CNS-Native Myeloid Cells Drive Immune Suppression in the Brain Metastatic Niche through Cxcl10 [R10_PD-L1+VISTA treatment]

We performed CITE-seq (10x Genomics-based) to profile and compare the transcriptomes and cell surface expression of immune epitopes in the brains of anti-VISTA+anti-PD-L1 treated (Dual TX) and control, non-treated mice. We sequenced a total of eight samples (4 control, 4 dual TX). We created an antibody pool consisting of 35 different antibodies and stained each sample individually with this antibody pool. Then, we stained each sample with it's own unique hashing antibody so that we could subsequently pool the samples for loading onto the 10x Chromium and later prepare one library consisting of all sight samples and finally separate each sample in silico by it's unique hashing antibody. The samples are as follows: (1) HTO1: Control/Non-treated (2) HTO2: Control/Non-treated ; (3) HTO3: Control/Non-treated ; (4) Control/Non-treated ; (5) HTO5: Dual TX; (6) Dual TX; (7) HTO7: Dual TX; (8) HTO8: Dual TX. The following sample comparisons were made: HTO1 - HTO4 were compared to HTO5 - HTO8. Overall design: Metastasis-burdened brains were obtained from 2-3 month old female mice with or without anti-VISTA + anti-PD-L1 treatment (Dual TX). These mice were administered Dual TX every other day starting 3 days-post metastasis injection. Brain metastasis was established by carotid injection of E0771 cells. Cell suspensions were prepared by percoll gradient centrifugation enrichment to obtain suspensions enriched for immune cells from the brain. We created an antibody pool consisting of 35 different antibodies and stained each sample individually with this antibody pool. Then, we stained each sample with it's own unique hashing antibody so that we could subsequently pool the samples for loading onto the 10x Chromium and later prepare one library consisting of all eight samples and finally separate each sample in silico by it's unique hashing antibody.

本研究采用基于10x Genomics平台的CITE-seq技术，对经抗VISTA联合抗PD-L1治疗（双重治疗，Dual TX）与未处理对照组小鼠的大脑转录组及免疫表位细胞表面表达谱进行分析与比较。本次实验共完成8例样本的测序，其中对照组4例、双重治疗组4例。我们制备了包含35种不同抗体的抗体混合液，对每例样本单独进行染色；随后为每例样本标记专属的哈希抗体（hashing antibody），以便后续将所有样本混合后上样至10x Chromium系统，构建包含全部8例样本的单一测序文库，最终通过每例样本的专属哈希抗体在生物信息学层面完成样本拆分。
样本信息如下：(1) HTO1：对照组/未处理组；(2) HTO2：对照组/未处理组；(3) HTO3：对照组/未处理组；(4) 对照组/未处理组；(5) HTO5：双重治疗组；(6) 双重治疗组；(7) HTO7：双重治疗组；(8) HTO8：双重治疗组。
本次研究设置如下样本比较组别：将HTO1至HTO4的对照组样本与HTO5至HTO8的双重治疗组样本进行对比分析。
实验整体设计如下：研究对象为2~3月龄雌性小鼠，通过颈动脉注射E0771细胞构建脑转移模型；荷瘤小鼠于肿瘤接种后第3天起，每隔一日接受一次双重治疗。采用Percoll密度梯度离心法处理脑组织，制备富含脑内免疫细胞的细胞悬液。我们制备了包含35种不同抗体的抗体混合液，对每例样本单独进行染色；随后为每例样本标记专属的哈希抗体，以便后续将所有样本混合后上样至10x Chromium系统，构建包含全部8例样本的单一测序文库，最终通过每例样本的专属哈希抗体在生物信息学层面完成样本拆分。