Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Immunology

Language

English (en)

Date of Award

Summer 9-1-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Todd A Fehniger

Abstract

Natural Killer (NK) cells are a crucial component of the immune system, with a role in both early host defense against pathogens and tumor immunosurveillance. NK cells develop in the bone marrow from a common lymphoid precursor, along with T and B cells, but undergo a separate education process. Additional maturation in the periphery makes NK cells fully competent to protect the host. NK cells protect the host via two primary effector pathways - direct cytotoxicity, and indirect cytokine/chemokine production (e.g. IFN-g), which shapes the subsequent adaptive immune response. The importance of this role of NK cells in human health is highlighted by the recurrence of herpesvirus infections in NK-deficient patients, as well as a clinical association between low NK cell function and an increased risk of developing cancer.

Micro-RNAs (miRNAs) regulate numerous cellular functions. MiRNA genes are transcribed as long primary (pri-miRNA) transcripts that are subsequently 'cropped' by the Drosha/Dgcr8 complex into precursor miRNAs (pre-miRNA) that have a characteristic stem-loop structure. The pre-miRNA is exported to the cytoplasm where it is further processed by Dicer1, yielding a mature 18-24 nucleotide miRNA. The mature miRNA is loaded into the RNA induced silencing complex (RISC), a component of which is Argonaute-2 (Ago2), and binds to a semi-complementary segment of the 3' untranslated region (UTR) of a target mRNA. The RISC directs downregulation of the protein levels of the targeted transcript, either through RNA degradation, segregation from translational machinery, or translational inhibition. Murine and human NK cells express more than 300 mature miRNAs. Here, we investigated the role of global miRNA deficiency, as well as the specific elimination of two abundant miRNA families (miR-155 and miR-15/16) in NK cells.

miR-155 is markedly upregulated following cytokine activation of human and mouse NK cells. Surprisingly, mature human and mouse NK cells transduced to overexpress miR-155, NK cells from mice with NK cell-specific miR-155 overexpression, and miR-155-/- NK cells all secreted more IFN-g compared to controls. Investigating further, we found that activated NK cells with miR-155 overexpression had increased per cell IFN-g with normal IFN-g+ percentages, whereas greater percentages of miR-155-/- NK cells were IFN-g+. In vivo MCMV-induced IFN-g expression by NK cells in these miR-155 models recapitulated the in vitro phenotypes. We performed unbiased RISC-Seq on WT and miR-155-/- NK cells, and found that mRNAs targeted by miR-155 were enriched in NK cell activation signaling pathways. Using specific inhibitors, we confirmed these pathways were mechanistically involved in regulating IFN-g production by miR-155-/- NK cells. These data indicate that miR-155 regulation of NK cell activation is complex, and that miR-155 functions as a dynamic tuner for NK cell activation via both setting the activation threshold as well as controlling the extent of activation in mature NK cells. In summary, miR-155-/- NK cells are more easily activated than WT NK cells, through increased expression of proteins in the PI3K, NF-kB, and calcineurin pathways, and miR-155-/- and 155-overexpressing NK cells exhibit increased IFN-g; production through distinct cellular mechanisms.

The miR-15/16 family is a miRNA family that is highly expressed in resting NK cells. Utilizing an NK cell-specific miR-15/16 deficient genetic model [15a/16-1LoxP-FlankedKnockOut (FKO)], we identified a critical role for miR-15/16 family miRNAs in the normal maturation of NK cells in vivo, with a specific reduction in mature CD11b+CD27- NK cells in multiple tissues. The mechanism responsible was a block in differentiation, since accelerated NK cell death was not evident, and earlier intermediates of NK cell maturation were expanded. Further, we identified Myb as a direct target of miR-15/16 in NK cells, with Myb expression increased in immature 15a/16-1FKO NK cells. Following adoptive transfer, immature 15a/16-1FKO NK cells exhibited defective maturation, which was rescued by ectopic miR-15/16 expression or Myb knockdown. Moreover, Myb overexpression resulted in defective NK cell maturation. Thus, miR-15/16 regulation of Myb controls the normal NK cell maturation program.

Collectively, these studies provide new insight into the diverse role of miRNAs in NK cells. These investigations further our understanding of NK cell biology, including activation and maturation, two processes vital to the function of these important immune cells.

Comments

This work is not available online per the author’s request. For access information, please contact digital@wumail.wustl.edu or visit http://digital.wustl.edu/publish/etd-search.html.

Permanent URL: http://dx.doi.org/10.7936/K7G15XV6

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