Endo-lysosomal assembly variations among human leukocyte antigen class I (HLA class I) allotypes

Data for Figure 1A–C. Donors were selected to be heterozygous for one Bw6 and one Bw4 allotype with one or less cross-reactive HLA-C allotype per HLA-B allotype of interest. Monocytes were isolated and differentiated into moDCs for 7 d, followed by treatment with bafilomycin A1 for 1, 2, or 4 hr. HLA-B expression was measured with anti-Bw6 or anti-Bw4 and normalized relative to the untreated condition.

Data for Figure 1D–F. Re-analyzed from previous studies (Rizvi et al., 2014; Geng et al., 2018; Sarkizova et al., 2020) to describe HLA-B assembly modes.

Data for Figure 1H–I. Monocyte-derived dendritic cells were differentiated and pulsed with control (non-binding) peptide for 4 hr in the presence or absence of bafilomycin. Cells were stained with either BBM.1 (anti-β₂m) or HC10 (anti-open human leukocyte antigen class I).

Data for Figure 2A–B. moDCs were incubated with brefeldin A for various time points, followed by staining with Bw6 antibody. Sample data in (A) normalized to 0.25 hr. Data in (B) analyzed as described in methods to determine half-life.

Data for Figure 2C. Monocyte-derived dendritic cells were differentiated and plated into 96 well plates. Cells were cultured at 37°C, or for 1 or 2 hr at room temperature and 42°C. Cells were then stained with anti-Bw6 to measure surface HLA-B, and normalized to 37°C.

Data for Figure 2D. HLA-B monomers were loaded with low-affinity peptide and washed, then exchanged with either no peptide, medium-affinity peptide, or high-affinity peptide in pH 4, 5, 6, or 7 buffer. Monomers were then bound to streptavidin beads and stained with HC10.

Data for Figure 2E–F. moDCs were pulsed with B*08:01 or B*35:01 specific or control peptides for 4 hr, followed by washing and staining with the HC10 antibody. Pulses were performed at 37 or 4 °C.

Data for Figure 3C–F. Monocytes or moDCs were plated onto poly-L-lysine-coated coverslips, fixed, and stained for Bw6 co-localization with either EEA1, Rab11, or LAMP1. Co-localization was assessed using object-based methods as described.

Data for Figure 3—figure supplement 1. Data from Figure 3 analyzed with the JACOP plugin on FIJI to measure co-localization.

Data for Figure 4A–D. Monocytes or moDCs were treated with bafilomycin for 1 or 4 hr, followed by staining with anti-Bw6 antibody. Expression normalized to untreated cells.

Data for Figure 4F. moDCs were plated for microscopy as in Figure 3, but treated with bafilomycin for 4 hr and stained for Bw6 co-localization with LAMP1.

Data for Figure 4G. moDCs were pulsed with specific or control peptides in the presence or absence of bafilomycin, followed by staining with the HC10 antibody.

Data for Figure 5A. Monocytes or moDCs were pulsed in 15 min increments with BSA labeled with Alexa Fluor 594. AF594 signal normalized to no Ag.

Data for Figure 5B–C. Monocytes or moDCs were pulsed with DQ-Ova antigen alone or in the presence of bafilomycin, MG132, or both. Pulses were also done at 4°C. DQ-Ova fluorescence was measured by flow cytometry and normalized to untreated (100%) and 4°C (0%).

Data for Figure 6A. B*08:01 or B*35:01 peripheral blood mononuclear cells were pulsed with tenfold increments of peptide, followed by co-culture with Ag-specific CTLs for 5 hr. CTLs were stained for surface CD107a degranulation and intracellular IFNγ. Data normalized to 100 µM concentration.

Data for Figure 6C–H. Monocytes or moDCs were pulsed with antigen for 6 hr, followed by co-culture with cytotoxic T lymphocytes (CTLs) for 5 hr. CTLs were stained for activation markers, and antigen cross-presentation data was normalized to activation with peptide.

Data for Figure 7D–E. Cross-presentation was performed as in Figure 6, except during antigen pulse the inhibitors MG132, lactacystin, or cathepsin inhibitor I were added. Data is normalized to untreated cross-presentation.