Background: We have been developing NKT cell-based immunotherapy for head and neck cancer (HNC) patients who achieved complete response after standard therapy. However, the existence of immunosuppressive cells, including regulatory T cells (Tregs), could be an important factor limiting the efficacy of NKT cell-based immunotherapy for advanced cancer-bearing patients. Therefore, we examined the association between the frequency of peripheral blood Tregs and the clinical outcome of HNC patients, as well as the influence of Tregs on NKT cell function.

Methods: Forty-six HNC patients who received standard therapy at Chiba University Hospital, 23 benign tumor patients, and 4 healthy volunteers were enrolled. We examined the frequencies of functional Treg subsets based on the expressions of Foxp3, CD4, and CD45RA in peripheral blood mononuclear cells of these patients during standard treatment.

NKT cells obtained from healthy volunteers were cultured in the presence of α-GalCer and IL-2 for 7 days. Tregs from Naïve CD4⁺ T cells were cultured in the presence of IL-2, TGF-b and anti-CD3/CD28 beads for 7 days. These induced NKT cells and Tregs were co-cultured with or without use of a Transwell chamber, and NKT cell function was analyzed. We also examined the effect of paclitaxel on the suppressive activity of Tregs on NKT cells.

Results: Treg frequency was significantly higher in HNC patients than that in benign tumor patients. Increased frequency of Tregs was decreased after the standard curative treatment which, in turn, increased again during the early phase of recurrence. Functional analysis in vitro indicated that Tregs inhibited the function of NKT cells by cell-to-cell contact. Paclitaxel restored the impaired NKT cell function by inhibiting Tregs proliferation.

Conclusion: Increased Tregs are associated with the clinical outcome of HNC patients. Although Tregs inhibited the NKT cell function, paclitaxel restored NKT cell activation.