Cell type-specific interaction analysis using doublets in scRNA-seq.

Summary: Doublets are usually considered an unwanted artifact of single-cell RNA-sequencing (scRNA-seq) and are only identified in datasets for the sake of removal. However, if cells have a juxtacrine interaction with one another in situ and maintain this association through an scRNA-seq processing pipeline that only partially dissociates the tissue, these doublets can provide meaningful biological information regarding the intercellular signals and processes occurring in the analyzed tissue. This is especially true for cases such as the immune compartment of the tumor microenvironment, where the frequency and the type of immune cell juxtacrine interactions can be a prognostic indicator. We developed Cell type-specific Interaction Analysis using Doublets in scRNA-seq (CIcADA) as a pipeline for identifying and analyzing biologically meaningful doublets in scRNA-seq data. CIcADA identifies putative doublets using multi-label cell type scores and characterizes interaction dynamics through a comparison against synthetic doublets of the same cell type composition. In performing CIcADA on several scRNA-seq tumor datasets, we found that the identified doublets were consistently upregulating expression of immune response genes. Availability and implementation: An R package implementing the CIcADA method is in development and will be released on CRAN, but for now it is available at https://github.com/schiebout/CAMML.

Cell type-specific Interaction Analysis using Doublets in scRNA-seq (CIcADA).

Motivation: Doublets are usually considered an unwanted artifact of single-cell RNA-sequencing (scRNA-seq) and are only identified in datasets for the sake of removal. However, if cells have a juxtacrine attachment to one another in situ and maintain this association through an scRNA-seq processing pipeline that only partially dissociates the tissue, these doublets can provide meaningful biological information regarding the interactions and cell processes occurring in the analyzed tissue. This is especially true for cases such as the immune compartment of the tumor microenvironment, where the frequency and type of immune cell juxtacrine interactions can be a prognostic indicator. Results: We developed Cell type-specific Interaction Analysis using Doublets in scRNA-seq (CIcADA) as a pipeline for identifying and analyzing biological doublets in scRNA-seq data. CIcADA identifies putative doublets using multi-label cell type scores and characterizes interaction dynamics through a comparison against synthetic doublets of the same cell type composition. In performing CIcADA on several scRNA-seq tumor datasets, we found that the identified doublets were consistently upregulating expression of immune response genes. Contact: Courtney.T.Schiebout.GR@Dartmouth.edu , Hildreth.R.Frost@Dartmouth.edu.

Idiopathic multicentric Castleman disease with TAFRO clinical subtype responsive to IL-6/JAK inhibition: A pediatric case series.

TAFRO (thrombocytopenia, anasarca, fever/elevated C-reactive protein, reticulin myelofibrosis, renal dysfunction, and organomegaly) clinical subtype of idiopathic multicentric Castleman disease (iMCD-TAFRO) is a rare lymphoproliferative disease characterized by systemic inflammation. First-line treatment for iMCD-TAFRO includes steroids and interleukin (IL)-6 blockade. Many patients have refractory disease, which is associated with significant morbidity and mortality, and treatment remains challenging. We present two pediatric cases of iMCD-TAFRO. One patient responded to IL-6 blockade; the other was refractory to siltuximab and chemotherapy, ultimately responding to JAK inhibition with ruxolitinib. This is the first reported pediatric case of refractory iMCD-TAFRO responding to JAK inhibition.

Foxo1 Serine 209 Is a Critical Regulatory Site of CD8 T Cell Differentiation and Survival.

Foxo1 is an essential transcription factor required for the survival and differentiation of memory CD8 T cells, yet it is unclear whether these Foxo1-dependent functions are inherently coupled. To address this question, we examined the effects of different Foxo1 posttranslational modifications. Phosphorylation of Foxo1 by Akt kinases at three distinct residues is well characterized to inhibit Foxo1 transcriptional activity. However, the effect of Foxo1 phosphorylation within its DNA-binding domain at serine 209 by Mst1 kinase is not fully understood. In this study, we show that an S209A phospho-null Foxo1 exhibited Akt-dependent nuclear trafficking in mouse CD8 T cells and augmented the expression of canonical Foxo1 target genes such as Il7r and Sell In contrast, an S209D phosphomimetic Foxo1 (SD-Foxo1) was largely excluded from the nucleus of CD8 T cells and failed to transactivate these genes. RNA sequencing analysis revealed that SD-Foxo1 was associated with a distinct Foxo1-dependent transcriptional profile, including genes mediating CD8 effector function and cell survival. Despite defective transactivation of canonical target genes, SD-Foxo1 promoted IL-15-mediated CD8 T cell survival in vitro and survival of short-lived effector cells in vivo in response to Listeria monocytogenes infection. However, SD-Foxo1 actively repressed CD127 expression and failed to generate memory precursors and long-lived memory T cells. Together, these data indicate that S209 is a critical residue for the regulation of Foxo1 subcellular localization and for balancing CD8 T cell differentiation and survival.

Interleukin 1α Is Critical for Resistance against Highly Virulent Aspergillus fumigatus Isolates.

Heterogeneity among Aspergillus fumigatus isolates results in unique virulence potential and inflammatory responses. How these isolates drive specific immune responses and how this affects fungally induced lung damage and disease outcome are unresolved. We demonstrate that the highly virulent CEA10 strain is able to rapidly germinate within the immunocompetent lung environment, inducing greater lung damage, vascular leakage, and interleukin 1α (IL-1α) release than the low-virulence Af293 strain, which germinates with a lower frequency in this environment. Importantly, the clearance of CEA10 was consequently dependent on IL-1α, in contrast to Af293. The release of IL-1α occurred by a caspase 1/11- and P2XR7-independent mechanism but was dependent on calpain activity. Our finding that early fungal conidium germination drives greater lung damage and IL-1α-dependent inflammation is supported by three independent experimental lines. First, pregermination of Af293 prior to in vivo challenge drives greater lung damage and an IL-1α-dependent neutrophil response. Second, the more virulent EVOL20 strain, derived from Af293, is able to germinate in the airways, leading to enhanced lung damage and IL-1α-dependent inflammation and fungal clearance. Third, primary environmental A. fumigatus isolates that rapidly germinate under airway conditions follow the same trend toward IL-1α dependency. Our data support the hypothesis that A. fumigatus phenotypic variation significantly contributes to disease outcomes.

A Forward Genetic Screen for Suppressors of Somatic P Granules in Caenorhabditis elegans.

In Caenorhabditis elegans, germline expression programs are actively repressed in somatic tissue by components of the synMuv (synthetic multi-vulva) B chromatin remodeling complex, which include homologs of tumor suppressors Retinoblastoma (Rb/LIN-35) and Malignant Brain Tumor (MBT/LIN-61). However, the full scope of pathways that suppress germline expression in the soma is unknown. To address this, we performed a mutagenesis and screened for somatic expression of GFP-tagged PGL-1, a core P-granule nucleating protein. Eight alleles were isolated from 4000 haploid genomes. Five of these alleles exhibit a synMuv phenotype, whereas the remaining three were identified as hypomorphic alleles of known synMuv B genes, lin-13 and dpl-1. These findings suggest that most suppressors of germline programs in the soma of C. elegans are either required for viability or function through synMuv B chromatin regulation.