Transcriptional profilling of pathogen-specific T helper cells

Introduction: We reported a in vitro pathogen-specific priming system to using pathogen lysate stimulated splenic CD11c+ DCs to differentiate pathogen-specific murine T helper cells. This method allowed us to compare side-by-side transcriptional profiles of bulk T helper cells that are specific to distinct pathogens and to study their composition and functionality. Method: We stimulated mouse splenic CD11c+ DCs with 10ug/ml Lm or Cr lysate for 5hours. The DCs were subsequently washed and co-cultured at 1:5 ratio to purified, CFSE-labeled CD62L+CD44- mouse naïve CD4 T cells. At day-7, CD90+CFSE- (Pathogen-specific Th cells) or CD90+CFSE+ (naive T cells) were sorted from the same culture and were subjected to RNA isolation, library prep and subsequent sequencing. Conclusion: We found that Lm- and Cr-specific Th cells have distinct transcriptomes and contained distinct Th subtypes. Lm-specific Th cells contained higher expressions of Th1-associated cytokines, while Cr-specific Th cells contained higher levels of Th17 associate cytokines and transcription factors. In addition, we have identified heterogeneity within the pathogen-specific Th cells, as they express various cytokines from multiple Th lineages. Lastly, we found a vast variety of genes differentially expressed between Lm- and Cr-specific Th cells, indicating that different pathogen-stimulated DCs denote distinct function and developmental cues to T cells during differentiation. Overall design: mRNA profiles of Lm- or Cr-specific Th cells were generated by Illumina sequencing in duplicate. CFSE+CD90+ naïve T cell from Lm and Cr condition cultures served as control.

引言：本研究报道了一种体外病原体特异性致敏体系——利用病原体裂解物刺激小鼠脾脏CD11c+树突状细胞（DCs），以诱导病原体特异性小鼠辅助性T细胞（Th细胞）分化。该方法可实现不同病原体特异性Th细胞群体的转录组平行比较，并可用于研究其细胞组成与功能特性。
方法：我们以10μg/ml的李斯特菌（Lm）或克罗诺杆菌（Cr）裂解物刺激小鼠脾脏CD11c+ DCs，刺激时长为5小时。随后洗涤DCs，并以1:5的比例与纯化的、羧基荧光素琥珀酰亚胺酯（CFSE）标记的CD62L+CD44-小鼠初始CD4+ T细胞共培养。培养至第7天，从同一共培养体系中分选CD90+CFSE-（病原体特异性Th细胞）或CD90+CFSE+（初始T细胞），随后进行RNA提取、文库制备及后续测序。
结论：本研究发现，Lm特异性与Cr特异性Th细胞具有截然不同的转录组特征，且各自包含独特的Th细胞亚群。Lm特异性Th细胞高表达Th1相关细胞因子，而Cr特异性Th细胞则高表达Th17相关细胞因子与转录因子。此外，我们还鉴定到病原体特异性Th细胞内部存在异质性：这类细胞可表达多个Th细胞谱系相关的多种细胞因子。最后，我们在Lm与Cr特异性Th细胞之间鉴定到大量差异表达基因，这表明不同病原体刺激的DCs在T细胞分化过程中，为其提供了截然不同的功能与发育调控信号。
整体实验设计：采用Illumina测序技术对Lm特异性或Cr特异性Th细胞的mRNA转录组进行双重复测序；同时将Lm与Cr刺激培养体系中分离得到的CFSE+CD90+初始T细胞作为对照。