Nasal anti-CD3 monoclonal antibody ameliorates traumatic brain injury, enhances microglial phagocytosis and reduces neuroinflammation via IL-10-dependent T_reg–microglia crosstalk
鼻腔抗 CD3 单克隆抗体通过 IL-10 依赖性调节性 T 细胞-小胶质细胞互作改善创伤性脑损伤、增强小胶质细胞吞噬功能并减轻神经炎症

Nasal aCD3 mAb improves cognitive and motor outcomes after TBI
鼻腔抗 CD3 单克隆抗体可改善创伤性脑损伤后的认知和运动功能

To investigate the therapeutic effect of nasal aCD3 in TBI, we employed the CCI model of TBI, which is known for its accuracy and reproducibility³³, to recapitulate features of moderate-to-severe TBI features, including cerebral contusion, neuroinflammation, blood–brain barrier (BBB) dysfunction and long-term behavioral outcomes. We investigated the therapeutic effects of nasal aCD3 on motor and behavioral outcomes in male mice treated at different times after injury: immediate (4-6 h post-injury), early (3 d post-injury) and delayed (14 d post-injury) (Fig. 1a). Treatment was continued once daily for 7 d, then 3× weekly for up to 1 month after injury (Fig. 1a). In the immediate treatment group, we found improvement in motor function and coordination assessed by the rotarod test (Fig. 1b). Using the Morris water maze (MWM) test, TBI mice treated with aCD3 exhibited near-complete restoration of spatial memory and increased time spent in the target quadrant during the probe trial (Fig. 1b) compared with isotype-treated TBI mice. In addition, we found that male mice treated with immediate nasal aCD3 exhibited less anxious behavior, using the open field test. We found a similar beneficial effect with early treatment (3 d post-injury), except for anxiety (Extended Data Fig. 1a).
为探究鼻腔给予 aCD3 对创伤性脑损伤（TBI）的治疗效果，我们采用具有高度准确性和可重复性的 CCI 模型 ³³ 来模拟中重度 TBI 特征，包括脑挫伤、神经炎症、血脑屏障（BBB）功能障碍及长期行为学改变。我们研究了不同时间窗（伤后立即[4-6 小时]、早期[3 天]和延迟期[14 天]）给予鼻腔 aCD3 对雄性小鼠运动及行为学结局的影响（图 1a ）。治疗方案为每日 1 次连续 7 天，之后每周 3 次持续至伤后 1 个月（图 1a ）。在立即治疗组中，通过转棒测试发现运动功能和协调性改善（图 1b ）。Morris 水迷宫（MWM）测试显示，与同型对照处理的 TBI 小鼠相比，aCD3 治疗组小鼠的空间记忆能力接近完全恢复，且在探测试验中目标象限停留时间显著延长（图 1b ）。此外，通过旷场实验发现，立即鼻腔给予 aCD3 的雄性小鼠表现出更少的焦虑行为。 我们发现早期治疗（伤后 3 天）具有类似的有益效果，但焦虑症状除外（扩展数据图 1a ）。

We then investigated the effect of delayed (14 d post-injury) nasal aCD3 treatment on behavioral outcomes in male mice and found no improvement in the treated the group compared with the TBI-iso control (Extended Data Fig. 1b). In addition to male mice, we investigated the effects of immediate nasal aCD3 treatment on TBI in female mice. We did not find notable differences in motor or spatial memory testing between the groups after moderate CCI. As reported in the literature, female rodents may outperform males in behavioral tasks after brain injury^34–36. Thus, we were unable to demonstrate the beneficial effect of nasal aCD3 treatment in female mice at this injury level (Extended Data Fig. 1c).
随后，我们研究了延迟（伤后 14 天）鼻腔给予 aCD3 治疗对雄性小鼠行为学结果的影响，发现与 TBI-iso 对照组相比，治疗组未见改善（扩展数据图 1b ）。除雄性小鼠外，我们还考察了即刻鼻腔 aCD3 治疗对雌性小鼠 TBI 的影响。在中度 CCI 后，我们未发现各组间在运动或空间记忆测试中存在显著差异。文献报道显示，脑损伤后雌性啮齿类动物在行为学任务中可能优于雄性 ^34–36 。因此，我们未能在此损伤程度下证明鼻腔 aCD3 治疗对雌性小鼠的获益效应（扩展数据图 1c ）。

We then investigated the effect of nasal aCD3 treatment on behavioral outcomes in a more severe form of TBI (3 mm tip diameter and 1.5 mm depth) in both male and female mice. We found improvement in motor function and coordination in the TBI-aCD3 group when compared with TBI-iso control in both sexes (Extended Data Fig. 1d,e). There was partial restoration in spatial memory and increased time spent in the target quadrant during the probe trial in the nasal aCD3-treated mice compared with TBI-iso control in both sexes. Male mice treated with nasal aCD3 exhibited less anxiety-like behavior after severe TBI, but we were unable to assess the beneficial effects of nasal aCD3 in female mice because they did not develop an anxiety phenotype after severe TBI (Extended Data Fig. 1d,e). These data clearly demonstrate that nasal aCD3 improves behavioral outcomes in moderate and severe CCI-induced TBI, with a greater effect being observed in male mice.
随后，我们研究了鼻腔给予 aCD3 抗体治疗对雌雄两性小鼠更严重型 TBI（撞击头端直径 3 毫米、深度 1.5 毫米）行为学结局的影响。与 TBI-iso 对照组相比，两性 TBI-aCD3 治疗组均表现出运动功能和协调能力的改善（扩展数据图 1d,e ）。鼻腔 aCD3 治疗组小鼠的空间记忆得到部分恢复，在探测试验中停留在目标象限的时间较 TBI-iso 对照组延长。在严重 TBI 后，鼻腔 aCD3 治疗的雄性小鼠表现出更少的焦虑样行为，但由于雌性小鼠在严重 TBI 后未出现焦虑表型，我们无法评估鼻腔 aCD3 对雌鼠的改善作用（扩展数据图 1d,e ）。这些数据明确表明，鼻腔 aCD3 能改善中重度 CCI 诱导型 TBI 的行为学结局，且在雄性小鼠中观察到的疗效更为显著。

Nasal aCD3 mAb ameliorates TBI neuropathology
鼻腔给予 aCD3 单抗改善创伤性脑损伤神经病理学改变

TBI induces BBB disruption, edema, neuronal death and tissue loss, in addition to the increased production of inflammatory mediators and gliosis²⁰. Therefore, we assessed the effects of nasal aCD3 on these neuropathological outcomes (Fig. 1a). We first measured the parenchymal lesion volume in sham-iso, TBI-aCD3 and TBI-iso groups at 7 d post-injury, using 3-tesla magnetic resonance imaging (MRI). We found a reduction in lesion volume in the nasal aCD3-treated group compared with TBI-iso controls (Fig. 1c and Extended Data Fig. 2a). We also measured lesion volume at 1 month post-CCI using hematoxylin and eosin (H&E) staining and, consistent with the MRI, we found a reduction in lesion volume in TBI-aCD3 mice compared with the TBI-iso control (Fig. 1d). Nasal aCD3 also improved BBB integrity at 3 d post-injury compared with the TBI-iso control (Extended Data Fig. 2b). The percentage of brain edema in the ipsilateral and contralateral hemispheres at 3 d post-CCI was reduced in the ipsilateral hemisphere for the TBI-aCD3 group compared with the TBI-iso control (Extended Data Fig. 2c). CCI increased cell death, as measured by TUNEL staining at 7 d after brain injury (Fig. 1e), which was reduced in nasal aCD3 treatment compared with TBI-iso controls. Consistent with previous reports, we found that CCI was associated with an increase in microglia or macrophage activation (Iba-1 staining) at 30 d post-injury compared with sham-iso controls (Fig. 1f)^20,37. We found that there was a reduction in microgliosis at 30 d post-injury in male mice in both the immediate and the early nasal aCD3 groups (Fig. 1f and Extended Data Fig. 2d). Of note, TBI has also been associated with changes in serum biomarkers³⁸ and we found a reduction in several TBI serum biomarkers in the immediate nasal aCD3 group, including glial fibrillary acidic protein (GFAP), UCH-L1 and the inflammatory cytokines IL-1b and CXCL1 versus controls. It is interesting that we also found an increase in the anti-inflammatory cytokine IL-10 in the immediate nasal aCD3 group versus control animals (Fig. 1g). These markers could be used to assess the efficacy of treatment in humans. As in male mice, we found that nasal aCD3 reduced lesion volume (Extended Data Fig. 2e) and microgliosis at the lesion site in female mice after severe TBI (Extended Data Fig. 2f). These data clearly demonstrate that the behavioral improvements observed in TBI mice treated with nasal aCD3 are associated with enhanced tissue integrity, as indicated by neuropathology and serum biomarkers.
创伤性脑损伤（TBI）除引发炎症介质增加和神经胶质增生外，还会导致血脑屏障破坏、水肿、神经元死亡及组织缺损 ²⁰ 。为此，我们评估了鼻腔给予 aCD3 抗体对这些神经病理学结局的影响（图 1a ）。首先采用 3 特斯拉磁共振成像（MRI）检测假手术组、TBI-aCD3 治疗组和 TBI 对照组在损伤后 7 天的脑实质病灶体积，发现鼻腔 aCD3 治疗组的病灶体积较 TBI 对照组显著减小（图 1c 及扩展数据图 2a ）。通过苏木精-伊红（H&E）染色检测损伤后 1 个月的病灶体积，结果与 MRI 一致：TBI-aCD3 组小鼠的病灶体积小于 TBI 对照组（图 1d ）。与 TBI 对照组相比，鼻腔 aCD3 在损伤后 3 天还能改善血脑屏障完整性（扩展数据图 2b ）。在损伤后 3 天，TBI-aCD3 组小鼠损伤同侧大脑半球的脑水肿比例较 TBI 对照组降低，而对侧半球无显著差异（扩展数据图 2c ）。通过 TUNEL 染色检测发现，控制性皮质撞击伤（CCI）在脑损伤后 7 天会加剧细胞死亡（图 1e ），与 TBI-iso 对照组相比，鼻内 aCD3 治疗组该指标降低。与既往报道一致，我们发现 CCI 损伤后 30 天小胶质细胞/巨噬细胞活化标志物（Iba-1 染色）较假手术组升高（图 1f ） ^20,37 。值得注意的是，在雄性小鼠中，无论是即刻还是早期鼻内 aCD3 治疗组，损伤后 30 天的小胶质细胞增生均有所减少（图 1f 及扩展数据图 2d ）。需特别指出的是，TBI 还与血清生物标志物改变相关 ³⁸ ，我们在即刻鼻内 aCD3 治疗组观察到多个 TBI 血清标志物水平降低，包括胶质纤维酸性蛋白（GFAP）、UCH-L1 以及炎症细胞因子 IL-1β和 CXCL1。有趣的是，与对照组相比，即刻鼻内 aCD3 治疗组抗炎细胞因子 IL-10 水平升高（图 1g ），这些标志物或可用于评估人类治疗效果。与雄性小鼠结果类似，我们发现鼻内 aCD3 能减少雌性小鼠严重 TBI 后的病灶体积（扩展数据图 2e ）及病灶区小胶质细胞增生（扩展数据图 2f ）。 这些数据清楚地表明，经鼻腔 aCD3 治疗的创伤性脑损伤小鼠所表现出的行为改善与组织完整性增强相关，神经病理学检查和血清生物标志物检测均证实了这一点。

Nasal aCD3 mAb expands central and peripheral T_reg cells after TBI
鼻腔给予抗 CD3 单克隆抗体(aCD3 mAb)在创伤性脑损伤(TBI)后促进中枢及外周调节性 T 细胞(Treg)扩增

TBI is associated with major changes in the cellular kinetics of both resident and infiltrating cells, which contribute to brain injury. Therefore, to characterize the effect of nasal aCD3 on TBI, we investigated the temporal roles of specific immune cell populations post-injury²⁰. We performed flow cytometry analysis on immune cells isolated from cervical lymph nodes (cLNs), meninges and the ipsilateral hemisphere at multiple time points post-TBI. We found that the immediate treatment of nasal aCD3 increased the percentage of total CD4⁺ T cells and CD4⁺FoxP3⁺ T_reg cells in cLNs at 1 d (Extended Data Fig. 3a) and increased the total number of those in the meninges at 2 d post-injury (Fig. 2a,b). Nasal aCD3 also expanded the total number of CD4⁺ T cells in the brain at 3 and 7 d post-injury and increased CD4⁺FoxP3⁺ T_reg cells in the first 30 d post-injury compared with TBI isotype controls (Fig. 2a,b and Extended Data Figs. 3a and 4a). Of note, we did not observe an increase in CD4⁺ T cells expressing latency-associated peptide (LAP), a membrane-bound transforming growth factor β (TGF-β) compared with the TBI-iso group (Fig. 2c and Extended Data Figs. 3b and 4a). Thus, nasal aCD3 expands T_reg cells to control neuroinflammation after TBI.
创伤性脑损伤（TBI）会导致驻留细胞和浸润细胞的细胞动力学发生重大变化，这些变化加剧了脑损伤。因此，为明确鼻腔给予 aCD3 抗体对 TBI 的作用机制，我们研究了损伤后特定免疫细胞亚群的时序性变化。通过流式细胞术分析 TBI 后多个时间点从颈淋巴结（cLNs）、脑膜及损伤同侧大脑半球分离的免疫细胞，发现鼻腔 aCD3 即时治疗可使损伤后 1 天 cLNs 中总 CD4+T 细胞及 CD4+FoxP3+T 细胞比例增加（扩展数据图），并在损伤后 2 天增加脑膜中这些细胞的总数（图）。与 TBI 同型对照组相比，鼻腔 aCD3 还使损伤后 3 天和 7 天脑内 CD4+T 细胞总数扩增，并在损伤后 30 天内持续增加 CD4+FoxP3+T 细胞数量（图及扩展数据图）。值得注意的是，与 TBI-iso 组相比，我们未观察到表达潜伏相关肽（LAP，一种膜结合型转化生长因子β）的 CD4+T 细胞增加（图。 2c 及扩展数据图 3b 和 4a ）。因此，鼻腔给予 aCD3 可通过扩增 T _reg 细胞来调控创伤性脑损伤后的神经炎症反应。

Nasal aCD3 mAb modulates the innate and adaptive immune response after TBI
鼻腔给予 aCD3 单抗可调节 TBI 后的先天性与适应性免疫应答

Nasal aCD3 also reduced the number of CD8⁺ cells at 14 and 30 d and helper T cells (T_H1 and T_H17 cells) at day 30 post-injury (Fig. 2c and Extended Data Figs. 3b and 4a). In addition, we found a significant reduction in neutrophil recruitment at day 1, monocytes at day 7 and natural killer cells at days 1 and 14 after CCI in the nasal aCD3 group compared with the TBI-iso controls (Fig. 2d and Extended Data Figs. 3c and 4b,c).
鼻腔给予 aCD3 还显著减少了伤后 14 天和 30 天的 CD8 ⁺ 细胞数量，以及伤后 30 天的辅助性 T 细胞（T _H 1 和 T _H 17 细胞）（图 2c 及扩展数据图 3b 和 4a ）。此外，与 TBI 对照组相比，鼻腔 aCD3 治疗组在 CCI 后第 1 天中性粒细胞浸润、第 7 天单核细胞浸润以及第 1 天和第 14 天的自然杀伤细胞数量均出现显著减少（图 2d 及扩展数据图 3c 和 4b,c ）。

T_reg cells induced by nasal aCD3 have a unique immunomodulatory profile
鼻腔给予 aCD3 诱导产生的 T 细胞具有独特的免疫调节特性

As shown in Fig. 2b, nasal aCD3 increased CD4⁺FoxP3⁺ T_reg cells in the first 30 d post-injury compared with TBI isotype controls (Fig. 2b). To elucidate the mechanisms whereby aCD3-induced T_reg cells may have contributed to post-TBI recovery, we performed RNA sequencing (RNA-seq) analysis on CD4⁺FoxP3(GFP)⁺ T_reg cells isolated from both the pericontusional brain tissue and blood of the sham and injured mice 7 d after CCI (Fig. 2f–k, Extended Data Fig. 5a and Supplementary Table 1). Principal component analysis (PCA) showed that the transcriptomic profile of brain FoxP3 T_reg cells was markedly different from blood FoxP3 T_reg cells after TBI (Fig. 2f). Consistent with recent reports²³, we found increased expression of multiple immunomodulatory and trophic factor genes (Il10, Spp1, Gas6, Igf1, Dab2, Lif, Areg, Il1r2, Irf8, Osm, Tgfa, Ccl8, and Hmox1) in brain-infiltrating T_reg cells from TBI mice compared with blood T_reg cells from sham mice (Extended Data Fig. 5b).
如图 2b 所示，与 TBI 同型对照组相比，鼻腔给予 aCD3 在损伤后 30 天内增加了 CD4 ⁺ FoxP3 ⁺ T _reg 细胞数量（图 2b ）。为阐明 aCD3 诱导的 T _reg 细胞促进 TBI 后恢复的机制，我们在 CCI 术后 7 天对假手术组和损伤组小鼠挫伤周边脑组织及血液中分离的 CD4 ⁺ FoxP3(GFP) ⁺ T _reg 细胞进行了 RNA 测序分析（图 2f–k 、扩展数据图 5a 及补充表 1 ）。主成分分析（PCA）显示，TBI 后脑部 FoxP3 T _reg 细胞的转录组特征与血液 FoxP3 T _reg 细胞存在显著差异（图 2f ）。与近期研究报道一致 ²³ ，我们发现相较于假手术组小鼠血液 T _reg 细胞，TBI 小鼠脑浸润 T _reg 细胞中多种免疫调节及神经营养因子基因（Il10、Spp1、Gas6、Igf1、Dab2、Lif、Areg、Il1r2、Irf8、Osm、Tgfa、Ccl8 和 Hmox1）表达上调（扩展数据图 5b ）。

We first examined the effects of TBI and nasal aCD3 treatment on blood FoxP3 T_reg cells (Fig. 2g, Supplementary Table 1). We found that FoxP3 T_reg cells isolated from nasally treated aCD3 TBI mice had a unique transcriptomic signature with upregulation of several genes involved in T_reg cell proliferation and homeostasis (Cd47 (ref. ³⁹), Ndfip1 (ref. ⁴⁰), Cd2 (ref. ⁴¹)) and T_reg cell function (Lef1 (ref. ⁴²), Lgals1 (ref. ⁴³) and Runx1 (ref. ⁴⁴)). Ingenuity pathway analysis (IPA) revealed IL-10 as a top activated upstream regulator in blood aCD3-induced FoxP3 T_reg cells compared with TBI-iso FoxP3 T_reg cells along with other transcription factors relevant for T_reg cell development and function (Foxo3 and Foxo4)⁴⁵ (Extended Data Fig. 5c). Notably, both Foxo4 and Stat3 have been reported to regulate IL-10 transcription in CD4⁺ T_reg cells⁴⁶.
我们首先检测了创伤性脑损伤（TBI）和鼻腔 aCD3 治疗对血液 FoxP3 T 细胞的影响（图 1，补充表 2）。研究发现，从鼻腔给予 aCD3 治疗的 TBI 小鼠中分离的 FoxP3 T 细胞具有独特的转录组特征，表现为多个参与 T 细胞增殖与稳态（Cd47（参考文献 5）、Ndfip1（参考文献 6）、Cd2（参考文献 7））以及 T 细胞功能（Lef1（参考文献 9）、Lgals1（参考文献 10）和 Runx1（参考文献 11））的基因表达上调。Ingenuity 通路分析（IPA）显示，与 TBI-iso FoxP3 T 细胞相比，血液 aCD3 诱导的 FoxP3 T 细胞中 IL-10 是最活跃的上游调节因子，同时还包括其他与 T 细胞发育和功能相关的转录因子（Foxo3 和 Foxo4）（扩展数据图 16）。值得注意的是，已有报道表明 Foxo4 和 Stat3 均可调控 CD4 T 细胞中 IL-10 的转录。

We then examined the effects of TBI and nasal aCD3 treatment on brain-infiltrating T_reg cells (Fig. 2h and Supplementary Table 1). We found both TBI groups (treated and untreated) had upregulated genes enriched in immune regulation (Spp1, Lgals3, Arg1, Ccl3, Ccl8, Hmox1, Ctsl, Lgals1, and Il1rn) (Fig. 2h). In addition, nasal aCD3-induced T_reg cells had further increased expression of genes involved in immunomodulation (Lrp1, Tyrobp, Cxcl10, and Itgam), regulation of phagocytosis (Rab31 and Rab7), neurotrophic factors (Igf1 and Psap), lipid homeostasis (Abca1 and Lpl) and other genes required for T_reg cell immunosuppressive function (Dab2 (ref. ⁴⁷), Plau⁴⁸ and Lgmn⁴⁹). Gene set enrichment analysis (GSEA) and IPA revealed enrichment of biological pathways involved in migration, regulation of immune response, phagocytosis, neurogenesis, homeostasis and secretory functions in brain TBI-aCD3-FoxP3 T_reg cells compared with brain sham-iso controls (Fig. 2i) and TBI-iso-FoxP3 T_reg cells (Fig. 2j). Similar to blood FoxP3⁺ T_reg cells, IPA identified IL-10 and Stat3 among the most activated upstream regulators of T_reg cells in the brain of TBI mice treated with nasal aCD3 (Fig. 2k and Extended Data Fig. 5d). The IL-10–Stat3 axis has been reported as playing a role in the immune tolerance conferred by T_reg cells⁵⁰; consistent with this, flow cytometry analysis of brain T_reg cells showed upregulation of IL-10 expression in aCD3-treated animals compared with TBI-iso controls (Fig. 2l). Taken together, these findings demonstrate that peripheral and central T_reg cells possess unique immunomodulatory profiles associated with the amelioration of TBI via nasal aCD3 treatment.
随后我们检测了 TBI 和鼻腔 aCD3 治疗对脑浸润 T _reg 细胞的影响（图 2h 和附表 1 ）。发现两个 TBI 组（治疗组与未治疗组）均出现免疫调节相关基因（Spp1、Lgals3、Arg1、Ccl3、Ccl8、Hmox1、Ctsl、Lgals1 和 Il1rn）上调（图 2h ）。此外，鼻腔 aCD3 诱导的 T _reg 细胞还进一步增加了以下基因表达：免疫调节相关基因（Lrp1、Tyrobp、Cxcl10 和 Itgam）、吞噬作用调控基因（Rab31 和 Rab7）、神经营养因子（Igf1 和 Psap）、脂质稳态基因（Abca1 和 Lpl）以及 T _reg 细胞免疫抑制功能所需的其他基因（Dab2（参考文献 ⁴⁷ ）、Plau ⁴⁸ 和 Lgmn ⁴⁹ ）。基因集富集分析（GSEA）和 IPA 显示，与假手术对照组（图 2i ）及 TBI-iso-FoxP3 T _reg 细胞组（图 2j ）相比，TBI-aCD3-FoxP3 T _reg 细胞中涉及迁移、免疫应答调控、吞噬作用、神经发生、稳态和分泌功能的生物通路显著富集。 与血液中的 FoxP3 ⁺ T _reg 细胞类似，IPA 分析发现 IL-10 和 Stat3 是鼻腔 aCD3 治疗的 TBI 小鼠脑内 T _reg 细胞最活跃的上游调节因子（图 2k 及扩展数据图 5d ）。已有报道表明 IL-10-Stat3 轴在 T _reg 细胞介导的免疫耐受中发挥作用 ⁵⁰ ；与此一致，脑部 T _reg 细胞的流式细胞分析显示，与 TBI-iso 对照组相比，aCD3 治疗组动物的 IL-10 表达上调（图 2l ）。综上所述，这些发现表明外周和中枢 T _reg 细胞具有独特的免疫调节特征，这些特征与鼻腔 aCD3 治疗改善 TBI 相关。

Nasal aCD3 mAb modulates the microglial inflammation after TBI
鼻腔给予 aCD3 单抗调控 TBI 后小胶质细胞炎症反应

Microglia play a critical role in TBI pathogenesis and their adherent activation contributes to long-term functional deficits after TBI²⁰. Nasal aCD3 treatment increased the migration of FoxP3⁺ T_reg cells to the meninges and CCI lesion site (Fig. 2e), where they were found to be in close contact with microglial dendrites after injury (Fig. 3a). Thus, to further elucidate the effects of TBI and immediate nasal aCD3 treatment on the microglial inflammatory profile post-injury, we performed RNA-seq analysis on sorted microglial single-cell suspensions from the ipsilateral hemisphere of the mouse brains, using the microglia-specific 4D4⁺ antibody⁵¹ (Extended Data Fig. 6a) at 7 and 30 d post-CCI (Fig. 3b and Supplementary Table 2). In analyzing the highest expressed genes in the sham-iso, TBI-iso and TBI-aCD3 microglial groups, we found that multiple microglial genes, including Cx3cr1, Hexb and Tmem119, were among the highest expressed (Extended Data Fig. 6b). A heatmap of the microglial gene signature demonstrated that the TBI-iso and TBI-aCD3 groups had overall similar transcriptomic profiles at 7 d, yet with demonstration of early upregulation of anti-inflammatory genes such as Cx3cr1, CD33, Hspa1a, and Hspa1b, whereas there was a clear modulation of the microglial transcriptomic signature in TBI-aCD3 group toward the sham-iso phenotype at 30 d post-injury (Fig. 3c and Supplementary Table 2), including the downregulation of microglial proinflammatory genes (Il6, Il18, Cd36, Ifitm3, and Lgals1) and upregulation of key homeostatic genes (Tgfbr2, Adgrg1, Mertk, Rhob, Atp8a2, Abcc3, Fscn1, Pde3b, Inpp4b, and Cmklr1).
小胶质细胞在 TBI 发病机制中起关键作用，其持续性激活会导致 TBI 后长期功能缺损 ²⁰ 。鼻腔 aCD3 治疗增加了 FoxP3 ⁺ T _reg 细胞向脑膜和 CCI 损伤部位的迁移（图 2e ），这些细胞被发现与损伤后的小胶质细胞树突密切接触（图 3a ）。为进一步阐明 TBI 及鼻腔 aCD3 即刻治疗对损伤后小胶质细胞炎症表型的影响，我们使用小胶质细胞特异性抗体 4D4 ^{+ 51} （扩展数据图 6a ），对 CCI 后 7 天和 30 天小鼠大脑同侧半球分选的小胶质细胞单细胞悬液进行 RNA-seq 分析（图 3b 及补充表 2 ）。在分析假手术组、TBI 对照组和 TBI-aCD3 治疗组小胶质细胞中高表达基因时，发现包括 Cx3cr1、Hexb 和 Tmem119 在内的多个小胶质细胞基因表达量最高（扩展数据图 6b ）。 小胶质细胞基因特征的热图分析显示，TBI-iso 组和 TBI-aCD3 组在损伤后 7 天具有总体相似的转录组特征，但已表现出抗炎基因（如 Cx3cr1、CD33、Hspa1a 和 Hspa1b）的早期上调。而到损伤后 30 天时，TBI-aCD3 组的小胶质细胞转录组特征明显向假手术组表型转变（图 3c 和补充表 2 ），表现为小胶质细胞促炎基因（Il6、Il18、Cd36、Ifitm3 和 Lgals1）的下调，以及关键稳态基因（Tgfbr2、Adgrg1、Mertk、Rhob、Atp8a2、Abcc3、Fscn1、Pde3b、Inpp4b 和 Cmklr1）的上调。

Microglia possess a unique transcriptomic signature that enables them to perform sensing, homeostatic and housekeeping functions, which vary according to the brain’s physiological or pathological state⁵². To determine the effects of TBI and nasal anti-CD3 on these essential microglial functions, we examined the microglial sensome dataset for genes and pathways involved in each of these functions⁵³. At 7 d post-TBI, we found that microglia from the nasal aCD3-treated group, compared with TBI-iso, were associated with increased expression of homeostatic and sensing genes involved in pattern recognition receptors (Tlr1), Fc receptors (Cmtm7), Siglec receptors (Cd33), cell–cell interaction (Cd84 and Lag3) and chemokine receptors (Cx3cr1) (Fig. 3d). Cd33 and Lag3 were among the most significantly differentially expressed genes (DEGs) in the TBI aCD3-treated group compared with TBI-iso control at 7 d post-injury. CD33 activity has been implicated in processes including microglial endogenous ligand receptors and sensors, adhesion processing of immune cells and inhibition of cytokines release by monocytes^54,55. Lymphocyte activation gene-3 (Lag3) regulates T cell expansion and limits the duration and intensity of the immune response⁵⁶. Moreover, the TBI aCD3-treated group exhibited downregulation of Cd14, a key regulator of microglial proinflammatory responses to injury⁵⁷. At 30 d post-injury, we found that nasal aCD3 treatment was associated with increased expression of several transforming growth factor β (TGF-β)-signaling genes, including Smad3, Tgfbr1 and Tgfbr2 compared with TBI-iso control (Fig. 3c,d and Supplementary Table 2)⁵⁸. TGF-β is required for maintaining the microglial homeostatic state⁵⁹ and modulating microglia-mediated inflammation after acute brain injury⁶⁰. Nasal aCD3 treatment was also associated with decreased expression of sensing genes involved in cytokine receptor (Tnfrsf17), FC receptor (Fcgr1, Fcgr4, Fcgr3, and Fcer1g) and pattern recognition receptors (Cd74, Tlr6, Upk1b, and Selplg) compared with TBI-iso control (Fig. 3d).
小胶质细胞具有独特的转录组特征，使其能够执行感知、稳态维持和管家功能，这些功能会随大脑生理或病理状态的变化而改变 ⁵² 。为探究创伤性脑损伤（TBI）和鼻腔抗 CD3 单抗对这些关键小胶质细胞功能的影响，我们检测了参与各项功能的微胶质感知组基因及通路 ⁵³ 。在 TBI 后第 7 天，与 TBI-iso 组相比，鼻腔 aCD3 治疗组的小胶质细胞显示出参与模式识别受体（Tlr1）、Fc 受体（Cmtm7）、Siglec 受体（Cd33）、细胞间相互作用（Cd84 和 Lag3）以及趋化因子受体（Cx3cr1）的稳态与感知基因表达上调（图 3d ）。其中 Cd33 和 Lag3 是 TBI 后 7 天 aCD3 治疗组相较于 TBI-iso 对照组差异最显著的基因（DEGs）。CD33 活性已被证实参与微胶质细胞内源性配体受体与传感器的调控、免疫细胞粘附处理以及单核细胞细胞因子释放抑制等过程 ^54,55 。 淋巴细胞激活基因-3（Lag3）可调节 T 细胞增殖并限制免疫反应的持续时间和强度 ⁵⁶ 。此外，创伤性脑损伤 aCD3 治疗组表现出 Cd14（小胶质细胞对损伤促炎反应的关键调节因子）的下调 ⁵⁷ 。在损伤后 30 天，我们发现与 TBI-iso 对照组相比，鼻腔 aCD3 治疗与多个转化生长因子β（TGF-β）信号通路基因（包括 Smad3、Tgfbr1 和 Tgfbr2）表达增加相关（图 3c,d 和补充表 2 ） ⁵⁸ 。TGF-β是维持小胶质细胞稳态 ⁵⁹ 以及调节急性脑损伤后小胶质细胞介导的炎症反应所必需的 ⁶⁰ 。与 TBI-iso 对照组相比，鼻腔 aCD3 治疗还导致细胞因子受体（Tnfrsf17）、FC 受体（Fcgr1、Fcgr4、Fcgr3 和 Fcer1g）及模式识别受体（Cd74、Tlr6、Upk1b 和 Selplg）相关感知基因表达降低（图 3d ）。

We then performed GSEA comparing the TBI groups with sham-iso controls at 7 and 30 d post-injury. At 7 d post-injury, we observed an upregulation in pathways involved in oxidative stress and neuron apoptosis in the TBI-iso group compared with the sham-iso group. The TBI-aCD3-treated animals had less upregulation of genes in these pathways and more upregulation in microglial pathways involved in the regulation of anti-inflammatory IL-10 production, phagocytosis and tolerance induction at 7 d post-injury (Fig. 3e). At 30 d post-TBI, we found enrichment of pathways involved in proinflammatory mechanisms (IL-6, IL-1 and tumor necrosis factor production), adaptive immune response, T cell cytotoxicity and cell killing pathways in the TBI-iso control compared with the sham-iso group. However, TBI-aCD3-treated animals had less upregulation of genes in these proinflammatory pathways and more upregulation in biological pathways involved in regulation of phagocytosis and cytokine production (Fig. 3e).
随后我们通过 GSEA 分析比较了创伤性脑损伤组与假手术对照组在伤后 7 天和 30 天的基因表达差异。伤后 7 天时，与假手术组相比，TBI-iso 组显示出氧化应激和神经元凋亡相关通路上调。而经 TBI-aCD3 治疗的动物在这些通路中的基因上调程度较轻，同时在调控抗炎因子 IL-10 生成、小胶质细胞吞噬功能及免疫耐受诱导等通路上调更为显著（图 3e ）。至创伤后 30 天，与假手术组相比，TBI-iso 对照组中促炎机制（IL-6、IL-1 及肿瘤坏死因子生成）、适应性免疫应答、T 细胞毒性及细胞杀伤相关通路呈现富集。但 TBI-aCD3 治疗组动物在这些促炎通路中的基因上调减弱，而在调控吞噬作用与细胞因子生成的生物学通路上调更为明显（图 3e ）。

Previous studies have suggested an association between microglia-mediated chronic inflammation after TBI and subsequent chronic neurodegeneration^20,52. To investigate this relationship, we analyzed the expression levels of inflammatory response genes and genes characteristic of disease-associated microglia (DAMs)⁶¹ and neurodegenerative microglia (MGnDs)⁵¹ in all three groups at 30 d post-injury. We found that the TBI-iso microglia had a unique proinflammatory response (Fig. 3f) and a DAM or MGnD signature (Fig. 3g), because we found increased expression of key proinflammatory (Casp1, Nfkbia, C5ar1, Lyz1, Ifitm3, Il6, Lyz2, Cd86, and Irgm1), DAM-1 and -2 genes (B2m, Cstb, Cd52, Cd9, Cst7, Fth1, Ccl6, and Tyrobp) and MGnD microglial genes (Cybb, Gpnmb, Lgals3, and Clec7a)^51,61. Importantly, several of these genes were downregulated in nasal aCD3-treated mice, approaching expression levels observed in the sham group (Fig. 3f-g).
先前研究表明，创伤性脑损伤（TBI）后小胶质细胞介导的慢性炎症与后续慢性神经退行性病变存在关联 ^20,52 。为探究这一关系，我们在损伤后 30 天检测了三组实验对象的炎症反应基因表达水平，以及疾病相关小胶质细胞（DAMs） ⁶¹ 和神经退行性小胶质细胞（MGnDs） ⁵¹ 的特征基因。研究发现 TBI-iso 组小胶质细胞呈现独特的促炎反应（图 3f ）及 DAM/MGnD 特征（图 3g ），表现为关键促炎基因（Casp1、Nfkbia、C5ar1、Lyz1、Ifitm3、Il6、Lyz2、Cd86 和 Irgm1）、DAM-1/-2 基因（B2m、Cstb、Cd52、Cd9、Cst7、Fth1、Ccl6 和 Tyrobp）及 MGnD 小胶质细胞基因（Cybb、Gpnmb、Lgals3 和 Clec7a）表达上调 ^51,61 。值得注意的是，经鼻腔 aCD3 单抗治疗的小鼠中，多个基因表达下调至接近假手术组水平（图 3f-g ）。

Consistent with the microglial transcriptomic data, quantitative PCR with reverse transcription (RT–qPCR) from ipsilateral hemisphere sorted microglia (Fig. 3h) and brain tissue (Extended Data Fig. 6c) revealed that, compared with the TBI-iso control, nasal aCD3 treatment increased the expression of the anti-inflammatory cytokine Il10 in both microglia and brain tissue at 7 d post-injury. Moreover, treatment reduced several proinflammatory markers in microglia (Clec7a, Tlr2, Il1b, Tnf, Cd86, and Il18) and brain tissue (Il6, Tnf, Ifng, Il17a, and Ccl5) at 30 d post-injury. Of note, mice treated with nasal aCD3 showed upregulation of brain-derived neurotrophic factor (Bdnf), a neurotrophin that has a critical role in neuronal survival and is involved in synaptic plasticity, learning and memory⁶², compared with the TBI-iso control, at 1 month post-injury (Extended Data Fig. 6c).
与小胶质细胞转录组数据一致，通过同侧半球分选小胶质细胞（图 3h ）及脑组织（扩展数据图 6c ）的反转录定量 PCR（RT-qPCR）显示：与 TBI-iso 对照组相比，鼻腔给予 aCD3 治疗在损伤后 7 天同时提升了小胶质细胞和脑组织中抗炎细胞因子 Il10 的表达水平。此外，该治疗在损伤后 30 天显著降低了小胶质细胞（Clec7a、Tlr2、Il1b、Tnf、Cd86 和 Il18）与脑组织（Il6、Tnf、Ifng、Il17a 和 Ccl5）中多种促炎标志物的表达。值得注意的是，损伤后 1 个月时（扩展数据图 6c ），鼻腔 aCD3 治疗组小鼠相比 TBI-iso 对照组显示出脑源性神经营养因子（Bdnf）的上调——这种神经营养素对神经元存活至关重要，并参与突触可塑性、学习与记忆过程 ⁶² 。

To further investigate whether delaying the therapeutic window of nasal anti-CD3 to 3 d post-injury would still modulate the microglial transcriptomic profile at 30 d post-injury, we performed bulk RNA-seq on isolated microglia from mice treated with nasal aCD3 or isotype control from day 3 to day 30 (early treatment) post-injury (Supplementary Table 2). We found that the early treatment paradigm also modulated the microglial transcriptomic signature in the TBI-aCD3 group toward the sham-iso phenotype (Extended Data Fig. 6d) and was associated with decreased expression of several proinflammatory and DAM or MGnD genes (Lpl, Lyz1, Nfkbia, Irgm1, Lyz2 and Apoe) (Extended Data Fig. 6e). Consistent with this, GSEA analysis revealed that the early nasal aCD3 treatment paradigm was also associated with downregulation in several inflammatory response and immune response-related pathways compared with TBI-iso (Extended Data Fig. 6f). In line with these results, RT–qPCR of ipsilateral brain tissue demonstrated a reduction in several proinflammatory genes including Il6, Il18 and Tnf in TBI-aCD3 compared with TBI-iso controls (Extended Data Fig. 6g).
为进一步探究伤后 3 天延迟给予鼻用抗 CD3 单抗治疗是否仍能调控伤后 30 天小胶质细胞的转录组特征，我们对伤后第 3 至 30 天接受鼻用 aCD3 或同型对照（早期治疗组）的小鼠分离小胶质细胞进行批量 RNA 测序（补充表 2 ）。研究发现早期治疗模式同样使 TBI-aCD3 组的小胶质细胞转录特征向假手术-同型对照组表型转变（扩展数据图 6d ），并伴随多种促炎基因及 DAM/MGnD 相关基因（Lpl、Lyz1、Nfkbia、Irgm1、Lyz2 和 Apoe）表达下调（扩展数据图 6e ）。与此一致的是，GSEA 分析显示相较于 TBI-同型对照组，早期鼻用 aCD3 治疗方案还导致多种炎症反应和免疫反应相关通路下调（扩展数据图 6f ）。与这些结果相印证，同侧脑组织的 RT-qPCR 检测显示，相比 TBI-同型对照组，TBI-aCD3 组中包括 Il6、Il18 和 Tnf 在内的多个促炎基因表达降低（扩展数据图 6g ）。

To assess the effect of nasal aCD3 treatment on microglia inflammation in female mice after severe TBI, we performed bulk RNA-seq on sorted microglia at 30 d post-injury (Supplementary Table 2). We found a similar therapeutic effect in female mice with the TBI-aCD3 microglial transcriptomic profile reverting to that of sham mice, associated with a reduced inflammatory and DAM or MGnD profile relative to TBI-iso mice controls (Extended Data Fig. 6h,i). RT–qPCR of ipsilateral brain tissue showed consistent findings (Extended Data Fig. 6j). Taken together, these findings demonstrate that nasal aCD3 shifts microglia from a pathogenic, disease-associated phenotype to a beneficial, homeostatic phenotype.
为评估鼻腔 aCD3 治疗对雌性小鼠严重 TBI 后小胶质细胞炎症的影响，我们在损伤后 30 天对分选的小胶质细胞进行了批量 RNA 测序（补充表 2 ）。研究发现雌性小鼠具有相似治疗效果，TBI-aCD3 组小胶质细胞转录组特征恢复至假手术组水平，相较于 TBI-iso 对照组表现出炎症反应及 DAM/MGnD 特征减弱（扩展数据图 6h,i ）。同侧脑组织 RT-qPCR 结果与此一致（扩展数据图 6j ）。综上表明，鼻腔 aCD3 能使小胶质细胞从致病性、疾病相关表型转变为有益的内稳态表型。

Nasal aCD3 increases microglia phagocytosis in an IL-10-dependent manner
鼻腔给予 aCD3 通过 IL-10 依赖性途径增强小胶质细胞吞噬功能

Acute brain injury leads to neuronal cell death and the release of substantial amounts of myelin and cell debris, which subsequently trigger a persistent and intense inflammatory response that may impede neurological recovery. Microglia and macrophages play an important role in debris removal and modulating the immune response post-injury²⁰. However, uncontrolled phagocytosis may lead to progressive brain damage and worsening cognitive and memory impairments⁶³. We found that TBI-aCD3-treated animals had increased upregulation in biological pathways involved in regulation of phagocytosis (Fig. 3e) and a distinct upregulated phagocytic signature at 7 d post-injury (Fig. 4a), with increased expression of several key microglial phagocytosis regulators at 30 d post-TBI, including Mertk⁶⁴, Sirpa⁶⁵ and Tlr4 (ref. ⁶⁶), compared with TBI-iso microglia (Supplementary Table 2). To functionally assess the phagocytic capacity of microglia after TBI (with and without nasal aCD3 treatment), we performed an in vivo experiment in which the TBI-induced lesion was injected with either labeled apoptotic neurons or phosphate-buffered saline (PBS) on day 6 post-injury for 16 h and on day 7 post-injury for 4 h post-injection experiments (Fig. 4b). In line with our microglial transcriptomic data, aCD3-treated animals had higher microglial phagocytic capacity to uptake apoptotic neurons at both 4 and 16 h post-injection compared with the TBI-iso group (Fig. 4c,d and Extended Data Fig. 7a,b). To elucidate the mechanism by which nasal aCD3 enhanced microglial phagocytic capacity after TBI, we performed bulk RNA-seq on phagocytic and nonphagocytic microglia isolated at 4 and 16 h post-injection from TBI-iso and TBI-aCD3 groups at 7 d post-TBI (Supplementary Table 3). At 4 h post-injection, we observed a distinct microglial transcriptomic signature for phagocytic TBI-aCD3 microglia, characterized by the upregulation of several key phagocytosis genes (Fig. 4e). Compared with nonphagocytic TBI-iso microglia, phagocytic TBI-aCD3 microglia were associated with increased expression of genes involved in the recognition and engulfment (eat me and find me signals) of apoptotic cells and debris (Mertk⁶⁷, Mrc1 (ref. ⁶⁸), Abca1 (ref. ⁶⁷), Lrp1 (ref. ⁶⁷), and Stab1 (ref. ⁶⁹)), digestion and degradation of engulfed material including lysosomal machinery (Rab27a⁷⁰, Smcr8 (ref. ⁷¹), Clec16a⁷², and Vps8 (ref. ⁷³)), lipid metabolism (Apoc1 (ref. ⁷⁴), Olr1 (ref. ⁷⁵) and Ldlr⁷⁶), cytoskeleton dynamics pathways (Myo1e⁷⁷) and regulation of microglial phagocytosis (Tspo⁷⁸, Qk⁷⁹ and Pik3cg⁸⁰) (Fig. 4e,f and Supplementary Table 3). Consistent with these findings, GSEA analysis of phagocytic TBI-aCD3 microglia compared with nonphagocytic TBI-iso microglia showed enrichment for pathways involved in phagocytosis, along with other pathways pertinent for the phagocytic process such as endocytosis, pattern recognition and cell migration, all of which were not upregulated in the phagocytic TBI-iso microglia (Fig. 4g). In addition, compared with phagocytic TBI-iso microglia, phagocytic TBI-aCD3 microglia had increased upregulation of antigen presentation and IL-10 pathways (Fig. 4h). IPA analysis revealed IL-10 as a top regulator and signaling pathway in aCD3-treated phagocytic microglia post-TBI (Fig. 4h,i). We found a similar pattern of increased expression of phagocytosis machinery-related genes and pathways at 16 h post-injection (Extended Data Fig. 7c) and also observed that phagocytic TBI-aCD3 microglia had a more homeostatic and a less inflammatory (disease-associated) profile compared with phagocytic TBI-iso microglia (Extended Data Fig. 7d,e and Supplementary Table 3).
急性脑损伤会导致神经元细胞死亡并释放大量髓鞘和细胞碎片，这些物质随后会引发持续而强烈的炎症反应，可能阻碍神经功能恢复。小胶质细胞和巨噬细胞在损伤后清除碎片及调节免疫反应中起重要作用 ²⁰ 。然而不受控制的吞噬作用可能导致进行性脑损伤及认知记忆功能恶化 ⁶³ 。我们发现 TBI-aCD3 治疗组动物在吞噬作用调控相关生物通路上调增加（图 3e ），并在损伤后 7 天呈现显著上调的吞噬特征（图 4a ），与 TBI-iso 组小胶质细胞相比，损伤后 30 天多个关键小胶质细胞吞噬调控因子（包括 Mertk ⁶⁴ 、Sirpa ⁶⁵ 和 Tlr4（参考文献 ⁶⁶ ））表达增加（补充表 2 ）。 为功能性评估创伤性脑损伤（TBI）后小胶质细胞的吞噬能力（含/不含鼻腔 aCD3 治疗），我们进行了体内实验：在损伤后第 6 天向 TBI 病灶注射标记的凋亡神经元或磷酸盐缓冲液（PBS）持续 16 小时，并于损伤后第 7 天进行注射后 4 小时实验（图 4b ）。与小胶质细胞转录组数据一致，与 TBI-iso 组相比，aCD3 治疗组动物在注射后 4 小时和 16 小时均表现出更强的凋亡神经元摄取能力（图 4c,d 及扩展数据图 7a,b ）。为阐明鼻腔 aCD3 增强 TBI 后小胶质细胞吞噬能力的机制，我们对 TBI 后 7 天从 TBI-iso 组和 TBI-aCD3 组分离的吞噬性与非吞噬性小胶质细胞进行了批量 RNA 测序（补充表 3 ）。注射后 4 小时，我们观察到吞噬性 TBI-aCD3 小胶质细胞具有独特的转录组特征，表现为多个关键吞噬基因的上调（图 4e ）。 与非吞噬型的 TBI-iso 小胶质细胞相比，吞噬型 TBI-aCD3 小胶质细胞表现出以下基因表达上调：参与凋亡细胞和碎片识别与吞噬（"吃我"和"找我"信号）的基因（Mertk ⁶⁷ 、Mrc1（参考文献 ⁶⁸ ）、Abca1（参考文献 ⁶⁷ ）、Lrp1（参考文献 ⁶⁷ ）和 Stab1（参考文献 ⁶⁹ ））、被吞噬物质的消化降解相关基因（包括溶酶体机制相关基因 Rab27a ⁷⁰ 、Smcr8（参考文献 ⁷¹ ）、Clec16a ⁷² 和 Vps8（参考文献 ⁷³ ））、脂质代谢相关基因（Apoc1（参考文献 ⁷⁴ ）、Olr1（参考文献 ⁷⁵ ）和 Ldlr ⁷⁶ ）、细胞骨架动态通路相关基因（Myo1e ⁷⁷ ）以及小胶质细胞吞噬调控相关基因（Tspo ⁷⁸ 、Qk ⁷⁹ 和 Pik3cg ⁸⁰ ）（图 4e,f 和补充表 3 ）。与这些发现一致的是，GSEA 分析显示吞噬型 TBI-aCD3 小胶质细胞相较于非吞噬型 TBI-iso 小胶质细胞在吞噬作用相关通路上呈现富集，同时还富集了与吞噬过程相关的其他通路如内吞作用、模式识别和细胞迁移——这些通路在吞噬型 TBI-iso 小胶质细胞中均未出现上调（图 4g ）。 此外，与吞噬型 TBI-iso 小胶质细胞相比，吞噬型 TBI-aCD3 小胶质细胞的抗原呈递和 IL-10 通路上调更为显著（图 4h ）。IPA 分析显示 IL-10 是 TBI 后 aCD3 处理吞噬型小胶质细胞中的顶级调节因子和信号通路（图 4h,i ）。我们在注射后 16 小时也发现了吞噬机制相关基因和通路表达增加的相似模式（扩展数据图 7c ），并观察到与吞噬型 TBI-iso 小胶质细胞相比，吞噬型 TBI-aCD3 小胶质细胞具有更稳态且炎症（疾病相关）特征更弱的表型（扩展数据图 7d,e 及补充表 3 ）。

To investigate the role of IL-10 in the regulation of microglia phagocytic capacity after TBI, we repeated the in vivo microglia phagocytosis assay using IL-10 knockout (KO) mice (Fig. 4j). We found that the increased phagocytic capacity of TBI-aCD3 microglia at 4 h post-injection was significantly attenuated in the IL-10 KO TBI-aCD3 microglial group. Collectively, these findings demonstrate that nasal aCD3 treatment increases the phagocytic machinery of microglia after acute TBI, in an IL-10-dependent manner. In addition, we found that the phagocytic capacity of wild-type (WT) TBI-iso microglia was significantly reduced in the IL-10 KO TBI-iso microglia group, demonstrating the importance of IL-10 in microglial phagocytic function after TBI.
为探究 IL-10 在创伤性脑损伤（TBI）后调控小胶质细胞吞噬功能中的作用，我们使用 IL-10 基因敲除（KO）小鼠重复了体内小胶质细胞吞噬实验（图 4j ）。研究发现，在 IL-10 KO TBI-aCD3 组中，注射后 4 小时 TBI-aCD3 小胶质细胞增强的吞噬能力显著减弱。这些结果共同表明，鼻腔给予 aCD3 通过 IL-10 依赖性机制增强了急性 TBI 后小胶质细胞的吞噬功能。此外，我们发现野生型（WT）TBI-iso 组小胶质细胞的吞噬能力在 IL-10 KO TBI-iso 组中显著降低，证实了 IL-10 对 TBI 后小胶质细胞吞噬功能的关键作用。

Nasal aCD3 ameliorates TBI outcomes via IL-10/IL-10R signaling in microglia
鼻腔给予 aCD3 通过小胶质细胞中 IL-10/IL-10R 信号通路改善 TBI 预后

IL-10 is a potent anti-inflammatory cytokine produced by T_reg cells that acts on many cell types as a result of the presence of IL-10 receptor (IL-10R) on almost all hematopoietic cells⁸¹. IL-10 signaling is critical to maintain microglia under a homeostatic phenotype, because genetic depletion of IL-10 under proinflammatory conditions results in increased release of proinflammatory cytokines and chemokines⁸². We found a clear upregulation of the microglial IL-10-cytokine gene expression signature in TBI-aCD3 microglia group at 7 days post-CCI as compared to Sham-Iso and TBI-Iso controls (Fig. 5a). We also found that nasal aCD3 induced IL-10-secreting T_reg cells (from day 3 to day 30 post-injury) (Fig. 5b and Extended Data Fig. 8a) and increased IL-10 expression in both microglia and brain tissue derived from the site of injury (Fig. 3h and Extended Data Fig. 6c). We then investigated whether IL-10 played a role in the beneficial effects of nasal aCD3 by administering anti-IL-10R (aIL-10R)-blocking antibody intraperitoneally (i.p.) every 3 d post-injury (Fig. 5c) and investigating the behavioral outcomes and BBB disruption in sham-iso, TBI-iso, TBI-aCD3 and TBI-aCD3+aIL-10R groups. We found that the improvements in BBB disruption (Fig. 5d) and motor coordination functions, spatial memory and anxiety-like behavior observed in TBI-aCD3 were abrogated by blocking IL-10R (TBI-aCD3+aIL-10R group) (Fig. 5e).
IL-10 是一种由 T _reg 细胞产生的强效抗炎细胞因子，由于几乎所有造血细胞 ⁸¹ 表面都存在 IL-10 受体（IL-10R），其可作用于多种细胞类型。IL-10 信号传导对维持小胶质细胞稳态表型至关重要，因为在促炎条件下 IL-10 基因缺失会导致促炎细胞因子和趋化因子释放增加 ⁸² 。我们发现，与 Sham-Iso 和 TBI-Iso 对照组相比，CCI 术后 7 天的 TBI-aCD3 小胶质细胞组中，小胶质细胞 IL-10 细胞因子基因表达特征明显上调（图 5a ）。研究还发现鼻腔给予 aCD3 可诱导 IL-10 分泌型 T _reg 细胞（从损伤后第 3 天持续至第 30 天）（图 5b 及扩展数据图 8a ），并提高损伤部位小胶质细胞和脑组织中的 IL-10 表达（图 3h 及扩展数据图 6c ）。随后我们通过腹腔注射（i.p.）抗 IL-10R（aIL-10R）阻断抗体（每 3 天一次）来研究 IL-10 是否参与鼻腔 aCD3 的治疗作用（图 5c ）并研究假手术组（sham-iso）、创伤性脑损伤组（TBI-iso）、抗 CD3 单抗治疗组（TBI-aCD3）及抗 CD3 单抗联合 IL-10 受体阻断组（TBI-aCD3+aIL-10R）的行为学结果与血脑屏障破坏情况。我们发现，抗 CD3 单抗治疗组（TBI-aCD3）在改善血脑屏障破坏（图 5d ）、运动协调功能、空间记忆及焦虑样行为方面的效果，均被 IL-10 受体阻断剂（TBI-aCD3+aIL-10R 组）所抵消（图 5e ）。

We next investigated the impact of blocking IL-10R on the microglial inflammatory transcriptomic profile by performing RNA-seq on sorted microglia from the ipsilateral hemisphere of the brain (Extended Data Fig. 6a) at 1 month post-CCI (Fig. 3b and Supplementary Table 4). We found that the modulatory effect of nasal aCD3 on microglia was abrogated by blocking IL-10 as shown in the microglial heatmap signature (Fig. 5f). At 1 month post-CCI, similar to the transcriptomic signature of TBI-iso control, microglia from the TBI-aCD3+aIL-10R group had a more proinflammatory profile compared with sham-iso and the TBI-aCD3 group and was associated with decreased expression of homeostatic markers such as Mertk, Tgfbr2, Atp8a2, and Adgrg1 (Fig. 5f and Supplementary Table 4).
我们随后通过 RNA 测序研究了阻断 IL-10R 对损伤同侧大脑半球小胶质细胞炎症转录组特征的影响（扩展数据图 6a ），检测时间为 CCI 术后 1 个月（图 3b 和补充表 4 ）。小胶质细胞热图特征显示（图 5f ），鼻腔给予 aCD3 对小胶质细胞的调节作用会因 IL-10 阻断而消除。CCI 术后 1 个月，与 TBI-iso 对照组相似，TBI-aCD3+aIL-10R 组的小胶质细胞较假手术组和 TBI-aCD3 组表现出更强的促炎特征，同时伴随稳态标志物（如 Mertk、Tgfbr2、Atp8a2 和 Adgrg1）表达水平下降（图 5f 和补充表 4 ）。

We hypothesized that the beneficial effect of the T_reg cells induced by nasal aCD3 was dependent on IL10R signaling in microglia. We thus investigated this hypothesis by using IL-10R^flox/floxTmem119^CreETR2 conditional and tamoxifen-induced KO mice and littermate controls (Extended Data Fig. 8b). We investigated the effects of microglial IL-10R ablation on the behavioral and microglial phagocytic capacity at 7 d post-TBI (Fig. 5g). We found that tamoxifen-treated TBI-aCD3-IL-10R^flox/floxTmem119^CreETR2 mice exhibited worse motor and cognitive outcomes (Fig. 5h) and reduced microglial phagocytic capacity (Fig. 5i) compared with tamoxifen-treated TBI-aCD3 littermate controls, further supporting the role for IL-10 in modulating the microglial gene signature after nasal aCD3 treatment. Taken together, these data clearly demonstrate a critical role for IL-10/IL-10R signaling in augmenting microglial phagocytic capacity and ameliorating disease in TBI.
我们假设鼻腔给予 aCD3 诱导的 T _reg 细胞的神经保护作用依赖于小胶质细胞中的 IL10R 信号通路。为此，我们使用 IL-10R ^flox/flox Tmem119 ^CreETR2 条件性敲除和他莫昔芬诱导的基因敲除小鼠及其同窝对照进行验证（扩展数据图 8b ）。研究发现在 TBI 后第 7 天，小胶质细胞 IL-10R 缺失对行为学表现和小胶质细胞吞噬功能的影响（图 5g ）。与他莫昔芬处理的 TBI-aCD3 同窝对照组相比，他莫昔芬处理的 TBI-aCD3-IL-10R ^flox/flox Tmem119 ^CreETR2 小鼠表现出更差的运动及认知功能（图 5h ）和降低的小胶质细胞吞噬能力（图 5i ），这进一步证实了 IL-10 在鼻腔 aCD3 治疗后调节小胶质细胞基因特征中的作用。综上所述，这些数据明确表明 IL-10/IL-10R 信号通路在增强小胶质细胞吞噬能力和改善 TBI 病理过程中具有关键作用。

Nasal aCD3-induced CD4⁺FoxP3⁺ T_reg cells ameliorate neuroinflammation and TBI outcomes
鼻腔给予 aCD3 诱导的 CD4 ⁺ FoxP3 ⁺ T _reg 细胞可改善神经炎症及创伤性脑损伤预后

In the days to weeks after TBI, lymphocytes appear at the lesion site and both effector T cells and T_reg cells infiltrate the injured brain^83–86. We found that nasal aCD3 expanded FoxP3⁺ T_reg cells at the injury site from day 3 to day 30 post-injury compared with the TBI-iso controls (Fig. 2b) and that nasal aCD3 improved behavioral and neuropathological outcomes of TBI by decreasing microglial inflammation (Figs. 1b and 3c). Thus, we next investigated whether these beneficial outcomes were dependent on T_reg cells by employing multiple experimental approaches including adoptive transfer experiments (discussed here) and an ex vivo T_reg cell–microglia transwell co-culture system and T_reg cell depletion experiments (discussed below). In the experiments involving adoptive transfer, we found that the adoptive transfer of CD4⁺FoxP3⁺ T_reg cells from aCD3-treated mice improved motor function and coordination and restored spatial memory when compared with mice that received either CD4⁺FoxP3⁺ T_reg cells from TBI-iso mice or CD4⁺ T cells depleted of T_reg cells from anti-CD3-treated mice at 7 d post-TBI (Fig. 6b). We then performed RNA-seq on microglia isolated from the ipsilateral hemisphere of TBI-aCD3-FoxP3⁺, TBI-iso-Foxp3⁺ and TBI-aCD3-FoxP3⁻ green fluorescent protein (GFP) groups at 7 d post-TBI (Supplementary Table 5). We found that TBI-aCD3-FoxP3⁺ microglia had a distinct transcriptomic profile when compared with other groups (Fig. 6c). GSEA analysis comparing TBI-aCD3-FoxP3⁺ microglia with TBI-aCD3-FoxP3⁻ microglia demonstrated that the TBI-aCD3-FoxP3⁺ microglia were associated with downregulation of several proinflammatory pathways including interferon (IFN)γ and IFNα (Fig. 6d). IPA revealed IL-10Ra as one of the top activated upstream regulators and IL-1b as one of the top inhibited upstream regulators in TBI-aCD3-FoxP3⁺ microglia compared with TBI-aCD3-FoxP3⁻ microglia (Fig. 6e). IFNγ was one of the most downregulated regulators in TBI-aCD3-FoxP3⁺ microglia compared with TBI-iso-FoxP3⁺ microglia (Fig. 6f,g). Consistent with the microglial transcriptomic data, RT–qPCR of TBI-aCD3-FoxP3⁺, microglia from the lesion site showed reduction in proinflammatory cytokines (Inf-γ, Cd14 and Tnf) and increased expression of homeostatic (Cd206) and anti-inflammatory cytokines (such as Il10) compared with the two other groups studied (Fig. 6h). Importantly, we also found that adoptive transfer of CD4⁺FoxP3⁺ T_reg cells from aCD3-treated mice increased microglial phagocytic capacity at 7 d post-injury compared with the two other groups (Fig. 6i). The microglial phagocytic capacity was also increased in mice that received CD4⁺FoxP3⁺ T_reg cells from TBI-iso-mice compared with mice that received FoxP3⁻ cells from aCD3-treated mice, indicating the importance of CD4⁺FoxP3⁺ T_reg cells in increasing microglial phagocytic function after TBI.
在创伤性脑损伤（TBI）后的数天至数周内，淋巴细胞出现在病变部位，效应 T 细胞和调节性 T 细胞（Treg）均会浸润受损脑组织。我们发现，与 TBI-iso 对照组相比，鼻腔给予 aCD3 单抗能在损伤后第 3 天至第 30 天期间促进损伤部位 FoxP3+ Treg 细胞的扩增（图 4），并通过减轻小胶质细胞炎症反应改善 TBI 的行为学和神经病理学结局（图 5 和图 6）。因此，我们随后采用多种实验方法（包括下文讨论的过继转移实验、离体 Treg-小胶质细胞 Transwell 共培养系统及 Treg 细胞清除实验）来验证这些有益效应是否依赖于 Treg 细胞。在过继转移实验中，我们发现：与接受 TBI-iso 小鼠 CD4+FoxP3+ Treg 细胞或 aCD3 处理小鼠中清除 Treg 的 CD4+ T 细胞的受体鼠相比，移植 aCD3 处理小鼠 CD4+FoxP3+ Treg 细胞能在 TBI 后 7 天显著改善运动功能、协调能力并恢复空间记忆（图 18）。 随后我们对创伤性脑损伤后 7 天时从 TBI-aCD3-FoxP3 ⁺ 、TBI-iso-Foxp3 ⁺ 和 TBI-aCD3-FoxP3 ⁻ 绿色荧光蛋白(GFP)组同侧半球分离的小胶质细胞进行 RNA 测序（补充表 5 ）。研究发现 TBI-aCD3-FoxP3 ⁺ 小胶质细胞与其他组相比具有独特的转录组特征（图 6c ）。通过 GSEA 分析比较 TBI-aCD3-FoxP3 ⁺ 与 TBI-aCD3-FoxP3 ⁻ 小胶质细胞显示，TBI-aCD3-FoxP3 ⁺ 小胶质细胞中包括干扰素(IFN)γ和 IFNα在内的多个促炎通路表达下调（图 6d ）。IPA 分析表明，与 TBI-aCD3-FoxP3 ⁻ 相比，IL-10Ra 是 TBI-aCD3-FoxP3 ⁺ 小胶质细胞中最显著激活的上游调节因子之一，而 IL-1b 则是受抑制程度最高的上游调节因子之一（图 6e ）。与 TBI-iso-FoxP3 ⁺ 组相比，IFNγ是 TBI-aCD3-FoxP3 ⁺ 小胶质细胞中下调最显著的调节因子（图 6f,g ）。 与小胶质细胞转录组数据一致，RT-qPCR 分析显示，与其他两组相比，TBI-aCD3-FoxP3 处理组病灶区小胶质细胞的促炎细胞因子（Inf-γ、Cd14 和 Tnf）表达降低，而稳态标志物（Cd206）和抗炎细胞因子（如 Il10）表达升高（图 6h ）。值得注意的是，我们还发现，与其他两组相比，在损伤后 7 天时，接受 aCD3 处理小鼠来源的 CD4 ⁺ FoxP3 ⁺ T _reg 细胞过继转移可增强小胶质细胞的吞噬能力（图 6i ）。与接受 aCD3 处理小鼠 FoxP3 ⁻ 细胞组相比，接受 TBI-同型对照小鼠 CD4 ⁺ FoxP3 ⁺ T _reg 细胞组的小胶质细胞吞噬能力也有所提升，这表明 CD4 ⁺ FoxP3 ⁺ T _reg 细胞对 TBI 后增强小胶质细胞吞噬功能具有重要作用。

To assess the beneficial effects of T_reg cells on behavioral outcomes and microglial inflammation at more chronic time points (30 d) post-TBI, we employed an alternative approach where total splenic T cells (CD45.2⁺CD4⁺) isolated from TBI-iso (iso-total CD4⁺) and TBI-nasal aCD3-treated mice (aCD3-total CD4⁺) and CD45.2⁺CD4⁺FoxP3⁻ GFP cells isolated from aCD3-treated animals (aCD3-FoxP3⁻ GFP) post-CCI were transferred i.p. into untreated but CCI-injured congenic CD45.1-expressing mice (Extended Data Figs. 8c and 9a). Adoptive transfer was performed at three time points post-CCI with each mouse receiving 2.5 × 10⁶ cells per injection. We found improvement in motor function and coordination, restoration of spatial memory and increased time spent in the target quadrant during the probe trial in mice that received total CD4⁺ T cells from aCD3-treated mice compared with mice that received CD4⁺ T cells depleted of T_reg cells at 30 d post-TBI (Extended Data Fig. 9b). We performed flow cytometric analyses to track adoptively transferred cells in recipient mice by transferring cells from CD45.2 mice into CD45.1 mice and found CD45.2 transferred cells in all organs analyzed (Extended Data Fig. 9c). We then performed RNA-seq on microglia isolated from the ipsilateral hemisphere of iso-total CD4⁺, aCD3-total CD4⁺ and aCD3-FoxP3⁻ GFP groups at 30 d post-TBI (Supplementary Table 5). A heatmap signature showed a distinct microglial transcriptomic signature of aCD3-total CD4⁺ compared with iso-total CD4⁺ and aCD3-FoxP3⁻ GFP groups at 30 d post-CCI (Extended Data Fig. 9d). GSEA pathway analysis demonstrated that the aCD3-total CD4⁺ group was associated with the upregulation of neuron development pathways and the downregulation of several proinflammatory pathways involved in innate and adaptive immune responses, immune effector processes and antigen presentation compared with the iso-total CD4⁺ group, all of which were not notably upregulated in the aCD3-FoxP3⁻ GFP group (Extended Data Fig. 9e). Similarly, the aCD3-total CD4⁺ group was associated with a downregulation of the innate immune response pathway when compared with the aCD3-FoxP3⁻ GFP group (Extended Data Fig. 9f,g), where IPA analysis demonstrated IL-10 as a top activated upstream regulator and IFNγ as a top inhibited upstream regulator (Extended Data Fig. 9h). Consistent with the microglial transcriptomic data, RT–qPCR of the ipsilateral hemisphere showed an increase in the expression of several anti-inflammatory cytokines (Il10 and Il2), and growth factors including Gdnf at 1 month post-CCI in the aCD3-total CD4⁺ group compared with the iso-total CD4⁺ and aCD3-FoxP3⁻ GFP groups (Extended Data Fig. 9i). Taken together, these data demonstrate a critical role for FoxP3⁺ T_reg cells in augmenting microglia phagocytic capacity and ameliorating disease in TBI.
为评估 T _reg 细胞在创伤性脑损伤（TBI）后较长期时间点（30 天）对行为学结果和小胶质细胞炎症的改善作用，我们采用替代方案：将分别从 TBI 同型对照组（iso-total CD4 ⁺ ）和鼻腔给予 aCD3 治疗的 TBI 小鼠（aCD3-total CD4 ⁺ ）分离的脾脏总 T 细胞（CD45.2 ⁺ CD4 ⁺ ），以及从 aCD3 治疗动物分离的 CD45.2 ⁺ CD4 ⁺ FoxP3 ⁻ GFP 细胞（aCD3-FoxP3 ⁻ GFP），通过腹腔移植至未经处理但接受 CCI 损伤的 CD45.1 同源基因小鼠体内（扩展数据图 8c 和 9a ）。移植在 CCI 术后三个时间点进行，每次注射 2.5×10 ⁶ 个细胞。研究发现，与接受去除 T _reg 细胞的 CD4 ⁺ T 细胞移植组相比，接受 aCD3 治疗小鼠来源的总 CD4 ⁺ T 细胞移植组在 TBI 后 30 天表现出运动功能与协调性改善、空间记忆恢复，且在探测试验中目标象限停留时间显著延长（扩展数据图 9b ）。 我们通过流式细胞术分析追踪过继转移细胞，将 CD45.2 小鼠的细胞转移至 CD45.1 受体小鼠后，在所有检测器官中均发现 CD45.2 来源的转移细胞（扩展数据图 9c ）。随后我们对创伤性脑损伤（TBI）后 30 天从同侧半球分离的小胶质细胞进行 RNA 测序，样本来自 iso-total CD4 ⁺ 组、aCD3-total CD4 ⁺ 组和 aCD3-FoxP3 ⁻ GFP 组（补充表 5 ）。热图特征显示，在 CCI 术后 30 天，aCD3-total CD4 ⁺ 组与 iso-total CD4 ⁺ 组及 aCD3-FoxP3 ⁻ GFP 组相比具有显著不同的小胶质细胞转录组特征（扩展数据图 9d ）。GSEA 通路分析表明，与 iso-total CD4 ⁺ 组相比，aCD3-total CD4 ⁺ 组与神经元发育通路的上调相关，同时下调了涉及先天性和适应性免疫应答、免疫效应过程及抗原呈递的多个促炎通路，而这些通路在 aCD3-FoxP3 ⁻ GFP 组均未显著上调（扩展数据图 9e ）。 同样，与 aCD3-FoxP3 ⁻ GFP 组相比，aCD3-total CD4 ⁺ 组显示出先天免疫反应通路的下调（扩展数据图 9f,g ），其中 IPA 分析表明 IL-10 是最活跃的上游调节因子，而 IFNγ则是受抑制程度最高的上游调节因子（扩展数据图 9h ）。与小胶质细胞转录组数据一致，RT-qPCR 显示在 CCI 后 1 个月，aCD3-total CD4 ⁺ 组损伤同侧大脑半球中多种抗炎细胞因子（Il10 和 Il2）及神经营养因子（如 Gdnf）的表达较 iso-total CD4 ⁺ 和 aCD3-FoxP3 ⁻ GFP 组显著增加（扩展数据图 9i ）。这些数据共同表明 FoxP3 ⁺ T _reg 细胞在增强小胶质细胞吞噬能力及改善 TBI 病理过程中发挥关键作用。

CD4⁺Foxp3⁺ T_reg cells suppress microglial inflammatory and homeostatic markers in vitro
CD4 ⁺ Foxp3 ⁺ T _reg 细胞在体外抑制小胶质细胞的炎症和稳态标志物

To further investigate the interaction between T_reg cells and microglia after TBI, we employed an ex vivo transwell co-culture system in which microglia were isolated from the ipsilateral hemisphere of CCI mice 24 h after injury and T_reg cells were isolated from spleens of a separate cohort of mice subjected to CCI and treated with nasal aCD3 or isotype control for 7 d (Fig. 6j). Microglia were placed in the lower chamber and T_reg cells in the upper chamber and we performed RT–qPCR of microglia 72 h after incubation. We found an increase in anti-inflammatory and homeostatic markers (Il10, Cx3cr1 and Cd206) and a reduction in the proinflammatory marker Il1b in the TBI-aCD3 microglia, which are consistent with our in vivo microglial transcriptomic data at 7 d post-TBI. This effect was lost by blocking IL-10 in vitro which is also consistent with what we observed following in vivo Il-10 neutralization (Fig. 5).
为深入探究创伤性脑损伤（TBI）后 T 细胞与小胶质细胞的相互作用，我们建立了体外 Transwell 共培养系统：小胶质细胞取自 CCI 小鼠损伤 24 小时后同侧大脑半球，T 细胞则来自另一组接受 CCI 造模并经鼻腔给予 aCD3 或同型对照抗体处理 7 天的小鼠脾脏（图 2）。实验中将小胶质细胞置于下室，T 细胞置于上室，培养 72 小时后对小胶质细胞进行 RT-qPCR 检测。结果显示 TBI-aCD3 组小胶质细胞的抗炎及稳态标志物（Il10、Cx3cr1 和 Cd206）表达升高，促炎标志物 Il1b 表达降低，这与我们体内实验中 TBI 后 7 天的小胶质细胞转录组数据一致。通过体外阻断 IL-10 可消除这种效应，该现象与我们体内 IL-10 中和实验的观察结果相符（图 4）。

IL-10-producing T_reg cells modulate microglial phagocytosis, neuroinflammation and TBI outcomes
产生 IL-10 的调节性 T 细胞通过调控小胶质细胞吞噬功能、神经炎症反应及创伤性脑损伤预后发挥作用

FoxP3 T_reg cells play a critical role in suppressing CNS inflammation in acute stroke and chronic neurological diseases⁸⁷ and, as shown above, we found that adoptive transfer of FoxP3 T_reg cells from nasal aCD3-treated mice ameliorated TBI and IL-10 is an important factor in this process. We then asked whether IL-10 produced by T_reg cells was responsible for this effect. To address this question, we first depleted FoxP3 T_reg cells using FoxP3-DTR transgenic mice that express the diphtheria toxin receptor (DTR) under control of the FoxP3 promoter and then investigated the role of lL-10-producing T_reg cells in TBI by using a dual reporter system that involved the gene encoding IL-10 and transcriptomic factor FoxP3 (10BiT.FoxP3^GFP)⁸⁸ which allowed us to sort IL-10⁺ and IL-10⁻ T_reg cells. In the first FoxP3 T_reg cell depletion experiment, DT was injected 3 d before CCI and repeated every 3 d until 7 d after CCI or sham operation (Fig. 7a and Extended Data Fig. 10a). We found that the depletion of FoxP3 T_reg cells worsened motor function as measured by rotarod behavioral testing (Fig. 7b) and exacerbated microglial inflammation (Fig. 7c). We also observed increased expression of proinflammatory microglial markers including Il1b, Tnf, Il6, and Il18, as measured by RT–qPCR. Most importantly, we found that the depletion of FoxP3 T_reg cells reduced microglial phagocytic capacity to uptake dead neurons (Fig. 7d), highlighting their pivotal role in regulating microglial phagocytosis. Based on these results, we then investigated the role of lL-10-producing T_reg cells in TBI by using a dual reporter system that involved the gene encoding IL-10 and 10BiT.FoxP3^GFP (ref. ⁸⁸) that allowed us to sort IL-10⁺ and IL-10⁻ T_reg cells (Fig. 7e-f). We assessed the effect of IL-10⁺ and IL-10⁻ T_reg cells by adoptive transfer of these cells to FoxP3-depleted mice (Fig. 7g and Extended Data Fig. 10b). We observed that DT has similar behavior outcomes in WT TBI DT mice and the FoxP3 TBI DT-PBS group (Fig. 7h). However, we found that the adoptive transfer of IL-10⁺ T_reg cells was associated with improvements in motor function measured by rotarod and spatial memory measured by the Y-maze compared with the mice that received IL-10⁻ T_reg cells (Fig. 7h). Moreover, IL-10⁺ T_reg cells attenuated the microglial inflammatory response with reduction in proinflammatory markers such as Il1b, Tnf, Il6, and Il18 compared with IL-10⁻ T_reg cells (Fig. 7i). Most importantly, adoptive transfer of IL-10⁺ T_reg cells enhanced the microglial phagocytosis of dead neurons (Fig. 7j), indicating their critical role in modulating microglial inflammation and augmenting microglial phagocytosis. Thus, IL-10 plays a critical role in the beneficial effect of nasal aCD3-induced FoxP3 T_reg cells on TBI.
FoxP3 T 细胞在抑制急性脑卒中及慢性神经系统疾病的中枢神经系统炎症中起关键作用[1]。如上所述，我们发现鼻腔给予 aCD3 处理小鼠的 FoxP3 T 细胞过继转移可改善创伤性脑损伤（TBI），而 IL-10 是这一过程中的重要因子。随后我们探究了 T 细胞产生的 IL-10 是否介导了这一效应。为解决该问题，我们首先使用 FoxP3-DTR 转基因小鼠（在 FoxP3 启动子控制下表达白喉毒素受体 DTR）清除 FoxP3 T 细胞，随后通过采用同时标记 IL-10 编码基因和转录因子 FoxP3 的双报告系统（10BiT.FoxP3[6]）[7]，对 IL-10+和 IL-10- T 细胞进行分选，以研究产 IL-10 T 细胞在 TBI 中的作用。在首个 FoxP3 T 细胞清除实验中，于 CCI 术前 3 天注射 DT，之后每 3 天重复注射直至 CCI 或假手术后 7 天（图[12]及扩展数据图[13]）。通过转棒行为学测试发现，FoxP3 T 细胞的清除会加重运动功能障碍（图[15]），并加剧小胶质细胞的炎症反应（图[16]）。 我们还通过 RT-qPCR 检测发现促炎性小胶质细胞标志物 Il1b、Tnf、Il6 和 Il18 的表达增加。最重要的是，我们发现 FoxP3 T _reg 细胞的耗竭会降低小胶质细胞吞噬死亡神经元的能力（图 7d ），这凸显了它们在调节小胶质细胞吞噬功能中的关键作用。基于这些结果，我们随后通过使用包含 IL-10 编码基因和 10BiT.FoxP3 ^GFP （参考文献 ⁸⁸ ）的双报告系统，研究了产 IL-10 的 T _reg 细胞在 TBI 中的作用，该系统使我们能够分选 IL-10 ⁺ 和 IL-10 ⁻ T _reg 细胞（图 7e-f ）。我们通过将这些细胞过继转移至 FoxP3 缺陷小鼠体内，评估了 IL-10 ⁺ 和 IL-10 ⁻ T _reg 细胞的影响（图 7g 及扩展数据图 10b ）。我们观察到 DT 在野生型 TBI DT 小鼠和 FoxP3 TBI DT-PBS 组中具有相似的行为学结果（图 7h ）。然而，与接受 IL-10 ⁻ T _reg 细胞的小鼠相比，过继转移 IL-10 ⁺ T _reg 细胞可改善转棒测试评估的运动功能和 Y 迷宫测试评估的空间记忆（图 7h ）。 此外，与 IL-10 ⁻ T _reg 细胞相比，IL-10 ⁺ T _reg 细胞通过降低促炎标志物（如 Il1b、Tnf、Il6 和 Il18）减轻了小胶质细胞的炎症反应（图 7i ）。最重要的是，过继转移 IL-10 ⁺ T _reg 细胞增强了小胶质细胞对死亡神经元的吞噬作用（图 7j ），表明其在调节小胶质细胞炎症和增强小胶质细胞吞噬功能中的关键作用。因此，IL-10 在鼻内 aCD3 诱导的 FoxP3 T _reg 细胞对 TBI 的有益效应中起着至关重要的作用。

Experimental animals  实验动物

Studies were performed using 8- to 12-week-old C57BL6J mice (Jackson Laboratories cat. no. 000664), B6.CD45.1 mice (Jackson Laboratory, cat. no. 002014), FoxP3-GFP mice (Jackson Laboratory, cat. no. 023800), FoxP3-DTR mice (Jackson Laboratory, cat. no. 016958), IL-10 KO mice (Jackson Laboratory, cat. no. 002251), Tmem119-Cre^ETR2 mice (Jackson Laboratory, cat. no. 031820), IL-10ra^Flx mice (Jackson Laboratory, cat. no. 028146) and 10BiT.FoxP3^GFP (kindly provided by V. Kuchroo)⁸⁸. All mice were housed under specific pathogen-free conditions, with free access to food and water. All animals were housed in temperature (20 °C) and humidity (60%)-controlled rooms, maintained on a 12 h:12 h light:dark cycle (lights on at 07:00). Mice were euthanized by CO₂ inhalation. The Institutional Animal Care and Use Committee at Harvard Medical School and Brigham and Women’s Hospital has all experimental procedures involving animals.
实验采用 8 至 12 周龄的 C57BL6J 小鼠（杰克逊实验室，货号 000664）、B6.CD45.1 小鼠（货号 002014）、FoxP3-GFP 小鼠（货号 023800）、FoxP3-DTR 小鼠（货号 016958）、IL-10 基因敲除小鼠（货号 002251）、Tmem119-Cre ^ETR2 小鼠（货号 031820）、IL-10ra ^Flx 小鼠（货号 028146）及 10BiT.FoxP3 ^GFP 小鼠（由 V. Kuchroo 教授惠赠） ⁸⁸ 。所有小鼠均在无特定病原体条件下饲养，自由摄食饮水。动物房环境参数为恒温 20℃、相对湿度 60%，光/暗周期 12 小时:12 小时（07:00 开灯）。采用二氧化碳 ₂ 吸入法实施安乐死。哈佛医学院及布莱根妇女医院的机构动物护理与使用委员会批准了所有动物实验方案。

Treatment with aCD3 mAb
抗 CD3 单克隆抗体治疗

Mice were nasally treated with a daily dose, immediately (4–6 hours) and for some experiments early (3 days) or delayed (7 days) post TBI, of 1 µg per mouse hamster immunoglobulin G (IgG) CD3-specific antibody (BioXCell, clone no. 145-2C11) or hamster IgG control antibody (BioXCell) dissolved in PBS and henceforth every other day after the first week until the experimental endpoint. For some experiments, mice were given 0.5 mg of monoclonal anti-IL-10R-blocking antibody (BioXCell, clone 1B1.3A), by intraperitoneal injection at the onset of TBI and henceforth every third day until the experimental endpoint.
小鼠经鼻给予每日剂量处理，在创伤性脑损伤（TBI）后立即（4-6 小时）进行，部分实验采用早期（3 天）或延迟（7 天）给药方案，每只小鼠使用 1μg 仓鼠免疫球蛋白 G（IgG）CD3 特异性抗体（BioXCell，克隆号 145-2C11）或溶解于 PBS 的仓鼠 IgG 对照抗体（BioXCell），首周后改为隔日给药直至实验终点。部分实验中，小鼠在 TBI 发生时通过腹腔注射给予 0.5mg 单克隆抗 IL-10R 阻断抗体（BioXCell，克隆 1B1.3A），之后每三日给药一次直至实验终点。

Conditional genetic deletion of IL-10Ra in microglia
小胶质细胞中 IL-10Ra 的条件性基因敲除

To induce Cre-recombinase expression, a dose of tamoxifen (150 mg per kg of body weight) in corn oil was injected i.p. for 5 d consecutively. IL-10Ra^flx/flx was crossed with Tmem119-Cre^ETR2 mice¹¹⁰. Recombination was induced in Tmem119-Cre^ETR2:IL-10Ra^flx/flx mice and Tmem119-Cre^WT:IL-10Ra^flx/flx littermates were used as controls. A washout period of 2 weeks was implemented after the last tamoxifen injection before starting any experiment.
为诱导 Cre 重组酶表达，连续 5 天腹腔注射玉米油溶解的他莫昔芬（150 mg/kg 体重）。将 IL-10Ra ^flx/flx 小鼠与 Tmem119-Cre ^ETR2 小鼠 ¹¹⁰ 杂交。在 Tmem119-Cre ^ETR2 :IL-10Ra ^flx/flx 小鼠中诱导重组，并以 Tmem119-Cre ^WT :IL-10Ra ^flx/flx 同窝仔作为对照。末次他莫昔芬注射后实施 2 周洗脱期方可开始实验。

FoxP3 cell depletion  FoxP3 细胞耗竭

Depletion of FoxP3 was done as previously described²³. In short, DT (0.05 μg per g body weight) was injected i.p. 3 d before TBI and was repeated every 3 d to maintain FoxP3 depletion until sacrifice.
FoxP3 耗竭操作如前所述 ²³ 。简言之，在创伤性脑损伤（TBI）前 3 天腹腔注射 DT（0.05 μg/g 体重），之后每 3 天重复注射一次以维持 FoxP3 耗竭状态直至处死。

Controlled cortical impact
控制性皮质撞击

A CCI model was used as previously described¹¹¹. Mice were anesthetized with 4.5% isoflurane (Anaquest) in 70% nitrous oxide and 30% oxygen using a Fluotec 3 vaporizer (Colonial Medical). The mice were placed in a stereotaxic frame and a 5-mm craniotomy was made over the right somatosensory cortex using a drill and a trephine. The bone flap was removed and discarded and a pneumatic cylinder with a 1.5- or 3-mm flat tip impounder and velocity 6 m s⁻¹, depth 1.0 or 1.5 mm and dwell time of 0.8 s was used to induce CCI (Impact One, Leica Biosystems). The scalp was sutured closed and the mice were returned to their cages to recover.
采用先前描述的 CCI 模型 ¹¹¹ 。使用 Fluotec 3 蒸发器（Colonial Medical）以 4.5%异氟烷（Anaquest）与 70%一氧化二氮和 30%氧气的混合气体麻醉小鼠。将小鼠固定于立体定位仪上，使用钻头和环钻在右侧体感皮层区域制作 5 毫米颅骨开窗。移除骨瓣后，采用配备 1.5 或 3 毫米平头撞击器、速度 6 米/秒 ⁻¹ 、深度 1.0 或 1.5 毫米、停留时间 0.8 秒的气动撞击装置（Impact One，Leica Biosystems）诱导 CCI 损伤。缝合头皮后将小鼠放回笼中恢复。

Behavioral studies  行为学研究

BBB permeability 70-kDa FITC-dextran
血脑屏障通透性检测用 70-kDa FITC-葡聚糖

A 70-kDa FITC-dextran (Sigma-Aldrich, cat. no. 46945) was used to measure BBB permeability as described in previous studies^116,117, with modifications: 0.2 mg of 70-kDa FITC-dextran per g of body weight was injected retro-orbitally^118,119. Then, 10 min post-70-kDa FITC-dextran administration, the mouse was euthanized with a lethal dose of xylazine and ketamine cocktail (450 mg kg⁻¹ and 45 mg kg⁻¹) i.p. with a 29-gauge insulin pen. The mouse was transcardially perfused with 50 ml of PBS. The whole brain was removed and stored at −80 °C (ref. ¹²⁰). Cryoprotected and flash-frozen brains were coronally sectioned (16-μm-thick serial sections, 300 μm apart)¹¹⁶. The brain sections were captured by a Leica DMi8 wide-field microscope. FITC-dextran visualization was done under a 488-nm excitation wavelength laser. For each mouse, we obtained five to six continued section slices from the brain tissue’s front, middle and posterior sections. An investigator blind to the experimental design manually measured the l mean gray value of the dextran tracer-positive area found in the brain parenchyma using ImageJ.
采用 70-kDa FITC-葡聚糖（Sigma-Aldrich，货号 46945）检测血脑屏障通透性，方法参照前期研究 ^116,117 并稍作改良：按每克体重 0.2 mg 剂量经眶后静脉注射 ^118,119 。注射 10 分钟后，使用 29G 胰岛素笔经腹腔注射致死剂量的赛拉嗪-氯胺酮混合液（450 mg/kg ⁻¹ 和 45 mg/kg ⁻¹ ）处死小鼠。经心脏灌注 50 ml PBS 后取出全脑，保存于-80°C（参考文献 ¹²⁰ ）。经冷冻保护处理的速冻脑组织进行冠状切片（连续 16μm 厚切片，间隔 300μm） ¹¹⁶ ，使用 Leica DMi8 宽场显微镜采集脑切片图像，在 488 nm 激发波长激光下观察 FITC-葡聚糖信号。每只小鼠取脑组织前、中、后部 5-6 张连续切片，由不知实验分组的研究人员采用 ImageJ 软件手动测量脑实质内葡聚糖示踪剂阳性区域的平均灰度值。

Brain edema  脑水肿检测

Brains were removed at 72 h after CCI, bisected into left and right hemispheres and each hemisphere was weighed (wet weight). Brains were then dried at 60 °C for 48 h and dry weights were obtained. The percentage of brain water content was expressed as ((wet − dry weight)/(wet weight)) × 100% as previously described¹²¹.
在 CCI 后 72 小时取出脑组织，将其分为左右半球并分别称重（湿重）。随后将脑组织置于 60°C 环境下干燥 48 小时以获得干重。按先前描述的方法 ¹²¹ ，脑含水量百分比计算公式为：（（湿重－干重）/湿重）×100%。

Magnetic resonance imaging
磁共振成像

Imaging was done using a 7.0T Bruker BioSpect USR. In brief, mice were gently handled and placed in an isoflurane anesthesia chamber. Then the mice were placed inside the imaging apparatus with their nose in front of tubes releasing 2% isoflurane. Electrocardiogram (ECG) leads were placed on the animal’s paws and a pneumatic pillow sensor placed under the abdomen for continuous ECG and respiratory rate monitoring of the anesthetized animal. These waveforms were closely monitored throughout magnetic resonance imaging (MRI) by the MRI operator. The animal was placed on an MRI-compatible bed, which was placed inside the magnet for imaging. The imaging sessions lasted between 15 min and 60 min. Mice were then returned to their cages and monitored continuously after being returned to their cages before returning to a fully alert status. The following parameters were obtained to generate the T2 sequence images: slice thickness: 0.5 mm; repetition time: 3,000 ms; echo time: 50 ms; no. of averages: 3; spacing between slices: 0.5 mm; echo train length: 8; acquisition matrix: 200 × 200; flip angle: 90°; and field of view: 20 mm. Serial Images were viewed and analyzed using the three-dimensional (3D) Slicer platform¹²².
采用 7.0T 布鲁克 BioSpect USR 进行影像采集。简要流程如下：轻柔固定小鼠后置于异氟烷麻醉舱，随后将其鼻部对准输送 2%异氟烷的导管安置于成像设备内。动物四肢连接心电图（ECG）导联，腹部下方放置气垫式呼吸传感器，持续监测麻醉状态下的心电及呼吸频率。磁共振成像（MRI）过程中由操作人员全程监控这些波形参数。实验动物被安置于磁共振兼容扫描床上，推入磁体进行成像，单次扫描时长 15-60 分钟。扫描完成后将小鼠放回笼具持续观察，直至完全恢复清醒状态。获取 T2 序列图像的参数设置为：层厚 0.5mm；重复时间 3000ms；回波时间 50ms；平均次数 3 次；层间距 0.5mm；回波链长度 8；采集矩阵 200×200；翻转角 90°；视野 20mm。 使用三维(3D)Slicer 平台对连续图像进行查看和分析 ¹²² 。

Immunohistochemistry  免疫组织化学

Animals were anesthetized with CO₂ until the respiration rate slowed and perfused transcardially with Hanks’ balanced salt solution (HBSS). Brains were post-fixed in 4% paraformaldehyde for 48 h, then transferred to a 15% sucrose solution for 24 h and finally transferred to a 30% sucrose solution for 24 h. Brains were then flash frozen in Tissue-Tek Oct (Sakura, compound 4583) and stored at −80 °C until the time of sectioning. Brains were subsequently sectioned at −20 °C using a cryostat at the bregma position for each targeted brain. Sections were cut at 0.2 mm in a fourfold series interval. Five total sections were placed on Colorfrost Plus-treated adhesion slides (Thermo Fisher Scientific) and stored at −20 °C until the time of staining. Whole meninges were processed as previously described¹²³. For immunofluorescence, sections were blocked in in a 10% normal horse serum solution, containing 0.1% Triton X-100, 1% glycine and 2% bovine serum albumin (BSA). Slides were incubated overnight at 4 °C with anti-Iba-1 (rabbit, 1:1,000, Wako). The following day sections were washed and incubated with an Alexa Fluor-647 goat anti-rabbit IgG (1:1,000, Abcam, cat. no. ab150075) for 1 h at room temperature. Sections were also stained with H&E (Abcam, cat. no. ab245880) and TUNEL (TUNEL Assay Kit, BrdU-Red, Abcam, cat. no. ab66110) according to their corresponding kit protocols. Iba-1- and TUNEL-stained slides were co-stained with DAPI mounting medium (Vector Laboratories, cat. no. UX-93952-24). FoxP3 GFP mice were used to visualize FoxP3 T_reg cells in the meninges and brain using the Cy2 channel of the microscope and CD3 was stained using Alexa Fluor-647 anti-mouse CD3 (BioLegend, cat. no. 17A2, 1:50). Images were taken using a Leica DMi8 wide-field microscope on the ×20 objective or the Zeiss LSM710 confocal microscope.
实验动物采用 CO ₂ 麻醉至呼吸频率减缓后，经心脏灌注汉克斯平衡盐溶液(HBSS)。脑组织在 4%多聚甲醛中后固定 48 小时，随后转入 15%蔗糖溶液 24 小时，最终转移至 30%蔗糖溶液 24 小时。脑组织随后用 Tissue-Tek OCT 包埋剂（Sakura，货号 4583）快速冷冻，保存于-80°C 直至切片。在-20°C 条件下使用冷冻切片机沿前囟定位进行脑组织切片，以 0.2mm 厚度进行四倍连续切片。将五组切片置于 Colorfrost Plus 处理过的防脱载玻片（赛默飞世尔科技）上，-20°C 保存待染色。脑膜处理参照先前方法 ¹²³ 。免疫荧光实验中，切片用含 0.1% Triton X-100、1%甘氨酸和 2%牛血清白蛋白(BSA)的 10%正常马血清封闭液处理，4°C 条件下与抗 Iba-1 抗体（兔源，1:1000，和光纯药）孵育过夜。次日洗涤后与 Alexa Fluor-647 标记的山羊抗兔 IgG 二抗（1:1000，艾博抗，货号 室温下使用抗 CD3 单克隆抗体（Abcam，货号 ab150075）孵育 1 小时。切片还根据相应试剂盒说明书进行了 H&E 染色（Abcam，货号 ab245880）和 TUNEL 染色（TUNEL 检测试剂盒，BrdU-红色，Abcam，货号 ab66110）。Iba-1 和 TUNEL 染色切片使用 DAPI 封片剂（Vector Laboratories，货号 UX-93952-24）进行共染。采用 FoxP3 GFP 小鼠通过显微镜 Cy2 通道观察脑膜和脑组织中 FoxP3 T _reg 细胞，并使用 Alexa Fluor-647 标记的小鼠抗 CD3 抗体（BioLegend，货号 17A2，1:50 稀释）进行 CD3 染色。图像采集使用 Leica DMi8 宽场显微镜（20 倍物镜）或 Zeiss LSM710 共聚焦显微镜完成。

Image analysis  图像分析

Analysis of the percentage Iba-1 and number of TUNEL-positive cells per surface area was performed on five photomicrographs per animal (n = 4 or 5). The sections analyzed were taken between 300 μm and 1,500 μm laterally from the coronal plane. Each scanned photomicrograph was used to produce images of the area of contusion. All the images were analyzed using ImageJ software (National Institutes of Health: https://imagej.nih.gov/ij). Images were split by color channel, the channel of interest was threshold using the Yen setting and the number of positive cells were quantified as previously described¹²⁴.
对每只动物（n=4 或 5）的五张显微照片进行 Iba-1 阳性率及单位面积 TUNEL 阳性细胞数分析。所选切片位于冠状面外侧 300μm 至 1500μm 区间。每张扫描显微照片均用于生成挫伤区域图像，所有图像均采用 ImageJ 软件（美国国立卫生研究院： https://imagej.nih.gov/ij ）进行分析。图像经颜色通道分离后，使用 Yen 设定对目标通道进行阈值处理，阳性细胞计数方法如文献 ¹²⁴ 所述。

Serum biomarkers  血清生物标志物检测

Levels of cytokines in the serum of mice were measured with the V-PLEX Plus Proinflammatory Panel1 Mouse Kit (Meso Scale Discovery, cat. no. K15048G-1). All sample controls were diluted 1:2 and run as duplicates according to the manufacturer’s protocol, as previously described¹²⁵.
采用 V-PLEX Plus 促炎因子 Panel1 小鼠检测试剂盒（Meso Scale Discovery，货号 K15048G-1）测定小鼠血清细胞因子水平。所有样本按 1:2 稀释，依照制造商方案进行双孔检测，具体方法参见文献 ¹²⁵ 。

Quanterix single-molecule array analysis
Quanterix 单分子阵列分析

Levels of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and GFAP were quantified in the serum using single-molecule array technology (SiMoA, Quanterix). The SiMoA Neurology 4-Plex B kit was run according to the manufacturer’s directions. Briefly, samples were thawed, vortexed and centrifuged at 10,000g for 5 min. All samples were run at a 4× dilution, along with eight calibrators run neat and two controls also run at a 4× dilution. The data were validated using a calibration curve with R² > 0.99. For both UCHL-1 and GFAP, the dynamic range was 0–40,000 pg ml⁻¹, with a lower limit of quantification of 9.38 pg ml⁻¹.
采用单分子阵列技术（SiMoA，Quanterix）定量检测血清中泛素羧基末端水解酶 L1（UCH-L1）和 GFAP 水平。严格按制造商说明操作 SiMoA Neurology 4-Plex B 试剂盒：样本解冻后涡旋震荡，10,000g 离心 5 分钟。所有样本按 4 倍稀释度检测，同时运行 8 个未稀释校准品和 2 个 4 倍稀释对照品。通过 R²>0.99 的校准曲线验证数据。UCHL-1 和 GFAP 的检测动态范围均为 0-40,000 pg/ml，定量下限为 9.38 pg/ml。

Flow cytometry microglial sorting
流式细胞术小胶质细胞分选

For microglial cell sorting, mice were anesthetized with CO₂ until their respiration rate slowed and then transcardially perfused with 50 ml of HBSS containing heparin (1:1,000). After perfusion, the ipsilateral hemisphere was homogenized using a Dounce glass tissue homogenizer. Cells were separated through a Percoll (GE Healthcare Life Sciences) 30% gradient centrifugation. Cells were isolated from the Percoll layer and stained on ice for 30 min with combinations of phycoerythrin (PE)/cyanine7 anti-mouse CD11b (BioLegend, cat. no. M1/70, 1:100), allophycocyanin (APC)/cyanine7 anti-mouse CD45 (BioLegend, cat. no. 30-F11, 1:100), FITC anti-mouse Ly6C (BD Bioscience, cat. no. AL-21, 1:200) and APC anti-mouse 4D4 (ref. ⁵¹) (marking resident microglia; 1:1,000) in blocking buffer containing 0.2% BSA (Sigma-Aldrich) in HBSS. Cell sorting was performed using a FACSAriaIII cell sorter (Becton Dickson). Microglial cells were identified as CD45⁺CD11b⁺Ly6C⁻4D4⁺ and dead cells were also excluded based on 7-aminoactinomycin D (7-AAD; BD Bioscience) staining. Cells were sorted directly in 1.5-ml Eppendorf tubes and stored at −80 °C. Phagocytic positive microglia were sorted as 4D4⁺ Alexa-405⁺.
为进行小胶质细胞分选，实验小鼠经 CO ₂ 麻醉至呼吸频率减缓后，经心脏灌注 50ml 含肝素(1:1000)的 HBSS 溶液。灌注完成后，使用 Dounce 玻璃组织匀浆器对同侧大脑半球进行匀浆处理。细胞通过 30% Percoll（GE Healthcare Life Sciences）梯度离心分离。从 Percoll 层分离出的细胞在冰上用以下抗体组合染色 30 分钟：藻红蛋白(PE)/花青素 7 标记的抗小鼠 CD11b（BioLegend，货号 M1/70，1:100）、别藻蓝蛋白(APC)/花青素 7 标记的抗小鼠 CD45（BioLegend，货号 30-F11，1:100）、FITC 标记的抗小鼠 Ly6C（BD Bioscience，货号 AL-21，1:200）以及 APC 标记的抗小鼠 4D4（参考文献 ⁵¹ ）（标记定居型小胶质细胞；1:1000），所用封闭缓冲液为含 0.2% BSA（Sigma-Aldrich）的 HBSS 溶液。细胞分选采用 FACSAriaIII 流式细胞分选仪（Becton Dickson）完成。小胶质细胞鉴定标准为 CD45 ⁺ CD11b ⁺ Ly6C ⁻ 4D4 ⁺ ，同时通过 7-氨基放线菌素 D（7-AAD；BD Bioscience）染色排除死细胞。分选后的细胞直接收集于 1.5ml EP 管中，保存于-80°C 环境。 吞噬活性阳性小胶质细胞被分选标记为 4D4 ⁺ Alexa-405 ⁺ 。

Flow cytometry intracellular staining
流式细胞术细胞内染色

Intracellular cytokine staining and cell isolation were done as previously described¹²⁶. The meninges were carefully removed from the skull and the ipsilateral brain was further isolated. The enzyme dissociation mix used for the ipsilateral brain hemisphere and meninges was collagenase P (0.5 mg ml⁻¹; Sigma-Aldrich, cat. no. 11213865001) and DNase-1 (250 U ml⁻¹, Worthington, cat. no. LK003172) diluted in Roswell Park Memorial Institute (RPMI)-1640 with 10 mM Hepes. The samples were then finely minced and incubated in a room temperature shaker for 1 h. After enzyme dissociation, the cells were separated using Percoll (GE Healthcare Life Sciences) as described above. Cells isolated from the brain and meninges were incubated for only 2 h instead of the 4 h for cLN cells. Acquisition was performed on a Symphony (BD Biosciences) using DIVA software (BD Biosciences) and the data were analyzed with FlowJo software v.9.9 or v.10.1 (TreeStar Inc.). Intracellular staining antibodies used Zombie Aqua Fixable Viability Kit (BioLegend, cat. no. 423102, 1:1,000) or Zombie UV (BioLegend, cat. no. 423108, 1:1,000) was used to exclude dead cells. The staining antibodies used PE/cyanine7 anti-mouse CD11b (BioLegend, cat. no. M1/70, 1:300), APC/cyanine7 anti-mouse TCR-β (BioLegend, cat. no. H57-597, 1:100), BUV661 anti-mouse CD45 (30-F11, BD Biosciences, 1:200), PE anti-mouse CD4 (BioLegend, cat. no. GK1.5, 1:100), BUV805 anti-mouse CD8 (BD Biosciences, cat. no. 53-6.7, 1:100), FITC anti-mouse FoxP3 (eBioscience, cat. no. FJK-16s, 1:100), BV421 anti-mouse LAP (BioLegend, cat. no. TW7-16B4, 1:100), PE/Dazzle 594 anti-mouse IL-10 (BioLegend, cat. no. JES5-16E3, 1:100), BUV395 anti-mouse IL-17a (BD Biosciences, cat. no. TC11-18H10, 1:100), BV785 anti-mouse IFNγ (BioLegend, cat. no. XMG1.2, 1:100), BV605 anti-mouse Ly6G (BioLegend, cat. no. 1A8, 1:300), BV711 anti-mouse NK-1.1 (BioLegend, cat. no. PK136, 1:100), AF700 anti-mouse Ly6C (BioLegend, cat. no. HK1.4, 1:200), BV711 anti-mouse Ly6C (BioLegend, cat. no. HK1.4, 1:200) and APC anti-mouse 4D4 (ref. ⁴⁷) (1:1,000) provided by Butovsky.
细胞内细胞因子染色及细胞分离操作参照先前文献方法 ¹²⁶ 。小心剥离颅骨脑膜后，进一步分离同侧脑组织。用于同侧大脑半球及脑膜酶解混合液成分为：胶原酶 P（0.5 mg/ml ⁻¹ ；Sigma-Aldrich 货号 11213865001）与 DNase-1（250 U/ml ⁻¹ ，Worthington 货号 LK003172 ）溶于含 10 mM Hepes 的 RPMI-1640 培养基。组织样本精细剪碎后置于室温摇床孵育 1 小时。酶解完成后采用前述 Percoll（GE Healthcare Life Sciences）梯度离心法分离细胞。脑组织及脑膜来源细胞仅孵育 2 小时（对比 cLN 细胞的 4 小时）。使用 Symphony 流式细胞仪（BD Biosciences）配合 DIVA 软件（BD Biosciences）进行数据采集，FlowJo 软件 v.9.9 或 v.10.1（TreeStar Inc.）分析数据。细胞内染色采用 Zombie Aqua 可固定活力检测试剂盒（BioLegend 货号 423102，1:1000）或 Zombie UV（BioLegend 货号 423108，1:1000）排除死细胞。 实验所用染色抗体包括：PE/cyanine7 标记的小鼠 CD11b 抗体（BioLegend，货号 M1/70，1:300）、APC/cyanine7 标记的小鼠 TCR-β抗体（BioLegend，货号 H57-597，1:100）、BUV661 标记的小鼠 CD45 抗体（30-F11，BD Biosciences，1:200）、PE 标记的小鼠 CD4 抗体（BioLegend，货号 GK1.5，1:100）、BUV805 标记的小鼠 CD8 抗体（BD Biosciences，货号 53-6.7，1:100）、FITC 标记的小鼠 FoxP3 抗体（eBioscience，货号 FJK-16s，1:100）、BV421 标记的小鼠 LAP 抗体（BioLegend，货号 TW7-16B4，1:100）、PE/Dazzle 594 标记的小鼠 IL-10 抗体（BioLegend，货号 JES5-16E3，1:100）、BUV395 标记的小鼠 IL-17a 抗体（BD Biosciences，货号 TC11-18H10，1:100）、BV785 标记的小鼠 IFNγ抗体（BioLegend，货号 XMG1.2，1:100）、BV605 标记的小鼠 Ly6G 抗体（BioLegend，货号 1A8，1:300）、BV711 标记的小鼠 NK-1.1 抗体（BioLegend，货号 PK136，1:100）、AF700 标记的小鼠 Ly6C 抗体（BioLegend，货号 HK1.4，1:200）、BV711 标记的小鼠 Ly6C 抗体（BioLegend，货号 HK1.4，1:200）以及 Butovsky 提供的 APC 标记的小鼠 4D4 抗体（文献编号 ⁴⁷ ，1:1,000）。

Flow cytometry FoxP3(GFP) T_reg cell sorting from the brain and blood
流式细胞术从脑组织和血液中分选 FoxP3(GFP) T _reg 细胞

The ipsilateral brain hemisphere was processed with the same enzyme dissociation kit described above and then processed as the microglial cell sorting above. The sample was purified and enriched using CD4 cell isolation microbead kit (Miltenyi Biotech, cat. no. 130-104-454) on a magnetic MACS separator before sorting. Cell sorting was performed using FACSAriaIII cell sorter (Becton Dickson). APC/cyanine7 anti-mouse CD45 (BioLegend, cat. no. 30-F11, 1:100), APC anti-mouse CD4 antibody (BioLegend, cat. no. GK1.5, 1:100) and 7-AAD (BD Bioscience) was used to identify the live CD4⁺ population and the FITC channel was used to identify the FoxP3⁺ population. Each RNA-seq sample is a pool of 5 ipsilateral hemispheres and 20 sham brain hemispheres. The blood samples were collected in heparin-coated tubes and then transferred to 15-ml tubes where ACK lysis buffer was used to lyse the erythrocytes. The sample was then strained through a 70-μm filter and stained as decribed above. Each RNA-seq sample is a pool of five mice.
同侧大脑半球采用上述相同的酶解试剂盒处理，随后按照小胶质细胞分选流程进行操作。样本在分选前使用 CD4 细胞分离磁珠试剂盒（Miltenyi Biotech，货号 130-104-454）通过 MACS 磁珠分选仪进行纯化富集。细胞分选采用 FACSAriaIII 流式细胞仪（Becton Dickson）完成，使用 APC/cyanine7 标记的小鼠 CD45 抗体（BioLegend，货号 30-F11，1:100）、APC 标记的小鼠 CD4 抗体（BioLegend，货号 GK1.5，1:100）及 7-AAD（BD Bioscience）鉴定活体 CD4 ⁺ 细胞群，FITC 通道用于检测 FoxP3 ⁺ 细胞群。每个 RNA 测序样本由 5 个同侧半球与 20 个假手术脑半球混合组成。血液样本采集于肝素抗凝管后转移至 15 毫升离心管，采用 ACK 裂解液溶解红细胞，经 70 微米滤网过滤后按上述方法进行染色。每个 RNA 测序样本由五只小鼠的样本混合制备。

Quantitative PCR  定量聚合酶链反应

RNA was extracted with RNeasy columns (QIAGEN), complementary DNA was prepared and used for qPCR (Applied Biosystems, cat. no. 437466) and the results were normalized to Gapdh (Mm99999915_g1). Applied Biosystems supplied: Il10 (cat. no. Mm01288386_m1), Il6 (cat. no. Mm00446190_m1), Tnf (cat. no. Mm00443258_m1), Il1b (cat. no. Mm00434228_m1), Il2 (cat. no. Mm00434256_m1), Il18 (cat. no. Mm00434226_m1), Ifng (cat. no. Mm01168134_m1), Bdnf (cat. no. Mm04230607_s1), Gdnf (cat. no. Mm00599849_m1), Ccl5 (cat. no. Mm01302427_m1), Cd14 (cat. no. Mm01158466_g1), Mrc1 (Cd206) (cat. no. Mn01329359_m1), Tgfb1 (cat. no. Mm01178820_m1), Il17a (cat. no. Mn00439618_m1), Cd86 (cat. no. Mm00444540_m1), Clec7a (cat. no. Mm01183349_m1), Tlr2 (cat. no. Mm00442346_m1), Cd33 (cat. no. Mm00491152_m1), Cx3cr1 (cat. no. Mm00438354_m1) and Il10ra (cat. no. Mm00434151_m1). The 2^−ΔΔCt method was used to calculate the relative expression of each gene.
使用 RNeasy 柱（QIAGEN）提取 RNA，制备互补 DNA 并用于 qPCR（Applied Biosystems，货号 437466），结果以 Gapdh（Mm99999915_g1）为内参进行标准化。Applied Biosystems 提供的引物包括：Il10（货号 Mm01288386_m1）、Il6（货号 Mm00446190_m1）、Tnf（货号 Mm00443258_m1）、Il1b（货号 Mm00434228_m1）、Il2（货号 Mm00434256_m1）、Il18（货号 Mm00434226_m1）、Ifng（货号 Mm01168134_m1）、Bdnf（货号 Mm04230607_s1）、Gdnf（货号 Mm00599849_m1）、Ccl5（货号 Mm01302427_m1）、Cd14（货号 Mm01158466_g1）、Mrc1（Cd206）（货号 Mn01329359_m1）、Tgfb1（货号 Mm01178820_m1）、Il17a（货号 Mn00439618_m1）、Cd86（货号 Mm00444540_m1）、Clec7a（货号 Mm01183349_m1）、Tlr2（货号 Mm00442346_m1）、Cd33（货号 Mm00491152_m1）、Cx3cr1（货号 Mm00438354_m1）和 Il10ra（货号 Mm00434151_m1）。采用 2^-ΔΔCt 法计算各基因的相对表达量。

Isolation of primary neurons
原代神经元分离

Primary neuron isolation was carried out as previously described⁵¹. In short, primary neurons were prepared from embryos at age E18. Cell density was determined using a hemocytometer and cells were seeded. Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) was used for initial plating and the medium was changed to neurobasal supplemented with 1× B27 (Invitrogen) 3 h later. The medium was changed every 3 d.
原代神经元分离操作如前所述 ⁵¹ 。简言之，从胚胎第 18 天(E18)制备原代神经元。使用血细胞计数器测定细胞密度后进行接种。初始培养采用含 10%胎牛血清(FBS)的杜氏改良 Eagle 培养基(DMEM)，3 小时后更换为添加 1×B27 补充剂(Invitrogen)的神经基础培养基。此后每 3 天更换一次培养基。

Induction of apoptosis and labeling of neurons
诱导细胞凋亡与神经元标记

Apoptosis and labeling of neurons were done as previously described⁵¹. Neurons were irradiated with ultraviolet light (302 nm) with an intensity of 6 × 15 W for 15 min. The apoptotic neurons were labeled with labeling dye (Alexa-405 NHS Ester, Life Technologies/Thermo Fisher Scientific). Neurons were resuspended at a density of 500,000 cells per 4 μl for stereotactic injections.
神经元凋亡及标记操作参照先前方法 ⁵¹ 。采用 302nm 紫外光源(强度 6×15W)照射神经元 15 分钟。凋亡神经元使用标记染料(Alexa-405 NHS 酯，Life Technologies/Thermo Fisher Scientific)进行标记。将神经元重悬至每 4μl 含 50 万个细胞的密度，用于立体定位注射。

Stereotactic injections  立体定向注射

Mice were anesthetized by intraperitoneal injection of ketamine (100 mg kg⁻¹). Apoptotic neurons or sterile Dulbecco’s PBS (DPBS) were injected into the lesion of TBI mice at two depths of 1.5 mm and 2 mm. Then, 2 μl was injected at each depth using stereotaxic equipment (Harvard Apparatus). After recovery from surgery, animals were returned to their cages. Post-surgery (4 or 16 h), mice were euthanized by CO₂ inhalation and brains were processed for flow cytometry analysis of phagocytic microglia.
小鼠通过腹腔注射氯胺酮（100 mg/kg）麻醉。将凋亡神经元或无菌杜氏磷酸盐缓冲液（DPBS）在 1.5 毫米和 2 毫米两个深度注射入 TBI 小鼠的损伤部位。每个深度使用立体定位仪（Harvard Apparatus）注射 2 微升。术后恢复期间，动物被放回笼中。手术后（4 或 16 小时），通过二氧化碳吸入法处死小鼠，取脑组织进行流式细胞术分析小胶质细胞的吞噬功能。

Adoptive transfer  过继性转移

Splenocytes and cLNs from FoxP3-GFP or 10BiT.FoxP3^GFP mice were purified and enriched using CD4 cell isolation microbead kit (Militenyi Biotech, cat. no. 130-104-454) on a magnetic MACS separator before sorting. Cell sorting was performed using FACSAriaIII cell sorter (Becton Dickson). PE anti-mouse CD3 (BioLegend, cat. no. 17A2, 1:100), APC anti-mouse CD4 (BioLegend, cat. no. GK1.5, 1:100) and 7-AAD were used to identify the live CD4⁺ population and the FITC channel was used to identify the FoxP3⁺ population. For the 10BiT.FoxP3^GFP, PE anti-mouse Thy1.1 (BioLegend, cat. no. S20007C, 1:1,500) was used to identify the FoxP3⁺Thy1.1⁺ IL-10-producing FoxP3 T_reg cells. All cells were injected i.p. at 2.5 million for total CD4, 1 million for CD4⁺FoxP3⁺ and 1 million for FoxP3⁺Thy1.1⁺ or FoxP3⁻Thy1.1⁻ populations.
采用 CD4 细胞分离微珠试剂盒（美天旎生物技术，货号 130-104-454）在 MACS 磁珠分选仪上对 FoxP3-GFP 或 10BiT.FoxP3 小鼠的脾细胞和颈淋巴结细胞进行纯化富集后分选。使用 FACSAriaIII 流式细胞分选仪（BD 公司）进行细胞分选。PE 标记抗小鼠 CD3 抗体（BioLegend，货号 17A2，1:100）、APC 标记抗小鼠 CD4 抗体（BioLegend，货号 GK1.5，1:100）及 7-AAD 用于鉴定活体 CD4+细胞群，FITC 通道用于检测 FoxP3+细胞群。对于 10BiT.FoxP3 小鼠，采用 PE 标记抗小鼠 Thy1.1 抗体（BioLegend，货号 S20007C，1:1500）鉴定 FoxP3+Thy1.1+IL-10 分泌型 FoxP3 T 细胞。所有细胞均通过腹腔注射：总 CD4 细胞 250 万，CD4+FoxP3+细胞 100 万，FoxP3+Thy1.1+或 FoxP3+Thy1.1-细胞各 100 万。

In vitro cell culture
体外细胞培养

Sorted 4D4⁺ microglia 24 h post-TBI were cultured as previously described¹²⁷ at 200,000 cells in a 24-well plate (Kemtec, cat. no. 4422A). The microglial culture medium was composed of 10% FBS (Gibco, cat. no. 10438026), 100 U ml⁻¹ of penicillin–streptomycin mixture (Lonza, cat. no. DE17-602E), supplemented in DMEM/F-12 Glutamax medium (Gibco, cat. no. 10565018). FoxP3-GFP was treated with nasal aCD3 or the isotype control for 7 d and the splenic/cLN CD4⁺FoxP3⁺ and CD4⁺FoxP3(GFP)⁻ population, specifically from aCD3-treated animals, was placed in a lymphocyte culture medium composed of 10% FBS, 100 U ml⁻¹ of penicillin–streptomycin mixture, 55 µM 2-mercaptoethanol (Gibco, cat. no. 21985023), 1% sodium pyruvate (Lonza, cat. no. BE13-115E) and 1% Hepes (Lonza, cat. no. BE17-737E), supplemented in RPMI-1640 medium (Gibco, cat. no. 11875119) and then placed on the top of the hanging cell culture 0.4-µm insert (Millicell, cat. no. PTHT24H48) at 600,000 cells per insert on top of the cultured microglia, and the assay was left for 72 h in a CO₂ Cell culture Incubator (InCusafe). There was a fourth group where IL-10-neutralizing antibody (BioXCell, cat. no. JES5-2A5) at a concentration of 50 μg ml⁻¹ was added with the CD4⁺FoxP3⁺ which was cell sorted from nasal aCD3-treated mice After 72 h the microglia were lysed with buffer RLT and RNA was extracted with RNeasy columns (QIAGEN).
创伤性脑损伤后 24 小时分选的 4D4 ⁺ 小胶质细胞按前述方法 ¹²⁷ 培养于 24 孔板（Kemtec，货号 4422A），每孔接种 200,000 个细胞。小胶质细胞培养基成分为：DMEM/F-12 Glutamax 培养基（Gibco，货号 10565018）中添加 10%胎牛血清（Gibco，货号 10438026）和 100 U/ml ⁻¹ 青霉素-链霉素混合液（Lonza，货号 DE17-602E）。FoxP3-GFP 小鼠经鼻腔给予 aCD3 或同型对照处理 7 天后，取其脾脏/颈淋巴结 CD4 ⁺ FoxP3 ⁺ 与 CD4 ⁺ FoxP3(GFP) ⁻ 细胞群（仅取自 aCD3 处理组），置于淋巴细胞培养基（RPMI-1640 培养基[Gibco，货号 11875119]中添加 10%胎牛血清、100 U/ml ⁻¹ 青霉素-链霉素混合液、55 µM 2-巯基乙醇[Gibco，货号 21985023]、1%丙酮酸钠[Lonza，货号 BE13-115E]和 1% Hepes[Lonza，货号 BE17-737E]）中，以每孔 600,000 个细胞的密度接种于 0.4 µm 悬式细胞培养插板（Millicell，货号 PTHT24H48）中，置于已培养小胶质细胞的上层，在 CO ₂ 细胞培养箱（InCusafe）中共培养 72 小时。另设第四组加入 IL-10 中和抗体（BioXCell，货号 将浓度为 50 μg/ml 的 JES5-2A5 抗体与从鼻腔给予 aCD3 治疗的小鼠中分选出的 CD4+FoxP3+细胞共同培养 72 小时后，使用 RLT 裂解缓冲液裂解小胶质细胞，并通过 RNeasy 离心柱（QIAGEN）提取 RNA。

Bulk RNA-seq and analysis
批量 RNA 测序与分析

Bulk RNA-seq was performed as previously described⁵⁹. Briefly, 2,000 isolated microglia CD45⁺CD11b⁺Ly6C⁻4D4⁺ were lysed in 5 μl of TCL buffer + 1% 2-mercaptoethanol. Between 800 and 1,000 isolated CD4⁺FoxP3(GFP⁺) cells from the brain and blood were processed. Smart-Seq2 libraries were prepared and sequenced using the Broad Genomic Platform. The cDNA libraries were generated from sorted cells using the Smart-seq2 protocol5. RNA-seq was performed using Illumina NextSeq500 with a High Output v.2 kit to generate reads of 2× 38 bp. Sequencing data were demultiplexed and provided by the Broad Institute in FASTQ format. The processing of the bulk RNA-seq data (transcript assembly and quantification) was based on an established computational pipeline (HISAT, Stringtie and Ballgown)¹²⁸ and sequencing quality was assessed using FastQC. Reads were aligned to the ‘mm10’ reference genome. Transcript abundances were then imported into R (v.4.1.2) and converted to gene-level estimated counts using the ‘tximport’ package (v.1.22.0) from Bioconductor. Low abundance genes that achieved <10 counts aggregated across all samples were filtered out. Raw read counts were then normalized using DESeq2 (v.1.34.0). Differential gene expression was performed using DESeq2 with a significance cutoff of false discovery rate (FDR)-adjusted P < 0.05. Analyses that did not pass the FDR-correction cutoff were denoted with P < 0.05. Pairwise group comparisons were conducted using Wald’s test with standard parameters, then log₂(fold-changes) were shrunk using the lfcShrink function in DESeq2. Gene expression comparisons of three or more groups were conducted using a likelihood ratio test (LRT). Heatmaps were created using ComplexHeatmap (v.2.10.1) or pheatmap (v.1.0.12) and clusters were identified via hierarchal clustering. Identified clusters were functionally annotated with Gene Ontology Biological Process (GOBP) gene-set terms using the enrichGO function of the clusterProfilerpackage (v.4.2.2), where GOBP terms with q-values < 0.05 were considered significantly enriched.
批量 RNA 测序按先前描述方法进行 ⁵⁹ 。简言之，将 2,000 个分选的 CD45 ⁺ CD11b ⁺ Ly6C ⁻ 4D4 ⁺ 小胶质细胞裂解于 5μl 含 1% 2-巯基乙醇的 TCL 缓冲液中。对脑组织和血液中分离的 800-1,000 个 CD4 ⁺ FoxP3(GFP ⁺ )细胞进行处理。采用 Smart-Seq2 方案构建文库并在 Broad 基因组平台完成测序。分选细胞的 cDNA 文库通过 Smart-seq2 流程 5 制备。使用 Illumina NextSeq500 测序仪配合 High Output v.2 试剂盒生成 2×38 bp 读长。测序数据经 Broad 研究所解复用后以 FASTQ 格式提供。批量 RNA-seq 数据处理（转录本组装与定量）基于成熟计算流程（HISAT、Stringtie 和 Ballgown） ¹²⁸ ，测序质量通过 FastQC 评估。读段比对至"mm10"参考基因组。随后将转录本丰度数据导入 R 语言(v.4.1.2)，通过 Bioconductor 的"tximport"包(v.1.22.0)转换为基因水平计数。过滤掉所有样本总计数<10 的低丰度基因。 随后使用 DESeq2（v.1.34.0）对原始读数进行标准化处理。差异基因表达分析采用 DESeq2 软件，显著性阈值设定为错误发现率（FDR）校正后的 P 值<0.05。未通过 FDR 校正阈值的结果标注为 P<0.05。组间两两比较采用 Wald 检验（标准参数），随后通过 DESeq2 中的 lfcShrink 函数对 log2（倍数变化）进行收缩。三组及以上基因表达比较采用似然比检验（LRT）。热图使用 ComplexHeatmap（v.2.10.1）或 pheatmap（v.1.0.12）生成，并通过层次聚类识别簇群。鉴定出的簇群通过 clusterProfiler 包（v.4.2.2）的 enrichGO 函数进行基因本体生物过程（GOBP）功能注释，q 值<0.05 的 GOBP 条目视为显著富集。

Pathway analysis  通路分析

GSEA was performed using the clusterProfiler package and the resulting enriched terms with q-values < 0.05 were visualized through dot plots and bar plots, which were generated using the ggplot2 and ggpubr (v.0.6.0) packages. For IPA (https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-ipa), DEGs from pairwise comparisons with corresponding log₂(fold-changes) and FDR-adjusted P values were used as input. Biological networks were generated in IPA based on canonical pathways and biological functions, as outlined in our group’s previous work⁵⁹.
采用 clusterProfiler 软件包进行 GSEA 分析，并通过 ggplot2 和 ggpubr（v.0.6.0）软件包生成点图和条形图来可视化 q 值<0.05 的显著富集结果。对于 IPA 分析（ https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-ipa ），输入数据为配对比较获得的差异表达基因及其对应的 log ₂ （倍数变化）和 FDR 校正 P 值。如本团队先前工作所述（ ⁵⁹ ），基于经典通路和生物学功能在 IPA 中构建了生物网络。

Statistical and reproducibility
统计分析与可重复性

Statistical analysis was performed using GraphPad Prism v.9 software. Data are presented as mean ± s.e.m and two-sided Student’s t-tests (unpaired), two-factor, repeated-measure, two-way analysis of variance (ANOVA) (group × time) or one-way ANOVA, followed by Tukey’s multiple-comparison analysis was used to assess statistical significance between the groups. All n and P values and statistical tests are indicated in the figure legends. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications^15,23. Data distribution was assumed to be normal, but this was not formally tested. Data for each experiment were collected and processed randomly and animals were assigned randomly to various experimental groups as well. The investigators were blinded to allocation during experiments and outcome assessment. No animals or data points were excluded from the analyses. Each experiment was repeated 2–3 times.
使用 GraphPad Prism v.9 软件进行统计分析。数据以均值±标准误表示，采用双侧非配对 t 检验、双因素重复测量双因素方差分析（ANOVA）（组别×时间）或单因素 ANOVA，随后通过 Tukey 多重比较分析评估组间统计学差异。所有样本量 n 值、P 值及统计检验方法均标注于图注中。未采用统计方法预先确定样本量，但样本量与既往文献报道相当 ^15,23 。假设数据呈正态分布但未进行正式检验。每项实验数据均采用随机方式收集处理，实验动物也随机分配至各实验组。研究人员在实验实施及结果评估阶段均采用盲法。所有实验动物及数据点均纳入分析。每项实验重复 2-3 次。

Reporting summary  报告摘要

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
关于研究设计的更多信息，请参阅本文关联的 Nature Portfolio Reporting Summary 。

Peer review information  同行评审信息

Nature Neuroscience thanks John Lukens, Edward Needham and Michal Schwartz for their contribution to the peer review of this work.
《自然-神经科学》感谢 John Lukens、Edward Needham 和 Michal Schwartz 对本论文同行评审工作作出的贡献。

Extended data  扩展数据

is available for this paper at 10.1038/s41593-025-01877-7.
可在 10.1038/s41593-025-01877-7 获取本文补充材料

Supplementary information
补充信息

The online version contains supplementary material available at 10.1038/s41593-025-01877-7.
在线版本包含补充材料，访问地址：10.1038/s41593-025-01877-7