HLA expression and multilineage human immune subsets in HLA class I Tg NSG humanized mice. (A) Reverse-transcription PCR for HLA-A*0201, HLA-A*2402, human β-2-microglobulin (hu-β2M), and murine β-2-microglobulin (ms-β2M) using complementary DNA from BM, spleen, and msCD45⁻EpCAM⁺ thymic epithelial cells (TEC) derived from nonengrafted mice. “A2Tg” (left) and “A24Tg” (right) indicate NSG-HLA-A2/HHD and NSG-HLA-A24/HHD, respectively. (B) Representative contour plots indicating the engraftment of hCD45⁺ leukocytes, hCD3⁺ T cells, and hCD19⁺ B cells in the spleen of an NSG-HLA-A24/HHD recipient at 18 weeks after transplant. (C) Engraftment levels of hCD45⁺ cells in each organ (n = 11 for PB, spleen, BM, MLN, and thymus; n = 10 for axillary lymph nodes [AxLN]). (D) In the CD8⁺ cell fraction of the spleen of HLA class I Tg NSG recipients, naïve, CM, EM, and EMRA T-cell subsets were identified. (E) The frequencies of CD8⁺ memory T-cell subsets in the spleen of HLA class I Tg NSG recipients (n = 10). (F) Cytoplasmic expression of granzyme A in CD8⁺ T-cell subsets from naïve to E phenotype in the spleen of HLA class I Tg NSG recipients (n = 4). (G) Representative contour plots of human myeloid subsets in the BM of an HLA class I Tg NSG recipient. (H) The frequencies of monocytes (Mono; CD14⁺CD33⁺HLA-DR⁺), pDCs (CD123⁺CD11c⁻), and cDCs (CD33⁺HLA-DR⁺CD11c⁺) among hCD45⁺ cells in BM (left) and spleen (right). (I) cDCs were further divided into BDCA1⁺ and BDCA3⁺ DCs in BM (n = 8) (left) and spleen (n = 7) (right). Bars indicate the mean value. E, effector; ms, mouse; Spl, spleen; Thy, thymus.