Also, we confirmed that CD161 is not expressed on NK92 cells using the DX12 anti-CD161 monoclonal antibody (Fig. 1B). CD161 expression on K562-CD161 clones was confirmed via flow cytometric analysis using the DX12 anti-CD161 monoclonal antibody (Fig. 2A,B). Compared to K562 transfected with the empty pCI-neo vector, K562-CD161 clone #3 (Fig. 2A) exhibits 51.4% surface CD161 expression, whereas K562-CD161 clone #12 (Fig. 2B) exhibits 66.9% surface CD161 expression. Flow cytometry confirms that K562 (data not shown) and NK92 do not naturally express CD161. When NK92 cells that were rested overnight without IL-2 and K562-CD161/-pCI-neo cells https://www.selleckchem.com/products/rxdx-106-cep-40783.html are co-incubated overnight in a

1:1 ratio, NK92 incubated with K562-CD161 consistently produce significantly greater amounts of IFN-γ than NK92 incubated with K562-pCI-neo. Additionally, two separate K562-CD161 clones exhibiting high (Fig. 2B) and low (Fig. 2A) levels of CD161 expression were employed as target cells. As we predicted, the K562-CD161 low expressing cells were associated with reduced IFN-γ production compared to K562-CD161 high expressing Fluorouracil order cells (Fig. 2C). To confirm this increase in IFN-γ production was associated with CD161 ligation

of LLT1 on NK92, we blocked CD161 on K562-CD161 with DX12 anti-CD161 monoclonal antibody and repeated our IFN-γ assay. Blocking K562-CD161 in this manner reduces associated IFN-γ production to levels comparable to that observed with K562-pCI-neo (data not shown). Taken together, these data indicate that NK92 express LLT1, and LLT1 is functional in the same manner as previously reported on the YT cell line and freshly isolated NK cells. With this in mind, we proceeded to analyse LLT1 signalling pathways using this NK92:K562-CD161 LLT1 ligation system. Except where indicated, in all instances K562-CD161 ALOX15 refers to the CD161 high expressing K562-CD161 clone. NK92 (rested overnight without IL-2):K562-CD161/-pCI-neo cells were co-incubated overnight and harvested at various times for RT-PCR analysis. IFN-γ mRNA was evaluated by PCR, and GAPDH was amplified to confirm

an equal amount of template was used for each reaction. We observed no significant difference in IFN-γ mRNA over the various time points in either the presence or absence of CD161 ligation (Fig. 3A,B). The calibrated density of IFN-γ mRNA divided by median calibrated density of GAPDH mRNA for NK92 incubated with K562-CD161 and K562-pCI-neo clearly demonstrates there is no increase or decrease in IFN-γ mRNA expression associated with LLT1 ligation and IFN-γ production (Fig. 3B). This suggests that IFN-γ production associated with CD161 ligation does not directly stimulate IFN-γ transcription. IFN-γ mRNA detected came from NK92 as K562 does not produce IFN-γ. This was confirmed by analysing K562 cDNA alone for IFN-γ mRNA (data not shown).