Multiplexed imaging of immune cells in staged multiple sclerosis lesions by mass cytometry
- University of Toronto, Canada
- Yale School of Medicine, United States
- Université de Montréal, Canada
- University Health Network, Canada
- Fluidigm Canada Inc, Canada
Figures
Figure 1 with 4 supplements
Comparing IMC to IF in MS lesions.
Two serial sections were assessed: one used for immunofluorescence (IF, a and a’) and one dedicated to Imaging Mass Cytometry (IMC, a’’). (a) The region of interest (a’) was guided by the …
Figure 1—figure supplement 1
Staging of MS lesions by IF.
General pathology: demyelinating lesions (arrows and arrows head in (a and b) seen in (a) hematoxylin and eosin (HE)/Luxol fast blue (LFB) stain of myelin and (b) oil red o (ORO) stain of neutral …
Figure 1—figure supplement 2
Workflow of Imaging Mass Cytometry.
A panel is designed using pathologist-verified antibodies conjugated to metals. The brain tissue is stained simultaneously with a cocktail of all the metal-conjugated antibodies and placed into the …
Figure 1—figure supplement 3
Validation of IMC staining patterns in MS lesions.
Core of an active demyelinating lesion, showing reduced proteolipid protein (PLP) stain by (a) immunohistochemistry, (b) immunofluorescence and (c) imaging mass cytometry and corresponding areas …
Figure 1—figure supplement 4
Correlating IMC to IF staining patterns in MS lesions.
(a) Spearman correlation coefficient, showing a significant positive correlation between the % of PLP+ area identified with fluorochrome-conjugated antibody by IF and % of PLP+ area dentified with …
Figure 2 with 1 supplement
Validation of IMC specificity in MS lesions.
(a) Overlay of CD3 (green) and CD45 (red) identifies CD3+CD45+ T cells (solid arrows) and CD3-CD45+ leukocytes other than T cells (dotted arrow). (b) Overlay of CD3 (green) and CD68 (red) identifies …
Figure 2—figure supplement 1
Example of active lesion in which 8 analytes are displayed simultaneously.
https://doi.org/10.7554/eLife.48051.009
Figure 3
Staging of MS lesions by IMC.
Representative mass cytometry images of white matter areas of (a, f) healthy control, (b, g) MS normal-appearing white matter (block no. CR4A), (c, h) MS (p)reactive lesion (block no. CR4A), (d–i) …
Figure 4
Pattern of microglia or macrophage activity in different stages of MS lesions by IMC.
Representative mass cytometry images of (a, a’, b, b’) control white matter, (c, c’, d, d’) normal-appearing white matter (block no. CR4A), (e, e’, f, f’) (p)reactive lesion (block no. CR4A), (g, …
Figure 5
Pattern of T cell subpopulations in different stages of MS lesions by IMC.
Representative mass cytometry images of (a, a’, b, b’) white matter of control, (c, c’, d, d’) normal-appearing white matter (block no. CR4A), (e, e’, f, f’) (p)reactive lesion (block no. CR4A), (g, …
Figure 6
Pattern of immunoglobulins and B cell subpopulations in different stages of MS lesions by IMC.
Representative mass cytometry images of (a, a’, b, b’) white matter of control, (c, c’, d, d’) normal-appearing white matter (block no. CR4A), (e, e’, f, f’) a (p)reactive lesion (block no. CR4A), (g…
Figure 7 with 6 supplements
Density of immune cell subsets in different stages of MS lesions and their distance from blood vessels by IMC.
(a) Cell counts are provided as number of cells per mm2 (Barnett and Prineas, 2004) of region of interest. The category of cells is defined according to the expression of cell-specific and …
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Figure 7—source data 1
Source file for quantitative data of all ROI.
ROI 2 from block 95–056 (white matter control) ROI one from block CL3a (active lesion) ROI 2.1 from block CL3a (mixed active-inactive lesion) ROI 2.2 from block CL3a (mixed active-inactive lesion) ROI three from block CL3a (mixed active-inactive lesion) ROI four from block CL3a (active lesion) ROI two from block CR4a (mixed active-inactive lesion) ROI four from block CR4a (active lesion) ROI eight from block CR4a (normal-appearing white matter) ROI three from block CR4a (active lesion) ROI one from block CR4a ((p)reactive lesion) ROI five from block CR4a (active lesion) ROI six from block CR4a (mixed active-inactive lesion).
- https://doi.org/10.7554/eLife.48051.021
Figure 7—figure supplement 1
Single cell segmentation and validation of approach using anti-CD3.
A Gaussian blur was applied to the DNA signal (nucleus detection - a), and the resulting blurred image was segmented to identify nuclear content corresponding to individual cell areas using a …
Figure 7—figure supplement 2
Manual selection of myeloid cells.
(a) Representation of manually-annotated cells in an active lesion, based on the detection of nuclei. (b) Segmented cells. (c–h) Identification of cells that express (for example CD45+HLA+) or do …
Figure 7—figure supplement 3
Manual selection of T cells.
(a) Representation of manually-annotated cells in an active lesion, based on the detection of nuclei. (b) Segmented cells. (c–h) Identification of cells that express (for example CD45+CD3+CD4+ or …
Figure 7—figure supplement 4
Manual selection of B cells.
(a) Representation of manually-annotated cells in a mixed active-inactive lesion, based on the detection of nuclei. (b) Segmented cells. (c–h) Identification of cells that express (for example CD45+I…
Figure 7—figure supplement 5
Gating strategy used for the identification of cell subsets.
Gating strategy for the identification of cell subset phenotypes and activation states of microglia, macrophages, T cells and B cells. In brief, the per-cell mean intensities of specific marker …
Figure 7—figure supplement 6
Gating strategy used for the generation of heat maps.
Using the quadrants that capture the appropriate positivity range of each cell phenotype shown in Figure 7—figure supplement 5, cells were subjected to the positive and negative gating strategies as …
Figure 8
Heat-diffusion Affinity-based Transition Embedding (PHATE) mapping of T cells.
(a–c) PHATE plots of all T cells analyzed in this study, colored by (a) cell class, (b) lesion type of residence and (c) relative marker expression intensity or distance to blood vessels. The …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-Nucleic Acid-Ir191/Ir193 | Fluidigm | Cat#:201192A RRID: AB_2810850 | IMC: (1/3000) |
| Antibody | Anti-Proteolipid Protein-141Pr (Mouse monoclonal) | Bio-Rad | Cat#: MCA839G RRID:AB_2237198 | IMC: (1/25), IF: (1/25), IHC: (1/100) |
| Antibody | Anti-human CD38-167Er (Mouse monoclonal) | Fluidigm | Cat#:3167001B RRID: AB_2802110 | IMC: (1/2000) |
| Antibody | Anti-human CD45-154Sm (Mouse monoclonal) | Fluidigm | Cat#: 3154001B RRID:AB_2810854 | IMC: (1/2000) |
| Antibody | Anti-human CD68-159Tb (Mouse monoclonal) | Fluidigm | Cat#: 3159035D RRID:AB_2810859 | IMC: (1/100) |
| Antibody | Anti-human HLA-147Sm (Mouse monoclonal) | Fluidigm | Cat#: Ab55152 RRID: AB_944199 | IMC: (1/100), IF: (1/50), IHC: (1/100) |
| Antibody | Anti-human TMEM119-155Gd (Rabbit polyclonal) | Sigma-Aldrich | Cat#: HPA051870 RRID: AB_2681645 | IMC: (1/50), IF: (1/100) |
| Antibody | Anti-human CD3-170Er (Mouse monoclonal | Fluidigm | Cat#: 3170001 RRID: AB_2661807 | IMC: (1/100) |
| Antibody | Anti-human CD4-176Yb (Mouse monoclonal) | BioLegend | Cat#:344602 RRID: AB_1937277 | IMC: (1/100), IF: (1/20) |
| Antibody | Anti-human CD8a-162Dy (Mouse monoclonal) | Fluidigm | Cat#: 3162015B RRID:AB_2661802 | IMC: (1/100) |
| Antibody | Anti-human Granzyme B-171Yb (Mouse monoclonal) | ThermoFisher Scientific | Cat#: MA1-80734 RRID:AB_931084 | IMC: (1/25), IF: (1/20) |
| Antibody | Anti-human IgKappa-160Gd (Mouse monoclonal) | Fluidigm | Cat#:3160005B RRID:AB_2810855 | IMC: (1/3000) |
| Antibody | Anti-human IgLambda-151-Eu (Mouse monoclonal) | Fluidigm | Cat#: 3151004B RRID:AB_2810853 | IMC: (1/3000) |
| Antibody | Anti-human IgM-172Yb (Mouse monoclonal) | Fluidigm | Cat#: 3172004B RRID:AB_2810858 | IMC: (1/500) |
| Antibody | Anti-human Collagen Type I-169Tm (Goat polyclonal) | Fluidigm | Cat#: 3169023D RRID:AB_2810857 | IMC: (1/4000) |
| Antibody | Anti-human CD31-145Nd (Mouse polyclonal) | LSBio | Cat#: LS-C390863 RRID:AB_2810860 | IMC: (1/100) |
| Antibody | Anti-human NFAT1-143Nd (Rabbit monoclonal) | Fluidigm | Cat#: 3143023A RRID:AB_2810851 | IMC: (1/50) |
| Antibody | Anti-human Ki67-168Er (Mouse monoclonal) | Fluidigm | Cat#: 3168001B RRID:AB_2810856 | IMC: (1/100) |
Table 1
Lesion types and regions of interest.
https://doi.org/10.7554/eLife.48051.023| Case | Tissue block (anatomical location) | Lesion type | Region of interest |
|---|---|---|---|
| C, 95–056 | A (superior frontal gyrus) | WMC | |
| MS, AB129 | CL3a (cerebellum) | NAWM | |
| 2x Active demyelinating (pattern II) | PPWM, center | ||
| 3x Mixed active/inactive demyelinating | PPWM, edge, core | ||
| CR4a (cerebellum) | NAWM | ||
| (p)reactive | |||
| 3x Active demyelinating (pattern II) | center | ||
| 2x Mixed active/inactive demyelinating | edge, core |
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C, control; MS, multiple sclerosis; WMC, white matter of control; NAWM, normal-appearing white matter; PPWM, periplaque white matter.
Table 2
Antibody panels for the identification of functional cell types by IMC.
https://doi.org/10.7554/eLife.48051.024| Functional cell type | Antibody panel |
|---|---|
| Macrophages and microglia | |
| Macrophages | CD45highHLA+TMEM119- |
| Activated macrophages | CD45highHLA+TMEM119-CD68+PLP- |
| Demyelinating macrophages | CD45highHLA+TMEM119-CD68+PLP+ |
| Microglia | CD45low/+HLA+TMEM119+ |
| Activated microglia | CD45low/+HLA+TMEM119+CD68+PLP- |
| Demyelinating microglia | CD45low/+HLA+TMEM119+CD68+PLP+ |
| T Cells | |
| CD8+ T cells | CD45+CD3+CD8α+CD4- |
| Proliferating CD8+ T cells | CD45+CD8α+CD3+CD4-Ki67+ |
| Activated CD8+ T cells | CD45+CD3+CD8α+CD4-NFAT+ |
| Activated and proliferating CD8+ T cells | CD45+CD3+CD8α+CD4-NFAT+Ki67+ |
| ‘Chronically activated’ CD8+ T cells | CD45+CD3+CD8α-CD8+CD38+HLA+ |
| CD4+ T cells | CD45+CD3+CD8α-CD4+ |
| Proliferating CD4+ T cells | CD45+CD8α-CD3+CD4+Ki67+ |
| Activated CD4+ T cells | CD45+CD3+CD8α-CD4+NFAT+ |
| Activated and proliferating CD4+ T cells | CD45+CD3+CD8α-CD4+NFAT+Ki67+ |
| ‘Chronically activated’ CD4+ T cells | CD45+CD3+CD8α-CD4+CD38+HLA+ |
| B Cells | |
| Naïve B Cells | CD45+Igκ/Igλ+IgM+CD38- |
| IgM memory B cells | CD45+Igκ/Igλ+IgM+CD38+ |
| Switched memory B cells | CD45+Igκ/Igλ+IgM-CD38+ |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.48051.025
