Effects of Therapeutic Drug Exposure on Natural Killer Cell Phenotype and Cytotoxicity Following Liver Transplantation

1Dr Haroon ur Rashid, ²Dr Javed Akhtar, ³Kashif Lodhi ,²Ali Hammad, ²Mohib Ali, ²Ali RAza

1Assistant professor, Department of Radiotherapy & Nuclear Medicine Dr Faisal Masood Teaching Hospital Sargodha.

²PIMS, Islamabad

³Department of Agricultural, Food and Environmental Sciences. Università Politécnica delle Marche Via Brecce Bianche 10, 60131 Ancona (AN) Italy

ABSTRACT:

Background: Natural killer (NK) cells constitute a major innate lymphocyte population in the human liver and play a critical role in allograft rejection, viral immunity, and post-transplant surveillance. Standard maintenance protocols in liver transplantation (LT) rely on combination regimens consisting of calcineurin inhibitors (CNIs; tacrolimus/cyclosporine), corticosteroids (CS), antimetabolites (mycophenolate mofetil [MMF]), and mammalian target of rapamycin inhibitors (mTORIs; sirolimus/everolimus). While these agents effectively prevent allograft rejection by suppressing T-cell activation, their off-target effects on recipient NK cell frequency, phenotype, and cytolytic function remain incompletely defined.

Objective: This study evaluated the quantitative and functional modulation of peripheral and graft-infiltrating NK cells in liver transplant recipients exposed to therapeutic plasma concentrations of single and combined immunosuppressive agents.

Methods: Peripheral blood mononuclear cells (PBMCs) were evaluated from stable LT recipients ($n = 24$) and healthy controls ($n = 19$). In vitro microenvironment simulations exposed PBMCs and sorted NK cell subsets ($\text{CD56}^{\text{bright}}$ and $\text{CD56}^{\text{dim}}$) to clinically effective trough concentrations of single agents (TAC $5\text{ ng/mL}$, CSA $125\text{ ng/mL}$, MMF $15\ \mu\text{g/mL}$, EVE $5\text{ ng/mL}$, SIR $5\text{ ng/mL}$, CS $0.5\ \mu\text{g/mL}$) and multidrug combination protocols. NK cell proliferation, phenotype changes, degranulation ($\text{CD107a}$ expression), cytokine secretion ($\text{IFN-}\gamma$), transcriptomic signatures, and target cell cytotoxicity were assessed via flow cytometry and RNA sequencing.

Results: In vivo LT recipients demonstrated a significant reduction in overall peripheral blood NK cell counts and proportion compared to healthy controls ($p < 0.01$). Single-agent exposure revealed that corticosteroids exerted the most pronounced impairment on NK cell cytolytic activity, followed by MMF and mTORIs. Both CS and CNI agents (TAC/CSA) significantly suppressed $\text{IFN-}\gamma$ release and $\text{CD107a}$ degranulation ($p < 0.001$). Combination therapeutic drug exposure marked a synergistic reduction in NK cell numbers and function, where NK cells displayed higher sensitivity to combination suppression than CD4+ or CD8+ T cells. MMF-containing regimens significantly upregulated inhibitory receptor transcripts (specifically Siglec-7 and Siglec-9 family genes). Conversely, mTORI-containing protocols preserved superior NK cell cytotoxic activity and functional capacity compared to MMF-based regimens.

Conclusion: Immunosuppressive drug exposure in liver transplant recipients significantly impairs NK cell survival, cytokine release, and cytolytic function, predominantly driven by corticosteroids and CNI combination therapies. The retention of functional capacity under mTORI-based protocols suggests that CNI-minimization regimens utilizing mTOR inhibitors may preserve innate anti-viral and anti-tumor immunosurveillance while maintaining adequate allograft protection.