Experiment Overview
| Repository ID: | FR-FCM-ZZD7 | Experiment name: | Experimental human pneumococcal carriage augments IL-17A-dependent T-cell defence of the lung. | MIFlowCyt score: | 100.00% |
| Primary researcher: | Adam Kelvin Alec Wright | PI/manager: | Adam Kelvin Alec Wright | Uploaded by: | Adam Kelvin Alec Wright |
| Experiment dates: | 2010-05-01 - 2012-03-31 | Dataset uploaded: | Jun 2014 | Last updated: | Jun 2014 |
| Keywords: | [human] [CD4 T cell] [Streptococcus pneumoniae] [IL-17A] [alveolar macrophage] [Lung cells] [Vaccine] [experimental human pneumococcal carriage] [BAL] [TNF] [IL-17RA] [IL-17RC] [phagocytosis] [serotype 6B] [intra-cellular cytokine staining] [IFNgamma] [Influenza] | Manuscripts: | [23555269] |
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| Organizations: |
Liverpool School of Tropical Medicine, Respiratory Infection Group, Liverpool, (UK)
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| Purpose: | We experimentally induced pneumococcal nasopharyngeal carriage in young healthy adults. Our aim was to determine whether carriage (compared to no carriage) induced the formation of antigen specific T cells in the lung (sampled using bronchoalveolar lavage (BAL)) and blood. Our second aim was to identify the phenotype of such cells. | ||||
| Conclusion: | We were able to obtain pneumococcal carriage in 11 healthy adults. We obtained BAL and blood from these subjects and compared them to adults without carriage. In those with carriage, we observed large increases in the frequency of antigen specific CD4 T cells in BAL and blood. pneumococcal specific CD4 T cells secreted IL-17 and TNF but not IFN-gamma. | ||||
| Comments: | None | ||||
| Funding: | see linked manuscript | ||||
| Quality control: | From an experimental design perspective we recruited age matched young healthy adults to serve as controls for our "experimental group" in whom carriage was established. The BAL cell profile was similar between groups. From a technical perspective BAL and blood from both groups was collected and treated using the same protocols. The same number of BAL lymphocytes were seeded between both groups. Cells from both groups were stimulated with equal concentrations of 1) Whole pneumococcal cells ("6B" serotype) that had been washed and killed and resuspended in PBS to a known concentration 2) a concentrated culture supernatant ("c/s") from 6B grown in vegetone broth. We ensured that the pneumococcus was grown in the same broth as that used to inoculate our volunteers. 3) To check for cell responses to the vegetone broth as opposed to the pneumococcus we stimulated cells with a broth control ("vehicle" or control"). Any responses detected in this sample were subtracted from 2) above and were described as pneumococcal specific responses. 4) staphylococcal enterotoxin B - this served as a positive control 5) Flu - this also served as a positive control for the assay (since most people should have detectable flu responses) but also as a negative experimental control since our hypothesis was that pneumococal carriage should only augment pneumococcal specific responses but not those to unrelated respiratory antigens such as flu. Some files may contain data from a 6th stimulus (WCA) where sufficient cells permitted but this data was not used in the final manuscript. Following stimulation cells were harvested and stained for T cell markers (CD4APCCy7, CD3 APC, CD45ROPEC7) and cell viability (aquavid) followed by fixation and permeabilisation. Fixed and permeabilised cells were stained with antibodies to TNF (FITC), IL-17 (PE) and IFNgamma (AF700) followed by analysis by flow cytometry. cells that were negative for the respective markers served as internal controls to assess positive or negative staining. Reference to positive controls (SEB or flu) and negative controls (vehicle or unstimulated cells) were useful to discriminate positive from negative cytokine responses. In experiments prior to this paper and in some cases where cell numbers allowed cells stimulated with SEB were stained for isotype controls to TNF/IFNgamma and IL-17 to check specificity. Instrument controls: All data files were acquired on the same BD LSR2 instrument. Performance (QC) beads were run on the instrument prior to acquisition. The voltages obtained from this test were used as the default voltages for each PMT detector. Compensation beads were run for this panel of antibodies and calculated before acquisition (Hardware compensation(?)). Where possible we attempeted to colect the same number of events for each batch of tubes within an experimental sample (sample = cells from 1 volunteer) | ||||
Experiment variables
| Conditions | |
|---|---|
| · non-stimulated | 08_115_NS.fcs · 100_111 NS.fcs · 124_112 NS.fcs · 125_114 Pre_NS.fcs · 126_116 NS.fcs · 130_121_NS.fcs · 131_122_NS.fcs · 132_123_NS.fcs · 174_135 BAL_NS.fcs · 176_136 BAL_NS.fcs · 177_137 BAL_NS.fcs · 17_113_NS.fcs · 189_05 BAL_NS.fcs · 190_06 BAL_001_NS.fcs · 193_08 BAL_NS.fcs · 198_13_NS.fcs · 203_18 BAL NS.fcs · 240_51_NS.fcs · 244_66 BAL_NS.fcs · 251_68_NS.fcs |
| · 6B pneumococcus | 08_115_6B.fcs · 100_111 6B.fcs · 124_112 6B.fcs · 125_114 Pre_6b.fcs · 126_116 6B.fcs · 130_121_6B.fcs · 131_122_6B.fcs · 132_123_6B.fcs · 174_135 BAL_6b.fcs · 176_136 BAL_6b.fcs · 177_137 BAL_6b.fcs · 17_113_6B.fcs · 189_05 BAL_6B.fcs · 190_06 BAL_001_6b.fcs · 193_08 BAL_6b.fcs · 198_13_6B.fcs · 198_13_6b TNF_IL17_IFN control.fcs · 203_18 BAL 6B.fcs · 203_18 BAL 6B_TNF_IL17_IFN isotype control.fcs · 240_51_6B.fcs · 244_66 BAL_6B.fcs · 251_68_6B.fcs · 251_68_6b iso.fcs |
| · influenza | 08_115_flu.fcs · 100_111 flu.fcs · 124_112 flu.fcs · 125_114 Pre_Flu.fcs · 126_116 flu.fcs · 130_121_flu.fcs · 131_122_Flu.fcs · 132_123_Flu.fcs · 174_135 BAL_Flu.fcs · 176_136 BAL_Flu.fcs · 177_137 BAL_Flu.fcs · 17_113_flu.fcs · 189_05 BAL_Flu.fcs · 190_06 BAL_001_Flu_1.fcs · 193_08 BAL_Flu_1.fcs · 198_13_flu.fcs · 203_18 BAL Flu.fcs · 240_51_flu.fcs · 244_66 BAL_flu.fcs · 251_68_flu.fcs |
| · 6B pneumococcal culture supernatant | 08_115_c_s.fcs · 100_111 c_s.fcs · 124_112 c_s.fcs · 125_114 Pre_c,2f,s.fcs · 126_116 c_s.fcs · 130_121_c_s.fcs · 131_122_c_s.fcs · 132_123_c_s.fcs · 174_135 BAL_c_s.fcs · 176_136 BAL_c_s.fcs · 177_137 BAL_c_s.fcs · 17_113_c_s.fcs · 189_05 BAL_c_s.fcs · 190_06 BAL_001_c_s.fcs · 198_13_c_s.fcs · 203_18 BAL c_s.fcs · 240_51_c_s.fcs · 244_66 BAL_c_s.fcs · 251_68_c_s.fcs |
| · vehicle control for culture supernatant | 08_115_vehicle.fcs · 100_111 vehicle.fcs · 124_112 vehicle.fcs · 125_114 Pre_vehicle.fcs · 126_116 vehicle.fcs · 130_121_vehicle.fcs · 131_122_vehicle.fcs · 132_123_vehicle.fcs · 174_135 BAL_vehicle.fcs · 176_136 BAL_vehicle.fcs · 177_137 BAL_Vehicle.fcs · 17_113_vehicle.fcs · 189_05 BAL_Vehicle.fcs · 190_06 BAL_001_vehicle.fcs · 198_13_vehicle.fcs · 203_18 BAL Vehicle.fcs · 240_51_vehicle.fcs · 244_66 BAL_vehicle.fcs · 251_68_vehicle.fcs |
| · staphylococcal enterotoxin B (SEB) | 100_111 SEB.fcs · 100_111 SEB_TNF_IL17_IFN Isotype control.fcs · 125_114 Pre_seb.fcs · 125_114 Pre_seb_ iso.fcs · 176_136 BAL_SEB.fcs · 177_137 BAL_SEB iso.fcs · 177_137 BAL_SEB.fcs · 17_113_SEB.fcs · 17_113_SEB_TNF_IL-17_IFNg_control.fcs · 189_05 BAL_SEB.fcs · 190_06 BAL_001_SEB Iso.fcs · 190_06 BAL_001_SEB.fcs · 198_13_SEB.fcs · 203_18 BAL SEB.fcs · 240_51_seb.fcs |
| · Whole Cell antigen (from pneumococcus) | 100_111 WCA.fcs · 124_112 WCA.fcs · 125_114 Pre_WCA.fcs · 126_116 WCA.fcs · 177_137 BAL_wca.fcs · 17_113_WCA.fcs · 203_18 BAL WCA.fcs |
| Sample Type | |
|---|---|
| · Pneumococcal carriage negative | 08_115_6B.fcs · 08_115_NS.fcs · 08_115_c_s.fcs · 08_115_flu.fcs · 08_115_vehicle.fcs · 100_111 6B.fcs · 100_111 NS.fcs · 100_111 SEB.fcs · 100_111 SEB_TNF_IL17_IFN Isotype control.fcs · 100_111 WCA.fcs · 100_111 c_s.fcs · 100_111 flu.fcs · 100_111 vehicle.fcs · 124_112 6B.fcs · 124_112 NS.fcs · 124_112 WCA.fcs · 124_112 c_s.fcs · 124_112 flu.fcs · 124_112 vehicle.fcs · 125_114 Pre_6b.fcs · 125_114 Pre_Flu.fcs · 125_114 Pre_NS.fcs · 125_114 Pre_WCA.fcs · 125_114 Pre_c,2f,s.fcs · 125_114 Pre_seb.fcs · 125_114 Pre_seb_ iso.fcs · 125_114 Pre_vehicle.fcs · 126_116 6B.fcs · 126_116 NS.fcs · 126_116 WCA.fcs · 126_116 c_s.fcs · 126_116 flu.fcs · 126_116 vehicle.fcs · 130_121_6B.fcs · 130_121_NS.fcs · 130_121_c_s.fcs · 130_121_flu.fcs · 130_121_vehicle.fcs · 131_122_6B.fcs · 131_122_Flu.fcs · 131_122_NS.fcs · 131_122_c_s.fcs · 131_122_vehicle.fcs · 132_123_6B.fcs · 132_123_Flu.fcs · 132_123_NS.fcs · 132_123_c_s.fcs · 132_123_vehicle.fcs · 17_113_6B.fcs · 17_113_NS.fcs · 17_113_SEB.fcs · 17_113_SEB_TNF_IL-17_IFNg_control.fcs · 17_113_WCA.fcs · 17_113_c_s.fcs · 17_113_flu.fcs · 17_113_vehicle.fcs |
| · Pneumococcal carriage positive | 174_135 BAL_6b.fcs · 174_135 BAL_Flu.fcs · 174_135 BAL_NS.fcs · 174_135 BAL_c_s.fcs · 174_135 BAL_vehicle.fcs · 176_136 BAL_6b.fcs · 176_136 BAL_Flu.fcs · 176_136 BAL_NS.fcs · 176_136 BAL_SEB.fcs · 176_136 BAL_c_s.fcs · 176_136 BAL_vehicle.fcs · 177_137 BAL_6b.fcs · 177_137 BAL_Flu.fcs · 177_137 BAL_NS.fcs · 177_137 BAL_SEB iso.fcs · 177_137 BAL_SEB.fcs · 177_137 BAL_Vehicle.fcs · 177_137 BAL_c_s.fcs · 177_137 BAL_wca.fcs · 189_05 BAL_6B.fcs · 189_05 BAL_Flu.fcs · 189_05 BAL_NS.fcs · 189_05 BAL_SEB.fcs · 189_05 BAL_Vehicle.fcs · 189_05 BAL_c_s.fcs · 190_06 BAL_001_6b.fcs · 190_06 BAL_001_Flu_1.fcs · 190_06 BAL_001_NS.fcs · 190_06 BAL_001_SEB Iso.fcs · 190_06 BAL_001_SEB.fcs · 190_06 BAL_001_c_s.fcs · 190_06 BAL_001_vehicle.fcs · 193_08 BAL_6b.fcs · 193_08 BAL_Flu_1.fcs · 193_08 BAL_NS.fcs · 198_13_6B.fcs · 198_13_6b TNF_IL17_IFN control.fcs · 198_13_NS.fcs · 198_13_SEB.fcs · 198_13_c_s.fcs · 198_13_flu.fcs · 198_13_vehicle.fcs · 203_18 BAL 6B.fcs · 203_18 BAL 6B_TNF_IL17_IFN isotype control.fcs · 203_18 BAL Flu.fcs · 203_18 BAL NS.fcs · 203_18 BAL SEB.fcs · 203_18 BAL Vehicle.fcs · 203_18 BAL WCA.fcs · 203_18 BAL c_s.fcs · 240_51_6B.fcs · 240_51_NS.fcs · 240_51_c_s.fcs · 240_51_flu.fcs · 240_51_seb.fcs · 240_51_vehicle.fcs · 244_66 BAL_6B.fcs · 244_66 BAL_NS.fcs · 244_66 BAL_c_s.fcs · 244_66 BAL_flu.fcs · 244_66 BAL_vehicle.fcs · 251_68_6B.fcs · 251_68_6b iso.fcs · 251_68_NS.fcs · 251_68_c_s.fcs · 251_68_flu.fcs · 251_68_vehicle.fcs |
