were then generated and used as neonatal or adult recipients of human HSCs [13–17]. Although both HSC-infused newborn and adult mice were highly reconstituted with human B cells, the development of B cells was arrested in an early stage and did not suffice for reconstitution of human immunoglobulins (natural antibodies) in serum, other than IgM [18]. Newborn recipient mice were much more efficient for development of human T cells than the adult recipients [19], but the human T cells were functionally impaired, as their response to in vitro polyclonal T cell stimulation with anti-CD3/CD28 Abs or PMA/ionomycin was poor [18]. Studies from several laboratories further indicated that these mice were sub-optimal, or alternatively failed to elicit cellular and/or humoral responses upon infection or vaccination [15,20–22]. Impairment of human T and B cell function in HSC- reconstituted IL2RccKO genetic stocks has been attributed to the lack of expression of Human Leukocyte Antigens (HLA) in the mouse thymus, since HLA molecules are required for development of human T cells [23] that in turn, are essential for stimulation of B cells towards immunoglobulin class switching and antibody secretion [24]. Indeed, mice transplanted with human fetal thymus and liver under the kidney capsule and at the same time infused with human HSCs (a model known as BLT mouse –Bone marrow, Liver, Thymus) [25] showed significant improvement in the function of human T and B cells, as these mice were able to elicit specific IgM responses upon vaccination [25,26]. Recently, humanized mice expressing HLA class I (HLA-A2) molecules on the NOD.scid.IL2RccKO background have been generated [27–29]. Contrary to expectations, the HLA-A2 transgenic (Tg) mice showed similar rates of human T cell reconstitution as non-Tg littermates [28], though some of the T cell reconstituted mice were able to elicit HLA-A2-restricted CD8 T cell responses upon infection with Epstein Barr or dengue virus [27–29]. However, the function of human B cells was not improved by the transgenic expression of HLA-A2 molecules in HSC-recipient mice, as these mice were unable to elicit specific IgG antibodies [27–29]. Herein, we show that a newly-generated strain of NOD.Rag1- KO.IL2RccKO mice expressing HLA-DR*0401 molecules (here- after referred to as DRAG mice) develop high rate of human T and B cell reconstitution upon infusion of HLA-DR-matched HSC during adulthood. Both CD4+ and CD8+ human T cell subsets as well as human B cells are highly functional as indicated by their ability to secrete human cytokines, reconstitute serum levels of human IgM, IgG, IgA, and IgE, and elicit specific IgG antibodies upon vaccination. The results demonstrate that expression of HLA class II molecules in humanized mice supports the development of a functional human adaptive immune system. Results DRAG mice infused with HLA-DR-matched HSC are highly reconstituted with human T cells Early lymphoid precursors derived from hematopoietic stem cells in the bone marrow migrate to the thymus where they differentiate into CD4+CD8+ double positive (DP) cells. DP cells undergo positive or negative selection upon recognition of self- peptides presented by thymic epithelial cells in the context of MHC molecules [23], with positively-selected DP thymocytes differentiating into CD4+ or CD8+ single positive (SP) thymocytes that then repopulate peripheral lymphoid organs. Thymic selection is a physiological process that prevents autoimmunity by eliminating self-reactive T cells and at the same time generates a broad spectrum of T cells reactive against foreign antigens. HLA Class II and Humanized Mice To test the hypothesis that constitutive expression of HLA class II molecules in humanized mice rescues development of human CD4 T cells, we generated NOD.Rag1KO.IL2RccKO mice expressing HLA-DR4 (0401) molecules (hereafter referred to as DRAG mice) (Fig. 1A upper panel). Four-to-six week-old DRAG mice were infused with CD34+ HSC from HLA-DR*0401-positive umbilical cord bloods and monitored by FACS for development of human T cells in the peripheral blood. As a specificity control, we used NOD.Rag1KO.IL2RccKO littermates lacking expression of HLA-DR4 molecules (Fig. 1A, lower panel) that were infused with HSC from the same donors. As illustrated in figure 1B, the rate of human T cell reconstitution in DRAG mice was considerably higher than in control mice, since 14 out of 15 (93.3%) DRAG mice developed human T cells by 25 weeks post-infusion with HSC as compared to only 4 out of 11 (36.4%) control littermates developing human T cells. In addition, the frequency of human T cells in the blood of reconstituted DRAG mice (n = 14) was considerably higher than in the reconstituted control littermates (n = 4) (Fig. 1C, p = 0.0044, Table S1). Thus, the results demonstrated that expression of HLA-DR4 molecules in DRAG mice promoted development of human T cells. T cell-reconstituted DRAG (n = 14) and control mice (n = 4) developed both CD4 and CD8 T cells in blood, but the frequency of CD4 T cells was significantly higher in DRAG mice (Fig. 1D, p = 0.0032). Of note, the frequency of blood circulating CD4 T cells in DRAG mice was comparable to that of human volunteers (Fig. 1D, upper and lower panels). On the other hand, the frequency of human CD8 T cells in blood of both groups of mice was similar (Fig. 1D, p = 0.36) and at the same time lower than that in blood of human volunteers. Since T cells are developed in thymus from bone marrow precursors, we next investigated the kinetics of human cell reconstitution in bone marrow, thymus, and spleen of DRAG mice at 2 and 6 months post-infusion of HSC. At 2 months post- infusion of HSC, the bone marrow of DRAG mice (n = 4) showed much higher numbers of CD45+ and CD34+ cells than the control mice (n = 4) (Fig. 2A). CD45 is a type I transmembrane protein expressed in all human hematopoietic cells except erythrocytes and plasma cells, and CD34 is a marker specific for human HSCs. The thymus of DRAG mice also showed considerably higher numbers of human CD45+ cells than the control mice, with most CD4+CD8+ DP, followed by CD4+ SP and CD8+ SP (Fig. 2B). Of note, all DRAG mice (n = 4) used for these analyses had human cells in thymus but only 1 out of 4 control mice had human cells in thymus. Thus, these results indicated that expression of HLA-DR4 molecules in DRAG mice favored HSC engraftment in bone marrow and more importantly, promoted the development of human thymocytes. At 6 months post-infusion of HSC, the bone marrow of DRAG and control mice contained similar numbers of human CD45+ and CD34+ cells (Fig. 2A), but still the thymuses from control mice were poorly reconstituted with human thymocytes (Fig. 2B, right panel). Furthermore, the spleens of all DRAG mice examined (n = 4) contained mature human T cells (Fig. 2C), of which most were CD4 T cells (23.8%) and, to a lower extent, CD8 T cells (5.0%) (CD4:CD8 average = 5.2). The frequency of CD4 and CD8 T cells in human spleen is approximately 17% and 14%, respectively [30]. On the other hand, the control mice (n = 2) that were selected based on the presence of human T cells in blood had human T cells in the spleen as well (Fig. 2C), but the CD4 T cell frequency (4.6%) was considerably lower than that in spleens of DRAG mice (CD4:CD8 average ratio = 1.5). Also, among control mice showing no reconstitution with human T cells in blood (n = 3) all lacked human T cells in spleen (data not shown). PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e19826