Neonatal CD8+ T cells have a distinct gene expression and epigenetic profile compared to adult CD8+ T cells. Neonatal cells show lower expression of genes involved in T cell receptor signaling and cytotoxicity. In contrast, neonatal cells express higher levels of genes related to cell cycle and innate immunity. Functional studies confirm that neonatal CD8+ T cells proliferate more, are less cytotoxic, and express antimicrobial peptides and reactive oxygen species, consistent with an innate immune response bias. This distinctive profile may explain neonates' vulnerability to infection.
I. Computational analysis identified novel B- and T- cell epitopes from the glycoprotein and nucleoprotein of Ebola virus, including AIGLAWIPY, YDDDDDIPF, SQDTTIPDV, VSHLTTLAT, DLVLFDLDE, LRQLANETTT, and DYHKILTAGL.
II. A novel B-cell epitope was extracted from the 3D structure of Ebola glycoprotein bound to an antibody, and its sequence motifs were predicted by multiple algorithms.
III. An epitope from the nucleoprotein contained a conserved protein fingerprint that defines the WW domain-containing WAC protein, and a potential WW domain was
Analysis of Human Embryonic Stem Cells with Regulatable Expression of the Cel...Christopher S Park
This document describes research on developing a human neural stem cell line with regulatable expression of the cell adhesion molecule L1 using a tetracycline-controlled gene expression system. Embryonic stem cell-derived neural stem cells were transfected with a plasmid containing the L1 gene under control of a doxycycline-regulatable promoter. In the presence of doxycycline, L1 expression was eliminated, while without doxycycline L1 was expressed. This cell line with regulatable L1 expression was then tested in a mouse model of spinal cord injury, where expression of L1 improved locomotor recovery compared to when L1 expression was turned off with doxycycline.
This study examines the interactions between dendritic cells (DCs) and naive T cells, and how the maturation state of DCs impacts those interactions. The key findings are:
1) Mature DCs form stable conjugates with naive T cells and induce organized immune synapses, while immature DCs form few stable conjugates without organized synapses.
2) Mature DCs can sustain long-lasting interactions with naive T cells even without antigen, while immature DCs only establish short intermittent contacts.
3) The premature termination of interactions by immature DCs prevents the formation of organized immune synapses and full T cell activation.
1. The document summarizes research on CLN7 disease, which is caused by mutations in the CLN7 gene encoding a lysosomal membrane protein.
2. A mouse model of CLN7 disease was generated that recapitulates some neuropathological features seen in human patients, including accumulation of autofluorescent storage material in the brain.
3. The researchers aim to identify the pathomechanisms of CLN7 disease, develop biomarkers, analyze the function of the CLN7 protein, and understand how mutations impact its localization and function to inform potential therapies.
The document summarizes research into the roles of genes ELL2 and ELL3 in plasma cell development. It describes how ELL2 expression increases and ELL3 decreases as B cells differentiate into antibody-secreting plasma cells. The research aimed to determine if ELL1 and ELL3 have similar functions to ELL2. Cell lines expressing only ELL2 or ELL3 were analyzed to confirm expression. A lentivirus was prepared to introduce genes into B cells from ELL2 knockout mice to test for functional complementation. The work established methods and preliminary results to investigate the roles of the ELL genes in plasma cell development.
This document reports on a study that found adult hippocampal neural stem and progenitor cells (NSPCs) secrete significant amounts of vascular endothelial growth factor (VEGF), which plays an important role in maintaining the adult neurogenic niche. The study showed that NSPCs in the adult hippocampus express VEGF both in vivo and in vitro. Inducible knockout of VEGF specifically in NSPCs led to a 20-30% reduction in total hippocampal VEGF levels and impaired stem cell maintenance over time, demonstrating the functional relevance of NSPC-derived VEGF. These findings reveal that NSPCs act as a previously unknown secretory cell type that helps regulate the neurogenic niche through VEGF secretion.
This document summarizes research finding that elevated activity of the Akt protein protects prostate cancer LNCaP cells from apoptosis induced by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). The researchers found that LNCaP cells have high constitutive Akt activity due to lack of the PTEN lipid phosphatase. Inhibiting PI3-kinase, which activates Akt, sensitized LNCaP cells to TRAIL-induced apoptosis. TRAIL alone activated caspases 8 and XIAP in LNCaP cells but failed to induce full apoptosis. Combining TRAIL with Akt inhibitors allowed cleavage of BID and subsequent mitochondrial apoptosis steps. Akt inhibition of BID cleavage appears to mediate the protective effect
This document discusses the role of somatic mutagenesis in the development of autoimmunity, specifically in systemic lupus erythematosus (SLE). It argues that:
1) Antinuclear antibodies (ANA) that arise in SLE predominantly originate from non-autoreactive B cells that undergo somatic hypermutation (SHM) and are transformed into autoreactive cells, rather than arising from autoreactive B cells escaping central tolerance.
2) SHM may create antigenic peptides in the B cell receptor that provide a route for unregulated T cell help to autoreactive B cells.
3) Spontaneous somatic mutagenesis of genes regulating B cell survival and activation may be a stochastic rate-
I. Computational analysis identified novel B- and T- cell epitopes from the glycoprotein and nucleoprotein of Ebola virus, including AIGLAWIPY, YDDDDDIPF, SQDTTIPDV, VSHLTTLAT, DLVLFDLDE, LRQLANETTT, and DYHKILTAGL.
II. A novel B-cell epitope was extracted from the 3D structure of Ebola glycoprotein bound to an antibody, and its sequence motifs were predicted by multiple algorithms.
III. An epitope from the nucleoprotein contained a conserved protein fingerprint that defines the WW domain-containing WAC protein, and a potential WW domain was
Analysis of Human Embryonic Stem Cells with Regulatable Expression of the Cel...Christopher S Park
This document describes research on developing a human neural stem cell line with regulatable expression of the cell adhesion molecule L1 using a tetracycline-controlled gene expression system. Embryonic stem cell-derived neural stem cells were transfected with a plasmid containing the L1 gene under control of a doxycycline-regulatable promoter. In the presence of doxycycline, L1 expression was eliminated, while without doxycycline L1 was expressed. This cell line with regulatable L1 expression was then tested in a mouse model of spinal cord injury, where expression of L1 improved locomotor recovery compared to when L1 expression was turned off with doxycycline.
This study examines the interactions between dendritic cells (DCs) and naive T cells, and how the maturation state of DCs impacts those interactions. The key findings are:
1) Mature DCs form stable conjugates with naive T cells and induce organized immune synapses, while immature DCs form few stable conjugates without organized synapses.
2) Mature DCs can sustain long-lasting interactions with naive T cells even without antigen, while immature DCs only establish short intermittent contacts.
3) The premature termination of interactions by immature DCs prevents the formation of organized immune synapses and full T cell activation.
1. The document summarizes research on CLN7 disease, which is caused by mutations in the CLN7 gene encoding a lysosomal membrane protein.
2. A mouse model of CLN7 disease was generated that recapitulates some neuropathological features seen in human patients, including accumulation of autofluorescent storage material in the brain.
3. The researchers aim to identify the pathomechanisms of CLN7 disease, develop biomarkers, analyze the function of the CLN7 protein, and understand how mutations impact its localization and function to inform potential therapies.
The document summarizes research into the roles of genes ELL2 and ELL3 in plasma cell development. It describes how ELL2 expression increases and ELL3 decreases as B cells differentiate into antibody-secreting plasma cells. The research aimed to determine if ELL1 and ELL3 have similar functions to ELL2. Cell lines expressing only ELL2 or ELL3 were analyzed to confirm expression. A lentivirus was prepared to introduce genes into B cells from ELL2 knockout mice to test for functional complementation. The work established methods and preliminary results to investigate the roles of the ELL genes in plasma cell development.
This document reports on a study that found adult hippocampal neural stem and progenitor cells (NSPCs) secrete significant amounts of vascular endothelial growth factor (VEGF), which plays an important role in maintaining the adult neurogenic niche. The study showed that NSPCs in the adult hippocampus express VEGF both in vivo and in vitro. Inducible knockout of VEGF specifically in NSPCs led to a 20-30% reduction in total hippocampal VEGF levels and impaired stem cell maintenance over time, demonstrating the functional relevance of NSPC-derived VEGF. These findings reveal that NSPCs act as a previously unknown secretory cell type that helps regulate the neurogenic niche through VEGF secretion.
This document summarizes research finding that elevated activity of the Akt protein protects prostate cancer LNCaP cells from apoptosis induced by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). The researchers found that LNCaP cells have high constitutive Akt activity due to lack of the PTEN lipid phosphatase. Inhibiting PI3-kinase, which activates Akt, sensitized LNCaP cells to TRAIL-induced apoptosis. TRAIL alone activated caspases 8 and XIAP in LNCaP cells but failed to induce full apoptosis. Combining TRAIL with Akt inhibitors allowed cleavage of BID and subsequent mitochondrial apoptosis steps. Akt inhibition of BID cleavage appears to mediate the protective effect
This document discusses the role of somatic mutagenesis in the development of autoimmunity, specifically in systemic lupus erythematosus (SLE). It argues that:
1) Antinuclear antibodies (ANA) that arise in SLE predominantly originate from non-autoreactive B cells that undergo somatic hypermutation (SHM) and are transformed into autoreactive cells, rather than arising from autoreactive B cells escaping central tolerance.
2) SHM may create antigenic peptides in the B cell receptor that provide a route for unregulated T cell help to autoreactive B cells.
3) Spontaneous somatic mutagenesis of genes regulating B cell survival and activation may be a stochastic rate-
Development of an_immunogenicity_score_for_hla-dq_Georgia Marcusso
Eplets are defined as distinct amino acid configurations on the surface of HLA
molecules. The aim of this study was to estimate the immunogenicity of HLADQ eplets in a cohort of 221 pregnancies with HLA-DQ mismatches
This document describes a protocol for differentiating mouse induced pluripotent stem cells (miPSCs) into oligodendrocyte progenitor cells (OPCs). The protocol utilizes a defined, stepwise differentiation process over 8 days to generate high yields of OPCs from three different miPSC lines. The OPCs produced from this protocol are capable of further differentiation into mature, myelinating oligodendrocytes both in vitro and in vivo, making them a potentially useful cell source for modeling demyelinating disorders and developing cell-based therapies.
This study investigated the role of the CLN5 and CLN8 proteins in sphingolipid metabolism and ceramide synthesis. The following key findings are reported:
1) CLN5-deficient fibroblasts showed decreased levels of ceramide, sphingomyelin, and glycosphingolipids as well as defects in adhesion, increased growth, and apoptosis.
2) Expression of CLN8 protein corrected the growth and apoptosis abnormalities in CLN5-deficient cells, suggesting CLN5 and CLN8 proteins interact and modulate ceramide synthase activity.
3) Both CLN5- and CLN8-deficient cells showed decreased de novo ceramide synthesis and specific ceramide
Cilia are present in the developing zebrafish heart and play an important role in cardiac development. The student investigated this role by analyzing cilia in the heart, examining cardiac development in ift54 mutants which lack cilia, and establishing a conditional rescue approach. The key findings were that cilia were observed in the heart from 1-3 days post fertilization, ift54 mutants had smaller cardiac chambers, and the conditional rescue allowing ift54 mutation only in endothelial cells showed increased survival compared to global mutants. Future work includes further analysis of cardiac function in mutants and repeating the conditional rescue experiment.
Li Yingrui Talk at the Beyond the Genome Meeting 2011. "Heading for a Full Solution to Now-Generation Bioinformatics" Covers BGI's missions using "tree" view of genome analysis for discovery.
This paper describes a novel vector system called pMINERVA that allows for the direct conversion of single-chain variable fragments (scFvs) selected from phage display into full-length immunoglobulin G (IgG) molecules without subcloning. The pMINERVA system takes advantage of site-specific bacteriophage integrases expressed in E. coli and intron splicing that occurs in mammalian cells. Using this system, a phage display vector contains regulatory elements to support antibody expression in both E. coli and mammalian cells. The scFv is expressed on phage as a fusion to coat protein III and can be converted to an IgG expressed in mammalian cells through recombination in E. coli. This eliminates the
The researchers are studying synaptic changes in a fruit fly model of variant infantile Batten disease, which is caused by mutations in the CLN7 gene. They found that fruit flies lacking the CLN7 gene had smaller nerves and fewer synapses compared to normal flies. When the CLN7 gene was restored, the nerve returned to normal size. This demonstrates that losing the CLN7 gene causes the changes seen in nerve size and number of synapses. The researchers aim to understand what the CLN7 gene does in nerve cells and how its mutation leads to neurodegeneration, in order to inform future therapy development.
The researchers developed a novel non-viral single doxycycline inducible system that can efficiently regulate gene expression in stem cells both in vitro and in vivo. They generated stable clonal cell lines from mouse neuroblastoma cells (N2a) and human embryonic stem cells (hNSC-ESC) that responded to doxycycline by eliminating GFP expression within 3-8 days. GFP expression was restored 9-10 days after doxycycline removal. When transplanted hNSC-ESCs labeled with quantum dots were subjected to doxycycline in mice, GFP expression was reduced by 61-56% compared to controls. This suggests the system can control gene expression in stem cells without using viral vectors
1) The document examines the effects of mutations on the cAMP signaling pathway in Dictyostelium discoideum. When food is scarce, D. discoideum cells secrete cAMP to signal each other to aggregate.
2) It compares a wild type strain (AX3) to a mutant strain (RI-9) that is defective in cAMP receptor gene cARA. PCR and gel electrophoresis showed that RI-9 lacks expression of cARA, preventing it from sensing cAMP and aggregating.
3) The study also tested how different pH levels and chemicals affect AX3 development. AX3 grew best at pH 4.4 and showed higher aggregation and fruit
This document discusses using a Hidden Markov Model to predict the genome structure of HIV. An HMM with 8 hidden states provided the best balance of accuracy and complexity. Analysis of the HMM revealed non-random patterns in the nucleotide sequence. States were found to have preferential bases and transition probabilities. CpG islands, which are biologically important regions, were found to occur only in 3 of the 8 states. The model accurately generated a sequence similar to the original HIV genome.
The document describes a new method called the mutagenic chain reaction (MCR) that uses the CRISPR/Cas9 system to efficiently generate homozygous loss-of-function mutations from an initial heterozygous mutation. The researchers developed an MCR construct containing Cas9, a guide RNA targeting a genomic sequence of interest, and flanking homology arms. This construct inserts into the target site and expresses Cas9 and guide RNA to cleave the opposite chromosome, converting it to homozygosity via homology-directed repair. Testing this method in Drosophila, they found it efficiently spread mutations from the initial chromosome to the homologous one in somatic and germline cells, demonstrating the potential of MCR for
Pluripotency describes cells that have the potential to give rise to cells from the three germ layers but not extraembryonic tissues. There are multiple types of pluripotent stem cells that can be isolated from different stages of embryonic development in rodents and humans. Naive pluripotent stem cells, like mouse embryonic stem cells, resemble the pre-implantation embryo state while primed pluripotent stem cells, like mouse epiblast stem cells, resemble the post-implantation embryo state. Growth conditions influence whether pluripotent stem cells attain a naive or primed state in vitro.
This document summarizes recent research on CLN8 disease variants in Latin America. It discusses:
1) The establishment of an NCL research program in Argentina in 2003 to study different forms of NCL in Latin America.
2) The identification of the first reported CLN8 mutation (c.1A>G) found in an Argentinean patient, expanding knowledge of CLN8 geographic distribution.
3) Ongoing efforts to investigate the cellular biology and metabolic pathways of the CLN8 protein, identify new biomarkers for disease monitoring, and characterize the c.1A>G mutation's effects.
The document summarizes a study that found:
1) Interleukin-12 (IL-12) in dendritic cells traffics through late endocytic vesicles marked by the SNARE protein VAMP7.
2) Dendritic cells from VAMP7 knockout mice show partially impaired secretion of bioactive IL-12/p70 dimer.
3) At the immune synapse between dendritic cells and T cells, IL-12 containing vesicles rapidly redistribute and their release is entirely dependent on VAMP7, leading to reduced T cell acquisition of effector functions when stimulated with VAMP7 knockout dendritic cells.
I. The study screened the CHRNG gene in 29 individuals with distal limb contractures and various diagnoses like Multiple Pterygium Syndrome (MPS), Sheldon Hall Syndrome (SHS), distal arthrogryposis type 5D (DA5D), and neurologic abnormalities and distal contractures not otherwise specified (NAC).
II. Two mutations were found in the CHRNG gene in two individuals with MPS - a single-base pair insertion and a two-base pair deletion. It is unclear if these mutations are pathogenic.
III. No mutations were found in the individuals with DA5D, SHS or NAC, indicating CHRNG is unlikely responsible for clinical overlap between
1) WASp-deficient dendritic cells (DCs) show impaired activation of naive CD8+ T cells, especially at low antigen doses.
2) This is partly due to altered trafficking of antigen-bearing DCs from the periphery to lymph nodes. However, correcting DC migration does not fully rescue T cell activation.
3) In vitro and in vivo imaging revealed that cytoskeletal alterations in WASp-null DCs reduce their ability to form and maintain conjugates with naive CD8+ T cells in lymph nodes, contributing to defective T cell priming.
Plasmacytoid dendritic cells (PDC) play an important role in antiviral immunity by secreting type I interferons. This study found that while PDC have a poor ability to endocytose soluble or particulate antigens compared to myeloid dendritic cells, they can efficiently capture and cross-present viral antigens from influenza-exposed cells. Specifically, PDC took up cellular material from live influenza-infected cells, matured, and very efficiently cross-presented viral antigens to activate CD8+ T cells. Therefore, during viral infections PDC function not only to secrete cytokines but also to recognize infected cells and trigger the antiviral immune response through antigen cross-presentation.
Viviparity is a unique characteristic of mammals. Gestational outcomes avoiding fetal defects or loss, maternal infection, or morbidity are contingent upon an intimate association between mother and developing fetus that nurtures the fetus without provoking maternal immune responses. Therefore the maintenance of optimal immunity is necessary for the successful gestation.
CAR-T cells are genetically modified T cells that are designed to target and destroy cancer cells. They contain chimeric antigen receptors (CARs) that allow them to recognize specific antigens on tumor cells independently of the normal T cell receptor. The CAR is composed of an extracellular antigen recognition domain attached to transmembrane and co-stimulatory domains, allowing the modified T cells to directly bind and kill cancer cells upon antigen recognition and initiate an immune response against the tumor.
This document summarizes the genome sequencing and analysis of six Staphylococcus epidermidis strains isolated from preterm neonates presenting with sepsis. Genomic DNA was isolated from the strains and sequenced using Illumina MiSeq. The sequences were rapidly assembled and annotated using the Simplicity pipeline in under an hour. Comparison of the genomes identified genes relevant to adhesion, biofilm formation, virulence and antibiotic resistance.
Viviparity is a unique characteristic of mammals. Gestational outcomes avoiding fetal defects or loss, maternal infection, or morbidity are contingent upon an intimate association between mother and developing fetus that nurtures the fetus without provoking maternal The immune responses.immune cells exist within the decidua to combat infection. The immunology of the maternal-fetal interface from the perspective of these diverse sets of demands, which, may not always be compatible with one another.
Development of an_immunogenicity_score_for_hla-dq_Georgia Marcusso
Eplets are defined as distinct amino acid configurations on the surface of HLA
molecules. The aim of this study was to estimate the immunogenicity of HLADQ eplets in a cohort of 221 pregnancies with HLA-DQ mismatches
This document describes a protocol for differentiating mouse induced pluripotent stem cells (miPSCs) into oligodendrocyte progenitor cells (OPCs). The protocol utilizes a defined, stepwise differentiation process over 8 days to generate high yields of OPCs from three different miPSC lines. The OPCs produced from this protocol are capable of further differentiation into mature, myelinating oligodendrocytes both in vitro and in vivo, making them a potentially useful cell source for modeling demyelinating disorders and developing cell-based therapies.
This study investigated the role of the CLN5 and CLN8 proteins in sphingolipid metabolism and ceramide synthesis. The following key findings are reported:
1) CLN5-deficient fibroblasts showed decreased levels of ceramide, sphingomyelin, and glycosphingolipids as well as defects in adhesion, increased growth, and apoptosis.
2) Expression of CLN8 protein corrected the growth and apoptosis abnormalities in CLN5-deficient cells, suggesting CLN5 and CLN8 proteins interact and modulate ceramide synthase activity.
3) Both CLN5- and CLN8-deficient cells showed decreased de novo ceramide synthesis and specific ceramide
Cilia are present in the developing zebrafish heart and play an important role in cardiac development. The student investigated this role by analyzing cilia in the heart, examining cardiac development in ift54 mutants which lack cilia, and establishing a conditional rescue approach. The key findings were that cilia were observed in the heart from 1-3 days post fertilization, ift54 mutants had smaller cardiac chambers, and the conditional rescue allowing ift54 mutation only in endothelial cells showed increased survival compared to global mutants. Future work includes further analysis of cardiac function in mutants and repeating the conditional rescue experiment.
Li Yingrui Talk at the Beyond the Genome Meeting 2011. "Heading for a Full Solution to Now-Generation Bioinformatics" Covers BGI's missions using "tree" view of genome analysis for discovery.
This paper describes a novel vector system called pMINERVA that allows for the direct conversion of single-chain variable fragments (scFvs) selected from phage display into full-length immunoglobulin G (IgG) molecules without subcloning. The pMINERVA system takes advantage of site-specific bacteriophage integrases expressed in E. coli and intron splicing that occurs in mammalian cells. Using this system, a phage display vector contains regulatory elements to support antibody expression in both E. coli and mammalian cells. The scFv is expressed on phage as a fusion to coat protein III and can be converted to an IgG expressed in mammalian cells through recombination in E. coli. This eliminates the
The researchers are studying synaptic changes in a fruit fly model of variant infantile Batten disease, which is caused by mutations in the CLN7 gene. They found that fruit flies lacking the CLN7 gene had smaller nerves and fewer synapses compared to normal flies. When the CLN7 gene was restored, the nerve returned to normal size. This demonstrates that losing the CLN7 gene causes the changes seen in nerve size and number of synapses. The researchers aim to understand what the CLN7 gene does in nerve cells and how its mutation leads to neurodegeneration, in order to inform future therapy development.
The researchers developed a novel non-viral single doxycycline inducible system that can efficiently regulate gene expression in stem cells both in vitro and in vivo. They generated stable clonal cell lines from mouse neuroblastoma cells (N2a) and human embryonic stem cells (hNSC-ESC) that responded to doxycycline by eliminating GFP expression within 3-8 days. GFP expression was restored 9-10 days after doxycycline removal. When transplanted hNSC-ESCs labeled with quantum dots were subjected to doxycycline in mice, GFP expression was reduced by 61-56% compared to controls. This suggests the system can control gene expression in stem cells without using viral vectors
1) The document examines the effects of mutations on the cAMP signaling pathway in Dictyostelium discoideum. When food is scarce, D. discoideum cells secrete cAMP to signal each other to aggregate.
2) It compares a wild type strain (AX3) to a mutant strain (RI-9) that is defective in cAMP receptor gene cARA. PCR and gel electrophoresis showed that RI-9 lacks expression of cARA, preventing it from sensing cAMP and aggregating.
3) The study also tested how different pH levels and chemicals affect AX3 development. AX3 grew best at pH 4.4 and showed higher aggregation and fruit
This document discusses using a Hidden Markov Model to predict the genome structure of HIV. An HMM with 8 hidden states provided the best balance of accuracy and complexity. Analysis of the HMM revealed non-random patterns in the nucleotide sequence. States were found to have preferential bases and transition probabilities. CpG islands, which are biologically important regions, were found to occur only in 3 of the 8 states. The model accurately generated a sequence similar to the original HIV genome.
The document describes a new method called the mutagenic chain reaction (MCR) that uses the CRISPR/Cas9 system to efficiently generate homozygous loss-of-function mutations from an initial heterozygous mutation. The researchers developed an MCR construct containing Cas9, a guide RNA targeting a genomic sequence of interest, and flanking homology arms. This construct inserts into the target site and expresses Cas9 and guide RNA to cleave the opposite chromosome, converting it to homozygosity via homology-directed repair. Testing this method in Drosophila, they found it efficiently spread mutations from the initial chromosome to the homologous one in somatic and germline cells, demonstrating the potential of MCR for
Pluripotency describes cells that have the potential to give rise to cells from the three germ layers but not extraembryonic tissues. There are multiple types of pluripotent stem cells that can be isolated from different stages of embryonic development in rodents and humans. Naive pluripotent stem cells, like mouse embryonic stem cells, resemble the pre-implantation embryo state while primed pluripotent stem cells, like mouse epiblast stem cells, resemble the post-implantation embryo state. Growth conditions influence whether pluripotent stem cells attain a naive or primed state in vitro.
This document summarizes recent research on CLN8 disease variants in Latin America. It discusses:
1) The establishment of an NCL research program in Argentina in 2003 to study different forms of NCL in Latin America.
2) The identification of the first reported CLN8 mutation (c.1A>G) found in an Argentinean patient, expanding knowledge of CLN8 geographic distribution.
3) Ongoing efforts to investigate the cellular biology and metabolic pathways of the CLN8 protein, identify new biomarkers for disease monitoring, and characterize the c.1A>G mutation's effects.
The document summarizes a study that found:
1) Interleukin-12 (IL-12) in dendritic cells traffics through late endocytic vesicles marked by the SNARE protein VAMP7.
2) Dendritic cells from VAMP7 knockout mice show partially impaired secretion of bioactive IL-12/p70 dimer.
3) At the immune synapse between dendritic cells and T cells, IL-12 containing vesicles rapidly redistribute and their release is entirely dependent on VAMP7, leading to reduced T cell acquisition of effector functions when stimulated with VAMP7 knockout dendritic cells.
I. The study screened the CHRNG gene in 29 individuals with distal limb contractures and various diagnoses like Multiple Pterygium Syndrome (MPS), Sheldon Hall Syndrome (SHS), distal arthrogryposis type 5D (DA5D), and neurologic abnormalities and distal contractures not otherwise specified (NAC).
II. Two mutations were found in the CHRNG gene in two individuals with MPS - a single-base pair insertion and a two-base pair deletion. It is unclear if these mutations are pathogenic.
III. No mutations were found in the individuals with DA5D, SHS or NAC, indicating CHRNG is unlikely responsible for clinical overlap between
1) WASp-deficient dendritic cells (DCs) show impaired activation of naive CD8+ T cells, especially at low antigen doses.
2) This is partly due to altered trafficking of antigen-bearing DCs from the periphery to lymph nodes. However, correcting DC migration does not fully rescue T cell activation.
3) In vitro and in vivo imaging revealed that cytoskeletal alterations in WASp-null DCs reduce their ability to form and maintain conjugates with naive CD8+ T cells in lymph nodes, contributing to defective T cell priming.
Plasmacytoid dendritic cells (PDC) play an important role in antiviral immunity by secreting type I interferons. This study found that while PDC have a poor ability to endocytose soluble or particulate antigens compared to myeloid dendritic cells, they can efficiently capture and cross-present viral antigens from influenza-exposed cells. Specifically, PDC took up cellular material from live influenza-infected cells, matured, and very efficiently cross-presented viral antigens to activate CD8+ T cells. Therefore, during viral infections PDC function not only to secrete cytokines but also to recognize infected cells and trigger the antiviral immune response through antigen cross-presentation.
Viviparity is a unique characteristic of mammals. Gestational outcomes avoiding fetal defects or loss, maternal infection, or morbidity are contingent upon an intimate association between mother and developing fetus that nurtures the fetus without provoking maternal immune responses. Therefore the maintenance of optimal immunity is necessary for the successful gestation.
CAR-T cells are genetically modified T cells that are designed to target and destroy cancer cells. They contain chimeric antigen receptors (CARs) that allow them to recognize specific antigens on tumor cells independently of the normal T cell receptor. The CAR is composed of an extracellular antigen recognition domain attached to transmembrane and co-stimulatory domains, allowing the modified T cells to directly bind and kill cancer cells upon antigen recognition and initiate an immune response against the tumor.
This document summarizes the genome sequencing and analysis of six Staphylococcus epidermidis strains isolated from preterm neonates presenting with sepsis. Genomic DNA was isolated from the strains and sequenced using Illumina MiSeq. The sequences were rapidly assembled and annotated using the Simplicity pipeline in under an hour. Comparison of the genomes identified genes relevant to adhesion, biofilm formation, virulence and antibiotic resistance.
Viviparity is a unique characteristic of mammals. Gestational outcomes avoiding fetal defects or loss, maternal infection, or morbidity are contingent upon an intimate association between mother and developing fetus that nurtures the fetus without provoking maternal The immune responses.immune cells exist within the decidua to combat infection. The immunology of the maternal-fetal interface from the perspective of these diverse sets of demands, which, may not always be compatible with one another.
1) The document discusses tumor immunology and mechanisms of tumor immune evasion. It describes how tumors can downregulate MHC expression, secrete immunosuppressive factors, inhibit T cell function through checkpoint pathways like PD-1/PD-L1, and recruit immunosuppressive cells like Tregs.
2) Checkpoint pathways like CTLA-4 and PD-1 normally regulate T cell activation, but tumors can exploit these pathways to evade immune destruction by overexpressing ligands that bind these inhibitory receptors.
3) Several immunotherapies targeting CTLA-4 and PD-1/PD-L1 have been developed including ipilimumab, nivolumab, pembrol
This study monitored in vitro immune responses in a healthy donor to various self-antigens presented using dendritic cells. Adenoviral vectors were used to deliver tumor antigens like melan A/MART-1, gp100, NY-ESO-1, HER2, and cytokeratin 18 or HLA-A2 epitope peptides to antigen presenting cells. Analysis of T cell surface markers, cytokine release, and cytolytic activity revealed that gp100 stimulation led to a predominant IFN+ CD8+ CD56- T cell population within three restimulations, whereas MART-1 stimulation resulted in an equal division of IFN+ cells between CD4+ and CD8+ CD56- T cells. Comprehen
This document describes a case study of a 5-year-old female Argentine patient with a mutation in the CD25 gene resulting in CD25 deficiency. Key findings include chronic inflammatory lung disease (follicular bronchiolitis), eczema, infections, extremely low levels of regulatory T cells, and a homozygous missense mutation (c.122a>c; p.Y41S) in the CD25αR gene. The mutation is predicted to compromise protein function and cause immune dysregulation similar to IPEX syndrome. CD25 deficiency should be considered in patients presenting with these clinical and laboratory features to facilitate early diagnosis and treatment.
This research article demonstrates the use of Wavelength Modulated Raman Spectroscopy (WMRS) to identify major immune cell subsets in an unlabeled and non-fixed state. Using WMRS, the researchers were able to distinguish between CD4+ T lymphocytes, CD8+ T lymphocytes, and CD56+ Natural Killer cells from multiple donors with up to 96% specificity. They also distinguished between CD303+ plasmacytoid and CD1c+ myeloid dendritic cell subsets. This label-free method opens new opportunities for analyzing immune systems and developing diagnostic technologies without altering or damaging the cells.
current understanding of the immunological changes and adaptations that occur in pregnancy enabling tolerance to the foreign paternal fetal antigens in the maternal uterus
Yayan T. Sundara, Dokter di Klinik Jejaring Padjadjaran dan Master of Bio Medical Science Research dari Leiden University Medical Centrum, juga staff Departemen Pelayanan PT Rumah Sakit Padjadjaran
This document summarizes research on anaplastic large cell lymphoma (ALCL) associated with breast implants. The researchers established three new cell lines from patient biopsy specimens to study the unique biology of this cancer. Characterization found chromosomal abnormalities but no common genetic translocations. The cancer cells showed markers of T-cells and antigen presentation. Studies revealed strong activation of STAT3 signaling related to increased interleukin-6 and decreased SHP-1 phosphatase, suggesting a mechanism of cell survival. Inhibiting STAT3 or treating with chemotherapy killed the cancer cells in vitro, indicating potential new therapies for this disease. The cell lines provide models to further understand breast implant-associated ALCL and identify effective treatments.
This document summarizes T lymphocyte development and activation. It describes how progenitor cells commit to the T cell lineage in the bone marrow and thymus. In the thymus, T cells undergo proliferation, rearrangement of T cell receptor genes, and positive and negative selection. This results in functionally distinct T cell subsets that migrate to lymph nodes upon activation. T cell activation requires three signals - engagement of the T cell receptor by peptide-MHC complexes, costimulatory molecules such as CD28 binding to B7, and cytokine signals. This leads to intracellular signaling cascades and expression of genes regulating T cell effector function.
This document summarizes a presentation on T-helper 9 (Th9) cells, a relatively new member of the Th cell family. The presentation outlines that Th9 cells differentiate from naive CD4+ T cells in response to TGF-β and IL-4 cytokines. Th9 cells secrete IL-9 and play roles in allergies, parasite expulsion, autoimmune diseases, cancer, and other conditions. While Th9 cells can promote inflammation and tissue damage, they also help expel parasites and inhibit tumor growth. Further research is needed to understand Th9 cell mechanisms and potential immunotherapy applications.
1) The document discusses how the neonatal Fc receptor for IgG (FcRn) expressed by dendritic cells in the intestinal mucosa plays a critical role in anti-cancer immunosurveillance by eliciting protective CD8+ T cell immune responses against colorectal carcinoma.
2) FcRn mediates this tumor immunosurveillance by facilitating the cross-presentation of tumor-associated antigens from immune complexes formed between tumor antigens and tumor-reactive IgGs to activate tumor-specific CD8+ T cells.
3) Manipulating this FcRn-dependent antigen processing pathway in dendritic cells is a promising strategy for cancer immunotherapy as it can overcome the immunosuppressive environment in m
T-lymphocyte cells originate from hematopoietic stem cells in the bone marrow. They migrate to the thymus for maturation and differentiation. In the thymus, they progress through distinct developmental stages defined by changing cell surface marker expression. This includes double negative (DN) stages where T-cell receptor gene rearrangement occurs. Positively selected cells further differentiate into double positive (DP) cells and undergo negative selection to eliminate self-reactive cells. Mature naive T-cells exit the thymus to participate in immune responses by recognizing antigens and triggering effector functions through cell-cell interactions and cytokine signaling.
This document describes the development of a method to detect human CD4+ T cells specific for influenza A virus using soluble human MHC class II tetramers. Key points:
1) Soluble MHC class II tetramers containing an immunodominant peptide from influenza hemagglutinin were constructed and used to identify antigen-specific T cells from influenza-immune individuals.
2) After 7 days of proliferation in response to stimulation by influenza peptide or vaccine, tetramer-positive cells had undergone extensive division and expressed markers of activated CD4+ T cells.
3) The influenza peptide-specific T cells represented a major subset of proliferating CD4+ T cells responding to whole influenza vaccine.
4)
This document summarizes a study examining the use of biodegradable nanoparticles loaded with TGF-β and IL-2 cytokines and targeted to CD4+ cells for inducing and maintaining regulatory T cells (Tregs). The nanoparticles were able to induce CD4+ Tregs in vitro and expand their numbers in vivo. Nanoparticle-induced Tregs demonstrated enhanced stability and retained their suppressive phenotype even in inflammatory conditions, highlighting the potential of this nanoparticle approach for stabilizing Tregs to treat autoimmune disease and inflammation.
This study aimed to determine if mutations in the human cystatin M/E gene (CST6) contribute to harlequin ichthyosis (HI) by sequencing the entire coding region and intron-exon boundaries of CST6 in 11 patients with HI. No mutations were found in CST6, indicating it is not a major gene for types 1 and 2 HI. However, cystatin M/E protein expression was normal in patient tissues by immunohistochemistry, suggesting regulatory or noncoding mutations were unlikely. While CST6 does not appear to cause HI, further study of its role in skin differentiation and other skin disorders may provide insights into cornification pathways.
1. Resource
CD8+
T Cells from Human Neonates Are Biased
toward an Innate Immune Response
Graphical Abstract
Highlights
d Human neonatal CD8+
T cells have a distinctive transcription
and chromatin landscape
d Human neonatal CD8+
T cells are less cytotoxic than their
adult counterparts
d Human neonatal CD8+
T cells are biased toward an innate
immune response
d This bias could explain the sensitivity of neonates to
infections and inflammation
Authors
Ariel O. Galindo-Albarra´ n,
Oscar H. Lo´ pez-Portales,
Darely Y. Gutie´ rrez-Reyna, ...,
Alfonso Valencia, Salvatore Spicuglia,
M. Ange´ lica Santana
Correspondence
salvatore.spicuglia@inserm.fr (S.S.),
santana@uaem.mx (M.A.S.)
In Brief
Galindo-Albarra´ n et al. examine the
distinct pattern of gene transcription and
histone modification landscape in human
neonatal CD8+
T cells. Their data suggest
that cells are skewed toward an innate
immune response and low cytotoxic
function. These properties could explain
the high susceptibility of neonates to
infections and inflammation. Explore the
consortium data at the Cell Press IHEC
webportal at www.cell.com/consortium/
IHEC.
Accession Numbers
GSE61570
Galindo-Albarra´ n et al., 2016, Cell Reports 17, 2151–2160
November 15, 2016 ª 2016 The Author(s).
http://dx.doi.org/10.1016/j.celrep.2016.10.056
2. Cell Reports
Resource
CD8+
T Cells from Human Neonates Are Biased
toward an Innate Immune Response
Ariel O. Galindo-Albarra´ n,1,2,3 Oscar H. Lo´ pez-Portales,1 Darely Y. Gutie´ rrez-Reyna,1 Otoniel Rodrı´guez-Jorge,1
Jose´ Antonio Sa´ nchez-Villanueva,1 Oscar Ramı´rez-Pliego,1 Aure´ lie Bergon,2,3,4 Be´ atrice Loriod,2,3,4 He´ le` ne Holota,2,3,4
Jean Imbert,2,3,4 Armando Herna´ ndez-Mendoza,1 Pierre Ferrier,5 Enrique Carrillo-de Santa Pau,6 Alfonso Valencia,6
Salvatore Spicuglia,2,3,* and M. Ange´ lica Santana1,7,*
1Centro de Investigacio´ n en Dina´ mica Celular (IICBA), Universidad Auto´ noma del Estado de Morelos, Av. Universidad 1001, Chamilpa,
Cuernavaca Morelos 62100, Mexico
2INSERM, TAGC UMR_S 1090, 13288 Marseille Cedex 09, France
3Aix-Marseille University, TAGC UMR_S 1090, 13288 Marseille Cedex 09, France
4Transcriptomique et Ge´ nomique de Marseille-Luminy (TGML), IBiSA platform, 13288 Marseille, France
5Centre d’Immunologie de Marseille-Luminy, Aix Marseille Universite´ , INSERM, Centre National de la Recherche Scientifique (CNRS), 13288
Marseille Cedex 09, France
6Structural Biology and BioComputing Program, Spanish National Cancer Research Center, CNIO, 28029 Madrid, Spain
7Lead Contact
*Correspondence: salvatore.spicuglia@inserm.fr (S.S.), santana@uaem.mx (M.A.S.)
http://dx.doi.org/10.1016/j.celrep.2016.10.056
SUMMARY
To better understand why human neonates show
a poor response to intracellular pathogens, we
compared gene expression and histone modification
profiles of neonatal naive CD8+
T cells with that of
their adult counterparts. We found that neonatal lym-
phocytes have a distinct epigenomic landscape
associated with a lower expression of genes involved
in T cell receptor (TCR) signaling and cytotoxicity and
a higher expression of genes involved in the cell cycle
and innate immunity. Functional studies corrobo-
rated that neonatal CD8+
T cells are less cytotoxic,
transcribe antimicrobial peptides, and produce reac-
tive oxygen species. Altogether, our results show
that neonatal CD8+
T cells have a specific genetic
program biased toward the innate immune response.
These findings will contribute to better diagnosis and
management of the neonatal immune response.
INTRODUCTION
Infant morbidity and mortality is a major health problem world-
wide. Reducing the mortality of children under 5 years old is a pri-
ority for the United Nations. Of the deaths of children under five,
37% are neonatal (from birth to 1 month of age); 25% of these are
caused by infections (PrabhuDas et al., 2011). Following immune
challenges, the neonatal response is often tolerant, skewed, or
deficient (Levy, 2007). This leads to a poor response to infection
and low immune memory in response to vaccines, which require
multiple booster shots to protect infants. Murine neonatal lym-
phocytes are, however, capable of adult-like responses under
particular conditions, suggesting full functionality but a rather
high activation threshold (Opiela et al., 2008). IL-12 is necessary
as a third signal to induce an efficient response of neonatal CD8+
T cells (McCarron and Reen, 2010).
In human neonates, epigenetic mechanisms, involving
impaired nucleosome remodeling, keep IL-12 levels very low;
IL-12 does not reach adult levels until the age of 12 (Vanden Eijn-
den et al., 2006). The neonatal immune system faces a particular
demand during the transition from the basically sterile conditions
of the womb to the outside world. This transition should permit
the colonization of the mucosal flora and the recognition of
non-dangerous antigens of the new environment, avoiding
strong inflammation that could damage still developing tissues.
Neutralizing antibodies from the mother, as well as antimicrobial
peptides and lipids, help to protect the newborn. Nonetheless,
no passive cellular immunity is transferred from the mother,
and neonates depend on their own immune system to fight intra-
cellular pathogens (Adkins et al., 2004; Levy, 2007).
T cell differentiation proceeds through developmental stages
in which gene expression is established by signal clues that pro-
gram the epigenetic landscape, affecting the functionality of the
cells (Taniuchi, 2013). In this work, we have examined the tran-
scriptome and analyzed the RNA sequencing (RNA-seq) and
epigenetic profiles of neonatal and adult human naive CD8+
T cells. The expression signature of the neonatal cells is
described along with functional assays that corroborated that
neonatal CD8+
T cells undergo homeostatic proliferation, are
less cytotoxic, and produce antimicrobial peptides and reactive
oxygen species. Analysis of epigenetic marks showed corre-
spondence between changes in specific modifications and
differentially expressed genes. Furthermore, a specific set of
transcription factors is associated with these cell types, with
maturation and innate immunity characteristics in neonatal cells
and with functional cytotoxic T lymphocytes in adult cells. Chro-
matin state analysis also showed a specific configuration of the
neonatal cells. Altogether our data show that human neonatal
Cell Reports 17, 2151–2160, November 15, 2016 ª 2016 The Author(s). 2151
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
3. CD8+
T cells have a specific gene expression program, resulting
in a bias toward innate immunity defense mechanisms.
RESULTS
Neonatal CD8+
T Cells Have a Distinctive Transcriptome
Signature
To investigate the nature of the high sensitivity of neonates to
intracellular pathogens, we assessed the transcriptome of naive
CD8+
T cells from four human neonatal and four adult donors
(GEO: GSE61570). To ensure that we were evaluating only the
naive circulating populations of CD8+
T cells, peripheral blood
mononuclear cells (PBMCs) or cord blood mononuclear cells
(CBMCs) were first submitted to negative selection to eliminate
monocytes, B cells, CD4+
T cells, gd T cells, and natural killer
(NK) and NKT cells. PBMCs also were depleted of effector and
memory populations with antibodies against CD44 and
CD45RO. CBMCs were negative for these markers. Cells were
then purified with anti-CD8 antibodies. The resulting populations
were about 98% CD3+CD8+, over 95% negative for CD45 RO
and 100% positive for CD45 RA. The percentage of TCRgd+
cells
was found comparable between adult and neonatal samples,
and it represented less than 1% of the cells (Figure S1C).
To assess the consistency of the transcriptome data, we per-
formed a Pearson correlation with all microarray probes,
showing a clear separation between the adult and neonatal sam-
ples (Figure S1A). Figure 1A shows the volcano plot of differen-
tially expressed genes among adult and neonatal cells, with
576 significantly overexpressed genes in the neonatal samples
and 659 in the adult cells (p < 0.05, Limma corrected t test;
Data S1). Differential expression of genes of immunological inter-
est (Figures 1B, 3B, and 4C) or randomly chosen (Figure S1D)
was validated by qRT-PCR, using independent samples. Protein
expression of TLR5 was validated by flow cytometry (Figures 1C
and S5D). It has been reported that human neonatal CD8+
T cells
A
MAF
BCL11A
TLR3
TLR5
CDK1
B
Adult Neonate
TLR5MFI
*
*
C
200
150
100
50
0
Fold change Neonate / Adult
-Log10
(Pvalue)
-9 -7 -5 -3 -1 0
6
4
2
0
1 3 5 7 9
0
1
2
3
4
5
8
10
12
14
Ratio to GAPDH
*
*
*
*
*
*
*
Adult
Neonate
BCL11AB3GAT1
MEOX1
TRPC1
TLR3
SERPINF1
CBX2
KIF5C
Figure 1. Neonatal and Adult CD8+
T Cells
Have Their Own Characteristic Transcription
Profile
(A) Volcano plot shows differentially expressed
genes between adult and neonatal CD8+
T cells.
(B) Validation of a group of genes by qRT-PCR is
shown.
(C) Flow cytometry analysis of TLR5 expression in
four adult and five neonatal samples of CD8+
T cells
(mean ± SEM). Statistical significance was as-
sessed by Mann-Whitney U test (*p < 0.05 and
**p < 0.01).
respond to TLR5 enhancing the produc-
tion of IFN-g (McCarron and Reen,
2009), suggesting that the overexpressed
TLR5 protein could be relevant for
neonatal CD8+
T cell functions.
We asked whether differentially ex-
pressed genes clustered in specific path-
ways. Common pathways were certainly
found, but each cell group also showed
enrichment in their own specific signaling pathways (Figure 2)
and gene ontology (GO) terms (Figure S2A). Neonatal cells
were enriched in cell cycle, hematopoietic cell lineage, and ho-
mologous recombination pathways. Adult cells were enriched
in T cell receptor (TCR)- and mitogen-activated protein kinase
(MAPK)-signaling pathways, outlining differences in the maturity
and basic functions between the two cell groups. Global analysis
of our samples using the gene set enrichment analysis (GSEA)
software, which calculates significance as related to the number
of genes altered in a given pathway, corroborated that neonatal
CD8+
T cells were enriched in pathways associated with cell cy-
cle, cell maturation, and inflammation, while adult cells were en-
riched in TCR signaling and function (Figures S2B and S2C).
Consistent with these observations, reactome network analysis
showed that adult-specific transcripts formed a network of path-
ways related to TCR signaling, while neonate-specific transcripts
formed webs associated with cell cycle, antiviral pathways, and
innate immunity (Figure S3). Differential expression of genes of
these main signature pathways also was validated by comparing
our microarray analysis with RNA-seq data from Blueprint
(neonatal CD8+
T cells) (Adams et al., 2012) and Roadmap (adult
CD8+
T cells) (Kundaje et al., 2015) international consortia (Fig-
ure S4). The analysis of these pathways is described below.
Neonatal CD8+
T Cells Are Less Cytotoxic than
Adult Cells
The signaling pathways enriched in adult CD8+
T cells were
related to cytotoxicity and TCR and MAPK signaling. Upregula-
tion of adult cell genes involved in these pathways is shown in
Figure 3A. The genes for the effector molecules directly involved
in cytotoxic death (GZMB, PRF1, and FASL), however, were not
differentially expressed between adult and neonatal cells after
Limma correction, although differences in the mean fluores-
cence were higher than 2-fold. We therefore examined the
expression of these three genes and the other three genes
2152 Cell Reports 17, 2151–2160, November 15, 2016
4. involved in cytotoxicity that were differentially expressed
(GZMH, KIR2DS3, and ITGAL) in four independent neonatal
and adult cells samples by qRT-PCR. We validated the signifi-
cant difference in the expression of IFN-g, a signature molecule
for the activation of the cytotoxic response. We observed highly
significant differences in the expression of these seven genes
between the two types of cells (Figure 3B).
We also evaluated the mRNA levels of IL-2 and GZMB, both
under basal conditions and after TCR stimulation in vitro with
anti-CD3/CD28 cross-linking for 6 hr. In both cases, the basal
levels of expression were lower in neonatal cells, and the adult
cells responded to stimulation with a higher increase in IL2
expression (Figure S5C). We also assessed the amounts of
IFN-g, Granzime B, the activation marker CD69, and IL-2 pro-
teins in basal and stimulated conditions. The base level amount
of CD69 was similar in both naive neonatal and adult cells, but
it was much higher in the adult cells after stimulation (Fig-
ure S5A). For IL-2 and IFN-g, changes after 6 hr of stimulation
were limited in the two cell populations, but the basal levels of
the two cytokines were significantly lower in the neonatal cells
(Figure S5B), in agreement with a previous report (Hodge et al.,
2001).
To assess whether the differential expression in the cytotox-
icity-related genes leads to a functional difference, we examined
the cytotoxicity induced in target cells by neonatal or adult CD8+
T cell populations. To avoid misinterpretation occasioned by the
use of cell lines and their increased resistance to apoptosis, we
used as targets PBMCs of an allogeneic donor (the same donor
in all assays). Figure 3C shows that adult T cells induced a
greater extent of apoptosis in the target cells than the neonatal
cells. Furthermore, we measured the amount of the degranula-
tion marker CD107 and of Granzyme B in both cell populations.
Although both neonatal and adult T cells were positive for CD107
at similar proportions, only adult cells were positive for Gran-
zyme B; neonatal cells were basically negative for this marker
(Figures 3D and S5D). The lower cytotoxicity of neonatal cells
also could be related to a lower expression of the TCR and
MAPK signaling in these cells, consistent with their more imma-
ture phenotype.
Neonatal CD8+
T Cells Proliferate Constitutively and Are
Biased toward Innate Immunity
A prominent characteristic of the neonatal CD8+
T cells was the
high expression of cell cycle genes (Figure 4A). These genes
included cell cycle regulators (E2F2 and RBBP4) or were associ-
ated with the formation of the replicative complex (MCM7,
MCM3, and CDT1), the maturation-promoting factor (CCNB2),
and the cyclin-dependent kinase (CDK1), which promotes
mitosis from the G2 phase. T cells undergo two types of prolifer-
ation: homeostatic proliferation and clonal expansion (den
Braber et al., 2012). In the former, cells proliferate without differ-
entiation and, therefore, maintain the pools of circulating cells. In
the latter, during clonal expansion, antigen-recognizing clones
proliferate due to TCR and co-stimulatory signals, and cells
differentiate into effector and memory cells.
To investigate whether neonatal cells were actually undergo-
ing homeostatic proliferation, we labeled the cells with carbox-
yfluorescein succinimidyl ester (CFSE) and evaluated cell
division, without any stimulus apart from that of the fetal calf
05101520
Log10(Benjamini)
Neonate
Adult
MAPKsignalingpathway
Pathwaysincancer
Endocytosis
Cytokine-cytokinereceptorinteraction
Calciumsignalingpathway
Celladhesionmolecules(CAMs)
Focaladhesion
Lysosome
Dilatedcardiomyopathy
Naturalkillercellmediatedcytotoxicity
Melanogenesis
Tightjunction
Regulationofactincytoskeleton
Hypertrophiccardiomyopathy(HCM)
Systemiclupuserythematosus
Neurotrophinsignalingpathway
Adipocytokinesignalingpathway
Viralmyocarditis
Insulinsignalingpathway
IntestinalimmunenetworkforIgAprod.
Tcellreceptorsignalingpathway
Arginineandprolinemetabolism
Vascularsmoothmusclecontraction
Wntsignalingpathway
Hematopoieticcelllineage
Glutathionemetabolism
Mismatchrepair
N-Glycanbiosynthesis
Leukocytetransendothelialmigration
Neuroactiveligand-receptorinteraction
FcgammaR-mediatedphagocytosis
Huntington'sdisease
Colorectalcancer
Pyrimidinemetabolism
Alzheimer'sdisease
Onecarbonpoolbyfolate
Purinemetabolism
Oocytemeiosis
Chemokinesignalingpathway
Cellcycle
p53signalingpathwayFigure 2. Differential Pathways Enriched in Neonatal or Adult T Cells
Differentially expressed genes were analyzed with DAVID software to asso-
ciate genes to KEGG signaling pathways. The top 25 more significant path-
ways are shown.
A
Cytotoxicity
D
MFI
10
30
50
100
140
180
*
GZMB CD107a
Adult
Neonate
C
0
10
20
30
40
50
%IPTargetCells
Adult Neonate
*
Adult Neonate
1 2 3 4 1 2 3 4
CD48
FYN
GZMH
IFNG
ITGAL
KIR2DS3
KIR3DL2
MICB
SHC1
TNFRSF10B
B
0
1
2
3
ITGAL
FASL
PRF1
GZMH
GZMB
IFNG
Ratio to GAPDH
**
**
**
**
**
*
KIR2DS3
*
Figure 3. Adult CD8+
T Cells Are Enriched in Cytotoxicity-Related
Genes
(A) Heatmaps show the differentially transcribed cytotoxicity-signaling-related
genes.
(B) Validation of a group of genes related to cytotoxicity function by qRT-PCR
is shown.
(C) Flow cytometry of cytotoxicity assay with activated CD8+
T cells. The
experiments were performed in triplicate (error bars, mean ± SEM).
(D) Flow cytometry analyses of Granzyme B and CD107a expression in
neonatal and adult CD8+
T cells. Statistical significance was assessed by
Mann-Whitney U test (*p < 0.05 and **p < 0.01).
Cell Reports 17, 2151–2160, November 15, 2016 2153
5. serum contained in the medium (5%). Within 4 days, the prolif-
eration rate of neonatal CD8+
T cells was significantly higher
than that of comparable cells from the adult counterpart (Fig-
ure 4B). As a control, we measured homeostatic proliferation
and clonal expansion of CD8+
CBMCs and PBMCs at basal
level and after CD3/CD28 cross-linking. Neonatal cells again
showed a much higher homeostatic proliferation; clonal expan-
sion after stimulation was significantly higher in the adult cells
(Figure S6).
The transcriptome data also showed that neonatal CD8+
T cells were enriched in gene expression signatures character-
istic of innate immunity, particularly antiviral and inflammatory
responses (Figure 4A). The genes enriched in the antiviral
pathway participate in nucleus-to-cytoplasm transport (RAE1,
NUP37, NUP133, and KPNA3) and RNA maturation (SRSF6,
WDR77, and GEMIN4), and they are important cellular targets
during viral infections (NEDD4) (Figure 4A).
Among the genes overexpressed in the neonatal T cells, we
found several genes associated with inflammatory responses,
such as those for defensins and cathelicidins. GSEA showed
that neonatal CD8+
T cells were significantly enriched in inflam-
mation-related genes (Figure S2C). Because some of these
genes are characteristic of the neutrophil immune response,
we used the database of inflammation-induced genes in human
neutrophils (Surmiak et al., 2012) to make a comparison with the
genes expressed by neonatal and adult T cells. We found enrich-
ment in 28 of the 147 genes upregulated in neutrophils during the
inflammatory response, including genes encoding for antimicro-
bial peptides (DEFA4, DEFB3, and CTSG), chemokines (CXCL8/
IL8, CCL2, and CXCL1), and RNases 2 and 3 (Figure 4A). It is well
documented that neonatal antigen-presenting cells are high pro-
ducers of antimicrobial peptides (Levy, 2007; McDonald et al.,
2013), but the transcription of these genes was not documented
in T lymphocytes. We therefore validated by qRT-PCR the mRNA
levels of defensin A (DEFA4), cathepsin G (CTSG), and the tran-
scription factor CEBPE, necessary for the development of gran-
ulocytes and the formation of secondary granules (Gombart and
Koeffler, 2002), and we found a highly increased transcription of
these genes in neonatal CD8+
T cells (Figure 4C). We also vali-
dated the CTSG protein levels at basal and stimulated condi-
tions, and we found increased values in neonatal CD8+
T cells,
particularly after CD3/CD28 activation (Figure S5B).
To see if this inflammatory signature had an impact on the cell
biology of neonatal cells, we assayed the respiratory burst that is
used by neutrophils as an effector mechanism to fight infections.
We therefore measured dihydroethidium oxidation to measure
production of free radicals by CD8+
T cells. The free radical pro-
duction of neonatal CD8+
T cells was much higher in response to
the dihydroethidium itself than was the corresponding response
in adult cells, although not as high as that of neutrophils (Fig-
ure 4D). Even though this result remains preliminary and further
examination is needed to clarify the meaning of the increased
reactive oxygen species in neonatal cells, the higher dihydroethi-
dium response is in agreement with a higher expression of the
genes involved in ROS production, such as the proton channel
HVCV1, NADPH oxidase subunits (NCF, NCF2, and NCF4),
and lipoxygenase genes (ALOX5 and ALOX12) in neonatal
T cells (Figure S4B). Altogether, these results point to a bias of
neonatal CD8+
T cells toward the use of innate immunity mech-
anisms, which could explain the high incidence of sepsis and
sterile inflammatory conditions of neonates and yet low cytotox-
icity (Ghazal et al., 2013; Piantino et al., 2013).
Regulatory Pathways Involved in the Genetic
Programming of Neonatal versus Adult CD8+
T Cells
To gain insight into the regulatory pathways involved in the
different expression profiles of CD8+
T cells from neonatal and
adult origins, we analyzed the transcription factors differentially
expressed in the two cell types (Figure 5). Differentially ex-
pressed transcription factors were associated with different
cell functions (Figure 5). We found that transcription factor genes
overexpressed in the neonatal cells were involved in early T cell
differentiation (BCL11a, LEF1, MYB, and GFI1) (Rothenberg
et al., 2008), neutrophil differentiation (CEBPE) (Gombart and
Koeffler, 2002), inflammation (NF-E2), and cell cycle (DACH1
A
CellCycle
C
Inflammation
0 10 20 40 50 60
*
NeutrophilNeonateAdult
% Dihydroethidium cells
0´
5´
D
-2 0 2
Adult
0´
5´
0´
5´
Neonate
Adult
Neonate 0
4
Days
CFSE
%ofMax
Adult
CFSE
Neonate
0
4
Days
0
40
60
80
100
20
100
101
102
103
104
0
40
60
80
100
20
100
101
102
103 104
ANP32A
BIRC5
BUB1
CCDC99
CCNB2
CDC16
CDK1
CDT1
CENPP
CEP135
CETN2
CHEK1
DHFR
E2F2
GINS2
IGF1R
KIF2C
MAPRE1
MCM3
MCM7
MRC2
MYBL2
NCAPH
NUP133
NUP37
ODF2
PCNT
PLK4
POLD1
PSMA4
PSMB2
PSMB3
PSMB7
PSMD1
RBBP4
RCC2
RRM2
SDCCA
SEC13
SET
SGOL1
SMC1A
SMC4
SPC25
TFDP1
TYMS
YWHAG
ZWINT
**
**
CTSGDEFA4
RatiotoGAPDH
0
1
2
3
6
8
10 Adult
Neonate
CEBPE
*
AntiviralResponse
ANXA3
DEFA4
ELANE
PTGS1
RNASE2
RNASE3
CTSG
IL1R2
HBB
CAMP
PLA2G7
HPGD
CXCL1
MMP9
DEFA3
PTAFR
PTGS2
ALOX5
PTGIR
IL8
PF4
CCL2
TBXA2R
PLA2G4C
CYSLTR1
SERPINA1
TNFSF13B
ITGAM
RAE1
TRIM25
NUP37
NUP133
SLC25A5
NEDD4
HK3
SRSF6
NUPL2
WDR77
KPNA3
GEMIN4
CPSF3
RAN
EIF4E
EIF4E2
0
20
40
60
80
100
Cells(%)
Gen0 Gen1 Gen2 Gen3 Gen4 Gen5
Adult
Neonate
B
Figure 4. Neonatal CD8+
T Cells Are Biased toward Innate Immunity
(A) Heatmaps show differentially transcribed genes involved in cell cycle,
inflammation, and antiviral response.
(B) Flow cytometry of CFSE dilution in CD8+
T cells (top panel) at 0 day (gray
shadow) and 4 days (black line). The bottom panel shows the percentage of
cells in five divisions of three independent experiments (mean ± SEM).
(C) Validation by qRT-PCR of two antimicrobial peptides and CEBPE tran-
scripts found increased in the neonatal CD8+
T cells is shown.
(D) Flow cytometry of hydroethidium oxidation of CD8+
T cells and neutrophils.
The experiment was performed in triplicate. Statistical significance was as-
sessed by Mann-Whitney U test (*p < 0.05 and **p < 0.01; mean ± SEM).
2154 Cell Reports 17, 2151–2160, November 15, 2016
6. and E2F2, among others). The overexpressed transcription
factor genes in adult cells were involved in CD8+
T cell function,
particularly effector cell differentiation, regulation of cytotoxicity,
and cytokine function (RORC, STAT4, MAF, and RUNX3) (Cruz-
Guilloty et al., 2009). We concluded that the differential expres-
sion of master transcription factors might be involved in the
distinct genetic programming of neonatal versus adult CD8+
T cells.
Differential Epigenetic Programming of Neonatal versus
Adult CD8+
T Cells
We took advantage of available data from the Roadmap (Kun-
daje et al., 2015) and Blueprint (Adams et al., 2012) interna-
tional consortia (which have generated extensive epigenomic
maps of naive CD8+
T cells from adult and neonatal origins)
to evaluate the epigenetic regulation of these two types of sam-
ples. First, we compared the expression of genes from our
adult and neonatal transcriptome signatures with the RNA-
seq data obtained from the above consortia (Figures 6A and
S1B). We observed a highly significant difference between
the relative expression of genes from the adult and neonatal
signatures (Figure 6A, left panel). We also found that the signa-
ture profile of neonatal versus adult CD8+
T cells from RNA-seq
data from Blueprint and Roadmap was very similar to our tran-
scriptome data, thus cross-validating the different datasets
(Figures S1B and S4). Consistently, the overexpressed genes
of adult cells were associated with significantly higher levels
of open chromatin marks (H3K4me3 and H4K27ac) and lower
levels of the repressive mark H3K27me3 (Figure 6A, right
panel). Representative examples of such epigenomic profiles
are shown in Figures 6B and S7. We next quantified the enrich-
ment of H3K27ac at distal genomic regions, which represent
potentially active enhancers (Creyghton et al., 2010). Significant
differences also were observed at distal peaks of H3K27ac,
associated with differentially expressed genes between adult
and neonatal CD8+
T cells (Figure 6C). Thus, differences in
gene expression levels between adult and neonatal CD8+
T cells globally correlate with epigenetic differences between
the two populations.
To extend our observations, we performed an independent
and unbiased analysis integrating several histone modifications
from three adult and two neonatal CD8+
T cells. We used the
ChromHMM tool (Ernst and Kellis, 2012) to define the different
chromatin states of both cell populations (Figure 6D). We
focused on the differential enhancer activities, that is, distal re-
gions associated with H3K27ac and H3K4me1 (state 9, active
enhancers) in one cell population and those associated with
H3K4me1 only in the other cell population (state 8, poised en-
hancers) (Data S2). We found 858 active enhancers in adult
CD8+
T cells that are in a poised/inactive state in the neonatal
cells and, vice versa, 439 active enhancers in neonatal cells
that are in a poised/inactive state in the adult cells, thus high-
lighting extensive epigenetic differences at distal regulatory
elements. Examples of differentially marked enhancers are
provided for the T cell cytotoxic genes GZMH and GZMB, ex-
pressed only in adult CD8+
T cells, and for the neonate-specific
gene, coding for the transcription factor BCL11A (Figure 6E).
Strikingly, GO terms for genes close to active enhancers
specifically in adult CD8+
T cells were enriched in T cell activa-
tion and function, while those specifically active in neonatal
CD8+
T cells were related to cell proliferation and apoptosis
(Figure 6F). In conclusion, the independent analysis of
specific enhancers points toward a distinct epigenomic
programming of neonatal versus adult CD8+
T cells, which is
closely related to the observed transcriptomic and functional
differences.
DISCUSSION
We have assessed the transcriptomic and epigenetic profiles of
naive CD8+
T cells from neonatal and adult human blood.
Despite the common pathways and variations between individ-
ual samples, the neonatal cells expressed a distinctive signature
of transcribed genes, including a particular set of transcription
factors, and they showed a characteristic configuration of chro-
matin states and epigenetic marking at both proximal and distal
regulatory regions, suggesting a specific epigenetic program-
ming of the neonatal cells.
The transcriptional profile of genes enriched in the neonatal
cells was associated with cell cycle and innate immune response
(Figures 4 and S3). Our functional studies confirmed that
neonatal CD8+
T cells undergo a higher rate of homeostatic
BACH2
BCL11A
CEBPE
DACH1
E2F2
EZH2
GATA1
GFI1B
GLI1
HLX
JDP2
KLF5
LEF1
LYL1
MEF2A
MEOX1
MYB
MYBL2
NFE2
NPAS2
PAWR
PAX6
PLAG1
RAX
RFX2
SMAD1
SOX4
SREBF1
TCF4
TFAP2C
TFDP1
ZNF135
ZNF154
ZNF268
ZNF542
ZNF543
ZNF547
ZNF572
ZNF649
ZNF667
ZNF711
Transcriptionfactors
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
-3 0 3
Adult Neonate
ARID5B
EBF4
FOXC1
FOXD2
GLI3
HIVEP3
HOXB9
HOXC4
JAZF1
KLF11
KLF8
KLF9
MAF
NFIA
NKX3-1
NR1D2
NR3C2
R4A2
POU6F1
PRDM1
RORC
RUNX3
SIX5
SMAD7
SOX13
STAT4
TFAP2E
TSHZ2
ZBTB32
ZBTB38
ZBTB43
ZBTB7A
ZNF296
ZNF365
ZNF483
ZNF532
ZNF540
ZNF680
ZNF80
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Adult Neonate
1 2 3 4 1 2 3 4
-3 0 3
Early
hematopoiesis
CellCycle
Tcellfunction
1 2 3 4 1 2 3 4
Early
hematopoiesis
CellCycle
Tcellfunction
Figure 5. Neonatal and Adult CD8+
T Cells Have Their Own Char-
acteristic Transcription Factor Profile
Heatmaps showing differentially transcribed transcription factor genes be-
tween adult and neonatal CD8+
T cells. The panel at the right of each heatmap
shows the association with biological functions based on the literature.
Cell Reports 17, 2151–2160, November 15, 2016 2155
7. cell division than do adult cells (Figure 4B). It has been described
that neonatal cells divide in response to IL-7 and IL-15 (Marchant
and Goldman, 2005) and spontaneously in mixed T cells popula-
tions (Scho¨ nland et al., 2003). We corroborated those results and
found that purified CD8+
T cells are continuously cycling. The
original stimulus that triggered proliferation could have been
the lymphopenic conditions of neonatal blood, in particular in
relation to the CD8 compartment, given that the ratio of CD4+
to CD8+
T cells is higher in neonatal blood (Zhu et al., 2001).
Among the specific signatures of neonatal CD8+
T cells, we
found an innate immunity-related antiviral response. Neonatal
cells might have an increased antiviral response to compensate
for the low cytotoxicity of their CD8+
T cells. It is also possible,
however, that the neonatal cells have an increased expression
RNA
H3K4me3
H3K27ac
H3K27me3
TLR5
chr1:223,276,987-223,322,103
Neonate
Adult
___________
| |
1e-74
-6
-4
-2
0
2
4
6
RNAseq H3K27me3
___________
| | ___________
| |
___________
| |
3e-216e-472e-393
2
1
0
-1
-2
-3
H3K27acH3K4me3
Up Adult
Up Neonate
A B
E
F
D
State
(Emissionorder)
H3K36me3
1
2
3
4
5
6
7
8
9
10
11
12
H3K4me1
H3K27ac
H3K4me3
H3K27me3
H3K9me3
Colorcode
GZMH GZMBNeonate
Adult
RNA
H3K27ac
H3K4me3
H3K4me1
State
chr14:25,061,918-25,175,137
25,150,000 25,170,000
Zoom area
8
9
Zoom area
60,100,000 60,150,000
8
9
chr2:60,067,793-60,852,955
BCL11A
1
3
5
7
-Log10
Benjamini
5
1
3
regulation
ofcellproliferation
regulation
ofcelldeath
regulation
ofprogram
m
ed
celldeath
negative
regulation
ofcellproliferation
regulation
ofapoptosis
R
eg.ofsm
allG
TPase
m
ediated
signaltransd.
cellm
otion
transcription
regulation
ofcellm
otion
regulation
oftranscription
positive
regulation
ofcellproliferation
regulation
ofR
N
A
m
etabolic
process
localization
ofcell
cellm
otility
positive
regulation
ofcelldeath
cellm
igration
R
as
protein
signaltransduction
im
m
une
system
developm
ent
7
-Log10
Benjamini
cellactivation
leukocyte
activation
transcription
regulation
oftranscription
lym
phocyte
activation
hem
opoiesis
R
eg.(+)ofm
acrom
olecule
m
etabolic
process
hem
opoietic
orlym
phoid
organ
developm
ent
cellm
otion
regulation
ofcellm
orphogenesis
leukocyte
differentiation
regulation
ofcellm
otion
im
m
une
response
reg.oftranscription
from
R
N
A
polIIprom
oter
regulation
ofphosphorus
m
etabolic
process
regulation
ofphosphate
m
etabolic
process
im
m
une
system
developm
ent
regulation
ofcellproliferation
State 9 (Neonate)State 9 (Adult)
chr8:22,877,391-22,929,713
TNFRSF10B
Log2
(Adult/Neonate)
4
2
0
-2
-4
7e-16_________________________________
| |
Distal
H3K27ac
C
Neonate
Adult
Log2
(Adult/Neonate)
Log2
(Adult/Neonate)
Figure 6. Epigenomic Analysis
(A) Comparison of RNA-seq (left panel) and histone marks (right panel) around the TSS between the set of genes differentially expressed in neonatal and adult
CD8+
T cells based on the microarray analysis. The ratio between the neonatal and adult signal is shown. Statistical significance was assessed by Mann-Whitney
U test.
(B) Example of two genes overexpressed in neonate (top panel) or in adult (bottom panel). Scales were adjusted with respect to control genes shown in
Figure S7A.
(C) Comparison of H3K27ac signal at distal genomic regions (>2 kb from any TSS) is shown.
(D) Chromatin states defined in CD8+
T cells. The last column represents the color code for each chromatin state.
(E) Example of two loci containing dynamic regions corresponding to enhancer transition from active state (nine) in adult to poised state (eight) in neonate (left
panel) and vice versa (right panel). Chromatin states are shown in the bottom of each panel. The bottom panels represent the chromatin states of a zoomed-in
area.
(F) GO (biological process) analysis is shown.
2156 Cell Reports 17, 2151–2160, November 15, 2016
8. of these genes because of a still ongoing cell maturation pro-
cess, as most of these genes are involved in nucleus-to-cyto-
plasm transport and RNA maturation.
We found that several genes involved in cytotoxicity were un-
der-expressed and epigenetically silenced in the neonatal CD8+
T cells (GZMB, TLFRSF10B, RUNX3, NR3C2, and CTLA4; Fig-
ures 6B, 6E, and S7). Functional studies showed that indeed
neonatal cells do not express granzyme B; but, they expressed
the degranulation marker CD107. This suggests that the gran-
ules exist in the neonatal cells, although the contents are
different. It is possible that the granule content in cord blood
CD8+
T cells comprises antimicrobial peptides and elastase.
We also show that neonatal CD8+
T cells were less cytotoxic
than the adult cells. Immaturity of the neonatal CD8+
T cell pop-
ulations was confirmed in the pathway analysis (Figures 2, S2,
and S3) and in the neonate-specific set of transcription factors
(Figure 5). This could be responsible for the high susceptibility
of neonates to intracellular pathogen infections (PrabhuDas
et al., 2011).
Neonatal CD8+
T cells shared a number of transcripts with the
neutrophil activation response (Surmiak et al., 2012), particularly
as related to microbial peptide expression, chemokines, and
RNases. This is suggestive of a role of the neonatal CD8+
T cells within the innate immunity response. We found that,
indeed, the neonatal CD8+
T cells produce reactive oxygen
species, and we also corroborated, with qPCR, the higher rate
of transcription of the antimicrobial peptides cathepsin G and
defensin A (Ganz, 1994). The expression of these neutrophil-
related genes could be associated with the higher expression
in neonatal cells of the neutrophil differentiation transcription
factor CEBPE (Figure 4C) (Cloutier et al., 2009; Halene et al.,
2010; Lekstrom-Himes, 2001; Ma et al., 2014).
Master transcription factors could be involved in the differen-
tial programming of the neonatal cells. As expected, the tran-
scription factors of adult CD8+
T cells were mostly associated
with T cell function and activation, and this could be due to
the higher expression in adult cells of the master transcription
factor RUNX3 (Cruz-Guilloty et al., 2009). On the contrary,
neonatal T cells express master regulators of early leucocyte
differentiation like BCL11A (Yu et al., 2012) and CEBPE
involved in neutrophil differentiation (Gombart and Koeffler,
2002). This is indicative of a particular genetic programming
of neonatal CD8+
T cells, with immature characteristics and
biased toward innate immunity. Supporting the idea that
neonatal cells are biased toward innate immunity, it has been
shown that one of the major responses of human neonatal
T cells is the production of IL-8, a pro-inflammatory molecule
associated with innate immunity, and that these cells respond
to the TLR5 ligand, flagellin, further inducing IL-8 (Gibbons
et al., 2014). Additionally, in a transcriptome study of neonates
with sepsis, the authors found that the major response of neo-
nates was innate immunity-associated gene expression (Smith
et al., 2014). The authors concluded that neutrophils were the
main responsive cells. Our data suggest, however, that it is
also possible that CD8+
T cells were responding with innate im-
munity mechanisms, stressing the need of neonatal immune
cell expression signatures for whole-blood transcription anal-
ysis involving neonates.
Altogether our data suggest that neonatal CD8+
T cells have a
transitional program to deal with the birth-specific immunolog-
ical demands, while they complete their maturation from recent
thymus emigrants to become mature naive CD8+
T cells (Fink
and Hendricks, 2011; Opiela et al., 2009; Yang and Bell, 1992).
It has been estimated that 90%–95% of naive CD4+
T cells
from human cord blood cells are recent thymus emigrants
(RTEs), and, in the age groups of our adult donors (22–55 years
old), recent RTEs are expected to represent a very small propor-
tion of the naive T cell pool (den Braber et al., 2012). Therefore,
part of the differences encountered in this analysis could be ex-
plained by this fact. However, in a transgenic mice model where
RTEs were labeled, it was reported that neonatal and adult CD4+
T cell RTEs have phenotypic and functional differences. Adult
cells did not proliferate in response to IL-7 and produced less
of the Th1/Th2 cytokines as compared to neonatal RTEs (Opiela
et al., 2009). Our work suggests a function of neonatal cells
within the innate immune response. The consistency between
our observations and the transcriptomic and epigenetic data
from the Roadmap and Blueprint consortia strengthens our con-
clusions. Further analysis will determine whether the innate im-
munity role of CD8+
T cells could be either an ancient role of
these cells, possible pointing to an evolutionary functional origin
of CD8+
T cells, or an acquired role adapted to the transition of
birth in mammals.
EXPERIMENTAL PROCEDURES
Cell Preparations
Adult T cells were obtained from leukocyte concentrates of healthy adult
donors who donated to Centro Estatal de la Transfusio´ n Sanguı´nea in Cuerna-
vaca. Neonatal blood was collected from the cord vein of full-term vaginal
deliveries of healthy babies at Hospital General Parres in Cuernavaca. Blood
was collected just after the delivery and before placenta expulsion. All samples
for the transcriptome evaluation were from male donors. The average age of
the adult blood donors was 29 ± 6.44 years old (range, 22–55), and 73%
were male donors. The procedure was approved by the Hospital Parres Ethical
Committee, and it was performed on informed mothers that consented to
donate their babies’ cord blood.
Total blood was separated through a ficoll-hypaque gradient and plastic
adhesion. PBMCs or CBMCs were further depleted of unwanted cell popula-
tions with a cocktail of antibodies followed by G protein-magnetic bead
(88847, Invitrogen) binding and depletion with a magnet. Antibodies used
were CD57 (NK1, Zymed), CD19 (HD37, Santa Cruz Biotechnology), CD16
(3G8, BioLegend), anti-TCRg/d (B1, BioLegend), and CD4 (MT310, Santa
Cruz Biotechnology). To eliminate memory and effector cells in PBMCs, anti-
bodies against CD45RO (UCH-L1, Santa Cruz Biotechnology) and CD44
(BJ18, BioLegend) also were used. Then CD8+
T cells were obtained with an
anti-CD8 (SK1, BioLegend) antibody. This method allowed cell purities
>98%. Cells were used immediately or were maintained in RPMI supple-
mented with glutamine, antibiotics, and 5% fetal calf serum under 5% CO2
at 37
C.
RNA Extraction and Integrity Evaluation
The purified cells were centrifuged and placed in Trizol (15596-018, Invitrogen)
before being shipped on dry ice to the TGML platform in Marseille. RNA was
extracted according to the manufacturer’s instructions. The integrity of the
RNA was determined with an Agilent Bionalyzer, and only samples with RNA
integrity (RIN) over 8.0 were used for transcriptome evaluation.
Transcriptome Analysis
The labeled RNA was hybridized and scanned on an Agilent platform in an
8X60K human Agilent microarray. Raw data are available at GEO: GSE61570.
Cell Reports 17, 2151–2160, November 15, 2016 2157
9. For bioinformatic analysis, first the raw fluorescence data were normalized
by the R package (AGIND). Later, analysis was performed using software
Limma, Multiple Experiment Viewer (MEV) and R program, with an alpha of
0.05 to obtain the significantly changed genes. The heatmaps were obtained
with the software MEV, with base 2 logarithmic transformations and analysis
of arithmetic average for each gene.
GSEA (Subramanian et al., 2005) was performed regardless of Limma
analysis. Reactome analysis, Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathways, and GO analysis was performed with the ClueGO
plug-in under Cytoscape 3.0 (Shannon et al., 2003), with the Database for
Annotation, Visualization and Integrated Discovery (DAVID) software (Huang
et al., 2009).
Flow Cytometry
Extracellular staining was performed to evaluate purity, double positive for
CD3-phicoeritrin (Hit3a, BioLegend), CD8-FitC (Hit8a, BioLegend), and
TCRg/d-FitC (B1, BioLegend). The naive state of the cells was evaluated by
low CD45RO (UCHL1, Tonbo) and by high CD45RA (T6D11, Miltenyi Biotec).
For cytokine staining, we used IL-2-APC (MQ1-17H12BD, Pharmingen) and
IFN-g-FITC (45-15, Miltenyi Biotec). To quantify TLR5 (85B152.5, Abcam),
extracellular staining was performed. For cytotoxicity markers, GZMB
(GB11, BioLegend) and CD107a (H4A3, Miltenyi Biotec) intracellular staining
was performed. For assays of cell division, PBMCs were specifically evaluated
by double staining with CFSE (21888, Sigma) 50 nM and CD8-Alexa Fluor 405
(MHCD0826, Life Technologies) or only CFSE in purified CD8+
T cells. For
assays of oxidative burst, non-fixed CD8+
T cells were stained with 50 nM
dihydroethidium (D7008, Sigma) for 20 min at 37
C, then analyzed immediately
on the cytometer.
Cytotoxicity Assays
Naive CD8+
T cells were stimulated with 5 ml anti-CD3-CD28 beads
(11161D, Invitrogen) or 1 mg/ml each anti-CD3 (OKT3, 70-0037-U100,
Tonbo Biosciences) and anti-CD28 (CD28.2, 70-0289-U100, Tonbo Biosci-
ences) antibodies, followed by 1 mg/ml goat anti-mouse antibody (405301,
BioLegend) for 6 hr. Meanwhile, PBMCs from the same donor were stained
with CFSE (50 nM) for 15 min. Finally, activated cytotoxic cells were co-
cultured with the labeled target cells for 2 hr, and apoptosis was measured
by propidium iodide staining (33-1200, Invitrogen). Only the CFSE-positive
cells were assessed with propidium iodide, as a measure of target cell
death.
Respiratory Burst Assay
Naive CD8+
T cells from neonatal and adult donors, as well as neutrophils,
were cultured in phenol red-free RPMI containing 5% fetal bovine serum
(FBS). Cells were treated with 50 nM dihydroethidium (D7008, Sigma-Aldrich)
for 20 min, and they were compared to cells treated with the same compound
immediately before reading (time zero). Changes in fluorescence due to oxida-
tion of the dye with reactive oxygen species were evaluated by flow cytometry.
qRT-PCR
The cDNA was synthesized from total RNA using the Superscript Vilo cDNA
synthesis kit (11755050, Life Technologies). The qPCRs were performed using
SYBR Green PCR Master Mix (4309155, Life Technologies). The mRNA levels
were calculated from standard curves for each gene and relative to those of the
GAPDH gene, used as a standard.
Processing of ChIP-Seq and RNA-Seq Data
RNA-seq and chromatin immunoprecipitation sequencing (ChIP-seq)
(H3K4me3, K3K27ac, and H3K27me3) samples from adult naive CD8+
T cells (Donor_101_8) were obtained from the ROADMAP project. The
RNA-seq (donor C002YM) and ChIP-seq (H3K4me3, K3K27ac, and
H3K27me3; donor C0066P12) samples from neonatal naive CD8+
T cells
were obtained from the Blueprint project (see Table S1 for more information).
Gene expression was assessed by quantifying RefSeq gene exons signal
from RNA-seq BigWig files using BigWig-Tools extract option (Pohl and
Beato, 2014); then, for each gene, the corresponding exon signal was
summed up and normalized using the Normalizer package (Glusman et al.,
2013) with the quartiles option. Histone modifications within promoter regions
(related to Figure 6A) were assessed by extracting the average of the top 25%
signal from ChIP-seq BigWig files from H3K4me3, H3K27ac, and H3K27me3
histone marks using BigWig-Tools, in a region around 1.5 kb to the transcrip-
tion start site (TSS) RefSeq genes, then the signal was normalized as
described above. For distal H3K27ac analysis (related to Figure 6C), peak-
calling for H3K27ac ChIP-seq from neonatal CD8+
T cells was obtained
directly from the Blueprint database. Peak-calling for H3K27ac ChIP-seq
from adult CD8+
T cells (Roadmap) was performed with MACS2 (Zhang
et al., 2008), using the Blueprint parameters. The unique no-overlapping
peaks between the two populations (each ChIP-seq data) were selected
using Bedops (Neph et al., 2012); the signal was extracted from BigWig files
using BigWig-Tools and normalized as described above. Each peak was
assigned to one gene using GREAT software (McLean et al., 2010) with
default settings. H3K27ac peaks were defined as distal if they were located
more than 2 kb upstream and 1 kb downstream the TSS of any annotated
gene.
Analysis of Epigenomic and Transcriptomic Signatures
We computed the ratio between the adult and neonatal RNA-seq and
ChIP-seq data for each set of adult and neonate-specific genes. Statistical
significance between the two sets of genes was computed using the Mann-
Whitney U test. Heatmaps were obtained with the software MEV, with a
base 2 logarithmic transformation, and compared to microarray heatmaps
(Figure S4). Examples of genomic profiles were obtained using the IGV 2.3
software (Robinson et al., 2011).
Chromatin Segmentation
CD8 neonatal segmentations and visualization with 12 states for samples
C002YMH1, C0066PH1, and S00C2FH1 were obtained from the
BLUEPRINT project (2015-01-28 release, more details are given in Table
S1). CD8 adult samples (naive primary cells: Donor_100_7 and
Donor_101_8) were obtained from ROADMAP. ChIP-seq data for six histone
modifications (H3K4me3, H3K4me1, H3K27ac, H3K36me3, H3K27me3,
and H3K9me3) were used (see Table S1). For the segmentation, we used
the implementation described in the ChromHMM software (version 1.10)
(Ernst and Kellis, 2012). The input data were the ChIP-seq bed files with
the genomic coordinates and strand orientation of mapped sequences (after
remove duplicate reads). The genome was divided in consecutive 200-bp
non-overlapping intervals and independently assigned present (1) or absent
(0) for each of the six chromatin modifications. The assignment was based
on the count of tags mapping to the interval and on the basis of a Poisson
background model using a threshold of 10-4, as explained (Ernst and Kellis,
2012), after binarization, and for segmentation we used the 12 states model
established by the Blueprint Consortium (2015-01-28 release, see Table
S1). Further, we computed the probability that each location is in a given
chromatin state, and then we assigned each 200-bp interval to its most
likely state for each sample. In addition, visualization files were generated
with ChromHMM software (version 1.10).
Differential Chromatin States between Adult and Neonatal CD8+
T Cells
For each cell type, the chromatin state assignment at each 200-bp interval was
compared. Intervals with differential chromatin states were defined as follows:
all CD8 adult samples must share the same state but must be different from
CD8 neonatal samples; in addition, all CD8 neonatal samples must have the
same state. Lastly, we computed the frequency of segments that changed
in chromatin states between adult and neonatal CD8 (Figure 6D). The GO
biological process chart was performed using the DAVID Bioinformatics
Resources (Huang et al., 2009).
ACCESSION NUMBERS
The accession number for the transcriptome of naive CD8+
T cells from four
human neonatal and four adult donors reported in this paper is GEO:
GSE61570.
2158 Cell Reports 17, 2151–2160, November 15, 2016
10. SUPPLEMENTAL INFORMATION
Supplemental Information includes seven figures, one table, and two data files
and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.
2016.10.056.
AUTHOR CONTRIBUTIONS
A.O.G.-A., S.S., and M.A.S. conceived experiments, analyzed the data, and
wrote the manuscript. S.S. and M.A.S. conceived the project, revised the
manuscript, and secured funding. A.B., B.L., H.H., and J.I. performed micro-
array hybridization and processed the raw data. A.O.G.-A., O.H.L.-P.,
D.Y.G.-R., O.R.-J., J.A.S.-V., O.R.-P., and A.H.-M. performed experiments.
J.I. and P.F. provided expertise and feedback. E.C.d.S.P. and A.V. contributed
to epigenomic analyses.
ACKNOWLEDGMENTS
This project was specifically supported by a joint EcosNord-Anuies-SEP-Con-
acyt project (M11S01). Work in the M.A.S. laboratory is supported by grants
from Consejo Nacional de Ciencia y Tecnologı´a (CONACYT; CB-2011-01
168182) and Programa de Mejoramiento del Profesorado (PROMEPSI-
UAEM/13/342). Work in the S.S. laboratory is supported by recurrent funding
from the Inserm and Aix-Marseille University and by specific grants from the
European Union’s FP7 Program (agreement 282510-BLUEPRINT), the Associ-
ation pour la Recherche contre le Cancer (ARC) (project SFI20111203756), and
the Aix-Marseille initiative d’excelence (A*MIDEX) project ANR-11-IDEX-0001-
02. We thank Centro Estatal de la Transfusio´ n Sanguı´nea in Cuernavaca for the
donation of leukocyte concentrates and the mothers and babies of Hospital
General Parres in Cuernavaca for the donation of cord blood. This study makes
use of data generated by the Blueprint and Roadmap consortia. A full list of the
investigators who contributed to the generation of the data is available
from www.blueprint-epigenome.eu and http://www.roadmapepigenomics.
org/. Funding for the Blueprint project was provided by the European Union’s
Seventh Framework Program (FP7/2007-2013) under grant agreement
282510 – BLUEPRINT. The Roadmap consortium is financed by the NIH. We
are grateful to Professor C.I. Pogson for critical reading of the manuscript.
Received: February 9, 2016
Revised: July 3, 2016
Accepted: September 22, 2016
Published: November 15, 2016
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