HLA Class II and Humanized Mice Figure 6. B cells from DRAG mice undergo immunoglobulin class switching. Panels A and B, immunoelectrophoresis of plasma proteins from DRAG mice at 6 months post-infusion of HSC or prior to infusion with HSC, respectively. Panel C, percentage of human IgG subclasses within the total human IgG fraction in plasma of six DRAG mice examined. Panel D, 12 out 14 (85.7%) DRAG mice that showed human B cells in blood, reconstituted human IgA in plasma. Data present mean values of mice analyzed individually. Panel E, 7 out of 14 (50%) DRAG mice also reconstituted human IgE in plasma. No human IgA or IgE was detected in plasma of control mice (p values ,0.0001 and 0.015, respectively by Mann-Whitney test). doi:10.1371/journal.pone.0019826.g006 measured by ELISA 14 days later. As illustrated in figure 7, immunized-DRAG mice were able to elicit anti-TT IgG antibodies, whereas the control mice failed to do so. These results clearly indicated that human B cells developed by DRAG mice are fully functional. In addition, the results demonstrated that the ‘‘human immune system’’ developed by DRAG mice is superior to that developed by BLT mice, currently referred as the golden standard for humanized mice, since the BLT mice cannot elicit specific IgG antibodies upon TT immunization [26]. Discussion The critical role of thymus in T cell development was revealed in the 1930s when a line derived from CB.17 mice, nicknamed ‘‘nude’’ mice due to alopecia, was found athymic and unable to repopulate peripheral lymphoid organs with mature T cells [38]. Subsequent studies demonstrated that surgical removal of the thymus in neonatal mice reduced the numbers of mature T cells in the periphery as well [39]. These studies also demonstrated the critical role of T cells in commanding humoral responses, since both ‘‘nude’’ and thymectomized neonatal mice are impaired in eliciting antibodies, though they develop normal numbers of B cells. Studies by Zinkernagel and others further demonstrated a critical role of MHC molecules in the development and function of T cells through a process of thymic selection [40]. In this study, we demonstrated that transgenic expression of HLA class II (DR4) molecules in NOD.Rag1KO.IL2RccKO mice rescues the devel- opment and function of human T and B cells upon infusion of adult mice with HSC from HLA-DR-matched umbilical cord bloods. The rates of T cell reconstitution and frequencies of human CD4 T cells in DRAG mice were significantly superior to those in control NOD.Rag1KO.IL2RccKO mice (lacking HLA- DR4 molecules) that were infused with HSC from the same donors. The significant difference between DRAG and control mice was solely associated with expression of HLA-DR4 molecules. The HLA-DR4 molecules thus promoted (i) efficient human HSC engraftment in bone marrow, (ii) thymic develop- ment of bone marrow-derived human pro-T cells, (iii) proficient thymic selection of human thymocytes, and (iv) export of high numbers of terminally differentiated human CD4 T cells and Tregs to the periphery. PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e19826