Cyclosporin Influences NK Cell Expansion, Activating and Inhibitory Receptor Expression and Cytokine Synthesis.
Authors: Wang Hongbo , Lee Alisa , Verneris Michael R. 
Published: October 2019

Abstract
Mouse models demonstrate that natural killer (NK) cells play an important role after allogeneic BMT by mediating GVL effects. Human haploidentical transplantation extends these observations since KIR receptor mismatch between donor and recipient is associated with improved DFS for AML patients. In contrast to the above observations, most patients undergoing hematopoietic cell transplantation receive GVHD prophylaxis with immune suppressive drugs, such as cyclosporin A (CsA). Little is known about the effects of CsA on NK cells and to investigate this, mature peripheral blood NK cells were cultured in IL-2 (1,000 U/ml) with either CsA (1 μg/ml or 10 μg/ml) or vehicle (EtOH) for 7 days. Under these conditions, CsA resulted in a significant inhibition in NK cell (CD3−CD56+) expansion (p<0.05). Cell cycle analysis showed that compared to EtOH, more CsA treated cells were in G1, and less cells were in G2-M phase, demonstrating that CsA reduces the number of NK cells in cycle. Since NK cells recognize malignant targets using both activating and inhibitory cell surface receptors, we used FACS to investigate the expression of KIR receptors (CD158a, CD158b and NKB1) and activating receptors (NKG2D, NKp30, NKp44 and NKp46) on cells cultured with and without CsA. CsA induced changes in the intensity of one or more of the above receptors for all donors tested (n=12). When analyzed in aggregate, we found that compared to EtOH control, NK cells cultured in CsA frequently had reduced expression of KIR receptors (66.7% for CD158a, 50% for CD158b and 33.3% for NKB1) and rarely increased KIR expression (0% for CD158a, 16.6% for CD158b and 0% for NKB1). In contrast, when cells were cultured in CsA the change in expression of NK cell activating receptors was more variable since some receptors increased (33.3% for NKG2D, 33.3% for NKp30, 75% for NKp44 and 16.7% for NKp46) while others receptors decreased (25% for NKG2D, 50% for NKp30, 0% for NKp44 and 16.7% for NKp46). Because CsA affected NK cell receptor density, we performed cytotoxicity assays using both NK cell sensitive (K562) and NK cell resistant, LAK sensitive targets (Raji). NK cells cultured with CsA (for 1 week) had a slightly reduced capacity to kill both targets (E:T 5:1, 60.9%, 36.2%, 35.2% for K562 and 72.4%, 53.3%, 40.7% for EtOH, CsA 1μg/ml and 10μg/ml, respectively). Since CsA changed the expression of NK cell inhibitory and activating receptors, we tested whether this drug would influence the expression of other receptors important in NK cell function. To do this, multiplex PCR was used to analyze the expression of the chemokine receptors SDF-1, CCR 1–4 and CXCR 1–5. Relative to a GADPH control, there was no significant change in chemokine receptor expression after culture with CsA. Lastly, we investigated the effect of CsA on NK cell cytokine synthesis and secretion. Fewer IFN-γ secreting NK cells were present after PMA/ionmycin treatment in CsA containing cultures compared to EtOH controls. Using multiplex PCR, we consistently found that CsA treatment lead to either an induction or an increase in IL-5, IL-6, IL-8, IL-13 and TGF-β transcripts. Taken together these results demonstrate that CsA alters NK cells by inhibiting expansion, changing the density of NK cell inhibitory and activating receptors and shifts cytokine synthesis to a Th2 like pattern.