In the past year, our research program has focused on: 1) understanding the role of microRNA-155 (miR-155) on CD8+ T cell biology and anti-tumor function, 2) understanding the role of transcription factor c-Myb on CD8+ T cell biology and anti-tumor function 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. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155 while constitutive Akt activation promoted sustained effector functions, indicating that Stat5 and Akt play non-redundant and distinctive roles in driving miR-155-mediated numeric and functional advantages. To gain further insights on the biology behind miR-155 activity, we performed massively parallel RNA sequencing (RNA-Seq) and compared the gene expression profiles of miR-155-overexpressing and control CD8+ T cells. Gene Set Enrichment Analysis revealed that miR-155 overexpressing cells downregulated numerous genes that are silenced in embryonic stem cells by the Polycomb Repressive Complex 2 (PRC2) through trimethylation of H3K27. Genome-wide H3K4 and H3K27 trimethylation analysis indicated that miR-155 enhanced H3K27me3 at promoters of genes involved in T cell senescence. We are currently exploring the possibility that miR-155 might prevent terminal differentiation in T cells by targeting the Jumonji- and AT-rich epigenetic regulator Jarid2, an important regulator of PRC2 activity or indirectly affecting other PRC2 components. Project 2: We have started investigating the role of the transcription factor c-Myb in CD8+ T cell differentiation and memory formation. We found that c-Myb is preferentially expressed in a subset of long-lived memory T cells that we recently identified, called T memory stem cells. Using transgenic mice and gene engineering approaches we found that c-Myb is a critical regulator CD8+ T cell stemness. T cells lacking c-Myb have a propensity to undergo terminal differentiation and senescence whereas cells overexpressing this transcription factor preferentially form long-lived memory cells. We are currently working to elucidate the molecular mechanisms downstream c-Myb activity. We identified downstream transcription factors influenced by c-Myb activity and we are currently exploring whether manipulation of these genes can rescue c-Myb-deficient T cells. 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 wouldlike now to bring these exciting discoveries into the clinical arena to improve the outcome of current T-based immunotherapies. We have initiated a collaboration with intramural investigators (James Kochenderfer, NCI, David Stroncek, CC and Mario Roederer, NIAID) with the goal of developing a phase I dose-escalation trial using anti-CD19 CAR-modified Tscm cells for the treatment of patients with metastatic CD19+ B cell malignancies refractory to prior allogeneic stem transplantation or with advanced B-cell malignancies who have never undergone allogeneic stem transplantation. Together with the Department of Transfusion Medicine, CC, we have recently established a manufacturing procedure for the generation of anti-CD19 CAR-modified Tscm cells at clinical scale under GMP conditions. In vitro-generated CD19-CAR modified Tscm cells were phenotypically, functionally and transcriptomically equivalent to their naturally occurring counterpart. Compared with T cell products currently under clinical investigation, CD19-CAR modified Tscm cells exhibited enhanced metabolic fitness, persistence and anti-tumor activity against systemic acute lymphoblastic leukemia xenografts (Sabatino, M et al. Blood 2016). We have now initiated in collaboration with Dr. Kochenderfer a phase 1 trial to test safety and efficacy of CD19-CAR modified Tscm cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011480-05
Application #
9556597
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Gattinoni, Luca; Speiser, Daniel E; Lichterfeld, Mathias et al. (2017) T memory stem cells in health and disease. Nat Med 23:18-27
Ji, Yun; Hocker, James D; Gattinoni, Luca (2016) Enhancing adoptive T cell immunotherapy with microRNA therapeutics. Semin Immunol 28:45-53
Fowler, Daniel H; Gattinoni, Luca (2016) T memory stem cell formation: Caveat mTOR. EBioMedicine 4:3-4
Crompton, Joseph G; Narayanan, Manikandan; Cuddapah, Suresh et al. (2016) Lineage relationship of CD8(+) T cell subsets is revealed by progressive changes in the epigenetic landscape. Cell Mol Immunol 13:502-13
Gattinoni, Luca (2016) Adoptive T cell transfer: Imagining the next generation of cancer immunotherapies. Semin Immunol 28:1-2
Sabatino, Marianna; Hu, Jinhui; Sommariva, Michele et al. (2016) Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies. Blood 128:519-28
Ji, Yun; Gattinoni, Luca (2015) miR-155 releases the brakes on antitumor T cells. Oncoimmunology 4:e1026533
Ji, Yun; Wrzesinski, Claudia; Yu, Zhiya et al. (2015) miR-155 augments CD8+ T-cell antitumor activity in lymphoreplete hosts by enhancing responsiveness to homeostatic ?c cytokines. Proc Natl Acad Sci U S A 112:476-81
Roychoudhuri, Rahul; Lefebvre, Francois; Honda, Mitsuo et al. (2015) Transcriptional profiles reveal a stepwise developmental program of memory CD8(+) T cell differentiation. Vaccine 33:914-23
Gattinoni, Luca (2015) The dark side of T memory stem cells. Blood 125:3519-20

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