CytoNorm : a normalization algorithm for cytometry data

High-dimensional flow cytometry has matured to a level that enables deep phenotyping of cellular systems at a clinical scale. The resulting high-content data sets allow characterizing the human immune system at unprecedented single cell resolution. However, the results are highly dependent on sample preparation and measurements might drift over time. While various controls exist for assessment and improvement of data quality in a single sample, the challenges of cross-sample normalization attempts have been limited to aligning marker distributions across subjects. These approaches, inspired by bulk genomics and proteomics assays, ignore the single-cell nature of the data and risk the removal of biologically relevant signals. This work proposes CytoNorm, a normalization algorithm to ensure internal consistency between clinical samples based on shared controls across various study batches. Data from the shared controls is used to learn the appropriate transformations for each batch (e.g., each analysis day). Importantly, some sources of technical variation are strongly influenced by the amount of protein expressed on specific cell types, requiring several population-specific transformations to normalize cells from a heterogeneous sample. To address this, our approach first identifies the overall cellular distribution using a clustering step, and calculates subset-specific transformations on the control samples by computing their quantile distributions and aligning them with splines. These transformations are then applied to all other clinical samples in the batch to remove the batch-specific variations. We evaluated the algorithm on a customized data set with two shared controls across batches. One control sample was used for calculation of the normalization transformations and the second control was used as a blinded test set and evaluated with Earth Mover's distance. Additional results are provided using two real-world clinical data sets. Overall, our method compared favorably to standard normalization procedures

Presence of innate lymphoid cells in allogeneic hematopoietic grafts correlates with reduced graft-versus-host disease.

BACKGROUND Allogeneic hematopoietic cell transplantation (HCT) can be devastating when graft-versus-host disease (GvHD) develops. GvHD is characterized by mucosal inflammation due to breaching of epithelial barriers. Innate lymphoid cells (ILCs) are immune modulatory cells that are important in the maintenance of epithelial barriers, via their production of interleukin (IL)-22 and their T cell suppressive properties. After chemo- and radiotherapy, ILCs are depleted, and recovery after remission-induction therapy and after allogeneic HCT is slow and incomplete in a significant number of patients, which is associated with an increased risk to develop acute GvHD. OBJECTIVE To investigate whether the presence of mature ILCs within G-CSF-mobilized HCT grafts is correlated with the development of acute GvHD after allogeneic HCT. STUDY DESIGN We analyzed ILCs in a cohort of 36 patients who received allogeneic HCT for a hematologic malignancy, by flow-cytometric immune-phenotyping of prospectively collected, cryopreserved peripheral blood mononuclear cells (PBMCs) and donor-derived HCT grafts collected for the same patients. Biased analysis, with ILCs defined as CD3-lineage-CD45+CD127+CD161+ lymphocytes, was performed using FlowJo version 10 software. Unbiased analysis was done using FlowSOM, which uses a self-organizing map (SOM) with a minimal spanning tree (MST) to define and visualize different clusters present in the samples. RESULTS Remission-induction therapy significantly depleted ILCs from the blood, and patients who had a relatively low percentage of ILCs before allogeneic HCT were significantly more prone to develop acute GvHD, confirming previous findings in a separate cohort. Allogeneic HCT grafts, which were all obtained from the blood of G-CSF-mobilized healthy donors, contained ILCs at a frequency very similar to the peripheral blood of healthy individuals. The ILC subset composition was also comparable to that of the blood of healthy individuals, with the exception of NKp44+ ILC3s, which were significantly more abundant in HCT grafts. The relative ILC content of the graft tended to correlate with ILC reconstitution after allogeneic HCT, suggesting that peripheral expansion of transplanted mature ILCs may contribute to early ILC reconstitution after allogeneic HCT. Patients who received a relatively ILC-poor HCT graft had a significantly increased risk to develop acute GvHD, compared with patients who received relatively ILC-rich allogeneic HCT grafts. Unbiased phenotypic analysis with the FlowSOM algorithm confirmed that allogeneic HCT grafts of patients who developed acute GvHD contained a lower frequency of ILCs that clustered in NKp44+ ILC3 signature groups. CONCLUSION The presence of ILCs in allogeneic HCT grafts is associated with a reduced risk to develop acute GvHD. These data suggest that enhancement of ILC reconstitution of ILC3s in particular, for example via adoptive transfer of ILCs, may prevent acute GvHD and has the potential to improve outcome of allogeneic HCT recipients

De novo variants disturbing the transactivation capacity of POU3F3 cause a characteristic neurodevelopmental disorder

POU3F3, also referred to as Brain-1, is a well-known transcription factor involved in the development of the central nervous system, but it has not previously been associated with a neurodevelopmental disorder. Here, we report the identification of 19 individuals with heterozygous POU3F3 disruptions, most of which are de novo variants. All individuals had developmental delays and/or intellectual disability and impairments in speech and language skills. Thirteen individuals had characteristic low-set, prominent, and/or cupped ears. Brain abnormalities were observed in seven of eleven MRI reports. POU3F3 is an intronless gene, insensitive to nonsense-mediated decay, and 13 individuals carried protein-truncating variants. All truncating variants that we tested in cellular models led to aberrant subcellular localization of the encoded protein. Luciferase assays demonstrated negative effects of these alleles on transcriptional activation of a reporter with a FOXP2-derived binding motif. In addition to the loss-of-function variants, five individuals had missense variants that clustered at specific positions within the functional domains, and one small in-frame deletion was identified. Two missense variants showed reduced transactivation capacity in our assays, whereas one variant displayed gain-of-function effects, suggesting a distinct pathophysiological mechanism. In bioluminescence resonance energy transfer (BRET) interaction assays, all the truncated POU3F3 versions that we tested had significantly impaired dimerization capacities, whereas all missense variants showed unaffected dimerization with wild-type POU3F3. Taken together, our identification and functional cell-based analyses of pathogenic variants in POU3F3, coupled with a clinical characterization, implicate disruptions of this gene in a characteristic neurodevelopmental disorder

Increased CCL2, CCL3, CCL5, and IL-1β cytokine concentration in piriform cortex, hippocampus, and neocortex after pilocarpine-induced seizures

BACKGROUND: Cytokines and chemokines play an important role in the neuroinflammatory response to an initial precipitating injury such as status epilepticus (SE). These signaling molecules participate in recruitment of immune cells, including brain macrophages (microglia), as well as neuroplastic changes, deterioration of damaged tissue, and epileptogenesis. This study describes the temporal and brain region pattern expression of numerous cytokines, including chemokines, after pilocarpine-induced seizures and discusses them in the larger context of their potential involvement in the changes that precede the development of epilepsy. FINDINGS: Adult rats received pilocarpine to induce SE and 90 min after seizure onset were treated with diazepam to mitigate seizures. Rats were subsequently deeply anesthetized and brain regions (hippocampus, piriform cortex, neocortex, and cerebellum) were freshly dissected at 2, 6, and 24 h or 5 days after seizures. Using methodology identical to our previous studies, simultaneous assay of multiple cytokines (CCL2, CCL3, CCL5, interleukin IL-1β, tumor necrosis factor (TNF-α)), and vascular endothelial growth factor (VEGF) was performed and compared to control rats. These proteins were selected based on existing evidence implicating them in the epileptogenic progression. A robust increase in CCL2 and CCL3 concentrations in the hippocampus, piriform cortex, and neocortex was observed at all time-points. The concentrations peaked with a ~200-fold increase 24 h after seizures and were two orders of magnitude greater than the significant increases observed for CCL5 and IL-1β in the same brain structures. TNF-α levels were altered in the piriform cortex and neocortex (24 h) and in the hippocampus (5 days) after SE. CONCLUSIONS: Pilocarpine-induced status epilepticus causes a rapid increase of multiple cytokines in limbic and neocortical regions. Understanding the precise spatial and temporal pattern of cytokines and chemokine changes could provide more viable therapeutic targets to reduce, reverse, or prevent the development of epilepsy following a precipitating injury

Single-cell sequencing reveals clonal expansions of pro-inflammatory synovial CD8 T cells expressing tissue-homing receptors in psoriatic arthritis.

Psoriatic arthritis (PsA) is a debilitating immune-mediated inflammatory arthritis of unknown pathogenesis commonly affecting patients with skin psoriasis. Here we use complementary single-cell approaches to study leukocytes from PsA joints. Mass cytometry demonstrates a 3-fold expansion of memory CD8 T cells in the joints of PsA patients compared to peripheral blood. Meanwhile, droplet-based and plate-based single-cell RNA sequencing of paired T cell receptor alpha and beta chain sequences show pronounced CD8 T cell clonal expansions within the joints. Transcriptome analyses find these expanded synovial CD8 T cells to express cycling, activation, tissue-homing and tissue residency markers. T cell receptor sequence comparison between patients identifies clonal convergence. Finally, chemokine receptor CXCR3 is upregulated in the expanded synovial CD8 T cells, while two CXCR3 ligands, CXCL9 and CXCL10, are elevated in PsA synovial fluid. Our data thus provide a quantitative molecular insight into the cellular immune landscape of psoriatic arthritis

Aberrant function of the C-terminal tail of HIST1H1E Aacelerates cellular senescence and causes premature aging

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging

De novo mutations of KIAA2022 in females cause intellectual disability and intractable epilepsy

Background Mutations in the KIAA2022 gene have been reported in male patients with X-linked intellectual disability, and related female carriers were unaffected. Here, we report 14 female patients who carry a heterozygous de novo KIAA2022 mutation and share a phenotype characterised by intellectual disability and epilepsy. Methods Reported females were selected for genetic testing because of substantial developmental problems and/or epilepsy. X-inactivation and expression studies were performed when possible. Results All mutations were predicted to result in a frameshift or premature stop. 12 out of 14 patients had intractable epilepsy with myoclonic and/or absence seizures, and generalised in 11. Thirteen patients had mild to severe intellectual disability. This female phenotype partially overlaps with the reported male phenotype which consists of more severe intellectual disability, microcephaly, growth retardation, facial dysmorphisms and, less frequently, epilepsy. One female patient showed completely skewed X-inactivation, complete absence of RNA expression in blood and a phenotype similar to male patients. In the six other tested patients, X-inactivation was random, confirmed by a non-significant twofold to threefold decrease of RNA expression in blood, consistent with the expected mosaicism between cells expressing mutant or normal KIAA2022 alleles. Conclusions Heterozygous loss of KIAA2022 expression is a cause of intellectual disability in females. Compared with its hemizygous male counterpart, the heterozygous female disease has less severe intellectual disability, but is more often associated with a severe and intractable myoclonic epilepsy

Hippocampal CA3 Transcriptome Signature Correlates with Initial Precipitating Injury in Refractory Mesial Temporal Lobe Epilepsy

Background: Prolonged febrile seizures constitute an initial precipitating injury (IPI) commonly associated with refractory mesial temporal lobe epilepsy (RMTLE). in order to investigate IPI influence on the transcriptional phenotype underlying RMTLE we comparatively analyzed the transcriptomic signatures of CA3 explants surgically obtained from RMTLE patients with (FS) or without (NFS) febrile seizure history. Texture analyses on MRI images of dentate gyrus were conducted in a subset of surgically removed sclerotic hippocampi for identifying IPI-associated histo-radiological alterations.Methodology/Principal Findings: DNA microarray analysis revealed that CA3 global gene expression differed significantly between FS and NFS subgroups. An integrative functional genomics methodology was used for characterizing the relations between GO biological processes themes and constructing transcriptional interaction networks defining the FS and NFS transcriptomic signatures and its major gene-gene links (hubs). Co-expression network analysis showed that: i) CA3 transcriptomic profiles differ according to the IPI; ii) FS distinctive hubs are mostly linked to glutamatergic signalization while NFS hubs predominantly involve GABAergic pathways and neurotransmission modulation. Both networks have relevant hubs related to nervous system development, what is consistent with cell genesis activity in the hippocampus of RMTLE patients. Moreover, two candidate genes for therapeutic targeting came out from this analysis: SSTR1, a relevant common hub in febrile and afebrile transcriptomes, and CHRM3, due to its putative role in epilepsy susceptibility development. MRI texture analysis allowed an overall accuracy of 90% for pixels correctly classified as belonging to FS or NFS groups. Histological examination revealed that granule cell loss was significantly higher in FS hippocampi.Conclusions/Significance: CA3 transcriptional signatures and dentate gyrus morphology fairly correlate with IPI in RMTLE, indicating that FS-RMTLE represents a distinct phenotype. These findings may shed light on the molecular mechanisms underlying refractory epilepsy phenotypes and contribute to the discovery of novel specific drug targets for therapeutic interventions