antigens requires two signals, the first from interaction of antigen with the B cell receptor and the second from soluble factors secreted by CD4 T cells, such as IL-4, IL-6, or IL-21 [46–48]. The role of these cytokines in promoting B cell activation and immunoglobulin class switching has been demonstrated in IL- 21R/IL-4 double deficient mice, which can only secrete IgM [49], and also in IL-6 transgenic mice that develop plasmocytosis and hyper-IgG globulinemia [50]. The ability of DRAG mice to generate functional human T cells able to secrete cytokines required for immunoglobulin class switching may account for the development of functional human B cells that reconstituted serum levels of human natural antibodies of all classes and subclasses. Importantly enough, our study also demonstrated that the human immune system developed by DRAG mice elicited specific IgG antibodies upon immunization with tetanus toxoid vaccine. Our vaccinated-control mice were however unable to do so, and this is in agreement with previous studies in IL2RccKO genetic stocks lacking HLA class II molecules that were also severely impaired for eliciting human IgG responses to vaccination [15,20–22,26–28]. In summary, our results demonstrated that expression of HLA class II molecules in humanized mice is critical for development of functional human T and B cells able to efficiently respond to vaccination. DRAG mice represent a new in vivo model for studies on human T and B cell development and function, vaccine testing, and generation of human IgG monoclonal antibodies for prophylactic and/or therapeutic use. Materials and Methods Ethics Statement All animal procedures reported herein were conducted under IACUC protocols (ID numbers: D03-07, D06-07 and D07-10) approved by WRAIR/NMRC and in compliance with the Animal Welfare Act and in accordance with the principles set forth in the ‘‘Guide for the Care and Use of Laboratory Animals,’’ Institute of Laboratory Animals Resources, National Research Council, National Academy Press, 1996. The protocol for human use was approved by the Naval Medical Research Center Institutional Review Board in compliance with all applicable Federal regulations governing protection of human subjects. De-identified human blood samples were obtained through commercial vendors. DRAG mice C57BL/6 mice expressing transgenically HLA-DR4 (HLA- DR*0401/I-Ed) molecules under the mouse I-Ed promoter were purchased from Taconic (Hudson, NY) and backcrossed with NOD.Rag1KO mice (The Jackson Labs, Bar Harbor, Maine) for 12 generations. Litters from F2-F12 generation were screened for HLA-DR4/I-Edab transgenes by PCR using specific primers for HLA-DR4a (forward, AATCCTGACCAATCAGGCGAG, re- verse, GCGGAAGAGGTGATCGTCCCT) and HLA-DR4b (forward, GTTTCTTGGAGCAGGTTAAAC; reverse, CTGC- ACTGTGAAGCTCTCAC) transgenes. Rag1KO litters were identified by the absence of CD3+ T cells upon staining of peripheral blood cells with anti-mouse CD3 (Beckton Dickinson, San Jose, CA). The level of inbreeding of F12 mice in NOD background was 100% as determined by microsatellite analysis (Mappairs, Diversified Biopharma Solutions Inc, Loma Linda, CA). F12: HLA-DR4 Tg. NOD.Rag1KO males were crossed with NOD.Rag1KO.IL2RccKO females (The Jackson Laboratories) and male progeny was screened for HLA-DR4 transgenic (the IL2Rcc gene is located in the X chromosome). Positive males were crossed with NOD.Rag1KO.IL2RccKO females and all progeny was screened for HLA-DR4ab transgenes. The HLA-DR4 (+/2) HLA Class II and Humanized Mice Tg.NOD.Rag1KO.IL2RccKO (referred as DRAG) mice were maintained in the pathogen-free WRAIR Veterinary Medicine Facility. Infusion of mice with HSC Umbilical cord bloods (UCB) were purchased from AllCells (Emeryville,CA) and Promocell (Heidelberg, Germany). Screening for HLA-DRB1*0401 allele was carried out using DRB1*04 SSP Unitray (Invitrogen, San Diego, CA). HLA-DRB1*0401-positive cord bloods were enriched for CD34+ stem cells using EasySep, Human Progenitor Cell Enrichment kit (StemCell Technologies, Vancouver, BC). Prior to enrichment, the frequency of CD34+ cells in UCBs was 1.060.5% and after enrichment 62.068.6%. Mice were irradiated (350 rads), and injected with 86104 CD34+- enriched HSC cells by the intravenous route. For longitudinal analyses, we used groups of DRAG mice (n = 15) and control littermates (n = 11) that were infused with HLA-DR4-positive cord bloods from the same donors. For cross-sectional analyses we used additional groups of mice that were euthanized at 2 months or 6 months post-infusion of HSCs. FACS analysis Blood drawn for the tail vein (20 ml) was collected with heparin- coated capillary tubes (Fisher Scientific, Pittsburgh, PA). Erythro- cytes were lysed using ACK lysis buffer (InVitrogen) and white blood cells were incubated with Fcblock (BD Biosciences, San Jose, CA) for 10 minutes at 4uC, and then stained with anti-human CD3, CD4, CD8, and CD19Abs (BD Biosciences) for 30 minutes at 4uC, washed with PBS/1% BSA/0.1% Na azide and analyzed by FACS on the mononuclear FSC/SSC scatter. As control, we used human peripheral blood from adult healthy volunteers that was collected in heparin-coated tubes and treated as above. Mouse splenic and thymic cells were obtained as described [31,33,51] and bone marrow cells were obtained by flushing both tibias. Cell populations were quantified by FACS as above using anti-human CD3, CD4, CD8, CD19, CD45, CD34, CD95, CD11c, CD16, HLA-DR (BD Biosciences) and FOXP3 (eBiosiences, San Diego, CA). T cell responses Splenic cells (36105) from mice or PBMCs (36105) from volunteers were stimulated for 24 h with anti-human CD3/CD28- coated magnetic beads (InVitrogen), 1 mg/ml PMA plus 50 nM iomycin (Sigma), or left unstimulated. Cytokines in supernatants were measured by Luminex (InVitrogen). Cell depletion and isolation Spleen cells adjusted to 36106 cells/ml were depleted of CD4 T cells, CD8 T cells, or both cell subsets with antibody-coated magnetic beads (Dynabeads, InVitrogen). The efficiency of cell depletion was .95% as measured by FACS using CD3, CD4, and CD8 Abs. Equal volumes (0.1 ml) of cell depleted cell suspensions or corresponding volumes of positively isolated cells on the magnetic beads were stimulated with PMA/iomycin for 24 h and cytokine secretion in cell culture supernatants were measured by Luminex (InVitrogen). ELISA Peripheral blood from mice or humans was collected in heparin- coated capillaries and plasma samples were obtained by removal of cells by centrifugation. Total plasma immunoglobulins (IgM, IgG, IgA and IgE) were measured by ELISA Quantitation Sets (Bethyl Labs, Montgomery, Texas). Human IgG subclasses were PLoS ONE | www.plosone.org 10 May 2011 | Volume 6 | Issue 5 | e19826