Identifying Ligands with Soluble Gamma/Delta TCRs

描述（由申请人提供）： 虽然γ δ T细胞已被证明在许多实验系统中具有免疫调节作用，但它们在免疫生物学中的作用仍不清楚。令人惊讶的是，可以基于γ δ T细胞表达的V γ和/或V δ元件将γ δ T细胞分成功能亚群。基于这些发现，我们假设某些可诱导的宿主分子作为γ δ T细胞受体（TCR）的配体，并且在引发其免疫调节作用中是重要的。然而，γ δ TCR识别的配体的性质仍然存在争议。此外，最近的研究结果表明，γ δ T细胞反应可以由TCR以外的分子诱发，例如趋化因子受体或先天免疫系统的Toll样受体，使得细胞活化用作TCR介导的刺激的读出的实验难以解释。我们相信γ δ T细胞受体本身可能是阐明TCR在γ δ T细胞功能中作用的关键。在这个建议中，我们提出了初步的证据，小鼠可溶性γ δ TCR的多聚化版本可以像单克隆抗体一样用于染色携带其假定配体的细胞。该提案的具体目的是利用这种可溶性TCR来进一步研究γ δ TCR的天然配体： 目的1 -研究γ δ TCR天然配体的性质。使用四种不同的γ δ可溶性TCR（sTCR）的标记版本，我们将使用流式细胞术，试图检测胸腺和某些外周器官中驱动不同γ δ T细胞亚群发育或分化的配体。 其次，使用来自TCR可变的子集的sTCR，我们将确定给定子集的不同成员是否可以识别相同的配体。第三，我们将试图确定这些配体是MHC I类或I类通过检查特定突变小鼠品系的细胞。 目的2 -纯化和鉴定γ δ TCR的天然配体。使用两种不同的方法，我们将试图分离和识别的V？6/Vd 1不变TCR，以及典型的多态性V γ 1/Vdelta6.3 TCR。第一种方法涉及筛选cDNA表达克隆的用V γ 6/Vdelta 1或V γ 1/Vdelta6.3 sTCR染色的能力。备用方法涉及产生配体特异性mAb，通过其阻断sTCR/配体结合的能力鉴定，作为亲和纯化配体的试剂。

DESCRIPTION (provided by applicant): Although the gammadelta T cells have been shown to have immunoregulatory effects in a number of experimental systems, their role in immunobiology is still not clear. Surprisingly, it is possible to divide gammadelta T cells into functional subsets based on the Vgamma and/or Vdelta elements they express. Based on such findings, we have hypothesized that certain inducible host molecules act as ligands for gammadelta T cell receptors (TCRs), and are important in eliciting their immunoregulatory effects. However, the nature of the ligands recognized by gammadelta TCRs remains controversial. Furthermore, recent findings have suggested that gammadelta T cell responses can be evoked by molecules other than the TCR, such as chemokine receptors or the Toll-like receptors of the innate immune system, making experiments in which cellular activation is used as a readout for TCR-mediated stimulation difficult to interpret. We believe that the gammadelta T cell receptor itself may hold the key to elucidating the role of the TCR in bringing about gammadelta T cell function. In this proposal, we present preliminary evidence that multimerized versions of mouse soluble gammadelta TCRs can be used like monoclonal antibodies to stain cells bearing their putative ligands. The specific aims of this proposal make use of such soluble TCRs to further investigate the natural ligands of gammadelta TCRs: Aim 1 - to investigate properties of natural ligands for gammadelta TCRs. Using labeled versions of four different gammadelta soluble TCRs (sTCRs), we will use flow cytometry in an attempt to detect the ligands in thymus and certain peripheral organs that drive the development or differentiation of distinct gammadelta T cell subsets. Second, using sTCRs from a subset whose TCRs are variable, we will determine whether different members of a given subset can recognize the same ligand. Third, we will attempt to determine if these ligands are MHC class I or class I-like by examining cells from particular mutant mouse strains. Aim 2 - to purify and identify natural ligands for gammadelta TCRs. Using two different approaches, we will attempt to isolate and identify the natural ligand for the V?6/Vd1 invariant TCR, as well as for a typical polymorphic Vgamma1/Vdelta6.3 TCR. The first approach involves screening cDNA expression clones for their ability to stain with either the Vgamma6/Vdelta1 or Vgamma1/Vdelta6.3 sTCR. The backup approach involves generation of ligand specific mAbs, identified by their ability to block sTCR/ligand binding, as reagents to affinity-purify the ligand.