HLA Class II and Humanized Mice Figure 1. Kinetics of human T cell reconstitution in blood of DRAG mice. Panel A, FACS analysis of splenic, thymic, and bone marrow cells from na¨ıve (non-HSC infused) DRAG and control mice (n = 3) stained with HLA-DR antibodies. Groups of DRAG (n = 15) and control (n = 11) mice that were infused intravenously with 80,000 enriched-HSC (CD34+) cells from umbilical cord blood of HLA-DR4 (*0401) newborns were examined in peripheral blood by FACS using anti-human CD3, CD4, and CD8. Panel B, percentage of human T cell reconstituted mice at various time points post- infusion of HSC. Fisher exact test indicated that the rate of human T-cell reconstitution in DRAG mice (14 out of 15) was significantly superior to that in control mice (4 out of 11) (p = 0.0025). Panel C, frequency of human CD3+ T cells in blood of reconstituted DRAG (n = 14) and control (n = 4) mice. The cut-off for positive human CD3+ T cells was calculated as three times the standard deviation over the background levels of cells from na¨ıve (non- HSC infused) DRAG mice that were stained with anti-human CD3 (.0.17%). Ranges are indicated over the plots. Mann-Whitney test indicated that the frequency of human T cells in blood of DRAG mice was significantly higher than that in control mice (p = 0.0044). Panel D, frequency of human CD4 and CD8 T cells in blood of T cell reconstituted DRAG mice (n = 14) and control mice (n = 4) at 25 weeks post-infusion of HSC (upper two panels). Lower histograms shows frequencies of CD4 and CD8 T cells in human blood (n = 2). P values were calculated using the Mann-Whitney test. doi:10.1371/journal.pone.0019826.g001 These results demonstrated that expression of HLA-DR4 molecules in DRAG mice promoted repopulation of human T cells in the periphery. CD4+FOXP3+ regulatory T cells (Tregs) are the archetype of immune regulatory cells that are naturally developed in the thymus [31]. Tregs serve as ‘‘guardians’’ of the immune system by maintaining immune homeostasis and at the same time restricting the CD4 and CD8 T-cell responses as well as humoral responses to self and non-self antigens. The frequency of Tregs in human blood is 2–5% among the CD4 T cell population [32]. At 6 months after infusion of HSC, a much higher frequency (p = 0.021) of human CD4+FOXP3+ Tregs was detected in spleens of DRAG mice as compared to control mice (Fig. 3A). Thus, our results indicated that a major regulatory cell compartment (human CD4+FOXP3+ Tregs) was also reconstituted in DRAG mice upon infusion of HLA-DR-matched HSC. Dendritic cells (DCs) are critical components of the innate immune system due to their potent ability to process antigen and present immunogenic peptides to T cells for activation [33]. The frequency of DCs in human spleen has been estimated as 2.3% of the mononuclear population [34]. As illustrated in figure 3B, both DRAG and control mice developed human DCs (CD11c+CD192). The frequency of human DCs in spleens of DRAG mice (2.9%) was slightly higher than that in control mice (1.5%), though it did not reach statistical significance. Natural killer (NK) cells are characterized by expression of CD16, an immunoglobulin Fc receptor that allows them to eliminate target cells through a process known as ‘‘antibody- dependent-cell-cytotoxicity’’ or ADCC [35]. The frequency of NK cells in human spleen has been estimated as 15% [30]. As shown in figure 3B, the numbers of human NK cells in both DRAG and control mice were insignificant (0.05% vs. 0.07%), which indicated PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e19826