HLA Class II and Humanized Mice Figure 4. Human T cells developed by DRAG mice are functional. At 6 months post-infusion of HSC, splenic cells (36105) from DRAG (n = 4) and control mice (n = 2) as in figure 2, were stimulated for 24 h with anti-human CD3/CD28 Abs (panel A) PMA/ionomycin (panel B) or left unstimulated, and cytokines secreted in supernatants were measured by Luminex. Non-stimulated cells secreted insignificant levels of human cytokines (,50 pg/ml). Data represent mean values of mice analyzed individually over the background levels of non-stimulated cultures. Ranges are indicated over the plots. T cells from DRAG mice secreted significantly higher levels of IL-2 upon stimulation with CD3/CD28 Abs or PMA/ionomycin as compared to control mice (p values = 0.013 and 0.025, respectively using the Independent sample t-test). Panel C, cell-depleted spleens or positively isolated CD4 T and CD8 T cells from pooled spleens of two additional DRAG mice were stimulated in vitro with PMA/ionomycin for 24 h or left unstimulated. Non-stimulated cell cultures produced insignificant levels of human cytokines (,50 pg/ml). Data represent the mean 6 SD of duplicated samples over the background levels of non-stimulated cells. Similar results were obtained in a second independent experiment. doi:10.1371/journal.pone.0019826.g004 control mice were reconstituted with human B (CD19+) cells by 10 weeks after HSC infusion (Fig. 5A). Although not statistically significant (p = 0.31), 25 weeks after infusion of HSC the overall frequency of blood circulating human B cells in DRAG mice was slightly lower than in control mice (Fig. 5B, Table S1). Noteworthy, spleens of DRAG mice contained similar numbers of human B cells as compared to control mice (B cell frequency: 47.9%, range [45.9–50] vs. 60% [55–65.9]; total number of B cells/spleen: 5.5610661.95 vs. 6.35610662.75, respectively). This indicated that expression of HLA-DR4 molecules in DRAG mice did not improve significantly the reconstitution with human B cells in the peripheral blood on in the spleen as compared to control mice. Human B cells developed by DRAG mice are functional and reconstitute plasma levels of all human immunoglobulin classes The major function of B cells is to secrete immunoglobulins (antibodies) that can be detected in blood and body fluids. The function of immunoglobulins is to neutralize and eliminate viruses, bacteria, and parasites as well as to control the homeostatic growth of commensal bacteria in the intestinal tract [24]. In mammalians, there are five classes of immunoglobulins, namely IgD, IgM, IgG, IgA, and IgE, from which IgM and IgG are the most abundant in blood. Previous studies in various humanized mouse models revealed an impaired function of human B cells reflected by poor reconstitution of human IgM serum levels and limited ability to undergo immunoglobulin class switch and to secrete immunoglobulins other than IgM [15,18,20–22]. Thus, we investigated the kinetics of human immunoglobulin reconstitution in blood of DRAG mice. As illustrated in figure 5C, the levels of human IgM in plasma from B cell reconstituted DRAG mice (n = 14) were significantly higher than those from reconstituted control mice (n = 9) (p = 0.0002). In addition, the DRAG mice were able to reconstitute plasma IgG whilst control mice failed to do so (Fig. 5D, Table S1).The results indicated that the mechanism of immunoglobulin class switch was preserved in DRAG mice. The human identity of Ig heavy and PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e19826