Figure 7. DRAG mice respond to tetanus toxoid (TT) vaccina- tion. Groups of DRAG and control mice (n = 3) were immunized intramuscularly with 1 flocculation unit of TT vaccine, and plasma levels of human anti-TT IgG titers were measured by ELISA at day 14 post- immunization. The frequency of human T and B cells in blood and plasma levels of human immunoglobulins prior to TT-immunization are shown in Table S1. doi:10.1371/journal.pone.0019826.g007 There is evidence indicating that administration of anti-MHC class II antibodies prevent bone marrow engraftment in dogs [41] and inhibit differentiation of cultured human HSCs in vitro [42]. Furthermore, human studies showed that HLA-DR compatibility increases the engraftment efficiency in bone marrow transplants [43]. As shown in our study, expression of HLA-DR4 molecules in the bone marrow of DRAG mice favored early engraftment and expansion of HLA-DR-matched HSC, while control mice lacking expression of HLA-DR4 and infused with the same HSCs failed to do so. The mechanisms by which HLA-DR4 expression in the mouse bone marrow environment promote early engraftment of human HSC remains to be investigated. Our results also argue against the hypothesis that the adult mouse thymus, which is characterized by a loss of the structural and hormonal milieu, is unable to sustain development and differentiation of human T cells [19]. This is because the adult thymus of HSC-infused DRAG mice allowed engraftment and development of a large number of human thymocytes and exported mature CD4 T cells to periphery. Of note, the low rate of human T cell reconstitution in our control (NOD.Rag1- KO.IL2RccKO) mice (36%) was similar to that reported in studies using NOD.scid.IL2RccKO mice [18,19], which further validates the superiority of DRAG mice for reconstitution of human T cells. Almost all DRAG mice (14 out of 15) developed human CD8 T cells as compared to only 4 out 11 control mice. This was an interesting finding, since DRAG mice do not express HLA class I molecules, the restriction element for thymic differentiation of human CD8 T cells. Recently, NOD.scid.IL2RccKO mice expressing HLA class I (A2) molecules have been generated [27– 29]. Interestingly enough, the transgenic expression of HLA-A2 molecules did not improve the rates of human CD8 T cell reconstitution as compared to non-transgenic littermates, though the human CD8 T cells developed by some of the transgenic mice were restricted by HLA-A2 molecules. Thus, expression of HLA class I molecules does not suffice for promoting engraftment of pro-T cells derived from bone marrow and efficient development of human T cells, whilst expression of HLA class II does. The superiority of DRAG mice in developing human CD8 T cells as HLA Class II and Humanized Mice compared to our control mice and HLA-A2 Tg mice [27–29] suggests the possibility that human CD8 T cells developed by DRAG could have been educated and restricted by HLA class II molecules. This remains to be further investigated. Secondly, human T cells developed by DRAG mice were as functional as those from healthy human volunteers, since they vigorously responded to CD3/CD28 cross-linking and PMA/ ionomycin stimulation by secretion of human Th1 and Th2 cytokines (IL-2, IFNc, TNFa, IL-6, IL-5, and IL-4), whereas human T cells from control mice failed to do so. Our results in control mice are in agreement with numerous studies on NOD.scid.IL2RccKO mice showing human T cell functional impairment [15,18,20–22]. CD3/CD28 Abs stimulate T cells by crosslinking TCR and CD28 co-receptor followed by activation of ZAP70 intracellular signaling pathway that ultimately leads to the transcriptional activation of genes involved in T cell differentiation and cytokine secretion [44]. On the other hand, PMA/ionomycin stimulates T cells by inducing calcium influx, thereby bypassing TCR signaling pathways [45]. Our results clearly indicated that expression of HLA-DR4 molecules in DRAG mice promoted the development of human CD4 and CD8 T cells with preserved integrity of both TCR and non-TCR signaling pathways. It has been suggested that low frequency and functional unresponsiveness of human T cells from humanized IL-2RccKO genetic stocks lacking expression of HLA class II molecules maybe due to a high level of FAS receptor (CD95) expression on T cells, thereby increasing susceptibility to T cell death [18]. We found that the level of CD95 expression on reconstituted human T cells from DRAG and control mice was similar (MFI CD3 T cells: 20.5561.06 vs. 18.460.42; CD4 T cells 2261.55 vs. 19.160.98; CD8 T cells: 19.260.98 vs. 15.961.94, respectively). Therefore, it is unlikely that Fas-FasL induced apoptosis plays an important role in elimination of mature T cells, which might account for the low rates of T cell reconstitution and T cell numbers in our control mice or other similar humanized mouse strains lacking expression of HLA-DR4 molecules. Rather, the low human T cell reconstitution rate in our control mice is attributed to inefficient engraftment of pro-T cells derived from bone marrow and poor thymic selection of human thymocytes. Thirdly, though expression of HLA-DR4 molecules in DRAG mice did not affect the reconstitution levels with human B cells as compared to control mice, the B cells developed by DRAG mice were functional and able to reconstitute serum levels of human IgM comparable to those in human sera, whereas the levels of human IgM in control mice were significantly lower (p = 0.0002). More importantly, the human B cells from DRAG mice were able to undergo immunoglobulin class switching and to secrete IgG, IgA, and IgE. In contrast, the B cells from our control mice, and other similar genetic stocks [15,18,20–22] could only secrete low levels of human IgM and were impaired for undergoing immunoglobulin class switching. Two categories of antigens can activate B cells, and they are defined as T-cell independent or T-cell dependent antigens based on their ability to induce antibodies in athymic (nude) mice that lack mature T cells [24]. T-cell independent antigens are polyclonal B cell activators, such as bacterial polysaccharides and other components of the bacteria cell wall that act mainly through toll-like-receptors (TLR) rather than through the B cell receptor (surface Ig). Humoral responses to T-cell independent antigens are mainly of IgM class as they do not required CD4 T cell help. On the other hand, most antigens are T-cell dependent, meaning that CD4 T cell help is required for immunoglobulin class switching and antibody production. Unlike the T-cell independent antigens, activation of B cells by T-cell dependent PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e19826