The unexpected and differential effect of cyclosporin A on CD56 Bright and CD56 Dim NK cell expansion, phenotype, function and development from progenitor cells

Abstract

Following allogeneic hematopoietic cell transplantation, NK cells play important roles in hematopoietic cell engraftment, anti-viral responses, graft vs. host disease (GVHD) and graft vs. leukemia (GVL) reactions. Calcineurin inhibitors, such as cyclosporine A (CsA), are frequently administered to prevent or treat GVHD. It is generally considered that these immune suppressive agents inhibit GVL reactions. To date, little is known about the impact of these immune suppressants on NK cell function. To investigate this, NK cells were isolated from normal donors by negative selection and cultured with IL-2 (100 U/ml), IL-15 (10 ng/ml) and either physiological levels of CsA (1μg/ml) or vehicle control. Under these conditions, we consistently found that CsA treated cultures showed a reduction in NK cell expansion at one week (4.88 vs. 1.87 fold expansion, n=10). The phenotype of CsA treated NK cells was markedly different than controls. More specifically, after 7–10 days of culture with CsA there were significantly more CD56 bright CD16 − cells and significantly less CD56 dim CD16 + cells compared to controls (p dim CD16 + and CD56 bright CD16 − cell division. There was no difference in the proliferation of CD56 bright CD16 − cells between the CsA and control treated cultures. In contrast, the CsA treated CD56 dim CD16 + cells had fewer cell divisions, demonstrating that CsA selectively inhibits CD56 dim CD16 + cell proliferation. These results were confirmed by determining the fold expansion of freshly isolated, FACS sorted CD56 dim CD16 + and CD56 bright CD16 − populations cultured with CsA or vehicle control. To investigate the cytotoxicity of CsA treated NK cells, we performed killing assays using K562 and Raji cells as targets. Surprisingly, we consistently found higher cytotoxicity in CsA treated NK cells compared to controls (K562, p + Lin − CD38 − ) using an in vitro differentiation system. Briefly, progenitor cells were cultured on a murine feeder cell line (AFT-024) for 42 days in the presence of IL-3, IL-7, IL-15, SCF and FLT3L +/− CsA. We found that CsA treated cultures had less KIR expressing cells compared to controls. Collectively these results show that physiological levels of CsA inhibit CD56 dim CD16 + cell growth and result in a population of NK cells that have less KIR receptor expression, higher cytotoxicity and more cytokine secretion. These findings may have important implications for both GVHD and GVL following hematopoietic cell transplantation.