Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1997 Nov 17;186(10):1663-76.
doi: 10.1084/jem.186.10.1663.

Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice

J Verdaguer  1 , D Schmidt, A Amrani, B Anderson, N Averill, P Santamaria
Affiliations

Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice

J Verdaguer et al. J Exp Med. .

Abstract

It has been established that insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a CD4+ and CD8+ T cell-dependent autoimmune process directed against the pancreatic beta cells. The precise roles that beta cell-reactive CD8+ and CD4+ T cells play in the disease process, however, remain ill defined. Here we have investigated whether naive beta cell-specific CD8+ and CD4+ T cells can spontaneously accumulate in pancreatic islets, differentiate into effector cells, and destroy beta cells in the absence of other T cell specificities. This was done by introducing Kd- or I-Ag7-restricted beta cell-specific T cell receptor (TCR) transgenes that are highly diabetogenic in NOD mice (8.3- and 4.1-TCR, respectively), into recombination-activating gene (RAG)-2-deficient NOD mice, which cannot rearrange endogenous TCR genes and thus bear monoclonal TCR repertoires. We show that while RAG-2(-/-) 4.1-NOD mice, which only bear beta cell-specific CD4+ T cells, develop diabetes as early and as frequently as RAG-2+ 4.1-NOD mice, RAG-2(-/-) 8.3-NOD mice, which only bear beta cell-specific CD8+ T cells, develop diabetes less frequently and significantly later than RAG-2(+) 8.3-NOD mice. The monoclonal CD8+ T cells of RAG-2(-/-) 8.3-NOD mice mature properly, proliferate vigorously in response to antigenic stimulation in vitro, and can differentiate into beta cell-cytotoxic T cells in vivo, but do not efficiently accumulate in islets in the absence of a CD4+ T cell-derived signal, which can be provided by splenic CD4+ T cells from nontransgenic NOD mice. These results demonstrate that naive beta cell- specific CD8+ and CD4+ T cells can trigger diabetes in the absence of other T or B cell specificities, but suggest that efficient recruitment of naive diabetogenic beta cell-reactive CD8+ T cells to islets requires the assistance of beta cell-reactive CD4+ T cells.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Expression of the TCR-α/β transgenes in 8.3-NOD mice. CD4, CD8, and Vβ8.1/8.2 profiles of thymocytes (A) and lymph node cells (B) from nontransgenic NOD, 8.3–TCR-β–transgenic NOD, and 8.3-NOD mice. (Top) CD4 versus CD8 contour plots of cell suspensions stained with anti-CD8-PE, anti-V β8.1/8.2-FITC, and anti-CD4-biotin plus Streptavidin-PerCP. The lower panels show the Vβ8.1/8.2 fluorescence histograms of each T cell subset after electronic gating. Numbers indicate the average percentage of cells (top) or the average number of Vβ8.1/8.2+ cells (bottom) in each subset. Data correspond to 3–9 mice/group. DP, double-positive cells; DN, double-negative cells.
Figure 2
Figure 2
Responsiveness and peripheral frequency of beta cell–specific CD8+ T cells in 8.3-NOD mice. (A) Proliferation of splenic CD8+ T cells from 8.3-NOD and 8.3–TCR-β–transgenic NOD mice to islet cells. 2 × 104 splenic CD8+ T cells were incubated with γ-irradiated islet cells for 3 d, pulsed with [3H]thymidine, harvested, and counted. Bars show the standard error of the means. (B) Peripheral frequency of beta cell–reactive CD8+ T cells in 8.3-NOD and 8.3–TCR-β–transgenic NOD mice. 12 replicate cultures of serial dilutions of splenocytes (101– 105 cells/well) were stimulated with irradiated NOD islets (8/well) for 4 d, expanded in rIL-2 (0.5 U/ml) for 10 d and restimulated once with islets and rIL-2. The cultures were then challenged with 104 NIT-1 or L929-Kd cells for 24 h, and the supernatants collected to measure their TNF-α content. Cultures that secreted TNF-α in response to NIT-1, but not L929-Kd, cells were considered to contain beta cell–reactive CD8+ T cells. (C) General proliferative activity of splenic CD8+ T cells of 8.3-NOD and 8.3–TCR-β–transgenic NOD mice. 2 × 104 splenic CD8+ T cells were incubated with 10-fold serial dilutions of plate-bound KJ16 in rIL-2–containing CM for 3 d, pulsed with [3H]thymidine, harvested, and counted.
Figure 3
Figure 3
8.3–TCR-α/β– transgene expression and diabetogenesis. (A) Incidence of IDDM in female (26 8.3–, 21 8.3–TCR-β–transgenic, and 114 nontransgenic) and male (24 8.3–, 30 8.3–TCR-β–transgenic, and 59 nontransgenic) NOD mice. (B) Progression of insulitis in nontransgenic, 8.3– TCR-β–transgenic, and 8.3-NOD mice (4–7 mice/age group; 15–30 islets/mouse). Bars show the standard deviation of the means. *, P <0.0001 (Mann-Whitney U test). (C) Flow cytometry profile of islet-derived T cells from diabetic 8.3-NOD mice. Islets from acutely diabetic 8.3-NOD mice were cultured in rIL-2–containing CM for 3–5 d and the resulting cells studied by flow cytometry as in Fig. 1. (D) Phenotype of islet-infiltrating T cells in 8.3-NOD versus nontransgenic NOD mice. Pancreas sections were stained with anti-CD8 (53.6-7) or anti-CD4 (GK1.5) mAbs and FITC-labeled anti–rat IgG, and observed under a fluorescence microscope. Original magnification: 200.
Figure 4
Figure 4
Diabetogenesis in TCR-transgenic RAG-2−/− NOD mice. (A) Flow cytometry profiles of lymph node cells from RAG-2−/− 4.1-NOD mice (left) and RAG-2−/− 8.3-NOD mice (right). (B) Incidence of diabetes in RAG-2−/− 4.1-NOD (n = 29 females and 35 males), RAG-2−/− 8.3-NOD (n = 12 females and 20 males), and RAG-2−/− NOD mice (n = 20 females and 20 males). The few cells of the opposite T cell subset that appear in the flow cytometry profiles of these mice are the result of nonspecific staining of dead cells. (C) Phenotype of islet-infiltrating T cells in RAG-2−/− 4.1-NOD and RAG-2−/− 8.3-NOD mice. Most of the few CD8+ T cells in RAG-2−/− 4.1-NOD mice, and the few CD4+ T cells in RAG-2−/− 8.3-NOD mice were the result of background staining, as they were also seen in anti–rat IgG-FITC–stained tissue.
Figure 5
Figure 5
Phenotype and functional activity of CD8+ T cells from RAG-2−/− versus RAG-2+ 8.3-NOD mice. (A) Flow cytometry profiles of CD4+CD8+ (left) and CD4−CD8+ thymocytes (right) from 8.3-NOD mice (dotted line) and RAG-2−/− 8.3-NOD mice (solid line) for maturation markers. Thymocytes were analyzed by three-color cytofluorometry as in Fig. 1. Panels show the fluorescence histograms of each marker on gated CD4+CD8+ and CD4−CD8+ thymocytes. (B) Flow cytometry profiles of splenic CD8+ T cells from 8.3-NOD mice (dotted line) and RAG-2−/− 8.3-NOD mice (solid line) for activation and memory markers. (C) Proliferative activity of splenic CD8+ T cells from 8.3-NOD mice and RAG-2−/− 8.3-NOD mice in response to islet cells. (D) Reverse transcription-PCR analysis for cytokine gene expression of islet-derived CD8+ T cells from 8.3-NOD mice and RAG-2−/− 8.3-NOD mice. M, 1Kb ladder. (E) Kinetics of insulitis in RAG-2−/− 8.3-NOD mice.
See this image and copyright information in PMC

References

    1. Serreze DV, Leiter EH. Genetic and pathogenic basis of autoimmune diabetes in NOD mice. Curr Opin Immunol. 1994;6:900–906. - PubMed
    1. Tisch R, McDevitt HO. Insulin-dependent diabetes mellitus. Cell. 1996;85:291–297. - PubMed
    1. Bendelac A, Carnaud C, Boitard C, Bach J-F. Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates. Requirement for both L3T4+ and Lyt-2+T cells. J Exp Med. 1987;166:823–832. - PMC - PubMed
    1. Miller BJ, Appel MC, O'Neil JJ, Wicker LS. Both the Lyt-2+ and L3T4+T cell subsets are required for the transfer of diabetes in non-obese diabetic mice. J Immunol. 1988;140:52–58. - PubMed
    1. Yagi H, Matsumoto M, Kunimoto K, Kawaguchi J, Makino S, Harada M. Analysis of the roles of CD4+ and CD8+ T cells in autoimmune diabetes of NOD mice using transfer to NOD athymic nude mice. Eur J Immunol. 1992;22:2387–2393. - PubMed

Publication types

MeSH terms

Substances