In vitro anti-tumour activity of α-galactosylceramide-stimulated human invariant Vα24+NKT cells against melanoma

α-galactosylceramide (KRN 7000, α-GalCer) has shown potent in vivo anti-tumour activity in mice, including against melanoma and the highly specific effect of inducing proliferation and activation of human Vα24+NKT-cells. We hypothesized that human Vα24+NKT-cells activated by α-GalCer might exhibit anti-tumour activity against human melanoma. To investigate this, Vα24+NKT-cells were generated from the peripheral blood of patients with melanoma after stimulation with α-GalCer pulsed monocyte-derived dendritic cells (Mo-DCs). Vα24+NKT-cells did not exhibit cytolytic activity against the primary autologous or allogeneic melanoma cell lines tested. However, proliferation of the melanoma cell lines was markedly suppressed by co-culture with activated Vα24+NKT-cells (mean ± SD inhibition of proliferation 63.9 ± 1.3%). Culture supernatants of activated Vα24+NKT-cell cultures stimulated with α-GalCer pulsed Mo-DCs exhibited similar antiproliferative activities against melanoma cells, indicating that the majority of the inhibitory effects were due to soluble mediators rather than direct cell-to-cell interactions. This effect was predominantly due to release of IFN-γ, and to a lesser extent IL-12. Other cytokines, including IL-4 and IL-10, were released but these cytokines had less antiproliferative effects. These in vitro results show that Vα24+NKT-cells stimulated by α-GalCer-pulsed Mo-DCs have anti-tumour activities against human melanoma through antiproliferative effects exerted by soluble mediators rather than cytolytic effects as observed against some other tumours. Induction of local cytokine release by activated Vα24+NKT-cells may contribute to clinical anti-tumour effects of α-GalCer. © 2001 Cancer Research Campaign http://www.bjcancer.co

CD 69 antigen of human lymphocytes is a calcium-dependent carbohydrate-binding protein

CD69 is a signal transducing molecule of hematopoietic cells. Previous molecular cloning of CD69 has revealed a type II transmembrane orientation and the presence of an extracellular domain related to the Ca(2+)-dependent (C-type) animal lectins. As the predicted amino acid sequence for the lectin-like domain is highly divergent from those of other C-type lectin-like proteins - a feature shared with NKR-P1 of natural killer cells - CD69 and NKR-P1 are among proteins assigned to a separate group, group V. To initiate ligand identification studies, we have prepared soluble forms of CD69 protein by bacterial expression of its extracellular portion. We show that cysteine 68 located in the short membrane-proximal neck region of CD69 which adjoins the C-terminal lectin-like domain is a critical element for dimerization. We have evidence that the soluble dimeric CD69 has a tight association with calcium, a feature shared with NKR-P1, and that it is a carbohydrate-binding protein with N-acetyl-D-glucosamine and N-acetyl-D-galactosamine as the best inhibitors: 4-8 x 10(-5) M giving 50% inhibition of binding to N-acetyl-D-glucosamine neoglycoprotein. Thus, the tight association with calcium and high affinities for carbohydrate binding appear to be features of at least two members of the C-type lectin group