Cell therapy with human IL-10-producing ILC2s limits xenogeneic Graft-versus-Host Disease by inhibiting pathogenic T cell responses

IL-10-producing group 2 innate lymphoid cells (ILC210) have important functions in regulating immune responses, yet their therapeutic potential remains largely unexplored. Here, we demonstrate that cell therapy with human ILC210 inhibits pathogenic T cell responses in humanized mouse models of Graft-versus-Host Disease (GVHD). Both prophylactic or post-onset adoptive transfer of human ILC210 significantly reduced GVHD severity and improved survival in NOD-scid IL2Rγnull (NSG) mice. Notably, ILC210 provided comparable protection to regulatory T cells (Tregs) without impairing T cell engraftment, instead decreasing intestinal T cell infiltration and suppressing CD4+Th1 and CD8+Tc1. Additionally, ILC210 conferred superior protection from GVHD than conventional IL-10-/lowILC2s, and blocking IL-10 and IL-4 abrogated the protective effects of ILC210 cell therapy. In GVHD-protected HSCT recipients, ILC2s with an ILC210 phenotype were present at normal levels and inversely correlated with Th1 cells, consistent with findings in humanized mouse models. Critically, ILC210 did not impair T cell-mediated graft-versus-leukemia effects in a humanized model. CITE-seq analysis identified CD49d and CD86 as novel markers that allow for enrichment of ILC210 from conventional ILC2s, and to track ILC210 in patient studies. These results establish a platform for >2000-fold expansion of human ILC210 with a stable phenotype and underscore ILC210 potential in cell therapies for GVHD and other immune-mediated diseases. ILC2s, ILC3, CD56bright and CD56dim NK cells were all stained and FACS sorted from a healthy donor. Cells were expanded for 28 days in distinct growth media for each cell type. Cells were stained with TotalSeqC antibodies including hashtags and then analyzed using CITE-seq. Stained ILC2s were mixed 1:1 with CD56dim NK cells and ILC3s mixed 1:1 with CD56dim NK cells. Each mixed sample was run on separate lanes and identified using hashtag antibodies. ILC2s and CD56dim NK cells were stained with Hashtag1, CD56bright NK cells with Hashtag2 and ILC3s with Hashtag3. *************************************************************** Submitter states that missing raw files are due to patient privacy concerns. ***************************************************************

产生IL-10的2型固有淋巴细胞（ILC210）在免疫应答调控中发挥关键作用，但其治疗潜力迄今尚未得到充分挖掘。本研究证实，采用人源ILC210进行细胞治疗，可在移植物抗宿主病（Graft-versus-Host Disease, GVHD）人源化小鼠模型中抑制致病性T细胞应答。无论是预防性还是发病后过继转输人源ILC210，均可显著降低NOD-scid IL2Rγnull（NSG）小鼠的GVHD严重程度并改善其存活率。值得注意的是，ILC210的保护效果与调节性T细胞（regulatory T cells, Tregs）相当，且不会损害T细胞植入，反而可减少肠道T细胞浸润，抑制CD4+Th1及CD8+Tc1细胞的活化。此外，相较于传统的IL-10低表达/阴性2型固有淋巴细胞（IL-10-/lowILC2s），ILC210的抗GVHD保护效果更优；而阻断IL-10与IL-4则会完全消除ILC210细胞治疗的保护作用。在经GVHD保护的造血干细胞移植（hematopoietic stem cell transplantation, HSCT）受者体内，具有ILC210表型的ILC2细胞水平处于正常范围，且与Th1细胞呈负相关，这与人源化小鼠模型中的研究结果一致。至关重要的是，在人源化模型中，ILC210并不会损害T细胞介导的移植物抗白血病（graft-versus-leukemia, GVL）效应。通过CITE-seq分析，本研究鉴定出CD49d与CD86作为新型标志物，可用于从传统ILC2中富集ILC210，并在临床患者研究中追踪ILC210。本研究结果构建了一套可将人源ILC210稳定表型扩增2000倍以上的技术平台，并凸显了ILC210在GVHD及其他免疫介导性疾病细胞治疗中的应用潜力。

研究人员从健康供体中分离并通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）得到ILC2、ILC3、CD56bright及CD56dim自然杀伤（natural killer, NK）细胞。针对不同细胞类型，分别在专属培养基中扩增培养28天。随后使用包含标签抗体的TotalSeqC抗体对细胞进行染色，并通过CITE-seq进行测序分析。将染色后的ILC2与CD56dim NK细胞以1:1比例混合，ILC3与CD56dim NK细胞同样以1:1比例混合。每组混合样本分别在不同测序泳道上样，并通过标签抗体进行样本鉴定：ILC2与CD56dim NK细胞使用Hashtag1染色，CD56bright NK细胞使用Hashtag2染色，ILC3使用Hashtag3染色。

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提交者声明：原始数据文件缺失系出于患者隐私保护考量。
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