BCR-Seq of Germinal Center B cells from mice infected with LCMV
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ABSTRACT: To study how acute or chronic viral infection (LCMV-WE or Docile) affect somatic hypermutation and/or affinity maturation of Germinal Center B cells, wild-type or Bmi1-deleted mice were infected with either the acute (WE) versus chronic (Docile) strains of LCMV, GC B cells (CD19+IgDloCD95+CD38loCD138-) were isolated 14 days post-infection and thei BCR was sequenced.
Project description:To study how chronic viral infection disrupts B cell differentiation pathways and identify key intrinsic regulators in an endogenous polyclonal response, we used the comparative LCMV model. Wild-type or Bmi1 conditional know-out in B cells mice were infected with either the acute (WE) versus chronic (Docile) strains of LCMV, GC B cells (CD19+IgDloCD95+CD38loCD138-) isolated 14 days post-infection, and RNA-seq was undertaken
Project description:CD4 and CD8 T cells display functional defects during chronic infection such as loss of certain cytokines. Recent studies have suggested that CD4 T cells may actually gain other functions, however. Here, we analyzed gene expression profiles from LCMV-specific CD4 and CD8 T cells throughout the response to either acute LCMV or chronic LCMV infection. This alllowed us to identify CD4-specific changes during chronic infection compared to acute infection but also revealed shared core regulators between CD4 and CD8 T cells. LCMV-specific CD4 and CD8 T cells were isolated 6, 8, 15 and 30 days post infection with LCMV Armstrong or LCMV clone 13. Naïve CD4 and CD8 T cells were also isolated from naïve mice as comparisons. Four replicates of each sample were hybridized. The only exception is LCMV-specific CD4 T cells isolated 6 days post infection with LCMV-Arm where only three replicates were hybridized.
Project description:To study how chronic viral infection disrupts B cell differentiation pathways and identify key intrinsic regulators in an endogenous polyclonal response, we used the comparative LCMV model. Wild-type mice were infected with either the acute (WE) versus chronic (Docile) strains of LCMV, GC B cells (CD19+IgDloCD95+CD38loCD138-) isolated 14 days post-infection, and RNA-seq was undertaken
Project description:Necroptosis is a lytic and inflammatory form of cell death that is highly constrained to mitigate detrimental collateral tissue damage and impaired immunity. These constraints make it difficult to define the relevance of necroptosis in diseases such as chronic and persistent viral infections and within individual organ systems. The role of necroptotic signalling is further complicated because proteins essential to this pathway, such as receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), have been implicated in roles outside of necroptotic signalling. We sought to address this issue by individually defining the role of RIPK3 and MLKL in chronic lymphocytic choriomeningitis virus (LCMV) infection. We investigated if necroptosis contributes to the death of LCMV-specific CD8+ T cells or virally infected target cells during infection. We provide evidence showing that necroptosis was redundant in the pathogenesis of acute forms of LCMV (Armstrong strain) and the early stages of chronic (Docile strain) LCMV infection in vivo. The number of immune cells, their specificity and reactivity towards viral antigens and viral loads are not altered in the absence of either MLKL or RIPK3 during acute and during the early stages of chronic LCMV infection. However, we identified that RIPK3 promotes immune dysfunction and prevents control of infection at later stages of chronic LCMV disease. This was not phenocopied by the loss of MLKL indicating that the phenotype was driven by a necroptosis-independent function of RIPK3. We provide evidence that RIPK3 signaling evoked a dysregulated type 1 interferone response which we linked to an impaired antiviral immune response and abrogated clearance of chronic LCMV infection.
Project description:To study how chronic viral infection disrupts B cell differentiation pathways and identify key intrinsic regulators in an endogenous polyclonal response, we used the comparative LCMV model. Wild-type mice were infected with either the acute (WE) versus chronic (Docile) strains of LCMV, GC B cells (CD19+IgDloCD95+CD38loCD138-) isolated 14 days post-infection, an ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) was undertaken.
Project description:CD4 and CD8 T cells display functional defects during chronic infection such as loss of certain cytokines. Recent studies have suggested that CD4 T cells may actually gain other functions, however. Here, we analyzed gene expression profiles from LCMV-specific CD4 and CD8 T cells throughout the response to either acute LCMV or chronic LCMV infection. This alllowed us to identify CD4-specific changes during chronic infection compared to acute infection but also revealed shared core regulators between CD4 and CD8 T cells.
Project description:Understanding the response of memory CD8 T cells to persistent antigen re-stimulation and the role of CD4 T cell help is critical to the design of successful vaccines for chronic diseases. However, studies comparing the protective abilities and qualities of memory and naïve cells have been mostly performed in acute infections, and little is known about their roles during chronic infections. Herein, we show that memory cells dominate over naïve cells and are protective when present in large enough numbers to quickly reduce infection. In contrast, when infection is not rapidly reduced, memory cells are quickly lost, unlike naïve cells. This loss of memory cells is due to (i) an early block in cell proliferation, (ii) selective regulation by the inhibitory receptor 2B4, and (iii) increased reliance on CD4 T cell help. These findings have important implications towards the design of T cell vaccines against chronic infections and tumors. 16 samples are analyzed: 3 replicates of secondary effector CD8 P14 T cells at day 8 post-acute lymphocytic choriomeningitis virus (LCMV) infection; 4 replicates of secondary effector CD8 P14 T cells at day 8 post-chronic LCMV infection; 4 replicates of primary effector CD8 P14 T cells at day 8 post-acute LCMV infection; and 5 replicates of primary effector CD8 P14 T cells at day 8 post-chronic LCMV infection.
Project description:Chronic viral infection disrupts memory B cell development. We used single cell ATAC sequencing (scATAC-seq) to analyze chromatin accessibility in antigen-specific B cells responding to acute versus chronic LCMV infection.
Project description:Chronic viral infection disrupts memory B cell development. We used single cell RNA sequencing (scRNA-seq) to analyze the diversity of antigen-specific B cells responding to acute versus chronic LCMV infection.