Surface expression of HLA class I molecules on DCs and T cells.

We first investigated the concentrations of anti-B5 MAb (4D12) and anti-HLA-C MAb (DT9) that bound to the same number of HLA-B*52:01 and HLA-C*12:02 molecules, respectively. HLA class I-deficient 721.221 cells transfected with HLA-B*52:01 or HLA-C*12:02 gene (721.221-B*52:01 or 721.221-C*12:02 cells) were used to evaluate these concentrations. These cells were stained with various concentrations of 4D12, DT9, and TP25.99 anti-HLA class I α3 domain MAb followed by PE-conjugated anti-mouse IgG (BioLegend). The MFI of 721.221-HLA-B*52:01 and 721.221-HLA-C*12:02 cells stained with these antibodies was measured by flow cytometry, and then the relative MFI was calculated as follows: MFI of cells stained with MAb/MFI of cells unstained with MAb. To quantify binding affinity of 4D12 (61) and DT9 (39), we normalized the relative MFI of cells stained with 4D12 and DT9 to that of cells stained with TP25.99 by calculating relative binding ratio as follows: the relative MFI of 4D12 or DT9 MAb/the relative MFI of TP25.99 MAb. The concentration of 4D12 and DT9 providing the same relative binding ratio was used to evaluate the surface expression of HLA-B*52:01 or HLA-C*12:02 on the cells.

PBMCs isolated from 3 individuals who were homozygous for the HLA-B*52:01-C*12:02 haplotype were stimulated with Flt3 ligand for 24 h, followed by stimulation with 3′3′-cGAMP for 24 h or 48 h. The cells stimulated with 3′3′-cGAMP for 0 h, 24 h or 48 h were collected and then stained with diluted anti-B5 MAb 4D12, anti-HLA-C MAb DT9, or isotype control followed by PE-conjugated anti-mouse IgG (BioLegend). These cells were stained with the reagents of a LIVE/DEAD fixable near-infrared (IR) dead cell stain kit (Invitrogen) and FITC-conjugated lineage cocktail 1 (Lin1, CD3/CD14/CD16/CD19/CD20/CD56; BD Bioscience), peridinin chlorophyll protein (PerCP)-conjugated HLA-DR MAb (BioLegend), PE-Cy7-conjugated CD123 MAb (BioLegend), APC-conjugated CD11c MAb (BioLegend) for DCs (62), or FITC-conjugated CD3 MAb for T cells. The MFI was measured by using the FACS Canto II. Gated PBMCs negative for expression of Lin markers and positive for expression of HLA-DR were analyzed for CD11c and CD123. HLA-DR⁺ Lin⁻ populations were divided into different subsets: CD11c⁺ CD123^lo (myeloid DCs [mDCs]) and CD11c⁻ CD123^hi (plasmacytoid DCs [pDCs]).