Comparison of 6B11 mAb and α-GalCer-loaded CD1d dextramers for detection of iNKT cells by flow cytometry.

Invariant natural killer T (iNKT) cells are a small population of thymus-derived T cells that are restricted by non-classical MHC class I molecule CD1d and express an evolutionary conserved TCR with an invariant α-chain. The frequency of iNKT cells in peripheral blood is very low, thus, accurate methods to identify and enumerate iNKT cells are needed. The aim of the study was to compare 6B11 mAb or α-GalCer-loaded CD1d dextramers usage in iNKT cell detection. The frequency of CD3+ CD56+ lymphocytes is much higher, with statistical significance (p<0,001), than real iNKT cells detected by 6B11 mAb or α-GalCer-loaded CD1d dextramers. The frequency of iNKT cells, recognized by 6B11 mAb or α-GalCer-loaded CD1d dextramers, was in a similar range. Nonetheless, when we compared whether 6B11+ and α-GalCer-loaded CD1d dextramers+ are the same populations, it turned out that by this approach we were able to identify three distinct subsets of iNKT cells: i) 6B11+ /α-GalCer-loaded dextramer- cells, ii) 6B11+ /α-GalCer-loaded dextramer+ cells, and iii) 6B11- /α-GalCer-loaded dextramer+ . Thus, although 6B11 mAb and α-GalCer-loaded dextramers may identify not exactly the same cells, application of these methods seems to give similar results of iNKT cell frequency in peripheral blood. It seems that both approaches for iNKT detection can be used for precise identification of these cells. Moreover, our results indicate that CD3+ CD56+ lymphocytes are a heterogeneous population of T cells, expressing activation markers of both NK and T lymphocytes, yet with not well characterized properties.