The C-type lectin receptor CD69 is expressed on a range of haematopoietic cells following activation. On natural killer (NK) cells CD69 may play a direct role in mediating cytotoxicity of tumour targets. We have previously shown that remission following chemotherapy or autologous bone marrow transplantation (BMT) for acute myeloid leukaemia (AML) is dependent on NK cell cytotoxicity and have observed CD69 capping the immune synapse between autologous NK cells and conjugated AML cells (
Lowdell et al, Br J Haematol, 2002; 117(4):821–7
). Tumour cells which are resistant to NK-mediated lysis are often susceptible to lysis by activated NK cells (e.g. after stimulation with IL-2); thus we hypothesized that the interaction between CD69 on activated NK cells and its unidentified ligand (CD69L) on target cells is required for target cell lysis. Here we use soluble recombinant CD69 (rCD69) to investigate the role of CD69-CD69L interaction in mediating activated-NK cytotoxicity of NK-resistant and NK-sensitive target cells. The extracellular domain of CD69 (amino acids 65–199) fused with an N-terminal biotinylation sequence (Avidity) was expressed in Escherichia coli and a multivalent rCD69 reagent was created by binding biotinylated CD69 protein to avidin coated fluorescent beads (Spherotech Inc). Binding of rCD69 to NK-resistant Raji and Daudi Burkitt’s lymphoma cell line targets was determined by flow cytometry (11.9%, 12.4% positive respectively) and confocal microscopy; rCD69 did not bind to 293 kidney epithelial cells, K562 chronic myeloid leukaemia (an NK-sensitive target) or normal peripheral blood mononuclear cells. rCD69 was used to block the interaction between activated-NK cells and target cells; pre-incubation of Raji target cells with rCD69 reduced specific cytotoxicity to the level of unactivated NK cells (31.2 +/−1.6% to 8.0 +/−0.7%, Figure 1). Furthermore, activation of the intracellular tyrosine kinase Syk, which is selectively phosphorylated following CD69 signalling on activated NK cells (Pisegna et al, JI, 2002; 169: 68–74
), was abrogated by rCD69 pre-incubation as determined by confocal microscopy. These data show that rCD69 binds NK-resistant target cells and blocks the killing of these cells by activated NK cells. We conclude that CD69 is required for activated NK-cell-mediated killing of resistant targets and that CD69L may be a tumour-restricted marker. Screening of primary tumours for CD69L is ongoing.Figure 1.
rCD69 fractions block lysis of Raji cells by activated NK cells. Killing of Raji target cells by activated NK cells (aNK) is reduced to that of unactivated NK cells by pre-incubating Raji with HPLC purified fractions of rCD69. F3 contains rCD69 in dimeric and monomeric form, F2 in monomeric form only.
Figure 1.
rCD69 fractions block lysis of Raji cells by activated NK cells. Killing of Raji target cells by activated NK cells (aNK) is reduced to that of unactivated NK cells by pre-incubating Raji with HPLC purified fractions of rCD69. F3 contains rCD69 in dimeric and monomeric form, F2 in monomeric form only.
