Our lab discovered that natural killer (NK) cells use a perforin-dependent mechanism to constrain antiviral T cell responses during infection (Waggoner et al. Nature 2011). This mechanism limits follicular helper CD4 T cell responses and associated germinal center reactions after infection (Rydyznski et al. Nature Communications 2015) or immunization (Rydyznski et al. Cell Reports 2018). As a result, the activity of NK cells is associated with weaker elicitation of antiviral neutralizing antibodies in mice, a phenomenon consistent with observations made during HIV infection and after administration of yellow fever or hepatitis B virus vaccines in humans.

We are focused on discovery of mechanisms and mediators involved in NK cell suppression of adaptive immunity. We aim to translate these discoveries into innovative interventions that can transiently limit this activity of NK cells during vaccination in order to amplify resulting T and B cell responses to a level that can prevent infection with HIV or other causes of human disease.

This project started with NIH Director’s Pioneer (DP1) funding in the form of a 2014 Avant-Garde Award from the National Institute on Drug Abuse, and continues with a 2019 R01 from the National Institute of Allergy and Infectious Diseases (collaboration with Barton Haynes and Derek Cain at Duke University). 

Currently, we are focused on eight major themes using mice, non-human primates, human cells, and samples from clinical trials (collaboration with Paul Spearman, David Bernstein, and the VTEU at Cincinnati Children’s). First, we are assessing application of small molecule inhibitors of NK cell killing activity during immunization as a means to enhance vaccine efficacy (Patent Pending). Second, we aim to therapeutically interfere with migration of immunoregulatory NK cells to sites of priming and follicular differentiation of T cells (Ali et al. Journal of Clinical Investigation 2021). Third, we are characterizing NK cell responses after vaccination (Gyurova et al. Frontiers in Immunology 2019). Fourth, we are characterizing newly discovered transcription programs governing key aspects of the immunosuppressive functions of NK cells. Fifth, we are exploring specific receptors implicated in the immunosuppressive interaction between NK cells and their T-cell targets. Sixth, we are using single-cell transcriptomics and high-dimensional flow to discover discrete subsets of NK and T cells involved in this phenomenon. Seventh, we are using the Collaborative Cross resource to identify genetic regulators of the immunosuppressive capacity of NK cells. Eighth, we are evaluating the ‘purpose’ of this activity of NK cells by assessing whether blockade of immunoregulatory NK cells increases risk for autoimmunity.

Overall, we believe these studies will continue to uncover exciting new biology with the potential to facilitate development of innovative methods to modulate NK cells to enhance the ability of vaccines to block infection and prevent or treat disease.

The studies in our lab are organized symmetrically, with the yin of NK cells restricting vaccine efficacy balanced by the yang of NK-cell dysfunction in autoimmunity contributing to pathogenic adaptive immune responses. The mediators and pathways discovered as contributors to NK cell suppression of vaccine responses in the project above are then explored as potential targets to restore dysfunctional activity of NK cells in patients with lupus or other autoimmune diseases. Likewise, this project focuses on identification of genetic and molecular mechanisms governing dysfunction of NK cells in patients with autoimmune disease that could be applied in the context of vaccines to limit immunosuppressive functions of NK cells.

We share a National Institute of Arthritis and Musculoskeletal and Skin Disease R01 with the labs of Matt Weirauch and Leah Kottyan that is focused on PU.1/SPI.1 as a transcriptional driver of NK cell dysfunction through intersection with non-coding genetic risk loci promoting the development of lupus. This project also involves dissection of the interplay between PU.1 and a coding risk variant in IRF7 regulating responses of NK cells and other cells to interferon. This project and a Center for Pediatric Genomics pilot grant facilitated generation of new ChIP-seq, ATAC-seq, and RNA-seq datasets in NK cells from both healthy individuals and patients with lupus.

These studies are being extended to patients with juvenile idiopathic arthritis (JIA, collaboration with Susan Thompson) and systemic JIA (collaboration with Grant Schulert, Alexei Grom, Hermine Brunner, Sherry Thornton, Sandra Andorf), most notably those with fatal lung or macrophage activation syndrome complications. Transcriptomic analysis of NK cells in pediatric patients with lupus facilitated discovery of at least two new transcriptional programs, the roles of which in NK cell dysfunction are currently under investigation.

Inspired by the natural immunoregulatory functions of NK cells, our lab constructed a new affinity-based chimeric antigen receptor (CAR) from programmed death-ligand 1 (PD-L1) that when expressed by NK cells (or other cytotoxic cells) permits selective killing of targets that express high (but not intermediate or low) levels of PD-1 (Reighard et al. Cell Reports Medicine 2020). Human NK cells and mouse T cells expressing this CAR exhibited a selective capacity to kill follicular helper subsets of T cells (PD-1high) without depleting PD-1int/low populations of T or B cells or other leukocytes (Patent Pending with Hermine Brunner). These CAR-expressing cells could selective eliminate follicular T cells in mice and in humanized mice (collaboration with Marat Khodoun) with beneficial reduction of measures of disease activity. Our lab continues to advance use of this and related innovative CAR constructs in therapy of autoimmune diseases and chronic virus infection in animal models. We continue to endeavor to translate the success of this strategy into the clinic.

In studies supported by an NIH R21 and using the Collaborative Cross mouse genetic resource (Collaboration with Leah Kottyan, Hermine Brunner, Prasad Devarajan, Michael Taylor, and Wen-Hai Shao), we are pursuing identification of genetic loci governing involvement of heart, kidney, skin, lung, joint, and heart in manifestation of induced lupus-like disease. This study is predicated on the idea that genetically distinct mice administered disease-inducing chemicals will reproducibly develop disease involving select organ systems but not others, permitting fine mapping of loci involved in specific manifestations of this heterogeneous autoimmune disease.

Investigation of additional non-canonical functions of NK cells during chronic virus infection uncovered a role for NK cells in immune resilience. A subset of NK cell develops a new functionality that acts to protect the marginal zone from undergoing complete T-cell dependent atrophy. In the absence of NK cells or their specific ability to exert this novel functionality, the populations of marginal zone macrophages and B cells completely collapse. This collapse makes virus-infected mice highly susceptible to secondary infections with Listeria monocytogenes or Streptococcus pneumoniae. Our lab continues to investigate the molecular mechanisms governing this newly discovered non-canonical function of NK cells and to explore the relevance of this phenomenon to clinical disease in humans.

Our transcriptomic studies of immunoregulatory NK cells have serendipitously revealed a number of factors not previously implicated in the general biology of NK cells. In one project, we are exploring the essential functions of a DEAD box RNA helicase in development and later tumorigenesis of NK cells. Mice lacking both copies of this gene lack NK cells, while somatic loss of this gene is associated with transformation of NK cells.

A second project focused on a Forkhead Box transcription factor not previously studied in NK cells revealed a critical role for this gene in development of a functional NK cell compartment in mice.

A third project implicates a basic helix-loop-helix in functional maturation of NK cells. Our lab continues to use cutting-edge omics (i.e. paired single-cell nuclear RNA-seq and ATAC-seq), newly generated conditional knockout mice, and parallel CRISPR editing of human NK cells to explore the function of these and other genes in NK cell biology.

Our vaccine studies branched out into an attempt to understand how cytokines induced by various adjuvants or vaccine platforms could modulate NK cell functional pleiotropy. A former postdoc, David Ohayon, identified multiple different modalities of cytokine synergy that resulting in hyperfunctionality of NK cells (Ohayon et al. Journal of Leukocyte Biology 2020). Through ChIP-seq and ATAC-seq, we uncovered transcriptional and epigenetic mechanisms linked to this synergy that have implications for various disease states.

With Michael Borchers, we are exploring how this synergy can contribute to pathogenic NK cell functionality in COPD. With Jonathan Bernstein, we recognized potential contributions of cytokine-elicited NK cell hyperfunctionality to severe asthma. With Neeru Hershey, we are exploring abnormalities of NK cell phenotype and function that may be central to development of allergic sensitivity and asthma in children with atopic dermatitis.

NK cells are one type of innate lymphoid cells, which also includes ILC1, ILC2, LTi, and ILC3. A balanced number of and functional contribution from each type of ILC at mucosal sites is critical for health, and is frequently perturbed in disease states (e.g. inflammatory bowel disease).

Our lab is using promoter capture Hi-C, single-cell RNA- and ATAC-seq, ChIP-seq, CRISPRa/i, and CUT&RUN in collaboration with Emily Miraldi, Mikhail Spivakov, Valeriya Malysheva, Gene Oltz, and Michael Rosen to identify new gene regulatory networks governing the functional identify and plasticity of these various lineages. These studies spawned from an initial gene regulatory network in mouse intestinal ILC at steady state in which BCL6 was found to be a critical regulatory of group 1 and group 3 ILC in the gut (Pokrovskii et al Immunity 2019). The current studies now employ unique strains of mice as well as spleen, tonsil, blood, and intestinal tissues from humans. Several transcription factors and a variety of disease-associated genes not previously linked to this type of cell are being studies in the context of autoimmune, inflammatory, and neuroimmune disease development.

The exceptional collegiality and collaborate-first mentality of our colleagues in Cincinnati has enabled our lab to expansively pursue not only the projects described in detail above, but also to assess novel immune mechanisms contributing to other diseases.

With funding from the Cincinnati Research Foundation (collaboration with Marc Rothenberg, Sean Lang, Ken Kaufman, and Mingxia Gu), we identified a peroxidase gene mutation linked to giant aneurysm formation in Kawasaki Disease.

In another project, we have discovered a unique proteomic mechanism by which exosomes derived from human NK cells can modulate survival of other lymphocytes in disease settings (collaboration with Kim Risma).

A new student in the lab, Hilal Cevik, has uncovered an inflammatory mechanism through which NK cells control inflammation and tissue repair after muscle injury (collaboration with Doug Millay, Jeff Molkentin, Mattia Quattrocelli).

We are collaborating with numerous colleagues at Children’s Hospital (Brian Turpin, Richard Lu, Ralph Vatner, Marc Wunderlich, Gang Huang, Yuting Tang) and the University of Cincinnati (Laura Conforti, Trisha Wise-Draper) to develop novel strategies for enhancing or better employing the inherent antitumor functions to attack sarcomas, brain tumors, and other difficult-to-treat malignancies.

We are also exploring unexpected interactions between the hemostatic systemic and the function of T as well as NK cells during virus infection or immune disease in collaboration with Joe Palumbo, Neeru Hershey, and Michael Sherenian.

We are very interested in how NK cells contribute to chronicity and resolution of pain in diseases like fibromyalgia (Christopher King, Michael Jankowski).