NK Cell Research – Basic Immunobiology

NK Cell Research – Basic Immunobiology

Natural Killer Cells

NK cells are innate lymphoid cells important for host defense against infection (especially viruses) and mediate anti-tumor responses.  One major focus of the laboratory is to advance our understand the cellular and molecular events that regulate NK cell development and function. This includes the expression of microRNAs (miRNAs) and their contribution to the molecular regulation of NK cell biology, as well as transcription factors, and signaling pathways.  Recent studies have demonstrated that NK cells remember their prior experiences, and exhibit memory-like responses following combined cytokine activation, viral infection, and hapten exposure.  A second focus of the laboratory is defining mechanisms whereby cytokine activation results in memory-like NK cell responses.

The role of microRNAs in regulating NK cell development and function

MicroRNA expression by NK cells

MicroRNAs (miRNAs) are a large family of small non-coding RNAs that limit the expression of proteins by binding to the 3’UTR of mRNAs as causing translational blockade or mRNA degradation.  There are thought to be >1000 miRNA sequences in mammalian genomes, and expression is highly regulated dependent on organ, tissue, and cell type.  Our laboratory, in collaboration with The Genome Institute at Washington University, developed a small RNA sequencing pipeline using next-generation Illumina-based sequencing (Fehniger et al., Genome Research, 2010).  We applied this approach to mouse NK cells isolated from the spleen, and cataloged the relative expression of known and novel miRNAs in resting and IL-15-activated NK cells.  This represented the first list of miRNAs expressed by primary resting NK cells, and identified >300 expressed mature miRNA sequences, although only 50-100 were abundantly expressed.  We have performed similar next-generation sequencing of primary human NK cells using Illumina smallRNA sequencing.  Based on these unbiased sequencing experiments, we have developed a custom multiplexed quantitative real-time PCR assay that provides quantification for 96 of the most abundant miRNAs in NK cells.  Ongoing studies are evaluating the expression of miRNAs in various NK cell subsets, tissue sites, and modes of activation, in both human and mouse NK cells.

MicroRNAs regulate NK cell development and function

While identifying the miRNAs present in NK cells is an important first step, we also have evaluated the overall importance of these small RNAs to NK cell biology using genetic mouse models.  We engineered mice to have defective miRNA biogenesis (Dicer1 floxed) in a lymphocyte restricted fashion (hCD2-Cre).  MiRNA-deficient NK cells have defective development and survival, manifest as many fewer NK cells in vivo.  However, those NK cells that did develop appeared ‘hyperfunctional’ with exaggerated IFN-γ and degranluation responses to cytokine, activation-receptor, and tumor cell stimulation.  This same enhanced function was evident during a model viral infection by murine cytomegalovirus (MCMV).  This study identified the key role of miRNAs in regulating NK cells, and pointed to specific NK cell functions that may be most sensitive to miRNA-mediated regulation (Sullivan RP et al., J Immunol 2012).

The role of specific microRNAs in regulating NK cell development and function

Based on expression in NK cells, regulation during activation, and predicted miRNA targets our lab is studying a number of specific miRNAs (e.g., miR-155, miR-15/16, miR-142, miR-146) as regulators of NK cell biology, both in human NK cells and model organisms.  Using this approach we will elucidate the mechanisms whereby miRNAs regulate the NK cell developmental and functional programs.

Mir-155 regulates NK cell activation through complex mechanisms. Sullivan RP et. al., J Immunol, 2013

Mir-15/16 regulates NK cell maturation by targeting c-Myb. Sullivan RP et al., J Immunol, 2015

Our group has published comprehensive reviews on the miRNA regulation of NK cells:

 The role of PTEN in regulating NK cell development and function

Signaling via cell surface receptors provides key survival and activation signals that control many aspects of NK cell biology.  PTEN is a negative regulator of the PI3K pathway, and we have recently identified it as a critical regulator of NK cell trafficking during homeostasis and malignancy.  Current research focuses on defining the impact and mechanisms whereby PTEN loss alters NK cell functionality.

Mechanisms controlling cytokine-induced memory-like NK cell differentiation

Human cytokine-induced memory-like NK cells are formed after a brief combined pre-activation with IL-12, IL-15, and IL-18.  We have established that memory-like NK cells express the high affinity IL-2Rαβγ allowing responses to low concentrations of IL-2.  In addition, memory-like NK differentiation rescues hypofunctional unlicensed / uneducated NK cells (those lacking a self-inhibitory KIR receptor).  Current studies are evaluating several molecular mechanisms controlling NK cell memory-like generation.  These memory-like NK cells have considerable promise as anti-cancer immune effectors, and translational and research efforts in the laboratory are exploring their enhanced anti-tumor properties, and testing memory-like NK cells in clinical trials for cancer patients.

 

FUNDING:

The work has been supported by a number of grants and awards:

  • Edward J. Mallinckrodt Foundation Award
  • American Society of Hematology Scholar Award (Junior Faculty, Basic Science)
  • Howard Hughes Medical Institute Physician-Scientist Early Career Award
  • K08 Award form the NIH/NHLBI
  • R01 Award from the NIH/NIAID
  • Siteman Cancer Center Investment Program (St. Louis Pedal the Cause)