Microengraving as a tool to profile immune responses

Microfabrication tool that allows to follow the progress of immune responses at the single cell level. Applications to mouse B cells are shown

Transcript

1. Microengraving as a tool to profile immune responses Eli Papa, Ploegh Lab
2. Glass slides coated with capture Ab Secreted Ab is captured Microengraving Glass slides with replicated microarrays of Ab Culture dish PDMS Microengraving can generate multidimensional data
3. Antigen specific spot Non-specific spot anti-mouse Ig OVA (var.conc, Green) anti-mouse Ig (10 nM, Red) Constructing
   Antigen-Antibody Ratios
4. Ratio (Ag/IgG) 0.0 0.5 1.0 1.5 OVA Concentration (nM) 0.01 0.1 1 10 100 [OVA] [IgG] Constructing
   Antigen-Antibody Ratios/2
5. Green = H-2Kb (1nM) 50μm = c136 = Y3 = c127 Red = anti mouse IgG (10nM) tetH-2Kb concentration (nM) Ratio (Ag/IgG) 0.001 0.01 0.1 1 10 100 1000 1.0 0.5 0.0 c127 c136 Y3 Microarray Cells Distinguish clones based on affinity “signature”
6. tetH-2Kb concentration (nM) 0.001 0.01 0.1 1 10 100 1000 tetH-2Kb concentration (nM) Hierarchical K-means 0.001 0.01 0.1 1 10 100 1000 −1 −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 Making microengraving truly high-throughput
7. −1 −0.8 -0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 First Principal Component Second Principal Component −1 −0.8 -0.6 −0.4 −0.2 0 0.2 0.4 0.6 -1.2 Cell staining C136 Y3 C127 −1 −0.8 -0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 First Principal Component Second Principal Component −1 −0.8 -0.6 −0.4 −0.2 0 0.2 0.4 0.6 -1.2 Hierarchical(city/ward) 1pM 10pM 100pM 1nM 10nM 100nM 500nM B Clustering is precise in distinguishing clones
8. Extracting the data with high throughput Stamp
9. Extracting the data with high throughput / analysis Prints Stamp b220+ -/- igM+ igM+ 0.234 1.2 0.234 ..... 0.99 1.2
10. Mapping the course of an hyperimmunization
11. 10pM 100pM 1nM 10nM 100nM Isotype 11.0 10.0 14.3 19.3 22.5 31.4 31.3 28.8 27.5 26.6 26.9 27.6 28.0 28.8 29.3 28.6 28.9 27.4 27.1 27.3 26.1 25.6 25.8 24.3 23.9 24.1 23.7 24.8 23.9 IgG1 IgM K d Affinity 1st boost IgG1 IgM 1x boost −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 -1 Resolving single antigen specific clones
12. −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 -1 Isotype Affinity 10pM 100pM 1nM 10nM 100nM 1.7 0.9 1.9 2.9 6.8 7.8 7.9 11.0 10.1 12.6 15.3 18.0 24.6 24.6 18.0 20.1 16.8 19.1 22.4 19.6 15.3 15.7 16.9 21.0 20.1 22.8 23.4 23.7 22.4 24.0 26.5 21.8 22.7 20.7 21.8 26.5 25.0 24.4 24.7 23.6 22.3 20.7 20.1 18.9 18.0 18.6 15.2 1.8 IgM IgG1 K d 2nd boost IgG1 IgM 2x boost Resolving single antigen specific clones/2
13. 0 15 30 0 10 20 30 40 K D (nM) days 1st boost 2nd boost Single cell resolution on affinity maturation
14. Resolve frequency, specificity and affinity of single lymphocytes Snapshots of the state of immune responses Can follow course of vaccines, infections Single cells maintain viability and can be employed in further analysis Acknowledgements Hidde Ploegh J.Chris Love Craig Story Chih-chi Hu Jehnna Ronan all the friends and patient teachers I found in the ploegh lab Conclusions