Type 1 diabetes induction in humanized mice

Abstract

There is an urgent and unmet need for humanized in vivo models of type 1 diabetes to study immunopathogenesis and immunotherapy, and in particular antigen-specific therapy. Transfer of patient blood lymphocytes to immunodeficient mice is associated with xenogeneic graft-versus-host reactivity that complicates assessment of autoimmunity. Improved models could identify which human T cells initiate and participate in beta-cell destruction and help define critical target islet autoantigens. We used humanized mice (hu-mice) containing robust human immune repertoires lacking xenogeneic graft-versus-host reactivity to address this question. Hu-mice constructed by transplantation of HLA-DQ8⁺ human fetal thymus and CD34⁺ cells into HLA-DQ8-transgenic immunodeficient mice developed hyperglycemia and diabetes after transfer of autologous HLA-DQ8/insulin-B:9-23 (InsB:9-23)-specific T-cell receptor (TCR)-expressing human CD4⁺ T cells and immunization with InsB:9-23. Survival of the infused human T cells depended on the preexisting autologous human immune system, and pancreatic infiltration by human CD3⁺ T cells and insulitis were observed in the diabetic hu-mice, provided their islets were stressed by streptozotocin. This study fits Koch's postulate for pathogenicity, demonstrating a pathogenic role of islet autoreactive CD4⁺ T-cell responses in type 1 diabetes induction in humans, underscores the role of the target beta-cells in their immunological fate, and demonstrates the capacity to initiate disease with T cells, recognizing the InsB:9-23 epitope in the presence of islet inflammation. This preclinical model has the potential to be used in studies of the pathogenesis of type 1 diabetes and for testing of clinically relevant therapeutic interventions.

Keywords: humanized mice; insulin; type 1 diabetes.

Fig. S1.

Schematic of the LV-insTCR vector. Shown is the B:9–23-specific TCR-AcGFP fragment cloned into the pRRLSIN lentiviral vector.

Fig. S2.

Transgenic TCR expression in lentivirally transduced human TCR-deficient T-cell line cells. J.RT3-T3.5 cells were transduced with LV-insTCR lentiviruses (at a multiplicity of infection = 25). Shown are representative FCM profiles of human CD3 (A), TCRαβ (B), and TCRv_β11 (C) expression on nontransduced control and virally transduced (GFP⁺) cells.

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.

Fig. 2.

Purification and lentiviral transduction of hu-mouse–derived human CD3⁺CD4⁺CD45RO⁻CD25⁻-naive T cells. Spleen cells were prepared from donor hu-mice between 15 and 17 wk after humanization, as indicated in Fig. 1A, and human CD4⁺-naive T cells were prepared by MACS negative selection, using antibodies against mouse cells (anti-mCD45 and Ter119) and human cells expressing CD8, CD14, CD19, CD25, or CD45RO and transduced with LV-insTCR. (A) FCM profiles of human CD45⁺, CD3⁺, CD19⁺, CD3⁺CD4⁺, and CD4⁺CD8⁺ cells in the pooled spleen cells from donor hu-mice. (B) Purity of the MACS-selected hu-mouse spleen cells. Shown are the percentage of human CD3⁺CD4⁺ T cells and their expression of CD45RO, CD45RA, and CD25. (C) Expression of human CD3, CD4, and TCRv_β11 on the in vitro expanded LV-insTCR–transduced GFP⁺ cells.

Fig. S3.

Expansion of lentivirally transduced human CD4⁺ T cells in vitro. LV-insTCR–transduced hu-mouse–derived human CD4 T cells were expanded in vitro for 13 d. Data from two representative experiments are shown and presented as fold of expansion in cell numbers.

Fig. S4.

Survival of infused human T cells and levels of blood glucose in mice after infusion of human CD4 T cells. (A and B) LV-insTCR–transduced hu-mouse–derived human CD4 T cells were expanded for 13 d and injected into HLA-DQ8–Tg NSG mice (n = 2) or HLA-DQ8–Tg hu-mice grafted 14 wk earlier with human CD34⁺ FLCs (n = 2). (A) FCM analysis of surviving LV-insTCR–transduced GFP⁺CD4⁺ human T cells in spleen and bone marrow from hu-mouse (Left) and NSG mouse (Right) recipients at the indicated times after human T-cell transfer. (B) Blood glucose levels. (C) HLA-DQ8–Tg hu-mice grafted 14 wk earlier with human CD34⁺ FLCs and FTHY were treated by two injections of low-dose streptozotocin, followed 1 d later by infusion of expanded LV-insTCR–transduced (n = 2) or control (n = 2) hu-mouse–derived human CD4 T cells. Shown are blood glucose levels.

Fig. 3.

Survival of the infused LV-insTCR–transduced human CD4⁺ T cells in hu-mice. (A) Survival of GFP⁺CD4⁺ T cells in peripheral blood from hu-mice at 3 and 6 d after cell transfer. Data from two hu-mice at each point are shown. (B) Presence of GFP⁺CD4⁺ human T cells in spleen, liver, and bone marrow cells of two representative hu-mice analyzed at 3 and 5 wk, respectively, after cell transfer. (C) Pancreatic islets were prepared from hu-mice 11 d after infusion of LV-insTCR–transduced human CD4⁺ T cells, digested with trypsin, and stained with the indicated antibodies to detect human cell infiltration by flow cytometry. (D) Pancreatic tissue sections were prepared from hu-mice between 3 and 4 wk after injection of LV-InsTCR–transduced GFP⁺ human T cells (n = 3), and stained with anti-GFP antibodies. Shown are representative immunohistochemistry images of pancreas sections from hu-mice receiving LV-InsTCR–transduced GFP⁺ (Top) or control (Bottom) human T cells.

Fig. 4.

Induction of diabetes in HLA-DQ8–Tg hu-mice by adoptive transfer of autologous diabetogenic human CD4⁺ T cells. HLA-DQ8–Tg hu-mice were treated with low-dose streptozotocin and injected 1–2 d later with 5 × 10⁶ expanded LV-insTCR–transduced or control (i.e., the same hu-mouse–derived human CD4⁺ T cells that were similarly expanded in vitro as the LV-insTCR–transduced CD4⁺ T cells) human CD4⁺ T cells, followed 1 d later by immunization with InsB:9–23 peptides. (A) Cumulative incidence of diabetes (Top) and levels of blood glucose (Bottom) in hu-mice receiving LV-insTCR–transduced (solid symbol; n = 7) or control (opened symbol; n = 6) human T cells. Mice were defined as hyperglycemia if two consecutive blood glucose measurements >200 mg/dL (B and C) Immunofluorescent staining of pancreas samples prepared between 3 and 4 wk after injection of CD4⁺ T cells from hu-mice receiving control (Left) or LV-InsTCR–transduced (Right) human T cells (n = 3 per group). (B) Staining of mouse insulin (yellow) and glucagon (red). (C) Staining of human CD3⁺ cells (green), mouse insulin (pink) and glucagon (red). Nuclear is stained blue by DAPI.

Fig. S5.

Comparison of the survival of infused human T cells in humanized versus nonhumanized NSG mice. Ex vivo expanded hu-mouse–derived human T cells, which were transduced with insB:9–23-specific TCR/GFP, were injected i.v. to hu-mice with autologous human lymphohematopoietic cells or nonhumanized NSG mice. Blood was collected at days 5, 11, and 19 after adoptive transfer, and numbers of the transferred (GFP⁺) T cells were determined. Shown are GFP⁺ T-cell counts (mean ± SEM; n = 5 per group).