Expression of HLA Class II Molecules in Humanized NOD.Rag1KO.IL2RgcKO Mice Is Critical for Development and Function of Human T and B Cells Rebecca Danner1, Snehal N. Chaudhari1, John Rosenberger1, Jacqueline Surls2, Thomas L. Richie1, Teodor-Doru Brumeanu2, Sofia Casares1,2* 1 US Military Malaria Vaccine Program, Naval Medical Research Center/Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America, 2 Department of Medicine, Uniformed Services University of Health Sciences, Bethesda, Maryland, United States of America Abstract Background: Humanized mice able to reconstitute a surrogate human immune system (HIS) can be used for studies on human immunology and may provide a predictive preclinical model for human vaccines prior to clinical trials. However, current humanized mouse models show sub-optimal human T cell reconstitution and limited ability to support immunoglobulin class switching by human B cells. This limitation has been attributed to the lack of expression of Human Leukocyte Antigens (HLA) molecules in mouse lymphoid organs. Recently, humanized mice expressing HLA class I molecules have been generated but showed little improvement in human T cell reconstitution and function of T and B cells. Methods: We have generated NOD.Rag1KO.IL2RccKO mice expressing HLA class II (HLA-DR4) molecules under the I-Ed promoter that were infused as adults with HLA-DR-matched human hematopoietic stem cells (HSC). Littermates lacking expression of HLA-DR4 molecules were used as control. Results: HSC-infused HLA-DR4.NOD.Rag1KO.IL-2RccKO mice developed a very high reconstitution rate (.90%) with long- lived and functional human T and B cells. Unlike previous humanized mouse models reported in the literature and our control mice, the HLA-DR4 expressing mice reconstituted serum levels (natural antibodies) of human IgM, IgG (all four subclasses), IgA, and IgE comparable to humans, and elicited high titers of specific human IgG antibodies upon tetanus toxoid vaccination. Conclusions: Our study demonstrates the critical role of HLA class II molecules for development of functional human T cells able to support immunoglobulin class switching and efficiently respond to vaccination. Citation: Danner R, Chaudhari SN, Rosenberger J, Surls J, Richie TL, et al. (2011) Expression of HLA Class II Molecules in Humanized NOD.Rag1KO.IL2RgcKO Mice Is Critical for Development and Function of Human T and B Cells. PLoS ONE 6(5): e19826. doi:10.1371/journal.pone.0019826 Editor: Lucienne Chatenoud, Universite´ Paris Descartes, France Received December 8, 2010; Accepted April 18, 2011; Published May 17, 2011 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by grants from NIH/NIDDK, Juvenile Diabetes Research Foundation, Military Infectious Diseases Research Program, and USAID to SC and a USUHS grant to TBD. This work was supported by work unit number 6000.RAD1.F. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sofia.casares@med.navy.mil Introduction Early models of humanized mice used for HIS reconstitution were generated in the late 1980s by infusion of human peripheral blood mononuclear cells into T- and B-cell deficient rodents such as scid mice [1–5], a strain derived from CB.17 mice that bears a mutation in the protein kinase, DNA-activated catalytic polypep- tide (prkdc) [6]. The level of reconstitution in scid mice was however low (,1%) and consisted mainly of human T cells, as very few human B cells were able to survive. These humanized mice also faced the challenge of human T cell functional impairment upon vaccination, and in some cases development of graft versus host disease (GVHD) as a consequence of human T cell reactivity against the mouse MHC antigens [4,5]. To increase the level of human cell reconstitution and to overcome GVHD, investigators used scid mice infused with human hematopoietic stem cells (HSC) [3,7,8]. This model known as Hu- HSC-scid was able to develop human B and myeloid cells but the life-span of these human cells was relatively short, and the model also failed to develop critical compartments of the human immune system such as T cells. This limitation was mostly attributed to the destructive activity of mouse natural killer (NK) cells, since the development of mouse NK cells is not affected by the scid mutation [6]. Inbreeding the scid mutation into the non-obese diabetic (NOD) mouse increased the rate of human cell reconstitution upon HSC infusion due to the reduced number and functional impairment of NK cells in the NOD mouse [9,10]. Yet, the NOD.scid mouse model still did not support high rates or long- lasting reconstitution with human immune cells, nor did it allow development of functional human T and B cells [11]. The generation of mice bearing a mutation in the IL2R gamma chain (IL2Rcc) was a major breakthrough in the field, as this strain is devoid of NK cells [12]. Genetic stocks of NOD.scid.IL2RccKO, NOD.RagKO.IL2RccKO, and BALB/c.RagKO.IL2RccKO mice PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e19826