TIGIT+ iTregs elicited by human regulatory macrophages control T cell immunity. Homo sapiens

Human regulatory macrophages (Mreg) have shown early clinical promise as a cell-based, adjunct immunosuppressive therapy in solid organ transplantation. It is hypothesised that recipient CD4+ T cell responses are actively regulated through direct allorecognition of donor-derived Mregs. Here we show that human Mregs convert allogeneic CD4+ T cells to IL-10-producing, TIGIT+ FoxP3+ induced regulatory T cells that non-specifically suppress bystander T cells and inhibit dendritic cell maturation. Differentiation of Mreg-induced Tregs (miTregs) relies on multiple, non-redundant mechanisms that are not exclusive to interaction of Mregs and T cells, including signals mediated by indoleamine 2,3-dioxygenase, TGF-β, retinoic acid, Notch and progestagen-associated endometrial protein. Preoperative administration of donor-derived Mregs to living-donor kidney transplant recipients results in an acute increase in circulating TIGIT+ Tregs. These results suggest a feed-forward mechanism by which Mreg treatment promotes allograft acceptance through rapid induction of direct pathway Tregs. Overall design: To better characterise the phenotype of human miTregs, whole genome gene expression profiling studies by microarray were performed. Mregs and IFN-γ Mφ were generated from CD14+ monocytes of 5 healthy donors. Seven days later, CD3+ T cells were isolated from 5 unrelated healthy donors and split into two lots: From the first lot, a pure population of fresh CD4+ T cells was obtained by flow-sorting and total RNA was extracted; the second lot of T cells was added into direct co-culture with allogeneic Mregs or IFN-γ Mφ at a 1:1 ratio for 5 days, or were cultured alone without stimulation for 5 days. After coculture, CD4+ and CD8+ T cells were flow-sorted and total RNA was extracted. Microarray analysis was performed on the Agilent Whole Human Genome Oligo Microarray 8x60K platform. Hence, a dataset was generated comprising eight sample groups (S1-S8) representing four CD4+ T cell populations and four CD8+ T cell populations from 5 independent donors produced by single-colour hybridisations.

人调节性巨噬细胞（Human regulatory macrophages, Mreg）作为实体器官移植领域的细胞辅助免疫抑制疗法，已展现出早期临床应用潜力。现有假说认为，受者CD4+ T细胞应答可通过供者来源的Mreg的直接同种识别被主动调控。本研究证实，人Mreg可将同种异体CD4+ T细胞转化为分泌白细胞介素10（IL-10）、表达T细胞免疫球蛋白和ITIM结构域蛋白（TIGIT）以及叉头框P3（FoxP3）的诱导性调节性T细胞，这类细胞可非特异性抑制旁观者T细胞，并阻断树突状细胞成熟。Mreg诱导的调节性T细胞（miTregs）的分化依赖于多种非冗余机制，且并非仅依赖于Mreg与T细胞的相互作用，包括吲哚胺2,3-双加氧酶（indoleamine 2,3-dioxygenase, IDO）、转化生长因子β（transforming growth factor-β, TGF-β）、视黄酸、Notch信号通路以及妊娠相关子宫内膜蛋白（progestagen-associated endometrial protein, PAEP）介导的信号通路。对活体供肾移植受者术前给予供者来源的Mreg，可导致循环中TIGIT+调节性T细胞水平快速升高。上述结果提示存在一种前馈调控机制：Mreg疗法可通过快速诱导直接通路调节性T细胞，从而促进移植物耐受。
实验设计：为更好地表征人miTregs的表型，本研究采用微阵列技术开展全基因组基因表达谱分析。从5名健康供者的CD14阳性单核细胞中分别诱导培养Mreg以及干扰素γ处理的巨噬细胞（IFN-γ Mφ）。培养7天后，从5名无关健康供者中分离CD3阳性T细胞并分为两部分：第一部分通过流式分选获得新鲜纯化的CD4+ T细胞，并提取总RNA；第二部分T细胞则与同种异体Mreg或IFN-γ Mφ以1:1的比例直接共培养5天，或在无刺激条件下单独培养5天。共培养结束后，通过流式分选分离CD4+与CD8+ T细胞，并提取总RNA。采用安捷伦（Agilent）全人类基因组寡核苷酸微阵列8x60K平台开展微阵列分析。最终本研究生成了包含8个样本组（S1-S8）的数据集，涵盖来自5名独立供者的4个CD4+ T细胞群与4个CD8+ T细胞群，所有样本均采用单色杂交技术制备。