Title CD4+ T-cell responses among adults and young children in response to Streptococcus pneumoniae...
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Transcript of Title CD4+ T-cell responses among adults and young children in response to Streptococcus pneumoniae...
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title
CD4+ T-cell responses among adults and young children in response to Streptococcus pneumoniae and Haemophilus influenzae vaccine candidate protein antigens.
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1.Introduction• Adults and children are frequently affected by infecti
ons caused by Steptococcus pneumoniae(Spn) and non-typeable Haemophilus influenzae(NTHi) , accounting for an increased incidence of pneumonia,invasive Spn disease ,acute sinusits and acute otitis media[1-3].
• Among the vaccine candidates ,pneumococcal surface protein A(PspA),PhtD and PhtE,a choiline binding protein-PcpA,a murein hydrolase (-LytB)and a non-toxic pneumolysin derivative PlyD1,have emerged as most likely to proceed to clinical trials in humans[7-11].
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CD4+T lymphocytes have been shown to be important for protective immumnity against Spn and NTHi infections inboth adult and mice [16-18].However,there are no date that demonstrate the nature of CD4+T lymphocyte responses to Sgn and NTHi among younger children ,and their comparative analysis with adults. In this study we characterized and compared circulating antigen-specific CD4+T lymphocyte populations responsive to six Spn and twoNTHi antigens in adults and young children.
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2.Materials and methods2.1 Subjects and samples Eleven healthy adults (five females and six males; median age 32.5 years ) and 17 young children( 6 months to three years old)None of the subjects had experienced invasive Spn infections or lobar pneumonia.PBMCs were isolated using a Ficoll gradient according to the manufacturer's instruction (GE Healthcare) and then washed with 1×phosphate buffered saline (PBS),re-suspended at a concentration of 1×107 cells/ml in cell recovery freezing media (Gibco) and frozen in liquid nitrogen until used.
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2.2 Antigens and antibodies
• Pneumococcal protein antigens that were used for T-cell stimulation included : PspA(EF5668) PhtD , PhtE LytB PcpA PlyD1
• Antibodies: CD3 Qdot 605 (cione UCHT1,Invitogen) CD3Qdot 605 anti-CD4 APC Alex Flor 750 (clone RPAT4, eBiosciences) PE-Cy5 anti-CD69(cloneFN50, BD biosciences)...
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2.3PBMC stimulation for detection of intracellular cytokine
• Thawing frozen PBMCs• Counting cells• Resting overnight in complete culture media in 2
4-well plates• Stimulating• Counting again and stimulating• Incubating ,after 2 h golgi transport inhibitors wer
e added to preserve cytokines intracellularly and incubated for an additional 4 h.anti -CD28 and anti-CD49d antibodies were also added to provide co -stimulation and enhance the detection of antigen specific response as described earlier [26,27]
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2.4. surface and intracellular staining for flow
cytometric analysis. • Transfering to 96-well V-bottom and washing• Incubating• Permeabilizing• Intracellular staing• Furtherwashing• Collecting 0.2-1 ×106 events for each sample and a
nalyzing with FLOW JO (Tree Star )software.
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2.5. Cytometric bead array (CBA )for detection of secreted cytokines
• For CBA assay, PBMCs were thawed and rested overnight as described previously before stimulating them with either 1µg/ml
• of individual antigens or SEB for 18-20h. After stimulation, super natants were collected and cytokines were meansured using a CBA kit for TH -1and TH -2 cytokine detection (BD Biosciences )as described by the manufacturer.
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• Acquisition template provided by BD was used to acquire cytometric beads on a LSR flow Ⅱcytometer and date was analyzed using BD FACSDIVA software and Flowjo.Standard graphs were plotted and unknown sample values wre extrapalates on linear regression plots with GraphPAD Prism 5 software.
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2.6.Statistical analysis
• We used a rank based procedure, related to the Friedman blocked rank design, to determine those antigens with consistently high or low responses to specific cytokines.
• To assess the statistical significance of the average ranks a bootstrap procedure was used.
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3.Results
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3.1.CD4 + T-cell responses to spn and NTHi antigens in adults
• The IL-4 IL-10and IL-13 responses were generally lower than IL-2 and IFNγ responses.No IL-17 responses were detected in adult (data not show)
• In addition,all cytokines exhibited significantly non-hemogeneous responses with respect to antigen exposures.
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3.2 Characteristics of CD4+T-cell responses to spn and NTHi protein antigens in very young childr
en
• Overall,compared with negative controls, IL-2 and IFNγ producing cells exhibited significantly higher responses to vaccine antigen stimulations in young children.
• IL-17 producing CD4+ Th-cell were infrequent and were present only in a few children( data not show)
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3.3Divergence of CD4+ T-cell responses to Spn and NTHi protein antigens in young children versus
adults.
Adults had vaccine antigen-specifc Th1 and Th2 cells responsive to all antigens evaluated whereas young childrenhad significant numbers of vaccine antigen-specific CD4+ T-cell producing IL-2
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3.4 Memory phenotypes of Spn and NTHi specific CD4+ T-cells among very young childr
en and adults
• Vaccine antigen-specificCD4+ T-cell populations in adults were largely of effector(TEM)and/or central memory (TCM)phenotypes as denfined by CD45RA-CCR7+orCD45RA-aCCRA-respectively; however among young children antigen-specific IL-2 producingCD4+ T-cells demonstrated CD45RA+ expression (non-memory cells)
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• Therefore,vaccination with the studied antigens would likely to boost higher CD4 T-cell responses among adults,whereas young children have a more limited response.
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4.Discussion
• . Adults,who have a more mature immune system and who have more accumulated natural exposures to Spn and NTHi,had significantly higher antigen-specific CD4+T-cells compared to young children .In fact,in adults all tested vaccine candidate antigens stimulatedCD4+ T-cell responses,predominantly with a Th-1profile
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• In conclusion , this report demonstrates the frequencies of Spn and NTHi antigen specific CD4+ T-cells among adults compared to young children following natural exposure to the bacteria.
• Identified aspects contributing to the divergence between the age populations should represent target areas for evaluation of immune-response enhancing approaches.
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