5b). However, by FACS analysis, CD8α− NK cells exhibited only a modest up-regulation of IFN-γ production following co-culture with target cells (Fig. 4c). The rapidity

of IFN-γ gene transcription is consistent with reports showing that unlike T cells, which exhibit a delay in T-cell activation and function, NK cells are designed for a very rapid response. In the murine system, IFN-γ production is observed after only 4 hr of cytokine stimulation.53 The difference observed here by flow cytometry in the two NK subpopulations suggests a difference in kinetics of IFN-γ protein expression that will require further investigation. It is important to mention that although a significant proportion of mDCs is present in the

enriched CD8α− NK cells selleck chemical used for the reverse transcription-PCR assays, mDCs do not up-regulate IFN-γ production even in the presence of strong chemical stimulation such as PMA and ionomycin.40 In terms of cytotoxic potential, both NK cell subsets were positive for perforin and granzyme B expression, although to different degrees (Fig. 2) and both exhibited transcription of perforin and granzyme B mRNA (Fig. 5b). Vadimezan The increased transcription observed between un-treated and cytokine-treated cells, however, was very low. Both perforin mRNA and protein have been reported to be constitutively present in human NK cells and other types of CTLs, with gene transcription only up-regulated under long-term stimulating conditions.54 Therefore, it appears that perforin mRNA transcripts were constitutively present in both

Urease the CD8α− and CD8α+ NK cells of macaques, but were absent from B cells. Moreover, the stimulation approach used in the present study did not further increase perforin gene transcription. With regard to granzyme B, no increase in transcription relative to that of B cells was observed (Fig. 5b). However, human B cells can produce granzyme B in response to cytokine stimulation,55 which may be the case for macaque B cells as well. Overall, NK cells rely on pre-formed granules of perforin and granzymes to respond rapidly and exert cytotoxic function.56,57 The co-expression of these two cytotoxic proteins in approximately 10% of CD8α− NK cells (Fig. 2c) provides the potential for cytotoxic activity. In fact, the CD8α− NK cells exhibited reduced, albeit significant, killing capacity when compared with similarly purified CD8α+ NK cells, both by direct lysis of cells lacking MHC class I expression (Fig. 5c) and by antibody-dependent killing (Fig. 5e). This decreased capacity to mediate cytotoxic function probably reflects the relatively large proportion of mDCs present in the enriched CD8α− NK cell population, which significantly alters the E : T ratios.