Effect of Soluble HLA Class I Molecule on NK/LAK Cells Activation Induced by Poly I:C

Background:  Natural Killer cells express killer inhibitory receptors specific for HLA-class I molecules.  These receptors could induce signals that determine NK cells ability to mediate cytotoxicity.  Purified soluble form of HLA class I molecules (sHLA) could bind to NK cell receptors and down-regulate the NK killer function. Objective:  To evaluate the influence of sHLA and two monoclonal antibodies (mAbs) against killer inhibitory receptors on the poly I:C-treated freshly and IL-2 activated NK cells (LAK cells).Methods:  Isolation of CD56+ NK cells and CD56– cells was performed using the magnetic cell separation technique.  CD56+ cells were activated by rIL-2 and anti-HLA-B7 specific cytotoxic T lymphocytes (CTLs) were established from CD56- cells.  Flow cytometry analysis was performed to determine the percentage of CD3, CD16/CD56 and CD8 positive cells.  LAK and specific CTLs were tested for cytotoxicity against M4 cells using the 51Cr-release assay.  Freshly isolated NK cells and LAK cells were pre-incubated with 0.7-11 µg/ml of sHLA-B7 fused to the Fc portion of IgG molecule and subsequently tested for killing activity.  The influence of the sHLA molecule on the cytotoxicity of the cells after treatment with poly I:C as an inducer of interferon was also studied.  LAK cells were pre-treated with 0.01 to 10 µg/ml of two mAbs against NK inhibitory receptors, NKB1 and NKAT2.  The effect of these mAbs on the killing activity of poly I:C and non-poly I:C treated LAK cells against K562 target cell was determined.  Results:  In flow cytometry analysis of LAK cells, 99.5% were found to be CD56+.  Analysis of generated CTLs showed 88.4 % positivity for CD8.  Cytotoxicity of M4 stimulated CTLs was 12.3% at 1.5/1 E/T ratio to 97.1% at 25/1 E/T ratio.  NK cells had no effect on M4 cells.  Anti-NKB1 and Anti-NKAT2 mAbs decreased the cytotoxic activity of LAK cells.  These mAbs showed no effect on the poly I:C activated LAK cells.  Increasing concentrations of sHLA molecule decreased cytotoxic activity of LAK cells from 17.2% to 13.8% and poly I:C activated NK cells from 62.5% to 51.8%.  Treatment of LAK cells with poly I:C and exposure of these cells with increasing concentrations of sHLA resulted in an increase in the target cell killing of LAK cells from 20.1% to 27.6%.Conclusion:  LAK cells were not able to kill M4 cells with high expression of HLA molecules, whereas CTLs were efficient in killing these cells.  Recombinant sHLA fusion protein inhibited NK/LAK activity against K562 cells, whereas poly I:C activation of LAK cells reverted this effect indicating that poly I:C treatment of LAK cells could change the expression of NK inhibitory receptors.