Our research program has recently focused on: 1) understanding the role of microRNA-155 (miR-155) on CD8+ T cell biology and anti-tumor function, 2) determining how glucose metabolism influence the formation of memory CD8+ T cells and their capacity to destroy tumor in vivo and 3) developing a clinical trial using anti-CD19 CAR-transduced CD8+ memory stem cells (Tscm). Project 1: We found that tumor-specific CD8+ T cells constitutively expressing miR-155 displayed enhanced proliferation and anti-tumor function compared to cells transduced with a scramble microRNA. Interestingly, the increased functionality of miR-155 overexpressing T cells was minimized following adoptive transfer into irradiated or genetically lymphodepleted hosts, suggesting that miR-155 enhances T cell activity only under conditions of limited homeostatic cytokines availability. Consistently, the increased functionality of miR-155 overexpressing CD8+ T cells was virtually abrogated in mice deficient of the homeostatic cytokines, interleukin-7 and interleukin-15. We identified numerous miR-155 targets, such as the Akt inhibitor Ship1 and several negative regulators of Stat signaling including Socs1 and Ptpn2. Accordingly, miR-155 overexpressing T cells exhibited enhanced activity of Stat5 and Akt, two pivotal pathways downstream homeostatic cytokine signaling. Enforced expression of a constitutive active Stat5a but not Akt variant recapitulated the functional advantage conferred by miR-155 suggesting that miR-155 was acting through Stat5 rather than Akt signaling. Our findings indicate that miR-155 enhances the anti-tumor activity of CD8+ T cells by acting as a Stat5 rheostat to facilitate signaling when homeostatic cytokines are limiting. We have recently published parts of these findings in Immunity together with our collaborators from the University of Lausanne (Dudda JC, Salaun B, Ji Y et al. Immunity, 2013). To translate these findings into the clinic arena we have received the NCI Director Intramural Innovation Award. We have generated a retroviral vector encoding for human miR-155 and the suicide gene, inducible caspase 9 as a safety measure. Indeed, miR-155 has been shown to be potentially oncogenic in certain settings, although in our mouse studies no transformation of transduced T cells was observed. We are currently evaluating the activity of miR-155 in human T cells using a variety of phenotypic and functional assays. We also plan to perform long-term safety studies in vivo using highly immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Project 2: Naive CD8+ T cells rely upon oxidation of fatty acids as a primary source of energy. Following antigen encounter, T cells shift to a glycolytic metabolism to sustain effector function. It is unclear, however, whether changes in glucose metabolism ultimately influence the ability of activated T cells to become long-lived memory cells. Using a fluorescent glucose analog, 2-NBDG, to quantify glucose uptake in activated CD8+ T cells, we found that cells exhibiting limited glucose incorporation had a molecular profile characteristic of memory precursor cells and an increased capacity to enter the memory pool compared with cells taking up high amounts of glucose. Accordingly, enforcing glycolytic metabolism by overexpressing the glycolytic enzyme phosphoglycerate mutase-1 severely impaired the ability of CD8+ T cells to form long-term memory. Conversely, activation of CD8+ T cells in the presence of an inhibitor of glycolysis, 2-deoxyglucose, enhanced the generation of memory cells and anti-tumor functionality. Thus, our findings indicate that augmenting glycolytic flux drives CD8+ T cells towards a terminally differentiated state, while its inhibition preserves the formation of long-lived memory CD8+ T cells. A manuscript describing these findings has just been accepted for publication in The Journal of Clinical Investigations. Project 3: We have recently described in mice and humans a new memory T cell population endowed with the stem cell-like attributes of self-renewal and multipotency (Gattinoni at al. Nature Med 2009 and 2011). These T cells, termed stem cell memory T (Tscm) cells were capable of reconstituting the full diversity of memory and effector T cell compartments on serial transplantation. Most importantly, Tscm cells displayed robust proliferative and survival capacities and eradicated large established tumors even when limited numbers of cells were transferred, a condition in which other memory T cell subsets had little or no impact. We would like now to bring these exciting discoveries into the clinical arena to improve the outcome of current T-based immunotherapies. We have initiated a 4-way collaboration with intramural (James Kochenderfer, NCI, David Stroncek, CC and Mario Roederer, NIAID) and extramural (Carl June, University of Pennsylvania) investigators with the goal of developing a phase I dose-escalation trial using allogeneic anti-CD19 CAR-modified Tscm cells for the treatment of patients with metastatic CD19+ B cell malignancies refractory to prior allogeneic stem transplantation. We are currently working together with the Department of Transfusion Medicine, CC, to establish a manufacturing procedure for the generation of anti-CD19 CAR-modified Tscm cells under GMP conditions.
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