 Mreg 抑制异种反应的能力,表明 IDO 活性上调在 Mreg 介导的异种反应抑制中具有重要作用。体外异种反应。
本研究表明,人源性 Mreg 能够通过 IDO 参与的机制在体外抑制异种免疫反应,这表明它们作为异种移植中有效免疫调节工具的潜在作用。