measured using human IgG Subclass Profile ELISA kits (InVitro- gen). To determine titers of anti-TT IgG titers, ninety-six-well plates were coated with 10 mg/ml of tetanus toxin (Calbiochem, La Jolla, CA) in carbonate buffer for 2 h at RT. Plates were washed (ELISA Wash Solution, Bethyl Labs), blocked (ELISA Blocking solution, Bethyl Labs) for 30 min at RT, washed again, and incubated with plasma samples at serial half dilutions starting at 1:50. After 1 h incubation at RT, plates were washed and incubated with anti-human IgG-HRP Abs (Bethyl Labs) for 1 h at RT, washed, and revealed with Enzyme substrate (TMB, Bethyl Labs) in dark for 15 minutes. The enzymatic reaction was stopped (ELISA stop solution, Bethyl Labs) and plates were immediately read at 450 nm. After plotting the OD values against plasma dilution, the titers were calculated as the prior greatest dilution over a two-fold standard deviation over the background. Immunoelectrophoresis Visualization of human immunoglobulin classes and immuno- globulin light (kappa and lambda) chains was carried out using IEF kits (Helena Labs, Beaumont, TX). Immunization with tetanus toxoid Tetanus toxid vaccine was obtained from the Naval Hospital, Bethesda, MD. Mice were injected intramuscularly with 1 floculation unit (fl). Statistical analysis Comparison of cell reconstitution rates was carried out using Fisher’s exact test. Comparison of cell frequencies and level of immunoglobulins was determined by Mann-Whitney test. For analysis of low number of samples we used the Independent Samples t-test. References 1. Torbett BE, Picchio G, Mosier DE (1991) Hu-PBL-SCID mice: a model for human immune function, AIDS, and lymphomagenesis. Immunol Rev 124: 139–164. 2. Mosier DE, Gulizia RJ, Baird SM, Wilson DB (1988) Transfer of a functional human immune system to mice with severe combined immunodeficiency. Nature 335: 256–259. 3. McCune JM, Namikawa R, Kaneshima H, Shultz LD, Lieberman M, et al. (1988) The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Science 241: 1632–1639. 4. Pflumio F, Lapidot T, Murdoch B, Patterson B, Dick JE (1993) Engraftment of human lymphoid cells into newborn SCID mice leads to graft-versus-host disease. Int Immunol 5: 1509–1522. 5. Huppes W, De Geus B, Zurcher C, Van Bekkum DW (1992) Acute human vs. mouse graft vs. host disease in normal and immunodeficient mice. Eur J Immunol 22: 197–206. 6. Bosma GC, Custer CR, Bosma MJ (1983) A severe combined immunodeficiency mutation in the mouse. Nature 301: 527–530. 7. Namikawa R, Weilbaecher KN, Kaneshima H, Yee EJ, McCune JM (1990) Long-term human hematopoiesis in the SCID-hu mouse. J Exp Med 172: 1055–1063. 8. Kyoizumi S, Baum CM, Kaneshima H, McCune JM, Yee EJ, et al. (1992) Implantation and maintenance of functional human bone marrow in SCID-hu mice. Blood 79: 1704–1711. 9. Shultz LD, Schweitzer PA, Christianson SW, Gott B, Schweitzer IB, et al. (1995) Multiple defects in innate and adaptive immunologic function in NOD/LtSz- SCID mice. J Immunol 154: 180–191. 10. Hesselton RM, Greiner DL, Mordes JP, Rajan TV, Sullivan JL, et al. (1995) High levels of human peripheral blood mononuclear cell engraftment and enhanced susceptibility to human immunodeficiency virus type 1 infection in NOD/LtSzscid/scid mice. J Infect Dis 172: 974–982. 11. Larochelle A, Vormoor J, Hanenberg H, Wang JC, Bhatia M, et al. (1996) Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med 2: 1329–1337. 12. Cao X, Shores EW, Hu-Li J, Anver MR, Kelsall BL, et al. (1995) Defective lymphoid development in mice lacking expression of the common cytokine receptor g chain. Immunity 2: 223–238. HLA Class II and Humanized Mice Supporting Information Table S1 Human reconstitution in blood of DRAG and control mice upon infusion of HLA-DR*0401 hemato- poietic stem cells. (*) human cell frequencies measured at 25 # weeks post-infusion of HSC. ( ) concentration of plasma levels of human IgM and IgG (32 weeks post-infusion of HSC) and IgA and & IgE (25 weeks post-infusion of HSC). ( ) mice were immunized with TT at week 31 post-infusion of HSC. (ND) not done. (DOCX) Acknowledgments We thank to Margaret Kehl for performing Luminex assays, to Hernan- Antonio Flores and Tesfaye Mekonnen for animal care, Grace Aaron for providing the TT vaccine, and Mr. Shi Meng and Ms. Cara Olsen for statistical analyses. SC and TDB are US Government employees and TLR is military service member. The work of these individuals was prepared as part of official government duties. Title 17 U.S.C. 1105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. 1101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties. The views expressed are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. Author Contributions Conceived and designed the experiments: SC TDB. Performed the experiments: RD JR SNC JS TDB SC. Analyzed the data: RD JR SNC JS TDB SC. Wrote the paper: SC. Commented on and edited the manuscript: TDB TLR. 13. Ito M, Hiramatsu H, Kobayashi K, Suzue K, Kawahata M, et al. (2002) NOD/ SCID/c(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood 100: 3175–3182. 14. Yahata T, Ando K, Nakamura Y, Ueyama Y, Shimamura K, et al. (2002) Functional human T lymphocyte development from cord blood CD34+ cells in nonobese diabetic/Shi-SCID, IL-2 receptor c mice. J Immunol 169: 204–209. 15. Traggiai E, Chicha L, Mazzucchelli L, Bronz L, Piffaretti JC, et al. (2004) Development of a human adaptive immune system in cord blood cell- transplanted mice. Science 304: 104–107. 16. Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, et al. (2005) Human lymphoid and myeloid cell development in NOD/LtSz-SCID IL2Rc mice engrafted with mobilized human hemopoietic stem cells. J Immunol 174: 6477–6489. 17. Legrand N, Weijer K, Spits H (2006) Experimental models to study development and function of the human immune system in vivo. J Immunol 176: 2053–2058. 18. Watanabe Y, Takahashi T, Okajima A, Shiokawa M, Ishii N, et al. (2009) The analysis of the functions of human B and T cells in humanized NOD/shi-scid/ gcnull (NOG) mice (hu-HSC NOG mice). Int Immunol 21: 843–858. 19. Brehm MA, Cuthbert A, Yang C, Miller DM, DiIorio P, et al. (2010) Parameters for establishing humanized mouse models to study human immunity: analysis of human hematopoietic stem cells engraftment in 3 immunodeficient strains of mice bearing the IL2Rgamma(null) mutation. Clin Immunol 135: 84–98. 20. Ishikawa F, Yasukawa M, Lyons B, Yoshida S, Miyamoto T, et al. (2005) Development of functional human blood and immune systems in NOD/SCID/ IL2 receptor {gamma} chain(null) mice. Blood 106: 1565–1573. 21. Matsumura T, Kametani Y, Ando K, Hirano Y, Katano I, et al. (2003) Functional CD5+ B cells develop predominantly in the spleen of NOD/SCID/ gammac(null) (NOG) mice transplanted either with human umbilical cord blood, bone marrow, or mobilized peripheral blood CD34+ cells. Exp Hematol 31: 789–797. 22. Baenziger S, Tussiwand R, Schlaepfer E, Mazzucchelli L, Heikenwalder M, et al. (2006) Disseminated and sustained HIV infection in CD34+ cord blood cell-transplanted Rag22/2gamma c2/2 mice. Proc Natl Acad Sci USA 103: 15951–15956. PLoS ONE | www.plosone.org 11 May 2011 | Volume 6 | Issue 5 | e19826