Background: Lifileucel, a one-time autologous tumor-infiltrating lymphocyte (TIL) cell therapy, has demonstrated potentially clinically meaningful activity and durable responses in patients with advanced (unresectable or metastatic) melanoma. The regimen includes nonmyeloablative lymphodepletion (NMA-LD) prior to lifileucel infusion, and a short course of interleukin-2 (IL-2) post infusion. The safety profile of the regimen is consistent with the underlying disease and the known safety profiles of NMA-LD and IL-2. Cytokine release syndrome (CRS) is an acute and severe adverse event commonly associated with chimeric antigen receptor T-cell therapy. Here, we report circulating cytokine levels during and after the lifileucel treatment to monitor immune activation, and the dynamics of cytokines and chemokines to explore mechanism of action and potential predictive clinical biomarkers. Methods: The full analysis set of C-144-01 (NCT02360579) includes 153 patients with unresectable or metastatic melanoma who have progressed on checkpoint inhibitors and, if applicable, BRAF/MEK inhibitors. After tumor resection, patients received NMA-LD (cyclophosphamide on Days −7 to −6 and fludarabine on Days −5 to −1) followed by a single lifileucel infusion (Day 0) and up to 6 doses of IL-2 (Days 0-4). Peripheral blood samples were collected at baseline (pre-NMA-LD and pre-lifileucel infusion) and post-lifileucel infusion on Days 1, 4, 14, 42, and 84. Serum cytokine and chemokine levels were measured with BioPlex and included a multiplex panel for IL-6, IL-7, IL-9, IL-10, IL-12, CCL2, CXCL10, IFN-γ, and TNF-α. Plasma samples also were tested for IL-6, IL-15, and IL-7 levels by ELISA. Results: IL-15 levels following NMA-LD peaked at Day 1 and returned to baseline by Day 42. This is consistent with prior reports that NMA-LD increases circulating IL-15 levels, which may promote TIL expansion in the absence of competing endogenous lymphocytes. Similarly, IL-7 and CCL2 also peaked at Day 1. IL-6 did not increase during or after the lifileucel regimen, consistent with absence of severe systemic inflammation including higher grade CRS after lifileucel treatment. There was no difference in cytokine and chemokine levels between responders and nonresponders in the full analysis set. Conclusions: These data support the hypothesized mechanism of action of NMA-LD in the lifileucel regimen. Cytokine levels measured during treatment are consistent with lack of severe systemic inflammation observed in patients treated with lifileucel. Cytokine and chemokine dynamics did not predict for response to lifileucel. Clinical trial information: NCT02360579.