Bcl11b: A Master Transcription Factor Controlling Human NK Cell Development

PROJECT SUMMARY/ABSTRACT Natural killer (NK) cells are innate lymphocytes that mediate cellular cytotoxicity against virally infected and malignant cells. Upon activation, they also release chemokines and cytokines that help prime and coordinate the adaptive immune response. Because of these functional properties, there is continued interest in developing NK cell products for immunotherapy. However, this effort is hindered by our limited understanding of human NK cell differentiation and the transcription factor networks that regulate this process. In work supported by current K99/R00 funding, we generated a considerable amount of preliminary data comprehensively characterizing the transcriptional and epigenetic landscapes in sorted NK and CD8+ T cell subsets isolated from cytomegalovirus (CMV) seropositive donors. To complement these analyses, we performed flow cytometry-based analyses of transcription factor expression and ChIP-seq on sorted peripheral blood NK cell subsets to construct transcription factor networks that potentially regulate key steps in NK cell differentiation. The culmination of this work revealed a central role for Bcl11b. This was completely unexpected given several detailed studies in mice showing that Bcl11b expression is restricted to the T, NKT, and ILC2 lineages and acts to enforce T cell identity. Our preliminary results also revealed a reciprocal relationship between Runx2 and Bcl11b, with Runx2 regulating a set of transcription factors enriched in immature NK cells and Bcl11b regulating genes enriched throughout canonical NK cell maturation and in adaptive NK cells exhibiting a T cell-like gene expression signature. In this application we will test the hypothesis that Runx2 is a major hub that enforces the epigenetic identity of immature CD56bright NK cells, while Bcl11b suppresses the Runx2-directed transcriptional program to drive canonical NK cell differentiation, maturation, and cytotoxic effector function. We also posit that high levels of Bcl11b contribute to the T cell-like features of CMV-induced adaptive NK cells. We propose two independent aims to test our hypotheses. In the first aim, we will use overexpression and knockdown strategies in sorted NK cell subsets to define the roles of Bcl11b and Runx2 during canonical NK cell differentiation. These results will guide subsequent experiments designed to determine whether Bcl11b overexpression in hematopoietic progenitor cells (HPCs) promotes NK cell maturation and effector function both in vitro and after adoptive transfer into mice with established tumors. In the second aim, we will determine the role of Bcl11b in promoting T cell-associated features of CMV-induced adaptive NK cells and test the hypothesis that high levels of Bcl11b along with activating receptor and cytokine receptor stimulation drives adaptive NK cell differentiation and maturation. We will also test the hypothesis that adaptive NK cells mediate superior antitumor function in vivo relative to canonical NK cells. Results generated from this proposal will define the role of Bcl11b in promoting canonical and adaptive NK cell differentiation, determine whether Bcl11b overexpression enhances NK cell-mediated antitumor activity, and definitively test the antitumor function of adaptive NK cells.