Research of synapse formation between nervous system and regulatory T cells in melanoma lesions

我们研究了Treg细胞和胸腺外γδ T细胞是否与神经系统发生交叉反应，这两种细胞都被报道在肿瘤的形成中起免疫抑制作用。除了Treg和γδ T细胞的已知分子外，我们还鉴定了三种细胞表面标志物，称为R1、R68和Y 5。用抗R1、R68和Y 5单克隆抗体与神经元相关抗体（GFAP，bIII tubulin）联合免疫组化技术，对黑色素瘤和黑色素瘤相关淋巴结组织进行染色，并通过CD 4 + CD 25 + T细胞（鼠和人）和γδT细胞（鼠）的cDNA差减法鉴定R1、R68和Y 5。扩增R1、R68和Y 5蛋白的高抗原性DNA序列，插入pDisplay载体，转染BALB 3 T3或COS细胞系（人基因转化体：3 T3 hR 1、3 T3 hR 68和3 T3 hY 5;鼠基因转化体：COSmR 1、COSmR 68和COSmY 5）。用3 T3 hR 1、3 T3 hR 68和3 T3 hY 5免疫BALB/c小鼠，用COSmR 1、COSmR 68和COSmY 5免疫大鼠，获得多克隆抗体。将每种转化体免疫的小鼠或大鼠的脾细胞通过融合骨髓瘤（P3-X63）进行mAb制备，从B16接种后1、2、3和4周获得的B6小鼠中分离脾、黑素瘤病变和黑素瘤相关淋巴结。用R1、R68或Y 5特异性的多克隆Ab或mAb以及GFAP或βIII微管蛋白特异性的神经元相关Ab对脾脏、黑素瘤病变和黑素瘤相关淋巴结以及人黑素瘤病变的冷冻切片进行双重染色。虽然在脾脏纤维囊和淋巴结皮质的部位检测到少量与神经细胞相互作用的抑制细胞，但在黑色素瘤病变的检查中没有显示出突触相互作用。需要进一步利用变态反应或自身免疫模型研究免疫抑制细胞与神经系统的突触相互作用。

We examined whether Tregcellsandextrathymicγδ T cells, both of which have been reported to play as an immune suppressor in the formation of tumors, crosstalk with nervous system. In addition to the known molecules of Treg and γδ T cells, we identified three cell surface markers termed as R1, R68 and Y5. Sections of melanoma and melanoma-related lymph nodes were stained with polyclonal Abs and mAbs specific for R1, R68 and Y5 in combination with neuron-related Ab (GFAP, bIII tubulin) to investigate interaction of immune suppressive system with nervous system.R1, R68 and Y5 were identified by subtraction method of cDNAs from CD4+CD25+ T cells (murine and human) and γδT cells (murine). The DNA sequences with high antigenicity of R1, R68 and Y5 proteins were amplified and inserted into pDisplay vector, and then transfected with BALB3T3 or COS cell line (transformats for human genes: 3T3hR1, 3T3hR68 and 3T3hY5; transformats for murine genes: COSmR1, COSmR68, COSmY5). Polyclonal Abs were obtained by immunization BALB/c mice with 3T3hR1, 3T3hR68 and 3T3hY5, and rats with COSmR1, COSmR68 and COSmY5. Splenocytes of mice or rat immunized with each transformat were subjected to mAb preparation by fusing myeloma (P3-X63).Spleen, melanoma lesions and melanoma-related lymph nodes were separated from B6 mice obtained at 1, 2, 3 and 4 weeks after B16 inoculation. Frozen sections of spleen, melanoma lesions and melanoma-related lymph nodes, and human melanoma lesions were dual stained with polyclonal Abs or mAbs specific for R1, R68 or Y5 and neuron-related Ab specific for GFAP or βIII tubulin. While low numbers of suppressor cells interacting with nervous cells were detected at the sites of fibrous capsules of spleens and cortexes of lymph nodes, no synaptic interaction was shown in examination of melanoma lesions. Further studies using allergy or autoimmune models were necessary to demonstrate the synaptic interaction of immune suppressor cells with nervous system.