Type 1 Diabetes Induction in Humanized Mice
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Type 1 diabetes induction in humanized mice Shulian Tana,b,c,d, Yang Lia,b,c,d, Jinxing Xiad, Chun-Hui Jina,d, Zheng Hua,b,c, Gaby Duinkerkene, Yuying Lia,d, Mohsen Khosravi Maharlooeid, Estefania Chavezd, Grace Naumand, Nichole Danzld, Maki Nakayamaf, Bart O. Roepe,g, Megan Sykesd, and Yong-Guang Yanga,b,c,d,1 aThe First Hospital, Jilin University, Changchun, China 130061; bInternational Center of Future Science, Jilin University, Changchun, China 130012; cInstitute of Immunology, Jilin University, Changchun, China 130061; dColumbia Center for Translational Immunology, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032; eDepartment of Immunohaematology & Blood Transfusion, Leiden University Medical Center, 2300 RC Leiden, The Netherlands; fBarbara Davis Center for Childhood Diabetes, University of Colorado School of Medicine, Aurora, CO 80045; and gDepartment of Diabetes Immunology, Diabetes & Metabolism Research Institute at the Beckman Research Institute, City of Hope, Duarte, CA 91010 Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved August 11, 2017 (received for review June 8, 2017) There is an urgent and unmet need for humanized in vivo models T cells engineered with a TCR that recognizes the HLA-DQ8– of type 1 diabetes to study immunopathogenesis and immuno- restricted insulin B chain peptide consisting of amino acids 9–23 therapy, and in particular antigen-specific therapy. Transfer of (InsB:9–23). InsB:9–23 is a dominant MHC class II-restricted antigen patient blood lymphocytes to immunodeficient mice is associated recognized by islet-infiltrating insulin-specific T cells and serves as an with xenogeneic graft-versus-host reactivity that complicates essential target of the immune destruction of pancreatic β cells in assessment of autoimmunity. Improved models could identify nonobese diabetic (NOD) mice (6, 7). Previous observations sug- which human T cells initiate and participate in beta-cell destruction gested this epitope may also serve as a key autoantigenic target in and help define critical target islet autoantigens. We used humans, as it does in mice. HLA class II-restricted T-cell response to humanized mice (hu-mice) containing robust human immune InsB:9–23 peptide is highly associated with T1D in humans (8). A repertoires lacking xenogeneic graft-versus-host reactivity to recent study using HLA-DQ8/B:11–23R22E tetramers further con- address this question. Hu-mice constructed by transplantation of firmed the presence of CD4 T cells recognizing the HLA-DQ8– + + – HLA-DQ8 human fetal thymus and CD34 cells into HLA-DQ8 restricted B:11–23 peptide in patients with T1D (9). More recently, transgenic immunodeficient mice developed hyperglycemia and 2.35% of CD4 T-cell clones isolated from inflamed islets of patients diabetes after transfer of autologous HLA-DQ8/insulin-B:9–23 – – + with T1D were found to recognize InsB:9 23 (10). However, there (InsB:9 23)-specific T-cell receptor (TCR)-expressing human CD4 – – MEDICAL SCIENCES – has been no direct evidence for human InsB:9 23-reactive T-cell T cells and immunization with InsB:9 23. Survival of the infused β human T cells depended on the preexisting autologous human mediated in vivo destruction of pancreatic cells in humans. + Our data showed that adoptive transfer of HLA-DQ8–restricted immune system, and pancreatic infiltration by human CD3 – T cells and insulitis were observed in the diabetic hu-mice, pro- InsB:9 23-specific human CD4 T cells is capable of inducing di- – vided their islets were stressed by streptozotocin. This study fits abetes in HLA-DQ8 Tg hu-mice, consistent with the potential of – Koch’s postulate for pathogenicity, demonstrating a pathogenic T-cell responses to the InsB:9 23 epitope to initiate T1D in humans. + role of islet autoreactive CD4 T-cell responses in type 1 diabetes Results induction in humans, underscores the role of the target beta-cells α β in their immunological fate, and demonstrates the capacity to ini- The TCR (Vα21)and (Vβ11) chain cDNA was extracted from an – tiate disease with T cells, recognizing the InsB:9–23 epitope in the InsB:9 23-specific human T-cell line (clone #5), which was estab- presence of islet inflammation. This preclinical model has the po- lished from blood from an 18-y-old Caucasoid HLA-DQ8 homozy- tential to be used in studies of the pathogenesis of type 1 diabetes gous man diagnosed with T1D at the age of 8 y (11, 12), and linked and for testing of clinically relevant therapeutic interventions. by a P2A self-cleaving peptide gene. The TCRα-P2A-β gene frag- ment was then linked to a F2A-AcGFP gene fragment, and then type 1 diabetes | insulin | humanized mice cloned into a lentiviral vector (LV-insTCR; Fig. S1). To assess the ype 1 diabetes mellitus (T1D) is an autoimmune disease Significance Tresulting from immune destruction of insulin-producing β cells in the pancreatic islets. Autoreactive T cells recognizing Type 1 diabetes (T1D) is known to be caused by immune de- β-cell antigens (e.g., insulin) are believed to play a critical role in struction of insulin-producing β cells, but the disease pathogenesis disease onset and progression, but direct evidence for this is still remains poorly understood largely because of limitations in ani- lacking (1). The loss of β cells and the subsequent reduction or mal models to study the immunopathology. Here we established lack of insulin result in chronic elevation of blood glucose levels, a humanized mouse T1D model, in which diabetes is driven by leading to severe complications such as heart disease, stroke, and human T cells recognizing the HLA-DQ8–restricted insulin B chain kidney failure. Patients with T1D rely on exogenous insulin to peptide consisting of amino acids 9–23 (InsB:9–23). This study not control the disease (2), but insulin therapy does not eliminate the only demonstrates the capacity of InsB:9–23-specific human CD4 risk for T1D complications and may be associated with life- T cells to initiate diabetes but also provides a preclinical threatening hypoglycemia (3). Thus, new therapies are urgently humanized mouse model that has the potential to be used in needed for T1D treatment. Recent insight into the human dis- studies of the immunopathogenesis and immunotherapy of T1D. ease lesion in the pancreas indicated profound differences in immunopathology between patients with T1D and the nonobese Author contributions: S.T., Yang Li, J.X., Z.H., and Y.-G.Y. designed research; S.T., Yang Li, J.X., C.-H.J., G.D., Yuying Li, M.K.M., E.C., G.N., N.D., and M.N. performed research; G.D., diabetic mouse model that is regarded as the nearest preclinical M.N., and B.O.R. contributed new reagents/analytic tools; S.T., Yang Li, J.X., C.-H.J., Z.H., model of autoimmune diabetes (4). These observations, com- M.K.M., B.O.R., M.S., and Y.-G.Y. analyzed data; and S.T., Z.H., B.O.R., M.S., and Y.-G.Y. bined with a range of other differences in the immune systems of wrote the paper. humans versus mice, prompted us to design preclinical models in The authors declare no conflict of interest. which the immune system is humanized to study both disease This article is a PNAS Direct Submission. mechanisms and immunotherapeutic strategies (5). 1To whom correspondence should be addressed. Email: [email protected]. In this study, we sought to overcome this issue, using humanized This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mice (hu-mice), by infusing autologous hu-mouse–derived human 1073/pnas.1710415114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1710415114 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 ABNaïve human CD3+CD4+ cells ‘Donor’ 15-17 W hu-mouse Spleen HLA-DQ8+ InsB:9-23 TCR FTHY Lentiviruses CD34+ FLCs 10-14d ‘Recipient’ ~14 W hu-mouse i.v. STZ; B:9-23/CFA Fig. 1. Preparation of hu-mice for generating InsB:9–23-specific T cells and for induction of diabetes. (A) Schematic showing preparation of both donor hu- + + mice with HLA-DQ8 human immune reconstitution, from which splenic human CD4 -naive T cells are prepared, transduced with LV-insTCR, expanded, and injected into recipient hu-mice for diabetes induction, and recipient HLA-DQ8–Tg hu-mice reconstituted with human immune cells autologous to the donor + hu-mice, which are used as the recipients of LV-insTCR–transduced CD4 T cells. (B). Levels (mean ± SEM) of human hematologic and immune cell chimerism in PBMCs of hu-mice from a representative experiment. efficacy of cell surface TCR expression of the engineered human T cells in PBMCs from a representative experiment, in which the + T cells, we measured TCR expression on the TCR-negative human level of human CD3 T cells in all hu-mice examined was greater cell line J.RT3-T3.5 cells after transduction with LV-insTCR. Flow than 30% in PBMCs by 15 wk after humanization. + cytometric (FCM) analysis revealed that all transduced, that is, To collect human CD4 T cells for genetic engineering, spleens + GFP , J.RT3-T3.5, cells gained cell surface expression of human were harvested from hu-mice between 15 and 17 wk after human- + CD3 (Fig. S2A)andαβTCR (Fig. S2B). All GFP cells were also ization. After confirming high levels of human T-cell reconstitution A + stained positively for human TCRvβ11, further confirming the ex- by FCM analysis of PBMCs and spleen cells (Fig. 2 ), human CD4 - pression of the engineered B:9–23-specific TCR on the transduced naive T cells were enriched by depletion of mouse cells and human + + + + + cells (Fig.