TCR Diagnostics in LGL Leukemia and Immune Cytopenias

描述（由申请人提供）：大颗粒淋巴细胞（LGL）白血病是一种慢性T细胞淋巴增生，与免疫介导的细胞减少和克隆细胞毒性T细胞（CTL）的扩增有关。LGL CTL不受自然调控，它们的增殖似乎是由外部触发维持的。此外，克隆CTL似乎是介导可能针对造血祖细胞的免疫攻击的效应器。作为一种实验模型，LGL白血病具有克隆效应细胞及其识别的靶细胞可用性的优势。LGL白血病的病理生理可能对应于极化克隆反应，这构成了表征单个致病性T细胞克隆作用的合适模型，但也作为与伴随免疫介导的骨髓衰竭状态的其他原因的多克隆T细胞反应比较的基础。正常情况下，在对特定抗原的免疫反应过程中，免疫优势T细胞克隆扩增。T细胞受体（TCR）独特的抗原特异性部分，即a链可变部分（VB）的互补决定区-3 (CDR3)，可以作为抗原特异性T细胞克隆的分子标记（克隆型）。假设：LGL中抗原识别的特异性可能决定了其血液学表现和类似LGL白血病的隐性ctl介导的扩增，也可能是其他形式的免疫细胞减少的原因。CDR3克隆型可能是研究克隆过程和开发新诊断工具的合适靶点。具体目的：1)鉴定LGL T细胞克隆型将使我们能够确定患者之间是否共享相似的克隆型（或可能在健康个体中也发现），以及设计一类新的基于克隆型PCR的致病克隆定量的诊断工具。2) LGL白血病抗原识别的特异性可能决定其临床表现的类型（如贫血、中性粒细胞减少）。克隆性LGL对自体和异体红细胞和髓细胞前体，以及细胞系和未分化的CD34+细胞的抑制和细胞毒性活性将进行测试。3) LGL克隆的分离将允许产生可溶性TCR分子，这些分子将用于鉴定LGL CTL的细胞靶细胞。4)鉴定LGL特异性克隆型将用于测量患者针对LGL和对照克隆的抗独特型反应，以确定抗独特型反馈在调节自主和生理性克隆扩增中的作用。我们建议的长期目标包括建立基于TCR分子分析的新型诊断策略。我们的研究主要集中在LGL白血病，但总的来说，T细胞库的分子分析可以应用于许多血液学疾病的研究，包括白血病进化的免疫监测，免疫介导的骨髓衰竭和移植物抗宿主病。

DESCRIPTION (provided by applicant): Large granular lymphocyte (LGL) leukemia is a chronic T cell lymphoproliferation associated with immune-mediated cytopenias and expansion of clonal cytotoxic T cells (CTL). LGL CTL defies natural regulation, and their proliferation appears to be maintained by extrinsic triggers. Additionally, the clonal CTL appear to be the effectors that mediate an immune attack presumably directed against hematopoietic progenitors. As an experimental model LGL leukemia offers an advantage of availability of clonal effector cells and the target cells they recognize. The pathophysiology of LGL leukemia may correspond to a polarized clonal response that constitutes a suitable model for characterizing the role of individual pathogenic T cell clones but also as a basis for comparison with the polyclonal T cell responses accompanying other causes of immune-mediated bone marrow failure states. Under normal circumstances, in the process of immune reaction to specific antigens, immunodominant T cell clones expand. The unique antigen-specific portion of the T cell receptor (TCR), the complementary determining region-3 (CDR3) of the variable portion of the a-chain (VB), can serve as a molecular signature (clonotype) for the antigen-specific T cell clones. Hypothesis: The specificity of the antigenic recognition in LGL may determine its hematologic presentation and cryptic CTL-mediated expansions, analogous to LGL leukemia, may be responsible also in other forms of immune cytopenias. CDR3 clonotypes may constitute suitable targets to study clonal processes and development of new diagnostic tools. Specific Aims: 1) Characterization of LGL T cell clonotypes will allow us to determine if similar clonotypes are shared between the patients (or possibly also found in healthy individuals), as well as to design a new class of diagnostic tools based on the quantitation of pathogenic clones using clonotypic PCR. 2) The specificity of antigenic recognition in LGL leukemia may determine the types of clinical presentation (e.g. anemia, neutropenia). Inhibitory and cytotoxic activity of clonal LGL will be tested against autologous and allogeneic erythroid and myeloid precursors, as well as cell lines and undifferentiated CD34+ cells. 3) Isolation of LGL clones will allow for the generation of soluble TCR molecules that will be applied to identify cellular target cells for LGL CTL. 4) Identification of LGL specific clonotypes will be applied to measure the anti-idiotypic responses directed against LGL and control clones in patients to determine the role of anti-idiotypic feedback in the regulation of autonomous and physiologic clonal expansions. The long-term goals of our proposal include establishment of novel diagnostic strategy based on molecular analysis of TCR. Our studies concentrate on LGL leukemia but in general, molecular analysis of the T cell repertoire can be applied to the study of many hematologic conditions, including immune surveillance of leukemia evolution, immune-mediated bone marrow failure and graft versus host disease.