Introduction of Differential KIR Activation Mediated Patters of NK Cell Cytotoxicity Against Myeloma

Natural Killer cells (NK cells) are part of the innate immune system. These cells have the ability to recognise and kill malignant cells, like myeloma cells. NK cell activation is tightly regulated by different activating or inhibiting receptors. Killer immunoglobulin like receptors (KIR, CD158) are a family of receptors which have activating as well as inhibitory function. KIR molecules are thought to recognize the HLA-C molecules, which then lead to NK cell signal transduction. Our knowledge about KIR expression and impact on tumor cell control has developed over the last years, but still our understanding of how the receptors are activated in multiple myeloma is limited. We therefore, investigated three different model systems for NK cell alloreactivity: 1) HLA-C/ HLA-C interaction model (KIR-Ligand model), 2) HLA-C/ KIR receptor interaction model, and 3) impact of donor KIR haplotype.

Three different myeloma cell lines (KMS12BM [C1/C1], MOLP8 [C1/C2] and RPMI8266 [C2/C1]) and a NK cell line (NKL) were cultured under standard conditions. NKL cells were transfected with human KIR2DL1 and KIR2DL3 alleles, respectively. For RNAi experiments two siRNA and two control siRNA were used. NK cells from healthy donors were isolated by magnetic end labeling. Enriched NK cells were HLA-typed for expression of HLA-C molecules, KIR receptor expression or KIR haplotype. Thereafter, NK cells were transiently transfected with the siRNA or control siRNA against the KIR2DL1 or KIR2DL3 receptors for up to 48 h. Functional analysis of the NK cell cytotoxicity was measured using a LDH release assay, based on their killing ability against the three fore mentioned myeloma cell lines.

Using NK cells that have been HLA-C genotyped as HLA-C1/C1, we observed a rescue of C1/C1 positive meyeloma cell lines in contrast to the C2/C1 myeloma cell line (12% cytotoxicity vs. 38% and 45% cytotoxicity, respectively, p <0.05). KIR receptors, by recognition of HLA-C molecules, are believed to reduce NK cell cytotoxicity. We used NK cells from healthy donors, which express KIR2DL1 (recognizing C2) to evaluate whether receptors` RNAi knockdown could increase the ability of NK cells to lyse target cells. Knockdown of KIR2DL1 in NK cells results in a highly efficient killing of a C2/C2 target cell line (RNAi treated NK cells showed a cytotoxicity up to 70%, p<0.05). To evaluate whether KIR receptors are major inhibitory molecules, we used the NK cell line (wt NKL, a non expressor of KIR receptors) that exhibited high cytotoxic potential against all tested target cell lines (>65% cytotoxicity). This NK cell line was transfected with KIR2DL1 or KIR2DL3 (recognizing C2, or C1). C2 positive myeloma cell lines were significantly rescued when exposed to KIR2DL1 transfected NK cells (wt NKL 72% vs. KIR2DL1 NKL 29%, p <0.05). Similar results were obtained when C1 positive target cell line was co- cultured with KIR2DL3 transfected NK cells. RNAi mediated silencing of KIR overexpression restores protection observed. As last model for the regulation of NK cell cytotoxicity we used KIR haplotyped NK cells and co-cultured them with a C1/C2 myeloma cell line. NK cells with an AA KIR haplotype showed the lowest killing capacity against the myeloma cell line, while BB KIR haplotype had higher killing potential. (46% vs 59%, p=0,005).

We have shown that NK cell cytotoxicity is regulated by the HLA-C expression on the target cell as well as the expression of a specific KIR receptor and various KIR haplotypes. All tested models showed significant killing effect on myeloma cells. These data underline the importance of KIR receptor modulation on NK cell alloreactivity against myeloma cells.

No relevant conflicts of interest to declare.