Date of Graduation

12-2017

Document Type

Dissertation (PhD)

Program Affiliation

Experimental Therapeutics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Eugenie Kleinerman, M.D.

Committee Member

Dean Lee, M.D.,Ph.D

Committee Member

Stephen Ullrich, Ph.D

Committee Member

Joya Chandra, Ph.D

Committee Member

Gheath Al-Atrash, D.O.,Ph.D

Committee Member

Ignacio Wistuba, M.D.

Abstract

Natural Killer (NK) cells are cells of the innate immune system that act as first line of defense against viral infections and participate in tumor immune surveillance. NK cells do not cause graft versus host disease (GvHD), or require prior antigen exposure to exert anti-tumor activity, hence are an attractive choice for immunotherapy applications. Owing to small numbers of NK cells in peripheral blood (1-32%, with a 6% median), ex vivo expansion of NK cells is critical for NK cell adoptive immunotherapy, various expansion platforms have been explored over the decades. We developed a robust platform for ex vivo expansion of human NK cells using K562 expressing membrane bound IL-21 (K562.mbIL21), and showed that K562.mbIL21 enables significantly higher expansion of functionally potent NK cells compared to K562.mbIL15. Both membrane bound IL15 (mbIL15) and membrane bound IL21 (mbIL21) expanded NK cells are currently in several Phase I/II clinical trials. K562.mbIL21 surpasses any other expansion to date not only in its rate of NK cell expansion, but also cytokine secretion, cytotoxicity, telomere length and MTOC synapse formation, yielding highly activated non-senescent cells.

IL-15 and IL-21 signaling are essential for proliferation and activation of NK cells in vivo, yet mbIL21 far exceeds that of mbIL15 based expansion ex vivo. It is possible that mbIL21 expansion is better due to regulation of telomere length by promotion of telomerase activity by Stat 3, however this may not be the only determining factor. MiRNAs, crucial regulators of post-transcriptional gene regulation could play a role in causing the observed differences in proliferation in these 2 expansion systems. The role of miRNA in NK cells biology is an emerging area of interest and their effect on regulation of protein expression and function during expansion of NK cells remain to be studied. We hypothesize that activating and expanding human NK cells on membrane bound IL15 and membrane bound IL21 will lead to differential gene expression mediated by miRNAs and subsequent observed differential expansion potential in these cells. Thus, my project’s goal is to identify miRNA that regulate the superior proliferation and function of NK cells produced by IL-21 signaling as well as to delineate the pathways that enable IL-21 signaling in achieving this effect.

To accomplish this, we initiated an exploratory study to identify differential gene, protein and miRNA that regulate these observed differences and evaluated the global changes between mbIL15 and mbIL21 expanded NK cells and identified BCL2L11, Bim and miR 124-3p to be the most differentially expressed factors, respectively (p < 0.01), with highest expression in mbIL15 NK cells. This indicated that miR 124-3p and Bim downregulation might mediate a proliferative and anti-apoptotic advantage in mbIL21 NK cells, and consequently better expansion. MiR 124-3p is recently shown to be a tumor suppressor miRNA, with high expression in normal cells; downregulation of miR 124-3p causes tumorigenesis due to increased proliferation and suppression of apoptosis. We further correlated and validated the findings by knockdown of miR124-3p in NK cells, to specifically study the role of miR124-3p in causing induction of BCL2L11, Bim and Stat3, and consequent expansion in NK cells. We discovered that knockdown of miR124-3p decreased BCL2L11 and Bim expression, and increased telomerase reverse transcriptase (TERT), Stat 3 and Stat 3 phosphorylation. The overall impact of these changes was increased proliferation, decreased apoptosis and increased overall expansion of NK cells.

Thus, this study establishes additional mechanisms for the superior proliferation in mbIL21-expanded NK cells, provides a potential approach to increase expansion of NK cells on mbIL15 for adoptive immunotherapy applications, and provides insight into the mechanistic role of miR124-3p in regulating NK cells proliferation and apoptosis.

Keywords

Natural Killer Cells, miRNA, IL21, IL15, NK cell expansion, Integrated data analysis, miR 124-3p, Bim, Stat 3, Tert

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