Indeed, we observed greater IFN-γ production by expanded rat CD8α+ iNKT cells, compared with that of DN and CD4+ iNKT cells, as it has been reported for expanded human CD8α+ iNKT cells [6]. The genetic basis for the low iNKT cell frequencies of F344 rats compared with that in mice remains unclear. However, different expression

levels of CD1d by thymocytes, which would affect iNKT cell selection, can be excluded since thymocytes of both species have nearly identical CD1d levels [13]. The same is true for the recognition signal sequences, which are identical for all AV14 gene segments Trichostatin A ic50 of mice and rats and for AJ18 genes differ in only two nucleotides (Supporting Information Fig. 1E and data not shown). The low frequency of iNKT cells probably explains the lack of a direct identification of thymic F344 iNKT cells. Although frequencies of peripheral and thymic iNKT cells do not necessarily correlate [32], an extrapolation from frequencies of C57BL/6 liver and thymic iNKT cells would predict

for F344 rats a frequency in the range of 0.01% of total thymocytes and 0.08% for CD8β-pregated cells. In humans, gating of vehicle-CD1d stained cells into a “dump” channel has been instrumental for better characterization of very low frequencies (about 0.01%) of (thymic) Lumacaftor supplier iNKT cells [32]. So far, this approach is not feasible since in this study the rat CD1d dimers used are detected with a secondary reagent (fluorochrome-labeled anti-mouse Ig), which binds both vehicle and α-GalCer-loaded dimers. Nevertheless, the identification of canonical iNKT-TCRα sequences among AV14AJ18 PCR products clearly indicates the existence Sorafenib of iNKT cells in the F344 thymus. The differences observed in iNKT cell numbers between F344 and LEW rats in this study are not completely understood, but cannot be accounted to strain-specific differences

in the amino acid sequence of the mature CD1d protein or its expression levels, which are identical for both rat strains [13]. Nonetheless, since the first step necessary for the development of iNKT cells is the rearrangement of the A14 and AJ18 gene segments, the lack of iNKT cells in LEW inbred rats might be the consequence of a very inefficient production of iTCRα chains by thymocytes. Furthermore, LEW and F344 rats do differ in their Tcrb haplotypes. In particular, BV8S2 and BV8S4 are distinct in their CDR2β. Consequently, they could very well affect iNKT-TCR affinity and, thereby, iNKT cell development as well [12].