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H. Nishihori, J.–H. Sohn, J.M. C. Cruz, H.J. Kaplan, N.S. Bora; Characterization of T Cell Function After 64Cu Labeling . Invest. Ophthalmol. Vis. Sci. 2004;45(13):620.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In microPET imaging studies, we use 64Cu–PTSM [64Cu pyruvaldehyde bis (N4–methylthiosemicarbazone] and 64Cu–DOTA [64Cu–1,4,7,10–tetraazacyclododecane–N, N’,N’’,N’’’–tetraacetic acid] to monitor the migration and localization of CD4+ T cells in normal Lewis rats. This study was undertaken to investigate the effect of 64Cu–PTSM or 64Cu–DOTA on the viability and function of rat CD4+ T cells. Methods: The spleen was harvested from naïve Lewis rats, a single–cell suspension of spleen cells was prepared in RPMI–1640 medium and CD4 +T cells were purified by negative selection using immuno–columns. Purified CD4+ T cells were cultured with 50 and 100 uCi of 64Cu–PTSM or 64Cu–DOTA. Cells were harvested from each group at 6, 18, 24 and 48 hours and stained with Trypan Blue for cell viability. Cells were also stained with FITC conjugated monoclonal antibody against rat CD25 or CD134 and analyzed by flow cytometry. T cell proliferation assay was used to analyze the ability of 64Cu–PTSM or 64Cu–DOTA labeled cells to proliferate in response to Concanavalin A. Results: The 64Cu–PTSM or 64Cu–DOTA labeling procedure did not interfere with the viability of naïve CD4+ T cells within 18 hours when 50µCi of label was used per one million T–cells. However, when 100 µCi of 64Cu–PTSM/64Cu–DOTA was used, the cell viability decreased to 50% at 18 h. Labeling with 64Cu–PTSM or 64Cu–DOTA did not affect the expression of activation markers – CD25 and CD134 – at 6, 18 and 24 hours. However, when CD4+ T cells were incubated with 64Cu–PTSM or 64Cu–DOTA for 48h, the expression of CD25 and CD134 was significantly reduced. 64Cu–PTSM or 64Cu–DOTA labeled naïve CD4+ T cells retained their ability to proliferate polyclonally in response to Concanavalin A. Conclusions: CD4 + T cells labeled with < 50µCi 64Cu–PTSM and 64Cu–DOTA are viable and retain their functional activity for 24 hours after labeling. We conclude that labeling with 64Cu–PTSM or 64Cu–DOTA serves as a useful tool to monitor the migration and localization of T cells in vivo.
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