Hla e

Fresh cultured cells were prepared into single cell suspensions were analyzed. Anti-human HLA-ABC, HLA-E, MICA/B, PD-L1 and EGFR antibodies were purchased from Biolegend. Primary antibodies were applied for 30 minutes at concentrations titrated for each antibody. Dead cells were excluded via 7AAD uptake and a “fluorescence-minus-one” technique was used to validate specific staining in all antibody combinations. Analysis was performed on a BD FACSCanto analyzer running FACSDiva software and interpreted using FlowJo (vX10.0.7r2).

For baboon cell populations, antibodies against CD3 (BD Pharmingen:
clone SP34–2), CD4 (BD Horizon: clone L200) and CD8 (BD Horizon RPA
–T8) for T cells, CD20 (BD Horizon: clone 2H7) and CD22 (Life
technology, MHCD 2201) for B cells, NHP CD45 (BD biosciences:
D058–1283) for all baboon leukocytes were used. In order to detect
pig cells specifically (macrochimerism), we used biotinylated
1030H1–19 (mouse anti-pan-pig tissue, IgM) and allophycocyanine
(APC)-Streptavidin (BD Biosciences). After incubation, cells were washed
twice and lysed with FACS lysing solution (BD Biosciences, San Jose, CA),
and then washed twice again. Cells were read on FACS Canto II (Becton
Dickinson, Mountain View, CA, USA) for detection of cell-bound antibodies,
and data was analyzed using FlowJo (V.10. 1, Tree Star, Ashland, OR, USA).
The percentage of pig cells (macrochimerism) staining with anti-pan-pig
antibody in gating for whole leukocytes was determined by comparing to PBMC
staining from the naïve pig cells as positive control, and
naïve baboon cells as negative control. To confirm Tg expression in
donor pigs’ PBMC and BM, anti-human hCD47Ab (BD Pharmingen, clone:
B6H12), hCD55 (Biolegend, clone: JS11), hCD46 (BD Pharmingen, clone: E4. 3)
and HLA-E (Biolegend, clone: 3D12) were used.