HLA Class II and Humanized Mice Figure 2. Kinetics of human T cell reconstitution in peripheral lymphoid organs. Groups of DRAG and control mice (n = 4) were euthanized at 2 months post-infusion of HSC and bone marrow (panel A) and thymus (panel B) were examined for the presence of human cells by FACS using specific antibodies. All DRAG mice reconstituted bone marrow and thymus with human cells. All control mice reconstituted bone marrow with human cells, though at much lower frequencies than DRAG mice, and only 1 of them was able to reconstitute thymus with human cells. No significant human T cell reconstitution was detected in spleens of DRAG or control mice at this time point (data not shown). Additional groups of DRAG (n = 4) and control (n = 2) mice that showed human T cells in blood at 6 months post-infusion of HSC, were examined by FACS in bone marrow (panel A), thymus (panel B) and spleen (panel C). Data represent mean values of mice analyzed individually. Ranges are indicated over the plots. P values were calculated using Independent Samples t-test. Of note, control mice (n = 3) that did not have human T cells in blood, also showed absence of human T cells in thymus and spleen (data not shown). doi:10.1371/journal.pone.0019826.g002 that expression of HLA-DR4 did not improve development of human NK cells. In aggregate, these results demonstrated that expression of HLA-DR4 molecules in DRAG mice favored engraftment of HSC in bone marrow, homing and development of bone-marrow- derived human T cell precursors in thymus, and proficient repopulation of peripheral lymphoid organs with mature CD4 and CD8 T cells. Expression of HLA-DR4 molecules however did not improve development of human NK cells. Human CD4 and CD8 T cells developed by DRAG mice are functional Previous studies in HSC-infused NOD and BALB/c genetic stocks bearing the IL2RccKO mutation revealed a functional impairment of human T cells reconstituted in these strains, as they responded weakly to polyclonal stimulation with anti-human CD3/CD28 antibodies or PMA/ionomycin [18]. It was thus important to investigate whether expression of HLA-DR4 molecules in DRAG mice may have rescued the function of human T cells. As illustrated in figure 4A&B, human T cells from spleens of DRAG mice (n = 4) responded vigorously to CD3/CD28 or PMA/ionomycin stimula- tion as indicated by secretion of high levels of human cytokines in cell cultures. Noteworthy, the magnitude of T cell responses in DRAG mice was comparable to that of human PBMCs from healthy volunteers. In contrast, human T cells from the spleens of control mice (n = 2) responded poorly to CD3/CD28 or PMA/ionomycin stimulation (Fig. 4A&B). These results demonstrated that the human T cell compartment reconstituted in DRAG mice is functional. Since DRAG mice express HLA class II (DR4) molecules but not HLA class I molecules, which is the restriction element for thymic selection of human CD8 T cells, we next compared the function of human CD4 vs. CD8 T cells developed by DRAG mice in a well-controlled in vitro system. For this comparison, spleen cells from DRAG mice were depleted of either CD4 T cells, CD8 T cells, or both T cell subsets using antibody-coated magnetic beads followed by stimulation with PMA/ionomycin. As illustrated in figure 4C, depletion of human CD4 T cells partially reduced the secretion of IL-2 and IFNc as compared to that of non-depleted splenic cells, and abrogated secretion of TNFa. Depletion of human CD8 T cells from spleens also reduced the secretion of IL- 2 and IFNc, but had little effect on secretion of TNFa. In addition, positively-isolated CD4 T cells from spleens of DRAG mice secreted high levels of IL-2, IFNc, and TNFa upon stimulation with PMA/ionomycin, and positively-isolated CD8 T cells secreted IL-2 and IFNc, but not TNFa. It is know that CD8 T cells secrete little or no TNFa upon PMA/ionomycin stimulation [36]. These results indicated that both human CD4 and CD8 T cells developed by DRAG mice are functional as they respond to stimulation through TCR and non-TCR signaling pathways. DRAG mice infused with HLA-DR-matched HSC are highly reconstituted with human B cells The longitudinal FACS analysis in blood of DRAG and control mice using anti-human CD19 revealed that the rate of human B-cell reconstitution was similar between the two strains. Thus, 14 out of 15 (93.3%) DRAG mice and 9 out of 11 (81.8%) Figure 3. Expression of HLA-DR4 molecules in DRAG mice fosters development of Tregs but not of myeloid and natural killer cells. Panel A, frequency of CD4+FOXP3+ Tregs among splenic T cells from DRAG (n = 4) and control (n = 2) mice that were examined at 6 months post- infusion of HSC (p = 0.021). Panel B, frequency of dendritic cells (CD11c+CD192) and NK cells (CD16+CD32) in spleens of DRAG (n = 3) and control (n = 2) mice at 6 months post-infusion of HSC. The cut-off for positive cells was 0.09% for DCs, and 0.01% for NK cells. P values were calculated using Independent Samples t-test. doi:10.1371/journal.pone.0019826.g003 PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e19826