The D409G pigs displayed severe anemia and immunodeficiency. (A) The Wright-Giemsa staining of peripheral blood smears showed that the mutants exhibited a higher number of NRBCs (arrowheads) per 100 WBCs than did the WT group (WT, n = 7; D409G, n = 6). Scale bars, 50 μm. The quantification of NRBCs per 100 WBCs on the right showed that the number of NRBCs in D409G pigs was markedly higher (187.0 ± 18.01) than that of WT pigs (3.7 ± 2.93). **P < .01. (B) The D409G pigs exhibited anemia with significantly lower levels of red blood cells (RBCs), HGB, and hematocrit (HCT) (WT, n = 15; D409G, n = 10; **P < .01), whereas WBC count was normal (WT, n = 7; D409G, n = 6; P > .05). (C) The D409G mutants had lower levels of CD3⁺ T cells, CD4⁺CD8⁺ cells, CD4⁻CD8⁻ cells, and the CD3⁺ subpopulation of CD4⁺CD8⁻ helper T cells (**P < .01), and no significant differences were observed in CD4⁻CD8⁺ cytotoxic T lymphocytes (P > .05) compared with WT pigs (WT, n = 15; D409G, n = 10). (D) The H&E staining results revealed atrophied lobules and decreased thymocytes in the thymuses (top left panel) of D409G mutants; the mutant spleens (bottom left panel) exhibited periarterial lymphatic sheath (PALS) and indistinguishable white pulp (WP) and red pulp (RP) compared with the WT spleens (central artery [CA]). The number of lymphocytes was also remarkably reduced in the spleens of mutant pigs. Scale bars, 50 μm. The relative spleen weight was increased in D409G pigs, whereas the thymus weight was notably decreased compared with that of WT pigs (right panel) (WT, n = 15; D409G, n = 10; **P < .01). The weights of organs were normalized by total body weight. The data are presented as the means ± SD.