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. 2018 May 9;7:e36341. doi: 10.7554/eLife.36341

Figure 5. Clustering of peptide-deficient HLA-I in cognate peptide-induced immunological synapses.

Expression levels of HLA-I on the surface of activated CD4+ T cells isolated from PBMCs of three healthy donors were assessed by flow cytometry after staining with HC10 (A), confirming that activated lymphocytes express peptide-deficient conformers of HLA-I molecules. The mean ± SEM of two replicates for each donor are shown. CTL line B8-RL8 was incubated with activated CD4+ T cells from Donor 25 (carrying B*08:01 and B*35:01) loaded with peptide RL8 (B, C, D and E) or not (F and G). Cells were fixed and stained with anti-CD8 and W6/32 or HC10 before analysis by confocal microscopy. Peptides were used at a concentration of 100 μM. Anti-CD8 staining (B, D and F) were shown in red and W6/32 (C) or HC10 (E and G) in green. Arrowheads indicate peptide-deficient conformers of HLA-I clustering at the interface between CD4+ T cells and CTL line. The intensity of HLA-I staining of the CD4+ T cells at the interface was compared with the membrane at a noncontact area and plotted as the fold increase above background (H). The results with a total of 20–30 conjugates (mean ± SEM) per condition are shown. CD8 clustering was derived from 30 conjugates under conditions shown in D. Statistical analyses were undertaken using a one-way ANOVA analysis with Fisher’s LSD test. ***, p<0.001; ****, p<0.0001.

Figure 5.

Figure 5—figure supplement 1. Clustering of peptide-deficient HLA-I in cognate peptide-induced immunological synapses.

Figure 5—figure supplement 1.

CTL line B8-RL8 was incubated with activated PBMCs from Donor 25 (carrying B*08:01 and B*35:01) loaded with peptide RL8 (A–D) or not (E and F). Cells were fixed and stained with anti-CD8 and W6/32 or HC10 before analysis by confocal microscopy. Peptides were used at a concentration of 100 μM. Anti-CD8 staining (A, C and E) is shown in red and W6/32 (B) or HC10 (D and F) in green. Arrowheads indicate peptide-deficient conformers of HLA-I clustering at the interface between PBMCs and CTL line. The intensity of HLA-I staining of the PBMCs at the interface was compared with the membrane at a noncontact area and plotted as the fold increase above background (G). The results with a total of 39 conjugates (for B and D) or 10 conjugates (for F) per condition are shown. CD8 clustering was derived from 39 conjugates as condition shown in C. The mean ± SEM was shown. Statistical analyses were undertaken using one-way ANOVA analysis with Fisher’s LSD test. ****, p<0.0001.