Project description:Memory B cell responses are more rapid and of greater magnitude than are primary antibody responses. The mechanisms by which these secondary responses are eventually attenuated remain unknown. We demonstrate that the transcription factor ZBTB32 limits the rapidity and duration of antibody recall responses. ZBTB32 is highly expressed by mouse and human memory B cells, but not by their naïve counterparts. Zbtb32-/- mice mount normal primary antibody responses to T-dependent antigens. However, Zbtb32-/- memory B cell-mediated recall responses occur more rapidly and persist longer than do control responses. Microarray analyses demonstrate that Zbtb32-/- secondary bone marrow plasma cells display elevated expression of genes that promote cell cycle progression and mitochondrial function relative to wild-type controls. BrdU labeling and adoptive transfer experiments confirm more rapid production and a cell-intrinsic survival advantage of Zbtb32-/- secondary plasma cells relative to wild-type counterparts. ZBTB32 is therefore a novel negative regulator of antibody recall responses. CD45.2 wild type and Zbtb32-/- splenocytes from NP-CGG-immune donors were transferred into CD45.1 recipients and challenged with NP-CGG. CD45.2 donor NP-specific memory B cells were isolated from the spleen 7 days later. 5-6 biological replicates of each genotype were performed.
Project description:Memory B cell responses are more rapid and of greater magnitude than are primary antibody responses. The mechanisms by which these secondary responses are eventually attenuated remain unknown. We demonstrate that the transcription factor ZBTB32 limits the rapidity and duration of antibody recall responses. ZBTB32 is highly expressed by mouse and human memory B cells, but not by their naïve counterparts. Zbtb32-/- mice mount normal primary antibody responses to T-dependent antigens. However, Zbtb32-/- memory B cell-mediated recall responses occur more rapidly and persist longer than do control responses. Microarray analyses demonstrate that Zbtb32-/- secondary bone marrow plasma cells display elevated expression of genes that promote cell cycle progression and mitochondrial function relative to wild-type controls. BrdU labeling and adoptive transfer experiments confirm more rapid production and a cell-intrinsic survival advantage of Zbtb32-/- secondary plasma cells relative to wild-type counterparts. ZBTB32 is therefore a novel negative regulator of antibody recall responses. CD45.2 wild type and Zbtb32-/- splenocytes from NP-CGG-immune donors were transferred into CD45.1 recipients and challenged with NP-CGG. CD45.2 donor NP-specific plasma cells B cells were isolated from the bone marrow 7 days later. 6 biological replicates of each genotype were performed.
Project description:Memory B cell responses are more rapid and of greater magnitude than are primary antibody responses. The mechanisms by which these secondary responses are eventually attenuated remain unknown. We demonstrate that the transcription factor ZBTB32 limits the rapidity and duration of antibody recall responses. ZBTB32 is highly expressed by mouse and human memory B cells, but not by their naïve counterparts. Zbtb32-/- mice mount normal primary antibody responses to T-dependent antigens. However, Zbtb32-/- memory B cell-mediated recall responses occur more rapidly and persist longer than do control responses. Microarray analyses demonstrate that Zbtb32-/- secondary bone marrow plasma cells display elevated expression of genes that promote cell cycle progression and mitochondrial function relative to wild-type controls. BrdU labeling and adoptive transfer experiments confirm more rapid production and a cell-intrinsic survival advantage of Zbtb32-/- secondary plasma cells relative to wild-type counterparts. ZBTB32 is therefore a novel negative regulator of antibody recall responses. Wild type and Zbtb32-/- resting polyclonal splenic memory B cells were purified by fluorescence activated cell sorting, RNA was extracted, and used for Affymetrix microarray analysis. 5 biological replicates of wild type and Zbtb32-/- cells were included for each cell type.
Project description:CD4 T follicular helper (Tfh) cells provide the required signals to B cells for germinal center reactions that are necessary for longlived antibody responses. However, it remains unclear whether there are CD4+ memory T cells committed to the Tfh lineage after antigen clearance. Using adoptive transfer of antigen-specific memory CD4+ subpopulations (based on CXCR5 and Ly6c expression)in the LCMV infection model, we found that there are distinct memory CD4+ T cell populations with commitment to the Tfh and Th1 lineages. Our conclusions are based on gene expression profiles, epigenetic studies and phenotypic and functional analysis. The gene expression profiles of virus-specific CD4 T cell subets at effector and memory stages is presented here. The SMARTA TCR transgenic / adptive transfer system was used to identify and sort subsets of antigen-specific CD4 T cells (based on their expression of Ly6c and CXCR5) elicited after acute infection with LCMV (Arm).
Project description:Sequencing of mRNA from ID8 tumor cells and ID8 tumor cells harvested from ascites of mice 11 weeks after intra peritoneal inoculation show acquisition of cancer stem cell-like features in ascitic tumor cells.
Project description:The study was a comparison of gene expression using RNA-seq. We analyzed the stem and progenitor cells from WT and Vav-cre+ Tet2fl/fl Flt3-ITD (T2F3) mice. We isolated stem cells LSK (lin- sca+ kit+) and granulocyte-macrophage progenitors GMP (lin- sca- kit+ fcgr+ cd34+) cells from bone marrow. Comparisons were made across genotypes WT vs. T2F3 and cell types LSK vs. GMP. Comparison of WT and Tet2-/-Flt3ITD bone marrow stem and progenitor cells.
Project description:We previously generated the Orex-HA mouse model in which orexin-producing neurons residing in the hypothalamus selectively expressed a model self-antigen, namely hemagglutinin (HA) from the H1N1 influenza virus. In this model the adoptive transfer of in vitro activated HA-specific CD8 T cells leads to the specific loss of orexinergic neurons located in the hypothalamus. To assess the phenotype and investigate the pathogenic mechanisms and the potential tissue-resident properties of autoreactive (HA-specific) CD8 T cells infiltrating the hypothalamus of Orex-HA mice , we performed a single-cell transcriptomic analysis (scRNA-seq) of HA-specific CD8+ T cells isolated from the hypothalamus and the spleen at day 30 after T cell transfer in Orex-HA mice.
Project description:Innate memory phenotype (IMP) CD4+ T cells are non-conventional αβ T cells exhibiting features of innate immune cells, characterized as CD44high and CD62Llow in periphery. It is recently reported by our group that bone marrow chimeric mice lacking thymic MHCI expression develop predominantly IMP CD8+ T cells, while those lacking hematopoietic MHCI develop predominantly naïve CD8+ T cells. Here we perform hirarchical clustering analysis and found that CD4+ T cells share similar property: chimeras lacking thymic MHCII gave rise to predominantly CD4+ T cells that resemble IMP CD4+ T cells observed in WT mice, and vice versa, chimeras lacking hematopoietic MHCII had a majority of naïve-like CD4+ T cells resembling naïveCD4+ T cells seen in WT mice. We used microarrays to compare the global programme of gene expression to determine whether the hematopoietic MHCII selected CD4+ T cells are IMP, and whether the thymic MHCII selected CD4+ T cells are naïve CD4+ T cells as observed in WT mice. Through hierarchical clustering and analysis of global gene differential expression, we determined that hematopoietic MHCII dependent IMP CD4+ T cells generated from WT bone marrow transplanted into irradiated MHCII-/- recipients, resemble IMP CD4+ T cells in WT mice, while naïve CD4+ T cells generated from MHCII-/- bone marrow transplanted into irradiated WT recipients, resemble naïve CD4+ T cells in WT mice. Cell Sorting was performed using a Cytopeia Influx Cell Sorter. Chimeric IMP (CD45.1+TCRβ+CD4+CD44highCD62Llow) CD4+ T cells were sorted from splenocytes of CD45.1+WTâCD45.2+MHCII-/- chimeras (WM IMP CD4), and chimeric naïve (CD45.2+TCRβ+CD4+CD44lowCD62Lhigh) CD4+ T cells were sorted from splenocytes of CD45.2+MHCII-/- â CD45.1+WT chimeras (MW naïve CD4) respectively, 8 weeks post transplantation. WT IMP (TCRβ+CD4+CD44highCD62Llow) and naïve (TCRβ+CD4+CD44lowCD62Lhigh) CD4+ T cells were sorted from splenocytes of 8-week old WT mice.
Project description:CSF1-cultured macrophages were collected and sorted into CD11b+CD206high populations which then were separated into each genotype. Total RNA was extracted for Poly A bulk sequencing.
Project description:The generation of naM-CM-/ve T lymphocytes is critical for immune function yet the mechanisms governing their maturation remain incompletely understood. We have identified a mouse mutant, bloto, that harbors a hypomorphic mutation in the zinc finger protein Zfp335. Mutant blt/blt mice exhibit a naM-CM-/ve T cell deficiency due to an intrinsic developmental defect that begins to manifest in the thymus and continues into the periphery, affecting T cells that have recently undergone thymic egress. Zfp335 binds to promoter regions via a consensus motif, and its target genes are enriched in categories related to protein metabolism, mitochondrial function and transcriptional regulation. Restoring the expression of one target, Ankle2, partially rescues T cell maturation. Our findings identify Zfp335 as a transcription factor and essential regulator of late-stage intrathymic and post-thymic T cell maturation. WT and blt/blt CD4+ RTEs were sorted from mixed hematopoietic chimeras for RNA extraction and gene expression analysis on Affymetrix arrays. 6 samples were analyzed in total, with 3 biological replicates per genotype.