Magnetic Separation of Melanoma-Specific Cytotoxic T Lymphocytes from a Vaccinated Melanoma Patient's Blood Using MHC/Peptide Complex-Conjugated Bacterial Magnetic Particles

Target antigen-specific cytotoxic T lymphocytes (CTLs) play a key role in anticancer and antivirus immunity in the body, and purification of CTLs from heterogeneous immune cells is desired for an efficient cancer immunotherapy and fundamental research. Herein, a novel magnetic nanoparticle conjugated with major histocompatibility complex (MHC)/peptide complexes was developed for the magnetic separation of melanoma-specific CTLs. To conjugate biotinylated MHC/peptide complexes on nanosized bacterial magnetic particles (BacMPs), which were synthesized intracellularly by magnetotactic bacteria, phased modification of biotin and streptavidin onto BacMPs was investigated. When biotin was modified on BacMPs, a polyethylene oxide (PEO)-linker contributed to the maintenance of the high dispersion properties of BacMPs. Furthermore, nonspecific binding of BacMPs to cell surface was prevented by controlling the level of streptavidin bound on BacMPs and through PEO blocking of the empty streptavidin sites on BacMPs. Finally, single-step magnetic separation of melanoma-specific CTLs was demonstrated using developed MHC/MAGE-1 A24 peptide complex-conjugated BacMPs. Melanoma-reactive cells and melanoma-specific CTLs were successfully separated from stimulated peripheral blood mononuclear cells derived from a vaccinated melanoma patient with 93.1% and 87.7% purity, respectively, and specificity of antigen recognition and cytokine secretion from separated CTLs were confirmed. The potential of MHC/peptide complex-conjugated BacMPs was indicated for efficient separation of antigen-specific CTLs in cancer immunotherapy and fundamental research.