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. 2018 Jul 10;7:e34961. doi: 10.7554/eLife.34961

Figure 4. HLA class I assembly differences between monocytes and lymphocytes.

A: Flow cytometry experiments measuring W6/32-based staining of cell surface HLA class I (fixed PBMCs) expressed as a ratio relative to W6/32-based staining of total HLA class I (fixed and permeabilized PBMCs). Each point represents an individual donor measurement, and a total of 33 donor samples were tested. B: PBMCs were fixed and permeabilized, then stained with either anti-tapasin or W6/32 antibodies. The ratio of tapasin MFI relative to the W6/32 MFI was calculated for each cell type, then normalized to the corresponding monocyte ratios. Each point represents an individual donor measurement, and a total of 29 donor samples were tested. C and D: Summary statistics from two ImageStream experiments with three donors in monocytes (C) or CD4+ T cells (D). Bw6 and AP-1 co-localization, Bw6 and CRT co-localization, and Bw6 and LAMP-1 co-localization were quantified for donors 94 and 64. Only Bw6 and AP-1 co-localization was measured for donor 237. E and F: Representative monocyte (E) or CD4+ T cell (F) images for the experiments summarized in Panels C and D. This figure has five supplementary figures and one source data table.

Figure 4—source data 1. Imaging cytometry co-localization source data.
The data represents one imaging cytometry experiment performed on two donors: 94 and 64. Genotypes for donors 94 and 64 are indicated in Figure 1—source data 1. In monocytes and CD4+ T cells, Bw6 colocalization is quantified with three different intracellular markers: AP-1 (top), calreticulin (middle), and LAMP-1 (bottom). In imaging cytometry experiments, co-localization is quantified as Bright Detail Similarity (BDS), which is the degree of overlap between the two markers of interest. The red columns represent cell population gates with a high degree of co-localization, yellow columns represent cells with intermediate co-localization, and blue columns represent cells with low co-localization. Intermediate co-localization was calculated only for Bw6/AP-1 co-localization. The first row for each donor is the quantification of the cell count within each gate, the second row is the percentage of cells within a gate, relative to the total number of cells in the previous gate, and the final row is the median BDS for each population. In each cell population, the Bw6+ M2+ column represents cells that are double positive for Bw6 and the second co-localization marker (Marker 2; M2). M2 is AP-1 for the top table, calreticulin for the middle table, and LAMP-1 for the bottom table.
elife-34961-fig4-data1.docx (20KB, docx)
DOI: 10.7554/eLife.34961.021

Figure 4.

Figure 4—figure supplement 1. Tapasin expression.

Figure 4—figure supplement 1.

Intracellular tapasin expression measured by flow cytometry with the monoclonal PaSta-1 antibody, and the data were normalized to monocyte expression levels. Each point represents a single measurement on 33 donors. One-way ANOVA analysis was performed, with p ****<0.0001.
Figure 4—figure supplement 2. Gating strategy for imaging cytometry experiments.

Figure 4—figure supplement 2.

PBMCs were analyzed on the Amnis ImageStreamX imaging cytometer and gated for cell populations. These populations were then analyzed for colocalization of Bw6 with either AP-1, calreticulin (CRT), or LAMP-1. Gating strategies for monocytes and CD4+ T cells are shown in panel R2. In this panel, CD3-Pacific Blue is plotted on the X axis, and CD8-Alexa Fluor 700 and CD14-Alexa Fluor 700 are plotted on the Y axis. CD4+ T cells were identified by gating on the CD3+, CD8- cells (gate R3), and monocytes were identified by gating CD3-, CD14+ cells (gate R4). These populations were then gated on cells that were double positive for the two co-localization markers of interest, and these double positive cells were analyzed for Bright Detail Similarity (BDS) in panels R5 and R8. BDS is a quantification of the degree of overlap between two markers. Thus, cells with a high BDS score have a high degree of co-localization between the two markers analyzed. Panels R5 and R8 show the gates used to quantify cells with high, intermediate, and low co-localization.
Figure 4—figure supplement 3. Representative image gallery for Donor 64 monocytes.

Figure 4—figure supplement 3.

PBMCs from donor 64 were analyzed by imaging cytometry for Bw6/AP-1 co-localization. The top panel represents gate R9—monocytes with high Bw6/AP-1 co-localization—while the bottom panel represents gate R12—monocytes with an intermediate degree of Bw6/AP-1 co-localization (defined as in Figure 4—figure supplement 2).
Figure 4—figure supplement 4. Representative image gallery for Donor 94 monocytes.

Figure 4—figure supplement 4.

PBMCs from donor 94 were analyzed by imaging cytometry for Bw6/AP-1 colocalization. The top panel represents gate R9—monocytes with high Bw6/AP-1 colocalization—while the bottom panel represents gate R12—monocytes with intermediate Bw6/AP-1 colocalization (defined as in Figure 4—figure supplement 2).
Figure 4—figure supplement 5. Representative image gallery for Donor 237 monocytes.

Figure 4—figure supplement 5.

PBMCs from donor 237 were analyzed by imaging cytometry for Bw6/AP-1 colocalization. The top panel represents gate R9—monocytes with high Bw6/AP-1 colocalization—while the bottom panel represents gate R12—monocytes with intermediate Bw6/AP-1 colocalization (defined as in Figure 4—figure supplement 2).