miR-196b target screen reveals mechanisms maintaining leukemia stemness with therapeutic potential.

We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27Kip1) is a direct miR-196b target whose repression enhanced an embryonic stem cell-like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo. Conversely, elevation of p27Kip1 significantly reduced MLL-r leukemia self-renewal, promoted monocytic differentiation of leukemic blasts, and induced cell death. Antagonism of miR-196b activity or pharmacologic inhibition of the Cks1-Skp2-containing SCF E3-ubiquitin ligase complex increased p27Kip1 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia.

A guide to choosing fluorescent protein combinations for flow cytometric analysis based on spectral overlap.

The advent of facile genome engineering technologies has made the generation of knock-in gene-expression or fusion-protein reporters more tractable. Fluorescent protein labeling of specific genes combined with surface marker profiling can more specifically identify a cell population. However, the question of which fluorescent proteins to utilize to generate reporter constructs is made difficult by the number of candidate proteins and the lack of updated experimental data on newer fluorescent proteins. Compounding this problem, most fluorescent proteins are designed and tested for use in microscopy. To address this, we cloned and characterized the detection sensitivity, spectral overlap, and spillover spreading of 13 monomeric fluorescent proteins to determine utility in multicolor panels. We identified a group of five fluorescent proteins with high signal to noise ratio, minimal spectral overlap, and low spillover spreading making them compatible for multicolor experiments. Specifically, generating reporters with combinations of three of these proteins would allow efficient measurements even at low-level expression. Because the proteins are monomeric, they could function either as gene-expression or as fusion-protein reporters. Additionally, this approach can be generalized as new fluorescent proteins are developed to determine their usefulness in multicolor panels. © 2018 International Society for Advancement of Cytometry.

Extrinsic allospecific signals of hematopoietic origin dictate iNKT cell lineage-fate decisions during development.

Invariant NKT (iNKT) cells are critical to the maintenance of tolerance toward alloantigens encountered during postnatal life pointing to the existence of a process for self-education. However, the impact of developmentally encountered alloantigens in shaping the phenotype and function of iNKT cells has not been described. To better understand this process, the current report examined naïve iNKT cells as they matured in an allogeneic environment. Following the prenatal transfer of fetal hematopoietic cells between age-matched allogeneic murine fetuses, cell-extrinsic signals appeared to dictate allospecific patterns of Ly49 receptor expression and lineage diversity in developing iNKT cells. Regulation for this process arose from cells of hematopoietic origin requiring only rare exposure to facilitate broad changes in developing iNKT cells. These findings highlight surprisingly asymmetric allospecific alterations in iNKT cells as they develop and mature in an allogeneic environment and establish a new paradigm for study of the self-education of iNKT cells.

Prenatal Allogeneic Tolerance in Mice Remains Stable Despite Potent Viral Immune Activation.

Transplanting stem cells before birth offers an unparalleled opportunity to initiate corrective treatment for numerous childhood diseases with minimal or no host conditioning. Although long-term engraftment has been demonstrated following in utero hematopoietic cellular transplantation during immune quiescence, it is unclear if prenatal tolerance becomes unstable with immune activation such as during a viral syndrome. Using a murine model of in utero hematopoietic cellular transplantation, the impact of an infection with lymphocytic choriomeningitis virus on prenatal allospecific tolerance was examined. The findings in this report illustrate that established mechanisms of donor-specific tolerance are strained during potent immune activation. Specifically, a transient reversal in the anergy of alloreactive lymphocytes is seen in parallel with the global immune response toward the virus. However, these changes return to baseline following resolution of the infection. Importantly, prenatal engraftment remains stable during and after immune activation. Collectively, these findings illustrate the robust nature of allospecific tolerance in prenatal mixed chimerism compared with models of postnatal chimerism and provides additional support for the prenatal approach to the treatment of congenital benign cellular disease.