Impact of Commonly Used Transplant Immunosuppressive Drugs on Human NK Cell Function Is Dependent upon Stimulation Condition

Lung transplantation is a recognised treatment for patients with end stage pulmonary disease. Transplant recipients receive life-long administration of immunosuppressive drugs that target T cell mediated graft rejection. However little is known of the impact on NK cells, which have the potential to be alloreactive in response to HLA-mismatched ligands on the lung allograft and in doing so, may impact negatively on allograft survival. NK cells from 20 healthy controls were assessed in response to Cyclosporine A, Mycophenolic acid (MPA; active form of Mycophenolate mofetil) and Prednisolone at a range of concentrations. The impact of these clinically used immunosuppressive drugs on cytotoxicity (measured by CD107a expression), IFN-γ production and CFSE proliferation was assessed in response to various stimuli including MHC class-I negative cell lines, IL-2/IL-12 cytokines and PMA/Ionomycin. Treatment with MPA and Prednisolone revealed significantly reduced CD107a expression in response to cell line stimulation. In comparison, addition of MPA and Cyclosporine A displayed reduced CD107a expression and IFN-γ production following PMA/Ionomycin stimulation. Diminished proliferation was observed in response to treatment with each drug. Additional functional inhibitors (LY294002, PD98059, Rottlerin, Rapamycin) were used to elucidate intracellular pathways of NK cell activation in response to stimulation with K562 or PMA-I. CD107a expression was significantly decreased with the addition of PD98059 following K562 stimulation. Similarly, CD107a expression significantly decreased following PMA-I stimulation with the addition of LY294002, PD98059 and Rottlerin. Ten lung transplant patients, not receiving immunosuppressive drugs pre-transplant, were assessed for longitudinal changes post-transplant in relation to the administration of immunosuppressive drugs. Individual patient dynamics revealed different longitudinal patterns of NK cell function post-transplantation. These results provide mechanistic insights into pathways of NK cell activation and show commonly administered transplant immunosuppression agents and clinical rejection/infection events have differential effects on NK cell function that may impact the immune response following lung transplantation

Cytomegalovirus replication is associated with enrichment of distinct g 8 T cell subsets following lung transplantation: A novel therapeutic approach?

BACKGROUND: Anti-viral treatments to control cytomegalovirus (CMV) after lung transplantation (LTx) are associated with toxicity and anti-viral resistance. Cellular immunotherapy with virus-specific cytotoxic T cells has yielded promising results but requires donor/recipient matching. γδ T cells are involved in anti-viral immunity and can recognize antigens independently of major histocompatibility complex molecules and may not require the same level of matching. We assessed the phenotype of circulating γδ T cells after LTx to identify the candidate populations for CMV immunotherapy. METHODS: Peripheral blood mononuclear cells were isolated from lung transplant recipients before transplantation and at routine bronchoscopies after LTx. Patients were stratified by risk of CMV disease into moderate risk (recipient CMV seropositive, n = 15) or high risk (HR) (recipient CMV seronegative/donor CMV seropositive, n = 10). CMV replication was classified as polymerase chain reaction positive (>150 copies/ml) in blood and/or bronchoalveolar lavage within the first 18 months. The phenotype of γδ T cells was assessed by multicolor flow cytometry, and T-cell receptor (TCR) sequences were determined by deep sequencing. RESULTS: In HR lung transplant recipients with CMV replication, we observed striking phenotypic changes in γδ T cells, marked by an increase in the proportion of effector Vδ1+ γδ T cells expressing the activating natural killer cell receptor NKG2C. Moreover, we observed a remarkable increase in TCR diversity. CONCLUSIONS: NKG2C+ Vδ1+ γδ T cells were associated with CMV replication and may indicate their potential to control infection. As such, we propose that they could be a potential target for cellular therapy against CMV