193%) whereas the background staining among TCRβ-positive cells was much lower (0.06%, data not shown).

Last, consistent with iNKT cells being the major PLZF-expressing T-cell population, most PLZF+ αβ T cells expressed NKR-P1A/B at intermediate levels (Fig. 2F). Apart from F344 inbred rats, we also examined the widely used LEW inbred rat strain. The LEW strain is well known for its susceptibility to induced organ-specific autoimmunity, which is not to be found in F344 rats [24-26]. As shown in Figure 2F LEW rats lack the PLZF+ NKR-P1A/B-intermediate T-cell AUY-922 in vitro population found in F344 and show no specific binding of α-GalCer-CD1d dimers (Fig. 2B). Nevertheless, the few cells stained with α-GalCer-CD1d dimers in the liver of LEW rats showed some increase of the DN fraction in comparison with the cells stained with vehicle-CD1d dimers (Fig. 2B). Therefore, it is conceivable that these DN cells are iNKT cells, which may Midostaurin mw be missed due to nonspecific staining of the vehicle control. However, even if it is postulated that all the DN α-GalCer-CD1d-stained cells would be bona fide iNKT cells, their frequency would be a maximum of 0.003% in IHLs (i.e., about 2% of the iNKT cells found in F344 liver). Next, we examined the presence

of iNKT cells in the thymus of both inbred rat strains by flow cytometry and compared it with that of C57BL/6 mice (Fig. 2G). We used both rat and mouse CD1d dimers, but none of them revealed a distinct iNKT-cell population among F344 or LEW thymocytes. In contrast, C57BL/6 thymocytes contained a distinct fraction of α-GalCer-CD1d dimer-stained cells. The analysis of iNKT cells in mouse thymi is commonly carried out after exclusion of HSAhigh (CD24) immature thymocytes. The commercially available anti-rat HSA many mAb does not stain rat thymocytes. Therefore, we analyzed CD8− cells (CD8αβ− in case of rat and CD8αα−/CD8αβ− in case of mouse), stained with anti-TCRβ mAb and CD1d dimers. This approach has been chosen to specifically enrich

the populations among which rat (CD4+, DN, and CD8αα+) or mouse (CD4+, DN) iNKT cells are expected and found to result in an eightfold increase of the relative iNKT-cell frequency among C57BL/6 thymocytes. However, we were still not able to detect a distinct iNKT-cell population among F344 or LEW thymocytes (Fig. 2G). In addition to flow cytometry experiments, we also examined the expression of AV14-containing TCRs by RT-PCR (Supporting Information Fig. 1F). First, we analyzed the expression of TCRα chains comprised by AV14 and AJ18 gene segments. The highest expression levels were found among F344 IHLs, followed by F344 splenocytes, and thymocytes. In contrast, analysis of LEW-derived RNA gave only very weak or no signals. Importantly, the differences between LEW and F344 were already found in thymocytes. AV14-AJ18 rearrangements were also analyzed by sequencing the RT-PCR products.