Perforin-2 dependent intracellular bacterial killing by skin γδ T cells

Abstract The skin serves as a first line of defense against physical, chemical insults and a vast array of potentially pathogenic microorganisms. Gamma delta (γδ) T cells are a subset of unconventional T cells residing in the skin that play a critical role in protection against bacterial infections, malignancies and in maintaining keratinocyte homeostasis. To characterize the mechanisms governing γδ T cell cytotoxic responses against intracellular pathogens, such as Methicillin Resistant Staphylococcus Aureus(MRSA), we investigated the role of Perforin-2 (P-2), a novel member of the pore-forming family of innate immune proteins. Skin cells (epidermis and dermis) were separated enzymatically from C57/Bl6 WT and P2 deficient (−/−) mouse dorsal skin using collagenase D. Cells were stained with anti-TCR γδ antibodies, sorted, infected with MRSA and used for intracellular gentamicin protection assays or electron microscopy analysis. γδ T cell lysates were plated and colony forming unit (CFU) count was determined. Frozen sections of dorsal mouse skin were used for immunofluorescence analysis. We found that P-2 deficient γδ T cells infected intracellularly with MRSA are unable to kill MRSA. Using electron microscopy we confirmed that MRSA resides within γδ T cells. In P2 −/− mice we observed a lower number of γδ T cells in the skin at steady state. Upon transcutaneous MRSA infection (109 CLP 153), in the absence of P2 (−/−), all mice die. We observed that P2 (−/−) γδ T cells migrate more rapidly in the blood than γδ T cells expressing P2. In summary, we showed for the first time that skin γδ T cells can directly kill an intracellular pathogen (MRSA) through a P-2 dependent mechanism. Our data confirms that Perforin-2 is a novel γδ T cell effector molecule in the skin.