Background: NK cells may play an important role in the control of CMV replication post-transplant. NK cells can be divided into subsets based on surface intensity of CD56 and CD16 as well as expression of NKG2C, NKp30, and other markers. Some markers have been implicated in memory function specific to CMV control (NKG2C). The effect of immunosuppression (IS) on the NK cell subsets in the setting of CMV replication is unknown. We studied the in-vitro effect of IS drugs on NK cell subsets in the setting of CMV stimulation.

Methods: Peripheral blood mononuclear cells (PBMCs) obtained from healthy volunteers (n=10, all CMV seropositive) were treated with varying doses of MMF (100-5000ng/ml), Tacrolimus (5-20ng/mL), Sirolimus (5-20ng/ml) alone or in combination. PBMCs then underwent stimulation with live CMV Towne strain virus at MOI 0.03 and culture for a period of 3-days with subsequent flow cytometry and surface and intracellular staining for CD3, CD56, CD16, NKG2C, NKG2D, NKp30, CD107a, and IFN-γ and the proliferation marker CFSE. Unstimulated and untreated controls were run concurrently.

Results: The CD56dimCD16+ NK cell population is normally more prevalent in the peripheral blood and displays high cytotoxicity. Immunosuppression resulted in a dose dependent inhibition of NK cell proliferation (by CFSE) which was most pronounced with MMF and least with TAC (p<0.01 vs. no IS). In the presence of viral stimulation and varying doses of IS, the portion of CD56dimCD16+ NK cells tended to remain relatively stable with the exception of MMF, where an increase in the proportion of CD56Bright cells was observed over time (a more immature NK cell subset). Viral stimulation resulted in an increased expression of NKG2C compared with unstimulated NK cells (p<0.01). The presence of IS generally did not lead to a significant change in the portion of NK cells expressing NKG2C. The CD56Bright subset was assessed for IFN-γ production in response to CMV. Viral stimulation resulted in a significant increase in IFN-γ from NK cells (p<0.01). Interestingly IS did not appear to inhibit IFN-γ production from CD56Bright NK cells and in some cases a significant increase in % IFN-γ production from this NK cell subset was observed with the addition of IS.

Conclusion: This study provides novel insights into the effect of standard immunosuppression agents on NK cell subsets in the setting of in-vitro CMV stimulation. Although IS decreases NK cell proliferation, IFN-γ production is maintained.

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