HLA Class II and Humanized Mice Figure 5. Kinetics of human B cell reconstitution in blood of DRAG and control mice. DRAG (n = 15) and control (n = 11) mice as in figure 1 were examined in peripheral blood by FACS using human CD19 Abs. The cut-off for positive human B cells was calculated as three times the standard deviation over the background of cells from na¨ıve (non-HSC infused) DRAG mice stained with CD19 antibodies (.0.18%). Panel A, percentage of human B cell reconstituted mice at various time points post-infusion of HSC. Fisher exact test indicated that the rate of human B-cell reconstitution in DRAG mice (14 out of 15) was similar to that of control mice (9 out 11) (p = 0.54). Panel B, frequency of human B cells in blood of reconstituted DRAG (n = 14) and control (n = 9) mice (p = 0.31, Mann-Whitney test). Panels C and D, plasma values of human IgM and IgG respectively in B-cell reconstituted DRAG (n = 14) and control (n = 9) mice at various time points post-infusion of HSC (p values 0.0002 and ,0.0001, Mann-Whitney test). Data represent mean values of mice analyzed individually. Ranges are indicated over the plots. doi:10.1371/journal.pone.0019826.g005 light chains in blood of DRAG mice was confirmed by immunoelectrophoresis (Fig. 6A). Human IgG class comprises four different subclasses that differ in structure and biological functions, namely (i) ability to cross placenta, (ii) ability to activate the complement cascade and (iii) affinity binding to Fc receptors expressed on phagocytic cells [37]. As illustrated in figure 6C, DRAG mice reconstituted plasma levels of all human IgG subclasses, with IgG2 as the most prevalent followed by IgG1, IgG3 and IgG4, whereas in the human blood the most prevalent subclass is IgG1, followed by IgG2, IgG3 and IgG4 [36]. In addition, 12 out of 14 (85.7%) DRAG mice reconstituted plasma levels of human IgA (Fig. 6D) and 7 out of 14 (50%) reconstituted plasma levels of human IgE (Fig. 6E). In contrast, control mice did not show detectable levels of human IgA or IgE (Fig. 6D&E, Table S1). The results demonstrated that human B cells developed by DRAG were functional as they secreted all human immunoglobulin classes, whereas those from control mice were impaired in their ability to undergo class switching and secreted only IgM. Human B cells develop by DRAG mice respond to tetanus toxoid immunization A major function of the immune system is to protect against infections by eliciting specific antibodies that bind to and eliminate pathogens. Tetanus toxoid, a formalin-detoxified form of Clostrid- ium tetani toxin adjuvanted in alumn, is a licensed human vaccine that induces neutralizing anti-tetanus toxin (TT) IgG antibodies. We investigated whether human B cells from DRAG mice could elicit specific humoral responses upon immunization with TT vaccine. Groups of HSC-infused DRAG and control mice were immunized with 1 flocculation unit of TT vaccine (Sanofi Pasteur) by the intramuscular route, and the titers of anti-TT IgG Abs were PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e19826