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 (p27) 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 p27 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 p27 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia.

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.

Transplanting stem cells before birth offers an unparalleled opportunity to initiate corrective treatment for numerous childhood diseases with minimal or no host conditioning. While long-term engraftment has been demonstrated following in utero hematopoietic cellular transplantation (IUHCT) 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 IUHCT, the impact of an infection with lymphocytic choriomenigitic virus (LCMV) 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 to models of postnatal chimerism and provides additional support for the prenatal approach to the treatment of congenital benign cellular disease.

Prenatal transplantation capitalizes on the unique fetal environment, allowing for life-long engraftment of allogeneic stem cells without the need for harsh conditioning regimens. A prerequisite for stable engraftment of allogeneic cells likely requires the negative selection of donor-specific host effector T cells (Teff) and the support of donor-specific regulatory T cells (Tregs). However, little is known about the interplay between these cell types during development. The purpose of this study was to characterize the dynamic relationship between donor-specific Teff and Tregs as they emerge during development. Prenatal allogeneic chimeras were established by in utero transplantation of E14 fetal liver light density cells into age-matched allogeneic fetal recipients (Balb/c to B6 or B6 to Balb/c). In this model, immature T cells from B6 mice expressing TCRv-beta-5, 11, and 12 are negatively selected by mtv-8 superantigen complexed with I-E class MHC II on Balb/c cells. As alpha/beta TCR rearrangement does not occur until E16, this established transplantation model allows for alloantigen to be present from the earliest stages of thymic selection. Kinetic analysis of donor-specific T cell populations was performed in peripheral blood paired with in depth analysis in thymus and spleen in control and chimeric mice. Negative selection of donor-specific Teff cells occurs at an unexpectedly slow pace in Balb/c to B6 prenatal chimeras. Donor-specific CD4 and CD8 Teff are significantly decreased in frequency at 4 weeks of age but do not reach maximal deletion until 12 weeks of age (TCRv-beta-5 data shown in Figure A). Further analysis demonstrated that this slow elimination of donor-specific Teff was paired with an early increase in the frequency of donor-specific Tregs at 4 weeks of age (TCRv-beta-5 data shown in Figure B.) This increase in donor-specific Tregs likely occurred as a result of peripheral expansion as there was no change in the frequency of donor-specific Tregs in the thymus (Figure B) and no change in the frequency of these cells that expressed the markers of thymically derived natural Tregs neuropilin-1 or helios (data not shown). In agreement with this hypothesis, donor-specific splenic Tregs incorporated BrdU at a higher rate than other Tregs in young mice indicating a potential expansion of donor-specific Tregs in the periphery (TCRv-beta-5 data shown in Figure C.) Collectively, these data demonstrate that prenatal transplantation is characterized by: 1) a surprisingly slow reduction of donor-specific Teff subsets; 2) an early expansion of donor-specific Tregs. Further studies will explore the role of donor-specific Tregs in controlling early immunity to prenatally encountered antigens. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.