The cellular and molecular basis of defects in Down syndrome fetal bone marrow B-lymphopoiesis

Background Trisomy of the chromosome 21 (T21) causes a broad clinical syndrome called Down syndrome (DS). Children with DS have an increased risk of acute lymphoblastic leukaemia of DS (DS-ALL) and reports show qualitative and functional abnormalities in B-cells within trisomic individuals as young as neonatal stages, which indicates these abnormalities begin before birth. Our lab has shown that T21 perturbs haematopoiesis in fetal liver (FL), causing a bias toward the megakaryocytic and erythroid lineages at the expense of B-progenitors. Preliminary work suggests this is also the case in fetal bone marrow (FBM), the main site of B-lymphopoiesis before birth. Thus far, most work on lymphoid defects of DS, particularly in fetal tissue, has focused on B-cell development despite reports that T21 disrupts both T/NK lymphocytes. Therefore, it is unclear whether impairments are specific to the B-lineage or the result of disrupted pan-lymphoid differentiation. The exact mechanisms for how T21 impacts haematopoiesis are unknown. Several genes which have roles in haematopoiesis including RUNX1, BACH1, ERG and DYRK1A, are encoded on chromosome 21 and their altered expression may disrupt haematopoiesis. More recent evidence suggests that epigenetic mechanisms may also play a role. Aim The aim of this project was to refine the existing model fetal B-lymphopoiesis use this as a reference point to i) identify the specific immunophenotypic and functional differences in B-progenitor differentiation between normal and DS FBM and ii) to understand these changes at the molecular level. Results Discovery of new lymphomyeloid progenitors in FBM helps refine the fetal B-lymphopoiesis model: Immunophenotypic profiling of normal and DS FBM revealed the existence of 3 newly described progenitors: CD10-/+ LMPP and Pre-ELP/GMP. Extensive functional characterisation places CD10-LMPP upstream of both CD10+LMPP and Pre-ELP/GMP in the differentiation hierarchy and reveals these populations support the expansion of B-cells in FBM. Whilst CD10- LMPP and Pre-ELP/GMP also show myeloid potential, CD10+LMPP are comparatively lymphoid primed. Preliminary immunophenotyping of cord blood (CB), suggests that Pre-ELP/GMP are enriched in FBM, like other CD10- progenitors: ELP and PreProB, and the frequency of CD10+LMPP is significantly expanded in CB relative to FBM. The functional impact of T21 on B-lymphopoiesis in FBM: Immunophenotyping of DS FBM shows large-scale alterations to HSPC frequencies including a significant depletion of B-progenitors, most severely the CD10- ELP and PreProB and a significant expansion of HSCs and MEPs. Interestingly, CD10+ LMPP, which are rare in FBM, are significantly expanded in DS and ProB show a less severe reduction, suggesting that CD10-/+ HSPCs are affected differently in DS. Functional assays reveal a severe B-cell defect in upstream and intermediate DS HSPCs. T-cell differentiation is also impaired, but less markedly and CD10-LMPP and Pre-ELP/GMP appear to be junctions through which myeloid biasing is propagated. Interestingly, almost all of the DS FBM HSPCs showed an NK bias, which is the first time this has been described in fetal tissue. DS FBM HSCs and MPPs showed a significant erythroid bias on methylcellulose. Understanding the impact of T21 on FBM HSPCs at the molecular level: Gene expression and chromatin accessibility profiles demonstrate that CD10-LMPP and Pre-ELP/GMP are distinct populations. Clustering based on gene expression and chromatin accessibility profiles resolves the proposed lineage hierarchies in FBM, but these are altered in DS where Pre-ELP/GMP and MPP show greater similarity to GMP and MEP respectively. RNA sequencing of several DS FBM HSPCs including the newly described progenitors revealed significantly reduced expression of several B-cell genes and elevated expression of genes associated with the myeloid, mega-erythroid and – to a lesser degree- NK lineages. Conclusions The identification and characterisation of new progenitor populations in FBM has facilitated existing models of fetal B-lymphopoiesis to be refined. In DS FBM, lymphopoiesis is disrupted with a severe block in B-cell differentiation that appears to occur upstream and is propagated through the newly described progenitors, as well as moderately impaired T-cell development and a skew toward NK lymphopoiesis. These findings were validated at the molecular level, with T21 causing broad changes in both the transcriptome and chromatin accessibility of FBM HSPCs.