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Ankur Gupta, Baoying Liu, William R Tucker, Denise Cunningham, Hirofumi Hanoaka, Rafael Villasmil, Hisataka Kobayashi, Peter L Choyke, Robert N Fariss, Robert B Nussenblatt; Development of an Imaging Approach for Inflammatory and Neoplastic Ocular Lesions by Establishing Specific Identification of T-cells In Vitro with Indocyanine Green Fluorescence. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1614.
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© ARVO (1962-2015); The Authors (2016-present)
To demonstrate that indocyanine green (ICG) dye can be used to label immune cell subtypes in vitro and in vivo, we utilized a method wherein ICG dye is conjugated to an antibody of choice and thereafter translocated intracellularly. The proposed mechanism entails the binding of a quenched ICG-antibody “complex” to a cell-specific surface marker. ICG fluorescence is then dequenched following endocytosis-mediated dissociation of the complex, thereby preventing extracellular background fluorescence. Since many ophthalmic imaging tools can already detect ICG (excitation/emission in near-infrared spectrum), we leveraged this conjugation method to specifically identify T-cells as an initial proof-of-concept.
Anti-CD25 antibody specific for IL-2 receptor α (basiliximab), a marker of activated T-cells, was incubated with ICG-sulfo-OSu in Na2HPO4 (pH 8.5) at room temperature to generate a quenched complex. The Heidelberg Spectralis’ “infrared” setting validated quenched and dequenched states. Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donor blood and stimulated for 3 days in the presence of concavalin A (conA). Quenched ICG-basiliximab complex (100 ug/mL) was added for 8 hours before ICG fluorescence detection using a 700 nm long-pass filter during flow cytometry analysis of CD4+CD25+ T-cells.
CD4+ T-cells stimulated with conA showed increased expression of CD25 (76%) compared to unstimulated CD4+ T cells (9%). Similarly, stimulated CD4+ T-cells showed 21 times more ICG fluorescence signal than unstimulated cells. These relative differences were observed for 48-hours after conA stimulation. CD4+ T-cells not incubated with the complex demonstrated low autofluorescence in the ICG spectral range; they emitted 146 times less signal than stimulated T-cells which had been incubated with the complex.
We demonstrated that an ICG-basiliximab complex can selectively identify stimulated human T-cells. This finding provides the foundation for a potential noninvasive, clinical approach to locate and characterize specific immune cell populations in vitro and in vivo with the goal of providing practitioners a tool for diagnosis and management of ocular neoplastic and inflammatory lesions before escalation to vitrectomy and biopsy.
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