Background: Patients with metastatic melanoma were treated with tremelimumab and IFN in a previously reported study (Tarhini. J Clin Oncol. 2012). Clonality of T-cell repertoire was analyzed in terms of clinical response both in TME and in peripheral blood. Methods: Patients received tremelimumab 15 mg/kg I.V. every 12 weeks. High dose IFN (HDI) was administered concurrently. Responses were assessed by RECIST as complete (CR) or partial (PR), stable disease (SD) or progression (PD). T-cell receptor beta chain (TCRB) repertoire was immunosequenced in peripheral blood mononuclear cells (PBMC) (N=33 patients) and tumor (N=18) utilizing Adaptive Biotechnologies immunoSEQ platform to determine repertoire clonality and T-cell fractions at pre-treatment (tumor, PBMC), one month (PBMC), and 3 months (PBMC). The clonality metric quantitates, the extent of mono- or oligo-clonal expansion by measuring the shape of the clone frequency distribution. Values range from 0 to 1; values approaching 1 indicate a nearly monoclonal population. Results: In pretreatment TME, T-cell clonality was significantly (p = 0.0008) different and greater in patients who achieved disease control (CR, PR, SD) versus those with PD. Further, there was a significant (p = 0.044) difference between the increased TCR fraction in TME in responders (CR, PR) and non-responders (SD, PD). There was a trend towards association between pretreatment TME T-cell clonality and overall survival (OS) (p = 0.24) and progression free survival (PFS) (p = 0.18) not reaching significance. Within the circulation (PBMC), no significant associations were found by examining the pretreatment samples. However, early on-treatment (day 29) there was significant association and decrease in T-cell clonality and OS (p = 0.005) and PFS (p = 0.003). Conclusions: T-cell clonality in the TME pretreatment is a promising biomarker of immunotherapeutic benefit in our study. While baseline PBMC clonality was not associated with clinical benefit, early on-treatment (day 29) was significantly associated. These findings require validation in an independent cohort and exploration in relation to other immunotherapeutics. Clinical trial information: NCT00610857