ABSTRACT Adoptive T cell transfer therapy mediates potent immunity in patients with bulky metastatic malignancies, but proves difficult to translate clinically due to production costs, time, and labor required to generate T cell infusion products. To overcome such obstacles, we proposed a method of shortened ex vivo expansion using Th17 cells to treat melanoma. Our new unpublished work indicates that Th17 cells expanded only four days ex vivo can eradicate tumors even when only very few cells (~200K) are infused into the animal. These day-4 Th17 cells mediate more potent antitumor responses than greater numbers (>25X more) of Th17 cells expanded long term. In contrast to long-term expanded cells, day-4 cells 1) persist at greater fold once infused in the animal, 2) induce significantly increased production of multiple cytokines (IL-6, G-CSF, MCP-1, KC), 3) express high levels of cytokine receptors and costimulatory molecules, and 4) provide long-lived protection against tumor recurrence. This proposal will determine the mechanism of enhanced day-4 Th17 cell antitumor efficacy and examine whether 4-days of ex vivo expansion will improve clinical protocols for generating tumor-infiltrating lymphocyte (TIL) products with superior efficacy. Based on our findings, we propose the central hypothesis that day-4 expanded Th17 cells possess enhanced efficacy in vivo due to their unique ability to recruit other immune cells to the tumor as well as form durable memory. To test this hypothesis, in Aim 1, we will examine the effects of IL- 6 on shaping memory and antitumor activity of Th17 cells via neutralization versus exogenous supplementation of IL-6. In Aim 2, we will determine whether the antitumor efficacy of very few day-4 Th17 cells is reliant upon host immunity through antibody depletion of host neutrophils, macrophages, NK cells, and lymphocytes. Finally, in Aim 3, we will assess the functionality of TIL products kinetically during ex vivo expansion and determine whether 4-days of rapid expansion is superior to clinically standard 14-days. These investigations are expected to expose key mechanisms underlying short-term expanded Th17 cell potency to 1) identify novel approaches to improve ACT with long-term expanded cells and 2) highlight shortened expansion as an efficient, less expensive method for future ACT (recently FDA approved at a cost of >$500,000/infusion).