Project description:We used scRNA seq to analyze mice organ cells (liver, spleen, thymus and bone marrow) as well as neonatal thymus organ cultures that were treated cytokines IL-12 and IL-18.
Project description:In this report, we show that VEGF-blockade in neonates induces rapid changes in all three stromal compartments of the thymus, and that these changes recapitulate the maturation of these cell types seen during the transition from the neonatal to the young adult thymus. These changes in the microenvironment occur prior to the loss of thymocytes, and include not only a loss of cTECs relative to mTECs but also depletion of CD146+CD140aneg cells, a novel population of pericytes we have identified in the neonatal thymus. Importantly, we show that based on receptor expression and receptor blockade, VEGF drives the fetal/neonatal phenotype of the thymus through distinct VEGFR2- and NRP1-dependent pathways in thymic endothelial and mesenchymal cells respectively. In addition, receptor signaling induces changes in critical pathways affecting the function of all three compartments, with consequences on thymopoieisis.
Project description:Herpesviruses, including the roseoloviruses, have been linked to autoimmune disease. The ubiquitous and chronic nature of these infections as well as the temporal dissociation between acute infection and autoimmune disease onset have made establishing a causal association challenging. We have shown that murine roseolovirus (MRV), which is highly related to human roseoloviruses, induces thymic atrophy and disruption of central tolerance after neonatal infection. Moreover, neonatal MRV infection results in development of autoimmunity in adult mice, long after resolution of acute infection. This suggests that MRV induces durable immune dysregulation. In these studies, we utilized single-cell RNA sequencing (scRNAseq) to study the tropism of MRV in the thymus and determine cellular processes in the thymus that were disrupted by neonatal MRV infection. We then utilized tropism data to establish a cell culture system. Herein, we describe how MRV alters the thymic transcriptome during acute neonatal infection.We found that MRV infection results in major shifts in inflammatory, differentiation and cell cycle pathways in the infected thymus. We also observed shifts in the relative number of specific cell populations. Moreover, utilizing expression of late viral transcripts as a proxy of viral replication we identified the cellular tropism of MRV in the thymus. This approach demonstrated that double negative, double positive, and CD4 single positive thymocytes, as well as medullary thymic epithelial cells were infected by MRV in vivo. Finally, by applying pseudotime analysis to viral transcripts, which we refer to as "pseudokinetics," we identified viral gene transcription patterns associated with specific cell types and infection status. We applied this information to establish the first cell culture systems susceptible to MRV infection in vitro. Our research provides the first complete picture of MRV tropism in the thymus after neonatal infection. Additionally, we identified major transcriptomic alterations in cell populations in the thymus during acute neonatal MRV infection. These studies offer important insight into the early events that occur after neonatal MRV infection that disrupt central tolerance and promote autoimmune disease
Project description:Innate lymphoid cell (ILC) subsets that mirror helper T cells in their effector cytokine profiles have recently emerged as central players in both homeostatic and inflammatory conditions. Like their Th1, Th2 and Th17/Th22 helper T cell counterparts, ILC subsets are categorized based on their expression of specific transcription factors and effector cytokines: group 1 ILC (ILC1) express T-bet and IFN-γ; group 2 ILC (ILC2) express GATA-3 and type 2 effector cytokines such as IL-13 and IL-5; and group 3 ILC (ILC3) express RORgt and the cytokines IL-22 and/or IL-17. Under this nomenclature, natural killer (NK) cells and lymphoid tissue inducers (LTi) are considered ILC1 and ILC3, respectively. ILC1 contain both CD4+ and CD4- populations, but whether this phenotypic characteristic reflects functional differences between these two populations is unknown. These studies examine the gene expression profiles of CD4+ vs CD4- ILC1 in a cohort of healthy control subjects. ILC subsets were isolated from the peripheral blood of healthy control subjects. cDNA was isolated and amplified from sorted populations, and gene expression was analyzed by RNAseq
Project description:The ligand for the c-Kit receptor, KitL, exists as a membrane-associated (mKitL) and a soluble form (sKitL). KitL functions outside c-Kit activation have not been identified. We show that co-culture of c-Kit– and mKitL–expressing NIH3T3 cells results in signaling through mKitL: c-Kit–bound mKitL recruits calcium-modulating cyclophilin ligand (CAML) to selectively activate Akt, leading to CREB phosphorylation, mTOR pathway activation, and increased cell proliferation. Activation of mKitL in thymic vascular endothelial cells (VECs) induces mKitL- and Akt-dependent proliferation, and genetic ablation of mKitL in thymic VECs blocks their c-Kit responsiveness and proliferation during neonatal thymic expansion. Therefore, mKitL–c-Kit form a bi-directional signaling complex that acts in the developing thymus to coordinate thymic VEC and early thymic progenitor (ETP) expansion by simultaneously promoting ETP survival and VEC proliferation. This mechanism may be relevant to both normal tissues and malignant tumors that depend on KitL–c-Kit signaling for their proliferation.
Project description:The effect of PDGF-DD on the gene expression of human tonsil ILC1 is unknown. We used microarray to determine the transcriptional differences between unstimulated and PDGF-DD-stimulated human tonsil ILC1.
Project description:Purpose: To understanding the effects of RORa deficiency on ILC1 cells, we conducted bulk mRNA-seqencing Method: Firstly, we purified liver ILC1 cells (CD45.2+CD3-CD19-NKp46+NK1.1+CD49a+CD49b-) via Fluorescence Activated Cell Sorting, then frozen in -80 °C ultra-low temperature refrigerator, followed by High-throughput sequencing, in three replicates for WT (Rorafl/fl) mice and one replicate for cKO (Ncr1Cre-Rorafl/fl) , using Illumina Hiseq 1500 platform.
Project description:Innate and adaptive immune cells can acquire “memory” of encounters with a diverse range of activating signals to tune their response to secondary stimuli. Group 1 innate lymphoid cells (ILC1) are recently discovered tissue-resident sentinels that are essential for early host protection from intracellular pathogens at initial sites of infection. However, whether ILC1 function as short-lived effectors or persist and refine their responsiveness following pathogen challenge is not well understood. Furthermore, whether pathogen-derived antigens directly modulate tissue-resident ILC responses remains unclear. Here, we found that liver-resident ILC1 expand locally and persist following the resolution of mouse cytomegalovirus (MCMV) infection. MCMVexperienced ILC1 acquired stable transcriptional, epigenetic, and phenotypic changes, with an enhanced protective effector response to secondary MCMV challenge. Protective memory ILC1 responses were dependent on the MCMV-encoded glycoprotein m12, but not formed during bystander cytokine activation following heterologous infection. Thus, liver ILC1 acquire adaptive features in a MCMV-specific manner.