CTLA-4+ regulatory t cells increased in cetuximab-treated head and neck cancer patients suppress nk cell cytotoxicity and correlate with poor prognosis

The EGFR-targeted antibody cetuximab is effective against head and neck cancer (HNSCC), but in only 15% to 20% of patients, and the variability and extent of cetuximab-mediated cellular immunity is not fully understood. We hypothesized that regulatory T cells (Treg) may exert a functional and clinical impact on antitumor immunity in cetuximab-treated individuals. The frequency, immunosuppressive phenotype, and activation status of Treg and natural killer (NK) cells were analyzed in the circulation and tumor microenvironment of cetuximab-treated patients with HNSCC enrolled in a novel neoadjuvant, single-agent cetuximab clinical trial. Notably, cetuximab treatment increased the frequency of CD4+FOXP3+ intratumoral Treg expressing CTLA-4, CD39, and TGFb. These Treg suppressed cetuximab-mediated antibodydependent cellular cytotoxicity (ADCC) and their presence correlated with poor clinical outcome in two prospective clinical trial cohorts. Cetuximab expanded CTLA-4+FOXP3+ Treg in vitro, in part, by inducing dendritic cell maturation, in combination with TGFb and T-cell receptor triggering. Importantly, cetuximab-activated NK cells selectively eliminated intratumoral Treg but preserved effector T cells. In ex vivo assays, ipilimumab targeted CTLA- 4+ Treg and restored cytolytic functions of NK cells mediating ADCC. Taken together, our results argue that differences in Tregmediated suppression contribute to the clinical response to cetuximab treatment, suggesting its improvement by adding ipilimumab or other strategies of Treg ablation to promote antitumor immunity.