Sphingolipids are membrane and bioactive lipids that are required for many aspects of normal mammalian development and physiology. However, the importance of the regulatory mechanisms that control sphingolipid levels in these processes is not well understood. The mammalian ORMDL proteins (ORMDL1, 2 and 3) mediate feedback inhibition of the de novo synthesis pathway of sphingolipids by inhibiting serine palmitoyl transferase in response to elevated ceramide levels. To understand the function of ORMDL proteins in vivo, we studied mouse knockouts (KOs) of the Ormdl genes. We found that Ormdl1 and Ormdl3 function redundantly to suppress the levels of bioactive sphingolipid metabolites during myelination of the sciatic nerve. Without proper ORMDL-mediated regulation of sphingolipid synthesis, severe dysmyelination results. Our data indicate that the Ormdls function to restrain sphingolipid metabolism in order to limit levels of dangerous metabolic intermediates that can interfere with essential physiological processes such as myelination.

The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro–B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro– to pre–B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1’s critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.

Background: Although chiefly a B-lymphocyte disorder, several research groups have identified common variable immunodeficiency (CVID) subjects with numeric and/or functional T H cell alterations. The causes, interrelationships, and consequences of CVID-associated CD4 1 T-cell derangements to hypogammaglobulinemia, autoantibody production, or both remain unclear. Objective: We sought to determine how circulating CD4 1 T cells are altered in CVID subjects with autoimmune cytopenias (AICs; CVID1AIC) and the causes of these derangements. Methods: Using hypothesis-generating, high-dimensional single-cell analyses, we created comprehensive phenotypic maps of circulating CD4 1 T cells. Differences between subject groups were confirmed in a large and genetically diverse cohort of CVID subjects (n 5 69) by using flow cytometry, transcriptional profiling, multiplex cytokine/chemokine detection, and a suite of in vitro functional assays measuring naive T-cell differentiation, B-cell/T-cell cocultures, and regulatory T-cell suppression. Results: Although CD4 1 T H cell profiles from healthy donors and CVID subjects without AICs were virtually indistinguishable, T cells from CVID1AIC subjects exhibited follicular features as early as thymic egress. Follicular skewing correlated with IgA deficiencyassociated endotoxemia and endotoxin-induced expression of activin A and inducible T-cell costimulator ligand. The resulting enlarged circulating follicular helper T-cell population from CVID1AIC subjects provided efficient help to receptive healthy donor B cells but not unresponsive CVID B cells. Despite this, circulating follicular helper T cells from CVID1AIC subjects exhibited aberrant transcriptional profiles and altered chemokine/cytokine receptor expression patterns that interfered with regulatory T-cell suppression assays and were associated with autoantibody production. Conclusions: Endotoxemia is associated with early commitment to the follicular T-cell lineage in IgA-deficient CVID subjects, particularly those with AICs.

RATIONALE: PU.1, encoded by the gene SPI1, is an essential hematopoietic pioneer transcription factor. Although PU.1 deficiency is lethal in mice and somatic SPI1 mutations are common in human myeloid leukemias, primary PU.1-mediated diseases have not been described. Here, we identify four unrelated, agammaglobulinemic subjects each harboring novel, non-synonymous heterozygous SPI1mutations. We sought to determine if and how SPI1 mutations cause PU.1-mutated agammaglobulinemia (PU.MA). METHODS: PU.MA subject DNA, peripheral blood and bone marrow samples were analyzed with whole exome sequencing, flow cytometry and immunohistochemistry. Effect of PU.1 mutation on chromatin accessibility, transcription, molecular interactions, nuclear localization and DNA binding were assessed using a custom PU.1 reporter cell line, immunoprecipitation, confocal microscopy and electrophoretic mobility shift assays. B-cell developmental arrest was modelled in genetically edited in human pro-B cell lines and primary stem cells. RESULTS: All PU.MA subjects lack circulating B cells and are deficient PU.1 high expressing myeloid cells. PU.MA marrow samples displayed developmental B-cell arrest. In HEK293 cells, PU.1 mutants failed to drive a PU.1-dependent promoter but did not interfere with wild type PU.1's ability to do so. Two of four mutant SPI1 alleles failed to produce detectable protein. Remaining PU.1 mutant proteins were expressed but did not bind their target DNA sequence. Genetically edited cells and lines modelled features of PU.MA B-cell development. CONCLUSIONS: We identified and molecularly described the molecular basis of a novel autosomal dominant form of congenital agammaglobulinemia caused by PU.1 haploinsufficiency. PU.MA underscores the essential, dose-dependent role of PU.1 in B-cell lymphopoiesis.