Functional analysis and the molecular mechanism of CCR5 in the CTL induction.

1. 免疫突触中的趋化因子受体调节抗原识别的敏感性：CCR5主要在静息记忆T细胞上表达，在活化T细胞上表达增强。尽管CCR5被认为是HIV的一种共受体，但有研究表明，CCR5除了趋化性外，还具有体外白细胞活化的共刺激分子功能。为了扩展这一观点，我们通过评估BRET（生物发光共振能量转移）信号，证明了CCR5在TCR刺激后在免疫突触中积累并实际上被激活。此外，一种CCR5拮抗剂TAK-779通过CD3和CD28包被板以及TCR刺激后通过Rapt通过LFA-1内向外信号传导强烈抑制人CD8+T细胞的增殖。我们通过培养CCR5和CXCR3双敲除小鼠，在体内证实了CCR5和CXCR3作为T细胞共刺激分子的功能。综上所述，CD8+T细胞上的CCR5和CXCR3与T细胞受体介导的信号具有共刺激信号或增强信号，通过免疫突触中的Rapl调节LFA-1的激活。这些发现解释了最近报道的CCR5delta32突变患者肾移植完全接受的机制。靶向趋化因子阻断GVHD和选择性诱导GVL/T方法的开发：在急性GVHD模型中，供体CD8T细胞在次级淋巴器官中增殖分化，在隐窝处诱导肠上皮细胞凋亡。令人惊讶的是，针对Fractalkine/MAdCAM-1的中和抗体抑制了肠道损伤。将肿瘤细胞P815 （H-2^d）注射到BDF-1 （H-2^d <bd>）中，并将C57BL/6 （H-2^b）的脾细胞转移到受体小鼠体内，观察到受体存活延长，保留了GVL的作用。因此，我们提出了一种靶向CX3CR-1-Fractalkine和a4b7-MadCAM-1的GVHD治疗方法，以保持GVL/T效应。

1. Chemokine receptors in the immunological synapse regulate the sensitivity of antigen recognition : CCR5 is predominantly expressed on resting memory T cells of which expression is enhanced on activated T cells. Although CCR5 is known as, a coreceptor of HIV, it has been suggested that CCR5 has also a function as a costimulatory molecule for leukocyte activation in vitro other than chemotaxis. To extend this idea, we have shown that CCR5 accumulated and actually was activated in the immunological synapse after TCR stimulation by evaluating BRET (Bioluminescence Resonance Energy Transfer) signals. Furthermore, a CCR5 antagonist, TAK-779 strongly inhibited human CD8+T cell proliferation by CD3 and CD28 coated plates and also LFA-1 inside-out signaling through Rapt after TCR stimulation. We confirmed that CCR5 and CXCR3 function as costimulatory molecules of T cells in vivo by developing double CCR5 and CXCR3 knockout mouse. Taken together, CCR5 and CXCR3 on CD8+T cells have a costimulatory signal or boosting signal with T cell receptor mediated-signal to regulate LFA-1 activation through Rapl in the immunological synapse. These findings explain a mechanism of recent report describing complete acceptance of kidney transplants in recipients with CCR5delta32 mutation.2.Blockade of GVHD by targeting chemokines and development of a way to selectively induce GVL/T : In an acute GVHD model, donor CD8T cells proliferated and differentiated in the secondary lymphoid organs and apoptosis of intestinal epithelium was induced at the crypts. Surprisingly, neutralizing antibodies against Fractalkine/MAdCAM-1 inhibited intestinal injury. When tumor cells P815 (H-2^d) were injected into BDF-1 (H-2^<bd>) and the splenocytes from C57BL/6 (H-2^b) were transferred to recipient mice, elongation of the survival of recipients was observed preserving GVL effect. Thus we propose a therapy for GVHD by targeting CX3CR-1-Fractalkine and a4b7-MadCAM-1 keeping GVL/T effect.