T Cell Differentiation and Diversification in Jawless Vertebrates

项目摘要 拟议的研究解决尚未解决的主要特点，适应性免疫系统，我们已经确定 七鳃鳗和八目鳗中。与所有有颌脊椎动物的免疫球蛋白（IG）结构域系统不同， 无颌脊椎动物的适应性免疫是由高度多样的富含亮氨酸重复序列（LRR）介导的 可变LRR受体（VLRs）。我们已经证明，七鳃鳗中的三种类型的VLR（VLRA， VLRB和VLRC），虽然在结构上与IG和T细胞受体（TCR）无关，但仍然由 具有与哺乳动物B细胞（VLRB）相似的功能特征和基因表达谱的细胞， αβT细胞（VLRA）和γδT细胞（VLRC）。在这里，我们将描述无颌症T样细胞的多样性， 它们的VLR信号复合物的结构。沿着VLRA和VLRC细胞，我们将表征两个 我们最近发现的新的VLRs，VLRD和VLRE，与VLRA和VLRC远缘。我们将 探讨VLRD和VLRE受体的多样性及淋巴细胞亚群的特征 表达他们。我们已经在所有六个目前可用的七鳃鳗基因组中发现了VLRD和VLRE的直系同源物。 我们将扩展我们的系统发育调查的存在和基因组组织这些新的受体， 更多分歧的七鳃鳗和八目鳗。与VLRA和VLRC一样，VLRD和VLRE是跨膜蛋白 具有短的细胞质尾，缺乏可识别的信号基序。这意味着存在与VLR相关的 信号分子，如有颌脊椎动物TCR信号。我们将研究信号伙伴的性质 在复合VLRA/C/D/E受体使用下拉/质谱策略与我们的单克隆 抗体的我们已经鉴定了几种具有ITAM基序的候选跨膜蛋白的转录物， 由VLRA+和VLRC+淋巴细胞表达。我们还鉴定了一种类似七鳃鳗ZAP 70的激酶 在VLRA和VLRC T样细胞中优先表达的基因。我们将使用海七鳃鳗ZAP 70串联 SH 2结构域作为诱饵从活化七鳃鳗裂解物中鉴定潜在的相互作用ITAM蛋白 淋巴细胞我们将使用单细胞RNA测序（scRNA-seq）来描绘T样细胞的亚群， 七鳃鳗幼虫的组织我们将使用CRISPR/Cas9进一步表征T样细胞的早期发育。 突变VLRA/C/D/E装配和信号传导的候选调节因子，然后评估VLR 表达、库多样性和淋巴细胞发育。我们还将描述T的出现- 用scRNA-seq分析从早期摄食幼虫中分离的细胞， VLR组装的开始。这些研究将阐明T样细胞在免疫球蛋白的一个不同分支中的功能。 脊椎动物生殖他们将强调在脊椎动物中获得性免疫的共同机制， 这些特征保留在来自共同脊椎动物祖先的无颌类谱系中，并揭示了新的 Agnathan创新总的来说，这些研究将有助于深入了解脊椎动物适应性的起源， 免疫和提供T细胞生物学的更深入的理解。

PROJECT SUMMARY The proposed studies address unresolved principal features of the adaptive immune system we have identified in lampreys and hagfish. Instead of the immunoglobulin (Ig) domain-based system of all jawed vertebrates, adaptive immunity in the jawless vertebrates is mediated by highly diverse sets of leucine-rich repeat (LRR) proteins, named Variable LRR Receptors (VLRs). We have shown that three types of VLR in lampreys (VLRA, VLRB and VLRC), while structurally unrelated to Ig and T cell receptors (TCRs), are nonetheless expressed by cells with functional characteristics and gene expression profiles similar to those of mammalian B cells (VLRB), αβT cells (VLRA), and γδT cells (VLRC). Here we will characterize the diversity of the agnathan T-like cells and the structures of their VLR signaling complexes. Along with VLRA and VLRC cells, we will characterize two novel VLRs that we recently identified, VLRD and VLRE, that are distantly related to VLRA and VLRC. We will investigate the receptor diversity of VLRD and VLRE and the characteristics of subpopulations of lymphocytes that express them. We have found orthologs of VLRD and VLRE in all six currently available lamprey genomes. We will extend our phylogenetic survey of the presence and genomic organization of these new receptors to more divergent lampreys and hagfishes. Like VLRA and VLRC, VLRD and VLRE are transmembrane proteins with short cytoplasmic tails that lack recognizable signaling motifs. This implies the presence of VLR-associated signaling molecules, as in jawed vertebrate TCR signaling. We will investigate the nature of signaling partners in the composite VLRA/C/D/E receptors using pulldown/mass spectrometry strategies with our monoclonal antibodies. We have identified transcripts for several candidate transmembrane proteins with ITAM motifs that are expressed by VLRA+ and VLRC+ lymphocytes. We have also identified a sea lamprey ZAP70-like kinase gene with preferential expression in the VLRA and VLRC T-like cells. We will use the sea lamprey ZAP70 tandem SH2 domains as bait to identify potential interacting ITAM proteins from lysates of activated lamprey lymphocytes. We will use single-cell RNA sequencing (scRNA-seq) to delineate the subsets of T-like cells from lamprey larval tissues. We will further characterize the early development of the T-like cells using CRISPR/Cas9 to mutate candidate regulators of VLRA/C/D/E assembly and signaling, followed by assessments of VLR expression, repertoire diversity, and lymphocyte development. We will also characterize the emergence of T- like lymphocytes in development with scRNA-seq analysis of cells isolated from early feeding larvae throughout the onset of VLR assembly. These investigations will elucidate T-like cell function in a divergent branch of vertebrate phylogeny. They will highlight common mechanisms of adaptive immunity across vertebrates, ancient features that are retained in the agnathan lineage from a common vertebrate ancestor, and reveal novel agnathan innovations. Collectively, these studies will contribute insight into the origins of vertebrate adaptive immunity and provide a deeper understanding of T cell biology.