ALBERT

Description: Normalizing the environment recapitulates adult human immune traits in laboratory mice Nature 532, 7600 (2016). doi:10.1038/nature17655 Authors: Lalit K. Beura, Sara E. Hamilton, Kevin Bi, Jason M. Schenkel, Oludare A. Odumade, Kerry A. Casey, Emily A. Thompson, Kathryn A. Fraser, Pamela C. Rosato, Ali Filali-Mouhim, Rafick P. Sekaly, Marc K. Jenkins, Vaiva Vezys, W. Nicholas Haining, Stephen C. Jameson & David Masopust Our current understanding of immunology was largely defined in laboratory mice, partly because they are inbred and genetically homogeneous, can be genetically manipulated, allow kinetic tissue analyses to be carried out from the onset of disease, and permit the use of tractable disease models. Comparably reductionist experiments are neither technically nor ethically possible in humans. However, there is growing concern that laboratory mice do not reflect relevant aspects of the human immune system, which may account for failures to translate disease treatments from bench to bedside. Laboratory mice live in abnormally hygienic specific pathogen free (SPF) barrier facilities. Here we show that standard laboratory mouse husbandry has profound effects on the immune system and that environmental changes produce mice with immune systems closer to those of adult humans. Laboratory mice—like newborn, but not adult, humans—lack effector-differentiated and mucosally distributed memory T cells. These cell populations were present in free-living barn populations of feral mice and pet store mice with diverse microbial experience, and were induced in laboratory mice after co-housing with pet store mice, suggesting that the environment is involved in the induction of these cells. Altering the living conditions of mice profoundly affected the cellular composition of the innate and adaptive immune systems, resulted in global changes in blood cell gene expression to patterns that more closely reflected the immune signatures of adult humans rather than neonates, altered resistance to infection, and influenced T-cell differentiation in response to a de novo viral infection. These data highlight the effects of environment on the basal immune state and response to infection and suggest that restoring physiological microbial exposure in laboratory mice could provide a relevant tool for modelling immunological events in free-living organisms, including humans.

Description: Fetal and adult hematopoietic stem cells (HSCs) have distinct gene expression profiles and distinct functional properties. Differences between fetal and adult HSCs have been extensively characterized, but the mechanisms that regulate the transition from fetal to adult identity remain unclear. These mechanisms are important because they underlie temporal changes in self-renewal, lineage commitment and leukemogenesis. In mice, HSCs are generally thought to transition from fetal- to adult-like states between 3 and 4 weeks after birth. The transition is thought to be regulated by a cell-intrinsic program, and it is thought to be rapid. This model raises an important mechanistic question. How do HSCs record developmental time so that they can transition from fetal to adult states on cue? To address this question, we performed single cell RNA-seq on HSCs and committed hematopoietic progenitors (HPCs) at various pre- and post-natal time points. We used quadratic programming to calculate adult and fetal identity scores for each HSC at each age. Our analyses revealed several surprising findings. HSCs began transitioning from fetal to adult transcriptional states shortly after birth rather than at 3 weeks after birth. This was well before they became quiescent, and it was independent of location (i.e. P0 liver and bone progenitors had similar transcriptional states). The fetal to adult transition appeared graded rather than bi-modal (Figure 1). In other words, HSCs became incrementally more "adult-like" with each passing week, and all HSCs had relatively synchronous changes in their fetal and adult identity scores. Similar patterns were observed in HPCs. The graded transition of HSCs from fetal to adult identity raises the question of how such a transition could be encoded within the cis-regulatory architecture of the genome. To answer this question, we performed ATAC-seq and ChIP-seq (H3K4me1 and H3K27ac) to identify fetal- and adult-specific enhancers. We performed the assays in highly purified HSCs and HPCs, and we obtained similar results for each cell population. Adult-specific enhancers (adult-specific ATAC-seq peaks with overlapping H3K4me1 peaks) were enriched near genes that are more highly expressed in adult HSCs than in fetal HSCs, as one might expect. The putative enhancer regions were highly enriched for ETS, RUNX1 and AP-1 binding sites. When we evaluated these enhancers at E16.5, P7, P14 and P21, we found that ATAC-seq peak heights gradually increased as development progressed. To achieve this pattern at a population level, individual HSCs must commission enhancers in a non-uniform manner. Otherwise, fetal to adult peak-height changes would be binary. Our data suggest a simple mechanism for coordinating slow, graded, changes in HSC identity. As time passes, or as HSCs divide, individual enhancers may flip, stochastically but irreversibly, from fetal- to adult-like states. In this model, the transition from fetal to adult identity is not guided by hard-wired developmental cues. Rather, the rate of change depends on the stochastic probability of activating adult enhancers and inactivating fetal enhancers. This system differs from other developmental circuits wherein well-defined inductive signals activate super-enhancers and feed-forward loops to induce robust, uniform changes in cell identity. A stochastic circuit is simple, but it leads to epigenomic and transcriptional heterogeneity that has implications for lineage priming and leukemia initiation, particularly in children. Disclosures No relevant conflicts of interest to declare.

Description: Under normal homeostatic conditions, adult hematopoietic stem cells (HSCs) are usually quiescent. Hematopoietic stress, such as blood loss, infection, inflammation, or chemotherapy, can drive HSCs into cycle. When adult HSCs divide multiple times, they lose self-renewal capacity. Inflammatory cytokines, such as interleukin-1 (IL-1), can accelerate the loss of HSC self-renewal capacity by activating PU.1 and promoting myeloid commitment. This raises the question of whether intrinsic tumor suppressor genes can modulate sensitivity to inflammatory cytokines, and whether loss of these tumor suppressors can allow HSCs to evade commitment programs that would otherwise limit self-renewal capacity. The KMT2C tumor suppressor is located on chromosome 7q within a region that is frequently deleted in myelodsplastic syndrome (MDS) and therapy-related acute myeloid leukemia (AML). It encodes MLL3, a histone methyltransferase that activates enhancer elements and promotes transcription. Haploid KMT2C deletion has previously been shown to activate self-renewal programs and accelerate AML formation. This raised the question of whether KMT2C/MLL3 regulates normal HSC self-renewal and whether KMT2C deletion conveys a selective advantage to HSCs in contexts that would otherwise deplete the HSC pool. By protecting HSCs from exhaustion, KMT2C deletions may indirectly facilitate 7q-deficient MDS/AML. To understand whether and how Kmt2c regulates HSC self-renewal, we developed novel germline and conditional Kmt2c knockout mouse alleles. Mono- and bi-allelic Kmt2c deletions led to a modest increase in adult HSC numbers and a significant reduction in committed hematopoietic progenitors (HPCs). Kmt2c deletions markedly enhanced HSC self-renewal capacity, but HSC proliferation rates were not altered. To mimic conditions that lead to therapy-related AML, we deleted a single Kmt2c allele in a minority of HSCs. We then tested whether the Kmt2c-deleted HSC population expanded, relative to wild type HSCs, under native and stressed conditions. Under native conditions, the percentage of Kmt2c-heterozygous HSCs remained stable. However, after several cycles of chemotherapy, the Kmt2c mutant HSCs predominated within the marrow. In mechanistic studies, RNA-sequencing showed that Kmt2c-deficient HSCs expressed genes associated with innate immune signaling, including the receptor for interleukin-1 (IL1R), at lower levels than wild type HSCs. This suggested that Kmt2c mutations might sustain self-renewal capacity in multiply divided HSCs by dampening IL-1 driven myeloid commitment. In support of this hypothesis, Kmt2c-deficient HSCs retained multilineage potential when they were cultured with IL-1, and they failed to activate JNK and p38 upon exposure to IL-1. Altogether, our data suggest a mechanism to explain how KMT2C deletions, in the context of larger 7q deletions, may promote therapy related MDS/AML. When HSCs acquire a KMT2C deletion, they can then escape IL-1-mediated exhaustion when they are driven into cycle by chemotherapy or other stressors. In lieu of chemotherapy-induced stress, the same clones may remain relatively indolent. Disclosures No relevant conflicts of interest to declare.

Description: The FLT3 Internal Tandem Duplication (FLT3ITD) is common somatic mutation in acute myeloid leukemia (AML). We have previously shown that FLT3ITD fails to induce changes in HSC self-renewal, myelopoiesis and leukemogenesis during fetal stages of life. FLT3ITD signal transduction pathways are hyperactivated in fetal progenitors, but FLT3ITD target genes are not. This suggests that postnatal-specific transcription factors may be required to help induce FLT3ITD target gene expression. Alternatively, repressive histone modifications may impose a barrier to FLT3ITD target gene activation in fetal HPCs that is relaxed during postnatal development. To resolve these possibilities, we used ATAC-seq, as well as H3K4me1, H3K27ac and H3K27me3 ChIP-seq, to identify cis-elements that putatively control FLT3ITD target gene expression in fetal and adult hematopoietic progenitor cells (HPCs). We identified many enhancer elements (ATAC-seq peaks with H3K4me1 and H3K27ac) that exhibited increased chromatin accessibility and activity in FLT3ITD adult HPCs relative to wild type adult HPCs. These elements were enriched near FLT3ITD target genes. HOMER analysis showed enrichment for STAT5, ETS, RUNX1 and IRF binding motifs within the FLT3ITD target enhancers, but motifs for temporally dynamic transcription factors were not identified. We cloned a subset of the enhancers and confirmed that they could synergize with their promoter to activate a luciferase reporter. For representative enhancers, STAT5 binding sites were required to activate the enhancer - as anticipated - and RUNX1 repressed enhancer activity. We tested whether accessibility or priming changed between fetal and adult stages of HPC development. FLT3ITD-dependent changes in chromatin accessibility were not observed in fetal HPCs, though the enhancers were primed early in development as evidenced by the presence of H3K4me1. Repressive H3K27me3 were not present at FLT3ITD target enhancers in either or adult HPCs. The data show that FLT3ITD target enhancers are demarcated early in hematopoietic development, long before they become responsive to FLT3ITD signaling. Repressive marks do not appear to create an epigenetic barrier to enhancer activation in the fetal stage. Instead, age-specific transcription factors are likely required to pioneer enhancer elements so that they can respond to STAT5 and other FLT3ITD effectors. Disclosures No relevant conflicts of interest to declare.