The transcriptional factor X-box-binding protein 1 (XBP1), a critical gene in the unfolded protein response (UPR), has recently been demonstrated to be essential for the development, survival and function of B cells, T cells, macrophages and dendritic cells. Here it has been demonstrated that it may also be important for NK cell effector function. Following activation of the UPR, xbp1 mRNA is converted to its active form (XBP1s) through splicing mediated by IRE1. Splicing of xbp1 mRNA was observed during NK cell-mediated direct cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC), indicating that XBP1s is being activated. Interestingly, using the SNK10 cell line, microarray data indicated that splicing of xbp1 during NK cell-mediated cytotoxicity was occurring independent of a canonical UPR and resulted in increased expression of only a handful of known XBP1s downstream targets, suggesting it may be acting under novel conditions independent to a canonical UPR.
To determine if splicing of xbp1 was required for NK cell effector function, splicing was inhibited using chemical means. Inhibition of IRE1-mediated splicing of xbp1 significantly impaired the ability of the SNK10 cell line to kill target cells by both ADCC and direct cytotoxicity. Furthermore, IRE1 inhibited SNK10 cells had reduced CD107a surface expression, suggesting a defect in cellular de-granulation. While addition of the inhibitor did result in a trending decrease in the ability of pNK cells to lyse target cells, this failed to reach statistical significance.
In addition to target cell lysis, following stimulation NK cells also secrete cytokines that help shape the immune response. IFNγ is the main cytokine produced by NK cells. It was demonstrated that inhibition of IRE1-mediated splicing of xbp1 greatly impaired the production and secretion of IFNγ by NK cells during both direct cytotoxicity and ADCC. Together these results indicate that IRE1-mediated splicing of XBP1s is likely involved in NK cell effector function.
To better understand the global impact that XBP1s may have on NK cell effector function, gene expression microarray analysis was used to identify genes showing differential expression following IRE1 inhibition in stimulated SNK10 cells. Supporting the hypothesis that XBP1s was acting independent to a canonical UPR, no changes were observed in the expression of genes previously described to be involved in an UPR following inhibition of IRE1-mediated splicing of xbp1 during either direct cytotoxicity or ADCC. Furthermore, there was no change in the expression of downstream targets of XBP1s, suggesting that it was acting in a novel role. To identify what this role could be pathway analysis software was used to determine the main pathways impacted following IRE1 inhibition during direct cytotoxicity and ADCC. Using this software, the gene set cytokine-cytokine receptor interaction was predicted to be enriched with genes showing significantly reduced expression following inhibition of IRE1-mediated splicing of xbp1 during either direct cytotoxicity or ADCC. Closer examination of genes in this gene set revealed select cytokines and cytokine receptors that were impacted by a loss of XBP1s, including those involved in interleukin signalling as well as cytokines involved in death-receptor mediated apoptosis of target cells. Collectively these results suggest a novel role for XBP1s in the expression of cytokines and cytokine receptors during NK cell-mediated cytotoxicity.
Further to its role in the function of immune cells, XBP1s has also been demonstrated to be important for the development of DCs and is spliced early during T cells and B cells development. Here it has been demonstrated that xbp1 is spliced in both immature NK (iNK, CD117+ CD94-) and mature NK (mNK, CD117lo/+ CD94+) cells isolated from human tonsil tissue. However, this was found to be independent to a canonical UPR and in the absence of increased expression of downstream targets. Furthermore, microarray analysis was also used to profile genes showing differential expression following NK cell maturation and to identify molecular regulators that influence these changes. Following maturation an increase in genes encoding proteins involved in NK cell-mediated cytotoxicity, including NK cell receptors, cytotoxic granules and cytokines, was observed. In addition, upstream regulator analysis was used to identify four transcription factors, T-Bet, GFI1, PIAS4 and HDAC5, which were predicted to be involved in the maturation of iNK cells to mNK cells.
An understanding of the molecular mechanisms that influence NK cell development and function is lacking. Here XBP1s has been identified as a transcription factor that is likely involved in NK cell effector function. Furthermore, it has been demonstrated that in NK cells, XBP1s is likely acting independent to a canonical UPR and is likely involved in the expression of several cytokines during direct cytotoxicity or ADCC. Finally, xbp1 was also demonstrated to be spliced in immature and mature NK cells, however again this appears to be independent of a canonical UPR. Collectively these results provide an important insight into how NK cells develop and function, an essential step in understanding possible aspects leading to NK cell dysfunction and disease.