No abstract

Cooperation between CD16 and NKG2D receptors has been demonstrated both in animal and NK cell line models leading to an enhanced cytotoxicity and tumor elimination. However, the mechanisms of the receptor cooperation are not known, and whether the receptors form co-clusters has not been determined either. We exposed human primary polyclonal NK cells with CD16 158V/V-genotype to planar lipid bilayers (PLB) presenting Herceptin and anti-NKG2D Ab in the presence of ICAM-1. NK cells interacting with Herceptin-containing PLBs + ICAM-1 formed mostly unstable synapse, i.e., kinapses, while stimulation with anti-NKG2D alone or Herceptin+anti-NKG2D bilayers + ICAM-1 led to formation of mature synapses showing complete segregation of the receptors from ICAM-1, increased synapse stability, and efficient NK cell cytotoxicity. The cells exposed to PLBs with both anti-NKG2D and Herceptin ligands were mostly confined within the initial contact area while the cells responding to Herceptin alone were significantly more motile. Regardless of stimulatory ligands displayed on PLB, the frequency of adhered cells was very similar, but the degranulation was observed within 40% of kinapses and 80% of mature synapses, suggesting that degranulation efficiency is linked to synapse stability. Importantly, more efficient degranulation was associated with rapid and sustained Ca2+ signaling as opposed to oscillating Ca2+ signaling observed for NK cells showing less efficient degranulation. Our data demonstrated that Herceptin alone functions as a weak agonist, while co-activation of CD16 and NKG2D results in rapid and sustained Ca2+ signaling that mediates stable synaptic interface and efficient cytolytic activity by human NK cells. Supported by Grant from NIH U01AI148117