1 / 23

title

title. CD4+ T-cell responses among adults and young children in response to Streptococcus pneumoniae and Haemophilus influenzae vaccine candidate protein antigens. 1 . Introduction.

sveta
Download Presentation

title

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. title CD4+ T-cell responses among adults and young children in response to Streptococcuspneumoniae and Haemophilus influenzae vaccine candidate protein antigens.

  2. 1.Introduction • Adults and children are frequently affected by infections 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].

  3. 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.

  4. 2.Materials and methods 2.1 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.

  5. 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)...

  6. 2.3PBMC stimulation for detection of intracellular cytokine • Thawing frozen PBMCs • Counting cells • Resting overnight in complete culture media in 24-well plates • Stimulating • Counting again and stimulating • Incubating ,after 2 h golgi transport inhibitors were 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]

  7. 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 analyzing with FLOW JO (Tree Star )software.

  8. 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.

  9. Acquisition template provided by BD was used to acquire cytometric beads on a LSRⅡ flow cytometer 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.

  10. 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.

  11. 3.Results

  12. 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.

  13. 3.2 Characteristics of CD4+T-cell responses to spn and NTHi protein antigens in very young children • 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)

  14. 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 children had significant numbers of vaccine antigen-specific CD4+ T-cell producing IL-2

  15. 3.4 Memory phenotypes of Spn and NTHi specific CD4+ T-cells among very young children 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)

  16. 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.

  17. 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

  18. 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.

  19. THE END!

More Related