AimWe have previously reported that polyfunctional T cell responses can be induced to the cancer testis antigen NY-ESO-1 in melanoma patients injected with mature autologous monocyte-derived dendritic cells (DCs) loaded with long NY-ESO-1-derived peptides together withα-galactosylceramide (α-GalCer), an agonist for type 1 Natural Killer T (NKT) cells.ObjectiveTo assess whether inclusion ofα-GalCer in autologous NY-ESO-1 long peptide-pulsed DC vaccines (DCV +α-GalCer) improves T cell responses when compared to peptide-pulsed DC vaccines withoutα-GalCer (DCV).Design, setting and participantsSingle-centre blinded randomised controlled trial in patients ≥ 18 years old with histologically confirmed, fully resected stage II–IV malignant cutaneous melanoma, conducted between July 2015 and June 2018 at the Wellington Blood and Cancer Centre of the Capital and Coast District Health Board.InterventionsStage I. Patients were randomised to two cycles of DCV or DCV +α-GalCer (intravenous dose of 10 × 106cells, interval of 28 days).Stage II.Patients assigned to DCV +α-GalCer were randomised to two further cycles of DCV +α-GalCer or observation, while patients initially assigned to DCV crossed over to two cycles of DCV +α-GalCer.Outcome measuresPrimary:Area under the curve (AUC) of mean NY-ESO-1-specific T cell count detected by ex vivo IFN-γ ELISpot in pre- and post-treatment blood samples, compared between treatment arms at Stage I.Secondary:Proportion of responders in each arm at Stage I; NKT cell count in each arm at Stage I; serum cytokine levels at Stage I; adverse events Stage I; T cell count for DCV +α-GalCer versus observation at Stage II, T cell count before versus after cross-over.ResultsThirty-eight patients gave written informed consent; 5 were excluded before randomisation due to progressive disease or incomplete leukapheresis, 17 were assigned to DCV, and 16 to DCV +α-GalCer. The vaccines were well tolerated and associated with increases in mean total T cell count, predominantly CD4+T cells, but the difference between the treatment arms was not statistically significant (difference − 6.85, 95% confidence interval, − 21.65 to 7.92;P= 0.36). No significant improvements in T cell response were associated with DCV +α-GalCer with increased dosing, or in the cross-over. However, the NKT cell response toα-GalCer-loaded vaccines was limited compared to previous studies, with mean circulating NKT cell levels not significantly increased in the DCV +α-GalCer arm and no significant differences in cytokine response between the treatment arms.ConclusionsA high population coverage of NY-ESO-1-specific T cell responses was achieved with a good safety profile, but we failed to demonstrate that loading withα-GalCer provided an additional advantage to the T cell response with this cellular vaccine design. Clinical trial registration: ACTRN12612001101875. Funded by the Health Research Council of New Zealand.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-023-03400-y.