| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
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.
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