Killer cell immunoglobulin-like receptors (KIRs) are known to modulate the cytotoxic ability of human Natural Killer (NK) cells, as well as a subset of T cells. To date, only a very small number of publications have discussed the role of KIRs on T cells, e.g. CMV-specific CD4+CD28-KIR+ cytotoxic T cells (van Bergen, J., J Immunol. 2004), so we investigated whether CD56+CD3+ NKT cells might also have KIR-positive subsets. Whole human blood as well as magnetically sorted human CD56+CD3+ NKT cells were analyzed for their expression of various KIR molecules using a novel panel of fluorochrome-conjugated, anti-KIR monoclonal antibodies (CD158a/h (KIR2DL1/DS1), CD158b (KIR2DL2), CD158e (KIR3DL1), CD158i (KIR2DS4), KIR2D; Miltenyi Biotec). KIR-positive CD56+CD3+ NKT cells were identified in every donor tested. Donors possessing NK cells of a specific KIR phenotype also possessed CD56+CD3+ NKT cells with the same KIR phenotype. KIRs were also expressed in a clonal fashion on CD56+CD3+ NKT cells, similarly to NK cells. The investigated KIRs were also shown to be expressed on unseparated NK and CD56+CD3+ NKT cells from whole blood. In addition, the ratio between KIR expression on NK and CD56+CD3+ NKT cells was calculated for each donor analyzed. The results show that there is no correlation between the frequencies of KIR expression on NK cells with that of CD56+CD3+ NKT cells. For example, the expression of CD158a/h in one donor was found to be the highest of all CD56+CD3+ NKT cells analyzed, but the lowest of all NK cells by comparison to the other donors tested. For all KIR phenotypes analyzed, the frequency of KIR+ NK cells was higher than the frequency of KIR+ CD56+CD3+ NKT cells in all samples (range: 1.1 to 25.3-fold higher). Interestingly, the frequency of KIR+ NK cells versus KIR+ CD56+CD3+ NKT cells differs significantly between donors: in one donor the frequency of KIR expression is between 7.3 to 25.3-fold higher in NK cells for multiple KIR phenotypes, while this range is more narrow in other donors (2.0–5.4-fold higher). The frequencies of CD56+CD3+ NKT cell subsets staining positive for particular KIRs differ significantly between donors, e.g. for CD158b, the number of positive CD56+CD3+ NKT cells fall within a range of 4.8% to 43.3%. For CD56+CD3+ NKT cells sorted with MACS® Technology, a similarly wide-ranging distribution of CD158b (KIR2DL2) expression was found (0.85%–5.82%), though at a lower level. Further research will be required to explore these differences as they may point to different mechanisms of KIR regulation. The identification of KIR-positive CD56+CD3+ NKT cells may also provide an opportunity for their use for functional KIR studies instead of NK cell clones, as the cloning of CD56+CD3+ NKT cells may prove easier (i.e. using standard T cell cloning methods) than that of NK cells.
Disclosure:Employment: All authors employed at Miltenyi Biotec GmbH.