In addition to the signs of liver and kidney damages, recruitment of immune cells and their activation in tissues and organs reflects systemic inflammatory response in mice (11). Statistical analysis showed that mice supplemented with drinking water containing 1000 mg nCu/l displayed an increased infiltration of innate immune cells into spleens as well as an elevated activation of splenic leukocytes as compared to control group (Figure 3). In contrast, percentages of immune cells and their activation in spleens of the other mouse groups were unaltered (Figure 3). Accordingly, the numbers of antigen-presenting cells including dendritic cells (CD45+CD11c+) and macrophages (CD45+CD11b+F4/80+) infiltrated into spleens were high, whereas the number of B cells (CD45+CD19+) was found significantly lower in the 1000 mg nCu/l-treated mouse group than that in control group (Figure 3A-B). Consequently, increased expressions of major histocompatibility complex (MHC) class II molecule on CD45+ cells (Figure 3C-D) as well as the upregulation of CD69 marker on both CD4 T cells (CD3+CD4+) and CD8 T cells (CD3+CD8+) (Figure 3E-F) were observed only in spleens of 1000 mg nCu/l-treated mice, but not in spleens of the other mouse groups. The evidences showed that the excessive accumulation of Cu in organs of the body such as liver and serum induced the infiltration of antigen-presenting cells and stimulated immune cell activation in mouse spleens.