A Novel Approach to Tracking Antigen-Experienced CD4 T Cells into Functional Compartments via Tandem Deep and Shallow TCR Clonotyping
A Novel Approach to Tracking Antigen-Experienced CD4 T Cells into Functional Compartments via Tandem Deep and Shallow TCR Clonotyping
复制标题
DOI：
10.4049/jimmunol.1300622
复制
发表时间：
2013-12-01
影响因子：
4.4
通讯作者：
Peakman, Mark
中科院分区：
文献类型：
--
作者：
Estorninho, Megan;Gibson, Vivienne B.;Peakman, Mark
人类 Ag TCR 的广泛多样性既是有效适应性免疫的基石，能够保护宿主免受多种病原体的侵害，也是引起潜在病理性自身反应的弱点。多样性带来的复杂性使得检测和跟踪参与这些免疫反应的单个 Ag 特异性 CD4 T 细胞 (AST) 具有挑战性。我们报告了一种串联、多步骤的过程，用于量化大型多克隆群体中 AST 的罕见 TCR β 链可变序列。该方法将功能性 CD4 T 细胞区室（例如幼稚/记忆细胞）内的深度高通量测序 (HTS) 与通过体外短期 Ag 刺激后激活标记物上调鉴定的浅层、基于多个标识符的 AST HTS 相结合。我们发现识别病原体来源的抗原和自身抗原的 HLA II 类限制性表位的克隆型是寡克隆的并且通常是私有的。正如预期的那样，个体内的克隆型跟踪揭示了内存群体中存在的私有 AST 克隆型，代表克隆扩展（相同的核苷酸序列；“超私有”）。其他 AST 克隆型通过聚合重组共享 CDR3 β 氨基酸序列，并在多个个体的记忆群体中发现。基于串联 HTS 的克隆分型将有助于研究疫苗接种后以及针对癌症和自身免疫性疾病的 Ag 特异性免疫疗法后出现的 AST 动态、表位扩散和库变化。
Extensive diversity in the human repertoire of TCRs for Ag is both a cornerstone of effective adaptive immunity that enables host protection against a multiplicity of pathogens and a weakness that gives rise to potential pathological self-reactivity. The complexity arising from diversity makes detection and tracking of single Ag-specific CD4 T cells (ASTs) involved in these immune responses challenging. We report a tandem, multistep process to quantify rare TCR beta-chain variable sequences of ASTs in large polyclonal populations. The approach combines deep high-throughput sequencing (HTS) within functional CD4 T cell compartments, such as naive/memory cells, with shallow, multiple identifier-based HTS of ASTs identified by activation marker upregulation after short-term Ag stimulation in vitro. We find that clonotypes recognizing HLA class II-restricted epitopes of both pathogen-derived Ags and self-Ags are oligoclonal and typically private. Clonotype tracking within an individual reveals private AST clonotypes resident in the memory population, as would be expected, representing clonal expansions (identical nucleotide sequence; "ultraprivate"). Other AST clonotypes share CDR3 beta amino acid sequences through convergent recombination and are found in memory populations of multiple individuals. Tandem HTS-based clonotyping will facilitate studying AST dynamics, epitope spreading, and repertoire changes that arise postvaccination and following Ag-specific immunotherapies for cancer and autoimmune disease.