Project description:Major Histocompatibility Complex I (MHC-I) CNS cellular localization and function is still being determined after previously being thought to be absent from the brain. MHC-I expression has been reported to increase with brain aging in mouse, rat, and human whole tissue analyses. Neuronal MHC-I has been proposed to regulate developmental synapse elimination and tau pathology in Alzheimer’s disease (AD). Here we report that across newly generated and publicly available ribosomal profiling, cell sorting, and single-cell data, microglia were the primary source of classical and non-classical MHC-I in mice and humans. Translating Ribosome Affinity Purification (TRAP)-qPCR analysis of 3-6 and 18-22 month old (m.o.) mice revealed significant age-related microglial induction of MHC-I pathway genes B2m, H2-D1, H2-K1, H2-M3, H2-Q6, and Tap1 but not in astrocytes and neurons. Across a timecourse (12-23 m.o.), microglial MHC-I gradually increased until 21 m.o. and then accelerated. MHC-I protein was also enriched in microglia and increased with aging. Microglial expression, and absence in astrocytes and neurons, of MHC-I binding Leukocyte Immunoglobulin-like (Lilrs) and Paired immunoglobin-like type 2 (Pilrs) receptor families could enable cell-autonomous MHC-I signaling and increased with aging in mice and humans. Increased microglial MHC-I, Lilrs, and Pilrs were observed in AD mouse models and human AD data is evident across numerous mouse models, methods, and studies. MHC-I expression correlated with p16INK4A, suggesting an association with cellular senescence. Conserved induction of MHC-I, Lilrs, and Pilrs with aging and AD opens the possibility of cell-autonomous MHC-I signaling to regulate microglial reactivation with aging and neurodegeneration.
Project description:A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur prior to any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Neural stem cells derived from mouse embryonic stem cells were differentiated into neurons and FACS purified based on RedStar fluorescence driven by the Tau promoter. Chromatin was prepared from NSCs and neurons (n=1), sonicated to roughly 300bp and immunoprecipitated with antibodies against H3K4me3, H3K27me3, total Histone H3 and total IgG, alongside a 5% input sample. K4/K27 and corresponding input samples were analysed by ChIPSeq
Project description:Human TAPBPR is known to function as a Major Histocompatibility Complex class I (MHC-I) peptide exchange catalyst which shapes the peptide repertoire presented to immune cells. Never14theless, investigations characterizing TAPBPR from other species are limited. Here, we characterize mouse TAPBPR, exploring its association partners in mouse cell lines and comparing its function to human TAPBPR. We find that mouse TAPBPR binds MHC-I and calnexin, with a notably sustained interaction with H2-Db compared to H2-Kb. We reveal mouse TAPBPR mediates peptide exchange on both H2-Db and H2-Kb, restricting the peptide repertoire presented on MC-38 cells. Intriguingly, mouse TAPBPR promotes the selection of peptides with a C-terminal methionine on H2-Kb. Together, our findings establish that mouse TAPBPR plays an important role in shaping the MHC-I immunopeptidome by functioning as a peptide editor, like its human counterpart.
Project description:Immune checkpoint inhibitors interfere with T cell exhaustion but often fail to cure or control cancer long-term in patients. Using a genetic screen in C57BL/6J mice, we discovered a mutation in host H2-Aa that caused strong immune-mediated resistance to mouse melanomas. H2-Aa encodes an MHC class II α chain, and its absence in C57BL/6J mice eliminates all MHC-II expression. H2-Aa deficiency, specifically in dendritic cells (DC), led to a quantitative increase in type 2 conventional DC (cDC2) and a decrease in cDC1. H2-Aa–deficient cDC2, but not cDC1, were essential for melanoma suppression and effectively cross-primed and recruited CD8 T cells into tumors. Lack of T regulatory cells, also observed in H2-Aa deficiency, contributed to melanoma suppression. Acute disruption of H2-Aa was therapeutic in melanoma-bearing mice, particularly when combined with checkpoint inhibition, which had no therapeutic effect by itself. Our findings suggest that inhibiting MHC-II may be an effective immunotherapeutic approach to enhance immune responses to cancer.
Project description:Primitive neural stem cells (NSCs) could be derived from pluripotent mouse embryonic stem (ES) cells, and then differentiate into definitive-type neural stem cells which resemble NSCs obtained from the central nervous system. Hence, primitive NSCs define an early stage of neural induction and provide a model to understand the mechanism that controls initial neural commitment. In this study, we performed microarray assay to analyze the global transcriptional profiles in mouse ES cell-derived primitive and definitive NSCs and to depict the molecular changes during the multi-staged neural differentiation process. Primitive NSCs derived directly from ESCs in Lif (p-NSC_L), primitive NSCs that were sub-cultured in the presence of Lif and FGF (p-NSC_LF), as well as definitive NSCs derived from primitive NSCs in medium containing FGF and EGF, were collected for RNA extraction and hybridization on Affymetrix microarrays. Mouse ESCs and NSCs obtained from mouse embryonic brain (E11.5) were included for controls. For each cell type, we collected two biological replicate samples for microarray analysis.
Project description:Leishmania parasites cause cutaneous leishmanina (CL), a pathologic disease characterized by disfiguring, ulcerative skin lesions. Both parasite and host gene expression following infection with various Leishmania species has been investigated in vitro, but global transcriptional analysis following L. major infection in vivo is lacking. Thus, we conducted a comprehensive transcriptomic profiling study combining bulk RNA sequencing and single-cell RNA sequencing (scRNA-Seq) to identify global changes in gene expression in vivo following L. major infection. Bulk RNA-Seq analysis revealed that host immune response pathways like the antigen processing and presentation pathway were significantly enriched amongst differentially expressed genes (DEGs) upon infection, while ribosomal pathways were significantly downregulated in infected mice compared to naive controls. scRNA-Seq analyses revealed cellular heterogeneity including distince resident and recruitment cell types in the skin following L. major infection. Within the individual immune cell types, several DEGs indicative of many interferon induced GTPases and antigen presentation molecules were significantly enhanced in the infected ears including macrophages (H2-K1, H2-D1, Gbp4, Gbp8, Gbp2), and inflammatory monocytes (Gbp2, Gbp5, Gbp7, Gbp3). Ingenuity Pathway Analysis of scRNA-Seq data indicated the antigen presentation pathway was increased with infection, while EIF2 signaling is the top downregulated pathway followed by eIF4/p70S6k and mTOR signaling in multiple cell types including macrophages, BECs, and LECs. Altogether, this transcriptomic profile highlights known recruitment of myeloid cells to lesions and recognizes a previously undefined role for EIF2 signaling in murine L. major infection in vivo.
Project description:Human TAPBPR is known to function as a Major Histocompatibility Complex class I (MHC-I) peptide exchange catalyst which shapes the peptide repertoire presented to immune cells. Never14theless, investigations characterizing TAPBPR from other species are limited. Here, we characterize mouse TAPBPR, exploring its association partners in mouse cell lines and comparing its function to human TAPBPR. We find that mouse TAPBPR binds MHC-I and calnexin, with a notably sustained interaction with H2-Db compared to H2-Kb. We reveal mouse TAPBPR mediates peptide exchange on both H2-Db and H2-Kb, restricting the peptide repertoire presented on MC-38 cells. Intriguingly, mouse TAPBPR promotes the selection of peptides with a C-terminal methionine on H2-Kb. Together, our findings establish that mouse TAPBPR plays an important role in shaping the MHC-I immunopeptidome by functioning as a peptide editor, like its human counterpart.
Project description:This study explored the potential of carbon nanotubes (CNTs) bioconjugated with antigenic epitopes from fucosyltransferase 4 (FUT4) to serve as adjuvants and carriers in ovarian cancer immunotherapy. We confirmed FUT4 overexpression via flow cytometry and confocal microscopy in an immunocompetent ovarian cancer model in which the ID8-Def29/Vegf-a cell line (ID8DVLuc) was inoculated into C57BL6 mice. Mice were immunized with nonconjugated peptide (PEP37), PEP37 bioconjugated CNTs (f-CNTs), or f-CNTs plus adjuvant, and nonimmunized mice were used as controls. Tumor development, the spleen, and ascitic fluid immune populations, the antibody response, and survival rates were evaluated. The results revealed reduced tumor development and ascitic fluid volume in immunized mice, with the best outcomes in the f-CNT group. Immunized mice presented increased infiltration of leukocytes, M1 macrophages, dendritic cells, T lymphocytes, and CD8+ T cells alongside reduced Tregs. Enhanced IgM, IgG1a, and IgG2a responses were observed in the f-CNT groups. Splenocytes from these groups also showed increased antigen-specific proliferation and enhanced cytotoxicity against ID8DVLuc cells mediated by CD8+ T cells. Survival analysis revealed median survival times of 6, 7.5, 11, and 8.5 weeks for the nonimmunized, PEP37, f-CNT, and f-CNT plus adjuvant groups, respectively. In addition, RNA-seq analysis of f-CNT-immunized mice revealed the overexpression of genes related to antigen processing and presentation, CD8+ T-cell activation, and Th1-type-mediated responses (H2-K1, H2-D1, B2m, Trex1 Cd80, Cd8a, Prf1, IL18r1, Ccr7, Stat4, Tbx21), among others. These findings suggest that f-CNTs enhance the antitumor immune response mediated by M1 macrophage polarization, enhance antigen processing and presentation to CD8+ T cells, and evoke a robust cytotoxic response against ID8DVLuc cells. These findings suggest the potential of this nanocarrier system in ovarian cancer immunotherapy
Project description:Quantitative phosphoproteome with OT-I TAP-/- mice DP thymocytes stimulated by H2-Kb tetramers for 2 min. The quantitative data were calculated by the spike-in standard lysates from DPK cells cultured in the heavy media containing 15N2 13C6-lysine and 15N4 13C6-arginine.