Engineered T cell therapy for central nervous system injury [control tissues TCR scRNA-seq]

Traumatic injuries to the central nervous system (CNS) affect millions of people worldwide yet lack an effective treatment. These injuries contain infiltrating immune cells that can promote tissue repair and could be exploited for therapeutic benefit. Here, using single-cell RNA-sequencing of T cells infiltrating the injured CNS we demonstrate their clonal expansion and antigen specificity towards CNS derived self-peptides. We confirm the beneficial effect of these injury-associated autoimmune CD4+ T cells in murine models of optic nerve and spinal cord injury. Subsequently, using mRNA-based transient T cell receptor (TCR) reconstitution, we demonstrate a therapeutic T cell strategy to alleviate CNS injury. Treatment of CNS-injured mice with this therapy improved locomotion and alleviated histological signs of damage, through regulation of myeloid cells, without detrimental autoimmune side effects. This strategy provides a means of developing custom-designed T cell therapies for CNS injury, and possibly for other neurodegenerative disorders. To FACS sort T cells, mice were given a lethal dose of anesthetics by i.p. euthasol (10% v/v) and transcardially perfused with ice-cold 0.025% (w/v) heparin in PBS. Before perfusion, peripheral blood was collected by retro-orbital bleeding (200-300ul) and diluted 1:1 with PBS containing heparin. Red blood cells were lysed twice by resuspension in ACK lysis buffer for 5 min at RT. Cells were washed with FACS buffer (2 mM EDTA, 25 mM HEPES, 1% BSA in 1X PBS) before being re-suspended for immunostaining. After perfusion, meninges and diaphragm were digested for 20 and 30 min, respectively, at 37 °C with 1.4 U/mL of Collagenase VIII (Sigma Aldrich) and 35 U/mL of DNAse I (Sigma Aldrich) in IMDM (Sigma Aldrich) media. Following the digestion step, the tissues were gently pressed through 70 μm nylon mesh cell strainers with a sterile plastic plunger. Cells were then centrifuged at 450g at 4°C for 4:30 min. The cell pellets were resuspended in ice-cold FACS buffer and stained at 1:300 dilution. The single-cell suspensions were immunostained with Live/Dead Fixable Aqua Dead (Biolegend; #Cat: 423102), anti-CD45 APC (BD Bioscience; Clone: 30-F11; #Cat: 559864), anti-TCR FITC (Biolegend; Clone: H57-597; #Cat: 109206), anti-CD4 PE (BD Bioscience; Clone: RM4-5; #Cat: 553049), and anti-CD8a PE (eBioscience; Clone: 53-6.7; #Cat: 12-0081-81) using the BD Influx Cell Sorter (BD Biosciences). CD4+ and CD8+ T cells were combined into a single tube (CD45+TCR +CD4+ and CD45+TCR +CD8+, respectively). Cells were sequenced in 4 separate runs to improve sequencing depth via 5' chemistry.

中枢神经系统（central nervous system，CNS）创伤性损伤在全球范围内影响数百万人群，目前尚无有效治疗方案。此类损伤病灶内存在浸润的免疫细胞，其可促进组织修复，有望被开发为治疗手段。本研究通过对损伤CNS内浸润的T细胞进行单细胞RNA测序，证实了这些细胞的克隆扩增以及针对CNS来源自身肽段的抗原特异性。我们在视神经与脊髓损伤的小鼠模型中，验证了此类损伤相关的自身免疫性CD4+ T细胞（CD4+ T cells）的有益作用。随后，我们借助基于信使RNA（mRNA）的瞬时T细胞受体（T cell receptor，TCR）重构技术，证实了一种可缓解CNS损伤的治疗性T细胞策略。采用该疗法对CNS损伤小鼠进行干预后，可通过调控髓系细胞改善小鼠运动功能，并减轻损伤的组织学特征，且未产生有害的自身免疫性副作用。该策略为开发针对CNS损伤的定制化T细胞疗法提供了可行路径，或可推广至其他神经退行性疾病的治疗。为通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting，FACS）分离T细胞，我们对小鼠腹腔注射致死剂量的安乐死注射液（euthasol，10% v/v），随后经心脏灌流冰冷的0.025%（w/v）肝素-磷酸盐缓冲液（phosphate-buffered saline，PBS）。灌流前，通过眼眶后采血采集外周血（200~300 μL），并以含肝素的PBS按1:1比例稀释。将细胞重悬于ACK裂解液中室温孵育5分钟以裂解红细胞，该步骤重复两次。随后用FACS缓冲液（含2 mM乙二胺四乙酸、25 mM 4-羟乙基哌嗪乙磺酸、1% 牛血清白蛋白（bovine serum albumin，BSA）的1×PBS）洗涤细胞，并重悬以进行免疫染色。灌流结束后，将脑膜与膈肌分别置于含1.4 U/mL VIII型胶原酶（Sigma Aldrich）与35 U/mL 脱氧核糖核酸酶I（Sigma Aldrich）的伊斯科夫改良达勒姆培养基（Iscove's Modified Dulbecco's Medium，IMDM，Sigma Aldrich）中，于37℃分别消化20分钟与30分钟。消化完成后，用无菌塑料推杆将组织轻轻压过70 μm尼龙网细胞筛。随后将细胞于4℃以450g离心4分30秒。将细胞沉淀重悬于冰冷的FACS缓冲液中，按1:300的稀释比例进行染色。采用BD Influx细胞分选仪（碧迪生物科技，BD Biosciences），将单细胞悬液与以下试剂进行免疫染色：死活染色Fixable Aqua Dead试剂盒（Biolegend；货号：423102）、APC标记抗CD45抗体（BD Bioscience；克隆号：30-F11；货号：559864）、FITC标记抗TCR抗体（Biolegend；克隆号：H57-597；货号：109206）、PE标记抗CD4抗体（BD Bioscience；克隆号：RM4-5；货号：553049）以及PE标记抗CD8a抗体（eBioscience；克隆号：53-6.7；货号：12-0081-81）。将CD4+与CD8+ T细胞分别收集至同一试管中（分别对应CD45+TCR+CD4+与CD45+TCR+CD8+细胞群）。我们通过4次独立上机测序，采用5'端测序化学法以提升测序深度。