CD160 is a cell surface antigen expressed on normal human NK cells, as well as a subset of T cells. We have previously reported that almost all cases of chronic lymphocytic leukaemic (CLL) express CD160 and the activation of CD160 by monoclonal anti-CD160 antibodies (mAb) induces cellular activation (proliferation and cytokine production). In this study, using fresh, peripheral blood mononuclear cells from patients with lymphocyte counts of > 40 x 109/L, we found that the activation of CD160 protected CLL cells from spontaneous cell death (n = 18). The rates of spontaneous cell death of CLL cells ranged from 5 to 64% (average 33 ± 18%) after culture for 18 hours. However, the spontaneous death rate was reduced to 20 ± 11% (p=0.012) when the CLL cells were incubated with anti-CD160 MAb. The protective effect on cell survival was more marked in the cases showing the greatest spontaneous cell death: in those cases with > 30% cell death (ie, greater than the average for the whole population), cell death was reduced from 48±9.9% to 28±8.2% (p=0.0001). In the cases which were resistant to spontaneous cell death (< 30%), CD160 reduced cell death from 16 ± 6.6% to 11 ± 5.9% (p = 0.15). The PI3- kinase/AKT pathway is a well known survival pathway and CD160 activity in NK cells has been shown to involve PI3-kinase. CLL cells were incubated with anti-CD160 mAb and found to show AKT phosphorylation, I-kB degradation, Bcl-2 upregulation, but also Bax upregulation. Interestingly, the PI3-kinase inhibitor, wortmannin, completely blocked AKT phosphorylation, but had no effect on CD160-mediated cell survival and did not prevent activation of NF-kB nor upregulation of Bcl-2 or Bax. In view of the upregulated Bax expression, we hypothesised that despite CD160-induced increases in Bcl-2 and the improved survival, CLL cells may have simultaneously become more sensitive to death signals. CLL cells (from 9 cases) were treated with chlorambucil (20mg/ml) with or without the anti-CD160 antibody overnight and apoptosis was determined with Annexin-V/propidium iodide by flow cytometry. Chlorambucil alone induced 63%±16 apoptosis with 34%±23 spontaneous cell death. Co-treatment with chlorambucil and anti-CD160 mAb increased apoptosis to 72±14% with spontaneous rate at 23%±13.
In summary, the activation of CD160 protected CLL cells from spontaneous cell death in vitro via a PI3-kinase/AKT independent pathway. However, this improved survival was also associated with increased Bax and sensitisation to chemotherapeutic agents. The mechanism by which the activation of CD160 plays dual roles in the destiny of CLL cells remains unclear, but this represents a potential novel therapeutic approach. Data will be presented showing the activity of a chimeric, humanised anti-CD160 mAb on CLL killing.
Disclosures: No relevant conflicts of interest to declare.