P80
QUANTITATIVE MEASUREMENT OF NATURAL KILLER (NK) CELL FUNCTION
IN EARLY AND LATE RENAL TRANSPLANT PATIENTS
Morteau O, Blundell L, Chakera A, Bennett S, Christou C, Mason PD, O'Callaghan C and
Cornall RJ
Nuffield Department of Clinical Medicine, University of Oxford and Oxford Kidney Unit,
Churchill Hospital, Oxford
INTRODUCTION: NK cells are an important component of both innate and adaptive arms of
immunity, particularly against viral infections. Despite this fact, the role of NK cells in the
transplant situation is poorly defined and the inhibitory effect of the immunosuppressive drugs
cyclosporin A (CsA) and FK506 on NK cell function is unclear. To start to explore these
questions we have developed a quantitative assay of NK function that measures NK cell
degranulation and interferon (IFN)  production in response to target cells. Here go on to report
our first results of NK function in early and late renal transplant patients receiving
immunosuppressive therapy. Knowledge of NK function in these situations may help us to
make a better balance of the risks of transplant rejection and viral infection and possibly identify
patients at higher risk of CMV and other pathogens.
METHODS: Blood samples were collected from healthy control subjects and early and late
transplant recipients (4-21 weeks and 4-13 years, respectively) and separated into samples of
peripheral blood mononuclear cells (PBMCs), serum and plasma. The PBMCs were rested
overnight to remove adherent cells in complete medium (10% FCS, RPMI) serum or plasma,
and then incubated for 6hr with a 5-fold excess of MHC class I-deficient target cells, which
were either K562 or Daudi cells transfected with the NKG2D activating ligand MICA (D860
cells). Cells were then analysed by flow cytometry for degranulation (LAMP-1, CD107a+) and
intracellular IFN production. In some experiments, PBMCs were incubated with interleukin
(IL)-2, CsA or FK506 overnight and during the 6-hr incubation.
RESULTS: During optimisation of our assay we showed that overnight treatment of PBMCs
from healthy controls with 0.1, 1 or 10 mM CsA or FK506 inhibited NK cell degranulation and
intracellular IFN production in a dose dependent manner (P<0.05, unpaired Student's t-test).
Inhibitory effects of 0.01 mM FK506 were detectable using K562 but not D860 cells; in
addition, incubation with IL-2 (100 U/ml) significantly increased the double positive
(CD107a+IFN+) NK cells in untreated, CsA-treated and FK506-treated PBMCs. Trough levels
of CsA or FK506 in both late and early transplant patient groups ranged from 7.5 to 105 ng/ml.
The absolute number of blood NK cells was significantly lower in early (P<0.05) but not late
transplant patients, probably due to the action of depleting antibodies. NK cell degranulation
and intracellular IFN in response to K562 cells were not significantly different in early (n=9)
and late (n=14) transplant groups compared to controls (n=9) (P>0.05 in ANOVA and unpaired
Student's t-test), wether the PBMCs were rested overnight in medium, or patient's plasma or
serum. However, our data suggest that overnight resting of PBMCs in the patient's own plasma
tends to lower NK cell function in early transplant patients compared to healthy controls and
late transplant patients. Overnight IL-2 treatment significantly increased the percentage of
CD107a+IFN+ NK cells in all three groups, irrespective of the resting conditions.
CONCLUSION: We have developed a quantitative assay of NK cell function sensitive to CsA
or FK506 inhibition that accurately measures NK degranulation and the induction of IFN
synthesis in response to target cells. A significant depletion and trend in functional inhibition of
NK cells can be measured in early transplant patients. The assay should make it possible to
titrate immunosuppression against NK function and anti-viral immunity during the period of
highest risk of infection, and assess risk of infection in prospective studies.