Format

Send to

Choose Destination
See comment in PubMed Commons below

Cy5.5-CGRRRQRRKKRG-Labeled T lymphocytes.

Authors

Zhang H.

Source

Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.
2008 Jul 10 [updated 2008 Aug 27].

Excerpt

T cells are responsible for regulating immune responses and maintaining immune tolerance via recognition of peptide antigens that are bound to human leukocytes (1). Some T cells possess autoimmunity or self-tolerance through recognition of self-antigens. Loss of this required self-tolerance can result in an autoimmune disorder. For instance, experimental allergic encephalomyelitis (EAE) is one of immune-mediated diseases in which immune cells become reactive against myelins, which leads to the destruction of myelin sheets (2). EAE can be induced in rodents by adoptive transfer of CD4+ T cells specific to myelin basic protein (MBP), an autoantigen of myelin (3). As an animal model for the inflammatory disease, EAE can reproduce many clinical neuropathological and immunological aspects of multiple sclerosis (MS) and thus has been widely used in therapeutic development for MS (4). The evolution of inflammatory lesions in EAE involves several steps (1, 3). After intravenous administration, the injected MBP-specific T cells cross the blood–brain barrier to recognize the T cell antigen located on perivascular microglia. This antigen-specific interaction produces a plethora of inflammatory cytokines and mediators, leading to amplification of the inflammatory reaction. The blood–brain barrier then opens to allow antigen-independent recruitment of various mononuclear inflammatory cells into the central nervous system, including additional T cells, macrophages, and granulocytes. Consequently, the inflammatory lesions evolve into severe neurological dysfunctions such as ascending paraparesis and paralysis. Because the activated T cells are involved in the entire process, the trafficking of the activated MBP-specific T cells in EAE can reflect their immune activity in every evolutionary phase (2). Labeling T cells with imaging probes will allow non-invasive tracking of the migration of T cells in vivo. The imaging probes can be internalized into cells with the use of cell-penetrating peptides (CPP) as vector/nuclear delivery vehicles of conjugated cargo (5). In general, CPP comprise a protein-transduction domain formed by small peptides (<20 amino acids) for cell membrane translocation. A commonly used CPP is a peptide truncated from the 86-mer transactivating transcriptional activator (Tat) protein in human immunodeficiency virus type 1 (HIV) (6). In particular, Tat(47-57) is widely used in cellular delivery of peptides, proteins, genetic material, antibodies, nanoparticles, and liposomes (5). Tat(47-57) contains an α-helical structure with a charged face formed by six arginine and two lysine residues (6). Tat-mediated internalization consists of multiple steps: the binding of Tat to the cell surface, stimulation of macropinocytotic uptake of Tat and transfer into macropinosomes, and finally endosomal escape into the cytoplasm (7). A T lymphocyte is labeled with Cy5.5-Cys-Gly-Arg-Arg-Arg-Gln-Arg-Arg-Lys-Lys-Arg-Gly (CGRRRQRRKKRG) (Cy5.5-Tat-T cell) for optical imaging (2). This agent contains a near-infrared fluorescence (NIRF) dye shuttled across the cell membrane of T cells via Tat(48-57). The fluorescence probe Cy5.5 is a cyanine dye consisting of two quaternized heteroaromatic bases (A and A’) joined by a polymethine chain with five carbons (8), and it is bound to cysteine-terminated Tat(48-57) via a maleimide group as a spacer (2). Cy5.5 has a delocalized positive charge in its chromophore and possesses high quantum yield (0.22 at 678 nm), good chemical stability, easy conjugation, and high sensitivity (mole extinction coefficient ~250,000 mol/cm) (9, 10). The excitation/emission wavelength is 674/692 nm for Cy5.5, where hemoglobin and water have their lowest absorption coefficient. The labeled cargo Cy5.5-Tat is translocated through the plasma membrane of cells by an energy-dependent process involving endocytosis (2). The produced Cy5.5-Tat-T cells can be adoptively transferred to EAE rats for visualization and quantification of T cells activity in the early inflammation stages of EAE.

PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons
    Write to the Help Desk