Toll-like receptors (TLR) expressed on inflammatory cells play a key role in host defense against pathogens, benefiting the host. TLR are also expressed on tumor cells. To evaluate the role of TLR in tumor cells, we investigated TLR4 signaling effects on human head and neck squamous cell carcinoma (HNSCC). Tumor tissues were obtained from 27 patients with laryngeal and 12 with oral cavity cancers. Normal mucosa was obtained from 10 patients with nonneoplastic disorders. Smears for bacteria were taken from all patients during surgery. TLR4 expression in tumors and HNSCC cell lines (PCI-1, PCI-13, and PCI-30) was detected by reverse transcription-PCR and immunohistochemistry. Cell growth, apoptosis, nuclear factor-κB (NF-κB) translocation, and MyD88 and IRAK-4 expression, as well as Akt phosphorylation were measured following tumor cell exposure to the TLR4 ligand lipopolysaccharide (LPS). Tumor cell sensitivity to NK-92–mediated lysis was evaluated in 4-hour 51Cr-release assays. Cytokine levels in HNSCC supernatants were measured in Luminex-based assays. TLR4 was expressed in all tumors, HNSCC cell lines, and normal mucosa. The TLR4 expression intensity correlated with tumor grade. LPS binding to TLR4 on tumor cells enhanced proliferation, activated phosphatidylinositol 3-kinase/Akt pathway, up-regulated IRAK-4 expression, induced nuclear NF-κB translocation, and increased production (P < 0.05) of interleukin (IL)-6, IL-8, vascular endothelial growth factor, and granulocyte macrophage colony-stimulating factor. TLR4 triggering protected tumor cells from lysis mediated by NK-92 cells. TLR4 ligation on tumor cells supports HNSCC progression.

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Human CD4+CD39+ regulatory T (Treg) cells hydrolyze exogenous ATP and participate in immunosuppressive adenosine production. They contain two T-cell subsets whose role in mediating suppression is not understood. Frequencies of both CD4+CD39+ subsets were evaluated in peripheral blood lymphocytes of 57 cancer patients and in tumor infiltrating lymphocytes (TILs) of 6 patients. CD4+CD39+ and CD4+CD39neg T cells isolated using immunobeads and cell sorting were cultured under various conditions. Their conversion into CD39+FOXP3+CD25+ or CD39+FOXnegCD25neg cells was monitored by multiparameter flow cytometry. Hydrolysis of exogenous ATP was measured in luminescence assays. Two CD4+CD39+ cell subsets differing in expression of CD25, FOXP3, CTLA-4, CD121a, PD-1, LAP, GARP and the cytokine profile accumulated with equal frequencies in the blood and tumor tissues of cancer patients. The frequency of both subsets was significantly increased in cancer. CD39 expression levels correlated with the subsets' ability to hydrolyze ATP. Conventional CD4+CD39neg T cells incubated with IL-2 + TGF-β expanded to generate CD4+CD39+FOXP3+ Treg cells, while CD4+CD39+FOXP3negCD25neg subset cells stimulated via the TCR and IL-2 converted to FOXP3+CTLA4+CD25+ TGF-β-expressing Treg cells. Among CD4+CD39+ Treg cells, the CD4+CD39+FOXP3negCD25neg subset serves as a reservoir of cells able to convert to Treg cells upon activation by environmental signals.

Background p53 accumulation in head and neck squamous cell carcinoma (HNSCC) cells creates a targetable tumor antigen. Adjuvant dendritic cell (DC)-based vaccination against p53 was tested in a phase I clinical trial. Methods Monocyte-derived DC from 16 patients were loaded with two modified HLA-class I p53 peptides (Arm 1); additional T-helper(Th) tetanus toxoid peptide (Arm 2) or additional Th wt p53-specific peptide (Arm 3). Vaccine DC (vDC) were delivered to inguinal lymph nodes at 3 time points. Vaccine (vDC) phenotype, circulating p53-specific T-cells and regulatory T-cells (Treg) were serially monitored by flow cytometry and cytokine production by Luminex. vDC properties were compared to those of DC1 generated with an alternative maturation regimen. Results No grade II-IV adverse events were observed. Two-year disease-free survival (DFS) of 88% was favorable. p53-specific T-cell frequencies were increased post vaccination in 11/16 patients (69%), with IFN-γ secretion detected in 4/16 patients. Treg frequencies were consistently decreased (p=0.006) relative to pre-vaccination values. The phenotype and function of DC1 were improved relative to vDC. Conclusion Adjuvant p53-specific vaccination of HNSCC patients was safe and associated with promising clinical outcome, decreased Treg levels, and modest vaccine-specific immunity. HNSCC patients’ DC required stronger maturation stimuli to reverse immune suppression and improve vaccine efficacy.